WO2014052613A2

WO2014052613A2 - Methods and compositions relating to next generation sequencing for genetic testing in alk related cancers

Info

Publication number: WO2014052613A2
Application number: PCT/US2013/061950
Authority: WO
Inventors: David R. Hout; Eric Dahlhauser; Adam Platt
Original assignee: Insight Genetics Inc
Current assignee: Insight Genetics Inc
Priority date: 2012-09-26
Filing date: 2013-09-26
Publication date: 2014-04-03
Anticipated expiration: 2015-03-26
Also published as: WO2014052613A3; CA2886397A1

Description

METHODS AND COMPOSITIONS RELATING TO NEXT GENERATION SEQUENCING FOR GENETIC TESTING IN ALATRELATED CANCERS

BACKGROUND

Mutations of anaplastic lymphoma kinase (ALK) gene are thought to be involved in the development of subsets of numerous cancers including i) non-small cell lung carcinoma (NSCLC); ii) diffuse large B-cell lymphoma; iii) esophageal squamous cell carcinoma; iv) anaplastic large-cell lymphoma (ALCL); v) neuroblastoma (a childhood cancer that arises from the developing peripheral nervous system); and vi) the sarcomas known as inflammatory myofibroblastic tumors (IMTs). Patient outcomes with many of these malignancies are poor, due in part to the late detection of the cancers because of the lack of efficient clinical diagnostic methods. Early detection and diagnosis oiALK- mediated cancers dramatically increases survival rates within the patient population; as an example, early detection of ALK-positb/Q anaplastic large-cell lymphoma can result in survival rates of up to 83% whereas late detection is associated in some instances with survival of only 50% of the patient population.

The critical role of deregulated ALK signaling as a driver of subsets of NSCLC, ALCL, and other ALK-dspsndsnt cancer types has been validated in clinical trials, with dramatic anti-tumor efficacy observed in response to the ALK small-molecule inhibitor crizotinib (XALKORI^®, Pfizer; approved by the US FDA in August 201 1).

Unfortunately, despite the marked anti-tumor responses to XALKORI^® seen in patients with ALK-drb/sn tumors, most patients eventually experience progression of their cancer as a consequence of treatment resistance. For example, the median duration of

progression-free survival in patients with ALK-positb/Q NSCLC treated with Xalkori is only about 10 months. What is needed are assays the can efficiently and reliably detect kinase inhibitor-resistance mutations and therefore predict which members of a patient population is likely to develop kinase inhibitor resistance. Additionally as new

generations of small-molecule inhibitors are developed, also need is a clinically applicable diagnostic test to identify resistance mutations in the ALK kinase domain and therefore to guide the rational use of these small-molecule inhibitors for the treatment of ALK-drwen cancers that have lost their responsiveness to -generation inhibitor therapy. Moreover, once several ALK small-molecule inhibitors are approved for clinical use, optimal management of patients with

tumors will require screening for de novo inhibitor resistance mutations by healthcare providers treating newly diagnosed patients in order to assess their inhibitor sensitivity and choose the best ALK inhibitor drug(s) for personalized therapy.

BRIEF SUMMARY

The methods, assays, and compositions disclosed herein relate to the field of detection or diagnosis of mutations that confer resistance to kinase inhibitors of a disease or condition such as cancer. In one aspect, the kinase inhibitors or ALK kinase inhibitors. Also disclosed herein are methods and assays for assessing the susceptibility or risk for developing resistance to an inhibitor, wherein the disease or condition is a cancer associated with expression of the ALK gene. It is understood and herein contemplated that the methods disclosed herein allow for rapid and sensitive detection of nucleic acid expression of mutations in ALK.

In another aspect, disclosed herein are kinase inhibitor resistance panels comprising one or more primer sets from each of the genes selected from group of genes comprising KRAS, BRAF, EGFR, ALK, and KIT.

In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, relates to an ALK kinase inhibitor resistance panel. In particular, the invention, in one aspect, relates to an ALK kinase inhibitor resistance panel comprising one or more primer sets for detecting the presence of a mutation in a gene that will confer resistance to the ALK kinase inhibitor.

Additional advantages of the disclosed methods and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice of the disclosed method and compositions. The advantages of the disclosed methods and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

BRIEF DESRIPTION OF THE DRAWINGS

Figure 1 shows XALKORI®-resistance mutations identified in patient specimens.

The figure depicts the XALKORI®-resistance mutations in the ALK kinase domain identified to date in patient cancer specimens.

DETAILED DESCRIPTION

Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

Ranges can be expressed herein as from "about" one particular value, and/or to

"about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is disclosed the "less than or equal to 10"as well as "greater than or equal to 10" is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15.

In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

"Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

An "increase" can refer to any change that results in a larger amount of a composition or compound, such as an amplification product relative to a control. Thus, for example, an increase in the amount in amplification products can include but is not limited to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 2500%, 3000%, 3500%, 4000%, 4500%, or 5000% increase. It is further contemplated herein that the detection an increase in expression or abundance of a DNA, mRNA, or protein relative to a control necessarily includes detection of the presence of the DNA, mRNA, or protein in situations where the DNA, mRNA, or protein is not present in the control.

"Obtaining a tissue sample" or "obtain a tissue sample" means to collect a sample of tissue either from a party having previously harvested the tissue or harvesting directly from a subject. It is understood and herein contemplated that tissue samples obtained directly from the subject can be obtained by any means known in the art including invasive and noninvasive techniques. It is also understood that methods of measurement can be direct or indirect. Examples of methods of obtaining or measuring a tissue sample can include but are not limited to tissue biopsy, tissue lavage, blood collection, aspiration, tissue swab, spinal tap, magnetic resonance imaging (MRI), Computed Tomography (CT) scan, Positron Emission Tomography (PET) scan, and X-ray (with and without contrast media). It is further understood that a "tissue" can include, but is not limited to any grouping of one or more cells or analytes to be used in a an ex vivo or in vitro assays. Such tissues include but are not limited to blood, saliva, sputum, lymph, cellular mass, and tissue collected from a biopsy.

Kinase Inhibitor Resistant Panels

In one aspect, disclosed herein are kinase inhibitor resistance panels such as, for example, an ALK kinase inhibitor panel. Kinase inhibitors are known in the art and have found use in the treatment of, amongst other things, the treatment of cancer. For example, cancers involving the overexpression or fusion of Analplastic Lymphoma Kinase can be treated through the use of a kinase inhibitor. Kinase inhibitors are known in the art and include, but are not limited to crizotinib, afatinib, Axitinib, bevacizumab, Bosutinib, Cetuximab, Dasatinib, Erlotinib, Fostamati nib, Gefitinib, Imatinib, Lapatinib, Lenvatinib, Nilotinib, Panitumumab, Pazopanib, Pegaptanib, Ranibizumab, Ruxolitinib, Sorafenib,

Sunitinib, Trastuzumab, and Vemurafenib. Thus, in one aspect, disclosed herein are kinase inhibitor resistance panels for detecting susceptibility or resistance to treatment in a subject to a kinase inhibitor comprising crizotinib, afatinib, Axitinib, bevacizumab, Bosutinib, Cetuximab, Dasatinib, Erlotinib, Fostamati nib, Gefitinib, Imatinib, Lapatinib, Lenvatinib, Nilotinib, Panitumumab, Pazopanib, Pegaptanib, Ranibizumab, Ruxolitinib, Sorafenib, Sunitinib, Trastuzumab, or Vemurafenib.

Unfortunately, mutations in the ALK sequence and other genes, such as, BRAF, KIT, KRAS, and EGFR can lead to kinase inhibitor resistance. These mutations can comprise any of the mutations to ALK, KIT, BRAF, KRAS, or EGFR listed in Tables 2, 3, 4, 5, or 6.

Accordingly, in a further aspect, disclosed herein are kinase inhibitor panels comprising one or more primer sets that selectively hybridize and can be used to amplify one of the genes selected from group of genes comprising KRAS(SEQ ID NO: 7718), BRAF(SEQ ID NO: 7717), EGFR (SEQ ID NO: 7716), ALK (SEQ ID NO: 7714 and SEQ ID NO: 7717

(cDNA)), and KIT. In one aspect, the kinase inhibitor resistance panel disclosed herein can comprise one or more primer set(s) that hybridizes and amplifies nucleic acid from exon 1 (SEQ ID NOs: 4601-4880 and 7181-7230) exon 2 (SEQ ID NOs: 4881-5200 and 7231- 7326) or both exons 1 and 2 (SEQ ID NOs: 7327-7610) ofKRAS; exon 18 (SEQ ID NOs: 1641 - 1760 and 5819-5934), exon 19 (SEQ ID NOs : 1761 - 1880), exon 20 (SEQ ID NOs : 1881-2000 and 5934-6042), exon 21 (SEQ ID NOs: 2001-2120 and 6043-6150), exon 22 (SEQ ID NOs: 2121-2240, 2321-2360, and 2401-2440), exons 18 and 19 (SEQ ID NOs: 2241-2280), exons 18, 19, and 20 (SEQ ID NOs: 6151-6274), exons 20 and 21 (SEQ ID NOs: 2281-2320 and 6275-6388), or exons 18, 19, 20, and 21 (SEQ ID NOs: 2361-2400 and 6389-6524) oiEGFR; exon 8 (SEQ ID NOs: 2441-2800), exon 9 (SEQ ID NOs: 2841- 3120), exon 10 (SEQ ID NOs: 3201-3360), exon 11 (SEQ ID NOs: 3361-3480), exon 12 (SEQ ID NOs: 3481-3640), exon 13 (SEQ ID NOs: 3641-3800), exon 17 (SEQ ID NOs: 4241-4600), exon 8 and 9 (SEQ ID NOs: 2801-2840), exons 9 and 10 (SEQ ID NOs: 3121- 3160), exons 9, 10, and 11 (SEQ ID NOs: 3161-3200); exons 10 and 11 (SEQ ID NOs: 3801-3960), exons 12 and 13 (SEQ ID NOs: 3961-4120), or exons 10, 11, 12, and 13 (SEQ ID NOs: 4121-4240) of KIT; exons 10 and 11 (SEQ ID NOs: 6525-6832) or exons 13, 14, or 15 (SEQ ID NOs: 66833-7180) oiBRAF, and/or exon 21 (SEQ ID NOs: 1-160), exon 22 (SEQ ID NOs: 401-560), exon 23 (SEQ ID NOs: 561-840 and 5311-5446), exon 24 (SEQ ID NOs: 921-1240), exon 25 (SEQ ID NOs: 1241-1600), exons 21 and 22 (SEQ ID NOs: 161-400 and 5201-5310), exons 21, 22, and 23 (SEQ ID NOs: 841-920), exons 24 and 25 (SEQ ID NOs: 1601-1640 and 5447-5576), or exons 21, 22, 23, 24, and 25 (SEQ ID NOs: 5577-5818) of ALK As disclosed herein "primer set" refers to a forward and reverse primer pair (i.e., a left and right primer pair) that can be used together to amplify a given region of a nucleic acid (e.g., DNA, RNA, or cDNA) of interest. Thus, panels with multiple primer sets include multiple primer pairs. It is understood and herein contemplated that some primer sets may have a common forward or reverse primer and thus have an odd number of primers.

It is further understood and herein contemplated that the disclosed kinase inhibitor resistant panels can comprise a single primer sets that hybridizes to a single gene, region, or exon of a gene selected from the group of genes comprising KRAS, BRAF, EGFR, ALK, and KIT (i. Q, a single primer sets for KRAS, BRAF, EGFR, ALK, or KIT); multiple primer sets that hybridize to a single gene, region, or exon of a gene selected from the group of genes comprising KRAS, BRAF, EGFR, ALK, and KIT (i.e, one or more primer sets for KRAS, BRAF, EGFR, ALK, or KIT); multiple primer sets comprising a single primer set that specifically hybridize to a single gene, region, or exon for each of the genes comprising KRAS, BRAF, EGFR, ALK, and KIT (i.e, a single primer set for each oiKRAS, BRAF, EGFR, ALK, and/or KIT); or multiple primer sets comprising where in there is more than one primer set for each gene, region or exon for each of the genes selected from the group of genes comprising KRAS, BRAF, EGFR, ALK, and KIT (i.e, one or more primer sets for each oiKRAS, BRAF, EGFR, ALK, and/or KIT). Thus, it is contemplated herein that the kinase inhibitor panel can comprise primer sets that recognize and specifically hybridize to a gene, region, or exon, of one or combination of the gene selected from the group consisting oiKRAS, BRAF, EGFR, ALK, and KIT. For example, the panel can comprise primer sets that hybridize to a gene, region, or exon oiKRAS, BRAF, EGFR, ALK, or KIT; KRAS and BRAF; KRAS and EGFR; KRAS and ALK; KRAS and KIT; BRAF and EGFR; BRAF and KIT; BRAF and ALK; EGFR and ALK; EGFR and KIT; ALK and KIT; KRAS, BRAF, and EGFR; KRAS, BRAF, and ALK; KRAS, BRAF, and KIT; KRAS, EGFR, and ALK; KRAS, EGFR, and KIT; KRAS, ALK, and KIT; BRAF, EGFR, and ALK, BRAF,

EGFR, and KIT; BRAF, ALK, and KIT; EGFR, ALK, and KIT; KRAS, BRAF, EGFR, and ALK; KRAS, BRAF, EGFR, and KIT, BRAF, EGFR, ALK, and KIT; and KRAS, BRAF, EGFR, ALK, and KIT. For example, the primer or primer sets in the kinase inhibitor resistance panel can detect any of the mutations in Tables 2-6. In another aspect, the primers or primer sets used in the inhibitor resistance panel can comprise one or more of the primers or primer sets listed in Tables 7-14 as disclosed herein and/or probes listed in Table 15 (i.e., SEQ ID NOs: 7611-7613).

Methods of detecting the presence of a kinase inhibitor resistant cancer

The disclosed kinase inhibitor resistant panels, in one aspect, contain primers or primer sets for the detection of mutations that confer kinase inhibitor resistance. Thus, in another aspect disclosed herein are methods and assays for the detection of kinase inhibitor resistant forms of an ALK-rslatsd cancer. For example, disclosed herein are methods and assays for the detection of kinase inhibitor resistance, such as, for example ALK kinase inhibitor resistance, comprising obtaining a tissue sample from a subject with a cancer, such as a kinase related cancer (e.g., ALK-related cancers); conducting a high throughput sequencing (also known as next generation sequencing) reaction on the sample, wherein the presence of a mutation in the nucleic acid sequence of a gene associated with kinase inhibitor resistance indicates that that the cancer is resistant or will become resistant to a kinase inhibitor. In one aspect, the mutation can be a nucleic acid mutation in ALK, EGFR, KRAS, BRAF, or KIT. For example, the mutation can be any mutation listed in Tables 2-6. In a further aspect, the disclosed methods and assays for detection of kinase inhibitor resistance can comprise performing next generation sequencing using a kinase inhibitor resistant panel as disclosed herein which comprises a primer or primer set that hybridizes and amplifies nucleic acid from exon 1 or 2 oiKRAS; exon 18, 19, 20, 21 or 22 oiEGFR; exon 8, 9, 10, 1 1, 12, 13, or 17 οΐΚΙΤ; exon 10, 11, 13, 14, or 15 oiBRAF, and/or exon 21 , 22, 23, 24, or 25 oiALK. For example, the primer or primer set can comprise any of the primers or primer sets disclosed in Tables 7-14. Thus, disclosed herein are methods wherein the one or more primer set(s) that hybridizes and amplifies nucleic acid from exon 1 (SEQ ID NOs: 4601-4880 and 7181-7230) exon 2 (SEQ ID NOs: 4881-5200 and 7231- 7326) or both exons 1 and 2 (SEQ ID NOs: 7327-7610) oiKRAS; exon 18 (SEQ ID NOs: 1641 - 1760 and 5819-5934), exon 19 (SEQ ID NOs : 1761 - 1880), exon 20 (SEQ ID NOs : 1881-2000 and 5934-6042), exon 21 (SEQ ID NOs: 2001-2120 and 6043-6150), exon 22 (SEQ ID NOs: 2121-2240, 2321-2360, and 2401-2440), exons 18 and 19 (SEQ ID NOs: 2241-2280), exons 18, 19, and 20 (SEQ ID NOs: 6151-6274), exons 20 and 21 (SEQ ID NOs: 2281-2320 and 6275-6388), or exons 18, 19, 20, and 21 (SEQ ID NOs: 2361-2400 and 6389-6524) oiEGFR; exon 8 (SEQ ID NOs: 2441-2800), exon 9 (SEQ ID NOs: 2841- 3120), exon 10 (SEQ ID NOs: 3201-3360), exon 11 (SEQ ID NOs: 3361-3480), exon 12 (SEQ ID NOs: 3481-3640), exon 13 (SEQ ID NOs: 3641-3800), exon 17 (SEQ ID NOs: 4241-4600), exon 8 and 9 (SEQ ID NOs: 2801-2840), exons 9 and 10 (SEQ ID NOs: 3121- 3160), exons 9, 10, and 11 (SEQ ID NOs: 3161-3200); exons 10 and 11 (SEQ ID NOs: 3801-3960), exons 12 and 13 (SEQ ID NOs: 3961-4120), or exons 10, 11, 12, and 13 (SEQ ID NOs: 4121-4240) οΐΚΙΤ; exons 10 and 11 (SEQ ID NOs: 6525-6832) or exons 13, 14, or 15 (SEQ ID NOs: 66833-7180) oiBRAF, and/or exon 21 (SEQ ID NOs: 1-160), exon 22 (SEQ ID NOs: 401-560), exon 23 (SEQ ID NOs: 561-840 and 5311-5446), exon 24 (SEQ ID NOs: 921-1240), exon 25 (SEQ ID NOs: 1241-1600), exons 21 and 22 (SEQ ID NOs: 161-400 and 5201-5310), exons 21, 22, and 23 (SEQ ID NOs: 841-920), exons 24 and 25 (SEQ ID NOs: 1601-1640 and 5447-5576), or exons 21, 22, 23, 24, and 25 (SEQ ID NOs: 5577-5818) oiALK.

It is understood that the disclosed methods can further comprise synthesizing cDNA from the nucleic acid extracted from a tissue sample before detection of a mutation in ALK, EGFR, KRAS, BRAF, or KIT. Thus, in one aspect, disclosed herein are methods for detecting kinase inhibitor resistance in a cancer in a subject, for example ALK kinase inhibitor resistance, comprising obtaining a tissue sample from a subject with a cancer, such as a kinase related cancer (e.g., ALK-rolatsd cancers); synthesixing cDNA from the tissue sample, and conducting a high throughput sequencing (also known as next generation sequencing) reaction on the sample, wherein the presence of a mutation in the nucleic acid sequence of a gene associated with kinase inhibitor resistance indicates that that the cancer is resistant or will become resistant to a kinase inhibitor.

It is further understood and herein contemplated that the subject of the disclosed methods can be a subject that has been previously diagnosed with a cancer including but not limited to inflammatory breast cancer, non-small cell lung carcinoma, esophageal squamous cell carcinoma, colorectal carcinoma, Inflammatory myofibroblastic tumor, familial and sporadic neuroblastoma. In yet another aspect, the subject has been previously diagnosed with a cancer that results from ALK, ROS1, RET, DEPDC1 overexpression, dysregulation, or fusion. Examples of such fusions include but are not limited to nucleophosmin-^Z T (NPM-ALK), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC-ALK), clathrin heavy chain-ALK (CLTC-ALK), kinesin-1 heavy chain gene-ALK (KIF5B-ALK); Ran-binding protein 2-ALK (RANBP2- ALK), SEC31L1- ALK, tropomyosin^-.^ (ΤΡΜ3-.4Ζ 0, tropomyos -4-ALK (TFM4-ALK), TRK-fused gene(Large) -ALK (TFG_L-ALK), TRK-fused gene(Small) -ALK (TFGs-ALK), CARS-ALK, EML4-^ ^, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase-ALK (ATIC-ALK), AL017 -ALK, moQsin- ALK (MSN -ALK), non-muscle myosin heavy chain gsns-ALK (MYH9-ALK), and TRK-fused gene(Extra Large) -ALK (TFGXL-ALK). In a further aspect, the present methods could not only be used to diagnose a kinase inhibitor resistant cancer , but diagnose the cancer itself as the subject with a kinase inhibitor resistant cancer would necessarily not only have a cancer, but have a kinase related cancer such as those disclosed herein .

Therefore, in one aspect, disclosed herein are methods for the detection of kinase inhibitor resistance comprising obtaining a tissue sample from a subject with a cancer and conducting a high throughput sequencing (also known as next generation sequencing) reaction on the sample using one or more primer sets or primer panels with primer sets that specifically hybridizes to one or more of the genes selected from the group consisting of ALK, KRAS, EGFR, KIT, and BRAF, wherein the presence of a mutation in the nucleic acid sequence of a gene associated with kinase inhibitor resistance indicates that that the cancer is resistant or will become resistant to a kinase inhibitor.

Also disclosed are methods, wherein at least one primer sets hybridizes and amplifies nucleic acid from exon 1 or 2 oiKRAS, wherein at least one primer sets hybridizes and amplifies nucleic acid from exon 18, 19, 20, 21 or 22 oiEGFR, wherein at least one primer sets hybridizes and amplifies nucleic acid from exon 21, 22, 23, 24, or 25 oiALK, wherein at least one primer sets hybridizes and amplifies nucleic acid from exon 8, 9, 10, 11, 12, 13, or 17 οΐΚΙΤ, and/or wherein at least one primer sets hybridizes and amplifies nucleic acid from exon 10, 11, 13, 14, or 15 oiBRAF.

In one aspect, disclosed are methods, wherein one or more KRAS hybridizing primers or primer sets comprise one or more of the primers of Tables 10 and/or 14 (SEQ ID NOs: 4601-5200 and 7181-7610); wherein one or more EGFR hybridizing primers or primer sets comprise one or more of the primers of Tables 8 and/or 12 (1641-2440 and 5819-6524); wherein one or more ALK hybridizing primers or primer sets comprise one or more of the primers of Tables 7 and/or 1 1 (SEQ ID NOs: 1-1640 and 5201-5818); wherein one or more KIT hybridizing primers or primer sets comprise one or more of the primers of Table 9 ( SEQ ID NOs: 2441-4600); and/or wherein one or more BRAF hybridizing primers or primer sets comprise one or more of the primers of Table 13 (SEQ ID NOs: 6525-7180).

In one aspect are methods comprising the use of a kinase inhibitor resistance panel, wherein the panel comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more primer sets for one or more of the genes selected from group of genes comprising KRAS, BRAF, EGFR, ALK, and KIT.

In another aspect, disclosed are methods wherein the panel comprises one or more primer sets for 2, 3, 4, of all 5 of the genes selected from group of genes comprising KRAS, BRAF, EGFR, ALK, and KIT

Also disclosed are methods, wherein the kinase inhibitor is selected from the group consisting of crizotinib, afatinib, Axitinib, bevacizumab, Bosutinib, Cetuximab, Dasatinib, Erlotinib, Fostamati nib, Gefitinib, Imatinib, Lapatinib, Lenvatinib, Nilotinib, Panitumumab, Pazopanib, Pegaptanib, Ranibizumab, Ruxolitinib, Sorafenib, Sunitinib, Trastuzumab, and Vemurafenib.

Methods, assays, and primer panels for assessing the suitability of ALK directed treatments

Though not wishing to be bound by current theories, it is believed that inhibition of these over-expression or aberrant expressions of ALK it small-molecule drug candidates abrogates related abnormal cell proliferation and promotes apoptosis in ALK-rslatsd tumor cell lines. Furthermore, both preclinical animal models and the early clinical experience with these inhibitors indicate that ALK small-molecule inhibitors not only possess marked antitumor activity against ^Z T-related cancers but are also very well tolerated with no limiting target-associated toxicities. Therefore, such small molecules can be used to treat ALK-AnvQn cancers.

However, the presence of a mutation in one of the genes associated with an ALK- related cancer can confer resistance to treatment with a kinase inhibitor, such as an ALK kinase inhibitor. Nevertheless, knowledge of the presence of said mutation can still be useful to the practicing physician in assessing the suitability of a treatment or prescribing a particular treatment regimen. For example, the presence of a mutation in a gene which confers kinase inhibitor resistance, such as, for example, ALK kinase inhibitor resistance, can inform the skilled artisan to choose a particular kinase inhibitor over another due to the presence of a mutation affecting one kinase inhibitor and not the other. Alternatively, the presence of a mutation can inform the physician to discontinue the course of treatment with one kinase inhibitor due to detection of kinase inhibitor resistance and select a different kinase inhibitor to which the patient is not yet resistant. Accordingly, disclosed herein are methods and assays for assessing the suitability of an ALK inhibitor treatment for a cancer, for example, NSCLC, in a subject comprising performing high throughput sequencing on nucleic acid from a tissue sample from the subject; wherein the presence of a mutation in ALK, EGFR, BRAF, KRAS, or KIT indicates a cancer that comprises resistance to an ALK kinase inhibitor. In one aspect, disclosed herein are methods and assays for assessing a subject's suitability for treatment with a kinase inhibitor comprising obtaining a tissue sample from a subject with a cancer, such as a kinase related cancer (e.g., ALK-rslatsd cancers); detecting the presence of a mutation through sequencing or other nucleic acid detection technique for the presence of a mutation in the nucleic acid sequence of a gene associated with kinase inhibitor resistance indicates that that the cancer is resistant or will become resistant to a kinase inhibitor and therefore continued use of an inhibitor to which the cancer has become resistant or to which the cancer is already resistant should be discontinued in favor of a cancer to which resistance has not developed.

It is understood and herein contemplated that any of the disclosed nucleic acid sequencing techniques disclosed herein can be used in these methods. Thus, disclosed herein are methods and assays assessing the suitability of an ALK kinase inhibitor treatment for an ALK related cancer in a subject comprising conducting high throughput sequencing (also known as next generation sequencing) on nucleic acid such as mRNA or DNA from a tissue sample from the subject; wherein the sequencing reaction reveals the nucleic acid sequence for one or more exons οΐΚΙΤ, BRAF, KRAS, EGFR, and ALK; and wherein the presence of one or more mutations in KIT, BRAF, KRAS, EGFR, and/or ALK indicates the presence of kinase inhibitor resistance. The mutations can occur in any exon οΐΚΙΤ, BRAF, KRAS, EGFR, and ALK. Thus, for example, the mutations can occur in and therefore the primers or primer sets can hybridize to exon 1 or 2 oiKRAS; exon 18, 19, 20, 21 r 22 of EGFR; exon 8, 9, 10, 1 1, 12, 13, or 17 οΐΚΙΤ; exon 10, 11, 13, 14, or 15 oiBRAF, and/or exon 21, 22, 23, 24, or 25 oiALK. In one aspect, the mutation can comprise any one or more of the mutations listed in Tables 2-6. It is further understood that the disclosed methods and assays can further comprise any of the primers disclosed herein in Tables 7-14 or probes listed in Table 15 and utilize the multiplexing PCR techniques disclosed.

In another aspect, two or more of the disclosed primers and primer sets can comprise a primer panel can be used in methods and assays for the assessment of the suitability of a kinase inhibitor for the treatment of a subjects' cancer. In one aspect, the primer panel comprises one or more primers that can detect a nucleic acid mutation in ALK, BRAF, EGFR, KRAS, or KIT. In a further aspect, the primers or primer sets that hybridizes and amplifies nucleic acid from exon 1 or 2 oiKRAS; exon 18, 19, 20, 21 or 22 oiEGFR; exon 8, 9, 10, 1 1, 12, 13, or 17 οΐΚΙΤ; exon 10, 1 1, 13, 14, or 15 oiBRAF, and/or exon 21, 22, 23, 24, or 25 oiALK. In another aspect, the disclosed primer panel can comprise any primer or primer set which detects one or more of the mutations found in Tables 2-6. For example, the primer or primer set can comprise any of the primers or primer sets disclosed in Tables 7-14.

In another aspect, knowledge of kinase inhibitor resistant cancer can be used to screen for a drug that is not a kinase inhibitor. Thus, in one aspect, disclosed herein are methods of screening for a drug to treat a subject with a cancer comprising obtaining a tissue sample from a subject with a cancer, such as a kinase related cancer (e.g., ALK- related cancers); conducting a high throughput sequencing (also known as next generation sequencing) reaction on the sample, wherein the presence of a mutation in the nucleic acid sequence of a gene, region, or exon associated with kinase inhibitor resistance indicates that that the subject has a cancer is resistant or will become resistant to a kinase inhibitor, and contacting a tissue sample from subject with a cancer with an agent; wherein an agent that inhibits or reduces the growth or development of a kinase inhibitor resistant cancer is not a kinase inhibitor. The disclosed methods can further comprise the sue of the kinase inhibitor resistant panels disclosed herein or any of the primers, primer sets or probes disclosed herein. The methods can also further comprise the treatment of a subject with a kinase inhibitor resistant cancer with an agent that is identified in the method as not being a kinase inhibitor or discontinuing treatment in a subject with kinase inhibitor resistant cancer with an agent that has been found to be a kinase inhibitor.

Methods of identifying subjects for participation in clinical trials to screen for new cancer treatments. In one aspect, it is contemplated herein that the identification of individuals with a kinase inhibitor resistant cancer can be useful for establishing clinical trials to screen for drugs that can be used to treat individuals with kinase inhibitor resistant cancers. Thus, in one aspect, disclosed herein are methods for identifying a subject for screening for a drug that can treat a cancer in a subject with a kinase inhibitor resistant cancer, for example ALK kinase inhibitor resistance, comprising obtaining a tissue sample from a subject with a cancer, such as a kinase related cancer (e.g., ALK-related cancers); and conducting a high throughput sequencing (also known as next generation sequencing) reaction on the sample, wherein the presence of a mutation in the nucleic acid sequence of a gene, region, or exon associated with kinase inhibitor resistance indicates that that the subject has a cancer is resistant or will become resistant to a kinase inhibitor and the subject can be used in trials to screen for a drug to which a kinase inhibitor resistant subject will respond. In one aspect, the mutation can be a nucleic acid mutation in ALK, EGFR, KRAS, BRAF, or KIT. For example, the mutation can be any mutation listed in Tables 2-6. In one aspect, said methods can further comprise synthesizing cDNA from the tissue sample of the subject.

It is understood and herein contemplated that the disclosed methods can be used in conjunction with any of the kinase inhibitor resistant panels, primer sets, or probes disclosed herein. For example, the disclosed methods can be performed using a primer or primer set that hybridizes and amplifies nucleic acid from exon 1 or 2 oiKRAS; exon 18, 19, 20, 21 or 22 oiEGFR; exon 8, 9, 10, 1 1, 12, 13, or 17 οΐΚΙΤ; exon 10, 1 1, 13, 14, or 15 oiBRAF, and/or exon 21, 22, 23, 24, or 25 oiALK. For example, the primer or primer set can comprise any of the primers or primer sets disclosed in Tables 7-14. Thus, disclosed herein are methods wherein the one or more primer set(s) that hybridizes and amplifies nucleic acid from exon 1 (SEQ ID NOs: 4601-4880 and 7181-7230) exon 2 (SEQ ID NOs: 4881- 5200 and 7231-7326) or both exons 1 and 2 (SEQ ID NOs: 7327-7610) ofKRAS; exon 18 (SEQ ID NOs: 1641-1760 and 5819-5934), exon 19 (SEQ ID NOs: 1761-1880), exon 20 (SEQ ID NOs: 1881-2000 and 5934-6042), exon 21 (SEQ ID NOs: 2001-2120 and 6043- 6150), exon 22 (SEQ ID NOs: 2121-2240, 2321-2360, and 2401-2440), exons 18 and 19 (SEQ ID NOs: 2241-2280), exons 18, 19, and 20 (SEQ ID NOs: 6151-6274), exons 20 and

21 (SEQ ID NOs: 2281-2320 and 6275-6388), or exons 18, 19, 20, and 21 (SEQ ID NOs: 2361-2400 and 6389-6524) oiEGFR; exon 8 (SEQ ID NOs: 2441-2800), exon 9 (SEQ ID NOs: 2841-3120), exon 10 (SEQ ID NOs: 3201-3360), exon 1 1 (SEQ ID NOs: 3361-3480), exon 12 (SEQ ID NOs: 3481-3640), exon 13 (SEQ ID NOs: 3641-3800), exon 17 (SEQ ID NOs: 4241-4600), exon 8 and 9 (SEQ ID NOs: 2801-2840), exons 9 and 10 (SEQ ID NOs: 3121-3160), exons 9, 10, and 1 1 (SEQ ID NOs: 3161-3200); exons 10 and 11 (SEQ ID NOs: 3801-3960), exons 12 and 13 (SEQ ID NOs: 3961-4120), or exons 10, 1 1, 12, and 13 (SEQ ID NOs: 4121-4240) of ATT; exons 10 and 1 1 (SEQ ID NOs: 6525-6832) or exons 13, 14, or 15 (SEQ ID NOs: 66833-7180) oiBRAF, and/or exon 21 (SEQ ID NOs: 1-160), exon

22 (SEQ ID NOs: 401-560), exon 23 (SEQ ID NOs: 561-840 and 5311-5446), exon 24 (SEQ ID NOs: 921-1240), exon 25 (SEQ ID NOs: 1241-1600), exons 21 and 22 (SEQ ID NOs: 161-400 and 5201-5310), exons 21, 22, and 23 (SEQ ID NOs: 841-920), exons 24 and 25 (SEQ ID NOs: 1601-1640 and 5447-5576), or exons 21, 22, 23, 24, and 25 (SEQ ID NOs: 5577-5818) οΐΑΣΚ.

Methods of detecting a kinase inhibitor resistance in an _^4ZJ£-related cancer

In another aspect, the disclosed methods and assays relate to the detection or diagnosis of the presence of a kinase inhibitor resistance, such as, for example, ALK kinase inhibitor resistance, in a disease or condition such as a cancer and methods and assays for the determination of susceptibility or resistance to therapeutic treatment for a disease or condition such as a cancer in a subject comprising detecting the presence or measuring the expression level of nucleic acid (for example, DNA, mRNA, cDNA, RNA, etc) through the use of next generation sequencing (NGS) from a tissue sample from the subject; wherein the presence of a mutations in the nucleic acid code of the KIT, BRAF, KRAS, EGFR, or ALK gene or the ALK gene portion of an ALK fusion construct indicates the presence of a cancer that is resistant to a kinase inhibitor. In one aspect, the cancer is associated with

amplification, overexpression, nucleic acid variation, truncation, or gene fusion oiALK. It is understood, that the kinase inhibitor resistance panels disclosed herein can be used to perform said methods and the detection of one or more of the mutations in Tables 2-6 indicates the presence of kinase inhibitor resistance. In one aspect, the disclosed methods can further comprise discontinuing use of a kinase inhibitor to treat a cancer in a subject that has been identified with a kinase inhibitor resistant cancer. In another embodiment, the disclosed methods can further comprise treating a subject with a kinase inhibitor resistant cancer with a chemotherapeutic that is not a kinase inhibitor. Thus, in one aspect, disclosed herein are methods of treating a subject with a kinase inhibitor resistant cancer (such as, for example, an ALK kinase inhibitor resistant cancer) comprising obtaining a tissue sample from a subject with a cancer, such as a kinase related cancer (e.g., ALK-rslatsd cancers); conducting a high throughput sequencing (also known as next generation sequencing) reaction on the sample, wherein the presence of a mutation in the nucleic acid sequence of a gene, region, or exon associated with kinase inhibitor resistance indicates that that the subject has a cancer is resistant or will become resistant to a kinase inhibitor; and treating the subject with a chemotherapeutic that is not a kinase inhibitor. Also disclosed are methods of treating a subject without a kinase inhibitor resistant cancer comprising obtaining a tissue sample from a subject with a cancer, such as a kinase related cancer (e.g., ALK-related cancers); conducting a high throughput sequencing (also known as next generation sequencing) reaction on the sample, wherein the absence of a mutation in the nucleic acid sequence of a gene, region, or exon associated with kinase inhibitor resistance indicates that that the subject does not have a cancer is resistant nor will become resistant to a kinase inhibitor; and treating the subject with a kinase inhibitor.

Anaplastic Lymphoma Kinase (ALK)

ALK (SEQ ID NO: 7714 (Genbank Accession No. U62540 (human coding sequence)) is a receptor tyrosine kinase (RTK) of the insulin receptor superfamily encoded by the ALK gene and is normally expressed primarily in the central and peripheral nervous systems. The I620aa ALK polypeptide comprises a 1030aa extracellular domain which includes a 26aa amino-terminal signal peptide sequence, and binding sites located between residues 391 and 401 for the ALK ligands pleiotrophin (PTN) and midkine (MK).

Additionally, the ALK polypeptide comprises a kinase domain (residues 1 1 16-1383) which includes three tyrosines responsible for autophosphorylation within the activation loop at residues 1278, 1282, and 1283. ALK amplification, overexpression, and mutations have been shown to constitutively activate the kinase catalytic function of the ALK protein, with the deregulated mutant ALK in turn activating downstream cellular signaling proteins in pathways that promote aberrant cell proliferation. In fact, the mutations that result in dysregulated ALK kinase activity are associated with several types of cancers.

ALK fusions represent the most common mutation of this tyrosine kinase. Such fusions include but are not limited to nucleophosmin-^Z T (NPM-ALK), 5-aminoimidazole- 4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC-ALK), clathrin heavy chain-ALK (CLTC-ALK), kinesin-1 heavy chain gsm-ALK (KIF 5B-ALK); Ran- binding protein 2-ALK (RANBP2-ALK), SEC3 I l-ALK, tropomyosin-3- ΖΑ^" (TPM3- ALK), tropomyosin^^ (TFM4-ALK), TRK-fused gene(Large) -ALK (TFG_L-ALK),

TRK-fused gene(Small) -ALK (TFG_S-ALK), CARS-ALK, LML4-ALK, 5-aminoimidazole-4- carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase-ALK (ATIC-ALK), ALO 17 -ALK, moesin-^Z T (MSN-ALK), non-muscle myosin heavy chain gsns-ALK (MYH9-ALK), and TRK-fused gene(Extra Large) -ALK (TFG_XL-ALK). Six ALK fusions, CARS-ALK. CLTC-ALK, RA BP2-^ ^, SEC3 -ALK, TFM3-ALK, and TFM4-ALK have been identified in IMTs. TPMS-ALK, TPM4-ALK and CLTC-ALK fusions have been detected in both classical T- or null-cell lymphomas and IMT sarcomas, whereas CARS- ALK, RA BP2-^ ^, and SEC3 ILI-ALK occur in IMT. CLTC-ALK and NFM-ALK also occur in B-cell plasmablastic/immunoblastic lymphomas. The TPM4-ALK fusion occurs in esophageal squamous cell carcinomas, and the ALK fusion LML4-ALK , TFG-ALK and

Y T5B-ALK are found in non-small cell lung cancers. EML4-ALK has also recently been identified in both colorectal and breast carcinomas as well. ALK fusions are associated with several known cancer types. It is understood that one or more ALK fusions can be associated with a particular cancer. It is further understood that there are several types of cancer associated with ALK fusions including but not limited to anaplastic large-cell lymphoma (ALCL), neuroblastoma, breast cancer, ovarian cancer, colorectal carcinoma, non-small cell lung carcinoma, diffuse large B-cell lymphoma, esophageal squamous cell carcinoma, anaplastic large-cell lymphoma, neuroblastoma, inflammatory myofibroblastic tumors, malignant histiocytosis, and glioblastomas.

ALCL. anaplastic large-cell lymphomas comprise -2.5% of all NHL; within the pediatric age group specifically, -13% of all NHL (30 - 40% of all childhood large-cell lymphomas) are of this type. Studies of ALCL patients now divide this NHL into ALK- positive and ALK-negative subsets; -60% of all ALCLs are caused by ALK fusions. For unclear reasons, ALK-positive ALCL patients fare significantly better following CHOP based multi-agent conventional chemotherapy than those with ALK-negative disease (with overall 5-year survivals of -75% vs. -35%, respectively). However, more than a third of patients suffer multiple relapses following chemotherapy, thus the 5-year disease-free survival of ALK-positive ALCL is only -40%.

ALK+ Diffuse large B-cell lymphoma. In 2003, ALK fusions were shown to occur in a non-ALCL form of NHL with the description of CLTC-ALK or NPM-ALK in diffuse large B-cell lymphomas (ALK+ DLBCLs). Consistent with their B-lineage, these NHLs express cytoplasmic IgA and plasma cell markers, and possess an immunoblastic morphology. Translational research studies revealed the t(2; 17) and CLTC-ALK mRN A in the majority of these lymphomas, while immunolabeling confirmed granular ALK staining identical to that observed in CLTC-ALK-positive ALCL. As for all other ALK fusion partner proteins, a self-association motif in the CLTC portion of CLTC-ALK mediates constitutive self-association and activation of the fusion kinase to drive lymphomagenesis. ALK+ DLBCLs occur predominately in adults; however, the t(2;5) and NPM-ALK mRNA in pediatric lymphomas are phenotypically identical to CLTC-ALK- positive adult B-NHLs. Approximately 0.5-1% of all DLBCL is thought to be ALK-positive. The identification of DLBCLs caused by mutant ALK is important because patients with these lymphomas have outcomes that are much inferior to ALK-negative DLBCL patients following CHOP-based treatments; thus, ALK+ DLBCL patients should strongly be considered as candidates for ALK-targeted kinase inhibitor therapy.

ALK+ systemic histiocytosis. ALK fusions were described in 2008 in another hematopoietic neoplasm, systemic histiocytosis. Three cases of this previously

uncharacterized form of histiocytosis, which presents in early infancy, exhibited ALK immunoreactivity and the one case analyzed molecularly expressed TPM3-ALK.

In addition to the aforementioned hematological malignancies in which

constitutively activated ALK fusions have been shown to be a causative mechanism in many cases, the genesis of subsets of various solid tumors in some instances, very common human tumors such as non-small cell lung cancer, colorectal and breast cancers has recently been demonstrated to be due to aberrantly activated ALK.

Inflammatory myofibroblasts tumor. The first non-hematopoietic tumor discovered to express ALK fusions was the sarcoma known as inflammatory myofibroblastic tumor (IMT), a spindle cell proliferation in the soft tissue and viscera of children and young adults (mean age at diagnosis ~10 years). Many IMTs are indolent and can be cured by resection. However, locally recurrent, invasive, and metastatic IMTs are not uncommon and current chemo- and radio-therapies are completely ineffective. Disclosed herein is the involvement of chromosome 2p23 (the location of the ALK gene) in IMTs, as well as ALK gene rearrangement. ALK immunoreactivity in 7 of 11 IMTs has been shown and TPM3-ALK and TPM4-ALK were identified in several cases. Additionally, two additional ALK fusions in IMT, CLTC- and RanBP2-ALK were identified. ALK fusions have also been examined by immunostaining in 73 IMTs, finding 60% (44 of the 73 cases) to be ALK-positive. Thus, ALK deregulation is of pathogenic importance in a majority of IMTs.

Non-small cell lung carcinoma. The role of ALK fusions in cancer expanded further with the description of the novel EML4-ALK chimeric protein in 5 of 75 (6.7%) Japanese non-small cell lung carcinoma patients. Shortly thereafter, the existence of ALK fusions in lung cancer was corroborated by a different group who found 6 of 137 (4.4%) Chinese lung cancer patients to express ALK fusions (EML4-ALK, 3 pts; TFG-ALK, 1 pt; X-ALK. Two common themes have emerged - 1) ALK fusions occur predominately in patients with adenocarcinoma (although occasional ALK-positive NSCLCs of squamous or mixed histologies are observed), mostly in individuals with minimal/no smoking history, and 2) ALK abnormalities usually occur exclusive of other common genetic abnormalities (e.g., EGFR and KRAS mutations). The exact percentage of NSCLCs caused by ALK fusions is not yet clear but estimates based on reports in the biomedical literature suggest a range of -5-10%.

Esophageal squamous cell carcinoma. In 45 Iranian patients, a proteomics approach identified proteins under or over-represented in esophageal squamous cell carcinomas (ESCCs); TPM4-ALK was among those proteins over-represented. A second proteomics- based ESCC study - in this case, in Chinese patients - identified TPM4-ALK in these tumors as well.

Colorectal carcinoma, breast cancer. Three human tumor types - colorectal, breast, and non-small cell lung cancers were surveyed for the presence of the EML4-ALK fusion (other ALK mutations were not assessed in this study). In addition to confirming the expression of EML4-ALK in NSCLC (in 12 of 106 specimens studied, 1 1.3%), a subsets of breast (5 of 209 cases, 2.4%) and colorectal (2 of 83 cases, 2.4%) carcinomas were EML4- ALK-positive. In addition to known EML4-ALK variants 1 (E13; A20) and 2 (E20; A20), a novel variant (E21 ; A20) was found in colorectal carcinoma.

ALK in familial and sporadic neuroblastoma. Neuroblastoma is the most common extracranial solid tumor of childhood, and is derived from the developing neural crest. A small subset (-1-2%) of neuroblastomas exhibit a familial predisposition with an autosomal dominant inheritance. Most neuroblastoma patients have aggressive disease associated with survival probabilities <40% despite intensive chemo- and radio-therapy, and the disease accounts for -15% of all childhood cancer mortality. ALK had previously been found to be constitutively activated also due to high-level over-expression as a result of gene amplification in a small number of neuroblastoma cell lines, in fact, ALK amplification occurs in -15% of neuroblastomas in addition to activating point mutations. These missense mutations in ALK have been confirmed as activating mutations that drive neuroblastoma growth; furthermore, incubation of neuroblastoma cell lines with ALK small-molecule inhibitors reveal those cells with ALK activation (but not cell lines with normal levels of expression of wild-type ALK) to exhibit robust cytotoxic responses.

The sensitive detection of a mutation at a known site in DNA is readily done with existing technologies. Allele specific primers can be designed to target a mutation at a known location such that its signal can be preferentially amplified over wild-type DNA.

Next Generation Sequencing for Genetic Testing

From a technical perspective High-throughput or Next Generation Sequencing (NGS) represents an attractive option for detecting the somatic mutations within a gene. Unlike PCR, microarrays, high-resolution melting and mass spectrometry, which all indirectly infer sequence content, NGS directly ascertains the identity of each base and the order in which they fall within a gene. The newest platforms on the market have the capacity to cover an exonic region 10,000 times over, meaning the content of each base position in the sequence is measured thousands of different times. This high level of coverage ensures that the consensus sequence is extremely accurate and enables the detection of rare variants within a heterogeneous sample. For example, in a sample extracted from FFPE tissue, relevant mutations are only present at a frequency of 1% with the wild-type allele comprising the remainder. When this sample is sequenced at 10,000X coverage, then even the rare allele, comprising only 1% of the sample, is uniquely measured 100 times over. Thus, NGS can provide reliably accurate results with very high sensitivity, making it ideal for clinical diagnostic testing of FFPEs and other mixed samples.

In one aspect, disclosed herein are methods and assays for detecting kinase inhibitor resistance or determining the susceptibility to a particular kinase inhibitor treatment in an ALK-related cancer comprising performing next generation sequencing on a tissue sample obtained from a subject with an ALK-related cancer, wherein the presence of a nucleic acid variation in the ALK, BRAF, EGFR, KIT, or KRAS sequence of the tissue sample at a nucleic acid residue indicates that presence of kinase inhibitor resistance. For example, the methods and assays for detecting kinase inhibitor resistance or determining the susceptibility or developing kinase inhibitor resistance in an ALK-related cancer or determining the suitability of a particular kinase inhibitor for use in treating an ALK-related cancer in a subject can comprise the detection of any of the mutations in Tables 2-6. It is understood that the methods and assays can further comprise comparing the sequence to known kinase inhibitor resistance mutations list and determining what if any kinase inhibitors are affected by the mutation and altering or maintaining treatment as appropriate to utilize kinase inhibitors that are unaffected by the mutation. As the disclosed methods and assays employ the use of primers or primer sets to detect mutations that confer kinase inhibitor resistance, also disclosed herein are primer panels for use in next generation sequencing for the determination of kinase inhibitor resistance comprising one or more primer sets from each οΐΚΙΤ, BRAF, KRAS, EGFR, and ALK, for example, the disclosed primer panels, methods, and assays can comprise one or more of the primers or primer sets listed in Tables 7-14.

Examples of Next Generation Sequencing techniques include, but are not limited to Massively Parallel Signature Sequencing (MPSS), Polony sequencing, pyrosequencing,

Reversible dye-terminator sequencing, SOLiD sequencing, Ion semiconductor sequencing, DNA nanoball sequencing, Helioscope single molecule sequencing, Single molecule real time (SMRT) sequencing, Single molecule real time (RNAP) sequencing, and Nanopore DNA sequencing.

MPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences. Polony sequencing, combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1/10 that of Sanger sequencing.

A parallelized version of pyrosequencing, the method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony. The sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes. Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs. This technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.

A sequencing technology based on reversible dye-terminators. DNA molecules are first attached to primers on a slide and amplified so that local clonal colonies are formed. Four types of reversible terminator bases (RT-bases) are added, and non-incorporated nucleotides are washed away. Unlike pyrosequencing, the DNA can only be extended one nucleotide at a time. A camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3' blocker is chemically removed from the DNA, allowing the next cycle.

SOLiD technology employs sequencing by ligation. Here, a pool of all possible oligonucleotides of a fixed length are labeled according to the sequenced position.

Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR. The resulting bead, each containing only copies of the same DNA molecule, are deposited on a glass slide. The result is sequences of quantities and lengths comparable to Illumina sequencing.

Ion semiconductor sequencing is based on using standard sequencing chemistry, but with a novel, semiconductor based detection system. This method of sequencing is based on the detection of hydrogen ions that are released during the polymerization of DNA, as opposed to the optical methods used in other sequencing systems. A micro well containing a template DNA strand to be sequenced is flooded with a single type of nucleotide. If the introduced nucleotide is complementary to the leading template nucleotide it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers a hypersensitive ion sensor, which indicates that a reaction has occurred. If homopolymer repeats are present in the template sequence multiple nucleotides will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.

DNA nanoball sequencing is a type of high throughput sequencing technology used to determine the entire genomic sequence of an organism. The method uses rolling circle replication to amplify small fragments of genomic DNA into DNA nanoballs. Unchained sequencing by ligation is then used to determine the nucleotide sequence. This method of DNA sequencing allows large numbers of DNA nanoballs to be sequenced per run.

Helicos's single-molecule sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface. The next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides (one nucleotide type at a time, as with the Sanger method). The reads are performed by the Helioscope sequencer.

SMRT sequencing is based on the sequencing by synthesis approach. The DNA is synthesized in zero-mode wave-guides (ZMWs) - small well-like containers with the capturing tools located at the bottom of the well. The sequencing is performed with use of unmodified polymerase (attached to the ZMW bottom) and fluorescently labeled nucleotides flowing freely in the solution. The wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected. The fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand. Single molecule real time sequencing based on RNA polymerase (RNAP), which is attached to a polystyrene bead, with distal end of sequenced DNA is attached to another bead, with both beads being placed in optical traps. RNAP motion during transcription brings the beads in closer and their relative distance changes, which can then be recorded at a single nucleotide resolution. The sequence is deduced based on the four readouts with lowered concentrations of each of the four nucleotide types (similarly to Sangers method).

Nanopore sequencing is based on the readout of electrical signal occurring at nucleotides passing by alpha-hemolysin pores covalently bound with cyclodextrin. The DNA passing through the nanopore changes its ion current. This change is dependent on the shape, size and length of the DNA sequence. Each type of the nucleotide blocks the ion flow through the pore for a different period of time.

VisiGen Biotechnologies uses a specially engineered DNA polymerase. This polymerase acts as a sensor - having incorporated a donor fluorescent dye by its active centre. This donor dye acts by FRET (fluorescent resonant energy transfer), inducing fluorescence of differently labeled nucleotides. This approach allows reads performed at the speed at which polymerase incorporates nucleotides into the sequence (several hundred per second). The nucleotide fluorochrome is released after the incorporation into the DNA strand.

Sequencing by hybridization is a non-enzymatic method that uses a DNA microarray. A single pool of DNA whose sequence is to be determined is fluorescently labeled and hybridized to an array containing known sequences. Strong hybridization signals from a given spot on the array identify its sequence in the DNA being sequenced.

Mass spectrometry may be used to determine mass differences between DNA fragments produced in chain-termination reactions.

Another NGS approach is sequencing by synthesis (SBS) technology which is capable of overcoming the limitations of existing pyrosequencing based NGS platforms.

Such technologies rely on complex enzymatic cascades for read out, are unreliable for the accurate determination of the number of nucleotides in homopolymeric regions and require excessive amounts of time to run individual nucleotides across growing DNA strands. The SBS NGS platform uses a direct sequencing approach to produce a sequencing strategy with very a high precision, rapid pace and low cost.

SBS sequencing is initialized by fragmenting of the template DNA into fragments, amplification, annealing of DNA sequencing primers, and finally affixing as a high-density array of spots onto a glass chip. The array of DNA fragments are sequenced by extending each fragment with modified nucleotides containing cleavable chemical moieties linked to fluorescent dyes capable of discriminating all four possible nucleotides. The array is scanned continuously by a high-resolution electronic camera (Measure) to determine the fluorescent intensity of each base (A, C, G or T) that was newly incorporated into the extended DNA fragment. After the incorporation of each modified base the array is exposed to cleavage chemistry to break off the fluorescent dye and end cap allowing additional bases to be added. The process is then repeated until the fragment is completely sequenced or maximal read length has been achieved.

mRNA detection and quantification

A number of widely used procedures exist for detecting and determining the abundance of a particular mRNA in a total or poly(A) RNA sample. For example, specific mRNAs can be detected using Northern blot analysis, nuclease protection assays (NPA), in situ hybridization (e.g., fluorescence in situ hybridization (FISH)), or reverse transcription- polymerase chain reaction (RT-PCR), and microarray.

In theory, each of these techniques can be used to detect specific RNAs and to precisely determine their expression level. In general, Northern analysis is the only method that provides information about transcript size, whereas NPAs are the easiest way to simultaneously examine multiple messages. In situ hybridization is used to localize expression of a particular gene within a tissue or cell type, and RT-PCR is the most sensitive method for detecting and quantitating gene expression.

RT-PCR allows for the detection of the RNA transcript of any gene, regardless of the scarcity of the starting material or relative abundance of the specific mRNA. In RT- PCR, an RNA template is copied into a complementary DNA (cDNA) using a retroviral reverse transcriptase. The cDNA is then amplified exponentially by PCR using a DNA polymerase. The reverse transcription and PCR reactions can occur in the same or difference tubes. RT-PCR is somewhat tolerant of degraded RNA. As long as the RNA is intact within the region spanned by the primers, the target will be amplified.

Relative quantitative RT-PCR involves amplifying an internal control

simultaneously with the gene of interest. The internal control is used to normalize the samples. Once normalized, direct comparisons of relative abundance of a specific mRNA can be made across the samples. It is crucial to choose an internal control with a constant level of expression across all experimental samples (i.e., not affected by experimental treatment). Commonly used internal controls (e.g., GAPDH, β-actin, cyclophilin) often vary in expression and, therefore, may not be appropriate internal controls. Additionally, most common internal controls are expressed at much higher levels than the mRNA being studied. For relative RT-PCR results to be meaningful, all products of the PCR reaction must be analyzed in the linear range of amplification. This becomes difficult for transcripts of widely different levels of abundance.

Competitive RT-PCR is used for absolute quantitation. This technique involves designing, synthesizing, and accurately quantitating a competitor RNA that can be distinguished from the endogenous target by a small difference in size or sequence. Known amounts of the competitor RNA are added to experimental samples and RT-PCR is performed. Signals from the endogenous target are compared with signals from the competitor to determine the amount of target present in the sample.

Northern analysis is the easiest method for determining transcript size, and for identifying alternatively spliced transcripts and multigene family members. It can also be used to directly compare the relative abundance of a given message between all the samples on a blot. The Northern blotting procedure is straightforward and provides opportunities to evaluate progress at various points (e.g., intactness of the RNA sample and how efficiently it has transferred to the membrane). RNA samples are first separated by size via electrophoresis in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane, crosslinked and hybridized with a labeled probe. Nonisotopic or high specific activity radiolabeled probes can be used including random-primed, nick-translated, or PCR-generated DNA probes, in vitro transcribed RNA probes, and oligonucleotides. Additionally, sequences with only partial homology (e.g., cDNA from a different species or genomic DNA fragments that might contain an exon) may be used as probes.

The Nuclease Protection Assay (NPA) (including both ribonuclease protection assays and SI nuclease assays) is a sensitive method for the detection and quantitation of specific mRNAs. The basis of the NPA is solution hybridization of an antisense probe (radiolabeled or nonisotopic) to an RNA sample. After hybridization, single-stranded, unhybridized probe and RNA are degraded by nucleases. The remaining protected fragments are separated on an acrylamide gel. Solution hybridization is typically more efficient than membrane-based hybridization, and it can accommodate up to 100 μg of sample RNA, compared with the 20-30 μg maximum of blot hybridizations. NPAs are also less sensitive to RNA sample degradation than Northern analysis since cleavage is only detected in the region of overlap with the probe (probes are usually about 100-400 bases in length).

NPAs are the method of choice for the simultaneous detection of several RNA species. During solution hybridization and subsequent analysis, individual probe/target interactions are completely independent of one another. Thus, several RNA targets and appropriate controls can be assayed simultaneously (up to twelve have been used in the same reaction), provided that the individual probes are of different lengths. NPAs are also commonly used to precisely map mRNA termini and intron/exon junctions.

In situ hybridization (ISH) is a powerful and versatile tool for the localization of specific mRNAs in cells or tissues. Unlike Northern analysis and nuclease protection assays, ISH does not require the isolation or electrophoretic separation of RNA.

Hybridization of the probe takes place within the cell or tissue. Since cellular structure is maintained throughout the procedure, ISH provides information about the location of mRNA within the tissue sample.

The procedure begins by fixing samples in neutral-buffered formalin, and embedding the tissue in paraffin. The samples are then sliced into thin sections and mounted onto microscope slides. (Alternatively, tissue can be sectioned frozen and post-fixed in paraformaldehyde.) After a series of washes to dewax and rehydrate the sections, a Proteinase K digestion is performed to increase probe accessibility, and a labeled probe is then hybridized to the sample sections. Radiolabeled probes are visualized with liquid film dried onto the slides, while non-isotopically labeled probes are conveniently detected with colorimetric or fluorescent reagents.

DNA detection and quantification

The methods, assays, and primer panels disclosed herein relate to the detection of nucleic acid variation that confer kinase inhibitor resistance in the form of, for example, point mutations and truncations of, KRAS, BRAF, KIT, EGFR, and/or ALK. Thus, in one aspect, disclosed herein are methods, assays, and use of the disclosed primer panels for diagnosing an anaplastic lymphoma kinase (ALK) related cancer in a subject is resistant to a kinase inhibitor comprise performing NGS which sequences DNA from a tissue sample from the subject. It is understood that high throughput sequencing methods (also known as next generation sequencing methods) can comprise any known amplification and detection method for DNA known in the art.

A number of widely used procedures exist for detecting and determining the abundance of a particular DNA in a sample. For example, the technology of PCR permits amplification and subsequent detection of minute quantities of a target nucleic acid. Details of PCR are well described in the art, including, for example, U.S. Pat. Nos. 4,683, 195 to Mullis et al, 4,683,202 to Mullis and 4,965, 188 to Mullis et al. Generally, oligonucleotide primers are annealed to the denatured strands of a target nucleic acid, and primer extension products are formed by the polymerization of deoxynucleoside triphosphates by a polymerase. A typical PCR method involves repetitive cycles of template nucleic acid denaturation, primer annealing and extension of the annealed primers by the action of a thermostable polymerase. The process results in exponential amplification of the target nucleic acid, and thus allows the detection of targets existing in very low concentrations in a sample. It is understood and herein contemplated that there are variant PCR methods known in the art that may also be utilized in the disclosed methods, for example,

Quantitative PCR (QPCR); microarrays, real-time PCR; hot start PCR; nested PCR; allele- specific PCR; and Touchdown PCR.

Microarrays

An array is an orderly arrangement of samples, providing a medium for matching known and unknown DNA samples based on base-pairing rules and automating the process of identifying the unknowns. An array experiment can make use of common assay systems such as microplates or standard blotting membranes, and can be created by hand or make use of robotics to deposit the sample. In general, arrays are described as macroarrays or microarrays, the difference being the size of the sample spots. Macroarrays contain sample spot sizes of about 300 microns or larger and can be easily imaged by existing gel and blot scanners. The sample spot sizes in microarray can be 300 microns or less, but typically less than 200 microns in diameter and these arrays usually contains\ thousands of spots.

Microarrays require specialized robotics and/or imaging equipment that generally are not commercially available as a complete system. Terminologies that have been used in the literature to describe this technology include, but not limited to: biochip, DNA chip, DNA microarray, GENECHIP® (Affymetrix, Inc which refers to its high density,

oligonucleotide-based DNA arrays), and gene array.

DNA microarrays, or DNA chips are fabricated by high-speed robotics, generally on glass or nylon substrates, for which probes with known identity are used to determine complementary binding, thus allowing massively parallel gene expression and gene discovery studies. An experiment with a single DNA chip can provide information on thousands of genes simultaneously. It is herein contemplated that the disclosed microarrays can be used to monitor gene expression, disease diagnosis, gene discovery, drug discovery (pharmacogenomics), and toxicological research or toxicogenomics.

There are two variants of the DNA microarray technology, in terms of the property of arrayed DNA sequence with known identity. Type I microarrays comprise a probe cDNA (500-5,000 bases long) that is immobilized to a solid surface such as glass using robot spotting and exposed to a set of targets either separately or in a mixture. This method is traditionally referred to as DNA microarray. With Type I microarrays, localized multiple copies of one or more polynucleotide sequences, preferably copies of a single

polynucleotide sequence are immobilized on a plurality of defined regions of the substrate's surface. A polynucleotide refers to a chain of nucleotides ranging from 5 to 10,000 nucleotides. These immobilized copies of a polynucleotide sequence are suitable for use as probes in hybridization experiments.

To prepare beads coated with immobilized probes, beads are immersed in a solution containing the desired probe sequence and then immobilized on the beads by covalent or non-covalent means. Alternatively, when the probes are immobilized on rods, a given probe can be spotted at defined regions of the rod. Typical dispensers include a micropipette delivering solution to the substrate with a robotic system to control the position of the micropipette with respect to the substrate. There can be a multiplicity of dispensers so that reagents can be delivered to the reaction regions simultaneously. In one embodiment, a microarray is formed by using ink-jet technology based on the piezoelectric effect, whereby a narrow tube containing a liquid of interest, such as oligonucleotide synthesis reagents, is encircled by an adapter. An electric charge sent across the adapter causes the adapter to expand at a different rate than the tube and forces a small drop of liquid onto a substrate.

Samples may be any sample containing polynucleotides (polynucleotide targets) of interest and obtained from any bodily fluid (blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. DNA or RNA can be isolated from the sample according to any of a number of methods well known to those of skill in the art. In one embodiment, total RNA is isolated using the TRIzol total RNA isolation reagent (Life Technologies, Inc., Rockville, Md.) and RNA is isolated using oligo d(T) column chromatography or glass beads. After hybridization and processing, the

hybridization signals obtained should reflect accurately the amounts of control target polynucleotide added to the sample.

The plurality of defined regions on the substrate can be arranged in a variety of formats. For example, the regions may be arranged perpendicular or in parallel to the length of the casing. Furthermore, the targets do not have to be directly bound to the substrate, but rather can be bound to the substrate through a linker group. The linker groups may typically vary from about 6 to 50 atoms long. Linker groups include ethylene glycol oligomers, diamines, diacids and the like. Reactive groups on the substrate surface react with one of the terminal portions of the linker to bind the linker to the substrate. The other terminal portion of the linker is then functionalized for binding the probes.

Sample polynucleotides may be labeled with one or more labeling moieties to allow for detection of hybridized probe/target polynucleotide complexes. The labeling moieties can include compositions that can be detected by spectroscopic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical or chemical means. The

32 33 35

labeling moieties include radioisotopes, such as ^JZP, or ^JJS, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers, such as fluorescent markers and dyes, magnetic labels, linked enzymes, mass spectrometry tags, spin labels, electron transfer donors and acceptors, biotin, and the like.

Labeling can be carried out during an amplification reaction, such as polymerase chain reaction and in vitro or in vivo transcription reactions. Alternatively, the labeling moiety can be incorporated after hybridization once a probe-target complex his formed. In one embodiment, biotin is first incorporated during an amplification step as described above. After the hybridization reaction, unbound nucleic acids are rinsed away so that the only biotin remaining bound to the substrate is that attached to target polynucleotides that are hybridized to the polynucleotide probes. Then, an avidin-conjugated fluorophore, such as avidin-phycoerythrin, that binds with high affinity to biotin is added. Hybridization causes a polynucleotide probe and a complementary target to form a stable duplex through base pairing. Hybridization methods are well known to those skilled in the art. Stringent conditions for hybridization can be defined by salt concentration, temperature, and other chemicals and conditions. Varying additional parameters, such as hybridization time, the concentration of detergent (sodium dodecyl sulfate, SDS) or solvent (formamide), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Additional variations on these conditions will be readily apparent to those skilled in the art.

Methods for detecting complex formation are well known to those skilled in the art. In one embodiment, the polynucleotide probes are labeled with a fluorescent label and measurement of levels and patterns of complex formation is accomplished by fluorescence microscopy, preferably confocal fluorescence microscopy. An argon ion laser excites the fluorescent label, emissions are directed to a photomultiplier and the amount of emitted light detected and quantitated. The detected signal should be proportional to the amount of probe/target polynucleotide complex at each position of the microarray. The fluorescence microscope can be associated with a computer-driven scanner device to generate a quantitative two-dimensional image of hybridization intensities. The scanned image is examined to determine the abundance/expression level of each hybridized target polynucleotide.

In a differential hybridization experiment, polynucleotide targets from two or more different biological samples are labeled with two or more different fluorescent labels with different emission wavelengths. Fluorescent signals are detected separately with different photomultipliers set to detect specific wavelengths. The relative abundances/expression levels of the target polynucleotides in two or more samples is obtained. Typically, microarray fluorescence intensities can be normalized to take into account variations in hybridization intensities when more than one microarray is used under similar test conditions. In one embodiment, individual polynucleotide probe/target complex hybridization intensities are normalized using the intensities derived from internal normalization controls contained on each microarray.

Type II microarrays comprise an array of oligonucleotides (20~80-mer oligos) or peptide nucleic acid (PNA) probes that is synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization. The array is exposed to labeled sample DNA, hybridized, and the identity/abundance of complementary sequences are determined. This method, "historically" called DNA chips, was developed at Affymetrix, Inc. , which sells its photolithographically fabricated products under the GENECHIP® trademark.

The basic concept behind the use of Type II arrays for gene expression is simple: labeled cDNA or cRNA targets derived from the mRNA of an experimental sample are hybridized to nucleic acid probes attached to the solid support. By monitoring the amount of label associated with each DNA location, it is possible to infer the abundance of each mRNA species represented. Although hybridization has been used for decades to detect and quantify nucleic acids, the combination of the miniaturization of the technology and the large and growing amounts of sequence information, have enormously expanded the scale at which gene expression can be studied.

Microarray manufacturing can begin with a 5 -inch square quartz wafer. Initially the quartz is washed to ensure uniform hydroxylation across its surface. Because quartz is naturally hydroxylated, it provides an excellent substrate for the attachment of chemicals, such as linker molecules, that are later used to position the probes on the arrays.

The wafer is placed in a bath of silane, which reacts with the hydroxyl groups of the quartz, and forms a matrix of covalently linked molecules. The distance between these silane molecules determines the probes' packing density, allowing arrays to hold over 500,000 probe locations, or features, within a mere 1.28 square centimeters. Each of these features harbors millions of identical DNA molecules. The silane film provides a uniform hydroxyl density to initiate probe assembly. Linker molecules, attached to the silane matrix, provide a surface that may be spatially activated by light. Probe synthesis occurs in parallel, resulting in the addition of an A, C, T, or G nucleotide to multiple growing chains simultaneously. To define which oligonucleotide chains will receive a nucleotide in each step, photolithographic masks, carrying 18 to 20 square micron windows that correspond to the dimensions of individual features, are placed over the coated wafer. The windows are distributed over the mask based on the desired sequence of each probe. When ultraviolet light is shone over the mask in the first step of synthesis, the exposed linkers become deprotected and are available for nucleotide coupling.

Once the desired features have been activated, a solution containing a single type of deoxynucleotide with a removable protection group is flushed over the wafer's surface. The nucleotide attaches to the activated linkers, initiating the synthesis process.

Although each position in the sequence of an oligonucleotide can be occupied by lof Nucleotides, resulting in an apparent need for 25 x 4, or 100, different masks per wafer, the synthesis process can be designed to significantly reduce this requirement. Algorithms that help minimize mask usage calculate how to best coordinate probe growth by adjusting synthesis rates of individual probes and identifying situations when the same mask can be used multiple times.

Some of the key elements of selection and design are common to the production of all microarrays, regardless of their intended application. Strategies to optimize probe hybridization, for example, are invariably included in the process of probe selection.

Hybridization under particular pH, salt, and temperature conditions can be optimized by taking into account melting temperatures and using empirical rules that correlate with desired hybridization behaviors.

To obtain a complete picture of a gene's activity, some probes are selected from regions shared by multiple splice or polyadenylation variants. In other cases, unique probes that distinguish between variants are favored. Inter-probe distance is also factored into the selection process.

A different set of strategies is used to select probes for genotyping arrays that rely on multiple probes to interrogate individual nucleotides in a sequence. The identity of a target base can be deduced using four identical probes that vary only in the target position, each containing one of the four possible bases.

Alternatively, the presence of a consensus sequence can be tested using one or two probes representing specific alleles. To genotype heterozygous or genetically mixed samples, arrays with many probes can be created to provide redundant information, resulting in unequivocal genotyping. In addition, generic probes can be used in some applications to maximize flexibility. Some probe arrays, for example, allow the separation and analysis of individual reaction products from complex mixtures, such as those used in some protocols to identify single nucleotide polymorphisms (SNPs).

Real-time PCR

Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production during each PCR cycle (i.e., in real time) as opposed to the endpoint detection. The real-time progress of the reaction can be viewed in some systems. Real-time PCR does not detect the size of the amplicon and thus does not allow the differentiation between DNA and cDNA amplification, however, it is not influenced by non-specific amplification unless SYBR Green is used. Real-time PCR quantitation eliminates post-PCR processing of PCR products. This helps to increase throughput and reduce the chances of carryover contamination. Real-time PCR also offers a wide dynamic range of up to 10⁷-fold. Dynamic range of any assay determines how much target concentration can vary and still be quantified. A wide dynamic range means that a wide range of ratios of target and normaliser can be assayed with equal sensitivity and specificity. It follows that the broader the dynamic range, the more accurate the quantitation. When combined with RT-PCR, a real-time RT- PCR reaction reduces the time needed for measuring the amount of amplicon by providing for the visualization of the amplicon as the amplification process is progressing.

The real-time PCR system is based on the detection and quantitation of a fluorescent reporter. This signal increases in direct proportion to the amount of PCR product in a reaction. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. A significant increase in fluorescence above the baseline value measured during the 3-15 cycles can indicate the detection of accumulated PCR product.

A fixed fluorescence threshold is set significantly above the baseline that can be altered by the operator. The parameter CT (threshold cycle) is defined as the cycle number at which the fluorescence emission exceeds the fixed threshold.

There are three main fluorescence-monitoring systems for DNA amplification: (1) hydrolysis probes; (2) hybridising probes; and (3) DNA-binding agents. Hydrolysis probes include TaqMan probes, molecular beacons and scorpions. They use the fluorogenic 5' exonuclease activity of Taq polymerase to measure the amount of target sequences in cDNA samples.

TaqMan probes are oligonucleotides longer than the primers (20-30 bases long with a Tm value of 10°C higher) that contain a fluorescent dye usually on the 5' base, and a quenching dye (usually TAMRA) typically on the 3' base. When irradiated, the excited fluorescent dye transfers energy to the nearby quenching dye molecule rather than fluorescing (this is called FRET = F5rster or fluorescence resonance energy transfer). Thus, the close proximity of the reporter and quencher prevents emission of any fluorescence while the probe is intact. TaqMan probes are designed to anneal to an internal region of a PCR product. When the polymerase replicates a template on which a TaqMan probe is bound, its 5' exonuclease activity cleaves the probe. This ends the activity of quencher (no FRET) and the reporter dye starts to emit fluorescence which increases in each cycle proportional to the rate of probe cleavage. Accumulation of PCR products is detected by monitoring the increase in fluorescence of the reporter dye (note that primers are not labelled). TaqMan assay uses universal thermal cycling parameters and PCR reaction conditions. Because the cleavage occurs only if the probe hybridises to the target, the origin of the detected fluorescence is specific amplification. The process of hybridisation and cleavage does not interfere with the exponential accumulation of the product. One specific requirement for fluorogenic probes is that there be no G at the 5' end. A 'G' adjacent to the reporter dye can quench reporter fluorescence even after cleavage.

Molecular beacons also contain fluorescent (FAM, TAMRA, TET, ROX) and quenching dyes (typically DABCYL) at either end but they are designed to adopt a hairpin structure while free in solution to bring the fluorescent dye and the quencher in close proximity for FRET to occur. They have two arms with complementary sequences that form a very stable hybrid or stem. The close proximity of the reporter and the quencher in this hairpin configuration suppresses reporter fluorescence. When the beacon hybridises to the target during the annealing step, the reporter dye is separated from the quencher and the reporter fluoresces (FRET does not occur). Molecular beacons remain intact during PCR and must rebind to target every cycle for fluorescence emission. This will correlate to the amount of PCR product available. All real-time PCR chemistries allow detection of multiple DNA species (multiplexing) by designing each probe/beacon with a spectrally unique fluor/quench pair as long as the platform is suitable for melting curve analysis if SYBR green is used. By multiplexing, the target(s) and endogenous control can be amplified in single tube.

With Scorpion probes, sequence-specific priming and PCR product detection is achieved using a single oligonucleotide. The Scorpion probe maintains a stem-loop configuration in the unhybridised state. The fluorophore is attached to the 5' end and is quenched by a moiety coupled to the 3' end. The 3' portion of the stem also contains sequence that is complementary to the extension product of the primer. This sequence is linked to the 5' end of a specific primer via a non-amplifiable monomer. After extension of the Scorpion primer, the specific probe sequence is able to bind to its complement within the extended amplicon thus opening up the hairpin loop. This prevents the fluorescence from being quenched and a signal is observed.

Another alternative is the double-stranded DNA binding dye chemistry, which quantitates the amplicon production (including non-specific amplification and primer-dimer complex) by the use of a non-sequence specific fluorescent intercalating agent (SYBR- green I or ethidium bromide). It does not bind to ssDNA. SYBR green is a fluorogenic minor groove binding dye that exhibits little fluorescence when in solution but emits a strong fluorescent signal upon binding to double-stranded DNA. Disadvantages of SYBR green-based real-time PCR include the requirement for extensive optimisation. Furthermore, non-specific amplifications require follow-up assays (melting point curve or dissociation analysis) for amplicon identification. The method has been used in HFE-C282Y genotyping. Another controllable problem is that longer amplicons create a stronger signal (if combined with other factors, this may cause CDC camera saturation, see below). Normally SYBR green is used in singleplex reactions, however when coupled with melting point analysis, it can be used for multiplex reactions.

The threshold cycle or the CT value is the cycle at which a significant increase in ARn is first detected (for definition of ARn, see below). The threshold cycle is when the system begins to detect the increase in the signal associated with an exponential growth of PCR product during the log-linear phase. This phase provides the most useful information about the reaction (certainly more important than the end-point). The slope of the log-linear phase is a reflection of the amplification efficiency. The efficiency (Eff) of the reaction can be calculated by the formula: Eff=10^{( 1/slope)}-l . The efficiency of the PCR should be 90 - 100% (3.6 > slope >3.1). A number of variables can affect the efficiency of the PCR. These factors include length of the amplicon, secondary structure and primer quality. Although valid data can be obtained that fall outside of the efficiency range, the qRT-PCR should be further optimised or alternative amplicons designed. For the slope to be an indicator of real amplification (rather than signal drift), there has to be an inflection point. This is the point on the growth curve when the log-linear phase begins. It also represents the greatest rate of change along the growth curve. (Signal drift is characterised by gradual increase or decrease in fluorescence without amplification of the product.) The important parameter for quantitation is the CT. The higher the initial amount of genomic DNA, the sooner accumulated product is detected in the PCR process, and the lower the CT value. The threshold should be placed above any baseline activity and within the exponential increase phase (which looks linear in the log transformation). Some software allows determination of the cycle threshold (CT) by a mathematical analysis of the growth curve. This provides better run-to-run reproducibility. A CT value of 40 means no amplification and this value cannot be included in the calculations. Besides being used for quantitation, the CT value can be used for qualitative analysis as a pass/fail measure.

Multiplex TaqMan assays can be performed using multiple dyes with distinct emission wavelengths. Available dyes for this purpose are FAM, TET, VIC and JOE (the most expensive). TAMRA is reserved as the quencher on the probe and ROX as the passive reference. For best results, the combination of FAM (target) and VIC (endogenous control) is recommended (they have the largest difference in emission maximum) whereas JOE and VIC should not be combined. It is important that if the dye layer has not been chosen correctly, the machine will still read the other dye's spectrum. For example, both VIC and FAM emit fluorescence in a similar range to each other and when doing a single dye, the wells should be labelled correctly. In the case of multiplexing, the spectral compensation for the post run analysis should be turned on (on ABI 7700: Instrument/Diagnostics/Advanced Options/Miscellaneous). Activating spectral compensation improves dye spectral resolution.

Nested PCR

The disclosed methods can further utilize nested PCR. Nested PCR increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are being used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.

Primers and Probes The disclosed methods and assays can use primers and probes. As used herein, "primers" are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur. A primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation.

As used herein, "probes" are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art.

Disclosed are assays and methods which include the use of primers and probes, as well as, the disclosed primer panels all of which are capable of interacting with the disclosed nucleic acids such as ALK (SEQ ID NO: 1), BRAF, EGFR, KIT, or KRAS or their complement. For example, any of the primers or primer sets from Table 7-14 can be used in the disclosed primer panels or any of the methods and assays disclosed herein. In certain embodiments the primers are used to support nucleic acid extension reactions, nucleic acid replication reactions, and/or nucleic acid amplification reactions. Typically the primers will be capable of being extended in a sequence specific manner. Extension of a primer in a sequence specific manner includes any methods wherein the sequence and/or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer. Extension of the primer in a sequence specific manner therefore includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions that amplify the primer in a sequence specific manner are disclosed. In certain embodiments the primers are used for the DNA amplification reactions, such as PCR or direct sequencing. It is understood that in certain embodiments the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner. Typically the disclosed primers hybridize with the disclosed nucleic acids or region of the nucleic acids or they hybridize with the complement of the nucleic acids or complement of a region of the nucleic acids. As an example of the use of primers, one or more primers can be used to create extension products from and templated by a first nucleic acid.

The size of the primers or probes for interaction with the nucleic acids can be any size that supports the desired enzymatic manipulation of the primer, such as DNA amplification or the simple hybridization of the probe or primer. A typical primer or probe would be at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides long.

In other embodiments a primer or probe can be less than or equal to 6, 7, 8, 9, 10, 11, 12 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides long.

The primers for the nucleic acid of interest typically will be used to produce extension products and/or other replicated or amplified products that contain a region of the nucleic acid of interest. The size of the product can be such that the size can be accurately determined to within 3, or 2 or 1 nucleotides.

In certain embodiments the product can be, for example, at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides long.

In other embodiments the product can be, for example, less than or equal to 20, 21,

22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides long.

It is understood and herein contemplated that there are situations where it may be advantageous to utilize more than one primer pair to detect the presence of mutations conferring inhibitor resistance in EGFR, BRAF, KIT, KRAS, or ALK. Such RT-PCR, real- time PCT or other PCR reactions can be conducted separately, or in a single reaction.

When multiple primer pairs are placed into a single reaction, this is referred to as "multiplex PCR." It is understood and herein contemplated that any combination of two or more or three or more the forward and/or reverse primers disclosed herein can be used in the multiplex reaction.

Fluorescent Change Probes and Primers

Fluorescent change probes and fluorescent change primers refer to all probes and primers that involve a change in fluorescence intensity or wavelength based on a change in the form or conformation of the probe or primer and nucleic acid to be detected, assayed or replicated. Examples of fluorescent change probes and primers include molecular beacons, Amplifluors, FRET probes, cleavable FRET probes, TaqMan probes, scorpion primers, fluorescent triplex oligos including but not limited to triplex molecular beacons or triplex FRET probes, fluorescent water-soluble conjugated polymers, PNA probes and QPNA probes.

Fluorescent change probes and primers can be classified according to their structure and/or function. Fluorescent change probes include hairpin quenched probes, cleavage quenched probes, cleavage activated probes, and fluorescent activated probes. Fluorescent change primers include stem quenched primers and hairpin quenched primers.

Hairpin quenched probes are probes that when not bound to a target sequence form a hairpin structure (and, typically, a loop) that brings a fluorescent label and a quenching moiety into proximity such that fluorescence from the label is quenched. When the probe binds to a target sequence, the stem is disrupted, the quenching moiety is no longer in proximity to the fluorescent label and fluorescence increases. Examples of hairpin quenched probes are molecular beacons, fluorescent triplex oligos, triplex molecular beacons, triplex FRET probes, and QPNA probes.

Cleavage activated probes are probes where fluorescence is increased by cleavage of the probe. Cleavage activated probes can include a fluorescent label and a quenching moiety in proximity such that fluorescence from the label is quenched. When the probe is clipped or digested (typically by the 5'-3' exonuclease activity of a polymerase during amplification), the quenching moiety is no longer in proximity to the fluorescent label and fluorescence increases. TaqMan probes are an example of cleavage activated probes.

Cleavage quenched probes are probes where fluorescence is decreased or altered by cleavage of the probe. Cleavage quenched probes can include an acceptor fluorescent label and a donor moiety such that, when the acceptor and donor are in proximity, fluorescence resonance energy transfer from the donor to the acceptor causes the acceptor to fluoresce. The probes are thus fluorescent, for example, when hybridized to a target sequence. When the probe is clipped or digested (typically by the 5'-3' exonuclease activity of a polymerase during amplification), the donor moiety is no longer in proximity to the acceptor fluorescent label and fluorescence from the acceptor decreases. If the donor moiety is itself a fluorescent label, it can release energy as fluorescence (typically at a different wavelength than the fluorescence of the acceptor) when not in proximity to an acceptor. The overall effect would then be a reduction of acceptor fluorescence and an increase in donor fluorescence. Donor fluorescence in the case of cleavage quenched probes is equivalent to fluorescence generated by cleavage activated probes with the acceptor being the quenching moiety and the donor being the fluorescent label. Cleavable FRET (fluorescence resonance energy transfer) probes are an example of cleavage quenched probes.

Fluorescent activated probes are probes or pairs of probes where fluorescence is increased or altered by hybridization of the probe to a target sequence. Fluorescent activated probes can include an acceptor fluorescent label and a donor moiety such that, when the acceptor and donor are in proximity (when the probes are hybridized to a target sequence), fluorescence resonance energy transfer from the donor to the acceptor causes the acceptor to fluoresce. Fluorescent activated probes are typically pairs of probes designed to hybridize to adjacent sequences such that the acceptor and donor are brought into proximity. Fluorescent activated probes can also be single probes containing both a donor and acceptor where, when the probe is not hybridized to a target sequence, the donor and acceptor are not in proximity but where the donor and acceptor are brought into proximity when the probe hybridized to a target sequence. This can be accomplished, for example, by placing the donor and acceptor on opposite ends of the probe and placing target complement sequences at each end of the probe where the target complement sequences are complementary to adjacent sequences in a target sequence. If the donor moiety of a fluorescent activated probe is itself a fluorescent label, it can release energy as fluorescence (typically at a different wavelength than the fluorescence of the acceptor) when not in proximity to an acceptor (that is, when the probes are not hybridized to the target sequence). When the probes hybridize to a target sequence, the overall effect would then be a reduction of donor fluorescence and an increase in acceptor fluorescence. FRET probes are an example of fluorescent activated probes.

Stem quenched primers are primers that when not hybridized to a complementary sequence form a stem structure (either an intramolecular stem structure or an intermolecular stem structure) that brings a fluorescent label and a quenching moiety into proximity such that fluorescence from the label is quenched. When the primer binds to a complementary sequence, the stem is disrupted; the quenching moiety is no longer in proximity to the fluorescent label and fluorescence increases. In the disclosed method, stem quenched primers are used as primers for nucleic acid synthesis and thus become incorporated into the synthesized or amplified nucleic acid. Examples of stem quenched primers are peptide nucleic acid quenched primers and hairpin quenched primers.

Peptide nucleic acid quenched primers are primers associated with a peptide nucleic acid quencher or a peptide nucleic acid fluor to form a stem structure. The primer contains a fluorescent label or a quenching moiety and is associated with either a peptide nucleic acid quencher or a peptide nucleic acid fluor, respectively. This puts the fluorescent label in proximity to the quenching moiety. When the primer is replicated, the peptide nucleic acid is displaced, thus allowing the fluorescent label to produce a fluorescent signal.

Hairpin quenched primers are primers that when not hybridized to a complementary sequence form a hairpin structure (and, typically, a loop) that brings a fluorescent label and a quenching moiety into proximity such that fluorescence from the label is quenched. When the primer binds to a complementary sequence, the stem is disrupted; the quenching moiety is no longer in proximity to the fluorescent label and fluorescence increases. Hairpin quenched primers are typically used as primers for nucleic acid synthesis and thus become incorporated into the synthesized or amplified nucleic acid. Examples of hairpin quenched primers are Amplifluor primers and scorpion primers.

Cleavage activated primers are similar to cleavage activated probes except that they are primers that are incorporated into replicated strands and are then subsequently cleaved. Labels

To aid in detection and quantitation of nucleic acids produced using the disclosed methods, labels can be directly incorporated into nucleotides and nucleic acids or can be coupled to detection molecules such as probes and primers. As used herein, a label is any molecule that can be associated with a nucleotide or nucleic acid, directly or indirectly, and which results in a measurable, detectable signal, either directly or indirectly. Many such labels for incorporation into nucleotides and nucleic acids or coupling to nucleic acid probes are known to those of skill in the art. Examples of labels suitable for use in the disclosed method are radioactive isotopes, fluorescent molecules, phosphorescent molecules, enzymes, antibodies, and ligands. Fluorescent labels, especially in the context of fluorescent change probes and primers, are useful for real-time detection of amplification.

Examples of suitable fluorescent labels include fluorescein isothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-l,3-diazol-4-yl ( BD), coumarin, dansyl chloride, rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin, BODIPY^®, CASCADE BLUE^®, OREGON GREEN^®, pyrene, lissamine, xanthenes, acridines, oxazines, phycoerythrin, macrocyclic chelates of lanthanide ions such as quantum dye™, fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer, and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. Examples of other specific fluorescent labels include 3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine (5-HT), Acid Fuchsin, Alizarin Complexon, Alizarin Red, Allophycocyanin, Aminocoumarin, Anthroyl Stearate, Astrazon Brilliant Red 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrine, Auramine, Aurophosphine, Aurophosphine G, BAO 9 (Bisaminophenyloxadiazole), BCECF, BerberineSulphate, Bisbenzamide, Blancophor FFG Solution, Blancophor SV, Bodipy Fl, Brilliant Sulpho flavin FF, Calcien Blue, Calcium Green, Calcofluor RW Solution, Calcofluor White, Calcophor White ABT Solution, Calcophor White Standard Solution, Carbostyryl, Cascade Yellow, Catecholamine, Chinacrine, Coriphosphine O, Coumarin-Phalloidin, CY3.1 8, CY5.1 8, CY7, Dans (1- Dimethyl Amino Naphaline 5 Sulphonic Acid), Dansa (DiaminoNaphtylSulphonic Acid), Dansyl NH-CH3, Diamino Phenyl Oxydiazole (DAO), Dimethylamino-5-Sulphonic acid, DipyrrometheneboronDifluoride, Diphenyl Brilliant Flavine 7GFF, Dopamine, Erythrosin ITC, Euchrysin, FIF (Formaldehyde Induced Fluorescence), Flazo Orange, Fluo 3, Fluorescamine, Fura-2, Genacryl Brilliant Red B, Genacryl Brilliant Yellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF, Gloxalic Acid, Granular Blue,

Haematoporphyrin, Indo-1, IntrawhiteCf Liquid, Leucophor PAF, Leucophor SF,

Leucophor WS, LissamineRhodamine B200 (RD200), Lucifer Yellow CH, Lucifer Yellow VS, Magdala Red, Marina Blue, Maxilon Brilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF, MPS (Methyl Green PyronineStilbene), Mithramycin, NBD Amine,

Nitrobenzoxadidole, Noradrenaline, Nuclear Fast Red, Nuclear Yellow, Nylosan Brilliant Flavin E8G, Oxadiazole, Pacific Blue, Pararosaniline (Feulgen), Phorwite AR Solution, Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphine 3R, Phthalocyanine,

Phycoerythrin R, Phycoerythrin B, PolyazaindacenePontochrome Blue Black, Porphyrin, Primuline, Procion Yellow, Pyronine, Pyronine B, Pyrozal Brilliant Flavin 7GF, Quinacrine Mustard, Rhodamine 123, Rhodamine 5 GLD, Rhodamine 6G, Rhodamine B, Rhodamine B 200, Rhodamine B Extra, Rhodamine BB, Rhodamine BG, Rhodamine WT, Serotonin, Sevron Brilliant Red 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange, Sevron Yellow L, SITS (Primuline), SITS (Stilbenelsothiosulphonic acid), Stilbene, Snarf 1, sulphoRhodamine B Can C, SulphoRhodamine G Extra, Tetracycline, Thiazine Red R, Thioflavin S, Thioflavin TCN, Thioflavin 5, Thiolyte, Thiozol Orange, Tinopol CBS, True Blue, Ultralite, Uranine B, Uvitex SFC, Xylene Orange, and XRITC.

The absorption and emission maxima, respectively, for some of these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm), thus allowing their simultaneous detection. Other examples of fluorescein dyes include 6-carboxyfluorescein (6-FAM), 2',4',1,4,-tetrachlorofluorescein (TET), 2',4',5',7',1,4-hexachlorofluorescein (HEX), 2',7'-dimethoxy-4', 5'-dichloro-6-carboxyrhodamine (JOE), 2'-chloro-5'-fluoro-7',8'- fused phenyl- l,4-dichloro-6-carboxyfluorescein (NED), and 2'-chloro-7'-phenyl-l,4- dichloro-6-carboxyfluorescein (VIC). Fluorescent labels can be obtained from a variety of commercial sources, including Amersham Pharmacia Biotech, Piscataway, NJ; Molecular Probes, Eugene, OR; and Research Organics, Cleveland, Ohio.

Additional labels of interest include those that provide for signal only when the probe with which they are associated is specifically bound to a target molecule, where such labels include: "molecular beacons" as described in Tyagi& Kramer, Nature Biotechnology (1996) 14:303 and EP 0 070 685 B l. Other labels of interest include those described in U.S. Pat. No. 5,563,037 which is incorporated herein by reference.

Labeled nucleotides are a form of label that can be directly incorporated into the amplification products during synthesis. Examples of labels that can be incorporated into amplified nucleic acids include nucleotide analogs such as BrdUrd, aminoallyldeoxyuridine, 5-methylcytosine, bromouridine, and nucleotides modified with biotin or with suitable haptens such as digoxygenin. Suitable fluorescence-labeled nucleotides are Fluorescein- isothiocyanate-dUTP, Cyanine-3-dUTP and Cyanine-5-dUTP. One example of a nucleotide analog label for DNA is BrdUrd (bromodeoxyuridine, BrdUrd, BrdU, BUdR, Sigma- Aldrich Co). Other examples of nucleotide analogs for incorporation of label into DNA are AA-dUTP (aminoallyl-deoxyuridine triphosphate, Sigma-Aldrich Co.), and 5-methyl-dCTP (Roche Molecular Biochemicals). One example of a nucleotide analog for incorporation of label into RNA is biotin- 16-UTP (biotin- 16-uridine-5'-triphosphate, Roche Molecular Biochemicals). Fluorescein, Cy3, and Cy5 can be linked to dUTP for direct labeling.

Cy3.5 and Cy7 are available as avidin or anti-digoxygenin conjugates for secondary detection of biotin- or digoxygenin-labeled probes.

Labels that are incorporated into amplified nucleic acid, such as biotin, can be subsequently detected using sensitive methods well-known in the art. For example, biotin can be detected using streptavidin-alkaline phosphatase conjugate (Tropix, Inc.), which is bound to the biotin and subsequently detected by chemiluminescence of suitable substrates (for example, chemiluminescent substrate CSPD: disodium, 3-(4-methoxyspiro-[l,2,- dioxetane-3-2'-(5'-chloro)tricyclo [3.3.1.1³'⁷]decane]-4-yl) phenyl phosphate; Tropix, Inc.). Labels can also be enzymes, such as alkaline phosphatase, soybean peroxidase, horseradish peroxidase and polymerases, that can be detected, for example, with chemical signal amplification or by using a substrate to the enzyme which produces light (for example, a chemiluminescent 1 ,2-dioxetane substrate) or fluorescent signal.

Molecules that combine two or more of these labels are also considered labels. Any of the known labels can be used with the disclosed probes, tags, and method to label and detect nucleic acid amplified using the disclosed method. Methods for detecting and measuring signals generated by labels are also known to those of skill in the art. For example, radioactive isotopes can be detected by scintillation counting or direct visualization; fluorescent molecules can be detected with fluorescent spectrophotometers; phosphorescent molecules can be detected with a spectrophotometer or directly visualized with a camera; enzymes can be detected by detection or visualization of the product of a reaction catalyzed by the enzyme; antibodies can be detected by detecting a secondary label coupled to the antibody. As used herein, detection molecules are molecules which interact with amplified nucleic acid and to which one or more labels are coupled.

The disclosed methods, assays, and primer panels can be used to diagnose any disease where uncontrolled cellular proliferation occurs herein referred to as "cancer". A non-limiting list of different types of ALK related cancers is as follows: lymphomas (Hodgkins and non-Hodgkins), leukemias, carcinomas, carcinomas of solid tissues, squamous cell carcinomas, adenocarcinomas, sarcomas, gliomas, high grade gliomas, blastomas, neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas, hypoxic tumours, myelomas, AIDS-related lymphomas or sarcomas, metastatic cancers, or cancers in general. In particular, the disclosed methods, assays, and kits relate to the diagnosis, detection, or prognosis of inflammatory breast cancer

A representative but non-limiting list of cancers that the disclosed methods can be used to diagnose is the following: lymphoma, B cell lymphoma (including diffuse large B-cell lymphoma), B-cell plasmablastic/immunoblastic lymphomas, T cell lymphoma (including T- or null-cell lymphomas), mycosis fungoides, Hodgkin's Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, kidney cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma/glioblastoma, , anaplastic large-cell lymphoma (ALCL), inflammatory myofibroblastic tumors, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, malignant histiocytosis, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer (including inflammatory breast cancer), and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal squamous cell carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon and rectal cancers, prostatic cancer, or pancreatic cancer.

Nucleic Acids

The disclosed method and compositions make use of various nucleic acids.

Generally, any nucleic acid can be used in the disclosed method. For example, the disclosed nucleic acids of interest and the disclosed reference nucleic acids can be chosen based on the desired analysis and information that is to be obtained or assessed. The disclosed methods also produce new and altered nucleic acids. The nature and structure of such nucleic acids will be established by the manner in which they are produced and manipulated in the methods. Thus, for example, extension products and hybridizing nucleic acids are produced in the disclosed methods. As used herein, hybridizing nucleic acids are hybrids of extension products and the second nucleic acid.

It is understood and contemplated herein that a nucleic acid of interest can be any nucleic acid to which the determination of the presence or absence of nucleotide variation is desired. Thus, for example, the nucleic acid of interest can comprise a sequence that corresponds to the wild-type sequence of the reference nucleic acid. It is further disclosed herein that the disclosed methods can be performed where the first nucleic acid is a reference nucleic acid and the second nucleic acid is a nucleic acid of interest or where the first nucleic acid is the nucleic acid of interest and the second nucleic acid is the reference nucleic acid. It is understood and herein contemplated that a reference nucleic acid can be any nucleic acid against which a nucleic acid of interest is to be compared. Typically, the reference nucleic acid has a known sequence (and/or is known to have a sequence of interest as a reference). Although not required, it is useful if the reference sequence has a known or suspected close relationship to the nucleic acid of interest. For example, if a single nucleotide variation is desired to be detected, the reference sequence can be usefully chosen to be a sequence that is a homolog or close match to the nucleic acid of interest, such as a nucleic acid derived from the same gene or genetic element from the same or a related organism or individual. Thus, for example, it is contemplated herein that the reference nucleic acid can comprise a wild-type sequence or alternatively can comprise a known mutation including, for example, a mutation the presence or absence of which is associated with a disease or resistance to therapeutic treatment. Thus, for example, it is contemplated that the disclosed methods can be used to detect or diagnose the presence of known mutations in a nucleic acid of interest by comparing the nucleic acid of interest to a reference nucleic acid that comprises a wild-type sequence (i.e., is known not to possess the mutation) and examining for the presence or absence of variation in the nucleic acid of interest, where the absence of variation would indicate the absence of a mutation.

Alternatively, the reference nucleic acid can possess a known mutation. Thus, for example, it is contemplated that the disclosed methods can be used to detect susceptibility for a disease or condition by comparing the nucleic acid of interest to a reference nucleic acid comprising a known mutation that indicates susceptibility for a disease and examining for the presence or absence of the mutation, wherein the presence of the mutation indicates a disease.

Herein, the term "nucleotide variation" refers to any change or difference in the nucleotide sequence of a nucleic acid of interest relative to the nucleotide sequence of a reference nucleic acid. Thus, a nucleotide variation is said to occur when the sequences between the reference nucleic acid and the nucleic acid of interest (or its complement, as appropriate in context) differ. Thus, for example, a substitution of an adenine (A) to a guanine (G) at a particular position in a nucleic acid would be a nucleotide variation provided the reference nucleic acid comprised an A at the corresponding position. It is understood and herein contemplated that the determination of a variation is based upon the reference nucleic acid and does not indicate whether or not a sequence is wild-type. Thus, for example, when a nucleic acid with a known mutation is used as the reference nucleic acid, a nucleic acid not possessing the mutation (including a wild-type nucleic acid) would be considered to possess a nucleotide variation (relative to the reference nucleic acid). Nucleotides

The disclosed methods and compositions make use of various nucleotides.

Throughout this application and the methods disclosed herein reference is made to the type of base for a nucleotide. It is understood and contemplated herein that where reference is made to a type of base, this refers a base that in a nucleotide in a nucleic acid strand is capable of hybridizing (binding) to a defined set of one or more of the canonical bases. Thus, for example, where reference is made to extension products extended in the presence of three types of nuclease resistant nucleotides and not in the presence of nucleotides that comprise the same type of base as the modified nucleotides, this means that if, for example, the base of the modified nucleotide was an adenine (A), the nuclease-resistant nucleotides can be, for example, guanine (G), thymine (T), and cytosine (C). Each of these bases (which represent the four canonical bases) is capable of hybridizing to a different one of the four canonical bases and thus each qualify as a different type of base as defined herein. As another example, inosine base pairs primarily with adenine and cytosine (in DNA) and thus can be considered a different type of base from adenine and from cytosine- which base pair with thymine and guanine, respectively-but not a different type of base from guanine or thymine-which base pair with cytosine and adenine, respectively-because the base pairing of guanine and thymine overlaps (that is, is not different from) the hybridization pattern of inosine.

Any type of modified or alternative base can be used in the disclosed methods and compositions, generally limited only by the capabilities of the enzymes used to use such bases. Many modified and alternative nucleotides and bases are known, some of which are described below and elsewhere herein. The type of base that such modified and alternative bases represent can be determined by the pattern of base pairing for that base as described herein. Thus for example, if the modified nucleotide was adenine, any analog, derivative, modified, or variant base that based pairs primarily with thymine would be considered the same type of base as adenine. In other words, so long as the analog, derivative, modified, or variant has the same pattern of base pairing as another base, it can be considered the same type of base. Modifications can made to the sugar or phosphate groups of a nucleotide. Generally such modifications will not change the base pairing pattern of the base.

However, the base pairing pattern of a nucleotide in a nucleic acid strand determines the type of base of the base in the nucleotide.

Modified nucleotides to be incorporated into extension products and to be selectively removed by the disclosed agents in the disclosed methods can be any modified nucleotide that functions as needed in the disclosed method as is described elsewhere herein. Modified nucleotides can also be produced in existing nucleic acid strands, such as extension products, by, for example, chemical modification, enzymatic modification, or a combination.

Many types of nuclease-resistant nucleotides are known and can be used in the disclosed methods. For example, nucleotides have modified phosphate groups and/or modified sugar groups can be resistant to one or more nucleases. Nuclease-resistance is defined herein as resistance to removal from a nucleic acid by any one or more nucleases. Generally, nuclease resistance of a particular nucleotide can be defined in terms of a relevant nuclease. Thus, for example, if a particular nuclease is used in the disclosed method, the nuclease-resistant nucleotides need only be resistant to that particular nuclease. Examples of useful nuclease-resistant nucleotides include thio-modified nucleotides and borano-modified nucleotides.

There are a variety of molecules disclosed herein that are nucleic acid based. Non- limiting examples of these and other molecules are discussed herein. It is understood that for example, a nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage. The base moiety of a nucleotide can be adenine-9-yl (adenine, A), cytosine- 1-yl (cytosine, C), guanine-9-yl (guanine, G), uracil- 1- yl (uracil, U), and thymin-l-yl (thymine, T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. A non- limiting example of a nucleotide would be 3'-AMP (3'-adenosine monophosphate) or 5'- GMP (5'-guanosine monophosphate).

A nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to the base moiety would include natural and synthetic modifications of A, C, G, and T/U as well as different purine or pyrimidine bases, such as uracil-5-yl (ψ), hypoxanthin-9-yl (inosine, I), and 2- aminoadenin-9-yl. A modified base includes but is not limited to 5-methylcytosine (5-me- C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5- propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5 -uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional base modifications can be found for example in U.S. Pat. No. 3,687,808, which is incorporated herein in its entirety for its teachings of base modifications. Certain nucleotide analogs, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5-methylcytosine can increase the stability of duplex formation. Often time base modifications can be combined with for example a sugar modification, such as 2'- O-methoxyethyl, to achieve unique properties such as increased duplex stability. Nucleotide analogs can also include modifications of the sugar moiety.

Modifications to the sugar moiety would include natural modifications of the ribose and deoxy ribose as well as synthetic modifications. Sugar modifications include but are not limited to the following modifications at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted CI to CIO, alkyl or C2 to CIO alkenyl and alkynyl. 2' sugar modifications also include but are not limited to -0[(CH2)n 0]m CH3, - 0(CH2)n 0CH3, -0(CH2)n NH2, -0(CH2)n CH3, -0(CH2)n -0NH2, and - 0(CH2)nON[(CH2)n CH3)]2, where n and m are from 1 to about 10.

Other modifications at the 2' position include but are not limited to: CI to CIO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, CF3, OCF3, SOCH3, S02 CH3, ON02, N02, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar

modifications may also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Modified sugars would also include those that contain modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include but are not limited to those that can be modified so that the linkage between two nucleotides contains a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3'-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. It is understood that these phosphate or modified phosphate linkage between two nucleotides can be through a 3'-5' linkage or a 2'-5' linkage, and the linkage can contain inverted polarity such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.

It is understood that nucleotide analogs need only contain a single modification, but may also contain multiple modifications within one of the moieties or between different moieties.

Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson- Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.

Nucleotide substitutes are nucleotides or nucleotide analogs that have had the phosphate moiety and/or sugar moieties replaced. Nucleotide substitutes do not contain a standard phosphorus atom. Substitutes for the phosphate can be for example, short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or

cycloalkylinternucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones;

methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

It is also understood in a nucleotide substitute that both the sugar and the phosphate moieties of the nucleotide can be replaced, by for example an amide type linkage

(aminoethylglycine) (PNA). It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an

octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

A Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute. The Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl, and C6 positions of a purine based nucleotide, nucleotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute.

A Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA. The Hoogsteen face includes the N7 position and reactive groups (NH2 or O) at the C6 position of purine nucleotides.

Hybridization/Selective Hybridization

The term hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene. Sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide specific manner. For example, G interacting with C or A interacting with T are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide. The hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize. Parameters for selective hybridization between two nucleic acid molecules are well known to those of skill in the art. For example, in some embodiments selective

hybridization conditions can be defined as stringent hybridization conditions. For example, stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps. For example, the conditions of

hybridization to achieve selective hybridization may involve hybridization in high ionic strength solution (6X SSC or 6X SSPE) at a temperature that is about 12-25°C below the Tm (the melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5°C to 20°C below the Tm. The temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies. Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA

hybridizations. The conditions can be used as described above to achieve stringency, or as is known in the art. A preferable stringent hybridization condition for a DNA:DNA hybridization can be at about 68°C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68°C. Stringency of hybridization and washing, if desired, can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for. Likewise, stringency of hybridization and washing, if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art.

Another way to define selective hybridization is by looking at the amount

(percentage) of one of the nucleic acids bound to the other nucleic acid. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid is bound to the non-limiting nucleic acid. Typically, the non-limiting primer is in for example, 10 or 100 or 1000 fold excess. This type of assay can be performed at under conditions where both the limiting and non-limiting primer are for example, 10 fold or 100 fold or 1000 fold below their ka, or where only one of the nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules are above their ka.

Another way to define selective hybridization is by looking at the percentage of primer that gets enzymatically manipulated under conditions where hybridization is required to promote the desired enzymatic manipulation. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer is enzymatically manipulated under conditions which promote the enzymatic manipulation, for example if the enzymatic manipulation is DNA extension, then selective hybridization conditions would be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer molecules are extended. Conditions also include those suggested by the manufacturer or indicated in the art as being appropriate for the enzyme performing the manipulation.

Just as with homology, it is understood that there are a variety of methods herein disclosed for determining the level of hybridization between two nucleic acid molecules. It is understood that these methods and conditions may provide different percentages of hybridization between two nucleic acid molecules, but unless otherwise indicated meeting the parameters of any of the methods would be sufficient. For example if 80%

hybridization was required and as long as hybridization occurs within the required parameters in any one of these methods it is considered disclosed herein.

It is understood that those of skill in the art understand that if a composition or method meets any one of these criteria for determining hybridization either collectively or singly it is a composition or method that is disclosed herein.

Kits Disclosed herein are kits that are drawn to reagents that can be used in practicing the methods disclosed herein. In particular, he kits can include any reagent or combination of reagents discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods. For example, the kits could include one or more primers from Tables 7-14 disclosed herein to perform the extension, replication and amplification reactions discussed in certain embodiments of the methods, as well as the buffers and enzymes required to use the primers as intended. The kit can also include other necessary reagents to perform any of the next generation sequencing techniques disclosed herein. In another aspect, the disclosed kits can include one or more of the probes listed in Table 15 in addition to or instead of one or more primers from Table 7-14.

It is understood and herein contemplated that the disclosed kits can comprise at least one primer set to detect the presence of nucleic acid variation in each οΐΚΙΤ, BRAF, KRAS, ALK, and EGFR. For example, the kits can comprise at least one primer or primer set for sequencing at least one of each of the KIT, BRAF, KRAS, ALK, and EGFR exons of Tables 1. In one aspect, the kits can comprise at least one primer or primer set from each of Tables 7-14. Alternatively, the kit can comprise a primer or primer set that will detect one or more of the specific mutations listed in Tables 2-6. Therefore, in one aspect disclosed herein are kits for performing a NGS sequencing reaction on a tissue sample to detect the presence of a mutation conferring kinase inhibitor resistance comprising at least one or more primer or primer set from each of Table 7-14. In another aspect, disclosed herein are kits for performing a NGS sequencing reaction on a tissue sample to detect the presence of a mutation conferring kinase inhibitor resistance comprising at least one or more primer or primer set capable of specifically hybridizing an amplifying any of the mutant sequences of KIT, BRAF, KRAS, ALK, and EGFR present in Tables 2-6.

Additionally, it is understood that the disclosed kits can include such other reagents and material for performing the disclosed methods such as enzymes (e.g., polymerases), buffers, sterile water, and/or reaction tubes. Additionally the kits can also include modified nucleotides, nuclease-resistant nucleotides, and or labeled nucleotides. Additionally, the disclosed kits can include instructions for performing the methods disclosed herein and software for enable the calculation of the presence of a kinase inhibitor mutation (i.e., a mutation in KIT, BRAF, KRAS, EGFR, and/or ALK).

The compositions disclosed herein and the compositions necessary to perform the disclosed methods can be made using any method known to those of skill in the art for that particular reagent or compound unless otherwise specifically noted.

Nucleic Acid Synthesis

The disclosed nucleic acids, such as, the oligonucleotides to be used as primers can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation to purely synthetic methods, for example, by the cyanoethylphosphoramidite method using a Milligen or Beckman System lPlus DNA synthesizer (for example, Model 8700 automated synthesizer of Milligen- Biosearch, Burlington, MA or ABI Model 380B).

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

Example 1: ALK Inhibitor Resistance

Using an in vitro assay known to predict clinically relevant kinase inhibitor- resistance mutations resistance selection studies were performed with XALKORI® and identified a large number of ALK kinase domain point mutations that confer high-level resistance to the Pfizer inhibitor (Figure 1). In response to the issue of resistance, a number of pharma and biotech companies currently have 2ⁿ -generation ALK small-molecule inhibitors in development.

The need for more comprehensive oncogene profiling in patients with ALK inhibitor resistance was observed in an ALK positive crizotinib resistant cohort of patients that ALK specific kinase mutations accounted for only a third of crizotinib resistance. The larger subset of crizotinib resistant cases indicated that second (co-expression in conjunction with ALK) or separate (complete absence of ALK) oncogenic drivers such as EGFR, BRAF, KRAS or cKIT can relieve the sensitivity to crizotinib and drive oncogenesis. It was also observed in a single case that the complete loss of ALK expression did not correspond to the presence of an identifiable alternate driver indicating the genetic profiling of ALK inhibitor resistance cases should be extended past EGFR, BRAF, KRAS or cKIT expression using more versatile testing platforms. The presence of multiple oncogenes present in a single tumor sample is by no means a new phenomenon as EGFR driven tumors resistant to EGFR tyrosine kinase inhibitors can be driven by secondary MET gene amplification.

Example 2: A Diagnostic Cancer Panel that Employs NGS

Applicants have designed and developed a next generation sequencing panel to amplify and sequence one or more exons within ALK and other oncogenes implicated in driving tumorigenesis in the presence of crizotinib (i.e. ALK, BRAF, EGFR, KIT and K- RAS. See Table 1 for an overarching description of the exons targeted for sequencing in the panel and Tables 2-6 for a more detailed list of each mutation detected by the Insight ALK resistance ZD™ panel. Primer sequences used to amplify each gene segment are depicted in Tables 7-14.

EGFR 19

EGFR 20

EGFR 21

EGFR 22

ALK 21

ALK 22

ALK 23

ALK 24

ALK 25

KIT 8

KIT 9

KIT 10

KIT 11

KIT 12

KIT 13

KIT 17

BRAF 10

BRAF 11

BRAF 13

BRAF 14

BRAF 15

Example 3: Targeted Next Generation Sequencing Insight ALK resistance ID™

Polymerase chain reaction is used to create amplicons that span the exonic regions mentioned above. The design described here is agnostic to the NGS platform used to perform the actual sequencing, and thus multiple PCR strategies can match the size of the PCR fragments to the read-length of the sequencing platform being employed. The PCR amplification can be done in a single-tube as a multiple reaction where all targets are covered at once. In the case of low coverage or ambiguous results, a single-plex PCR can be performed as a confirmatory step to ensure accurate mutation calling. This is also true in the case of highly-degraded samples where the template DNA has fragmented and large- amplicons cannot be extracted from the DNA that remains. See Tables 7-14 for a full list of the primers that have been designed and the general size of fragments each set produces. There are a large number of primers in the list to ensure that there is flexibility to run various multiplex PCR reactions where there is very little sequence overlap in the primers, which can lead to dimerization, and allow melting temperatures of all the oligos in a particular reaction to be matched. The amplification parameters of each PCR reaction consist of 95°C 15-min heat denaturation phase followed by 40 cycles of denaturation at 95°C for 15 sec and 55°C annealing for 30 sec and 72°C extension for 1 min and finally a 72°C final extension step for 5 minutes. At the end of the PCR step a diverse set of fragments that cover the exons of interest can be synthesized. The fragments can then be adapted for sequencing on any commercially available NGS platform. Since there is a very wide range of read-lengths that the different NGS instruments produce, from as low as 35 bases to as high as 1500 and expectations of lOOkb read length in the near future, the Insight ALK resistance ID™ is designed to be able to produce fragments as short as 150 bases to as high as 5kb. This ensures for efficient sequencing where the size of each amplicon can be matched to the output of long-read and middle-read technologies (150-1000 bases) or have large enough fragments (5kb) that can be effectively sheared, either sonically or enzymatically, to be compatible with short-read sequencers (<150 bases).

The ALK resistance ID™ takes advantage of the very high-throughput offered by modern sequencers to cover the regions of interest at very high coverage (depth > 5,000X) and thus enable the detection of rare variants only present in the sample at a frequency of 1% or less. The sequence reads that are generated can be compared to a reference sequence examined for the presence of any of the mutations listed in Tables 2-6.

Table 2. ALK Mutations That Are Covered Amino Acid Mutation Nucleotide Mutation p.V1471fs*45 c.4409_4422delCCGTGGAAGGGGGA p.Y1584Y c.4752C>T p.T1597T c.4791T>A p.L1062l c.3185A>T p.T1087l c.3260C>T p.D1091N c.3271G>A

P.G1128A c.3383G>C

P.M 1166 c.3497T>G

P.A1168P .3502OG

P.I 1171N c.3512T>A

P.F1174I c.3520T>A

P.F1174L c.3522C>A

P.R1192P c.3575G>C p.F1245C c.3734T>G p.F1245V c.3733T>G p.F1245L c.3735C>G p.F1245l c.3733T>A

P.I 1250T c.3749T>C p.R1275Q c.3824G>A

Table 3. EGFR Mutations That Are Covered

Amino Acid Mutation Nucleotide Mutation p.L747_T751>S c.2240_2251del l2

P.L861Q c.2582T>A p.L747_E749del c.2239_2247del9 p.E746_S752>D c.2238_2255del l8 p.E746_A750del c.2235_2249del l5 p.L858R c.2573T>G p.E746_A750del c.2236_2250del l5 p.R776C c.2326C>T p.H835L c.2504A>T p.G719A c.2156G>C p.T790M c.2369C>T p.S768l c.2303G>T p.V769L c.2305G>T p.G719S c.2155G>A p.G719C c.2155G>T p.L747_T751del c.2239_2253dell5 p.L747_S752del c.2239_2256dell8 p.S752J759del c.2254_2277del24 p.P753S c.2257C>T p.L858M c.2572C>A p.E746_S752>A c.2237_2254dell8 p.L747_T751del c.2240_2254dell5 p.L747_P753>S c.2240_2257dell8 p.E709V c.2126A>T

P.I715S c.2144T>G p.S720F c.2159C>T

P.L861 c.2582T>G p.V769_D770insASV c.2307_2308ins9 p.H773_V774insH c.2319_2320insCAC p.D770_N771insG c.2310_2311insGGT p.V769_D770insCV c.2307_2308insTGCGTG p.H773_V774insPH c.2319_2320insCCCCAC p.H773_V774insNPH c.2319_2320ins9 p.L747_A750>P c.2239_2248TTAAGAGAAG>C p.L747_T751>P c.2239_2251>C p.E746_S752>V c.2237_2255>T p.E746_S752>l c.2235_2255>AAT p.E746_T751>V c.2237_2252>T p.L747_P753>Q c.2239_2258>CA p.H773>NPY c.2317_2317C>AACCCCT p.V774_C775insHV c.2322_2322G>CCACGTG p.L747_S752>Q c.2239_2256>CAA p.E746_T751>l c.2229_2252>AATTAAGA p.T751J759>S c.2252_2275>G p.E746_A750> P c.2236_2248>AGAC p.E746_T751>VA c.2237_2253>TTGCT p.L747_T751>Q c.2238_2252>GCA p.L747_T751>Q c.2239_2252>CA p.L747_S752>QH c.2238_2255>GCAACA p.L747_A750>P c.2238_2248>GC p.l744_K745insKIPVAI c.2231_2232insl8 p.D761_E762insEAFQ c.2283_2284insl2 p.A767_S768insTLA c.2302_2303ins9 p.V769_D770insASV c.2308_2309ins9 p.D770>GY c.2308_2309insGTT p.E709H c.2125_2127GAA>CAT p.L858R c.2573_2574TG>GT p.A859T c.2575G>A p.E746_T751>A c.2237_2251dell5 p.Y727C c.2180A>G p.V851l c.2551G>A p.E746_T751del c.2236_2253dell8 p.D770_N771>AGG c.2309_2312ACAA>CTGGTGG p.G857R c.2569G>A p.L858R c.2573.T>G p.E746_A750del c.2235_2249dell5 p.E746_A750>QP c.2236_2248>CAAC

P.G810D c.2429G>A p.E709K c.2125G>A p.D770_N771insN c.2310_2311insAAC p.D770_N771insG c.2310_2311insGGC p.H773L c.2318A>T p.V774M c.2320G>A p.G779F c.2335_2336GG>TT p.A871G c.2612C>G p.E709G c.2126A>G

P.L861Q c.2582T>A p.L730F c.2188C>T p.P733L c.2198C>T p.G735S c.2203G>A p.V742A c.2225T>C p.E746K c.2236G>A p.T751l c.2252C>T p.S752Y c.2255C>A p.H850N c.2548C>A p.D761N c.2281G>A p.S784F c.2351C>T p.L792P c.2375T>C p.L798F c.2392C>T

P.G810S c.2428G>A p.N826S c.2477A>G p.T847l c.2540C>T p.V851A c.2552T>C

P.I853T c.2558T>C p.A864T c.2590G>A

p.E866K c.2596G>A

p.G873E c.2618G>A

p.E746_P753>LS c.2236_2257>CTCT

p.V819V c.2457G>A

p.Y764Y c.2292C>T

p.L833V c.2497T>G

p.V769M c.2305G>A

p.L838V c.2512C>G

p.E709A c.2126A>C

p.D770_N771insSVD c.2311_2312ins9

p.A839T c.2515G>A

p.H773 c.2318A>G

p.P772P c.2316C>T

p.E746_T751>A c.2235_2251>AG

p.E746_A750>IP c.2235_2248>AATTC p.E746_T751>l c.2235_2252>AAT

p.E746_T751>IP c.2235_2251>AATTC

p.L858R c.2572_2573CT>AG

p.N771_P772>SVDNR c.2312_2315ACCC>GCGTGGACAACCG p.D770_P772>ASVDNR c.2308_2315GACAACCC>CCAGCGTGGATAACCG p.S752J759del c.2253_2276del24

p.E746_A750>QP c.2236_2248>CAAC

p.V769_D770insASV c.2309_2310AC>CCAGCGTGGAT p.M766_A767insAI c.2298_2299insGCCATA

p.G724S c.2170G>A

p.D770N c.2308G>A

p.T783l c.2348C>T p.G863D c.2588G>A p.V897l c.2689G>A p.K745 c.2234A>G p.P741L c.2222C>T p.E734K c.2200G>A p.E746del c.2234_2236delAGG p.E746_T751>VP c.2237_2251>TTC p.Q787R c.2360A>G p.V834L c.2500G>T p.A755A c.2265C>T p.G719D c.2156G>A p.E746_S752>V c.2237_2256>TC p.E746_P753>VS c.2237_2257>TCT p.E746_A750>DP c.2238_2249>TCC p.V769_D770insGSV c.2308_2309ins9 p.V769_D770insGVV c.2308_2309ins9 p.N771>GF c.2311_2312AA>GGGTT p.V774_C775insHV c.2321_2322insCCACGT p.G719C c.2154_2155GG>TT p.L747_R748>FP c.2241_2244AAGA>CCCG p.E872 c.2614G>T p.G873G c.2619A>T p.P753P c.2259G>A p.G719fs*29 c.2156delG p.L747_K754>ST c.2240_2261>CGAC p.S768_V769insVAS c.2303_2304ins9 p.V769_D770insDNV c.2307_2308ins9 p.D770_N771insAPW c.2310_2311ins9

p.1744V c.2230A>G p.S784P c.2350T>C p. 832L c.2495G>T p.V802F c.2404G>T p.E746_E749del c.2235_2246del l2 p.T854A c.2560A>G p.E884K c.2650G>A p.F712S c.2135T>C

P.I744M c.2232C>G p.V765M c.2293G>A p.R836C c.2506C>T p.A871T c.2611G>A p.D855G c.2564A>G p.E868G c.2603A>G p.L798H c.2393T>A

P.K806E c.2416A>G p.L814P c.2441T>C p.E746_A750>VP c.2237_2250>TCCCT p.V769_D770insMASVD c.2307_2308insl5 p.F723S c.2168T>C p.T785N c.2354C>A p.V845M c.2533G>A p.M766T c.2297T>C p.S752P c.2254T>C p.T725T c.2175G>A p.D855N c.2563G>A p.L858Q c.2573T>A p.H870R c.2609A>G p.F712L c.2134T>C ρ.Ι821Τ c.2462T>C p.V834A c.2501T>C p.L718P c.2153T>C p.D770_N771insNPH c.2310_231 linsAACCCCCAC p.D770_N771insGL c.2310_2311insGGGTTA p.D770_N771insSVD c.2311_2312insGCGTGGACA p.P772_H773insTHP c.2315_2316insGACACACCC p.S720T c.2158T>A p.E746V c.2237_2238AA>TT p.E746_P753>VQ c.2237_2258>TTCA p.E709_T710>D c.2127_2129delAAC p.E746_T751>IP c.2236_2253>ATTCCT p.L747_T751>Q c.2239_2253>CAA p.H773_V774insGNPH c.2320_2321insl2

P.I732T c.2195T>C p.N756Y c.2266A>T p.L844P c.2531T>C

P.I740T c.2219T>C p.E746_T751>VP c.2237_2253>TTCCT p.W731L c.2192G>T p.E734Q c.2200G>C p.T785A c.2353A>G p.C797Y c.2390G>A p. 831H c.2492G>A p.N771>GY c.2311_2311A>GGTT p.P733S c.2197C>T p.R748l c.2243G>T

p.E709_T710>A c.2126_2128delAAA p.E746_S752>V c.2235_2255>GGT p.l744_A750>VK c.2230_2249>GTCAA p.L747_K754>N c.2239_2264>GCCAA p.l740_P741insPVAIKI c.2219_2220insl8 p. 836 c.2508C>T p.V843l c.2527G>A p.K754R c.2261A>G p.A840T c.2518G>A p.K754E c.2260A>G p.A859D c.2576C>A

P.Y801C c.2402A>G

P.I744T c.2231T>C p.T854l c.2561C>T p.G863S c.2587G>A p.H850R c.2549A>G p.K754A c.2260_2261AA>GC p.D807N c.2419G>A p.S720P c.2158T>C p.K757M c.2270A>T p.L862Q c.2585T>A ρ.Τ751_Ι759>Ν c.2252_2276>A p.P772R c.2315C>G p.A839V c.2516C>T p.K716R c.2147A>G p.H773_V774insQ c.2319_2320insCAG p.E711V c.2132_2133AA>TT p.T710A c.2128A>G p.K714N c.2142G>C p.V717A c.2150T>C p.G729E c.2186G>A p.l744_E749>LK c.2230_2247>CTTAAGAGA p.E746_T751>L c.2236_2253>CTA p.E746_S752>l c.2236_2255>AT p.E746_S752del c.2236_2256del21 p.E746_S752>l c.2236_2256>ATC p.E746_P753>IS c.2236_2259>ATCTCG p.E746_T751>V c.2237_2253>TA p.E746_T751>V c.2237_2253>TC p.L747_A750>P c.2239_2250>CCA p.L747_S752>QH c.2239_2256>CAACAT p.L747_P753>S c.2239_2257>T p.R748K c.2243G>A p.E749G c.2246A>G p.T751J759>S c.2251_2277>TCC p.P772_H773insHV c.2316_2317insCACGTG p.G779D c.2336G>A p.V802A c.2405T>C p.L833W c.2498T>G p.D837G c.2510A>G p.L844V c.2530C>G p.T751J759del c.2252_2275del24 p.V765G c.2294T>G p.G796D c.2387G>A p.R836H c.2507G>A p.K757R c.2270A>G p.E872K c.2614G>A p.L858L c.2574G>A

P.I780S c.2339T>G ρ.Τ785Ρ c.2353A>C p.Y801fs* l c.2402_2403insG p.L858 c.2573_2574TG>GA p.N771_P772insRH c.2311_2312insACCGGC p.H850Y c.2548C>T p.E868K c.2602G>A

P.I780T c.2339T>C p.E866D c.2598G>T p.L833F c.2499G>T p.A864V c.2591C>T p.K745_A750del c.2232_2249del l8 p.P794H c.2381C>A p.E804K c.2410G>A p.G857E c.2570G>A

p.S476l c.l427G>T

p.D479fs*2 .1434_1462del29

p.S480fs*47 c.l439delC

p.N486D c.l456A>G

p.V489l c.l465G>A

p.V489A c.l466T>C

p.E490G c.l469A>G

p.E490_F504>DHIVVSLTF c.l470_1512>CCACATCGTTGTAAGCCTTACATTC p.N495l c.l484A>T

p.N495l c.l484A>T

p.D496V c.l487A>T

p.V50M c.l48G>A

p.V497V c.l491G>A

p.G498D c.l493G>A

p.K499K c.l497G>A

p.A502_Y503insSA c.l504_1505insCTTCTG p.A502_Y503insSA c.l505_1506insTTCTGC p.Y503_F504insSA c.l507_1508insCTGCCT p.A502_Y503insFA c.l507_1508insTTGCCT p.Y503_F504insAY c.l509_1510insGCCTAT

p.F504L c.l510T>C

p.N505H c.l513A>C

p.F506L c.l516T>C

p.F506_A507insAYFNF .1518_1519insl5

p.F508_K509insNFAF .1524_1525insl2

p.K509l c.l526A>T

p.G510del c.l528_1530delGGT p.N512D c.l534A>G

p.P551_V555>L .1652_1663dell2 p.P551_V559del>L .1652_1678del27 p.P551L c.l652C>T p.M552_Y553del c.l653_1658delCATGTA p.M552_Q556> c.l653_1667>TCT p.M552_W557del .1653_1670dell8 p.M552_Y553del c.l654_1659delATGTAT p.M552_E554del .1654_1662del9 p.M552_V555del .1654_1665dell2 p.M552_Q556del .1654_1668dell5 p.M552_W557del .1654_1671dell8 p.M552_K558del .1654_1674del21 p.M552_D572del .1654_1716del63 p.M552L c.l654A>C p.M552L c.l654A>C p.M552_Y553>N c.l655_1657delTGT p.M552_E554>K c.l655_1660delTGTATG p.M552_V555>l .1655_1663del9 p.M552_Q556>K .1655_1666dell2 p.M552_W557> .1655_1669dell5 p.M552_W557del .1655_1672dell8 p.M552_K558>T c.l655_1674>CN p.M552_E561>K .1655_1681del27 p.M552_T574>TESA c.l655_1720>CAGAATCAG p.M552K c.l655T>A p.M552T c.l655T>C p.Y553_W557del .1656_1670dell5 p.Y553_K558> .1656_1673dell8 p.Y553V c.l657_1658TA>GT p.Y553_Q556del C.1657_1668dell2 p.Y553_W557del C.1657_1671dell5 p.Y553_K558del C.1657_1674dell8 p.Y553_V559>E c.l657_1677>GAA p.Y553_V559del C.1657_1677del21 p.Y553N c.l657T>A p.Y553_T574>S C.1658_1720del63 p.E554_K558del C.1660_1674dell5 p.E554_E562del C.1660_1686del27 p.E554_N564del C.1660_1692del33 p.E554J571del C.1660_1713del54 p.E554_D572del C.1660_1716del57 p.E554K c.l660G>A p.E554K c.l660G>A p.E554_K558del C.1661_1675dell5 p.E554G c.l661A>G p.V555_E562del C.1662_1685del24 p.E554D c.l662A>T p.V555_Q556del c.l663_1668delGTACAG p.V555_K558del C.1663_1674dell2 p.V555_V559del C.1663_1677dell5 p.V555_V560del C.1663_1680dell8 p.V555J563del C.1663_1689del27 p.V555_G565del C.1663_1695del33 p.V555_Y570del C.1663_1710del48 p.V555J571del C.1663_1713del51 p.V555_P573del C.1663_1719del57 p.V555l c.l663G>A

p.V555_N566>D .1664_1696del33 p.Q556_V559del .1665_1676dell2 p.V555_V560>V .1665_1679dell5 p.Q556_N566>SNNLQLY c.l665_1696>TTCCAACAACCTTCCACTGT p.Q556_D572del c.l665_1716>T p.Q556_W557del c.l666_1671delCAGTGG p.Q556_V559del .1666_1677dell2 p.Q556_V560>F c.l666_1678>T p.Q556_V560>TTF c.l666_1680>ACAACCTTC p.Q556_V560del .1666_1680dell5 p.Q556_E561>HH c.l666_1683>CATCAT p.Q556_E561del .1666_1683dell8 p.Q556_D572>PS c.l666_1716>CCATCC p.Q556_P573del .1666_1719del54 p.Q556_T574del .1666_1722del57 p.Q556_L576del .1666_1728del63 p.Q556_W557> c.l667_1669delAGT p.W557_K558del c.l667_1672delAGTGGA p.Q556_K558>R c.l667_1673AGTGGAA>G p.W557_E561del .1667_1681dell5

p.Q556R c.l667A>G

p.W557_K558del c.l668_1673delGTGGAA p.Q556_K558>HPCR c.l668_1673GTGGAA>CCCCTGCAG p.Q556_K558>H c.l668_1674GTGGAAG>Y p.Q556_V559>H .1668_1676del9 p.Q556_V559>HT c.l668_1677GTGGAAGGTT>TACT p.Q556_V560>HNLQLY c.l668_1679>CAACCTTCCACTGTA p.Q556_V560>H .1668_1679dell2 p.W557J571del .1668_1712del45 p.Q556_D572>H .1668_1715del48 p.W557_Q575del .1668_1724del57 p.W557del c.l669_1671delTGG p.W557_K558>E c.l669_1672TGGA>G p.W557_K558del c.l669_1674delTGGAAG p.W557_K558>S c.l669_1674TGGAAG>C p.W557_V559>l c.l669_1675TGGAAGG>A p.W557_V559del .1669_1677del9 p.W557_V560del .1669_1680dell2 p.W557_E561del .1669_1683dell5 p.W557_E562del .1669_1686dell8 p.W557_Q575del .1669_1725del57 p.W557 c.l669T>A p.W557R c.l669T>C p.W557G c.l669T>G p.W557_K558>SS c.l670_1673GGAA>CTTC p.W557_K558>FP c.l670_1674GGAAG>TTCCT p.W557_V559>F c.l670_1675delGGAAGG p.W557_V560>F .1670_1678del9 p.W557_P573>S .1670_1717del48 p.W557S c.l670G>C p.W557_K558>CT c.l671_1673GAA>CAC p.W557_K558>CP c.l671_1673GAA>TCC p.W557_K558>C c.l671_1674GAAG>C p.W557_V559>C c.l671_1676delGAAGGT p.W557_V560>C .1671_1679del9 p.W557 c.l671G>A p.W557C c.l671G>T p.K558_V559>SS c.l672_1676AAGGT>TCTTC p.K558_V559del c.l672_1677delAAGGTT p.K558_V560del .1672_1680del9 p.K558_E562del .1672_1686dell5 p.K558_N564del .1672_1692del21 p.K558_G565del .1672_1695del24 p.K558_D572del .1672_1716del45 p.K558_Q575del .1672_1725del54 p.K558E c.l672A>G p.K558* c.l672A>T p.K558>NP c.l673_1674insTCC p.K558_V560>l c.l673_1678delAGGTTG p.K558_V560>M c.1673_1680AGGTTGTT>TG p.K558_E562del .1673_1687dell5 p.K558_G565> .1673_1693del21 p.K558R c.l673A>G p.K558>NP c.l674_1674G>TCCT p.K558_V559>N c.l674_1676delGGT p.K558_V560>N c.l674_1679delGGTTGT p.K558_Y570>N .1674_1709del36 p.K558_L576>NV c.l674_1726>CG p.K558K c.l674G>A p.K558N c.l674G>C p.K558N c.l674G>Y p.V559del c.l675_1677delGTT p.V559K c.l675_1677GTT>AAG p.V559_V560del c.l675_1680delGTTGTT p.V559_E561del C.1675_1683del9 p.V559_G565del C.1675_1695del21 p.V559J571del C.1675_1713del39 p.V559_L576del C.1675_1728del54 p.V559l c.l675G>A p.V559_E561del C.1676_1684del9 p.V559_E562del C.1676_1687dell2 p.V559_P573>A C.1676_1717del42 p.V559D c.l676T>A p.V559A c.l676T>C p.V559G c.l676T>G p.V560del c.l678_1680delGTT p.V560_L576del C.1678_1728del51 p.V560E c.l679_1680TT>AG p.V560E c.l679_1680TT>A p.V560del c.l679_1681delTTG p.V560J571del C.1679_1714del36 p.V560D c.l679T>A p.V560A c.l679T>C p.V560G c.l679T>G p.E561del c.l680_1682delTGA p.V560V c.l680T>G p.E561del c.l681_1683delGAG p.E561_P577del C.1681_1731del51 p.E561K c.l681G>A p.E561G c.l682A>G p.E561E c.l683G>A p.E562_P573del .1684_1719del36 p.E562K c.l684G>A p.E562V c.l685A>T p.E562_V569>D .1686_1706del21 p.l563_D572del .1687_1716del30 p.l563_L576del .1687_1728del42

P.I563V c.l687A>G p.N564_T574del .1690_1722del33 p.N564_L576del .1690_1728del39 p.N564_P577del .1690_1731del42 p.N564_Y578del .1690_1734del45 p.N564H c.l690A>C p.N564_P573>TS c.l691_1717>CCT p.N564_P573>T .1691_1717del27 p.N564S c.l691A>G p.N564K c.l692T>G p.G565 c.l693G>A p.G565E c.l694G>A p.G565V c.l694G>T p.N566D c.l696A>G p.N566S c.l697A>G p.N567_L576>E c.l698_1728>CGAA p.N566N c.l698C>T p.N567_P573del .1699_1719del21 p.N567H c.l699A>C p.N567K c.l701T>A p.Y568_T574del .1702_1722del21 p.Y568D c.l702T>G

p.T574_Q575insl2 .1721_1722ins36 p.T574l c.l721C>T p.Q575del c.l723_1725delCAA p.Q575_P577>T c.l723_1731CAACTTCCT>ACA p.L576del c.l726_1728delCTT p.L576F c.l726C>T p.L576del c.l727_1729delTTC p.L576P c.l727T>C p.L576_P577insQL c.l728_1729insCAACTT p.P577_Y578del c.l729_1734delCCTTAT p.P577S c.l729C>T p.P577_D579del .1730_1738del9 p.P577H c.l730C>A p.P577L c.l730C>T p.D579del c.l735_1737delGAT p.D579_H580insPTQLPYD .1737_1738ins21 p.D579_H580insSYD .1737_1738ins9 p.H580del c.l737_1739delTCA p.H580Y c.l738C>T p.H580_K581insHPYD .1739_1740insl2 p.H580_K581insPYDH .1740_1741insl2 p.H580_K581insPTQLPYDH .1740_1741ins24 p.H580_K581inslDPTQLPYDH .1740_1741ins30 p.H580_K581insYDH .1740_1741ins9 p.K581 c.l742A>G p.W582* c.l745G>A p.W582* c.l746G>A p.E583_F584insPYDHKWE .1748_1749ins21

p.E635G c.l904A>G p.A636V c.l907C>T p.L637F c.l909C>T p.S639P c.l915T>C p.K642Q c.l924A>C p.K642E c.l924A>G p.V643A c.l928T>C p.S645N c.l934G>A p.L647F c.l939C>T p.L647P c.l940T>C p.G648S c.l942G>A p.N649_H650insN c.l947_1948insAAT

P.I653T c.l958T>C p.V654A c.l961T>C p.N655K c.l965T>G p.G663V c.l988G>T p.G664 c.l990G>A p.T670E c.2008_2009AC>GA p.T670l c.2009C>T p.L682fs*l c.2045delT p.S692L c.2075C>T p.E695K c.2083G>A p.H697Y c.2089C>T p.H697fs*28 c.2089delC p.R815_D816insVI c.2445_2446insGTCATA

P.D816I c.2446_2447GA>AT

P.D816F c.2446_2447GA>TT

P.D816N c.2446G>A P.D816H c.2446G>C

P.D816Y c.2446G>T p.D816>GP c.2447_2448AC>GGCCA p.D816>VVA c.2447_2448AC>TCGTTGCA

P.D816A c.2447A>C

P.D816G c.2447A>G

P.D816V c.2447A>T

P.D816E c.2448C>G

P.I817V c.2449A>G

P.I817T c.2450T>C

P.K818 c.2453A>G

P.K818K c.2454G>A p.N819Y c.2455A>T p.D820N c.2458G>A p.D820H c.2458G>C p.D820H c.2458G>C p.D820Y c.2458G>T p.D820Y c.2458G>T p.D820A c.2459A>C p.D820G c.2459A>G p.D820V c.2459A>T p.D820E c.2460T>A p.D820E c.2460T>G p.N822H c.2464A>C p.N822Y c.2464A>T p.N822Y c.2464A>T p.N822S c.2465A>G p.N822K c.2466T>A p.N822N c.2466T>C p.N822K c.2466T>G p.N822K c.2466T>R p.Y823N c.2467T>A p.Y823D c.2467T>G p.Y823C c.2468A>G p.V825l c.2473G>A p.V825A c.2474T>C p.A829P c.2485G>C p.A829V c.2486C>T p. 830* c.2488C>T p.R830* c.2488C>T p.L831P c.2492T>C p.V833L c.2497G>C p.V833V c.2499G>T p.E839K c.2515G>A p.C844Y c.2531G>A p.Y846H c.2536T>C p.F848L c.2542T>C p.E849* c.2545G>T p.W853* c.2558G>A p.S854P c.2560T>C p.L859P c.2576T>C p.L859L c.2577T>G

P.E861E c.2583G>A p.L862L c.2586G>C

p.Q61P c.l82A>C p.Q61 c.l82A>G p.Q61L c.l82A>T p.Q61H c.l83A>C p.Q61H c.l83A>T p.D69fs*4 c.205delG p.G12fs*3 c.35delG p.G13V c.38_39GC>TT p.V14l c.40G>A p.Q61K c.l80_181TC>CA

P.G258V c.773G>T p.H298Y c.892C>T p.BOOV c.898A>G p.A305V c.914C>T p.E309* c.925G>T

P.T310I C.9290T

P.S323S c.969G>A p.I326V c.976A>G p.I326T c.977T>C p.F357S c.l070T>C

P.G358G C.1074G>C

P.S364L C.1091OT p.S365L c.l094C>T p.P367R cllOOOG p.S394* C.11810G

P.T401I C.1202OT p.P403fs*8 c.l208delC p.A404fs*9 c.l208_1209insC

P.G421V c.l262G>T

P.G421G c.l263A>G p.K439Q c.l315A>C p.K439T c.l316A>C p.T440P c.l318A>C p.T440A c.l318A>G p.T440T c.l320A>G

P.G442S c.l324G>A p.R444W C.1330OT p.R444Q c.l331G>A p.R444L c.l331G>T p.R444R c.l332G>A p.R444R c.l332G>T

P.S447S c.l341T>C p.W450* c.l349G>A p.W450L c.l349G>T ρ.Ρ453Τ C.13570A p.P453P c.l359T>C p.G455R c.l363G>A p.G455E c.l364G>A p.Q456* C.13660T p.Q456R c.l367A>G p.Q456Q c.l368G>A ρ.Ι457Τ c.l370T>C p.V459L c.l375G>C p.V459A c.l376T>C p.V459V c.l377G>A p.G460* c.l378G>T

P.G460G c.l380A>G

P.R462G c.l384A>G p.R462K c.l385G>A p.R462I c.l385G>T p.R462R c.l386A>G p.I463V c.l387A>G

P.I463S c.l388T>G p.14631 c.l389T>C P.G464R C.1390OA

P.G464R c.l390G>C

P.G464E c.l391G>A

P.G464V c.l391G>T

P.S465S c.l395T>C

P.G466R c.l396G>A

P.G466R c.l396G>C

P.G466E c.l397G>A

P.G466A c.l397G>C

P.G466V c.l397G>T

P.G466G c.l398A>G

P.S467P c.l399T>C

P.S467L C.1400OT

p.F468L c.l402T>C

P.F468S c.l403T>C

P.F468C c.l403T>G

p.F468F c.l404T>C

P.G469R C.1405OA

P.G469R C.1405OC

p.G469>? c.l405_1406GG>CT

P.G469S C.1405_1406GG>TC

P.G469L c.l405_1406GG>TT

P.G469S c.l405_1407GGA>AGC

P.G469S c.l405_1407GGA>AGT

P.G469E C.1406OA

P.G469A c.l406G>C

P.G469V c.l406G>T P.G469G C.1407A>G

P.V471I c.l411G>A p.V471F c.l411G>T p.V471A c.l412T>C

P.Y472S c.l415A>C

P.Y472C c.l415A>G p.K475R c.l424A>G p.K475M c.l424A>T ρ.Κ475Κ c.l425G>A p.D479Y c.l435G>T p.L485L c.l453T>C p.L485S c.l454T>C p.L485_P490>Y c.l454_1469>A p.L485F c.l455G>T p.N486_P490del C.1457_1471dell5 p.V487V c.l461G>A p.L505H c.l514T>A

P.R509* C.15250T p.L514P c.l541T>C p.W531C c.l593G>T p.L537S c.l610T>C p.H539P c.l616A>C p.H542Y c.l624C>T p.K570K c.l710G>A p.H574N C.1720OA p.H574Q C.17220A p.N581S c.l742A>G P.N581I c.l742A>T p.I582M c.l746A>G p.F583S c.l748T>C p.F583F c.l749T>C p.L584F C.1750OT p.L584P c.l751T>C p.L584L c.l752T>C p.H585H c.l755T>C p.E586K c.l756G>A p.E586E c.l758A>G p.D587N c.l759G>A p.D587A c.l760A>C p.D587G c.l760A>G p.D587E c.1761 OA p.D587E c.l761C>G p.L588P c.l763T>C p.L588R c.l763T>G p.L588L c.l764C>T p.T589A c.l765A>G ρ.Τ589Ι C.17660T ρ.Τ589Τ c.l767A>G p.V590I c.l768G>A p.V590A c.l769T>C p.V590fs*3 c.l769delT p.V590V c.l770A>G p.K591E c.l771A>G p.K591R c.l772A>G p.I592V c.l774A>G p.I592M c.l776A>G p.15921 c.l776A>T

P.G593S c.l777G>A

P.G593C c.l777G>T p.G593D c.l778G>A p.D594N c.l779_1780TG>GA p.D594N C.1780OA p.D594H c.l780G>C p.D594G c.l781A>G p.D594V c.l781A>T p.D594E c.l782T>A p.D594D c.l782T>C p.D594E c.l782T>G p.F595L c.l783T>C p.F595S c.l784T>C p.F595L c.l785T>A p.F595F c.l785T>C p.F595L c.l785T>G p.G596R c.l786G>C p.G596fs*2 c.l786delG p.G596D c.l787G>A

P.G596G c.l788T>C p.L597V C.17890G p.L597S c.l789_1790CT>TC p.L597Q C.1790T>A p.L597P c.l790T>C p.L597R C.1790T>G

p.L597L c. l791A>G

p.A598T c. l792G>A

p.A598V C.17930T

p.A598A c. l794T>A

p.A598_T599insV c. l794_1795insGTT p.T599del c. l794_1796delTAC p.T599I C.17960T

p.T599_V600insT c.l796_1797insTAC p.T599_V600>IAL c.l796_1798CAG>TAGCTT p.T599_R603>I C.1796_1809>TC p.T599T c. l797A>B

p.T599T c. l797A>G

p.T599T c. l797A>T

p.T599_V600insTT c.l797_1797A>TACTACG p.T599_V600insTT c.l797_1798ins? p.T599_V600insT c. l797_1798insACA p.T599_V600insDFGLAT C.1798_1799insl8

P.V600R c. l797_1799AGT>GAG

P.V600M c. l798G>A

P.V600L c. l798G>C

P.V600L c. l798G>T

p.V600>YM c.l798_1798G>TACA

P.V600K c.l798_1799GT>AA

P.V600R c. l798_1799GT>AG

P.V600Q c.l798_1799GT>CA

P.V600E c. l799T>A P.V600A c.l799T>C

P.V600G c.l799T>G

P.V600E c.l799_1800TG>AA

P.V600D c.l799_1800TG>AC

P.V600D c.l799_1800TG>AT p.V600fs*ll c.l799_1800delTG p.V600_K601>E c.l799_1801delTGA p.V600_S602>DT c.1799_1804TGAAAT>ATA p.V600_S605>D c.l799_1814>A

p.V600_S605>DV c.l799_1814>ATGT p.V600_S605>EK c.l799_1815>AAAAG

P.V600V C.1800OA

p.V600? c.(1798-1800)?

P.K601E c.l801A>G

p.K601del c.l801_1803delAAA

P.K601R c.l802A>G

P.K601I c.l802A>T

P.K601N c.l803A>C

P.K601K c.l803A>G

P.K601N c.l803A>T

P.S602S c.l806T>G

p.R603R C.1807OA

p.R603* C.1807OT

p.R603L C.1808OT

p.R603R c.l809A>G

p.W604del c.l808_1810delGAT p.W604R c.l810T>A p.W604G c.l810T>G

p.W604* C.18HOA

p.W604* c.l812G>A

P.S605G c.l813A>G

p.S605F c.l813_1814AG>TT p.S605N c.l814G>A

p.S605R c.l815T>A

P.G606R C.I8I6OA

P.G606S c.l816_1818GGG>AGT

P.G606E c.l817G>A

P.G606A c.l817G>C

P.G606V c.l817G>T

P.G606G C.I8I8OA

P.S607P c.l819T>C

P.H608R c.l823A>G

P.H608H c.l824T>C

p.Q609R c.l826A>G

P.Q609Q c.l827G>A

P.F610L c.l828T>C

P.F610S c.l829T>C

P.F610F C.1830T>C

P.E611G c.l832A>G

P.E611D c.l833A>C

P.E611E c.l833A>G

p.Q612E c.l834C>G

p.Q612* C.18340T

P.S614P c.l840T>C P.S614S c.l842T>C p.G615R c.l843G>A

P.S616P c.l846T>C

P.S616F C.18470T

P.I617T C.1850T>C

P.L618L c.l852T>C

P.L618S c.l853T>C

P.L618W c.l853T>G p.W619R c.l855T>C p.Q636E C.1906OG p.Q636* C.1906OT p.Q636R c.l907A>G

P.S637P c.l909T>C p.S637* C.1910OG p.S637L C.1910OT

P.S657S c.l971A>G p.R671Q c.2012G>A p.R682W c.2044C>T p.R682Q c.2045G>A p.K698R c.2093A>G p.A718V C.21530T

P.P731S C.21910T p.P731P C.21930T

111

1515739

Seq. 905 Left ATGTTGGCTTACATTAACTCCCATA

Primer Sequence

ID 906 Right TTTGCAAAGTCCCTCTCCTTT

891 Left GCAGACTCCTCTAGCCACAAAAG

892 Right TACAACTTTCTCTCCTTAAGCCTCA 907 Left GTTATCACAGCACCGCAGACT

908 Right TACAACTTTCTCTCCTTAAGCCTCA

893 Left GCAGACTCCTCTAGCCACAAAA

894 Right TACAACTTTCTCTCCTTAAGCCTCA 909 Left GCAGACTCCTCTAGCCACAAA

910 Right TACAACTTTCTCTCCTTAAGCCTCA

895 Left AGACTCCTCTAGCCACAAAAGG

896 Right TACAACTTTCTCTCCTTAAGCCTCA 911 Left GCAGTGTAGGGGCTGAATGTTA

912 Right TACAACTTTCTCTCCTTAAGCCTCA

897 Left CAGTGTAGGGGCTGAATGTTATC

898 Right GCATGCATACAACTTTCTCTCCTT 913 Left TAGGGGCTGAATGTTATCACAGC

914 Right TACAACTTTCTCTCCTTAAGCCTCA

899 Left GCAGTGTAGGGGCTGAATGTTATC

900 Right TACAACTTTCTCTCCTTAAGCCTCA 915 Left CAGACTCCTCTAGCCACAAAAGG

916 Right GCATGCATACAACTTTCTCTCCTTA

901 Left GTAGGGGCTGAATGTTATCACAGC

902 Right TACAACTTTCTCTCCTTAAGCCTCA 917 Left GCAGTGTAGGGGCTGAATGTTAT

918 Right ACAACTTTCTCTCCTTAAGCCTCA

903 Left GAGGACAAGCCTTGACATTCAG

904 Right TACAACTTTCTCTCCTTAAGCCTCA 919 Left ATGTTGGCTTACATTAACTCCCATA

920 Right CAAAGTCCCTCTCCTTTGCAT

ALK Exoii24 130-150 bases

Seq. 939 Left AGTGGCCCGCTTCTGTCT

Primer Sequence

ID 940 Right ATGACAGGAAGAGCACAGTCAC

921 Left AGTGGCCCGCTTCTGTCT

922 Right GATGACAGGAAGAGCACAGTCAC Seq.

Primer Sequence

ID

923 Left CGCTTCTGTCTCCCCACAG 941 Left CGCTTCTGTCTCCCCACAG

924 Right GATGACAGGAAGAGCACAGTCAC 942 Right ATGACAGGAAGAGCACAGTCAC

925 Left CGCTTCTGTCTCCCCACA 943 Left CGCTTCTGTCTCCCCACA

926 Right GATGACAGGAAGAGCACAGTCAC 944 Right ATGACAGGAAGAGCACAGTCAC

927 Left AGTGGCCCGCTTCTGTCT 945 Left AGTGGCCCGCTTCTGTCT

928 Right AGGATGACAGGAAGAGCACAGT 946 Right AGGATGACAGGAAGAGCACAGTC

929 Left CGCTTCTGTCTCCCCACAG 947 Left CGCTTCTGTCTCCCCACAG

930 Right AGGATGACAGGAAGAGCACAGT 948 Right AGGATGACAGGAAGAGCACAGTC

931 Left CGCTTCTGTCTCCCCACA 949 Left CGCTTCTGTCTCCCCACA

932 Right AGGATGACAGGAAGAGCACAGT 950 Right AGGATGACAGGAAGAGCACAGTC

933 Left AGTGGCCCGCTTCTGTCT 951 Left AGTGGCCCGCTTCTGTCT

934 Right GATGACAGGAAGAGCACAGTCA 952 Right GGATGACAGGAAGAGCACAGTC

935 Left CGCTTCTGTCTCCCCACAG 953 Left CGCTTCTGTCTCCCCACAG

936 Right GATGACAGGAAGAGCACAGTCA 954 Right GGATGACAGGAAGAGCACAGTC

937 Left CGCTTCTGTCTCCCCACA 955 Left CGCTTCTGTCTCCCCACAG

938 Right GATGACAGGAAGAGCACAGTCA 956 Right GACAGGATGACAGGAAGAGCAC 957 Left CGCTTCTGTCTCCCCACA 959 Left CGCTTCTGTCTCCCCACA

958 Right GGATGACAGGAAGAGCACAGTC 960 Right GACAGGATGACAGGAAGAGCAC Ζ ΓΕχοη24 161-200 bases

Seq.

Primer Sequence

ID J81 Left ATTTCAGATTTCCCTCCTCTCACT

961 Left ATTTCAGATTTCCCTCCTCTCACT J82 Right AGGATGACAGGAAGAGCACAGTC

962 Right GATGACAGGAAGAGCACAGTCAC

Seq. Left ATTTCAGATTTCCCTCCTCTCACT

Primer equence

ID J84 Right GGATGACAGGAAGAGCACAGTC

963 Left ATTTCAGATTTCCCTCCTCTCACT

964 Right AGGATGACAGGAAGAGCACAGT ?85 Left TTTCAGATTTCCCTCCTCTCACT

965 Left ATTTCAGATTTCCCTCCTCTCACT J86 Right GATGACAGGAAGAGCACAGTCA

966 Right GATGACAGGAAGAGCACAGTCA

?87 Left ATTTCAGATTTCCCTCCTCTCAC

967 Left ATTTCAGATTTCCCTCCTCTCAC J88 Right AGGATGACAGGAAGAGCACAGTC

968 Right GATGACAGGAAGAGCACAGTCAC

m Left TTTCAGATTTCCCTCCTCTCACT

969 Left ATTTCAGATTTCCCTCCTCTCAC ?90 Right ATGACAGGAAGAGCACAGTCAC

970 Right AGGATGACAGGAAGAGCACAGT

m Left ATTTCAGATTTCCCTCCTCTCAC

971 Left ATTTCAGATTTCCCTCCTCTCACT m Right GGATGACAGGAAGAGCACAGTC

972 Right ATGACAGGAAGAGCACAGTCAC

m Left ATTTCAGATTTCCCTCCTCTCACT

973 Left ATTTCAGATTTCCCTCCTCTCAC m Right AGGATGACAGGAAGAGCACAG

974 Right GATGACAGGAAGAGCACAGTCA

Left ATTTCCCTCCTCTCACTGACAA

975 Left ATTTCAGATTTCCCTCCTCTCAC m Right GATGACAGGAAGAGCACAGTCAC

976 Right ATGACAGGAAGAGCACAGTCAC

Left ATTTCCCTCCTCTCACTGACAA

977 Left TTTCAGATTTCCCTCCTCTCACT m Right AGGATGACAGGAAGAGCACAGT

978 Right GATGACAGGAAGAGCACAGTCAC

Left CATTTCAGATTTCCCTCCTCTCACT

979 Left TTTCAGATTTCCCTCCTCTCACT 1 000 Right GATGACAGGAAGAGCACAGTCAC

980 Right AGGATGACAGGAAGAGCACAGT

ALK Exon24 201-300 bases

Seq.

Primer Sequence

ID 1011 Left ATTTCAGATTTCCCTCCTCTCACT

1001 Left ATTTCAGATTTCCCTCCTCTCACT 1012 Right AGGGAGACCTAGTATTCTGCTCTGA

1002 Right GAGACCTAGTATTCTGCTCTGAAGG

1013 Left ATTTCAGATTTCCCTCCTCTCACT

1003 Left ATTTCAGATTTCCCTCCTCTCACT 1014 Right TCTGGAGGGAGACCTAGTATTCTG

1004 Right GGAGACCTAGTATTCTGCTCTGAAG

1015 Left ATTTCAGATTTCCCTCCTCTCAC

1005 Left ATTTCAGATTTCCCTCCTCTCACT 1016 Right CTCTGGAGGGAGACCTAGTATTCTG

1006 Right CTCTGGAGGGAGACCTAGTATTCTG Seq.

Primer Sequence

ID

1007 Left ATTTCAGATTTCCCTCCTCTCAC 1017 Left ATTTCAGATTTCCCTCCTCTCACT

1008 Right GAGACCTAGTATTCTGCTCTGAAGG 1018 Right GGGAGACCTAGTATTCTGCTCTGA

1009 Left ATTTCAGATTTCCCTCCTCTCAC 1019 Left ATTTCAGATTTCCCTCCTCTCAC

1010 Right GGAGACCTAGTATTCTGCTCTGAAG 1020 Right AGGGAGACCTAGTATTCTGCTCTGA

1084 Right ACAATAAAACAAAGCTGAATCATCC

103 Left TGCACAATAAATTAAAAGGGAAAGA

1085 Left GTGCACAATAAATTAAAAGGGAAAG 104 Right GAGACCTAGTATTCTGCTCTGAAGG

1086 Right AAACAAAGCTGAATCATCCTACATC

105 Left TGCACAATAAATTAAAAGGGAAAGA

1087 Left TGCACAATAAATTAAAAGGGAAAGA 106 Right GGAGACCTAGTATTCTGCTCTGAAG

1088 Right AAAACAAAGCTGAATCATCCTACAT

Seq. 107 Left TGTGCACAATAAATTAAAAGGGAAA

Primer Sequence

ID 108 Right ACAATAAAACAAAGCTGAATCATCC

1089 Left ATCATATTACCTGGGAAGACTTCAA

1090 Right AAACAAAGCTGAATCATCCTACATC 109 Left TGTTTATAAATTGGGGGTATTCAAA

110 Right GAGACCTAGTATTCTGCTCTGAAGG

1091 Left GTGCACAATAAATTAAAAGGGAAAG

1092 Right ACAATAAAACAAAGCTGAATCATCC 1 1 1 Left TTGTTTATAAATTGGGGGTATTCAA

112 Right GAGACCTAGTATTCTGCTCTGAAGG

1093 Left TGTGCACAATAAATTAAAAGGGAAA

1094 Right AAACAAAGCTGAATCATCCTACATC 1 13 Left TGTTTATAAATTGGGGGTATTCAAA

114 Right GGAGACCTAGTATTCTGCTCTGAAG

1095 Left TGCACAATAAATTAAAAGGGAAAGA

1096 Right TAAAACAAAGCTGAATCATCCTACA 1 15 Left TTGTTTATAAATTGGGGGTATTCAA

116 Right GGAGACCTAGTATTCTGCTCTGAAG

1097 Left TAAATTAAAAGGGAAAGAACACCTG

1098 Right AAACAAAGCTGAATCATCCTACATC 117 Left GTGCACAATAAATTAAAAGGGAAAG

118 Right AAAACAAAGCTGAATCATCCTACAT

1099 Left GCACAATAAATTAAAAGGGAAAGAA

1 100 Right AAACAAAGCTGAATCATCCTACATC 119 Left TAAATTAAAAGGGAAAGAACACCTG

120 Right ACAATAAAACAAAGCTGAATCATCC

1 101 Left TGGGAAGACTTCAAATGTACAAATA

1 102 Right AAACAAAGCTGAATCATCCTACATC

ALK Exoii24 601-800 bases

Seq.

Primer Sequence

ID 135 Left CTTGTTTATAAATTGGGGGTATTCA

1 121 Left TGTTTATAAATTGGGGGTATTCAAA 136 Right AAACAAAGCTGAATCATCCTACATC

1 122 Right AAACAAAGCTGAATCATCCTACATC

137 Left TGCACAATAAATTAAAAGGGAAAGA

1 123 Left TTGTTTATAAATTGGGGGTATTCAA 138 Right GCAAGTGAATCCCTGATAGAATAAG

1 124 Right AAACAAAGCTGAATCATCCTACATC

139 Left CTGGATCTGCTTGAAGAAAATTAGT

1 125 Left TGTTTATAAATTGGGGGTATTCAAA 140 Right AAACAAAGCTGAATCATCCTACATC

1 126 Right ACAATAAAACAAAGCTGAATCATCC

1 127 Left TGCACAATAAATTAAAAGGGAAAGA >eq.

Primer Sequence

1 128 Right AGTTACCATCTCAAAGACAAAGCTG ID

141 Left CAAACTTGTTTATAAATTGGGGGTA

1 129 Left TGTTTATAAATTGGGGGTATTCAAA 142 Right ACAATAAAACAAAGCTGAATCATCC

1 130 Right AGTTACCATCTCAAAGACAAAGCTG

143 Left TTTATAAATTGGGGGTATTCAAATG

1 131 Left TTGTTTATAAATTGGGGGTATTCAA 144 Right AAACAAAGCTGAATCATCCTACATC

1 132 Right AGTTACCATCTCAAAGACAAAGCTG

145 Left TGTTTATAAATTGGGGGTATTCAAA

1 133 Left CAAACTTGTTTATAAATTGGGGGTA 146 Right AAAACAAAGCTGAATCATCCTACAT

1 134 Right AAACAAAGCTGAATCATCCTACATC

Ζ Γ Εχοη25 161-200 bases

1270 Right GAGGGGTGAGGCAGTCTTTACTCA : 275 Left CCTAGGGATAAAATTAGGAAATGC

: 276 Right GAGGGGTGAGGCAGTCTTTACT

1271 Left TTCCTAGGGATAAAATTAGGAAATGC

1272 Right GGGGTGAGGCAGTCTTTACTC : 277 Left TTCCTAGGGATAAAATTAGGAAATG

: 278 Right GGGTGAGGCAGTCTTTACTCA

1273 Left CCTAGGGATAAAATTAGGAAATGC

1274 Right AGGGGTGAGGCAGTCTTTACTC : 279 Left TTCCTAGGGATAAAATTAGGAAATG

280 Right AGGGGTGAGGCAGTCTTTACT

1588 Right TGGTCACTAATTAAGGTTTCCCATA

] 595 Left TGTAGCTTAGCAAGGGCTTTAGATA

1589 Left AACTTCAGCTTGGAGATAAAATCCT ] 596 Right GGAACTAGAGGCTAGGAAGAGAAGA

1590 Right GTGCTGAGGACATAAATAGGTCAGT

] 597 Left GGACAGTAATAGCACCTTGTGTCTT

1591 Left TGTAGCTTAGCAAGGGCTTTAGATA ] 598 Right TCAGTGACACAAATGAAGAATTGAT

1592 Right AGTCTCACTTATTCCCCAAAGAGTT

] 599 Left TTAATCATTTCCCCTAATCCTTTTC

1593 Left TGTAGCTTAGCAAGGGCTTTAGATA ] 600 Right TGCATTGCAATATAGAAAACACAGT

1594 Right GGTGTCTGGATCAGTCTCACTTATT

ALK Έχοη24-25 5kb

Seq. 622 Right AGCCTGAAAAGGAACTTAGTGAAAT

Primer Sequence

ID

1601 Left ATCTCCTTTTGAATGAAAGAGACCT

1602 Right TGGTCACTAATTAAGGTTTCCCATA

Seq.

Primer Sequence 623 Left TGCACAATAAATTAAAAGGGAAAGA ID 624 Right TGGTCACTAATTAAGGTTTCCCATA

1603 Left ATCTCCTTTTGAATGAAAGAGACCT

1604 Right AGCCTGAAAAGGAACTTAGTGAAAT 625 Left TGTTTATAAATTGGGGGTATTCAAA

1605 Left ACGTCAGGGATTTAGGAATTAAAAG 626 Right TGGTCACTAATTAAGGTTTCCCATA

1606 Right TGGTCACTAATTAAGGTTTCCCATA

627 Left TTGTTTATAAATTGGGGGTATTCAA

1607 Left ATGTGAATCATACTCCTCCAGGTAA 628 Right TGGTCACTAATTAAGGTTTCCCATA

1608 Right TGGTCACTAATTAAGGTTTCCCATA

629 Left TGAATCATACTCCTCCAGGTAAATC

1609 Left ATCTCCTTTTGAATGAAAGAGACCT 630 Right TGGTCACTAATTAAGGTTTCCCATA

1610 Right GTATCCAAGTTATCCCATGTCTCAG

631 Left AGGTATGTGAATCATACTCCTCCAG

1611 Left ATCTCCTTTTGAATGAAAGAGACCT 632 Right AGCCTGAAAAGGAACTTAGTGAAAT

1612 Right TAGCCTGAAAAGGAACTTAGTGAAA

633 Left ACGTCAGGGATTTAGGAATTAAAAG

1613 Left ACGTCAGGGATTTAGGAATTAAAAG 634 Right TAGCCTGAAAAGGAACTTAGTGAAA

1614 Right AGCCTGAAAAGGAACTTAGTGAAAT

635 Left AAGAGTCACCAGCTTAAACAAACAC

1615 Left ATCTCCTTTTGAATGAAAGAGACCT 636 Right TAGCCTGAAAAGGAACTTAGTGAAA

1616 Right CATAGCCTGAAAAGGAACTTAGTGA

637 Left ATGTGAATCATACTCCTCCAGGTAA

1617 Left AGGTATGTGAATCATACTCCTCCAG 638 Right TAGCCTGAAAAGGAACTTAGTGAAA

1618 Right TGGTCACTAATTAAGGTTTCCCATA

639 Left TGCACAATAAATTAAAAGGGAAAGA

1619 Left AAGAGTCACCAGCTTAAACAAACAC 640 Right AGCCTGAAAAGGAACTTAGTGAAAT

1620 Right AGCCTGAAAAGGAACTTAGTGAAAT

1621 Left ATGTGAATCATACTCCTCCAGGTAA

Table 8. EGFR Capture Primer List for NGS Panel

EGFR Exonl8 100-200 bases

Seq. ] 644 Right TTCTTGACGAGGTCCATGTG

Primer Sequence

ID

1641 Left TGCCAAAGAAGTAGAATGAG Ί 645 Left TGCCAAAGAAGTAGAATGAG

1642 Right AAAGCATCTTCACCCACAGC J 646 Right GTCAGAAATGCAGGAAAGCA

1643 Left TGCCAAAGAAGTAGAATGAG ] 647 Left TGCCAAAGAAGTAGAATGAG 1713 Left TGCCAAAGAAGTAGAATGAG 717 Left GCCAAAGAAGTAGAATGAGA

1714 Right AACCAGCTGGGCAGTCTCT 718 Right CCAGCACTGTGTGTCCAACT

1715 Left TGCCAAAGAAGTAGAATGAG 719 Left GCCAAAGAAGTAGAATGAGA

1716 Right GAAACCCTGGCTGAGGGTAG 720 Right TCCCTCCACTGAGGACAAAG

EGFR Exonl8 400-1000 bases

Seq.

Primer Sequence

ID 739 Left TGCCAAAGAAGTAGAATGAG

1721 Left TGCCAAAGAAGTAGAATGAG 740 Right ACGCCATCGAGAGTAACACC

1722 Right CAGTGTGGAGTGGGGAAGTT

J 741 Left TGCCAAAGAAGTAGAATGAG

1723 Left TGCCAAAGAAGTAGAATGAG J 742 Right AGGAGCATGCCAAAATGAAG

1724 Right ACTCCCCTATGCTGGAGGTT

Ί 743 Left TGCCAAAGAAGTAGAATGAG

1744 Right TGTTGAAGGAAGCCCTTTTG

Seq. 745 Left TGCCAAAGAAGTAGAATGAG

Primer Sequence

ID 746 Right CCAATGGGGTAAGTGGACAG

1725 Left TGCCAAAGAAGTAGAATGAG

1726 Right TGGGAAAGAAAGCAAGGAGA 747 Left TGCCAAAGAAGTAGAATGAG

748 Right TTGCCTTCTTCCTCGATCAT

1727 Left TGCCAAAGAAGTAGAATGAG

1728 Right TCTGGGAAAGAAAGCAAGGA 749 Left TGCCAAAGAAGTAGAATGAG

750 Right CATCGAACAGAAAGGCCACT

1751 Left TGCCAAAGAAGTAGAATGAG

1729 Left TGCCAAAGAAGTAGAATGAG J 752 Right GGTGGCAGGAGAGAGAGCTA

1730 Right ACCAATGGGGTAAGTGGACA

753 Left TGCCAAAGAAGTAGAATGAG

1731 Left TGCCAAAGAAGTAGAATGAG J 754 Right ATGGGACCAATGGGGTAAGT

1732 Right CCTCGATCATGTGACACTGG

Ί 755 Left TGCCAAAGAAGTAGAATGAG

1733 Left TGCCAAAGAAGTAGAATGAG 756 Right TGGAGGTTGTCATCGAACAG

1734 Right AAAATGGCAAACAGGTGCTC

Ί 757 Left TGCCAAAGAAGTAGAATGAG

1735 Left TGCCAAAGAAGTAGAATGAG 758 Right CTGGAGGTTGTCATCGAACA

1736 Right AACTGGCCAGAGCTGATGTT

759 Left TGCCAAAGAAGTAGAATGAG

1737 Left TGCCAAAGAAGTAGAATGAG J 760 Right TATGCTGGAGGTTGTCATCG

1738 Right AAACTGGCCAGAGCTGATGT

EGFR Exonl9 100-200 bases

Seq.

Primer Sequence

ID 767 Left CTTGTTCCTCCACCTCATTCC

1761 Left CTTCCTTGTTCCTCCACCTCAT 768 Right ACCCAGGACTGGCACTCAC

1762 Right ACCCAGGACTGGCACTCAC

Ί 769 Left CCTTGTTCCTCCACCTCATTC

1763 Left CTTCCTTGTTCCTCCACCTCAT 770 Right ACCCAGGACTGGCACTCAC

1764 Right CCCAGGACTGGCACTCAC

J 771 Left TCCTTGTTCCTCCACCTCATT

1765 Left CTTCCTTGTTCCTCCACCTCAT J 772 Right ACCCAGGACTGGCACTCAC

1766 Right ACCCAGGACTGGCACTCA

1840 Right ACATTCAGAGATTCTTTCTGCATCA

EGFR Exonl9 400-1000 bases

Seq.

Primer Sequence

ID 861 Left CTCGTTCAGAGAGTATTTCACACAA

1841 Left AATACCAATCCATGAAAAAGCATTA 862 Right AACATGTCACCAACTGGGTATAACT

1842 Right AACATGTCACCAACTGGGTATAACT

J 863 Left TCCAAGATCATTCTACAAGATGTCA

1843 Left CCTATTCCTTTATAACCCCTTTCAA J 864 Right ACTGAACAGCTACCTTTCAACAAAC

1844 Right AACATGTCACCAACTGGGTATAACT

Ί 865 Left CCTATTCCTTTATAACCCCTTTCAA

1845 Left TCCAAGATCATTCTACAAGATGTCA j 866 Right AACTGCACATTCAGAGATTCTTTCT

1846 Right AACATGTCACCAACTGGGTATAACT

Ί 867 Left TCCAAGATCATTCTACAAGATGTCA

1847 Left TTTCAAGCTCGTTCAGAGAGTATTT j 868 Right AACTGCACATTCAGAGATTCTTTCT

1848 Right AACATGTCACCAACTGGGTATAACT

869 Left ACTCTTGGAATGAACAAAATACCAA

1849 Left TTCAGAGAGTATTTCACACAATCCA j 870 Right AACATGTCACCAACTGGGTATAACT

1850 Right AACATGTCACCAACTGGGTATAACT

Seq. 871 Left CCTATTCCTTTATAACCCCTTTCAA

Primer Sequence

ID 872 Right GGGTATAACTGCACATTCAGAGATT

1851 Left GTGTCTCACTTTCCAAGATCATTCT

1852 Right AACATGTCACCAACTGGGTATAACT 873 Left TCCAAGATCATTCTACAAGATGTCA

874 Right GGGTATAACTGCACATTCAGAGATT

1853 Left AGTGTCTCACTTTCCAAGATCATTC

1854 Right AACATGTCACCAACTGGGTATAACT 875 Left AGTGTCTCACTTTCCAAGATCATTC

876 Right ACTGAACAGCTACCTTTCAACAAAC

1855 Left CCATGAAAAAGCATTATTGAAGTCT

1856 Right AACATGTCACCAACTGGGTATAACT 877 Left GTGTCTCACTTTCCAAGATCATTCT

878 Right ACTGAACAGCTACCTTTCAACAAAC

1857 Left TATTCCTTTATAACCCCTTTCAAGC

1858 Right AACATGTCACCAACTGGGTATAACT 879 Left AGTGTCTCACTTTCCAAGATCATTC

880 Right AACTGCACATTCAGAGATTCTTTCT

1859 Left ATGGAAATACTCTTGGAATGAACAA

1860 Right AACATGTCACCAACTGGGTATAACT

EGFR Exon20 100-200 bases

Seq. 891 Left AGGTGACCCTTGTCTCTGTGTT

Primer Sequence

ID 892 Right CCTGTGCCAGGGACCTTAC

1881 Left GTGACCCTTGTCTCTGTGTTCTT

1882 Right CCTGTGCCAGGGACCTTAC 893 Left GACCCTTGTCTCTGTGTTCTTGT

894 Right CCTGTGCCAGGGACCTTA

1883 Left ACCCTTGTCTCTGTGTTCTTGTC

1884 Right CCTGTGCCAGGGACCTTAC 895 Left GTGACCCTTGTCTCTGTGTTCTT

896 Right CCTGTGCCAGGGACCTTA

1885 Left GACCCTTGTCTCTGTGTTCTTGT

1886 Right CCTGTGCCAGGGACCTTAC 897 Left ACCCTTGTCTCTGTGTTCTTGTC

898 Right CCTGTGCCAGGGACCTTA

1887 Left GACCCTTGTCTCTGTGTTCTTGTC

1888 Right CCTGTGCCAGGGACCTTAC 899 Left GACCCTTGTCTCTGTGTTCTTGTC

900 Right CCTGTGCCAGGGACCTTA

1889 Left GTGACCCTTGTCTCTGTGTTCTTGT

1890 Right CCTGTGCCAGGGACCTTAC >eq.

Primer Sequence

ID 1901 Left GTGACCCTTGTCTCTGTGTTCTTG 911 Left CTGAGGTGACCCTTGTCTCTGT

1902 Right CCTGTGCCAGGGACCTTAC 912 Right CCTGTGCCAGGGACCTTAC

1903 Left TGACCCTTGTCTCTGTGTTCTTGT 913 Left AGGTGACCCTTGTCTCTGTGTTCTT

1904 Right CCTGTGCCAGGGACCTTAC 914 Right CCTGTGCCAGGGACCTTAC

1905 Left GAGGTGACCCTTGTCTCTGTGT 915 Left GGTGACCCTTGTCTCTGTGTTCT

1906 Right CCTGTGCCAGGGACCTTAC 916 Right CCTGTGCCAGGGACCTTAC

1907 Left TGACCCTTGTCTCTGTGTTCTTG 917 Left AGGTGACCCTTGTCTCTGTGTTC

1908 Right CCTGTGCCAGGGACCTTAC 918 Right CCTGTGCCAGGGACCTTAC

1909 Left AGGTGACCCTTGTCTCTGTGTTCT 919 Left GAGGTGACCCTTGTCTCTGTGTT

1910 Right CCTGTGCCAGGGACCTTAC 920 Right CCTGTGCCAGGGACCTTAC

EGFR Exon20 200-400 bases

Seq.

Primer Sequence

ID _ 941 Left TACATTTGTCCTTCCAAATGAGC

1921 Left AAGCTCTGTAGAGAAGGCGTACAT 942 Right AAATATACAGCTTGCAAGGACTCTG

1922 Right AAATATACAGCTTGCAAGGACTCTG

Seq. 943 Left CAAGCTCTGTAGAGAAGGCGTACAT

Primer Sequence

ID 944 Right AAATATACAGCTTGCAAGGACTCTG

1923 Left AGCTCTGTAGAGAAGGCGTACATT

1924 Right AAATATACAGCTTGCAAGGACTCTG 945 Left AGCTCTGTAGAGAAGGCGTACATT

1925 Left TCTGTAGAGAAGGCGTACATTTGTC 946 Right GGAAATATACAGCTTGCAAGGACTC

1926 Right AAATATACAGCTTGCAAGGACTCTG

947 Left AAGCTCTGTAGAGAAGGCGTACAT

1927 Left GTAGAGAAGGCGTACATTTGTCCT 948 Right GGAAATATACAGCTTGCAAGGACTC

1928 Right AAATATACAGCTTGCAAGGACTCTG

949 Left GTCAAGCTCTGTAGAGAAGGCGTA

1929 Left AAGCTCTGTAGAGAAGGCGTACATT 950 Right AAATATACAGCTTGCAAGGACTCTG

1930 Right AAATATACAGCTTGCAAGGACTCTG

: 951 Left TCTGTAGAGAAGGCGTACATTTGTC

1931 Left GCTCTGTAGAGAAGGCGTACATTT 952 Right GGAAATATACAGCTTGCAAGGACTC

1932 Right AAATATACAGCTTGCAAGGACTCTG

: 953 Left TGTAGAGAAGGCGTACATTTGTCCT

1933 Left CTCTGTAGAGAAGGCGTACATTTG 954 Right AAATATACAGCTTGCAAGGACTCTG

1934 Right AAATATACAGCTTGCAAGGACTCTG

: 955 Left GTAGAGAAGGCGTACATTTGTCCT

1935 Left CTGTAGAGAAGGCGTACATTTGTC 956 Right GGAAATATACAGCTTGCAAGGACTC

1936 Right AAATATACAGCTTGCAAGGACTCTG

957 Left AAGCTCTGTAGAGAAGGCGTACATT

1937 Left TTCTGTCAAGCTCTGTAGAGAAGG 958 Right GGAAATATACAGCTTGCAAGGACTC

1938 Right AAATATACAGCTTGCAAGGACTCTG

959 Left AGCTCTGTAGAGAAGGCGTACATT

1939 Left AAGCTCTGTAGAGAAGGCGTACA 960 Right ATGGAAATATACAGCTTGCAAGGAC

1940 Right AAATATACAGCTTGCAAGGACTCTG

EGFR Exon20 400-1000 bases

Seq.

Primer Sequence

ID j 963 Left GTTTCTACCAACTTCTGTCAAGCTC

1961 Left TTTCTACCAACTTCTGTCAAGCTCT j 964 Right ATCTAGAAGAAGCAAACGAAGATGA

1962 Right ATCTAGAAGAAGCAAACGAAGATGA 1965 Left GTTTCTACCAACTTCTGTCAAGCTC 983 Left AATCTCTGAATGTGCAGTTATACCC

1966 Right GATCTAGAAGAAGCAAACGAAGATG 984 Right AAATATACAGCTTGCAAGGACTCTG

1967 Left GTTTCTACCAACTTCTGTCAAGCTC 985 Left TTTCTACCAACTTCTGTCAAGCTCT

1968 Right CTATGACAGAGAGAGAAGGAAGACC 986 Right GTTATAAAGTCCGTGTGGATCATTT

1969 Left CTGTGTTTCTACCAACTTCTGTCAA 987 Left CTGTGTTTCTACCAACTTCTGTCAA

1970 Right GATCTAGAAGAAGCAAACGAAGATG 988 Right AAATATACAGCTTGCAAGGACTCTG

1971 Left TGTGTTTCTACCAACTTCTGTCAAG 989 Left TTTCTACCAACTTCTGTCAAGCTCT

1972 Right ATCTAGAAGAAGCAAACGAAGATGA 990 Right TTATAAAGTCCGTGTGGATCATTTC

1973 Left TGTGTTTCTACCAACTTCTGTCAAG 991 Left GAAATTGTGTTTGTTGAAAGGTAGC

1974 Right GATCTAGAAGAAGCAAACGAAGATG 992 Right AAATATACAGCTTGCAAGGACTCTG

1975 Left CTGTGTTTCTACCAACTTCTGTCAA 993 Left TTTCTACCAACTTCTGTCAAGCTCT

1976 Right ATCTAGAAGAAGCAAACGAAGATGA 994 Right CTGTTATAAAGTCCGTGTGGATCAT

Seq.

Primer Sequence

ID 995 Left GTTTCTACCAACTTCTGTCAAGCTC

1977 Left AGAAAGAATCTCTGAATGTGCAGTT J 996 Right GTTATAAAGTCCGTGTGGATCATTT

1978 Right AAATATACAGCTTGCAAGGACTCTG

Ί 997 Left TTTCTACCAACTTCTGTCAAGCTCT

1979 Left GAAATTGTGTTTGTTGAAAGGTAGC 998 Right TCTAGAAGAAGCAAACGAAGATGAG

1980 Right ATCTAGAAGAAGCAAACGAAGATGA

j 999 Left TTTCTACCAACTTCTGTCAAGCTCT

1981 Left GAAATTGTGTTTGTTGAAAGGTAGC 000 Right CTCCACGAATCACACTGATTATTTA

1982 Right GATCTAGAAGAAGCAAACGAAGATG

EGFR Exon21 100-200 bases

Seq. 016 Right ACACAGCAAAGCAGAAACTCAC

Primer Sequence

ID

2001 Left ACGTCTTCCTTCTCTCTCTGTCATA 017 Left CAGTTAACGTCTTCCTTCTCTCTCT

2002 Right ACACAGCAAAGCAGAAACTCAC 018 Right ACACAGCAAAGCAGAAACTCAC

2003 Left TTAACGTCTTCCTTCTCTCTCTGTC 019 Left AGTTAACGTCTTCCTTCTCTCTCTG

2004 Right ACACAGCAAAGCAGAAACTCAC 020 Right ACACAGCAAAGCAGAAACTCAC

2005 Left TAACGTCTTCCTTCTCTCTCTGTCA 5eq.

Primer Sequence

2006 Right ACACAGCAAAGCAGAAACTCAC ID

021 Left ACGTCTTCCTTCTCTCTCTGTCATA

2007 Left ACGTCTTCCTTCTCTCTCTGTCATA 022 Right CACACAGCAAAGCAGAAACTCA

2008 Right CACACAGCAAAGCAGAAACTCAC

023 Left GTTAACGTCTTCCTTCTCTCTCTGT

2009 Left ACGTCTTCCTTCTCTCTCTGTCAT 024 Right ACACAGCAAAGCAGAAACTCAC

2010 Right ACACAGCAAAGCAGAAACTCAC

025 Left TTAACGTCTTCCTTCTCTCTCTGTC

2011 Left CCAGTTAACGTCTTCCTTCTCTCTC 026 Right CACACAGCAAAGCAGAAACTCAC

2012 Right ACACAGCAAAGCAGAAACTCAC

027 Left GTTAACGTCTTCCTTCTCTCTCTGTC

2013 Left ACGTCTTCCTTCTCTCTCTGTCATA 028 Right ACACAGCAAAGCAGAAACTCAC

2014 Right CCACACAGCAAAGCAGAAACT

029 Left AGTTAACGTCTTCCTTCTCTCTCTGT

2015 Left AACGTCTTCCTTCTCTCTCTGTCAT 030 Right ACACAGCAAAGCAGAAACTCAC 036 Right CACACAGCAAAGCAGAAACTCA

2031 Left TTAACGTCTTCCTTCTCTCTCTGTC

2032 Right CCACACAGCAAAGCAGAAACT 037 Left TAACGTCTTCCTTCTCTCTCTGTCA

038 Right CACACAGCAAAGCAGAAACTCAC

2033 Left CCAGTTAACGTCTTCCTTCTCTCT

2034 Right ACACAGCAAAGCAGAAACTCAC 039 Left CGTCTTCCTTCTCTCTCTGTCATA

040 Right ACACAGCAAAGCAGAAACTCAC

2035 Left TTAACGTCTTCCTTCTCTCTCTGTC

EGFR Exon21 200-400 bases

Seq.

Primer Sequence

ID 061 Left ACGTCTTCCTTCTCTCTCTGTCATA

2041 Left ACGTCTTCCTTCTCTCTCTGTCATA 062 Right AAGTGAACATTTAGGATGTGGAGAT

2042 Right TGTCTCTAAGGGGAGGGAGTTATAC

063 Left ACGTCTTCCTTCTCTCTCTGTCATA

2043 Left ACGTCTTCCTTCTCTCTCTGTCATA 064 Right GTCAAGAAACTAGTGCTGGGTAGAT

2044 Right GAAAGTGAACATTTAGGATGTGGAG

065 Left TTAACGTCTTCCTTCTCTCTCTGTC

2045 Left ACGTCTTCCTTCTCTCTCTGTCATA 066 Right GTGTCAAGAAACTAGTGCTGGGTAG

2046 Right AAAGTGAACATTTAGGATGTGGAGA

067 Left TTAACGTCTTCCTTCTCTCTCTGTC

2047 Left ACGTCTTCCTTCTCTCTCTGTCATA 068 Right AGTGAACATTTAGGATGTGGAGATG

2048 Right AGAAAGTGAACATTTAGGATGTGGA

Seq. 069 Left TTAACGTCTTCCTTCTCTCTCTGTC

Primer Sequence

ID 070 Right AAGTGAACATTTAGGATGTGGAGAT

2049 Left TTAACGTCTTCCTTCTCTCTCTGTC

2050 Right TGTCTCTAAGGGGAGGGAGTTATAC 071 Left ACGTCTTCCTTCTCTCTCTGTCATA

072 Right TCAAGAAACTAGTGCTGGGTAGATG

2051 Left ACGTCTTCCTTCTCTCTCTGTCATA

2052 Right GTGTCAAGAAACTAGTGCTGGGTAG 073 Left TTAACGTCTTCCTTCTCTCTCTGTC

074 Right GTCAAGAAACTAGTGCTGGGTAGAT

2053 Left TTAACGTCTTCCTTCTCTCTCTGTC

2054 Right AAAGTGAACATTTAGGATGTGGAGA 075 Left ACGTCTTCCTTCTCTCTCTGTCATA

GAAAGGGAAAGACATAGAAAGTGAA

Right

2055 Left TTAACGTCTTCCTTCTCTCTCTGTC 076 C

2056 Right GAAAGTGAACATTTAGGATGTGGAG

077 Left ACGTCTTCCTTCTCTCTCTGTCATA

2057 Left TTAACGTCTTCCTTCTCTCTCTGTC 078 Right TGTCAAGAAACTAGTGCTGGGTAG

2058 Right AGAAAGTGAACATTTAGGATGTGGA

079 Left TAACGTCTTCCTTCTCTCTCTGTCA

2059 Left ACGTCTTCCTTCTCTCTCTGTCATA 080 Right TGTCTCTAAGGGGAGGGAGTTATAC

2060 Right AGTGAACATTTAGGATGTGGAGATG

EGFR Exon21 400-1000 bases

Seq.

Primer Sequence

ID 087 Left ACGTCTTCCTTCTCTCTCTGTCATA

2081 Left ACGTCTTCCTTCTCTCTCTGTCATA 088 Right TGAGGTAATAAGTCAGCCATTTTTC

2082 Right CAAAGTAACAATCAACAGACACTGG

089 Left ACGTCTTCCTTCTCTCTCTGTCATA

2083 Left ACGTCTTCCTTCTCTCTCTGTCATA 090 Right CCACAAAGTAACAATCAACAGACAC

2084 Right AAAGATGAGATAACTTGGTGGAGTG

091 Left TTCTAGATCCTCTTTGCATGAAATC

2085 Left ACGTCTTCCTTCTCTCTCTGTCATA 092 Right AAAGATGAGATAACTTGGTGGAGTG

2086 Right CAAAGATGAGATAACTTGGTGGAGT 2093 Left TTCTAGATCCTCTTTGCATGAAATC

2094 Right CAAAGATGAGATAACTTGGTGGAGT 107 Left ACGTCTTCCTTCTCTCTCTGTCATA

108 Right CCATTTCAAAGATGAGATAACTTGG

2095 Left ACGTCTTCCTTCTCTCTCTGTCATA

2096 Right GGTAATAAGTCAGCCATTTTTCCTT 109 Left AGGCTTTACAAGCTTGAGATTCTTT

110 Right CAAAGTAACAATCAACAGACACTGG

2097 Left ACGTCTTCCTTCTCTCTCTGTCATA

2098 Right AATTTCTTTATGCCTCCATTTCTTC 111 Left TTCTAGATCCTCTTTGCATGAAATC

112 Right AATTTCTTTATGCCTCCATTTCTTC

2099 Left ACGTCTTCCTTCTCTCTCTGTCATA

2100 Right GATGAGATAACTTGGTGGAGTGAAT 113 Left ACGTCTTCCTTCTCTCTCTGTCATA

114 Right AGATAACTTGGTGGAGTGAATTGAA

Seq.

Primer Sequence

ID 115 Left TTCTAGATCCTCTTTGCATGAAATC

2101 Left ACGTCTTCCTTCTCTCTCTGTCATA 116 Right GATGAGATAACTTGGTGGAGTGAAT

2102 Right GAATTTTCCAAGAACTTATTCCACA

117 Left TTCTAGATCCTCTTTGCATGAAATC

2103 Left ACGTCTTCCTTCTCTCTCTGTCATA 118 Right CCATTTCAAAGATGAGATAACTTGG

2104 Right TGTGGAATTTTCCAAGAACTTATTC

119 Left AGGCTTTACAAGCTTGAGATTCTTT

2105 Left ACGTCTTCCTTCTCTCTCTGTCATA 120 Right AAAGATGAGATAACTTGGTGGAGTG

2106 Right AATAAGTCAGCCATTTTTCCTTTTC

EGFR Exon22 100-200 bases

Seq. 140 Right CCGTATCTCCCTTCCCTGATTAC

Primer Sequence

ID

2121 Left CACACTGACGTGCCTCTCC 5eq.

Primer Sequence

2122 Right CGTATCTCCCTTCCCTGATTAC ID

141 Left CGAAGCCACACTGACGTG

2123 Left CCACACTGACGTGCCTCTC 142 Right CCGTATCTCCCTTCCCTGATTA

2124 Right CGTATCTCCCTTCCCTGATTAC

143 Left CATGCGAAGCCACACTGAC

2125 Left CACACTGACGTGCCTCTCC 144 Right CGTATCTCCCTTCCCTGATTAC

2126 Right CCGTATCTCCCTTCCCTGATTAC

145 Left CATGCGAAGCCACACTGA

2127 Left CCACACTGACGTGCCTCTC 146 Right CGTATCTCCCTTCCCTGATTAC

2128 Right CCGTATCTCCCTTCCCTGATTAC

147 Left CATGCGAAGCCACACTGAC

2129 Left CACACTGACGTGCCTCTCC 148 Right CCGTATCTCCCTTCCCTGATTAC

2130 Right CCGTATCTCCCTTCCCTGATTA

149 Left CACACTGACGTGCCTCTCC

2131 Left CCACACTGACGTGCCTCTC 150 Right TATCTCCCCTCCCCGTATCT

2132 Right CCGTATCTCCCTTCCCTGATTA

151 Left CATGCGAAGCCACACTGA

2133 Left CGAAGCCACACTGACGTG 152 Right CCGTATCTCCCTTCCCTGATTAC

2134 Right CGTATCTCCCTTCCCTGATTAC

153 Left CACACTGACGTGCCTCTCC

2135 Left CGAAGCCACACTGACGTG 154 Right CCGTATCTCCCTTCCCTGAT

2136 Right CCGTATCTCCCTTCCCTGATTAC

155 Left CCACACTGACGTGCCTCTC

2137 Left ACCATGCGAAGCCACACT 156 Right CCGTATCTCCCTTCCCTGAT

2138 Right CGTATCTCCCTTCCCTGATTAC

157 Left ACCATGCGAAGCCACACT

2139 Left ACCATGCGAAGCCACACT 158 Right CCGTATCTCCCTTCCCTGATTA 160 Right TATCTCCCCTCCCCGTATCTC

2159 Left CACACTGACGTGCCTCTCC

EGFR Exon22 200-400 bases

Seq. 180 Right CCGTATCTCCCTTCCCTGATTAC

Primer Sequence

ID

2161 Left GTATTTTGAAACTCAAGATCGCATT

2162 Right CGTATCTCCCTTCCCTGATTAC

181 Left CCATGAGTACGTATTTTGAAACTCA

2163 Left CCTCCATGAGTACGTATTTTGAAAC 182 Right CATGGCAAACTCTTGCTATCC

2164 Right CGTATCTCCCTTCCCTGATTAC

183 Left TATTTTGAAACTCAAGATCGCATTC

2165 Left GTATTTTGAAACTCAAGATCGCATT 184 Right CGTATCTCCCTTCCCTGATTAC

2166 Right CCGTATCTCCCTTCCCTGATTAC

185 Left GTATTTTGAAACTCAAGATCGCATT

2167 Left CCTCCATGAGTACGTATTTTGAAAC 186 Right CTTATCTCCCCTCCCCGTATCT

2168 Right CCGTATCTCCCTTCCCTGATTAC

187 Left GTATTTTGAAACTCAAGATCGCATT

2169 Left GTATTTTGAAACTCAAGATCGCATT 188 Right ACATATCCCCATGGCAAACTCT

2170 Right CATGGCAAACTCTTGCTATCC

189 Left CTTTTCCTCCATGAGTACGTATTTT

2190 Right CGTATCTCCCTTCCCTGATTAC

Seq. 191 Left TATTTTGAAACTCAAGATCGCATTC

Primer Sequence

ID 192 Right CCGTATCTCCCTTCCCTGATTAC

2171 Left GTATTTTGAAACTCAAGATCGCATT

2172 Right ATATCCCCATGGCAAACTCTT 193 Left GTATTTTGAAACTCAAGATCGCATT

194 Right ACATATCCCCATGGCAAACTCTT

2173 Left CCATGAGTACGTATTTTGAAACTCA

2174 Right CGTATCTCCCTTCCCTGATTAC 195 Left CCTCCATGAGTACGTATTTTGAAAC

196 Right CTTATCTCCCCTCCCCGTATCT

2175 Left GTATTTTGAAACTCAAGATCGCATT

2176 Right CCGTATCTCCCTTCCCTGATTA 197 Left CCATGAGTACGTATTTTGAAACTCA

198 Right CCGTATCTCCCTTCCCTGATTA

2177 Left CCTCCATGAGTACGTATTTTGAAAC

2178 Right CCGTATCTCCCTTCCCTGATTA 199 Left GTATTTTGAAACTCAAGATCGCATT

200 Right ACATATCCCCATGGCAAACTC

2179 Left CCATGAGTACGTATTTTGAAACTCA

EGFR Exon22 400-1000 bases

Seq. 209 Left GTATTTTGAAACTCAAGATCGCATT

Primer Sequence

ID 210 Right ATCCAAAATAAAGGAATGTGTGTGT

2201 Left GTATTTTGAAACTCAAGATCGCATT

2202 Right TTGAATCCAAAATAAAGGAATGTGT 211 Left GTATTTTGAAACTCAAGATCGCATT

212 Right GCTTACCTTGTTATCAAGTCCTGAA

2203 Left GTATTTTGAAACTCAAGATCGCATT

2204 Right CACACTGAGCACTCAATAAAGAGAA 213 Left TGGTCTATTGAAAGAGCTTATCCAG

214 Right TTGAATCCAAAATAAAGGAATGTGT

2205 Left GTATTTTGAAACTCAAGATCGCATT

2206 Right TTCTCCACTACAAATCACCACAGTA 215 Left CCTCCATGAGTACGTATTTTGAAAC

216 Right TTGAATCCAAAATAAAGGAATGTGT

2207 Left GTATTTTGAAACTCAAGATCGCATT

2208 Right ATTCTTCAAAGGTAGCTGATTGATG 217 Left CCTCCATGAGTACGTATTTTGAAAC

218 Right CACACTGAGCACTCAATAAAGAGAA 229 Left GTATTTTGAAACTCAAGATCGCATT

2219 Left CCTCCATGAGTACGTATTTTGAAAC 230 Right TATTCCTTCTCCACTACAAATCACC

2220 Right TTCTCCACTACAAATCACCACAGTA

231 Left GTATTTTGAAACTCAAGATCGCATT

Seq. 232 Right CCACTACAAATCACCACAGTATTCA

Primer Sequence

ID

2221 Left TGGTCTATTGAAAGAGCTTATCCAG 233 Left GTATTTTGAAACTCAAGATCGCATT

2222 Right ATCCAAAATAAAGGAATGTGTGTGT 234 Right CTTGATTGAATCCAAAATAAAGGAA

2223 Left CCTCCATGAGTACGTATTTTGAAAC 235 Left GTATTTTGAAACTCAAGATCGCATT

2224 Right ATCCAAAATAAAGGAATGTGTGTGT 236 Right TAAGAACAGAGACATCAGACCACAC

2225 Left CCTCCATGAGTACGTATTTTGAAAC 237 Left CCTCCATGAGTACGTATTTTGAAAC

2226 Right GCTTACCTTGTTATCAAGTCCTGAA 238 Right CAAAGGTAGCTGATTGATGAGAGTT

2227 Left GTATTTTGAAACTCAAGATCGCATT 239 Left GTATTTTGAAACTCAAGATCGCATT

2228 Right CAAAGGTAGCTGATTGATGAGAGTT 240 Right AAATTCTTCAAAGGTAGCTGATTGA

EGFR Exonl8-19 2kb

Seq.

Primer Sequence

ID 2 261 Left AATCTCCAAAATATATGCCAAAGAA

2241 Left ATGCCAAAGAAGTAGAATGAGAAAA 2 262 Right GGGTATAACTGCACATTCAGAGATT

2242 Right AACATGTCACCAACTGGGTATAACT

Seq. 2 263 Left AAATCTCCAAAATATATGCCAAAGA

Primer Sequence

ID 2 264 Right GGGTATAACTGCACATTCAGAGATT

2243 Left ATGCCAAAGAAGTAGAATGAGAAAA

2244 Right GGGTATAACTGCACATTCAGAGATT 2 265 Left ATCTCCAAAATATATGCCAAAGAAG

2 266 Right AACATGTCACCAACTGGGTATAACT

2245 Left TCTCCAAAATATATGCCAAAGAAGT

2246 Right AACATGTCACCAACTGGGTATAACT 2 267 Left AAAAATCTCCAAAATATATGCCAAAG

2 268 Right AACATGTCACCAACTGGGTATAACT

2247 Left TGCCAAAGAAGTAGAATGAGAAAAA

2248 Right AACATGTCACCAACTGGGTATAACT 2 269 Left AAAATCTCCAAAATATATGCCAAAG

2 270 Right AACATGTCACCAACTGGGTATAACT

2249 Left AATCTCCAAAATATATGCCAAAGAA

2250 Right AACATGTCACCAACTGGGTATAACT 2 271 Left ATGCCAAAGAAGTAGAATGAGAAAA

2 272 Right ACTGGGTATAACTGCACATTCAGAG

2251 Left AAATCTCCAAAATATATGCCAAAGA

2252 Right AACATGTCACCAACTGGGTATAACT 2 273 Left ATCTCCAAAATATATGCCAAAGAAG

2 274 Right AACTGCACATTCAGAGATTCTTTCT

2253 Left TCTCCAAAATATATGCCAAAGAAGT

2254 Right AACTGCACATTCAGAGATTCTTTCT 2 275 Left AATCTCCAAAATATATGCCAAAGAAG

2 276 Right AACATGTCACCAACTGGGTATAACT

2255 Left TCTCCAAAATATATGCCAAAGAAGT

2256 Right GGGTATAACTGCACATTCAGAGATT 2 277 Left ATCTCCAAAATATATGCCAAAGAAG

2 278 Right GGGTATAACTGCACATTCAGAGATT

2257 Left TGCCAAAGAAGTAGAATGAGAAAAA

2258 Right GGGTATAACTGCACATTCAGAGATT 2 279 Left AAAAATCTCCAAAATATATGCCAAAG

2 280 Right AACTGCACATTCAGAGATTCTTTCT

2259 Left AAATCTCCAAAATATATGCCAAAGA

2260 Right AACTGCACATTCAGAGATTCTTTCT

EGFR Exon20-21 2kb Seq. 300 Right AAAGATGAGATAACTTGGTGGAGTG

Primer Sequence

ID

2281 Left TTTCTACCAACTTCTGTCAAGCTCT 301 Left AGAAAGAATCTCTGAATGTGCAGTT

2282 Right TGCTATGTATTCTGTGGGTTAGACA 302 Right CAAAGATGAGATAACTTGGTGGAGT

2283 Left TTTCTACCAACTTCTGTCAAGCTCT 303 Left AGAAAGAATCTCTGAATGTGCAGTT

2284 Right TGAGGTAATAAGTCAGCCATTTTTC 304 Right TTTTCCAAGAACTTATTCCACAAAG

2285 Left TTTCTACCAACTTCTGTCAAGCTCT 305 Left AATCTCTGAATGTGCAGTTATACCC

2286 Right GGTAATAAGTCAGCCATTTTTCCTT 306 Right AAAGATGAGATAACTTGGTGGAGTG

2287 Left AGAAAGAATCTCTGAATGTGCAGTT 307 Left AATCTCTGAATGTGCAGTTATACCC

2288 Right CAAAGTAACAATCAACAGACACTGG 308 Right CAAAGATGAGATAACTTGGTGGAGT

2289 Left TTTCTACCAACTTCTGTCAAGCTCT

2290 Right ATAAAGGCCCATGTTCTCTTTACTT

309 Left TTTCTACCAACTTCTGTCAAGCTCT

2291 Left TTTCTACCAACTTCTGTCAAGCTCT 310 Right AATAAGTCAGCCATTTTTCCTTTTC

2292 Right CCTTCTTGGCTGTAAGATCAACTAA

311 Left TTTCTACCAACTTCTGTCAAGCTCT

2293 Left AATCTCTGAATGTGCAGTTATACCC 312 Right CTGCCCAGAGAAAATTAAACTGTAG

2294 Right CAAAGTAACAATCAACAGACACTGG

313 Left AATCTCTGAATGTGCAGTTATACCC

2314 Right TTTTCCAAGAACTTATTCCACAAAG

Seq. 315 Left GTTTCTACCAACTTCTGTCAAGCTC

Primer Sequence

ID 316 Right GGTAATAAGTCAGCCATTTTTCCTT

2295 Left TTTCTACCAACTTCTGTCAAGCTCT

2296 Right ATTACTCTCTGGCTTTTGTCCTTCT 317 Left TGTGTTTCTACCAACTTCTGTCAAG

318 Right TGCTATGTATTCTGTGGGTTAGACA

2297 Left GTTTCTACCAACTTCTGTCAAGCTC

2298 Right TGAGGTAATAAGTCAGCCATTTTTC 319 Left GTTTCTACCAACTTCTGTCAAGCTC

320 Right ATAAAGGCCCATGTTCTCTTTACTT

2299 Left AGAAAGAATCTCTGAATGTGCAGTT

EGFR Exon22 2kb

Seq. 331 Left GTCTGTAGGTTACACACAAATGCTG

Primer Sequence

ID 332 Right TTCTCCACTACAAATCACCACAGTA

2321 Left CACATAGCATTTGCACTGTATTAGG

2322 Right TTGAATCCAAAATAAAGGAATGTGT 333 Left AGGTAATCAGGAGATGCTGTAGATG

334 Right CACACTGAGCACTCAATAAAGAGAA

2323 Left ATTTTGATATTTAAGGGAGGTCCTG

2324 Right TTCTCCACTACAAATCACCACAGTA 335 Left GTCTGTAGGTTACACACAAATGCTG

336 Right ATTCTTCAAAGGTAGCTGATTGATG

2325 Left CACATAGCATTTGCACTGTATTAGG

2326 Right TTCTCCACTACAAATCACCACAGTA 337 Left ATTTTGATATTTAAGGGAGGTCCTG

338 Right GCTTACCTTGTTATCAAGTCCTGAA

2327 Left GTCTGTAGGTTACACACAAATGCTG

2328 Right CACACTGAGCACTCAATAAAGAGAA 339 Left CACATAGCATTTGCACTGTATTAGG

340 Right GCTTACCTTGTTATCAAGTCCTGAA

2329 Left CACATAGCATTTGCACTGTATTAGG

2330 Right ATTCTTCAAAGGTAGCTGATTGATG 5eq.

Primer Sequence

ID 2341 Left AGGTAATCAGGAGATGCTGTAGATG 351 Left CTCTGAGAAAGAGTCTGCTAAGGAA

2342 Right ATTCTTCAAAGGTAGCTGATTGATG 352 Right TTGAATCCAAAATAAAGGAATGTGT

2343 Left GTCTGTAGGTTACACACAAATGCTG 353 Left TCGGTACTGAACATATACGGACTTT

2344 Right GCTTACCTTGTTATCAAGTCCTGAA 354 Right TTGAATCCAAAATAAAGGAATGTGT

2345 Left AGGTAATCAGGAGATGCTGTAGATG 355 Left TCGGTACTGAACATATACGGACTTT

2346 Right GCTTACCTTGTTATCAAGTCCTGAA 356 Right CACACTGAGCACTCAATAAAGAGAA

2347 Left GTATTTTGAAACTCAAGATCGCATT 357 Left CACATAGCATTTGCACTGTATTAGG

2348 Right TTATACACATAGCGGAGTGATCAAA 358 Right CAAAGGTAGCTGATTGATGAGAGTT

2349 Left TCTGAGAAAGAGTCTGCTAAGGAAG 359 Left TCTGAGAAAGAGTCTGCTAAGGAAG

2350 Right TTGAATCCAAAATAAAGGAATGTGT 360 Right ATCCAAAATAAAGGAATGTGTGTGT

EGFR Exonl8-21 5kb

Seq.

Primer Sequence

ID 379 Left TGCCAAAGAAGTAGAATGAGAAAAA

2361 Left ATGCCAAAGAAGTAGAATGAGAAAA 380 Right TGCTATGTATTCTGTGGGTTAGACA

2362 Right TGCTATGTATTCTGTGGGTTAGACA

381 Left ATGCCAAAGAAGTAGAATGAGAAAA

2363 Left ATGCCAAAGAAGTAGAATGAGAAAA 382 Right AGACATTTTTATAAAGGCCCATGTT

2364 Right CCTTCTTGGCTGTAAGATCAACTAA

383 Left ATGCCAAAGAAGTAGAATGAGAAAA

2384 Right ATTGTAAGTGAAAGGCTTCACAGAT

Seq. 385 Left TCTCCAAAATATATGCCAAAGAAGT

Primer Sequence

ID 386 Right GGTAATAAGTCAGCCATTTTTCCTT

2365 Left ATGCCAAAGAAGTAGAATGAGAAAA

2366 Right GTGCACTTAACTTTTAAGCCTTGAC 387 Left AATCTCCAAAATATATGCCAAAGAA

388 Right ATCTATCTTCTACCCCATTTCCAAC

2367 Left ATGCCAAAGAAGTAGAATGAGAAAA

2368 Right AGATTGTAAGTGAAAGGCTTCACAG 389 Left AAATCTCCAAAATATATGCCAAAGA

390 Right ATCTATCTTCTACCCCATTTCCAAC

2391 Left AAATCTCCAAAATATATGCCAAAGA

2369 Left TCTCCAAAATATATGCCAAAGAAGT 392 Right TGCTATGTATTCTGTGGGTTAGACA

2370 Right ATCTATCTTCTACCCCATTTCCAAC

393 Left TCTCCAAAATATATGCCAAAGAAGT

2371 Left ATGCCAAAGAAGTAGAATGAGAAAA 394 Right CCTTCTTGGCTGTAAGATCAACTAA

2372 Right CTGCCCAGAGAAAATTAAACTGTAG

395 Left TCTCCAAAATATATGCCAAAGAAGT

2373 Left TCTCCAAAATATATGCCAAAGAAGT 396 Right GTGCACTTAACTTTTAAGCCTTGAC

2374 Right TGCTATGTATTCTGTGGGTTAGACA

397 Left AATCTCCAAAATATATGCCAAAGAA

2375 Left TGCCAAAGAAGTAGAATGAGAAAAA 398 Right TGAGGTAATAAGTCAGCCATTTTTC

2376 Right ATCTATCTTCTACCCCATTTCCAAC

399 Left AAATCTCCAAAATATATGCCAAAGA

2377 Left TCTCCAAAATATATGCCAAAGAAGT 400 Right TGAGGTAATAAGTCAGCCATTTTTC

2378 Right TGAGGTAATAAGTCAGCCATTTTTC

EGFR Exon22 5kb

Seq. Primer Sequence ID 2401 Left GTTGGAAATGGGGTAGAAGATAGAT 421 Left CACATAGCATTTGCACTGTATTAGG

2402 Right TTGAATCCAAAATAAAGGAATGTGT 422 Right GGGTCAAATAAACCTCCACTTATCT

2403 Left GTTGGAAATGGGGTAGAAGATAGAT 423 Left GTTGGAAATGGGGTAGAAGATAGAT

2404 Right CACACTGAGCACTCAATAAAGAGAA 424 Right ATTCTTCAAAGGTAGCTGATTGATG

2405 Left ATTTTGATATTTAAGGGAGGTCCTG 425 Left ATTTTGATATTTAAGGGAGGTCCTG

2406 Right CTCTCCCATCAACATTTAGAAGAAA 426 Right TATAAGCCAATAAATCCCATTTTGA

2407 Left CACATAGCATTTGCACTGTATTAGG 427 Left CACATAGCATTTGCACTGTATTAGG

2408 Right CTCTCCCATCAACATTTAGAAGAAA 428 Right TATAAGCCAATAAATCCCATTTTGA

2409 Left ATTTTGATATTTAAGGGAGGTCCTG 429 Left GTCTGTAGGTTACACACAAATGCTG

2410 Right TACAACAAACACAAGAATGGCTTTA 430 Right TACAACAAACACAAGAATGGCTTTA

2411 Left CACATAGCATTTGCACTGTATTAGG 431 Left TACAGATTATGATGACTGCCTCAAA

2412 Right TACAACAAACACAAGAATGGCTTTA 432 Right TACAACAAACACAAGAATGGCTTTA

2413 Left GTTGGAAATGGGGTAGAAGATAGAT 433 Left AGGAAAATAACACACACTCTCCTTG

2414 Right TTCTCCACTACAAATCACCACAGTA 434 Right TTACTGGGAGATGATTAAGAACAGC

2415 Left ATATCTGAATAAAAGGTCACCACCA 435 Left GTCTGTAGGTTACACACAAATGCTG

2416 Right CTCTCCCATCAACATTTAGAAGAAA 436 Right GGGTCAAATAAACCTCCACTTATCT

2417 Left CCATATCTGAATAAAAGGTCACCAC 437 Left ATATCTGAATAAAAGGTCACCACCA

2418 Right CTCTCCCATCAACATTTAGAAGAAA 438 Right TATAAGCCAATAAATCCCATTTTGA

Seq.

Primer Sequence

ID 439 Left CCATATCTGAATAAAAGGTCACCAC

2419 Left TATCTGAATAAAAGGTCACCACCAT 440 Right TATAAGCCAATAAATCCCATTTTGA

2420 Right CTCTCCCATCAACATTTAGAAGAAA

Table 9. KIT Capture Primer List for NGS Panel

KIT Exon8 150-175 bases

Seq. 452 Right ATAAGCAGTGCCAAAAATAATCATC

Primer Sequence

ID

2441 Left ATATGGCCATTTCTGTTTTCCTGTA 453 Left TGACATATGGCCATTTCTGTTTT

2442 Right ATAAGCAGTGCCAAAAATAATCATC 454 Right ATAAGCAGTGCCAAAAATAATCATC

2443 Left CTGACATATGGCCATTTCTGTTT 455 Left ATATGGCCATTTCTGTTTTCCTGTA

2444 Right ATAAGCAGTGCCAAAAATAATCATC 456 Right TTATAAGCAGTGCCAAAAATAATCA

2445 Left GACATATGGCCATTTCTGTTTTC 457 Left TATGGCCATTTCTGTTTTCCTGTA

2446 Right ATAAGCAGTGCCAAAAATAATCATC 458 Right ATAAGCAGTGCCAAAAATAATCATC

2447 Left CTGACATATGGCCATTTCTGTTTT 459 Left GACATATGGCCATTTCTGTTTTC

2448 Right ATAAGCAGTGCCAAAAATAATCATC 460 Right TTATAAGCAGTGCCAAAAATAATCA

2449 Left ATATGGCCATTTCTGTTTTCCTGTA 5eq.

Primer Sequence

2450 Right GCATTATAAGCAGTGCCAAAAATAA ID

461 Left CTGACATATGGCCATTTCTGTTTTC

2451 Left TATGGCCATTTCTGTTTTCCTGTAG 462 Right ATAAGCAGTGCCAAAAATAATCATC 2463 Left ATATGGCCATTTCTGTTTTCCTGTA 473 Left TATGGCCATTTCTGTTTTCCTGTAG

2464 Right TATAAGCAGTGCCAAAAATAATCATC 474 Right TTATAAGCAGTGCCAAAAATAATCA

2465 Left GGCCATTTCTGTTTTCCTGTAG 475 Left TGACATATGGCCATTTCTGTTTTC

2466 Right ATAAGCAGTGCCAAAAATAATCATC 476 Right ATAAGCAGTGCCAAAAATAATCATC

2467 Left TATGGCCATTTCTGTTTTCCTGTAG 477 Left ATATGGCCATTTCTGTTTTCCTGTA

2468 Right GCATTATAAGCAGTGCCAAAAATAA 478 Right TAAGCAGTGCCAAAAATAATCATC

2469 Left ATATGGCCATTTCTGTTTTCCTGT 479 Left GACATATGGCCATTTCTGTTTTC

2470 Right ATAAGCAGTGCCAAAAATAATCATC 480 Right TATAAGCAGTGCCAAAAATAATCATC

2471 Left ACATATGGCCATTTCTGTTTTCCT

2472 Right ATAAGCAGTGCCAAAAATAATCATC

KIT Exon8 176-200 bases

Seq.

Primer Sequence

ID 501 Left GACATATGGCCATTTCTGTTTTC

2481 Left AGGTTTTCCAGCACTCTGACATA 502 Right GCATTATAAGCAGTGCCAAAAATAA

2482 Right ATAAGCAGTGCCAAAAATAATCATC

503 Left AGGTTTTCCAGCACTCTGACATA

2483 Left ACTCTGACATATGGCCATTTCTGTT 504 Right TTATAAGCAGTGCCAAAAATAATCA

2484 Right ATAAGCAGTGCCAAAAATAATCATC

505 Left ACTCTGACATATGGCCATTTCTGTT

2485 Left CTGACATATGGCCATTTCTGTTT 506 Right TTATAAGCAGTGCCAAAAATAATCA

2486 Right ATAAGCAGTGCCAAAAATAATCATC

507 Left GAGGTTTTCCAGCACTCTGACATA

2487 Left CTGACATATGGCCATTTCTGTTTT 508 Right ATAAGCAGTGCCAAAAATAATCATC

2488 Right ATAAGCAGTGCCAAAAATAATCATC

Seq. 509 Left CTGACATATGGCCATTTCTGTTT

Primer Sequence

ID 510 Right TTATAAGCAGTGCCAAAAATAATCA

2489 Left ATATGGCCATTTCTGTTTTCCTGTA

2490 Right GCATTATAAGCAGTGCCAAAAATAA 51 1 Left TCTGACATATGGCCATTTCTGTTT

512 Right ATAAGCAGTGCCAAAAATAATCATC

2491 Left CTCTGACATATGGCCATTTCTGTT

2492 Right ATAAGCAGTGCCAAAAATAATCATC 513 Left GACATATGGCCATTTCTGTTTTC

514 Right TTATAAGCAGTGCCAAAAATAATCA

2493 Left CTCTGACATATGGCCATTTCTGTTT

2494 Right ATAAGCAGTGCCAAAAATAATCATC 515 Left CTGACATATGGCCATTTCTGTTTTC

516 Right ATAAGCAGTGCCAAAAATAATCATC

2495 Left AGGTTTTCCAGCACTCTGACATA

2496 Right GCATTATAAGCAGTGCCAAAAATAA 517 Left TCTGACATATGGCCATTTCTGTTTT

518 Right ATAAGCAGTGCCAAAAATAATCATC

2497 Left ACTCTGACATATGGCCATTTCTGTT

2498 Right GCATTATAAGCAGTGCCAAAAATAA 519 Left CTGACATATGGCCATTTCTGTTTT

520 Right TTATAAGCAGTGCCAAAAATAATCA

2499 Left TCTGACATATGGCCATTTCTGTT

2500 Right ATAAGCAGTGCCAAAAATAATCATC

KIT Exon8 201-300 bases

Seq. 7 522 Right ATAAGCAGTGCCAAAAATAATCATC

Primer Sequence

ID

2521 Left TTAGAGAGGGAGTGAAGTGAATGTT 7 523 Left GATTAGAGAGGGAGTGAAGTGAATG

2589 Left TAGAGAGGGAGTGAAGTGAATGTTG 2595 Left TAGAGAGGGAGTGAAGTGAATGTTG

2590 Right TCATTCAGTAATGATTTTTCAGCAA 2 596 Right TTCAGTAATGATTTTTCAGCAAACA

2591 Left ATTAGAGAGGGAGTGAAGTGAATGT' Γ2 597 Left ATTAGAGAGGGAGTGAAGTGAATGTT

2592 Right TCATTCAGTAATGATTTTTCAGCAA 2 598 Right TCAGTAATGATTTTTCAGCAAACAA

2593 Left TAGAGAGGGAGTGAAGTGAATGTTG 2 599 Left CAGGAAGGTTGTAGGGATTAGAGAG

2594 Right TCAGTAATGATTTTTCAGCAAACAA 2 600 Right AATTATCCCTTCTAAAAAGCCACAT

KIT Exon8 401-500 bases

Seq. 620 Right AAATTGCATGATAAATCCAGAAAGA

Primer Sequence

ID

2601 Left GATTAGAGAGGGAGTGAAGTGAATG 621 Left GGATTAGAGAGGGAGTGAAGTGAAT

2602 Right TCATTCAGTAATGATTTTTCAGCAA 622 Right GAAATTGCATGATAAATCCAGAAAG

2603 Left GGATTAGAGAGGGAGTGAAGTGAAT 623 Left GTAGGGATTAGAGAGGGAGTGAAGT

2604 Right TCATTCAGTAATGATTTTTCAGCAA 624 Right AAATTGCATGATAAATCCAGAAAGA

2605 Left GTAGGGATTAGAGAGGGAGTGAAGT 625 Left GTAGGGATTAGAGAGGGAGTGAAGT

2606 Right TCATTCAGTAATGATTTTTCAGCAA 626 Right GAAATTGCATGATAAATCCAGAAAG

2607 Left GTAGGGATTAGAGAGGGAGTGAAGT 627 Left TTAGAAGCAGTCTTCAGATCCCTAC

2608 Right TCAGTAATGATTTTTCAGCAAACAA 628 Right ATAAGCAGTGCCAAAAATAATCATC

2609 Left GTAGGGATTAGAGAGGGAGTGAAGT 629 Left GATTAGAGAGGGAGTGAAGTGAATG

2610 Right TTCAGTAATGATTTTTCAGCAAACA 630 Right GTCATTCAGTAATGATTTTTCAGCA

2611 Left TTAGAGAGGGAGTGAAGTGAATGTT 631 Left GGATTAGAGAGGGAGTGAAGTGAAT

2612 Right AAATTGCATGATAAATCCAGAAAGA 632 Right GTCATTCAGTAATGATTTTTCAGCA

Seq. 633 Left AGATTTTTACCTGTGGAACACTTTG

Primer Sequence

ID 634 Right GCATTATAAGCAGTGCCAAAAATAA

2613 Left TTAGAGAGGGAGTGAAGTGAATGTT

2614 Right GAAATTGCATGATAAATCCAGAAAG 635 Left GTAGGGATTAGAGAGGGAGTGAAGT

636 Right GTCATTCAGTAATGATTTTTCAGCA

2615 Left GATTAGAGAGGGAGTGAAGTGAATG

2616 Right AAATTGCATGATAAATCCAGAAAGA 637 Left CAGGAAGGTTGTAGGGATTAGAGAG

638 Right TCATTCAGTAATGATTTTTCAGCAA

2617 Left GATTAGAGAGGGAGTGAAGTGAATG

2618 Right GAAATTGCATGATAAATCCAGAAAG 639 Left CTCAGGAAGGTTGTAGGGATTAGAG

640 Right TCATTCAGTAATGATTTTTCAGCAA

2619 Left GGATTAGAGAGGGAGTGAAGTGAAT

KIT Exon8 501-600 bases

Seq. 646 Right CGATCATTACTTTTTGGTAACTTGG

Primer Sequence

ID

2641 Left TTAGAGAGGGAGTGAAGTGAATGTT 647 Left TTAGAGAGGGAGTGAAGTGAATGTT

2642 Right CGATCATTACTTTTTGGTAACTTGG 648 Right CATTACTTTTTGGTAACTTGGCAAT

2643 Left GATTAGAGAGGGAGTGAAGTGAATG 649 Left TTAGAGAGGGAGTGAAGTGAATGTT

2644 Right CGATCATTACTTTTTGGTAACTTGG 650 Right GCGATCATTACTTTTTGGTAACTTG

2645 Left GGATTAGAGAGGGAGTGAAGTGAAT 651 Left TTAGAGAGGGAGTGAAGTGAATGTT 2652 Right TGCGATCATTACTTTTTGGTAACTT 666 Right ATAAGCAGTGCCAAAAATAATCATC

2653 Left GATTAGAGAGGGAGTGAAGTGAATG 667 Left TTAGAAGCAGTCTTCAGATCCCTAC

2654 Right CATTACTTTTTGGTAACTTGGCAAT 668 Right AATTATCCCTTCTAAAAAGCCACAT

2655 Left GATTAGAGAGGGAGTGAAGTGAATG 669 Left TTAGAGAGGGAGTGAAGTGAATGTT

2656 Right GCGATCATTACTTTTTGGTAACTTG 670 Right GATAACTACAGTCACATTTCCCACA

2657 Left GGATTAGAGAGGGAGTGAAGTGAAT 671 Left AACAACTCCTAATTTCATCCATTCC

2658 Right CATTACTTTTTGGTAACTTGGCAAT 672 Right ATAAGCAGTGCCAAAAATAATCATC

2659 Left ACTCCTAATTTCATCCATTCCAGTT 673 Left GATTAGAGAGGGAGTGAAGTGAATG

2660 Right ATAAGCAGTGCCAAAAATAATCATC 674 Right GATAACTACAGTCACATTTCCCACA

Seq. 675 Left TTAGAGAGGGAGTGAAGTGAATGTT

Primer Sequence

ID 676 Right TAACTACAGTCACATTTCCCACACA

2661 Left AACTCCTAATTTCATCCATTCCAGT

2662 Right ATAAGCAGTGCCAAAAATAATCATC 677 Left GGATTAGAGAGGGAGTGAAGTGAAT

678 Right GATAACTACAGTCACATTTCCCACA

2663 Left GTAGGGATTAGAGAGGGAGTGAAGT

2664 Right CATTACTTTTTGGTAACTTGGCAAT 679 Left CACTTTGGAGTCCTAGAGTTTGATT

680 Right AATTATCCCTTCTAAAAAGCCACAT

2665 Left GGCAGGAATCCTTTAAAGTAGATTT

KIT Exon8 601-800 bases

Seq.

Primer Sequence

ID 699 Left GGGCTTATCTTTTCCTCTAACAACT

2681 Left AGATTTTTACCTGTGGAACACTTTG 700 Right TCATTCAGTAATGATTTTTCAGCAA

2682 Right TCATTCAGTAATGATTTTTCAGCAA

701 Left GGGCTTATCTTTTCCTCTAACAACT

2683 Left AGATTTTTACCTGTGGAACACTTTG 702 Right TCAGTAATGATTTTTCAGCAAACAA

2684 Right TTCAGTAATGATTTTTCAGCAAACA

Seq. 703 Left GGGCTTATCTTTTCCTCTAACAACT

Primer Sequence

ID 704 Right TTCAGTAATGATTTTTCAGCAAACA

2685 Left AGATTTTTACCTGTGGAACACTTTG

2686 Right TCAGTAATGATTTTTCAGCAAACAA 705 Left AGATTTTTACCTGTGGAACACTTTG

706 Right AATTATCCCTTCTAAAAAGCCACAT

2687 Left GGAGAAAATTCATGTAAGAGCAAAA

2688 Right ATAAGCAGTGCCAAAAATAATCATC 707 Left AGGCTGGTTTTCTTTTCTAGTTTTC

708 Right ATAAGCAGTGCCAAAAATAATCATC

2689 Left AATTCATGTAAGAGCAAAAGAGTGG

2690 Right ATAAGCAGTGCCAAAAATAATCATC 709 Left GGCTGGTTTTCTTTTCTAGTTTTCT

710 Right ATAAGCAGTGCCAAAAATAATCATC

2691 Left GGAGAAAATTCATGTAAGAGCAAAA

2692 Right AATTATCCCTTCTAAAAAGCCACAT 711 Left ACTCCTAATTTCATCCATTCCAGTT

712 Right TCATTCAGTAATGATTTTTCAGCAA

2693 Left AGATTTTTACCTGTGGAACACTTTG

2694 Right AAATTGCATGATAAATCCAGAAAGA 713 Left AACTCCTAATTTCATCCATTCCAGT

714 Right TCATTCAGTAATGATTTTTCAGCAA

2695 Left AGATTTTTACCTGTGGAACACTTTG

2696 Right GAAATTGCATGATAAATCCAGAAAG 715 Left AGGAGAAAATTCATGTAAGAGCAAA

716 Right ATAAGCAGTGCCAAAAATAATCATC

2697 Left AATTCATGTAAGAGCAAAAGAGTGG

2698 Right AATTATCCCTTCTAAAAAGCCACAT 717 Left AAGGAGAAAATTCATGTAAGAGCAA 2718 Right ATAAGCAGTGCCAAAAATAATCATC 719 Left AAAGGAGAAAATTCATGTAAGAGCA

720 Right ATAAGCAGTGCCAAAAATAATCATC

KIT Exon8 801-1000 bases

Seq.

Primer Sequence

ID 741 Left AGGCTGGTTTTCTTTTCTAGTTTTC

2721 Left GGAGAAAATTCATGTAAGAGCAAAA 742 Right TCATTCAGTAATGATTTTTCAGCAA

2722 Right TCATTCAGTAATGATTTTTCAGCAA

743 Left GGCTGGTTTTCTTTTCTAGTTTTCT

2723 Left GGAGAAAATTCATGTAAGAGCAAAA 744 Right TCATTCAGTAATGATTTTTCAGCAA

2724 Right TCAGTAATGATTTTTCAGCAAACAA

745 Left AATTCATGTAAGAGCAAAAGAGTGG

2725 Left GGAGAAAATTCATGTAAGAGCAAAA 746 Right AAATTGCATGATAAATCCAGAAAGA

2726 Right TTCAGTAATGATTTTTCAGCAAACA

747 Left AATTCATGTAAGAGCAAAAGAGTGG

2727 Left AATTCATGTAAGAGCAAAAGAGTGG 748 Right GAAATTGCATGATAAATCCAGAAAG

2728 Right TCATTCAGTAATGATTTTTCAGCAA

749 Left AGATTTTTACCTGTGGAACACTTTG

2729 Left AATTCATGTAAGAGCAAAAGAGTGG 750 Right TGTTGTAATTGTGCGATCATTACTT

2730 Right TCAGTAATGATTTTTCAGCAAACAA

751 Left AGGCTGGTTTTCTTTTCTAGTTTTC

2731 Left AATTCATGTAAGAGCAAAAGAGTGG 752 Right TTCAGTAATGATTTTTCAGCAAACA

2732 Right TTCAGTAATGATTTTTCAGCAAACA

753 Left AGATTTTTACCTGTGGAACACTTTG

2733 Left TTAGAGCATTTCTGCTGTTACAGTG 754 Right CGATCATTACTTTTTGGTAACTTGG

2734 Right ATAAGCAGTGCCAAAAATAATCATC

755 Left TTAGAGAGGGAGTGAAGTGAATGTT

2735 Left GGAGAAAATTCATGTAAGAGCAAAA 756 Right GAACCCTACTTAGTATTCCCCAAAA

2736 Right AAATTGCATGATAAATCCAGAAAGA

757 Left AGGAGAAAATTCATGTAAGAGCAAA

2737 Left GGAGAAAATTCATGTAAGAGCAAAA 758 Right TCATTCAGTAATGATTTTTCAGCAA

2738 Right GAAATTGCATGATAAATCCAGAAAG

Seq. 759 Left AAGGAGAAAATTCATGTAAGAGCAA

Primer Sequence

ID 760 Right TCATTCAGTAATGATTTTTCAGCAA

2739 Left ATACCAAATTAGAGCATTTCTGCTG

2740 Right ATAAGCAGTGCCAAAAATAATCATC

KIT Exon8 2kb

Seq.

Primer Sequence

ID 771 Left AATTCATGTAAGAGCAAAAGAGTGG

2761 Left GGAGAAAATTCATGTAAGAGCAAAA 772 Right GGACTGAAGTTTGAGTTCTAAGCAG

2762 Right GGACTGAAGTTTGAGTTCTAAGCAG

773 Left AATTCATGTAAGAGCAAAAGAGTGG

2763 Left GGAGAAAATTCATGTAAGAGCAAAA 774 Right AGGACTGAAGTTTGAGTTCTAAGCA

2764 Right AGGACTGAAGTTTGAGTTCTAAGCA

775 Left GGAGAAAATTCATGTAAGAGCAAAA

2765 Left TTAGAGCATTTCTGCTGTTACAGTG 776 Right GACCAGAAAATAGTCAAAGTGAGGA

2766 Right CCTGTTTCCTTTCTTAACACCTACA

777 Left TTCTGATCTGTCAGTCTTTCCTTCT

2767 Left GGAGAAAATTCATGTAAGAGCAAAA 778 Right TCATTCAGTAATGATTTTTCAGCAA

2768 Right CCTGTTTCCTTTCTTAACACCTACA

779 Left ATACCAAATTAGAGCATTTCTGCTG

2769 Left AATTCATGTAAGAGCAAAAGAGTGG 780 Right CCTGTTTCCTTTCTTAACACCTACA

2770 Right ACTGAAGTTTGAGTTCTAAGCAGGA Seq.

Primer Sequence

ID 791 Left AATTCATGTAAGAGCAAAAGAGTGG

2781 Left TTAGAGCATTTCTGCTGTTACAGTG 792 Right GACCAGAAAATAGTCAAAGTGAGGA

2782 Right GCTATTCCCTGTTTCCTTTCTTAAC

793 Left TTCCTTCTCACTGCATATATTTTCC

2783 Left ATAAGTGCATCTTCCTTTCACTTTG 794 Right TCATTCAGTAATGATTTTTCAGCAA

2784 Right GAACCCTACTTAGTATTCCCCAAAA

795 Left ATACCAAATTAGAGCATTTCTGCTG

2785 Left GGAGAAAATTCATGTAAGAGCAAAA 796 Right GCTATTCCCTGTTTCCTTTCTTAAC

2786 Right GCTATTCCCTGTTTCCTTTCTTAAC

797 Left TGAAATTGGTTATCCAAGAAAGGTA

2787 Left TTCTGATCTGTCAGTCTTTCCTTCT 798 Right TCATTCAGTAATGATTTTTCAGCAA

2788 Right TTCAGTAATGATTTTTCAGCAAACA

799 Left AGATTTTTACCTGTGGAACACTTTG

2789 Left TTCTGATCTGTCAGTCTTTCCTTCT 800 Right ACTGAAGTTTGAGTTCTAAGCAGGA

2790 Right TCAGTAATGATTTTTCAGCAAACAA

JOT Exon8-9 5kb

Seq.

Primer Sequence

ID 821 Left AGTTGCCCATGATAATTAAATGAAA

2801 Left AGTTGCCCATGATAATTAAATGAAA 822 Right GCTTCCTTTATGGACGGTTTATATT

2802 Right AGGCAGTGTTAACTTTTGGATACAG

823 Left CCCATGATAATTAAATGAAACTTGC

2803 Left CCCATGATAATTAAATGAAACTTGC 824 Right GCTTCCTTTATGGACGGTTTATATT

2804 Right AGGCAGTGTTAACTTTTGGATACAG

825 Left TGCCCATGATAATTAAATGAAACTT

2805 Left GCCCATGATAATTAAATGAAACTTG 826 Right GCTTCCTTTATGGACGGTTTATATT

2806 Right AGGCAGTGTTAACTTTTGGATACAG

827 Left TTGCCCATGATAATTAAATGAAACT

2807 Left TGCCCATGATAATTAAATGAAACTT 828 Right CCCCTTAAATTGGATTAAAAAGAAA

2808 Right AGGCAGTGTTAACTTTTGGATACAG

829 Left AGTTGCCCATGATAATTAAATGAAA

2809 Left TTGCCCATGATAATTAAATGAAACT 830 Right CCCCTTAAATTGGATTAAAAAGAAA

2810 Right AGGCAGTGTTAACTTTTGGATACAG

Seq. 831 Left GCCCATGATAATTAAATGAAACTTG

Primer Sequence

ID 832 Right CCCCTTAAATTGGATTAAAAAGAAA

2811 Left AGTTTAGGCTTGCTTAGAAAAGGAG

2812 Right AGGCAGTGTTAACTTTTGGATACAG 833 Left CCCATGATAATTAAATGAAACTTGC

834 Right CCCCTTAAATTGGATTAAAAAGAAA

2813 Left AGAGTTTAGGCTTGCTTAGAAAAGG

2814 Right AGGCAGTGTTAACTTTTGGATACAG 835 Left TGCCCATGATAATTAAATGAAACTT

836 Right CCCCTTAAATTGGATTAAAAAGAAA

2815 Left GATTTCTTGTGTCGTGTCCTACTTT

2816 Right AGGCAGTGTTAACTTTTGGATACAG 837 Left AGTTTAGGCTTGCTTAGAAAAGGAG

838 Right CCCCTTAAATTGGATTAAAAAGAAA

2817 Left GCCCATGATAATTAAATGAAACTTG

2818 Right GCTTCCTTTATGGACGGTTTATATT 839 Left AGAGTTTAGGCTTGCTTAGAAAAGG

840 Right CCCCTTAAATTGGATTAAAAAGAAA

2819 Left TTGCCCATGATAATTAAATGAAACT

2820 Right GCTTCCTTTATGGACGGTTTATATT

KIT Exon9 200-250 bases

Seq. 841 Left GGCTTTTGTTTTCTTCCCTTTAG

Primer Sequence

ID 842 Right CATCCCCTTAAATTGGATTAAAAAG

2903 Left CCACATCCCAAGTGTTTTATGTAT 913 Left CTAGAGTAAGCCAGGGCTTTTGTTT

2904 Right ATCCCCTTAAATTGGATTAAAAAGA 914 Right AACATCCCCTTAAATTGGATTAAAA

2905 Left CCACATCCCAAGTGTTTTATGTATT 915 Left CTAGAGTAAGCCAGGGCTTTTGTT

2906 Right CCCCTTAAATTGGATTAAAAAGAA 916 Right AACATCCCCTTAAATTGGATTAAAA

2907 Left GGCTTTTGTTTTCTTCCCTTTAG 917 Left CCACATCCCAAGTGTTTTATGTATT

2908 Right TATGGTAGACAGAGCCTAAACATCC 918 Right TCCCCTTAAATTGGATTAAAAAGAAA

2909 Left CTAGAGTAAGCCAGGGCTTTTGTTT 919 Left GGCTTTTGTTTTCTTCCCTTTAG

2910 Right TATGGTAGACAGAGCCTAAACATCC 920 Right TCATGACTGATATGGTAGACAGAGC

2911 Left CTAGAGTAAGCCAGGGCTTTTGTT

2912 Right TATGGTAGACAGAGCCTAAACATCC

KIT Exon9 301-400 bases

Seq.

Primer Sequence

ID 941 Left TTTGTTTTAAAAGTATGCCACATCC

2921 Left CTCACTAGGTCACCAAAGTGCTTAT 942 Right TATGGTAGACAGAGCCTAAACATCC

2922 Right CCCCTTAAATTGGATTAAAAAGAAA

943 Left CTCACTAGGTCACCAAAGTGCTTAT

2923 Left CTCACTAGGTCACCAAAGTGCTTAT 944 Right ATCCCCTTAAATTGGATTAAAAAGA

2924 Right AACATCCCCTTAAATTGGATTAAAA

945 Left CCACATCCCAAGTGTTTTATGTATT

2925 Left CCACATCCCAAGTGTTTTATGTATT 946 Right TCATGACTGATATGGTAGACAGAGC

2926 Right TATGGTAGACAGAGCCTAAACATCC

947 Left CACTAGGTCACCAAAGTGCTTATTC

2927 Left CTCACTAGGTCACCAAAGTGCTTAT 948 Right AACATCCCCTTAAATTGGATTAAAA

2928 Right CATCCCCTTAAATTGGATTAAAAAG

949 Left TCACTAGGTCACCAAAGTGCTTATT

2929 Left CCACATCCCAAGTGTTTTATGTATT 950 Right AACATCCCCTTAAATTGGATTAAAA

2930 Right GTGATGCATGTATTACCAGAAATGA

951 Left TTTGTTTTAAAAGTATGCCACATCC

2931 Left CCACATCCCAAGTGTTTTATGTATT 952 Right GTGATGCATGTATTACCAGAAATGA

2932 Right AACATCCCCTTAAATTGGATTAAAA

Seq. 953 Left TCACCAAAGTGCTTATTCTTAGACA

Primer Sequence

ID 954 Right CCCCTTAAATTGGATTAAAAAGAAA

2933 Left CACTAGGTCACCAAAGTGCTTATTC

2934 Right CCCCTTAAATTGGATTAAAAAGAAA 955 Left ACTCACTAGGTCACCAAAGTGCTTA

956 Right CCCCTTAAATTGGATTAAAAAGAAA

2935 Left TCACTAGGTCACCAAAGTGCTTATT

2936 Right CCCCTTAAATTGGATTAAAAAGAAA 957 Left TTTGTTTTAAAAGTATGCCACATCC

958 Right AACATCCCCTTAAATTGGATTAAAA

2937 Left CTCACTAGGTCACCAAAGTGCTTAT

2938 Right TCCCCTTAAATTGGATTAAAAAGAA 959 Left AGGTCACCAAAGTGCTTATTCTTAG

960 Right CCCCTTAAATTGGATTAAAAAGAAA

2939 Left TTTGTTTTAAAAGTATGCCACATCC

2940 Right CCCCTTAAATTGGATTAAAAAGAAA

KIT Exon9 401-500 bases

Seq. 962 Right TATGGTAGACAGAGCCTAAACATCC

Primer Sequence

ID

2961 Left CTCACTAGGTCACCAAAGTGCTTAT 963 Left TACAGTCGTAGAAACTCAGTGTTGG 2964 Right CCCCTTAAATTGGATTAAAAAGAAA 982 Right GTGATGCATGTATTACCAGAAATGA

2965 Left CTCACTAGGTCACCAAAGTGCTTAT 983 Left TCACTAGGTCACCAAAGTGCTTATT

2966 Right GTGATGCATGTATTACCAGAAATGA 984 Right GTGATGCATGTATTACCAGAAATGA

2967 Left CTCACTAGGTCACCAAAGTGCTTAT 985 Left TACAGTCGTAGAAACTCAGTGTTGG

2968 Right TCATGACTGATATGGTAGACAGAGC 986 Right ATCCCCTTAAATTGGATTAAAAAGA

2969 Left TACAGTCGTAGAAACTCAGTGTTGG 987 Left TCACCAAAGTGCTTATTCTTAGACA

2970 Right CATCCCCTTAAATTGGATTAAAAAG 988 Right TATGGTAGACAGAGCCTAAACATCC

2971 Left CACTAGGTCACCAAAGTGCTTATTC 989 Left ACTCACTAGGTCACCAAAGTGCTTA

2972 Right TATGGTAGACAGAGCCTAAACATCC 990 Right TATGGTAGACAGAGCCTAAACATCC

2973 Left TCACTAGGTCACCAAAGTGCTTATT 991 Left ACAGTCGTAGAAACTCAGTGTTGGT

2974 Right TATGGTAGACAGAGCCTAAACATCC 992 Right AACATCCCCTTAAATTGGATTAAAA

2975 Left ACAGTCGTAGAAACTCAGTGTTGGT 993 Left CTAGGTCACCAAAGTGCTTATTCTT

2976 Right CCCCTTAAATTGGATTAAAAAGAAA 994 Right TATGGTAGACAGAGCCTAAACATCC

2977 Left TACAGTCGTAGAAACTCAGTGTTGG 995 Left AGGTCACCAAAGTGCTTATTCTTAG

2978 Right TCCCCTTAAATTGGATTAAAAAGAA 996 Right TATGGTAGACAGAGCCTAAACATCC

2979 Left CTCACTAGGTCACCAAAGTGCTTAT 997 Left TCACCAAAGTGCTTATTCTTAGACA

2980 Right GGTCAATGTTGGAATGAACTTAAAA 998 Right GTGATGCATGTATTACCAGAAATGA

Seq. 999 Left CACTAGGTCACCAAAGTGCTTATTC

Primer Sequence

ID 000 Right TCATGACTGATATGGTAGACAGAGC

2981 Left CACTAGGTCACCAAAGTGCTTATTC

KIT Exon9 501-600 bases

Seq.

Primer Sequence

ID 015 Left TCCTCCTCTATGCTATTTCTTTTCA

3001 Left CCTCTATGCTATTTCTTTTCAACCA 016 Right TATGGTAGACAGAGCCTAAACATCC

3002 Right TATGGTAGACAGAGCCTAAACATCC

017 Left ATTTTATTGAATTCCTTTCCAATCC

3003 Left CTCACTAGGTCACCAAAGTGCTTAT 018 Right GTGATGCATGTATTACCAGAAATGA

3004 Right AGGCAGTGTTAACTTTTGGATACAG

Seq. 019 Left CTCACTAGGTCACCAAAGTGCTTAT

Primer Sequence

ID 020 Right GTAAATATATTCCCCCATTTGCTTT

3005 Left TTTTATGCTTTCCTCCTCTATGCTA

3006 Right CCCCTTAAATTGGATTAAAAAGAAA 021 Left GCTTTCCTCCTCTATGCTATTTCTT

022 Right AACATCCCCTTAAATTGGATTAAAA

3007 Left CTCACTAGGTCACCAAAGTGCTTAT

3008 Right GGCAGTGTTAACTTTTGGATACAGT 023 Left TTTTATGCTTTCCTCCTCTATGCTA

024 Right AACATCCCCTTAAATTGGATTAAAA

3009 Left GCTTTCCTCCTCTATGCTATTTCTT

3010 Right TATGGTAGACAGAGCCTAAACATCC 025 Left CCACATCCCAAGTGTTTTATGTATT

026 Right GGCAGTGTTAACTTTTGGATACAGT

3011 Left TTTTATGCTTTCCTCCTCTATGCTA

3012 Right TATGGTAGACAGAGCCTAAACATCC 027 Left TTTAGTAGAGACGAGGTTTCACCAT

028 Right CCCCTTAAATTGGATTAAAAAGAAA

3013 Left CCACATCCCAAGTGTTTTATGTATT

3014 Right AGGCAGTGTTAACTTTTGGATACAG 029 Left GCTGAGATTACAGGTGTGAGCTACT 3030 Right CCCCTTAAATTGGATTAAAAAGAAA 035 Left GCTGAGATTACAGGTGTGAGCTACT

036 Right TATGGTAGACAGAGCCTAAACATCC

3031 Left CTCACTAGGTCACCAAAGTGCTTAT

3032 Right GATTGTTCTAATTCTGTTTGGGTGT 037 Left CACTAGGTCACCAAAGTGCTTATTC

038 Right AGGCAGTGTTAACTTTTGGATACAG

3033 Left TACAGTCGTAGAAACTCAGTGTTGG

3034 Right GTAAATATATTCCCCCATTTGCTTT 039 Left TCACTAGGTCACCAAAGTGCTTATT

040 Right AGGCAGTGTTAACTTTTGGATACAG

KIT Exon9 801-1000 bases

Seq.

Primer Sequence

ID 061 Left CCCTGTTTTACAGTCGTAGAAACTC

3041 Left ATTCCTTTCCAATCCTTTCAGTAAC 062 Right AGGCAGTGTTAACTTTTGGATACAG

3042 Right AGGCAGTGTTAACTTTTGGATACAG

063 Left ATTCCTTTCCAATCCTTTCAGTAAC

3043 Left ATTTTATTGAATTCCTTTCCAATCC 064 Right GTAAATATATTCCCCCATTTGCTTT

3044 Right AGGCAGTGTTAACTTTTGGATACAG

065 Left TACATCCTTGATTTTGTTGTTGTTG

3045 Left TACAGTCGTAGAAACTCAGTGTTGG 066 Right AACATCCCCTTAAATTGGATTAAAA

3046 Right AGGCAGTGTTAACTTTTGGATACAG

067 Left AACCCTCTGCAATGGGTATTACTAT

3047 Left TACATCCTTGATTTTGTTGTTGTTG 068 Right AGGCAGTGTTAACTTTTGGATACAG

3048 Right CCCCTTAAATTGGATTAAAAAGAAA

069 Left TTATTGAATTCCTTTCCAATCCTTT

3049 Left ATTCCTTTCCAATCCTTTCAGTAAC 070 Right AGGCAGTGTTAACTTTTGGATACAG

3050 Right GGCAGTGTTAACTTTTGGATACAGT

071 Left TTTTATTGAATTCCTTTCCAATCCT

3051 Left CCTCTATGCTATTTCTTTTCAACCA 072 Right AGGCAGTGTTAACTTTTGGATACAG

3052 Right GTAAATATATTCCCCCATTTGCTTT

073 Left TTTATTGAATTCCTTTCCAATCCTT

3053 Left ATTTTATTGAATTCCTTTCCAATCC 074 Right AGGCAGTGTTAACTTTTGGATACAG

3054 Right GGCAGTGTTAACTTTTGGATACAGT

075 Left ATTTTATTGAATTCCTTTCCAATCC

3055 Left TTCTGGTCTACATCCTTGATTTTGT 076 Right GTAAATATATTCCCCCATTTGCTTT

3056 Right CCCCTTAAATTGGATTAAAAAGAAA

077 Left CCTCTATGCTATTTCTTTTCAACCA

3057 Left TACAGTCGTAGAAACTCAGTGTTGG 078 Right TGCTTTCTCTAGCTCTTTTTAATGG

3058 Right GGCAGTGTTAACTTTTGGATACAGT

Seq. 079 Left CCACATCCCAAGTGTTTTATGTATT

Primer Sequence

ID 080 Right ACTACTCAAAACCTGAGAAAACACG

3059 Left CCTCTATGCTATTTCTTTTCAACCA

3060 Right GATTGTTCTAATTCTGTTTGGGTGT

KIT Exon9 2kb

Seq.

Primer Sequence

ID 087 Left TGTAGGTGTTAAGAAAGGAAACAGG

3081 Left TGTAGGTGTTAAGAAAGGAAACAGG 088 Right ACTACTCAAAACCTGAGAAAACACG

3082 Right GCTTCCTTTATGGACGGTTTATATT

089 Left AAGTAATGATCGCACAATTACAACA

3083 Left TCCTCACTTTGACTATTTTCTGGTC 090 Right CCCCTTAAATTGGATTAAAAAGAAA

3084 Right GCTTCCTTTATGGACGGTTTATATT

091 Left CCAAGTTACCAAAAAGTAATGATCG

3085 Left GTTAAGAAAGGAAACAGGGAATAGC 092 Right CCCCTTAAATTGGATTAAAAAGAAA

3086 Right GCTTCCTTTATGGACGGTTTATATT 3093 Left TGGATAAGCTTGTTCTAGTGGGTAG

3094 Right ACTACTCAAAACCTGAGAAAACACG 107 Left GGTCTACATCCTTGATTTTGTTGTT

108 Right GCTTCCTTTATGGACGGTTTATATT

3095 Left TGTAGGTGTTAAGAAAGGAAACAGG

3096 Right CCTCACTACTCAAAACCTGAGAAAA 109 Left GGAATAAGCCTCTTTATCACAACAA

110 Right GTAAATATATTCCCCCATTTGCTTT

3097 Left ATAACTAGGCCTTCCTGCTTAGAAC

3098 Right GCTTCCTTTATGGACGGTTTATATT 111 Left TGTAGGTGTTAAGAAAGGAAACAGG

112 Right TCTTTAAGCTTTCCTGTATTTTCCA

3099 Left GTTAAGAAAGGAAACAGGGAATAGC

3100 Right CCTCACTACTCAAAACCTGAGAAAA 113 Left AAGTAATGATCGCACAATTACAACA

114 Right AACATCCCCTTAAATTGGATTAAAA

Seq.

Primer Sequence

ID 115 Left TAACTAGGCCTTCCTGCTTAGAACT

3101 Left TGGATAAGCTTGTTCTAGTGGGTAG 116 Right GCTTCCTTTATGGACGGTTTATATT

3102 Right CCTCACTACTCAAAACCTGAGAAAA

117 Left TTCATGGAATAAGCCTCTTTATCAC

3103 Left TCTGGTCTACATCCTTGATTTTGTT 118 Right GTAAATATATTCCCCCATTTGCTTT

3104 Right GCTTCCTTTATGGACGGTTTATATT

119 Left TTCTTTTGGGGAATACTAAGTAGGG

3105 Left TTCTGGTCTACATCCTTGATTTTGT 120 Right GTAAATATATTCCCCCATTTGCTTT

3106 Right GCTTCCTTTATGGACGGTTTATATT

JOT Exon9-10 2kb

Seq. 139 Left AACCCTCTGCAATGGGTATTACTAT

Primer Sequence

ID 140 Right ATTAGAGCACTCTGGAGAGAGAACA

3121 Left CCTCTATGCTATTTCTTTTCAACCA

3122 Right ATTAGAGCACTCTGGAGAGAGAACA 141 Left GCTGAGATTACAGGTGTGAGCTACT

142 Right ATTAGAGCACTCTGGAGAGAGAACA

3123 Left ATTCCTTTCCAATCCTTTCAGTAAC

3124 Right AGCACTCTGGAGAGAGAACAAATAA 143 Left ATTTTATTGAATTCCTTTCCAATCC

144 Right CTCTGGAGAGAGAACAAATAAATGG

3125 Left ATTCCTTTCCAATCCTTTCAGTAAC

3126 Right ATTAGAGCACTCTGGAGAGAGAACA 145 Left TTATTGAATTCCTTTCCAATCCTTT

146 Right ATTAGAGCACTCTGGAGAGAGAACA

3127 Left ATTTTATTGAATTCCTTTCCAATCC

3128 Right ATTAGAGCACTCTGGAGAGAGAACA 147 Left TTTATTGAATTCCTTTCCAATCCTT

148 Right ATTAGAGCACTCTGGAGAGAGAACA

3129 Left GCTTTCCTCCTCTATGCTATTTCTT

3130 Right ATTAGAGCACTCTGGAGAGAGAACA 149 Left TTTTATTGAATTCCTTTCCAATCCT

Seq. 150 Right ATTAGAGCACTCTGGAGAGAGAACA

Primer Sequence

ID

3131 Left AACCCTCTGCAATGGGTATTACTAT 151 Left ATGCTTTCCTCCTCTATGCTATTTC

3132 Right AGCACTCTGGAGAGAGAACAAATAA 152 Right AGCACTCTGGAGAGAGAACAAATAA

3133 Left GCTGAGATTACAGGTGTGAGCTACT 153 Left TTTTATGCTTTCCTCCTCTATGCTA

3134 Right AGCACTCTGGAGAGAGAACAAATAA 154 Right CTCTGGAGAGAGAACAAATAAATGG

3135 Left ATTCCTTTCCAATCCTTTCAGTAAC 155 Left ATGCTTTCCTCCTCTATGCTATTTC

3136 Right GCACTCTGGAGAGAGAACAAATAAA 156 Right ATTAGAGCACTCTGGAGAGAGAACA

3137 Left ATTCCTTTCCAATCCTTTCAGTAAC 157 Left ATTCCTTTCCAATCCTTTCAGTAAC

3138 Right CTCTGGAGAGAGAACAAATAAATGG 158 Right GAGCACTCTGGAGAGAGAACAAATA

3223 Left CACATTTCTCTTCCATTGT 233 Left CACATTTCTCTTCCATTGTA

3224 Right GAGAGAACAAATAAATGGTTAC 234 Right GAGAGAACAAATAAATGGTTA

3225 Left CCACATTTCTCTTCCATTGT 235 Left CCACATTTCTCTTCCATTGTA

3226 Right AGAGAACAAATAAATGGTTA 236 Right GAGAACAAATAAATGGTTA

3227 Left CCACATTTCTCTTCCATTGT 237 Left CACATTTCTCTTCCATTGTA

3228 Right GAGAACAAATAAATGGTTAC 238 Right AGAGAACAAATAAATGGTTAC

3229 Left CATTTCTCTTCCATTGTA 239 Left TCCACATTTCTCTTCCATTG

3230 Right GAGAGAGAACAAATAAATGGTTAC 240 Right GAGAACAAATAAATGGTTA

3231 Left ACATTTCTCTTCCATTGTA

3232 Right AGAGAGAACAAATAAATGGTTAC

KIT ΈχοηΙΟ 151-200 bases

Seq.

Primer Sequence

ID 3 261 Left AAAGTTTGTGATTCCACATTTCTCT

3241 Left AGTTTGTGATTCCACATTTCTCTTC J 262 Right GAGCACTCTGGAGAGAGAACAAATA

3242 Right AGCACTCTGGAGAGAGAACAAATAA

3 263 Left CAAAGTTTGTGATTCCACATTTCTC

3243 Left AGTTTGTGATTCCACATTTCTCTTC J 264 Right AGCACTCTGGAGAGAGAACAAATAA

3244 Right GCACTCTGGAGAGAGAACAAATAAA

3 265 Left CAAAGTTTGTGATTCCACATTTCTC

3245 Left AGTTTGTGATTCCACATTTCTCTTC J 266 Right GCACTCTGGAGAGAGAACAAATAAA

3246 Right GAGCACTCTGGAGAGAGAACAAATA

1 267 Left CAAAGTTTGTGATTCCACATTTCTC

3247 Left AGTTTGTGATTCCACATTTCTCTTC 3 268 Right CTCTGGAGAGAGAACAAATAAATGG

3248 Right TCTGGAGAGAGAACAAATAAATGGT

1 269 Left AGTTTGTGATTCCACATTTCTCTTC

3249 Left AAGTTTGTGATTCCACATTTCTCTT J 270 Right CTCTGGAGAGAGAACAAATAAATGGT

3250 Right AGCACTCTGGAGAGAGAACAAATAA

1 271 Left CAAAGTTTGTGATTCCACATTTCTC

3251 Left AAAGTTTGTGATTCCACATTTCTCT J 272 Right GAGCACTCTGGAGAGAGAACAAATA

3252 Right AGCACTCTGGAGAGAGAACAAATAA

1 273 Left CAAAGTTTGTGATTCCACATTTCTC

3253 Left GATTCCACATTTCTCTTCCATTGTA J 274 Right TCTGGAGAGAGAACAAATAAATGGT

3254 Right AGCACTCTGGAGAGAGAACAAATAA

3 275 Left CAAAGTTTGTGATTCCACATTTCT

3255 Left AAGTTTGTGATTCCACATTTCTCTT J 276 Right AGCACTCTGGAGAGAGAACAAATAA

3256 Right GCACTCTGGAGAGAGAACAAATAAA

Seq. 3 277 Left GTGATTCCACATTTCTCTTCCATT

Primer Sequence

ID 3 278 Right AGCACTCTGGAGAGAGAACAAATAA

3257 Left AAAGTTTGTGATTCCACATTTCTCT

3258 Right GCACTCTGGAGAGAGAACAAATAAA 3 279 Left CAAAGTTTGTGATTCCACATTTCT

3 280 Right ATTAGAGCACTCTGGAGAGAGAACA

3259 Left AAGTTTGTGATTCCACATTTCTCTT

3260 Right GAGCACTCTGGAGAGAGAACAAATA

KIT ΈχοηΙΟ 201-300 bases

Seq. 3 282 Right AGCACTCTGGAGAGAGAACAAATAA

Primer Sequence

ID

3281 Left GTACAATGTAACCAAGGTGAAGCTC 1 283 Left GAGTACAATGTAACCAAGGTGAAGC 3284 Right AGCACTCTGGAGAGAGAACAAATAA 3302 Right GAGCACTCTGGAGAGAGAACAAATA

3285 Left TACAATGTAACCAAGGTGAAGCTCT 3 303 Left GAGTACAATGTAACCAAGGTGAAGC

3286 Right AGCACTCTGGAGAGAGAACAAATAA 3 304 Right GAGCACTCTGGAGAGAGAACAAATA

3287 Left TACAATGTAACCAAGGTGAAGCTCT 3 305 Left GTACAATGTAACCAAGGTGAAGCTC

3288 Right ATTAGAGCACTCTGGAGAGAGAACA 3 306 Right TCTGGAGAGAGAACAAATAAATGGT

3289 Left GTACAATGTAACCAAGGTGAAGCTC 3 307 Left GAGTACAATGTAACCAAGGTGAAGC

3290 Right GCACTCTGGAGAGAGAACAAATAAA 3 308 Right TCTGGAGAGAGAACAAATAAATGGT

3291 Left GAGTACAATGTAACCAAGGTGAAGC 3 309 Left TACAATGTAACCAAGGTGAAGCTCT

3292 Right GCACTCTGGAGAGAGAACAAATAAA 3 310 Right GAGCACTCTGGAGAGAGAACAAATA

3293 Left GTACAATGTAACCAAGGTGAAGCTC 3 311 Left TACAATGTAACCAAGGTGAAGCTCT

3294 Right CTCTGGAGAGAGAACAAATAAATGG 3 312 Right TCTGGAGAGAGAACAAATAAATGGT

3295 Left GAGTACAATGTAACCAAGGTGAAGC 3 313 Left CTCTGAGACTCACATAGCTTTGCAT

3296 Right CTCTGGAGAGAGAACAAATAAATGG 3 314 Right AGCACTCTGGAGAGAGAACAAATAA

3297 Left TACAATGTAACCAAGGTGAAGCTCT 3 315 Left GTACAATGTAACCAAGGTGAAGCTC

3298 Right GCACTCTGGAGAGAGAACAAATAAA 3 316 Right TAGAGCACTCTGGAGAGAGAACAAA

3299 Left TACAATGTAACCAAGGTGAAGCTCT 3 317 Left CTCTGAGACTCACATAGCTTTGCAT

3300 Right CTCTGGAGAGAGAACAAATAAATGG 3 318 Right ATTAGAGCACTCTGGAGAGAGAACA

Seq. 3 319 Left TACAATGTAACCAAGGTGAAGCTC

Primer Sequence

ID 3 320 Right AGCACTCTGGAGAGAGAACAAATAA

3301 Left GTACAATGTAACCAAGGTGAAGCTC

KIT ΈχοηΙΟ 301-400 bases

Seq.

Primer Sequence

ID 335 Left TCTGCAGTATTGTGGTTTCAAGTTA

3321 Left TCTATTCTGCAGTATTGTGGTTTCA ^r. 336 Right GCACTCTGGAGAGAGAACAAATAAA

3322 Right AGCACTCTGGAGAGAGAACAAATAA

337 Left TATTCTGCAGTATTGTGGTTTCAAG

3323 Left TCTGCAGTATTGTGGTTTCAAGTTA 338 Right AGCACTCTGGAGAGAGAACAAATAA

3324 Right AGCACTCTGGAGAGAGAACAAATAA

339 Left CTATTCTGCAGTATTGTGGTTTCAA

3325 Left TCTATTCTGCAGTATTGTGGTTTCA 340 Right AGCACTCTGGAGAGAGAACAAATAA

3326 Right ATTAGAGCACTCTGGAGAGAGAACA

341 Left TCTGCAGTATTGTGGTTTCAAGTTA

3327 Left TCTGCAGTATTGTGGTTTCAAGTTA 342 Right CTCTGGAGAGAGAACAAATAAATGG

3328 Right ATTAGAGCACTCTGGAGAGAGAACA

Seq. 343 Left GAGTACAATGTAACCAAGGTGAAGC

Primer Sequence

ID -. 344 Right ATTAGAGCACTCTGGAGAGAGAACA

3329 Left TCTATTCTGCAGTATTGTGGTTTCA

3330 Right GCACTCTGGAGAGAGAACAAATAAA 345 Left GTACAATGTAACCAAGGTGAAGCTC

346 Right ATTAGAGCACTCTGGAGAGAGAACA

3331 Left ATTCTGCAGTATTGTGGTTTCAAGT

3332 Right AGCACTCTGGAGAGAGAACAAATAA 347 Left ATTCTGCAGTATTGTGGTTTCAAGT

348 Right ATTAGAGCACTCTGGAGAGAGAACA

3333 Left TCTATTCTGCAGTATTGTGGTTTCA

3334 Right CTCTGGAGAGAGAACAAATAAATGG

g t g t

JOT Exonll 201-300 bases

Seq. 405 Left GTTCTCTCTCCAGAGTGCTCTAATG

Primer Sequence

ID 406 Right TTATGTGTACCCAAAAAGGTGACAT

3401 Left TTATTTGTTCTCTCTCCAGAGTGCT

3402 Right TTATGTGTACCCAAAAAGGTGACAT 407 Left CAGAGTGCTCTAATGACTGAGACAA

408 Right TTATGTGTACCCAAAAAGGTGACAT

3403 Left TGTTCTCTCTCCAGAGTGCTCTAAT

3404 Right TTATGTGTACCCAAAAAGGTGACAT 409 Left CTCTCTCCAGAGTGCTCTAATGACT

410 Right TTATGTGTACCCAAAAAGGTGACAT 425 Left GTTCTCTCTCCAGAGTGCTCTAATG

341 1 Left TTATTTGTTCTCTCTCCAGAGTGCT 426 Right TGTTATGTGTACCCAAAAAGGTGAC

3412 Right TGTTATGTGTACCCAAAAAGGTGAC

427 Left TGTTCTCTCTCCAGAGTGCTCTAAT

3413 Left TTATTTGTTCTCTCTCCAGAGTGCT 428 Right GTTATGTGTACCCAAAAAGGTGACA

3414 Right GTTATGTGTACCCAAAAAGGTGACA

429 Left GTTCTCTCTCCAGAGTGCTCTAATG

3415 Left AAGGTGATCTATTTTTCCCTTTCTC 430 Right GTTATGTGTACCCAAAAAGGTGACA

3416 Right TTATGTGTACCCAAAAAGGTGACAT

431 Left GTTCTCTCTCCAGAGTGCTCTAATG

3417 Left AAGGTGATCTATTTTTCCCTTTCTC 432 Right CTGTTATGTGTACCCAAAAAGGTG

3418 Right GCAATTTCACAGAAAACTCATTGTT

433 Left TGTTCTCTCTCCAGAGTGCTCTAAT

3419 Left TTATTTGTTCTCTCTCCAGAGTGCT 434 Right CTGTTATGTGTACCCAAAAAGGTG

3420 Right CTGTTATGTGTACCCAAAAAGGTG

435 Left CAGAGTGCTCTAATGACTGAGACAA

Seq. 436 Right GTTATGTGTACCCAAAAAGGTGACA

Primer Sequence

ID

3421 Left TCTCTCTCCAGAGTGCTCTAATGAC 437 Left CAGAGTGCTCTAATGACTGAGACAA

3422 Right TTATGTGTACCCAAAAAGGTGACAT 438 Right TGTTATGTGTACCCAAAAAGGTGAC

3423 Left TGTTCTCTCTCCAGAGTGCTCTAAT 439 Left TTATTTGTTCTCTCTCCAGAGTGCT

3424 Right TGTTATGTGTACCCAAAAAGGTGAC 440 Right CTGTTATGTGTACCCAAAAAGGTGA

KIT Έχοηΐΐ 301-400 bases

Seq. 455 Left CAGAGTGCTCTAATGACTGAGACAA

Primer Sequence

ID : 456 Right GTTCTCTATGGCAAACCTATCAAAA

3441 Left TTATTTGTTCTCTCTCCAGAGTGCT

3442 Right TTCTCTATGGCAAACCTATCAAAAG 457 Left TTATTTGTTCTCTCTCCAGAGTGCT

458 Right ATGTTGTCCAGAGACATTTTCCTAC

3443 Left TTATTTGTTCTCTCTCCAGAGTGCT

3444 Right GTTCTCTATGGCAAACCTATCAAAA J 459 Left TGTTCTCTCTCCAGAGTGCTCTAAT

460 Right ATGTTGTCCAGAGACATTTTCCTAC

3445 Left TGTTCTCTCTCCAGAGTGCTCTAAT

3446 Right TTCTCTATGGCAAACCTATCAAAAG

3447 Left GTTCTCTCTCCAGAGTGCTCTAATG 461 Left GTTCTCTCTCCAGAGTGCTCTAATG

3448 Right TTCTCTATGGCAAACCTATCAAAAG 462 Right ATGTTGTCCAGAGACATTTTCCTAC

3449 Left CAGAGTGCTCTAATGACTGAGACAA 463 Left TTATTTGTTCTCTCTCCAGAGTGCT

3450 Right TTCTCTATGGCAAACCTATCAAAAG 464 Right CATTTTCCTACGATGTTCTCTATGG

3465 Left CTCTCTCCAGAGTGCTCTAATGACT

3466 Right TTCTCTATGGCAAACCTATCAAAAG

Seq.

Primer Sequence

ID 467 Left TTATTTGTTCTCTCTCCAGAGTGCT

3451 Left GTTCTCTCTCCAGAGTGCTCTAATG 468 Right ATGTTCTCTATGGCAAACCTATCAA

3452 Right GTTCTCTATGGCAAACCTATCAAAA

469 Left CAGAGTGCTCTAATGACTGAGACAA

3453 Left TGTTCTCTCTCCAGAGTGCTCTAAT 470 Right ATGTTGTCCAGAGACATTTTCCTAC

3454 Right GTTCTCTATGGCAAACCTATCAAAA

: 471 Left TTATTTGTTCTCTCTCCAGAGTGCT

548 Right CATGCAGTACCATACAGGAACTTAC

3535 Left ACTTACCTTGTTGTCTTCCTTCCTA

3536 Right CATGCAGTACCATACAGGAACTTAC : 549 Left CATCACCACTTACCTTGTTGTCTT

: 550 Right CATGCAGTACCATACAGGAACTTAC

3537 Left CATCACCACTTACCTTGTTGTCTTC

3538 Right CATGCAGTACCATACAGGAACTTAC : 551 Left ACCTTGTTGTCTTCCTTCCTACAG

: 552 Right GCATGCAGTACCATACAGGAACTTA

3539 Left CACTTACCTTGTTGTCTTCCTTCCT

3540 Right CATGCAGTACCATACAGGAACTTAC : 553 Left ACCACTTACCTTGTTGTCTTCCTTC

: 554 Right GCATGCAGTACCATACAGGAACTTA

3541 Left CCACTTACCTTGTTGTCTTCCTTC

3542 Right CATGCAGTACCATACAGGAACTTAC : 555 Left TACCTTGTTGTCTTCCTTCCTACA

: 556 Right CATGCAGTACCATACAGGAACTTAC

3543 Left CACTTACCTTGTTGTCTTCCTTCC

3544 Right CATGCAGTACCATACAGGAACTTAC : 557 Left CTTACCTTGTTGTCTTCCTTCCTAC

558 Right GCATGCAGTACCATACAGGAACTTA

3545 Left ACTTACCTTGTTGTCTTCCTTCCTAC

3546 Right CATGCAGTACCATACAGGAACTTAC : 559 Left ACTTACCTTGTTGTCTTCCTTCCTA

: 560 Right GCATGCAGTACCATACAGGAACTTA

3547 Left ACCACTTACCTTGTTGTCTTCCTT

KIT ExonU 201-300 bases

Seq.

Primer Sequence

ID 581 Left GCCATAGAGAACATCGTAGGAAAAT

3561 Left CTTTTGATAGGTTTGCCATAGAGAA 582 Right CATGCAGTACCATACAGGAACTTAC

3562 Right CATGCAGTACCATACAGGAACTTAC

583 Left TTGATAGGTTTGCCATAGAGAACA

3563 Left AGAACATCGTAGGAAAATGTCTCTG 584 Right CATGCAGTACCATACAGGAACTTAC

3564 Right CATGCAGTACCATACAGGAACTTAC

585 Left AATTCCTTTATTGATTTTGAAACTGC

3565 Left CCATAGAGAACATCGTAGGAAAATG 586 Right CATGCAGTACCATACAGGAACTTAC

3566 Right CATGCAGTACCATACAGGAACTTAC

587 Left TTTGATAGGTTTGCCATAGAGAACA

3567 Left CTTTTGATAGGTTTGCCATAGAGAA 588 Right CATGCAGTACCATACAGGAACTTAC

3568 Right GCATGCAGTACCATACAGGAACTTA

589 Left TCTCTGGACAACATTGTTTTTAATTC

3569 Left TCCTTTATTGATTTTGAAACTGCAC 590 Right CATGCAGTACCATACAGGAACTTAC

3570 Right CATGCAGTACCATACAGGAACTTAC

591 Left TAGGTTTGCCATAGAGAACATCGTA

3571 Left TGATAGGTTTGCCATAGAGAACATC 592 Right CATGCAGTACCATACAGGAACTTAC

3572 Right CATGCAGTACCATACAGGAACTTAC

593 Left TGTCTCTGGACAACATTGTTTTTAAT

3573 Left AGAACATCGTAGGAAAATGTCTCTG 594 Right CATGCAGTACCATACAGGAACTTAC

3574 Right GCATGCAGTACCATACAGGAACTTA

595 Left AATGTCTCTGGACAACATTGTTTTTA

3575 Left CCATAGAGAACATCGTAGGAAAATG 596 Right CATGCAGTACCATACAGGAACTTAC

3576 Right GCATGCAGTACCATACAGGAACTTA

Seq. 597 Left TGGACAACATTGTTTTTAATTCCTT

Primer Sequence

ID 598 Right GCATTTTAGCAAAAAGCACAACT

3577 Left ATTCCTTTATTGATTTTGAAACTGC

3578 Right CATGCAGTACCATACAGGAACTTAC 599 Left TCCTTTATTGATTTTGAAACTGCAC

600 Right GCATGCAGTACCATACAGGAACTTA

3579 Left ATGTCTCTGGACAACATTGTTTTTA

3580 Right CATGCAGTACCATACAGGAACTTAC

3659 Left ATGCATGTTTCCAATTTTAG 670 Right AGCTTGGACACGGCTTTA

3660 Right GCTTGGACACGGCTTTAC

671 Left ATGCATGTTTCCAATTTT

3661 Left TGCATGTTTCCAATTTTA 672 Right AGCTTGGACACGGCTTTAC

3662 Right CAGCTTGGACACGGCTTTA

673 Left GCATGTTTCCAATTTTAG

3663 Left ATGCATGTTTCCAATTTTA 674 Right CAGCTTGGACACGGCTTTAC

3664 Right AGCTTGGACACGGCTTTAC

675 Left TGCATGTTTCCAATTTTAG

3665 Left AATGCATGTTTCCAATTTT 676 Right GCTTGGACACGGCTTTAC

3666 Right GCTTGGACACGGCTTTAC

677 Left GCATGTTTCCAATTTTAG

3667 Left TGCATGTTTCCAATTTTA 678 Right CAGCTTGGACACGGCTTTA

3668 Right CAGCTTGGACACGGCTTT

679 Left TGCATGTTTCCAATTTTA

3669 Left TGCATGTTTCCAATTTTAG 680 Right AGCTTGGACACGGCTTTAC

JOT Exonl3 151-200 bases

Seq.

Primer Sequence

ID 3 701 Left TGCTAAAATGCATGTTTCCAAT

3681 Left TGCTAAAATGCATGTTTCCAAT J 702 Right TGTTTTGATAACCTGACAGACAATAA

3682 Right CATGTTTTGATAACCTGACAGACAA

1 703 Left TAAAATGCATGTTTCCAATTTTAG

3683 Left TAAAATGCATGTTTCCAATTTTAG 3 704 Right ATGTTTTGATAACCTGACAGACAAT

3684 Right CATGTTTTGATAACCTGACAGACAA

1 705 Left TGCTAAAATGCATGTTTCCAAT

3685 Left TGCTAAAATGCATGTTTCCAAT 3 706 Right CATGTTTTGATAACCTGACAGACA

3686 Right TTGATAACCTGACAGACAATAAAAG(

1 707 Left TAAAATGCATGTTTCCAATTTTAG

3687 Left TAAAATGCATGTTTCCAATTTTAG J 708 Right TGTTTTGATAACCTGACAGACAATAA

3688 Right TTGATAACCTGACAGACAATAAAAG(

1 709 Left TAAAATGCATGTTTCCAATTTTAG

3689 Left TGCTAAAATGCATGTTTCCAAT J 710 Right CATGTTTTGATAACCTGACAGACA

3690 Right TGATAACCTGACAGACAATAAAAGG

1 711 Left TGCTAAAATGCATGTTTCCAAT

3691 Left TAAAATGCATGTTTCCAATTTTAG J 712 Right CTGACAGACAATAAAAGGCAGCTT

3692 Right TGATAACCTGACAGACAATAAAAGG

3 713 Left TAAAATGCATGTTTCCAATTTTAG

3693 Left AAAATGCATGTTTCCAATTTTAG J 714 Right CTGACAGACAATAAAAGGCAGCTT

3694 Right CATGTTTTGATAACCTGACAGACAA

1 715 Left TGCTAAAATGCATGTTTCCAAT

3695 Left TGCTAAAATGCATGTTTCCAAT 3 716 Right TGTTTTGATAACCTGACAGACAATA

3696 Right CATGTTTTGATAACCTGACAGACAAT

3697 Left TAAAATGCATGTTTCCAATTTTAG

3698 Right CATGTTTTGATAACCTGACAGACAAT 3 717 Left TAAAATGCATGTTTCCAATTTTAG

3718 Right TGTTTTGATAACCTGACAGACAATA

Seq. 3 719 Left AAAATGCATGTTTCCAATTTTAG

Primer Sequence

ID 3 720 Right TTGATAACCTGACAGACAATAAAAGG

3699 Left TGCTAAAATGCATGTTTCCAAT

3700 Right ATGTTTTGATAACCTGACAGACAAT

ID 302 Right AAATGTGTGATATCCCTAGACAGGA

4285 Left GTTTTCTTTTCTCCTCCAACCTAAT

4286 Right TGTGTGATATCCCTAGACAGGATTT 303 Left TTTTCTTTTCTCCTCCAACCTAATA

304 Right TGTGTGATATCCCTAGACAGGATTT

4287 Left GTTTTCTTTTCTCCTCCAACCTAAT

4288 Right CACAGGAAACAATTTTTATCGAAAG 305 Left TGAATTTAAATGGTTTTCTTTTCTCC

4306 Right CACAGGAAACAATTTTTATCGAAAG

307 Left TGAATTTAAATGGTTTTCTTTTCTCC

4289 Left GTTTTCTTTTCTCCTCCAACCTAAT 308 Right AAAATGTGTGATATCCCTAGACAGG

4290 Right AAAATGTGTGATATCCCTAGACAGG

309 Left TTTTCTTTTCTCCTCCAACCTAATA

4291 Left TGAATTTAAATGGTTTTCTTTTCTCC 310 Right CACAGGAAACAATTTTTATCGAAAG

4292 Right TGTGTGATATCCCTAGACAGGATTT

311 Left TAAATGGTTTTCTTTTCTCCTCCA

4293 Left TGAATTTAAATGGTTTTCTTTTCTCC 312 Right ATCACAGGAAACAATTTTTATCGAA

4294 Right AAATGTGTGATATCCCTAGACAGGA

313 Left TAAATGGTTTTCTTTTCTCCTCCA

4295 Left TTTTCTTTTCTCCTCCAACCTAATA 314 Right AAATGTGTGATATCCCTAGACAGGA

4296 Right ATCACAGGAAACAATTTTTATCGAA

315 Left TAAATGGTTTTCTTTTCTCCTCCA

4297 Left GTTTTCTTTTCTCCTCCAACCTAAT 316 Right TGTGTGATATCCCTAGACAGGATTT

4298 Right ATGTGTGATATCCCTAGACAGGATT

317 Left TTTTCTTTTCTCCTCCAACCTAATA

4299 Left GTTTTCTTTTCTCCTCCAACCTAAT 318 Right AAAATGTGTGATATCCCTAGACAGG

4300 Right AATGTGTGATATCCCTAGACAGGAT

319 Left TAAATGGTTTTCTTTTCTCCTCCAA

4301 Left TTTTCTTTTCTCCTCCAACCTAATA 320 Right ATCACAGGAAACAATTTTTATCGAA

KIT Exonll 301-400

Seq. 335 Left TTCAAGGCGTACTTTTGATTTTTAT

Primer Sequence

ID 336 Right TCGAAAGTTGAAACTAAAAATCCTTT

4321 Left ATTCAAGGCGTACTTTTGATTTTTA

4322 Right ATCACAGGAAACAATTTTTATCGAA 337 Left ATTCAAGGCGTACTTTTGATTTTTA

338 Right TCACAGGAAACAATTTTTATCGAA

4323 Left TCAAGGCGTACTTTTGATTTTTATT 5eq.

Primer Sequence

4324 Right ATCACAGGAAACAATTTTTATCGAA ID

339 Left TCAAGGCGTACTTTTGATTTTTATT

4325 Left TTCAAGGCGTACTTTTGATTTTTAT 340 Right TCACAGGAAACAATTTTTATCGAA

4326 Right ATCACAGGAAACAATTTTTATCGAA

341 Left TTCAAGGCGTACTTTTGATTTTTAT

4327 Left ATTCAAGGCGTACTTTTGATTTTTA 342 Right TCACAGGAAACAATTTTTATCGAA

4328 Right CACAGGAAACAATTTTTATCGAAAG

343 Left ATTCAAGGCGTACTTTTGATTTTT

4329 Left GTTTTCTTTTCTCCTCCAACCTAAT 344 Right ATCACAGGAAACAATTTTTATCGAA

4330 Right TAGTAATGTTCAGCATACCATGCAA

345 Left TTCAAGGCGTACTTTTGATTTTTATT

4331 Left ATTCAAGGCGTACTTTTGATTTTTAT 346 Right ATCACAGGAAACAATTTTTATCGAA

4332 Right ATCACAGGAAACAATTTTTATCGAA

347 Left AGTCCTGAGAAGAAAACAGCATTTA

4333 Left ATCATTCAAGGCGTACTTTTGATTT 348 Right ACTGTCAAGCAGAGAATGGGTACT

4334 Right ATCACAGGAAACAATTTTTATCGAA

349 Left ATTCAAGGCGTACTTTTGATTTTT

4543 Left TATTTCCCTATGAATGAAAGCAGTC 552 Right GGCAGAGAATATTATAAAGGGCAAT

4544 Right AAAACCCACAATTACTTTTACACCA

553 Left AGAGCCATAGTTAAAATGCAGAATG

4545 Left AACCAAAAGCAGAGGAAATTTAGTT 554 Right GGCAGAGAATATTATAAAGGGCAAT

4546 Right ATTACATTATCATAAGGGGCACAAA

555 Left AGCCATAGTTAAAATGCAGAATGTC

4547 Left GAAGGTTAGGAATGGAAAGAATGAT 556 Right ATTACATTATCATAAGGGGCACAAA

4548 Right ATTCAGAGGTATTGGACAACTCTTG

557 Left GAGCCATAGTTAAAATGCAGAATGT

4549 Left AGCCATAGTTAAAATGCAGAATGTC 558 Right ATTACATTATCATAAGGGGCACAAA

4550 Right GGCAGAGAATATTATAAAGGGCAAT

559 Left AGAGCCATAGTTAAAATGCAGAATG

4551 Left GAGCCATAGTTAAAATGCAGAATGT 560 Right ATTACATTATCATAAGGGGCACAAA

JOT Exonl7 5kb

Seq. 5eq.

Primer Sequence Primer Sequence

ID ID

4561 Left ATGGTTCAGAAAATCATCCAAATTA 581 Left GTACCTACCTATCAAGCAACCAAGA

4562 Right TCTGCCATAAAAAGCTAAATCAATC 582 Right GGCCACTAAGTTGTAAGTGCTGTAT

4563 Left TCAGAAAATCATCCAAATTATTGGT 583 Left GAGTACCTACCTATCAAGCAACCAA

4564 Right TCTGCCATAAAAAGCTAAATCAATC 584 Right GGCCACTAAGTTGTAAGTGCTGTAT

4565 Left GTATATTGCTGCAGTTGTGTGGTAG 585 Left TCATGGTGTTTCTATGCTAATCAGA

4566 Right TAAGGGCTCCTAACCTGAGATCTAT 586 Right TAAGGGCTCCTAACCTGAGATCTAT

4567 Left TCATGGTGTTTCTATGCTAATCAGA 587 Left GTAACCCAGCCTAGGATTGTTAAAT

4568 Right TCTGCCATAAAAAGCTAAATCAATC 588 Right CTTGAGTACCATCTCACAAAAACCT

4569 Left GTACCTACCTATCAAGCAACCAAGA 589 Left TCATGGTGTTTCTATGCTAATCAGA

4570 Right CTTGAGTACCATCTCACAAAAACCT 590 Right GGCCACTAAGTTGTAAGTGCTGTAT

4571 Left GAGTACCTACCTATCAAGCAACCAA 591 Left AACCAAAAGCAGAGGAAATTTAGTT

4572 Right CTTGAGTACCATCTCACAAAAACCT 592 Right CAGTGTGTCATAAAGAATCCAAGTG

4573 Left TCTATGCTAATCAGAAGCAGGAAGT 593 Left TCTATGCTAATCAGAAGCAGGAAGT

4574 Right TCTGCCATAAAAAGCTAAATCAATC 594 Right TAAGGGCTCCTAACCTGAGATCTAT

4575 Left ATGGTTCAGAAAATCATCCAAATTA 595 Left ATGTTTTTGTGCCTGAGTATCTTTC

4576 Right GGCCACTAAGTTGTAAGTGCTGTAT 596 Right CAGTGTGTCATAAAGAATCCAAGTG

4577 Left TCAGAAAATCATCCAAATTATTGGT 597 Left TCTATGCTAATCAGAAGCAGGAAGT

4578 Right GGCCACTAAGTTGTAAGTGCTGTAT 598 Right GGCCACTAAGTTGTAAGTGCTGTAT

4579 Left GTATATTGCTGCAGTTGTGTGGTAG 599 Left AACCAAAAGCAGAGGAAATTTAGTT

4580 Right AAAACCCACAATTACTTTTACACCA 600 Right CAATTTGCAACCTAAGATTAGGAGA

Table 10. KRAS Capture Primer List for NGS Panel

KRAS Exonl 169-300 bases

Seq. 601 Left CTCGGAGCTCGATTTTCCTA

Primer Sequence

ID 602 Right GGGACCCCTAATTCATTCACTC

4793 Left TACGCCCGTCTGAAGAAGAAT 797 Left TACGCCCGTCTGAAGAAGAAT

4794 Right TAACCTCAAACAGTGGTCTCTAAGC 798 Right ACACAATAACCTCAAACAGTGGTCT

4795 Left CCCGTCTGAAGAAGAATCGAG 799 Left CCCGTCTGAAGAAGAATCGAG

4796 Right ACAGTGGTCTCTAAGCACTTTCCTA 800 Right AAACACAATAACCTCAAACAGTGGT

ERAS Exonl 2kb

Seq.

Primer Sequence

ID 821 Left GGGATTTAAATTCAGCTTTATTGGT

4801 Left GGGATTTAAATTCAGCTTTATTGGT 822 Right TAACCTCAAACAGTGGTCTCTAAGC

4802 Right ACAGTGGTCTCTAAGCACTTTCCTA

Seq. 823 Left TCAAGACTCTCCCAAGATACATTTC

Primer Sequence

ID 824 Right ATAAGAAATAGGGGAAAGGACAAGA

4803 Left GGGATTTAAATTCAGCTTTATTGGT

4804 Right CATCTGGGATTAACTTTTTCCTTTT 825 Left TCAAGACTCTCCCAAGATACATTTC

4805 Left TCAAGACTCTCCCAAGATACATTTC 826 Right TAACCTCAAACAGTGGTCTCTAAGC

4806 Right TTTGCTATTGCTGTCTACACTCAAC

827 Left TTCAGCTTTATTGGTGGTTTATGAT

4807 Left TCAAGACTCTCCCAAGATACATTTC 828 Right ACAGTGGTCTCTAAGCACTTTCCTA

4808 Right ACAGTGGTCTCTAAGCACTTTCCTA

829 Left ATTCAGCTTTATTGGTGGTTTATGA

4809 Left GGGATTTAAATTCAGCTTTATTGGT 830 Right ACAGTGGTCTCTAAGCACTTTCCTA

4810 Right AAACACAATAACCTCAAACAGTGGT

831 Left GGGATTTAAATTCAGCTTTATTGGT

4811 Left TCAAGACTCTCCCAAGATACATTTC 832 Right ACACAATAACCTCAAACAGTGGTCT

4812 Right CATCTGGGATTAACTTTTTCCTTTT

833 Left TTCAGCTTTATTGGTGGTTTATGAT

4813 Left TCAAGACTCTCCCAAGATACATTTC 834 Right CATCTGGGATTAACTTTTTCCTTTT

4814 Right AAACACAATAACCTCAAACAGTGGT

835 Left ATTCAGCTTTATTGGTGGTTTATGA

4815 Left GTAGAAAGGAAAGGATGACAGTTGA 836 Right CATCTGGGATTAACTTTTTCCTTTT

4816 Right AAACACAATAACCTCAAACAGTGGT

837 Left ATTCAGCTTTATTGGTGGTTTATGA

4817 Left GATTACAGCCCGTGTAAGAGTAGAA 838 Right AAACACAATAACCTCAAACAGTGGT

4818 Right ACAGTGGTCTCTAAGCACTTTCCTA

839 Left TTCAGCTTTATTGGTGGTTTATGAT

4819 Left GATTACAGCCCGTGTAAGAGTAGAA 840 Right AAACACAATAACCTCAAACAGTGGT

4820 Right AAACACAATAACCTCAAACAGTGGT

ERAS Exonl 5kb

Seq.

Primer Sequence

ID 849 Left TGCTTTGAATGTTAGTCACAGAGAG

4841 Left GGGATTTAAATTCAGCTTTATTGGT 850 Right TGATGGATCTCAAGATTTAAGAAGG

4842 Right CAAAGCAATTAGGAATAGATGAGGA

851 Left ACGTAAGTAAGGAAGGGAGAACAGT

4843 Left ACGTAAGTAAGGAAGGGAGAACAGT 852 Right AACAGTTCTCAAAATGTGGTCTAGG

4844 Right CAAAGCAATTAGGAATAGATGAGGA

853 Left ACGTAAGTAAGGAAGGGAGAACAGT

4845 Left AGCAGTAAATGAAACAGACCAAAAC 854 Right TCCTTTCCCTCATGTAACACATAAT

4846 Right CAAAGCAATTAGGAATAGATGAGGA

855 Left CGAATCATGAGCCTAGATGATAACT

4847 Left GGGATTTAAATTCAGCTTTATTGGT 856 Right ATCCAACAATTTTGTAATGGAAGAA

4848 Right TCCTTTCCCTCATGTAACACATAAT 5eq. Primer Sequence

4973 Left GGTACTGGTGGAGTATTTGATAGTG

4974 Right CCAAGGAAAGTAAAGTTCCCATATT 4S 87 Left GGTACTGGTGGAGTATTTGATAGTGT

4988 Right CCAAGGAAAGTAAAGTTCCCATATT

5036 Right CGAAACTCTGAAATACACTTCCAAT

5( 139 Left TGAAGTACAGTTCATTACGATACACG

5037 Left TGAAGTACAGTTCATTACGATACACC 5( 140 Right TGACATACTCCCAAGGAAAGTAAAG

5038 Right CTGACATACTCCCAAGGAAAGTAAA

ERAS Exon2 601-800 bases

Seq. 5( 159 Left AGTCATGATATGATCCTTTGAGAGC

Primer Sequence

ID 5( 160 Right GAAACCCAAGGTACATTTCAGATAA

5041 Left AGTCATGATATGATCCTTTGAGAGC

5042 Right CCAAGGAAAGTAAAGTTCCCATATT 5( 161 Left CTACTGCCATGATGCTTTAAAAGTT

5( 162 Right TAACTTGAAACCCAAGGTACATTTC

5043 Left AGTCATGATATGATCCTTTGAGAGC

5044 Right CGAAACTCTGAAATACACTTCCAAT 5( 163 Left AGAGCACTGTGAAGTCTCTACATGA

5064 Right CCAAGGAAAGTAAAGTTCCCATATT

Seq. 5( 165 Left AGTCATGATATGATCCTTTGAGAGC

Primer Sequence

ID 5( 166 Right TACAAATTTCTACCCTCTCACGAAA

5045 Left AGTCATGATATGATCCTTTGAGAGC

5046 Right TGACATACTCCCAAGGAAAGTAAAG 5( 167 Left AGTCATGATATGATCCTTTGAGAGC

5( 168 Right AAATTTCTACCCTCTCACGAAACTC

5047 Left AGTCATGATATGATCCTTTGAGAGC

5048 Right CTGACATACTCCCAAGGAAAGTAAA 5( 169 Left CTACTGCCATGATGCTTTAAAAGTT

5070 Right GAAACCCAAGGTACATTTCAGATAA

5( 171 Left AGAGCACTGTGAAGTCTCTACATGA

5049 Left AGTCATGATATGATCCTTTGAGAGC 5( 172 Right CTGACATACTCCCAAGGAAAGTAAA

5050 Right AATTTCTACCCTCTCACGAAACTCT

5( 173 Left AGAGCACTGTGAAGTCTCTACATGA

5051 Left AGTCATGATATGATCCTTTGAGAGC 5( 174 Right TGACATACTCCCAAGGAAAGTAAAG

5052 Right GATACAAATTTCTACCCTCTCACGA

5( 175 Left TCTGTAGCTGTTGCATATTGACTTC

5053 Left TCTGTAGCTGTTGCATATTGACTTC 5( 176 Right TAACTTGAAACCCAAGGTACATTTC

5054 Right CCAAGGAAAGTAAAGTTCCCATATT

5( 177 Left AGAGCACTGTGAAGTCTCTACATGA

5055 Left TTTTTCTGTAGCTGTTGCATATTGA 5( 178 Right AATTTCTACCCTCTCACGAAACTCT

5056 Right CCAAGGAAAGTAAAGTTCCCATATT

5( 179 Left AGTCATGATATGATCCTTTGAGAGC

5057 Left AGTCATGATATGATCCTTTGAGAGC 5( 180 Right ACGAAACTCTGAAATACACTTCCAA

5058 Right TAACTTGAAACCCAAGGTACATTTC

ERAS Exon2 801-1000 bases

Seq. 5( 188 Right TGACATACTCCCAAGGAAAGTAAAG

Primer Sequence

ID

5081 Left ATCCAGCTTTATTTGACACTCATTC 5( 189 Left AATATTGTTCTTCTTTGCCTCAGTG

5082 Right CCAAGGAAAGTAAAGTTCCCATATT 5( 190 Right CTGACATACTCCCAAGGAAAGTAAA

5083 Left CTACTGCCATGATGCTTTAAAAGTT 5( 191 Left ATCCAGCTTTATTTGACACTCATTC

5084 Right CCAAGGAAAGTAAAGTTCCCATATT 5( 192 Right TGACATACTCCCAAGGAAAGTAAAG

5085 Left ATCCAGCTTTATTTGACACTCATTC 5( 193 Left ATCCAGCTTTATTTGACACTCATTC

5086 Right CGAAACTCTGAAATACACTTCCAAT 5( 194 Right CTGACATACTCCCAAGGAAAGTAAA

5087 Left AATATTGTTCTTCTTTGCCTCAGTG 5( 195 Left CTACTGCCATGATGCTTTAAAAGTT

ERAS Exon2 5kb

Seq.

Primer Sequence

ID 181 Left TCCTCATCTATTCCTAATTGCTTTG

5161 Left CAGCCAATAAGTCTAGGTAGAGCAG 182 Right TTGAACTGAATTATAAGTGCCACAA

5162 Right TAAAGATGAAACAAACCAATCCAAT

183 Left TTTGCTTTTAAGAGATGGTAGATGG

5163 Left AGCCTTCTTAAATCTTGAGATCCAT 184 Right TCCTAACCCACTTTATCACATTCAT

5164 Right TCTTTGCAAATAGGCATTATTTCTC

185 Left GCACATTCATTAATTTGGAGCTACT

5165 Left ATTATGTGTTACATGAGGGAAAGGA 186 Right TAAAGATGAAACAAACCAATCCAAT

5166 Right TAAAGATGAAACAAACCAATCCAAT

187 Left GACTTAAACATGTGCATCTCCTTTT

5167 Left GGCTAGTAAACTTTTTGGCCTTAAC 188 Right AAATGACAACAAAGCAAAGGTAAAG

5168 Right TCTTTGCAAATAGGCATTATTTCTC

189 Left TCCTCATCTATTCCTAATTGCTTTG

5169 Left AGCCTTCTTAAATCTTGAGATCCAT 190 Right CCTTACTGAATAGGAAACTGTTCCA

5170 Right AATTAGACTGTTCCCCTTTACTGCT

Seq. 191 Left TTTGCTTTTAAGAGATGGTAGATGG

Primer Sequence

ID 192 Right AATTAGACTGTTCCCCTTTACTGCT

5171 Left GGCTAGTAAACTTTTTGGCCTTAAC

5172 Right TCCTAACCCACTTTATCACATTCAT 193 Left AGTCATGATATGATCCTTTGAGAGC

194 Right AATTAGCATGATTGCCTAGAAACAC

5173 Left GGCTAGTAAACTTTTTGGCCTTAAC

5174 Right AATTAGACTGTTCCCCTTTACTGCT 195 Left AGCCTTCTTAAATCTTGAGATCCAT

196 Right ACCAAAAATATGTGACGTTTCCTTA

5175 Left TTTGCTTTTAAGAGATGGTAGATGG

5176 Right TCTTTGCAAATAGGCATTATTTCTC 197 Left AGCCTTCTTAAATCTTGAGATCCAT

198 Right TACCAAAAATATGTGACGTTTCCTT

5177 Left CAGCCAATAAGTCTAGGTAGAGCAG

5178 Right TTGAACTGAATTATAAGTGCCACAA 199 Left AGCCTTCTTAAATCTTGAGATCCAT

200 Right TCAATTAGACTGTTCCCCTTTACTG

5179 Left GGCTAGTAAACTTTTTGGCCTTAAC

5180 Right TTGGAAACAAAGTGTAATGGAATTT

221 Left atgaggagcagcagtgag

5213 Left tccaagcacaccatcctgag 222 Right gattcttccctggagcactgtccaa

5214 Right ccaaccatgcttccctgga

223 Left catcctgagtccgtggat

5215 Left ctgcacactggccgtct 224 Right agcactgtccaaccatgct

5216 Right ctcgaaatgggttgtctggac

225 Left tgagcactgcacact

5217 Left ccgtggatgaggagcagc 226 Right cccgattcttccctggagc

5218 Right gattcttccctggagcactgtc

Seq. 227 Left atgaggagcagcagt

Primer Sequence

ID 228 Right gacgcccgattcttccct

5219 Left gagcactgcacactggc

5220 Right cgattcttccctggagcact 229 Left gtgagcactgcac

230 Right aatgggttgtctggacgcc

ALK Regionl 176-225 bases

Seq.

Primer Sequence

ID 251 Left accatcctgagtccgtggat

5231 Left ctcctttctccttctcaacacct 252 Right ggccactcgaaatgggttg

5232 Right attcttccctggagcactgtc

Seq. 253 Left ccgtggatgaggagcagc

Primer Sequence

ID 254 Right ggagatgtattccagggcca

5233 Left cttctcaacacctcagctgact

5234 Right cgattcttccctggagcact 255 Left gagcactgcacactggc

5235 Left tttctccttctcaacacctcagct 256 Right gacaagctgcggtttccac

5236 Right gcactgtccaaccatgcttc

257 Left gcacaccatcctgagtcc

5237 Left ctgactccaagcacaccatc 258 Right gaaatgggttgtctggacgcc

5238 Right ctcgaaatgggttgtctggac

259 Left atgaggagcagcagtgag

5239 Left tccaagcacaccatcctgag 260 Right agggccactcgaaatggg

5240 Right ccactcgaaatgggttgtctg

261 Left ccgtggatgaggagc

5241 Left cctgagtccgtggatgagg 262 Right cccgattcttccctggagc

5242 Right gagatgtattccagggccactc

c 263 Left tgagcactgcacact

5243 Left ttctcaacacctcagctgactccaa s 264 Right aagctgcggtttccactgg

5244 Right gattcttccctggagcactgtccaa

s 265 Left atgaggagcagcagt

5245 Left gaggctcctttctccttctca s 266 Right ggacgcccgattcttccc

5246 Right ctggagcactgtccaacca

s 267 Left gtgagcactgcac

5247 Left ctcagctgactccaagcaca ί 268 Right tccactggagatgtattcca

5248 Right gagcactgtccaaccatgc

ί 269 Left ttctccttctcaaca

5249 Left acaccatcctgagtccgtg ί 270 Right gcccgattcttccctgg

5250 Right ttccagggccactcgaaat

ALK Regionl 226-275 bases

Seq. 5273 Left gaggctcctttctccttctcaa

Primer Sequence

ID 5274 Right ccactcgaaatgggttgtctg

5271 Left ctcctttctccttctcaacacct

5272 Right ctcgaaatgggttgtctggac ¾75 Left cttctcaacacctcagctgactc

¾76 Right gagatgtattccagggccactc

g t caaagaagtccactgcagacaag 5337 Left gaggctgcaagagagatcct 5352 Right agacaagctgcggttt

5338 Right cttcactgcagttcttcaggg

5353 Left tcctcctgatgccca

5339 Left cacaacgaggctgcaagag 5354 Right gatgttccttcactgcagtt

5340 Right gcaaagaagtccactgcagac

5355 Left gccccacaacgaggct

5341 Left ctctggaaggtacattgcccag 5356 Right tccactggagatgtattc

5342 Right caagctgcggtttccactg

5357 Left gctgctgccccacaac

5343 Left gaaggtacattgcccagctg 5358 Right ggcaaagaagtccact

5344 Right agacaagctgcggtttcca

5359 Left ctctggaaggtacattgcc

5345 Left tcctcctgatgcccactc 5360 Right agacaagctgcgg

5346 Right ctgcagttcttcagggcaaag

5361 Left cacctgcagccctct

5347 Left cccacaacgaggctgcaa 5362 Right tccactggagatgta

5348 Right ggcaaagaagtccactgca

Seq. 5363 Left agagatcctcctga

Primer Sequence

ID 5364 Right gatgttccttcactgca

5349 Left tgctgccccacaacgag 5365 Left ctgctgccccac

5350 Right ttcagggcaaagaagtcc 5366 Right tcagggcaaagaag

5351 Left gtacattgcccagctgctg

ALK Region2 176-225 bases

Seq. 5385 Left gagcactgcacactggc

Primer Sequence

ID 5386 Right gacaagctgcggtttccac

5367 Left gaaggtacattgcccagctg

5368 Right gatgttccttcactgcagttctt Seq.

Primer Sequence ID

5369 Left gaggctgcaagagagatcct 5387 Left actgcacactggccgtc

5370 Right gttccttcactgcagttcttcag 5388 Right aagctgcggtttccactgg

5371 Left gtacattgcccagctgctg 5389 Left tgctgccccacaacgag

5372 Right caaagaagtccactgcagacaag 5390 Right gctctgcagggccatct

5373 Left ctcctgatgcccactccag 5391 Left gccccacaacgaggct

5374 Right cattccaacaagtgaaggagctc 5392 Right gatgttccttcactgcagtt

5375 Left ctctggaaggtacattgccca 5393 Left cacctgcagccctct

5376 Right actgcagttcttcagggcaa 5394 Right gcaaagaagtccactgcagac

5377 Left cacaacgaggctgcaagagagat 5395 Left tcctcctgatgccca

5378 Right ctgcagttcttcagggcaaaga 5396 Right gactgtcccattccaacaagtg

5379 Left tcctcctgatgcccactc 5397 Left ctctggaaggtacattgc

5380 Right cccattccaacaagtgaaggag 5398 Right ggcaaagaagtccactgca

5381 Left acaacgaggctgcaagaga 5399 Left gctgctgccccacaac

5382 Right atcttggagcctggggatgttc 5400 Right ttcagggcaaagaagtcc

5383 Left ccacaacgaggctgcaag 5401 Left cgtctcggtgcacagg

5384 Right atcttggagcctggggatg 5402 Right agacaagctgcggtttc

5455 Left tcgagtggccctggaatac 5465 Left ggaatacatctccagtg 5466 Right cattccaacaagtgaag

ALK Region3 126-175 bases

Seq. 5481 Left gaagaatcgggcgtccaga

Primer Sequence

ID 5482 Right gactgtcccattccaacaa

5467 Left gtccagacaacccatttcgag

5468 Right cattccaacaagtgaaggagctc 5483 Left tcgagtggccctggaata

5484 Right ggaagtcacaggcctgcc

5469 Left gagtggccctggaatacatctc

5470 Right cccattccaacaagtgaaggag 5485 Left catttcgagtggccctgga

i486 Right cccattccaacaagtgaag

5471 Left cgagtggccctggaatacat

5472 Right gactgtcccattccaacaagtg 5487 Left ggcgtccagacaacccat

5488 Right caagctggaggactgtc

5473 Left gacagtgctccagggaagaat

5474 Right caacaagtgaaggagctctgc 5489 Left gaagaatcgggcgtccagacaa

5490 Right gactgtcccattccaa

5475 Left ggaagcatggttggacagtg 5491 Left ccatttcgagtggccct

5476 Right gctctgcagggccatct 5492 Right caagctggaggact

5477 Left tgctccagggaagaatcgg 5493 Left ggaagaatcgggcgtcc

5478 Right tgaaggagctctgcaggg 5494 Right ggactgtcccattc

Seq. 5495 Left ccatttcgagtggc

Primer Sequence

ID 5496 Right ggcctgcccaag

5479 Left gcgtccagacaacccatttc

5480 Right gagctctgcagggcca

ALK Region3 176-225 bases

Seq. 5512 Right ctctcatcttctccctgggc

Primer Sequence

ID

5497 Left gagtggccctggaatacatctc 5513 Left aacgaggctgcaagagagat

5498 Right acatctggctctcatcttctcc 5514 Right gctctgcagggccatct

5499 Left gacagtgctccagggaagaat 5515 Left gcatggttggacagtgctc

5500 Right cattccaacaagtgaaggagctc 5516 Right tgaaggagctctgcaggg

5501 Left ggaagcatggttggacagtg Seq.

Primer Sequence

5502 Right cccattccaacaagtgaaggag ID

5517 Left atggttggacagtgctccag

5503 Left ccagacaacccatttcgagtg 5518 Right ggaagtcacaggcctgcc

5504 Right atctggctctcatcttctccctg

5519 Left agggaagcatggttggaca

5505 Left cactccagggaagcatggtt 5520 Right gagctctgcagggcca

5506 Right gactgtcccattccaacaagtg

5521 Left tgctccagggaagaatcgg

5507 Left gaggctgcaagagagatcct 5522 Right gactgtcccattccaacaa

5508 Right caacaagtgaaggagctctgc

5523 Left tcgagtggccctggaat

5509 Left cgagtggccctggaataca 5524 Right gcagtttccggcacatctg

5510 Right gcacatctggctctcatcttc

5525 Left ctcctgatgcccactccag

551 1 Left cgtccagacaacccatttcg 5526 Right cccattccaacaagtgaag

5650 Right gcagggccatcttg

5655 Left cccagaggctcctttctcc

5651 Left gctgactccaagcacaccat 5656 Right catcttggagcctg

5652 Right gactgtcccattccaa

5657 Left gcacaccatcctgagtcc

5653 Left ccgtggatgaggagcagc 5658 Right caagctggaggact

5654 Right tggaagtcacaggcc

ALK Region4 376-425 bases

Seq.

Primer Sequence

ID 5679 Left cccagaggctcctttctcc

5659 Left ctcctttctccttctcaacacct 5680 Right aaggagctctgcagggc

5660 Right gactgtcccattccaacaagtg

5681 Left ttctcaacacctcagctgactccaa

5661 Left gaggctcctttctccttctcaac 5682 Right gactgtcccattccaacaa

5662 Right cattccaacaagtgaaggagctc

5683 Left ccgtggatgaggagcagc

5663 Left ctccttctcaacacctcagct i684 Right cacaggcagtttccggc

5664 Right cccattccaacaagtgaaggag

5685 Left ctcagctgactccaagcaca

5665 Left c cagaggctc ctttctc cttc i686 Right caagctggaggactgtc

5666 Right caacaagtgaaggagctctgc

i687 Left tggggcagagcgttct

5667 Left acaccatcctgagtccgtg 1688 Right cccattccaacaagtgaag

5668 Right acatctggctctcatcttctcc

Seq. 5689 Left gcacaccatcctgagtcc

Primer Sequence

ID i690 Right ttctccctgggcaca

5669 Left ctccaagcacaccatcctga

5670 Right ctggctctcatcttctccctg 5691 Left ccgtggatgaggagc

5692 Right gcacatctggctctcatc

5671 Left accatcctgagtccgtggat

5672 Right gcacatctggctctcatcttc 5693 Left agagcgttctaaggagatg

5694 Right gactgtcccattccaa

5673 Left ctgactccaagcacaccatc

5674 Right ctctcatcttctccctgggc 5695 Left atgaggagcagcagt

5696 Right gcagtttccggcacat

5675 Left cttctcaacacctcagctgactc

5676 Right ggaagtcacaggcctgcc 5697 Left cccagaggctcctttc

5698 Right caagctggaggact

5677 Left cctgagtccgtggatgagg

5678 Right gcagtttccggcacatctg

ALK Region4 426-475 bases

Seq. 5705 Left cttctcaacacctcagctgactc

Primer Sequence

ID 5706 Right gcacatctggctctcatcttc

5699 Left ctcctttctccttctcaacacctc

5700 Right acatctggctctcatcttctcc 5707 Left acaccatcctgagtccgtg

5708 Right aagccatcttcaaagttgcagta

5701 Left gactccaagcacaccatcct

5702 Right agccatcttcaaagttgcagtaaaa 5709 Left agatggacttgctggatggg

5710 Right cattccaacaagtgaaggagctc

5703 Left gctcctttctccttctcaacac

5704 Right atctggctctcatcttctccctg 571 1 Left gaggctcctttctccttctcaa

5712 Right ctctcatcttctccctgggc 5725 Left cagagctggtcctggcg

5713 Left tttctccttctcaacacctcagct 5726 Right aacaagtgaaggagctctgca

5714 Right gcagtttccggcacatctg

5727 Left cagatggacttgctggat

5715 Left ccgcatcccctccgag 5728 Right gactgtcccattccaacaagtg

5716 Right cccattccaacaagtgaaggag

5729 Left ccatcctgagtccgtggat

5717 Left cctgagtccgtggatgagg 5730 Right gtccagccacagaagcc

5718 Right tccagccacagaagccatc

5731 Left ccgtggatgaggagcagc

Seq. 5732 Right tgtgccttgggtccagc

Primer Sequence

ID

5719 Left ccagaggctcctttctccttc 5733 Left ctcagctgactccaagcaca

5720 Right ggaagtcacaggcctgcc 5734 Right gcacatctggctctcatc

5721 Left ttctcaacacctcagctgactccaa 5735 Left tcctggcgccgcatc

5722 Right cacaggcagtttccggc 5736 Right tgaaggagctctgcaggg

5723 Left tccaagcacaccatcctgag 5737 Left gctgactccaagcacaccat

5724 Right aagccatcttcaaagttgca 5738 Right gcagtttccggcacat

ALK Region4 476-525 bases

Seq. 5758 Right tgacagtgtgccttgggtc

Primer Sequence

ID

5739 Left ctcctttctccttctcaacacct 5759 Left ccagaggctcctttctccttc

5740 Right agccatcttcaaagttgcagtaaaa 5760 Right gcagtttccggcacatctg

Seq.

Primer Sequence

ID 5761 Left aacacctcagctgactccaag

5741 Left gaggctcctttctccttctcaac 5762 Right agtgtgccttgggtccag

5742 Right aagccatcttcaaagttgcagta

5763 Left ctccaagcacaccatcctga

5743 Left gagtattcccctccactgcat 5764 Right agtgtggggtgacagtgtg

5744 Right cattccaacaagtgaaggagctc

5765 Left gtattcccctccactgcatgacctc

5745 Left tattcccctccactgcatgac 5766 Right tgaaggagctctgcaggg

5746 Right cccattccaacaagtgaaggag

5767 Left acaccatcctgagtccgtg

5747 Left agatggacttgctggatggg 5768 Right gtcctgacctgccattgag

5748 Right acatctggctctcatcttctcc

5769 Left ctcagctgactccaagcaca

5749 Left cactgcatgacctcaggaac 5770 Right ggggtgacagtgtgcctt

5750 Right gactgtcccattccaacaagtg

5771 Left cccagaggctcctttctcc

5751 Left gcatgacctcaggaaccaga 5772 Right aagccatcttcaaagttgca

5752 Right caacaagtgaaggagctctgc

5773 Left agagcgttctaaggagatg

5753 Left cttctcaacacctcagctgactc 5774 Right gcacatctggctctcatcttc

5754 Right tccagccacagaagccatc

5775 Left accatcctgagtccgtggat

5755 Left ctgactccaagcacaccatc 5776 Right gtccagccacagaagcc

5756 Right attgaggagtgtggggtgac

5777 Left tccactgcatgacctcagg

5757 Left ctccttctcaacacctcagct 5778 Right gactgtcccattccaacaa ALK Region4 750-1250 bases

Seq.

Primer Sequence

ID 5799 Left cctgattattttacatggaatctcacc

5779 Left tggaatctcacctggataatgaaag 5800 Right tgtcagacacatcgaggagag

5780 Right ttttgttctccactagcaccaag

5801 Left ctcctttctccttctcaacacctc

5781 Left gaatcaccaacaaacatgccttc 5802 Right ctccttcccggttttgttctc

5782 Right agccatcttcaaagttgcagtaaaa

5803 Left gctcctttctccttctcaacac

5783 Left cacctggataatgaaagactccttc 5804 Right tttgttctccactagcaccaaggac

5784 Right tggtcactgtagcactttcagaa

5805 Left aaagactccttccctttcctgt

5785 Left tggataatgaaagactccttccctt 5806 Right ctcaagactccacgaatgagc

5786 Right caatagagcatggtcttggtgg

5787 Left attttacatggaatctcacctggat

5788 Right gaaacgtagcactggtcactgtag 5807 Left aatcaccaacaaacatgccttctcc

5808 Right ctagcaccaaggacacgtttc

5789 Left atctcacctggataatgaaagactc

5790 Right cttcccggttttgttctccactag 5809 Left cttctccttctcctgattattttaca

5810 Right cttcc cggttttgttctc cac

5791 Left tccttctcctgattattttacatgga

5792 Right aagccatcttcaaagttgcagta 5811 Left tctcctgattattttacatggaatctc

5812 Right gtagcactggtcactgtagcacttt

Seq. 5813 Left gactggtcatagctccttgga

Primer Sequence

ID 5814 Right gcacatctggctctcatcttc

5793 Left acatggaatctcacctggataatga

5794 Right gaaacgtagcactggtcactg 5815 Left gagaagaaggcgtcggaagt

5816 Right cattccaacaagtgaaggagctc

5795 Left gactggtcatagctccttggaatc

5796 Right ggttttgttctccactagcacc 5817 Left gatcttcgggactggtcatagctc

5818 Right atctggctctcatcttctccctg

5797 Left cagatcttcgggactggtcatag

5798 Right acatctggctctcatcttctcc

841 Left TGCTGCAGGAGAGGGA

5829 Left CTTGTGGAGCCTCTTACACCCAGT 842 Right GGGATCCAGAGTCCCTTAT

5830 Right TGATAGCGACGGGAATTTTAACT

843 Left CTTGTGGAGCCTCTT

5831 Left GGAGAGGGAGCTTGTGGAG 844 Right CCTTGATAGCGACGGGAATTTTA

5832 Right GGGATCCAGAGTCCCTTATACA

845 Left GAGAAGCTCCCAA

5833 Left CTTGTGGAGCCTCTTACA 846 Right GCGACGGGAATTTTAACTTTCT

5834 Right CGACGGGAATTTTAACTTTCTCAC

847 Left GCACGCTGCGGA

5835 Left CTGCAGGAGAGGGAGCTTG 848 Right TGGGATCCAGAGTCCC

5836 Right AATTTTAACTTTCTCACCTTCTG

849 Left CCCAGTGGAGAAG

5837 Left CCCAGTGGAGAAGCTC 850 Right TCTCTTAATTCCTTGATAGCG

5838 Right TTCTCTTAATTCCTTGATAGCGACG

851 Left CGGAGGCTGCTG

5839 Left GAGAAGCTCCCAACCA 852 Right TGGGATCCAGAGT

5840 Right CTTAATTCCTTGATAGCGACGGGAA

Seq. 853 Left TGCTGCAGGAGAG

Primer Sequence

ID 854 Right CTTCTGGGATCCA

EGFR Regionl 176-225 bases

Seq. 872 Right TCCTTGATAGCGACGGGAATTT

Primer Sequence

ID

5855 Left TTGTGGAGCCTCTTACACCC 873 Left GCGCCACATCGTTCGGAA

5856 Right TTCTCTTAATTCCTTGATAGCGACG 874 Right AATTTTAACTTTCTCACCTTCTG

5875 Left GGATCGGCCTCTTCATGC

Seq. 876 Right GGGATCCAGAGTCCCTTAT

Primer Sequence

ID

5857 Left CAGGAGAGGGAGCTTGTGG 877 Left CCCAGTGGAGAAGCTC

5858 Right ACGGGAATTTTAACTTTCTCACCTT 878 Right ACGTAGGCTTCATCGAGGATTT

5879 Left CTTGTGGAGCCTCTTACA

5859 Left CTGCAGGAGAGGGAGCTTG 880 Right CCTTGTTGGCTTTCGGAGA

5860 Right TGATAGCGACGGGAATTTTAACTT

881 Left GAGAAGCTCCCAACCA

5861 Left CTTGTGGAGCCTCTTACACCCAGT 882 Right CACGTAGGCTTCATCGAGGA

5862 Right CTTAATTCCTTGATAGCGACGGGAA

883 Left CGAAGGCGCCACATCG

5863 Left CCACATCGTTCGGAAGCG 884 Right TGGGATCCAGAGTCCC

5864 Right CGACGGGAATTTTAACTTTCTCAC

885 Left TGCTGCAGGAGAGG

5865 Left CATCGTTCGGAAGCGCAC 886 Right AGATGTTGCTTCTCTTAATTCC

5866 Right CCTTGATAGCGACGGGAATTTTAA

887 Left GCACGCTGCGGA

5867 Left ATCGGCCTCTTCATGCGAA 888 Right TTCTCTTAATTCCTTGATAGCG

5868 Right GGGATCCAGAGTCCCTTATACA

889 Left CCCAGTGGAGAAG

5869 Left GAAGGCGCCACATCGTTC 890 Right GGCCATCACGTAGGCTTCAT

5870 Right GCGACGGGAATTTTAACTTTCT

891 Left CTTGTGGAGCCTCTT

5871 Left TGCTGCAGGAGAGGGAG 892 Right TCGGAGATGTTGCTTCT

5948 Right ATTGTCTTTGTGTTCCCGGACATA i 956 Right AGGAGGCAGCCG

5949 Left TCGCTATCAAGGAATTAAGAGAAGC I 957 Left TTAAAATTCCCGTCGCTATCAAGG

5950 Right CCAGGAGGCAGCCGAAG I 958 Right CGAAGGGCATGAG

5951 Left GGAAATCCTCGATGAAGCC I 959 Left GGAAATCCTCGATGAA

5952 Right GGGAGCCAATATTGTCTTTGTGT I 960 Right TGGGAGCCAATATTGTCTTTG

Seq.

Primer Sequence

ID I 961 Left CAACAAGGAAATCC

5953 Left CAAGGAATTAAGAGAAGCAACATCT I 962 Right TGGGAGCCAATATTGTCT

5954 Right GACATAGTCCAGGAGG

5955 Left CCGTCGCTATCAAGGAATTAAGAG

EGFR Region2 176-225 bases

Seq.

Primer Sequence

ID 5 983 Left TTAAAATTCCCGTCGCTATCAAGG

5963 Left GAAAGTTAAAATTCCCGTCGCTATC J 984 Right CCAGGAGGCAGCCGAAG

5964 Right GAGCCAATATTGTCTTTGTGTTCC

J 985 Left GAAGCAACATCTCCGAAAGCCAA

5965 Left GAAGGTGAGAAAGTTAAAATTCCCG J 986 Right CGATCTGCACACACCAGTT

5966 Right ATTGTCTTTGTGTTCCCGGACATAG

J 987 Left AATTCCCGTCGCTATCAAGGAATTA

5967 Left GTGAGAAAGTTAAAATTCCCGTCG J 988 Right TGGGAGCCAATATTGTCT

5968 Right AATATTGTCTTTGTGTTCCCGGAC

1 989 Left AAATCCTCGATGAAGCCT

5969 Left CCGTCGCTATCAAGGAATTAAGAG 5 990 Right CCTCCAAGTAGTTCATGCCCTTT

5970 Right GGGAGCCAATATTGTCTTTGTGT

1 991 Left TCAAGGAATTAAGAGAAGCAACATCT

5971 Left GAAATCCTCGATGAAGCCTACG J 992 Right GACATAGTCCAGGAGG

5972 Right GACGGTCCTCCAAGTAGTTCAT

Seq. 1 993 Left TATCAAGGAATTAAGAGAAGCAACA

Primer Sequence

ID 5 994 Right GGTACTGGGAGCCA

5973 Left TCGCTATCAAGGAATTAAGAGAAGC

5974 Right TGGGAGCCAATATTGTCTTTG 5 995 Left CCCAGAAGGTGAGAAAGTTAAAAT

5 996 Right AGGAGGCAGCCG

5975 Left AGAAGCAACATCTCCGAAAGC

5976 Right GATCTGCACACACCAGTTGAG 5 997 Left AGAAGCAACATCTCCGAA

5 998 Right CGATCTGCACACACCA

5977 Left GAAATCCTCGATGAAGCCTACGTGA

5978 Right CGACGGTCCTCCAAGTAGTT 5 999 Left GAAATCCTCGATGAAG

6 000 Right GCGACGGTCCTCCAAGTA

5979 Left CTCGATGAAGCCTACGTGATGG

5980 Right TCCTCCAAGTAGTTCATGCCC 6 001 Left GGCACGGTGTATAAGGGAC

6 002 Right CGAAGGGCATGAG

5981 Left CAACATCTCCGAAAGCCAACAA

5982 Right CACACACCAGTTGAGCAGG

EGFR Region2 226-275 bases

Seq. 6 005 Left GAAAGTTAAAATTCCCGTCGCTATC

Primer Sequence

ID _6 006 Right TGGGAGCCAATATTGTCTTTGTG

6003 Left AAGGTGAGAAAGTTAAAATTCCCGT

6004 Right GGAGCCAATATTGTCTTTGTGTTC 6 007 Left TCGCTATCAAGGAATTAAGAGAAGC

6 008 Right GACGGTCCTCCAAGTAGTTCAT 6025 Left CCCAGAAGGTGAGAAAGTTAAAAT

6009 Left GTGAGAAAGTTAAAATTCCCGTCGC 6 026 Right GTCTTTGTGTTCCCGGACATA

6010 Right CCAATATTGTCTTTGTGTTCCCG

6 027 Left AATTCCCGTCGCTATCAAGGAAT

6011 Left CCGTCGCTATCAAGGAATTAAGAG 6 028 Right CGATCTGCACACACCAGTT

6012 Right CCTCCAAGTAGTTCATGCCCTTT

6 029 Left AACATCTCCGAAAGCCAACAAG

6013 Left TCAAGGAATTAAGAGAAGCAACATC Γ6 030 Right AAGCGACGGTCCTCCAAGTA

6014 Right TCCTCCAAGTAGTTCATGCCC

6 031 Left GGCACGGTGTATAAGGGAC

6015 Left TTAAAATTCCCGTCGCTATCAAGG 6 032 Right CTTTGTGTTCCCGGACATAGTC

6016 Right GATCTGCACACACCAGTTGAG

6 033 Left TCGATGAAGCCTACGTGATGG

6017 Left GAAATCCTCGATGAAGCCTACG 6 034 Right GACATGCTGCGGTGTTTTCA

6018 Right TGTTTTCACCAGTACGTTCCTG

6 035 Left GAAATCCTCGATGAAGCCTACGTGA

6019 Left AGAAGGTGAGAAAGTTAAAATTCC 6 036 Right AGTACGTTCCTGGCTGCC

6020 Right ATATTGTCTTTGTGTTCCCGGAC

6 037 Left AGAAGCAACATCTCCGAAAGC

6021 Left TATCAAGGAATTAAGAGAAGCAACA 6 038 Right CAAGCGACGGTCCTCCAA

6022 Right CGACGGTCCTCCAAGTAGTT

6 039 Left AAAAGATCAAAGTGCTGGGCTC

Seq. 6 040 Right CCAGGAGGCAGCCGAAG

Primer Sequence

ID

6023 Left ATTCCCGTCGCTATCAAGGAATTAA ~ 041 Left GAAGCAACATCTCCGAAAGCCAAC

6024 Right CACACACCAGTTGAGCAGG 6 042 Right CTGCCAGGTCGCGGT

EGFR Region3 75-125 bases

Seq. ( 045 Left AAAGGGCATGAACTACTTGGAGGAC

Primer Sequence

ID ( 046 Right CACCCAGCAGTTTG

6043 Left CAAAGGGCATGAACTACTTGGAG ( 047 Left GCATGAACTACTTG

6044 Right CCAGCAGTTTGGCC ( 048 Right TTCCGCACCCAG

Seq.

Primer Sequence

ID

EGFR Region3 126-175 bases

Seq. ( 059 Left AAAGGGCATGAACTACTTG

Primer Sequence

ID J 060 Right GCCTCCTTCTGCATGGTATTCT

6049 Left CAAAGGGCATGAACTACTTGGAG Seq.

Primer Sequence

6050 Right CCTTCTGCATGGTATTCTTTCTCTT ID

( 061 Left GGCTCCCAGTACCTGCTC

6051 Left AAAGGGCATGAACTACTTGGAGGAC ( 062 Right CCAGCAGTTTGGCC

6052 Right CCTCCTTCTGCATGGTATTCTTTC

( 063 Left CAAAGGGCATGAACTAC

6053 Left CAACTGGTGTGTGCAGATCG ( 064 Right CACTTTGCCTCCTTCTGC

6054 Right CTGCATGGTATTCTTTCTCTTCCG

( 065 Left GCTCAACTGGTGTGTGCAGA

6055 Left CTCCCAGTACCTGCTCAACT ( 066 Right CACCCAGCAGTTTG

6056 Right CTTTCTCTTCCGCACCCAG

( 067 Left CAGATCGCAAAGGG

6057 Left GCTCAACTGGTGTGTGC ( 068 Right GCCTCCTTCTGCATGGTAT

6058 Right CATGGTATTCTTTCTCTTCCGCAC

( 069 Left CAAAGGGCATGAA 6070 Right GCCTCCTTCTGCATGG

EGFR Region3 176-225 bases

Seq. 090 Right TTTCTCTTCCGCACCCAG

Primer Sequence

ID

6071 Left CAAAGGGCATGAACTACTTGGAG 1 091 Left CAAAGGGCATGAACTACTTG

6072 Right CTCTGGTGGGTATAGATTCTGTGTA J 092 Right AGATTCTGTGTAAAATTGATTCCA

6073 Left CTATGTCCGGGAACACAAAGAC 1 093 Left TGGCTCCCAGTACCTG

6074 Right CCTTCTGCATGGTATTCTTTCTCTT J 094 Right CACTTTGCCTCCTTCTGC

6075 Left GACTATGTCCGGGAACACAAA 095 Left GACTATGTCCGGGAACAC

6076 Right CCTCCTTCTGCATGGTATTCTTTC J 096 Right GCCTCCTTCTGCATGG

6077 Left AAAGGGCATGAACTACTTGGAGGAC 1 097 Left CTTCGGCTGCCTCCTGG

6078 Right ACTCTGGTGGGTATAGATTCTGT 098 Right CCAGCAGTTTGGCC

6079 Left CTCCCAGTACCTGCTCAACT 1 099 Left AAAGGGCATGAACTAC

6080 Right CTGCATGGTATTCTTTCTCTTCCG J 100 Right ACTCTGGTGGGTATAGATTC

6081 Left CTCAACTGGTGTGTGCAGATC 1 101 Left CTGCTCAACTGGTGT

6082 Right GCCTCCTTCTGCATGGTATTCT J 102 Right TTCCAATGCCATCCACTT

6083 Left GGCTCCCAGTACCTGCTC 1 103 Left CCCTTCGGCTGCCTCC

6084 Right CATGGTATTCTTTCTCTTCCGCAC J 104 Right CACCCAGCAGTTTG

6085 Left GTCCGGGAACACAAAGACAATATT 1 105 Left ACAATATTGGCTCCCA

6086 Right GCCTCCTTCTGCATGGTAT J 106 Right ATCCACTTGATAGGCAC

6087 Left CTGCTCAACTGGTGTGTGC 107 Left CAGATCGCAAAGGG

6088 Right TTCCAATGCCATCCACTTGAT J 108 Right AGATTCTGTGTAAAATTGAT

Seq. 1 109 Left CAAAGGGCATGAAC

Primer Sequence

ID ( 110 Right CTCTGGTGGGTATAGA

6089 Left GTCCGGGAACACAAAGACAAT

EGFR Region3 226-275 bases

Seq. ( 120 Right CCTCCTTCTGCATGGTATTCTTTC

Primer Sequence

ID

6111 Left GTCCGGGAACACAAAGACAATATT 1 121 Left CTCCACCGTGCAGCTCAT

6112 Right ATTCCAATGCCATCCACTTGAT 122 Right CTGCATGGTATTCTTTCTCTTCCG

6113 Left CTCCCAGTACCTGCTCAACT 1 123 Left CGTGCAGCTCATCACGC

6114 Right CTCTGGTGGGTATAGATTCTGTGTA J 124 Right CATGGTATTCTTTCTCTTCCGCAC

6115 Left GCTCATCACGCAGCTCATG 1 125 Left CTATGTCCGGGAACACAAAGAC

6116 Right CCTTCTGCATGGTATTCTTTCTCTT J 126 Right GATTCTGTGTAAAATTGATTCCA

6117 Left CAACTGGTGTGTGCAGATCG 1 127 Left GCTCAACTGGTGTGTGCAG

6118 Right ACTCTGGTGGGTATAGATTCTGT J 128 Right CCGTAGCTCCAGACATCACT

6119 Left CAGCTCATCACGCAGCTC ( 129 Left CTATGTCCGGGAACACAAAGACAAT

6189 Left GAGAAGCTCCCAACC 193 Left TGTGGAGCCTCT

6190 Right TGGGAGCCAATATTGT t 194 Right TACTGGGAGCCA

6191 Left TCGGAAGCGCACG

6192 Right AGGAGGCAGCCG

EGFR Region4 376-425 bases

Seq.

Primer Sequence

ID 5215 Left GAGAAGCTCCCAACCAAG

6195 Left AGGAGAGGGAGCTTGTGGA 5216 Right CGACGGTCCTCCAAGTAGTT

6196 Right GAGCCAATATTGTCTTTGTGTTCC

5217 Left CTTGTGGAGCCTCTTACA

6197 Left GAAGGCGCCACATCGTTC 5218 Right CACACACCAGTTGAGCAGG

6198 Right AATATTGTCTTTGTGTTCCCGGAC

5219 Left CCCAGTGGAGAAGCTC

6199 Left TTGTGGAGCCTCTTACACCC 5220 Right AAGCGACGGTCCTCCAAGTA

6200 Right CTCCAAGTAGTTCATGCCCTTT

5221 Left CGCCACATCGTTCGGAA

6201 Left CCACATCGTTCGGAAGCG 5222 Right TGGGAGCCAATATTGTCT

6202 Right GGGAGCCAATATTGTCTTTGTGT

5223 Left CTCCCAACCAAGCTCT

6203 Left CTGCAGGAGAGGGAGCTTG 5224 Right CAAGCGACGGTCCTCCAA

6204 Right TTGTCTTTGTGTTCCCGGACATAG

5225 Left GAGAAGCTCCCAACC

6205 Left TGGAGCCTCTTACACCCAGT 5226 Right CGATCTGCACACACCAGTT

6206 Right TCCTCCAAGTAGTTCATGCCC

5227 Left TCTCTTGAGGATCTTGA

6207 Left CTCCCAACCAAGCTCTCTT 5228 Right GTACGTTCCTGGCTGCCA

6208 Right GACGGTCCTCCAAGTAGTTCAT

5229 Left GGATCGGCCTCTTCATGC

6209 Left TGCTGCAGGAGAGGGAG 5230 Right GACATAGTCCAGGAGG

6210 Right GATCTGCACACACCAGTTGAG

Seq. 5231 Left CTGGTGGTGGCCCTGG

Primer Sequence

ID 5232 Right AGGAGGCAGCCG

621 1 Left CATCGTTCGGAAGCGCAC

6212 Right TGGGAGCCAATATTGTCTTTG 5233 Left CCTCTTGCTGCTGGTGGT

5234 Right CGAAGGGCATGAG

6213 Left ATCGGCCTCTTCATGCGAA

6214 Right CCAGGAGGCAGCCGAAG

EGFR Region4 426-475 bases

Seq. t 242 Right GGGAGCCAATATTGTCTTTGTGT

Primer Sequence

ID

6235 Left ATCGGCCTCTTCATGCGAA 1 243 Left CTTGTGGAGCCTCTTACACCCAGT

6236 Right GAGCCAATATTGTCTTTGTGTTCC J 244 Right CCTCCAAGTAGTTCATGCCCTTT

6237 Left CCTCCTCTTGCTGCTGGT ( 245 Left CCTCTTGCTGCTGGTGGT

6238 Right AATATTGTCTTTGTGTTCCCGGAC J 246 Right ATTGTCTTTGTGTTCCCGGACATAG

6239 Left TTGTGGAGCCTCTTACACCC ( 247 Left GGAGAGGGAGCTTGTGGAG

6240 Right GACGGTCCTCCAAGTAGTTCAT ( 248 Right TCCTCCAAGTAGTTCATGCCC

6241 Left GAAGGCGCCACATCGTTC 1 249 Left CCACATCGTTCGGAAGCG

6371 Left AACATCTCCGAAAGCCAACAAG 6381 Left GAAGCAACATCTCCGAAAGCCAAC

6372 Right CATCAACTCCCAAACAGTCACCCC 6 382 Right CACCCCGTAGCTCCAGAC

6373 Left AAATCCTCGATGAAGCCTACGTGA 6 383 Left TTAAAATTCCCGTCGCTATCAAGG

6374 Right AAACAGTCACCCCGTAGCTC 6 384 Right ACTCTGGTGGGTATAGATTC

6375 Left AAAGATCAAAGTGCTGGGCTCCGG 6 385 Left CAGAAGGTGAGAAAGTTAAAATTCC

6376 Right TGCATGGTATTCTTTCTCTTCCG 6 386 Right CACTTTGCCTCCTTCTGC

6377 Left GGAAATCCTCGATGAAGCCT 6 387 Left GGGCTCCGGTGCGTT

6378 Right AAGGTCATCAACTCCCAAACAG 6 388 Right GCCTCCTTCTGCATGGTATTCT

6379 Left GAGAAAGTTAAAATTCCCGTCGCTA

6380 Right ATTCCAATGCCATCCACTT

EGFR Region6 476-525 bases

Seq. ( )400 Right GCCTCCTTCTGCATGGTATTC

Primer Sequence

ID

6389 Left GGAGCCTCTTACACCCAGT j )401 Left TCTCTTGAGGATCTTGA

6390 Right TTCTGCATGGTATTCTTTCTCTTCC ( )402 Right ATTCCAATGCCATCCACTTGAT

6391 Left CTCCCAACCAAGCTCTCTT j i403 Left CTCCCAACCAAGCTCT

6392 Right CTCCTTCTGCATGGTATTCTTTCTC j )404 Right GCCTCCTTCTGCATGGTA

6393 Left CTTGTGGAGCCTCTTACAC ( )405 Left CTGCAGGAGAGGGAGCTTG

6394 Right CATGGTATTCTTTCTCTTCCGCAC j )406 Right CCAGCAGTTTGGCC

6395 Left GAGAAGCTCCCAACCAAG j )407 Left GAGAAGCTCCCAACC

6396 Right CCTCCTTCTGCATGGTATTCTTT j v408 Right CACTTTGCCTCCTTCTGC

6397 Left GGAGAGGGAGCTTGTGGA

6398 Right TTTCTCTTCCGCACCCAG

6409 Left TGCTGCAGGAGAGGGA

( 3410 Right CACCCAGCAGTTTG

Seq.

Primer Sequence

ID 411 Left CCCAGTGGAGAAG

6399 Left CCCAGTGGAGAAGCTC $412 Right GCCTCCTTCTGCATG

EGFR Region6 526-575 bases

Seq. )421 Left CTCCCAACCAAGCTCTCTT

Primer Sequence

ID >422 Right ATTCCAATGCCATCCACTTGAT

6413 Left CTTGTGGAGCCTCTTACACCCAG

6414 Right CCTTCTGCATGGTATTCTTTCTCTT >423 Left GAGAAGCTCCCAACCAAG

>424 Right GCCTCCTTCTGCATGGTATTCT

6415 Left CAGGAGAGGGAGCTTGTGG

6416 Right CTGCATGGTATTCTTTCTCTTCCG 3425 Left TGCAGGAGAGGGAGC

3426 Right CTTTCTCTTCCGCACCCAG

6417 Left CTGCAGGAGAGGGAGCTTG

6418 Right CCTCCTTCTGCATGGTATTCTTTC 3427 Left CCCAGTGGAGAAGCTC

3428 Right GCCTCCTTCTGCATGGTAT

6419 Left CTTGTGGAGCCTCTTACAC Seq.

Primer Sequence

6420 Right CATGGTATTCTTTCTCTTCCGCAC ID

3429 Left TCTCTTGAGGATCTTGA 6430 Right AGATTCTGTGTAAAATTGATTCCA >437 Left CATCGTTCGGAAGCGCAC

438 Right CACCCAGCAGTTTG

6431 Left CTCCCAACCAAGCTCT

6432 Right CACTTTGCCTCCTTCTGC >439 Left CTTGTGGAGCCTCTTA

)440 Right GCCTCCTTCTGCATGG

6433 Left CCACATCGTTCGGAAGCG

6434 Right CCAGCAGTTTGGCC 441 Left CCCAGTGGAGAAG

>442 Right ATCCACTTGATAGGCAC

6435 Left GAGAAGCTCCCAACC

6436 Right ATTCCAATGCCATCCACTT >443 Left CTTGTGGAGCCTC

>444 Right CACTTTGCCTCCTTC

EGFR Region6 576-625 bases

Seq.

Primer Sequence

ID 465 Left GAGAAGCTCCCAACCAAG

6445 Left CTTGTGGAGCCTCTTACACCCAG J 466 Right AGATTCTGTGTAAAATTGATTCCA

6446 Right CTCTGGTGGGTATAGATTCTGTGTA

1 467 Left GCGCCACATCGTTCGGAA

6447 Left CCACATCGTTCGGAAGCG j 468 Right GCCTCCTTCTGCATGGTAT

6448 Right CCTTCTGCATGGTATTCTTTCTCTT

469 Left CGAAGGCGCCACATCG

6449 Left ATCGGCCTCTTCATGCGAA j 470 Right CACTTTGCCTCCTTCTGC

6450 Right CATGGTATTCTTTCTCTTCCGCAC

471 Left TGCTGCAGGAGAGGGAG

6451 Left GAAGGCGCCACATCGTTC j 472 Right ATTCCAATGCCATCCACTT

6452 Right CCTCCTTCTGCATGGTATTCTTTC

1 473 Left CCCAGTGGAGAAGCTC

6453 Left CTTGTGGAGCCTCTTACACC 474 Right CCCGTAGCTCCAGACATCAC

6454 Right ACTCTGGTGGGTATAGATTCTGT

1 475 Left TCTCTTGAGGATCTTGA

6455 Left CAGGAGAGGGAGCTTGTGG J 476 Right AAAGGTCATCAACTCCCAAACAG

6456 Right ATTCCAATGCCATCCACTTGAT

1 477 Left CTCCCAACCAAGCTCT

6457 Left CGTTCGGAAGCGCACG J 478 Right AAACAGTCACCCCGTAGCTC

6458 Right CTGCATGGTATTCTTTCTCTTCCG

Seq.

Sequence 1 479 Left GAGAAGCTCCCAACC

Primer

ID ( 480 Right ACCCCGTAGCTCCAGACAT

6459 Left CTGCAGGAGAGGGAGCTTG

6460 Right GCCTCCTTCTGCATGGTATTCT ( 481 Left CTTGTGGAGCCTCTTAC

( 482 Right ACTCTGGTGGGTATAGATTC

6461 Left CTCCCAACCAAGCTCTCTT

6462 Right CGTAGCTCCAGACATCACTCT ( 483 Left CTCTTGCTGCTGGTGGTG

( 484 Right CCAGCAGTTTGGCC

6463 Left GGATCGGCCTCTTCATGC

6464 Right CTTTCTCTTCCGCACCCAG

EGFR Region6 750-1250 bases

Seq.

Primer Sequence

ID ( 489 Left ACCAAAATTATAAGCAACAGAGGTG

6485 Left TTTGTGGAGAACTCTGAGTGCATA ( 490 Right CCTCCTTCTGCATGGTATTCTTT

6486 Right CTCTGGTGGGTATAGATTCTGTGTA

1 491 Left TATAAGCAACAGAGGTGAAAACAGC

6487 Left CAGGAGTCATGGGAGAAAACAAC J 492 Right ACTCTGGTGGGTATAGATTCTGT

6488 Right CCTTCTGCATGGTATTCTTTCTCTT

6508 Right CATTTCTATCAATGCAAGCCACG

6612 Right ttttgaaggcttgtaactgct ( 622 Right gttttcctgagtactcctac

Seq. ( 623 Left cccctgcctcattacctg

Primer Sequence

ID ( 624 Right tgctgtcacattcaacattt

6613 Left cctcattacctggctcactaa

6614 Right cattttcactgccacatcaccat ( 625 Left aagtcatcttcatcctcag

( 626 Right tgaagagtaggatattcacatg

6615 Left gaaatctccaggacctcagcgagaa

6616 Right ctgaggtgtaggtgctgtca ( 627 Left ctcagcgagaaaggaagtca

( 628 Right tttgaaggcttgtaact

6617 Left agtcatcttcatcctcagaag

6618 Right gcccatgaagagtaggatattc ( 629 Left cctcattacctggctcac

( 630 Right tgctgtcacattcaaca

6619 Left ctcagcgagaaaggaagtcatct

6620 Right catgtcgtgttttcctga ( 631 Left caggtttgtctgctacccc

( 632 Right catcaccatgcca

6621 Left aaatctccaggacctcagcg

BRAF Regionl 276-325 bases

Seq.

Primer Sequence

ID

6633 Left aagtcatcttcatcctcagaagaca

6634 Right ggaatagcccatgaagagtaggata ( 653 Left aagccttacagaaatctccagga

t 654 Right ggaatagcccatgaagagtagg

6635 Left actaactaacgtgaaagccttacag

6636 Right tgaagagtaggatattcacatgtcg t 655 Left tattgatgacttgattagagac ca

( 656 Right caacattttcactgccacatcac

6637 Left ctcattacctggctcactaactaac

6638 Right ttttgaaggcttgtaactgctgag ( 657 Left ttgattagagaccaaggatttcgtg

( 658 Right tgctgtcacattcaacatttt

6639 Left aacgtgaaagccttacagaaatctc

6640 Right atagcccatgaagagtaggatattc ( 659 Left gctcactaactaacgtgaaagcctt

( 660 Right ttttgaaggcttgtaactgct

6641 Left ttgatgacttgattagagaccaagg

6642 Right attttcactgccacatcaccat ( 661 Left cttacagaaatctccaggacctca

6662 Right tgaagagtaggatattcacatg

Seq. t 663 Left cccctgcctcattacctgg

Primer Sequence

ID t 664 Right gtaactgctgaggtgtaggtgctg

6643 Left gatttcgtggtgatggaggatc

6644 Right gctgtcacattcaacattttcactg t 665 Left aaatctccaggacctcagcgagaaa

t 666 Right cagttgtggctttgtggaat

6645 Left attacctggctcactaactaacgtg

6646 Right gcttgtaactgctgaggtgtag t 667 Left ctcagcgagaaaggaagtcatct

t 668 Right ggtaacaatagccagttgtg

6647 Left caaggatttcgtggtgatggag

6648 Right tcacattcaacattttcactgcca t 669 Left atgacttgattagagaccaaggatt

t 670 Right aacattttcactgccacat

6649 Left cctcattacctggctcactaact

6650 Right cttgtaactgctgaggtgtaggtg 671 Left ctaacgtgaaagc cttacagaaat

( 672 Right catgtcgtgttttcctgag

6651 Left ctggctcactaactaacgtgaaag

6652 Right ttgaaggcttgtaactgctgaggtg

6736 Right tgatatggagatggtgatacaag

( 745 Left actaacgtgaaagccttacagaaat

6737 Left ttacctggctcactaactaacgt ( 746 Right ctggagccctcacaccac

6738 Right ttggtctcaatgatatggagatg

( 747 Left aatctccaggacctcagcgagaaag

6739 Left gatttcgtggtgatggaggatc ( 748 Right ctgtcgtgcaatatctataagtttg

6740 Right ggaatagcccatgaagagtagg

( 749 Left gagaccgatcctcatcagctc

6741 Left ataaacacaatagaacctgtcaata ( 750 Right aacattttcactgccacatcaccat

6742 Right cttgtaactgctgaggtgtaggt

( 751 Left gaaatctccaggacctcagcgaga

6743 Left agaaatctccaggacctcagc ( 752 Right tgatcatctcaaatttggtctcaa

6744 Right cgtgcaatatctataagtttgatca

BRAF Regionl 426-475 bases

Seq.

Primer Sequence

ID ( 773 Left aagtcatcttcatcctcagaagaca

6753 Left actaacgtgaaagccttacagaaat ( 774 Right ggatgattgacttggcgtgtaag

6754 Right gcagtctgtcgtgcaatatctataa

t 775 Left gatcatcgaaatcaatttgggcaac

6755 Left ccttcaaaatccattccaattccac ( 776 Right cttgtaactgctgaggtgtaggt

6756 Right gctgtcacattcaacattttcactg

( 777 Left ctcactaactaacgtgaaagcctta

6757 Left attgatgacttgattagagaccaagg ( 778 Right gcagtctgtcgtgcaatatcta

6758 Right ggaatagcccatgaagagtaggata

( 779 Left gtccgtctccttcaaaatccatt

6759 Left actaactaacgtgaaagccttacag t 780 Right caacattttcactgccacatcac

6760 Right agtctgtcgtgcaatatctataagtt

t 781 Left attacctggctcactaactaacgtg

6761 Left gaagatcatcgaaatcaatttgggc ( 782 Right tgatcatctcaaatttggtctcaa

6762 Right ttttgaaggcttgtaactgctga

( 783 Left atattgatgacttgattagagacca

6763 Left tgcatataaacacaatagaacctgtc t 784 Right aatagcccatgaagagtaggatattc

6764 Right tgaagagtaggatattcacatgtcg

e 785 Left ctcagcgagaaaggaagtcatc

6765 Left caaaatccattccaattccacagc t 786 Right gaggtctctgtggatgattgactt

6766 Right cacattcaacattttcactgccac

t 787 Left ttacagaaatctccaggacctca

6767 Left aacgtgaaagccttacagaaatctc t 788 Right cttggcgtgtaagtaatccatgc

6768 Right gtgtaagtaatccatgccctgtg

Seq. 789 Left caaattctcaccagtccgtctc

Primer Sequence

ID 790 Right aacattttcactgccacatcaccat

6769 Left gatttcgtggtgatggaggatc

6770 Right gatatggagatggtgatacaagctg 791 Left ctggctcactaactaacgtgaaa

792 Right cgtgcaatatctataagtttgatcatct

6771 Left ctcattacctggctcactaactaac

6772 Right cgtgcaatatctataagtttgatca

BRAF Regionl 476-525 bases

Seq. t 795 Left tcactaactaacgtgaaagccttac

Primer Sequence

ID t 796 Right ttattactcttgaggtctctgtgga

6793 Left cttgattagagaccaaggatttcgt

6794 Right gatatggagatggtgatacaagctg t 797 Left gaagatcatcgaaatcaatttgggc

798 Right ggaatagcccatgaagagtaggata

6923 Left cacagggcatggattactta ( 932 Right cacaaaatggatccag

6924 Right tccagtcatcaattcatacaga

( 933 Left ttgagac caaatttgagatgatc

6925 Left gagatgatcaaacttatagatattgc ( 934 Right attctgatgacttctgg

6926 Right tctgactgaaagctgtatg

( 935 Left ctccagcttgtatcaccatctc

6927 Left tgtatcaccatctccatatcattga ( 936 Right tgccatccacaaa

6928 Right tgccatccacaaaatg

( 937 Left cacagggcatggat

6929 Left cacagggcatggattac ( 938 Right acagaacaattccaaatgca

6930 Right caattccaaatgcatatacatct

( 939 Left gagatgatcaaacttatagatat

6931 Left c cage ttgtatcac catctc cat ( 940 Right tttatcttgcattctga

BRAF Region2 276-325 bases

Seq.

Primer Sequence

ID ( 961 Left ttgagaccaaatttgagatgatc

6941 Left cagcttgtatcaccatctccatatc ( 962 Right acagaacaattccaaatgcatataca

6942 Right tctgactgaaagctgtatggatttt

( 963 Left cacagggcatggattactta

6943 Left atcac catctc catatcattgagac ( 964 Right ggtccctgttgttgatgtttgaa

6944 Right atgcatatacatctgactgaaagct

( 965 Left tgatcaaacttatagatattgcacgac

6945 Left acttatagatattgcacgacagactg ( 966 Right ctgtccagtcatcaattcata

6946 Right ctgtccagtcatcaattcatacaga

( 967 Left ccacaactggctattgttacc

6947 Left cacagggcatggattacttacac ( 968 Right attctgatgacttctggtgc

6948 Right aaaattatctggtccctgttgttga

( 969 Left ttgagac caaatttgagatg

6949 Left caaacttatagatattgcacgacaga ( 970 Right acaattccaaatgcatatacatctg

6950 Right ccaaatgcatatacatctgactgaa

i 971 Left ctccagcttgtatcaccatctcc

6951 Left gcatggattacttacacgccaa t 972 Right tctgactgaaagctgtatg

6952 Right attatctggtccctgttgttgatgt

t 973 Left gagatgatcaaacttatagat

6953 Left cacaactggctattgttacccag t 974 Right acagaacaattccaaatgcatat

6954 Right attctgatgacttctggtgccat

t 975 Left cacagggcatggattac

6955 Left gcttgtatcaccatctccatatcatt t 976 Right aaaattatctggtccctgttgt

6956 Right tctgactgaaagctgtatggat

( 977 Left tatcctactcttcatgggctattcc

6957 Left gcacgacagactgcacag t 978 Right cacaaaatggatccag

6958 Right cctgttgttgatgtttgaataaggt

Seq. t 979 Left attccacaaagccacaactg

Primer Sequence

ID t 980 Right tgccatccacaaaatg

6959 Left gagatgatcaaacttatagatattgc

6960 Right attccaaatgcatatacatctgact

BRAF Region2 326-375 bases

Seq. t 983 Left atcaccatctccatatcattgagac

Primer Sequence

ID 984 Right ctgtccagtcatcaattcatacaga

6981 Left ttatagatattgcacgacagactgc

6982 Right aaaattatctggtccctgttgttga ί 985 Left ccagcttgtatcaccatctccatat

986 Right tccaaatgcatatacatctgactga 003 Left agatgatcaaacttatagatattgcac

6987 Left cacaactggctattgttacccag 004 Right ggtccctgttgttgatgtttgaa

6988 Right tctgactgaaagctgtatggatttt

005 Left gcttgtatcaccatctccatatcatt

6989 Left tatcctactcttcatgggctattcc 006 Right ctgtccagtcatcaattcatac

6990 Right attctgatgacttctggtgccat

007 Left ccacaactggctattgttacc

6991 Left aacttatagatattgcacgacagac 008 Right acaattccaaatgcatatacatctgac

6992 Right tatctggtccctgttgttgatgttt

009 Left tgaatatcctactcttcatgggcta

6993 Left gcatggattacttacacgccaa 010 Right attctgatgacttctggtgc

6994 Right ttttggacagttactccgtacctta

on Left agatgatcaaacttatagatattg

6995 Left cacagggcatggattacttacac 012 Right c ctgttgttgatgtttgaataaggt

6996 Right ccgtaccttactgagatctggag

013 Left gcacgacagactgcacag

6997 Left tcaaacttatagatattgcacgaca 014 Right aggtatcctcgtcccaccata

6998 Right ggtatcctcgtcccaccataaaa

015 Left ttgagac caaatttgagatgatc

6999 Left ctccagcttgtatcaccatctcc 016 Right aaaattatctggtccctgttgt

7000 Right acagaacaattccaaatgcatataca

017 Left ccagtggtgtgagggctc

Seq. 018 Right tctgactgaaagctgtatggat

Primer Sequence

ID

7001 Left gctattccacaaagccacaac 019 Left cacagggcatggattactta

7002 Right aatgcatatacatctgactgaaagc 020 Right caggtatcctcgtcccacc

BRAF Region2 376-425 bases

Seq. 036 Right cttttggacagttactccgtacc

Primer Sequence

ID

7021 Left cagcttgtatcaccatctccatatc 037 Left ctccagcttgtatcaccatctcc

7022 Right aaaattatctggtccctgttgttga 038 Right attatctggtccctgttgttgatgt

7023 Left tgaatatcctactcttcatgggcta 039 Left acgacatgtgaatatcctactct

7024 Right tctgactgaaagctgtatggatttt 040 Right tccaaatgcatatacatctgactga

7025 Left tatcctactcttcatgggctattcc 041 Left tcagcagttacaagccttcaaaa

7026 Right ctgtccagtcatcaattcatacaga 042 Right ttctgatgacttctggtgccat

7027 Left cgacatgtgaatatcctactcttca 043 Left tgatcaaacttatagatattgcacg

7028 Right atgcatatacatctgactgaaagct 044 Right ccgtaccttactgagatctggag

7029 Left acttatagatattgcacgacagact 045 Left cacagggcatggattacttacac

7030 Right ttttggacagttactccgtacctta 046 Right ggcttttggacagttactccg

Seq.

Primer Sequence

ID 047 Left gcttgtatcaccatctccatatcatt

703 1 Left atcaccatctc catatcattgagac 048 Right aaaattatctggtccctgttgt

7032 Right ggtccctgttgttgatgtttgaa

049 Left gagatgatcaaacttatagatattgc

7033 Left ccacaactggctattgttaccc 050 Right ggtatcctcgtcccaccataaaa

7034 Right cctgttgttgatgtttgaataaggt

051 Left tgaatatcctactcttcatggg

7035 Left tcaaacttatagatattgcacgaca 052 Right acagaacaattccaaatgcatataca

71 16 Right ggtatcctcgtcccaccataaaa

129 Left ccagcttgtatcaccatctccatat

7117 Left catggtgatgtggcagtgaaaat 130 Right cactctgccattaatctcttcat

71 18 Right tctgactgaaagctgtatggatttt

13 1 Left tggaacagtctacaagggaaagt

71 19 Left tcagcagttacaagccttcaaaa 132 Right ttctgatgacttctggtgccat

7120 Right attatctggtccctgttgttgatgt

133 Left aacagtctacaagggaaagtggc

7121 Left gtctacaagggaaagtggcatg 134 Right attccaaatgcatatacatctgact

7122 Right atgcatatacatctgactgaaagct

135 Left ctacacctcagcagttacaagcc

7123 Left aacttatagatattgcacgacagac 136 Right ggtccctgttgttgatgtttgaa

7124 Right aatagaggcgagaatttgggga

137 Left tgacagcacctacacctcag

7125 Left ccacaactggctattgttaccc 138 Right acagaacaattccaaatgcatataca

7126 Right cttttggacagttactccgtacc

139 Left cacagggcatggattacttacac

7127 Left acctacacctcagcagttaca 140 Right aaggagggttctgatgcactg

7128 Right cctgttgttgatgtttgaataaggt

BRAF Region2 750-1250 bases

Seq.

Primer Sequence

ID 161 Left tgacttgattagagaccaaggattt

7141 Left taaatttcaccagcgttgtagtaca 162 Right gagaatttggggaaagagtggtc

7142 Right aaaattatctggtccctgttgttga

163 Left aattatgaccaacttgatttgctgt

7143 Left catgtggttataaatttcaccagcg 164 Right ggtccctgttgttgatgtttgaa

7144 Right ctgtccagtcatcaattcatacaga

165 Left cttgatttgctgtttgtctccaag

7145 Left cttgattagagaccaaggatttcgt 166 Right ggtatcctcgtcccaccataaaa

7146 Right ttttggacagttactccgtacctta

167 Left cctcattacctggctcactaactaa

7147 Left tatgaccaacttgatttgctgtttg 168 Right aatagaggcgagaatttgggga

7148 Right cctgttgttgatgtttgaataaggt

169 Left caaaatccattccaattccacagc

7149 Left gaagatcatcgaaatcaatttgggc 170 Right gcatatagactaaaatcctctgtttgg

7150 Right agtggtctctcatctcttttctttt

171 Left cgttgtagtacagaagttccactg

7151 Left ccttcaaaatccattccaattccac 172 Right tgttgttgatgtttgaataaggtaac

7152 Right attatctggtccctgttgttgatgt

173 Left gtctccttcaaaatccattccaatt

7153 Left tgatgacttgattagagaccaagga 174 Right ggcttttggacagttactccg

7154 Right cttttggacagttactccgtacc

175 Left cgtctccttcaaaatccattcca

7155 Left tggttataaatttcaccagcgttgt 176 Right agcatatagactaaaatcctctgtt

7156 Right acagaacaattccaaatgcatataca

Seq. 177 Left caacttgatttgctgtttgtctcc

Primer Sequence

ID 178 Right aggtatcctcgtcccaccata

7157 Left ttgtagtacagaagttccactgatg

7158 Right ccgtaccttactgagatctggag 179 Left tgcatataaacacaatagaacctgt

180 Right gcgagaatttggggaaagagtg

7159 Left gatcatcgaaatcaatttgggcaac

7160 Right aatagaggcgagaatttggggaaag

Seq. Primer Sequence

ID ??7 Left TCAGCGGCTCCC

7211 Left CCAGGTGCGGGAGAGAG 228 Right AGGAATCCTCTATTGTT

7212 Right ATCATATTCGTCCACAAAATGATTC

KRAS Regionl 176-225 bases

Seq. Primer Sequence ID 7229 Left ATTTCGGACTGGGAGCGAG ID

7230 Right ATTGTTGGATCATATTCGTCCACAA 247 Left AGCGGCTCCCAGGTGC

248 Right GCTGTGTCGAGAATATCCAAGAG

7231 Left CTCGGCCAGTACTCCCG

7232 Right TGGATCATATTCGTCCACAAAATGA 249 Left TCCCAGGTGCGGGAGAG

250 Right CTCTTGACCTGCTGTGTCGA

7233 Left CAGGTGCGGGAGAGAGG

7234 Right TCGAGAATATCCAAGAGACAGGTTT 251 Left CCAGAGGCTCAGCGG

252 Right CCTGCTGTGTCGAGAATATCCAA

7235 Left CTCAGCGGCTCCCAGG

7236 Right TCCATCAATTACTACTTGCTTCCTG 253 Left GCTCGGCCAGTACTC

254 Right TCATATTCGTCCACAAAATGATTCT

7237 Left GCCAGTACTCCCGGCC

7238 Right ATTCGTCCACAAAATGATTCTGAAT 255 Left CCCCGCCATTTCG

256 Right TCCTCTATTGTTGGATCATATTCGT

7239 Left TGCGGGAGAGAGGCCT

7240 Right TGTGTCGAGAATATCCAAGAGACAG 257 Left TCAGCGGCTCCC

258 Right TTGACCTGCTGTGTCGAGAATATC

7241 Left GCACTGAAGGCGGCG

7242 Right TCAATTACTACTTGCTTCCTGTAGG 259 Left GGCACTGAAGGCG

260 Right ATCCTCTATTGTTGGATCATATT

7243 Left GCTCCCAGGTGCGGGA

7244 Right GTTTCTCCATCAATTACTACTTGCT 261 Left CATTTCGGACTGGG

262 Right AGGAATCCTCTATTGTTGGA

7245 Left CATTTCGGACTGGGAGC 229

7246 Right TCTATTGTTGGATCATATTCGTCCA 230 Left CCAGAGGCTCAG

Right GTTTCTCCATCAATTACTACTT

Seq. Primer Sequence

ERAS Region2 75-125 bases

Seq. 242 Right TCCCCAGTCCTCATGTA

Primer Sequence

ID

7231 Left CTTGGATATTCTCGACACAGCAG 243 Left CTACAGGAAGCAAGTAGTAATTGA

7232 Right TTATGGCAAATACACAAAGAAAGCC 244 Right TCCCCAGTCCTCAT

7233 Left AACCTGTCTCTTGGATATTCTCGAC 245 Left AGAAACCTGTCTCTTGGATATT

7234 Right AAATACACAAAGAAAGCCCTCCC 246 Right GATTTAGTATTATTTATGGC

7235 Left GGAAGCAAGTAGTAATTGATGGAGA 247 Left AGAAACCTGTCTCTTGGA

7236 Right AATACACAAAGAAAGCCCTCCCCAG 248 Right GGCAAATACACAAAGAAA

7237 Left AGAAACCTGTCTCTTGGATATTCTC 249 Left AGAAACCTGTCTCTT

7238 Right TCCCCAGTCCTCATGTACTG 250 Right TTTATGGCAAATACACAAAG

7239 Left TGTCTCTTGGATATTCTCGACACAG 251 Left CAAGTAGTAATTGATGG

7240 Right AAAGAAAGCCCTCCCCAGTCC 252 Right ATGGCAAATACACA

Seq.

Primer Sequence

ID

7241 Left AGCAAGTAGTAATTGATGGAGAAAC

RAS Region2 126-175 bases

Seq. Primer Sequence ID 7253 Left TTTGTGGACGAATATGATCCAACAA ID

7254 Right TTATGGCAAATACACAAAGAAAGCC 273 Left CAGGAAGCAAGTAGTAATTGATGGA

274 Right GATTTAGTATTATTTATGGC

Left TGGACGAATATGATCCAACAATAGA

7255 G 275 Left AGAAACCTGTCTCTTGGATATTCT

7256 Right AAATACACAAAGAAAGCCCTCCC 276 Right AATTTGTTCTCTATAATGGT

7257 Left CTTGGATATTCTCGACACAGCAG 277 Left CTACAGGAAGCAAGTAGTAATTG

7258 Right AGGTACATCTTCAGAGTCCTTAAC 278 Right TTTATGGCAAATACACAAAG

7259 Left TTCAGAATCATTTTGTGGACGAATA 279 Left AGAAACCTGTCTCTTGGATAT

7260 Right AATACACAAAGAAAGCCCTCCCCAG 280 Right GTCCTTAACTCTTTTAATTTG

7261 Left CCTTGACGATACAGCTAATTCAGAA 281 Left ACAGCTAATTCAGAATCATTTTGTGG

7262 Right TCCCCAGTCCTCATGTACTG 282 Right TCCCCAGTCCTCAT

7263 Left CATTTTGTGGACGAATATGATCCAA 283 Left AGAAACCTGTCTCTTGG

7264 Right GAAAGCCCTCCCCAGTCC 284 Right ACTCTTTTAATTTGTTCTCT

7265 Left TGTCTCTTGGATATTCTCGACACAG 285 Left CTACAGGAAGCAAGTAGTAA

7266 Right GTCCTTAACTCTTTTAATTTGTTC 286 Right TATAATGGTGAATATCTTC

7267 Left AACCTGTCTCTTGGATATTCTCGA 287 Left TCCAACAATAGAGGATTCC

7268 Right TAATTTGTTCTCTATAATGGTGAA 288 Right TTTATGGCAAATACACA

7269 Left GCAAGTAGTAATTGATGGAGAAAC 289 Left AGAAACCTGTCTCT

7270 Right TTTATGGCAAATACACAAAGAAA 290 Right GTCCTTAACTCTTTTAAT

7271 Left TGACGATACAGCTAATTCAGAATCA

7272 Right TCCCCAGTCCTCATGTA

Seq. Primer Sequence

RAS Region2 176-225 bases

Seq.

Primer Sequence

ID 301 Left TACAGGAAGCAAGTAGTAA

7291 Left CCTTGACGATACAGCTAATTCAGAA 302 Right CCATAGGTACATCTTCAGAGTCCTT

7292 Right TTATGGCAAATACACAAAGAAAGCC

303 Left GCTAATTCAGAATCATTTTGTGGACG

7293 Left TTCAGAATCATTTTGTGGACGAATA 304 Right TTTATGGCAAATACACAAAG

7294 Right GCAAATACACAAAGAAAGCCCTC

305 Left GCAAGTAGTAATTGATGGAGAA

7295 Left CAGGAAGCAAGTAGTAATTGATGGA 306 Right GTACATCTTCAGAGTCCTTAAC

7296 Right CCATAGGTACATCTTCAGAGTC

307 Left GTGGACGAATATGATCCAACAATAG

7297 Left TGACGATACAGCTAATTCAGAATCA 308 Right AACTCTTTTAATTTGTTCTC

7298 Right TTTATGGCAAATACACAAAGAAA

309 Left CTACAGGAAGCAAGTAGTAATTGA

310 Right CCATAGGTACATCTTCAGA

Seq.

Primer Sequence

ID 311 Left TCATTTTGTGGACGAATATGATCCA

Left GGACGAATATGATCCAACAATAGAG 312 Right GATTTAGTATTATTTATGGC

7299 G

7300 Right TTTAATTTGTTCTCTATAATGGTG 313 Left ACAGCTAATTCAGAATCATTTTGTGG

7432 Right tgttctagaaggcaaatcacatttat 7445 Left GGCGGCGAAGGT

7446 Right atacacaaagaaagccctccccagt

7433 Left AGCGGCTCCCAGGTGC

7434 Right gcctgttttgtgtctactgttc 7447 Left GCGAAGGTGGCG

7448 Right caaagaaagccctccccagtcct

7435 Left GCCAGTACTCCCGGCC

7436 Right taatttgttctctataatggtgaa 7449 Left TCAGCGGCTCCC

7450 Right tctactgttctagaaggcaaat

7437 Left CATTTCGGACTGGGAGC 7419

7438 Right gtccttaactcttttaatttgttc 7420 Left CGGAGGCAGCAG

Right tttatggcaaatacacaaagaaa

7439 Left CCAGAGGCTCAGCGG 7421

7440 Right ccataggtacatcttcagagtc 7422 Left CCCCGCCATTTCG

Right gtccttaactcttttaatttg

7441 Left GCTCGGCCAGTACTC 7423

7442 Right ttatggcaaatacacaaagaaagccctc 7424 Left GGCACTGAAGGCG

Right ccataggtacatcttcaga

7443 Left AAGGTGGCGGCG 7425

7444 Right aaatacacaaagaaagccctcccc 7426 Left CATTTCGGACTGGG

Right actcttttaatttgttctct

KRAS Region3 426-475 bases

Seq. 7446 Right ttgctgatgtttcaataaaaggaat

Primer Sequence

ID

7427 Left ATTTCGGACTGGGAGCGAG 7447 Left CATTTCGGACTGGGAGC

7428 Right actgttctagaaggcaaatcacatt 7448 Right tgttctagaaggcaaatcacatttat

7429 Left GCTCGGCCAGTACTCCC 7449 Left GGCACTGAAGGCG

7430 Right ccataggtacatcttcagagtcctt 7450 Right ttcttgctaagtcctgagcctgttt

7431 Left GTGCGGGAGAGAGGCC 7451 Left GCTCGGCCAGTACT

7432 Right gtcttgtctttgctgatgtttcaat 7452 Right ccataggtacatcttcagagtc

7433 Left GCCAGTACTCCCGGCC 7453 Left AAGGTGGCGGCG

7434 Right taggtacatcttcagagtccttaac 7454 Right gtccttaactcttttaatttgttc

7435 Left CTCAGCGGCTCCCAGG 7455 Left GGCGGCGAAGGT

7436 Right agcctgttttgtgtctactgttc 7456 Right taatttgttctctataatggtgaa

7437 Left GCACTGAAGGCGGCG 7457 Left TCAGCGGCTCCC

7438 Right tctactgttctagaaggcaaatcac 7458 Right gaattccataacttcttgctaag

7439 Left CCAGGTGCGGGAGAGAG 7459 Left CATTTCGGACTGGG

7440 Right cttcttgctaagtcctgagcct 7460 Right tctactgttctagaaggcaaat

7441 Left GCTCCCAGGTGCGGGA 7461 Left CCCCGCCATTTCG

7442 Right tgctgatgtttcaataaaaggaattcc 7462 Right ccataggtacatcttcaga

7443 Left CCAGAGGCTCAGCGG 7463 Left GCGAAGGTGGCG

7444 Right gttttgtgtctactgttctagaagg 7464 Right gtccttaactcttttaatttg

Seq.

Primer Sequence

ID 7465 Left CCAGAGGCTCAG

7445 Left AGCGGCTCCCAGGTGC 7466 Right agcctgttttgtgtctactg

Table 15: IRID Hybridization-based capture probes for NGS panel

7624 TTAGCCATTGGTCAAGATCTTCACAAAAGGGTTTGATAAGTTCTAGCTGTGGTGGGTTA TGGTCTTCAAAAGGATATTGTGCAACT

7625 AAGTGAAGATGACAATCATGTTGCAGCAATTCACTGTAAAGCTGGAAAGGGACGAACT GGTGTAATGATATGTGCATATTTATTACATCGGGGCAA

7626

CCCATAGAAATCTAGGGCCTCTTGTGCCTTTAAAAATT

7627

CTCTTTTTTTTCTGTCCACCAGGGAGTAACTA

7628 ACATCATCTTGTGAAACAACAGTGCCACTGGTCTATAATCCAGATGATTCTTTAACAGG TAGCTATAATAATACACATAGCGCCTCTGACTGGGAAT

7629 TTTGAAACTATTCCAATGTTCAGTGGCGGAACTTGCAGTAAGTGCTTGAAATTCTCATC CTTCCATGTATTGGAACAGTTTTCTTAACCATATCTAGAAGTTTACATAAAAATTTAGA AAGAAATTT

7630

AAGGCATTTCCTGTGAAATAATAC

7631 CGTGTGGGTCCTGAATTGGAGGAATATATCTTCACCTTTAGCTGGCAGACCACAAACTG AGGATCTGCATGGTTAAATACATACCAGTATT

7632 GACGGGAAGACAAGTTCATGTACTTTGAGTTCCCTCAGCCGTTACCTGTGTGTGGTGAT ATCAAAGTAGAGTTCT

7633 TTATAGTTCCTTACATGTCATAAAATAAAATATAGCTTTTAATCTGTCCTTATTTTGGAT

7634 TAACATAGGTGACAGATTTTCTTTTTTAAAAAAATAAAACATCATTAATTAAATATGTC ATTTCATTTCTTTTTCTTTTCTTTTTTTTTTTTTTTAGGACAAAATGTTTCACTTTTGGGTA

7635

TGTATTTA

7636 TATGTGATCAAGAAATCGATAGCATTTGCAGTATAGAGCGTGCAGATAATGACAAGGA ATATCTAGTACTTACTTTAACAAAAAATGATCTTGACAAAGCAAATAAAGACAA

7637 TACAAGTCAACAACCCCCACAAAATGTTTAATTTAACTGACCTTAAAATTTGGAGAAAA GTATCGGTTGGCTT

7638 AGAGAAACCTTTATCTGTATCAAAGAATGGTCCTGCACCAGTAATATGCATATTAAAAC AAGATTTACCTCTATTGTTGGATCA

7639 CTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGAC GAATATG 7640 GCTCCAACTACCACAAGTTTATATTCAGTCATTTTCAGCAGGCCTTATAATAAAAATAA TGAAAATGTGACTATATTAGAACATGTCACACATAAGGTTAATACA

7641 GCCCATGCCGTGGCTGCTGGTCCCCCTGCTGGGCCATGTCTGGCACTGCTTTCCAGCAT GGTGAGGGCTGAGGTGACCCTTGTCTCTGTGTTCTTGTCCCCCCCAGCTTGTGGAGCCT CTTACACC

7642 CAGAGGCCTGTGCCAGGGACCTTACCTTATACACCGTGCCGAACGCACCGGAGCCCAG TTCTCCACTGG

7643 CCTTCGGGGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGGCACCAT CTCACAATTGCCAGTTAACGTCTTCCT

7644 TCTCTTAATTCCTTGATAGCGACGGGAATTTTAACTTTCTCACCTTCTGGGATCCAGAGT CCCTATGACAGAGAGAGAAG

7645 AAGCAACATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTG TGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGC

7646 ATTTTGAAACTCAAGATCGCATTCATGCGTCTTCACCTGGAAGGGGTCCATGTGCCCCT CCTTCTGGCCACCATGCGAAG

7647 GAGGTGAGGCAGATGCCCAGCAGGCGGCACACGTGGGGGTTGTCCACGCTGGCCATCA CGTAGGCTTCCTGGAGGGAGGGAGAGGCACGTCAGTGTGGC

7648 CCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGG GAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGTGTGCAGATCGCAA AGGTAATCAGGGAAGGGA

7649 CGTGGAGAGGCTCAGAGCCTGGCATGAACATGACCCTGAATTCGGATGCAGAGCTTCT TCCCATGATGATCTGTCCCTCACAGCAGGGT

7650 GCGGTGTTTTCACCAGTACGTTCCTGGCTGCCAGGTCGCGGTGCACCAAGCGACGGTCC TCCAAGTAGTTCATGCCCTGAAACAGAGAAGACCCTGC

7651 CAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTGCTGGGTGCGGAAGAGAAAG AATACCATGCAGAAGGAGGCAAAGTAAGGAGGTGGCTTTAGGTCAGCCAGCATTT

7652 GATTATCTGTCTGGCCCCAGACCTGGAGCTTTCTTTCCATGATAGGAGTACTTCTTTGGG TTGACTTCTCTGGTGACAG

7653 CTCATCAAGCTCTAGCTCCTCCAGCTTCTTCTGCAAGGCCTCCAAGTTGGTCCTGTTCCA AAGTGGGGAGCACAAGTCAATACT

7654 GCAGCAGCGAAAGCGCCTTGAGGCCTTTCTTACCCAGAAGCAGAAGGTGGGAGAACTG AAGGATGACGACTTTGAGAAGATCAGTGAGCTGGGGGCTGGCAATGGCGGTGTGGTGT TCAAG

7655 CCCCAGGCTTCTAAGTACCCTGAGAAATAATCCAATTACCTGTTAATCAAGGCAAACTC ACCTTTCTGGCCATGACCAGGCCAGAAGGCTTGTGGGAGACC

7656 AAAATACTATAGTTGAGACCTTCAATGACTTTCTAGTAACTCAGCAGCATCTCAGGGCC AAAAATTTAATCAGTGGAAAAATAGCCTCA

7657 CTACAGTGAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATT TTGTGGATGGTAAGAATT

7658 GCTAGACCAAAATCACCTATTTTTACTGTGAGGTCTTCATGAAGAAATATATCTGAGGT GTAGTAAGTAAAGGAAAACAGTAGAT

7659 ATATATACATAAGAGAGAAGGTTTGACTGCCATAAAAAATATCTAATTTATGACAATA AAAACCTTACTTTATTTGGATTTGAT

7660 ATATATAATAGCTTTTCTTCCATCTCTTAGGAAACTCCATGCTTAGAGTTGGAGTTTGAC TGGTTCAGCAGTGTGGTAAAGT

7661 TTATAATTGATACTTAATAAACTCAGTGATTTGCCTTACCAGTCCTGCGTGGGAATAGC TAAATCCTGCTTCTCGGGATACAGACCAATTGGCATGCTCTTCAATCACTGACATATCT GGGAA

7662 TTTCTCCTGCAGCTGTGTGGACCTGGATGACAAGGGCTGCCCCGCCGAGCAGAGAGCC AGGTTGGCCTGGACCC

7663 GCTAGGGACAACACGATTTCCCTTGGAGATATCGATCTGTTAGAAACCTCTCCAGGTTC TTTGGGGGCAGAG

7664 TCATGGAAGCCCTGATCATCAGGTAAAGCCACAGAGAGACACCCTCACCCCAACTCCC CTCTGCCCCCAAAGAAC

7665 GAGGAAATCCAGTTCGTCCTGTTCAGAGCACACTTCAGGCAGCGTCTGGGCAGAGAAG GGGAGGGTGGGGAGGAGGAGGAGGCTGTGA

7666 CAAGTGGCTGTGAAGGTAAGAAGTGGCTCACTCTTGAGCCTGCCCTTGGCTTGCGGACT CTGTAGGCTGCAGTTCTCAGCTC

7667 GCAGGGGGCTTGGGTCGTTGGGCATTCCGGACACCTGGCCTTCATACACCTCCCCAAAG GCGCCATGGCCCAGACCCCTGTGCAAAGGAGAAGACAAGAGG

7668 CCCTCTAGGGTTGTCAATGAAATGAATTCACCAACATAAAATGGTTTTGAAAAATCCTA AAGAGCTCTACCAATGTGAGTGACCATTATCACTCCT

7669 GCTGGGCTTTACACACAGAATCTACCCACTGAATCACAATTTTGTTCTGGCTTCCATGG AGTTTGCCTTCCAGAACATCCTCACATGTA

7670 CCCCCCAACACATGGGCCAGGGCAAATGAGTCACCCGCTATGTGCTCAGTTCCCTCCTC TATGCAATGGACCGACCGTGATCAGATTAGGGTTACCTGAGGATCGAATGAATTGAAA TG

7671

GTTTTAGAAAATGAATAGTTGTTAAACCTGAATT

7672 AGAACCTTATTGAAATATGGCCAAAGTAAGTCAATAAACAGCCCTTAGTCCTCTTGAAA AAATTCAGGTTTAACAACTATTCATTTT

7673 TACCTTCCCATTTTGAACCTGGTAACTAATTCTGTAGGACATCATCTTCCAAAAAGCTTT ACTCTTGCTCTGCTAGCCTATCTAGCTAAAGAGCTTCTCTG

7674 GGCCACTTCCCAGCTGGCGCGGACACGGCAGGCTGGAGAGCCATGAGGCAGAGCATAC GCAGCCTGTACCCAGTGGTGCCGAGCCTCTGGCG

7675 CGATGAAGGAGAAGAGGACAGCGGCTGCGATCACCGTGCGGCACAGCTCGTCGCACA GTGGATCTGTGGGTGGGGGTGGTGTGAGGCTTGGCACCG

7676 CATCGTCTCGGTGCTGCTGTCTGCCTTCTGCATCCACTGCTACCACAAGTTTGCCCACAA GCCACCCATCTCCTCAGCTGAGATGAC

7677 GAGACCTGGTTCTCCATGGAGTCCAGCGAGGGCCGGCGGGCACCGGAAGAGGAGTAGC TGACCGGGAAGGCCTGGGCGGGCCTCCGGAAGG

7678 CCGTGGATGCCTTCAAGATCCTGGTGAGGGTCCCTGCGGGGCAGGGAAGATCCCCTGC CCTCCCCAGCTGCCTTCCAGGGAGGGAGGCCAGCTGG

7679 CCAGGAGTGTCTACAGCACTCCTCTGGTTACTGAAAGCTCAGGGATAGGGCCTGGCCTT CTCCTTTACCCCTCCTTCCTAGAGAGTTAGA

7680 AAAAGGGATTCAATTGCCATCCATTTAACTGGAATCCGACCCTAAAGAGAAGATGGAA TAAAGACATTGAAGTTACTC

7681 TTGATCATATCTACACCACGCAAAGTGATGTGTAAGTGTGGGTGTTGCTCTCTTGGGGT GGAGGTTACAGAAACACCCTTATACATGTAGTGGGGCCACGACGCCCGTCTGTGCAGC TTGGCCAG

7682 TCTTATTCCTTTAATACAGAATATGGGTAAAGATGATCCGACAAGTGAGAGACAGGAT CAGGTCAGCGGGCTACCACTGGGCC

7683 CAGGTGGTGTTGGGAAAAGCGCACTGACAATCCAGCTAATCCAGAACCACTTTGTAGA TGAATATGATCCCACCATAGAGGTGAGG

7684 GCTCCAACCACCACCAGTTTGTACTCAGTCATTTCACACCAGCAAGAACCTGTTGGAAA CCAGTAATCAGGGTTAATTGGCGAGCCACATCTACAGTACTTTAAAGCTTTCTATAATC

A

7685 AAGAAGAGTACAGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCCTCTG TGTATTTGCCAT

7686 GTACTCTTCTTGTCCAGCTGTATCCAGTATGTCCAACAAACAGGTTTCACCATCTATAAC CACTTGTTTTCTGTAAGAATCCTGGGGGTGT

7687 CTTGGCAATAGCATTGCATTCCCTGTGGTTTTTAATAAAAATTGAACTTCCCTCCCTCCC TGCCCCCTTACCCTCCA

7688 GGTTAAATAAGCATCTAACTATTCAAGCCCATTTCTGCCTATCTGGTTTGTCCCTCAAAT

TGCTAATATATAATCACAAACAAAAAGTATCCAATATCACCCTACATAAAAGAAAACC

C

7689 GGGCTTTCTTTGTGTATTTGCCATAAATAATACTAAATCATTTGAAGATATTCACCATTA

TAGGTGGGTTTAAATTGAATATAATAAGCTGACATTAAGGAGTAATTATAGTTTTTATT

TTTTGAG

7690 CCTCCCCAGTCCTCATGTACTGGTCCCTCATTGCACTGTACTCCTCTTGACCTGCTGTGT CGAGAATATCCAAGAGACAGGT

7691

GCACTGTAATAATCCAGACTGTGTTTCTCCCTTCTCAGGATTCCTACAGGAAGCAAGTA GTAATTGATGGAGAAA

7692 GCTTGTTGCTTGCCAGCCCAGGACTTGGAGGCTCCAGGGGACCCCCATCGTGGGGCCTG GTGGGCAAAGAGGGCTCCAGCCAACCCCCCAAAT

7693 CTACAAGGAGCGGCCGCAGGATGTGGACCAACGTGAGGCTCCCCTCAACAACTTCTCT GTGGCGCGTAAGTATCCCCTTGGCCTCTCGGGATTCAGATTTGGGGGGTTGGCTGGAGC

cc

7694 GCCAATGAAGGTGCCATCATTCTTGAGGAGGAAGTAGCGTGGCCGCCAGGTCTTGATG

TACTCCCCTACAGACGTGCGGGTGGTGAGAGCCACGCACACTCTACCCGTCAGACCCTC

GCCAGGCAGCCAGG

7695

AAAATTAGAACAGTAGATGCTTAGTTTA

7696 TTTCATGCCTTTGGCTACTTGAAGACCAAAGCCAATAAGATCTTTTACAGTTGGATTCT GCAGTCAAAGAGAGTTAGAAAAGCAT

7697 ATATCTTGCAAGCAAAAAGTTTGTCCACAGAGACTTGGCTGCAAGAAACTGTATGTAA GTATCAGAATCTCTGTGCCACAATCCAAATTAAGTGACAAGGAGGAATCTG

7698 AAGTAGTAATAACAGTGCTGTTGATATTGAGACAACACCAGCAATCAATCCTGTGAAA TTCTGATCTGGTTGAA

7699 TTGGGTTTTTCCTGTGGCTGAAAAAGAGAAAGCAAATTAAAGGTGCATTTTTGTTACTG TTCATTTTTAGAAGTTA

7700 GGTTTTATGGACTACATATAAGACAGCACACAAGAATCGACGACAATCTTAAACTGTA ATGACTGTGTTCTTAAGGTA

7701 ATAAAACCCATGAGTTCTGGGCACTGGGTCAAAGTCTCCTGGGGCCCATGATAGCCGTC TTTAACAAGCTCTTT

7702 CTTCGGGCACTTACAAGCCTATCCAAATGAGGAGTGTGTACTCTTGCATCGTAGCGAAC TAATTCACTGCCCAGATCTTAAAACAGAGAGAAAGA

7703 GTGTAAGCCCAACTACAGAAATGGTTTCAAATGAATCTGTAGACTACCGAGCTACTTTT CCAGAAGGTATATTTCAGTTTATTGTTCTGAGAAATACCTATACAT

7704 GCAGCAGCGAAAGCACCTTGAGGCCTTTCTTACCCAGAGATAGAAGGTGGGAGAACTA AAGGATGACGACTTTGAGAAGATCAGTGAGCTGGGGGCTGGCAATGGCGGTGTGGTGT CCAAA

7705 TATGTGATCAAGAAATTGATAGCATTTGCAGTATAGAGCGTGCAGATAATGACAAGGA GTATCTAGTACTTACTTTAACAAAAAATGATCTTGACAAAGCAAATAAAGACAA

7706

7707 GATGGGAGGACAAGTTCATGTATTTTGAGTTCCCTCAGCCGTTACCTGTGTGTGGTGAT ATCAAAGTAGAGTTCTTCCACAAACAGAACAAGATGCTAAAAAAGGACAAAATGTTTC ACTTTTGGGTA

7708 TTTGAAACTATTCCAATGTTCAGTGGCGGAACTTGCAATCCTCAGTTTGTGGTCTGCCA GCTAAAGGTGAAGATGTATTCCTCCAATTCAGGACCCACACGATGGGAGGACAAGTTC ATGTATTTTGAGTTCCCTCAGCCGTTACCTGTGTGTGGTGATATCAAAGTAGAGTTCT

7709 ACATCATCTTGTGAAACAACAGTGCCACTGGTCTATAATCCACATGATTCTTTACCAGG GTCCTTACTTCCCCATAGAAATCTAGGGCCTCTTGTGCCTTTAAAAATT

7710 AAGTGAAGATGACAATCATGTTGCAGCAATTCACTGTAAAGCTGGAAAGGGACGAACT GGTATAATGATTTATGCATATTTATTACATCGGGGCAA

7711 CGGTCCAGAGCCAAGCAGCGGCTGAGCGAGGGGCATCAGCTACCGCCAAGTCCAGAGC CATTTCCATCCTGCAGAGCCCCGCCACCAG

7712 CTGGTGGCGTAAGCAAAAGTGTCTTGACGATACAGCTAATTCAGAATCATTTTGTGGAC CAATATGATCCAACAATAGAGAATTCCTACAGGAAGCAAGTAGTAATTGATGGAGAAA

7713 CCCCCCCCCCCCCCCGCCCAGTCCTCATGTACTGGTCCTCATTGCATTGTACTCTTCTTG ACCTGTTGTGTCAAGAATATCCAAGAGACAGGT References

Bang Y, Kwak EL, Shaw AT, Camidge DR, Iafrate AJ, Maki RG, Solomon BJ, Ou R, Salgia R, Clark JW. Activity of the oral ALK inhibitor PF-02341066 in ALK- positive patients with non-small cell lung cancer (NSCLC). J Clin Oncol 28: 18s (suppl; abstr 3), 2010.

Butrynski JE, DAdamo DR, Hornick JL, Dal Cin P, Antonescu CR, Jhanwar SC, Ladanyi M, Capelletti M, Rodig SJ, Ramaiya N, Kwak EL, Clark JW, Wilner KD, Christensen JG, Janne PA, Maki RG, Demetri GD, Shapiro GI. Crizotinib in ALK- rearranged inflammatory myofibroblastic tumor. N Engl J Med. 2010 Oct 28;363(18): 1727-33.

Camidge DR, Doebele RC.Treating ALK-positive lung cancer-early successes and future challenges. Nat Rev Clin Oncol. 2012 Apr 3;9(5):268-77.

Cheng M, Ott GR. Anaplastic lymphoma kinase as a therapeutic target in anaplastic large cell lymphoma, non-small cell lung cancer and neuroblastoma. Anticancer Agents Med Chem 10: 236-249, 2010.

Choi YL, Soda M, Yamashita Y, Ueno T, Takashima J, Nakajima T, Yatabe Y, Takeuchi K, Hamada T, Haruta H, Ishikawa Y, Kimura H, Mitsudomi T, Tanio Y, Mano H; ALK Lung Cancer Study Group. EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors. N Engl J Med. 2010 Oct 28;363(18): 1734- 9.

Choi YL, Soda M, Yamashita Y, Ueno T, Takashima J, Nakajima T, Yatabe Y, Takeuchi K, Hamada T, Haruta H, Ishikawa Y, Kimura H, Mitsudomi T, Tanio Y, Mano H. ALK Lung Cancer Study Group. EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors. N Engl J Med. 2010 Oct 28;363(18): 1734- 9.

Christensen JG, Zou HY, Arango ME, Li Q, Lee JH, McDonnell SR, Yamazaki S, Alton GR, Mroczkowski B, Los G. Cytoreductive antitumor activity of PF-2341066, a novel inhibitor of anaplastic lymphoma kinase and c-Met, in experimental models of anaplastic large-cell lymphoma. Mol Cancer Ther 6 (12, Pt. 1): 3314-3322, 2007. Cui JJ et al. Structure based drug design for the discovery of clinical candidate PF- 2341066 as potent and highly selective c-Met inhibitor. Abstracts of Papers, 235th ACS National Meeting, New Orleans, LA, United States, April 6-10, 2008, 2008: p. MEDI-177.

Doebele RC, Pilling AB, Aisner DL, Kutateladze TG, Le AT, Weickhardt AJ, Kondo KL, Linderman DJ, Heasley LE, Franklin WA, Varella-Garcia M, Camidge DR. Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer. Clin Cancer Res. 2012 Mar 1; 18(5): 1472-82.

Doebele RC, Pilling AB, Aisner DL, Kutateladze TG, Le AT, Weickhardt AJ, Kondo KL, Linderman DJ, Heasley LE, Franklin WA, Varella-Garcia M, Camidge DR. Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer. Clin Cancer Res. 2012 Mar 1; 18(5): 1472-82. Epub 2012 Jan 10.

11. Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO, Lindeman N, Gale CM, Zhao X, Christensen J, Kosaka T, Holmes AJ, Rogers AM, Cappuzzo

F, Mok T, Lee C, Johnson BE, Cantley LC, Janne PA. Science. 2007 May 18;316(5827): 1039-43. Epub 2007 Apr 26.

12. Hallberg B, Palmer RH. Crizotinib— latest champion in the cancer wars? N Engl J Med 363 : 1760-1762, 2010.

13. Koivunen JP, Mermel C, Zejnullahu K, Murphy C, Lifshits E, Holmes AJ, Choi HG, Kim J, Chiang D, Thomas R, Lee J, Richards WG, Sugarbaker DJ, Ducko C, Lindeman N, Marcoux JP, Engelman JA, Gray NS, Lee C, Meyerson M, Janne PA. EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer. Clin Cancer Res 14: 4275-4283, 2008.

14. Lovly CM, Pao. Escaping ALK inhibition: mechanisms of and strategies to overcome resistance. W. Sci Transl Med. 2012 Feb 8;4(120): 120ps2.

15. McDermott U, lafrate AJ, Gray NS, Shioda T, Classon M, Maheswaran S, Zhou W, Choi HG, Smith SL, Dowell L, Ulkus LE, Kuhlmann G, Greninger P, Christensen JG, Haber DA, Settleman J. Genomic alterations of anaplastic lymphoma kinase may sensitize tumors to anaplastic lymphoma kinase inhibitors. Cancer Res 68:

3389-3395, 2008.

16. Sasaki T, Okuda K, Zheng W, Butrynski J, Capelletti M, Wang L, Gray NS, Wilner K, Christensen JG, Demetri G, Shapiro GI, Rodig SJ, Eck MJ, Janne PA. The neuroblastoma associated F 1174L ALK mutation causes resistance to an ALK kinase inhibitor in ALK translocated cancers. Cancer Res. 2010 Dec

15;70(24): 10038-43.

17. Sasaki T, Rodig SJ, Chirieac LR, Janne PA. The biology and treatment of EML4- ALK non-small cell lung cancer. Eur J Cancer 46: 1773-1780, 2010.

18. Shaw AT, Yeap BY, Mino-Kenudson M, Digumarthy SR, Costa DB, Heist RS, Solomon B, Stubbs H, Admane S, McDermott U, Settleman J, Kobayashi S, Mark

EJ, Rodig SJ, Chirieac LR, Kwak EL, Lynch TJ, lafrate AJ. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK. J Clin Oncol 27: 4247-4253, 2009.

19. Wood AC, Laudenslager M, Haglund EA, Attiyeh EF, Pawel B, Courtright J, Plegaria J, Christensen JG, Mosse YP. Inhibition of ALK mutated neuroblastomas by the selective inhibitor PF-02341066. J Clin Oncol 27: 15s, 2009 (suppl; abstr 10008b).

0. Zou HY, Li Q, Lee JH, Arango ME, McDonnell SR, Yamazaki S, Koudrakova TB, Alton G, Cui JJ, Kung PP, Nambu MD, Los G, Bender SL, Mroczkowski B, Christensen JG. An orally available small-molecule inhibitor of c-Met, PF-2341066, exhibits cytoreductive antitumor efficacy through antiproliferative and antiangiogenic mechanisms. Cancer Res 67: 4408-4417, 2007.

SEQUENCES

anaplastic lymphoma kinase (ALK) SEQ ID NO: 7714

1 mgaigllwllplllstaavgsgmgtgqragspaagpplqpreplsysrlqrkslavdfvv

61 pslfrvyardlllppssselkagrpeargslaldcapllrllgpapgvswtagspapaea

121 rtlsrvlkggsvrklrrakqlvlelgeeailegcvgppgeaavgllqfniseifswwirq

181 gegrlrirlmpekkasevgregrlsaairasqpr11fqifgtghsslesptnmpspspdy

241 ftwnltwimkdsfpflshrsryglecsfdfpceleyspplhdlrnqswswrripseeasq

301 mdlldgpgaerskemprgsflllntsadskhtilspwmrsssehctlavsvhrhlqpsgr

361 yiaqllphneaareillmptpgkhgwtvlqgrigrpdnpfrvaleyissgnrslsavdff

421 alkncsegtspgskmalqssftcwngtvlqlgqacdfhqdcaqgedesqmcrklpvgfyc

481 nfedgfcgwtqgtlsphtpqwqvrtlkdarfqdhqdhalllsttdvpasesatvtsatfp

541 apiksspcelrmswlirgvlrgnvslvlvenktgkeqgrmvwhvaayeglslwqwmvlpl

601 ldvsdrfwlqmvawwgqgsraivafdnisisldcyltisgedkilqntapksrnlfernp

661 nkelkpgensprqtpifdptvhwlfttcgasgphgptqaqcnnayqnsnlsvevgsegpl

721 kgiqiwkvpatdtysisgygaaggkggkntmmrshgvsvlgifnlekddmlyilvgqqge

781 dacpstnqliqkvcigennvieeeirvnrsvhewaggggggggatyvfkmkdgvpvplii

841 aaggggraygaktdtfhperlennssvlglngnsgaagggggwndntsllwagkslqega

901 tgghscpqamkkwgwetrggfggggggcssggggggyiggnaasnndpemdgedgvsfis

961 plgilytpalkvmeghgevnikhylncshcevdechmdpeshkvicfcdhgtvlaedgvs

1021 civsptpephlplslilsvvtsalvaalvlafsgimivyrrkhqelqamqmelqspeykl

1081 skirtstimtdynpnycfagktssisdlkevprknitlirglghgafgevyegqvsgmpn

1141 dpsplqvavktlpevcseqdeldfImealiiskfnhqnivrcigvslqslprfillelma

1201 ggdlksflretrprpsqpssiamldllhvardiacgcqyleenhfihrdiaarnclltcp

1261 gpgrvakigdfgmardiyrasyyrkggcamlpvkwmppeafmegiftsktdtwsfgvllw

1321 eifslgympypsksnqevlefvtsggrmdppkncpgpvyrimtqcwqhqpedrpnfaiil

1381 erieyctqdpdvintalpieygplveeeekvpvrpkdpegvppllvsqqakreeerspaa

1441 ppplpttssgkaakkptaaeisvrvprgpavegghvnmafsqsnppselhkvhgsrnkpt

1501 slwnptygswftekptkknnpiakkephdrgnlglegsctvppnvatgrlpgasllleps

1561 sltanmkevplfrlrhfpcgnvnygyqqqglpleaatapgaghyedtilksknsmnqpgp

ALK cDNA Sequence Reference SEQ ID NO: 7715

atgggagccatcgggctcctgtggctcctgccgctgctgctttccacggcagctgtgggc

tccgggatggggaccggccagcgcgcgggctccccagctgcggggccgccgctgcagccc

cgggagccactcagctactcgcgcctgcagaggaagagtctggcagttgacttcgtggtg

ccctcgctcttccgtgtctacgcccgggacctactgctgccaccatcctcctcggagctg

aaggctggcaggcccgaggcccgcggctcgctagctctggactgcgccccgctgctcagg

ttgctggggccggcgccgggggtctcctggaccgccggttcaccagccccggcagaggcc

cggacgctgtccagggtgctgaagggcggctccgtgcgcaagctccggcgtgccaagcag

ttggtgctggagctgggcgaggaggcgatcttggagggttgcgtcgggccccccggggag

gcggctgtggggctgctccagttcaatctcagcgagctgttcagttggtggattcgccaa

ggcgaagggcgactgaggatccgcctgatgcccgagaagaaggcgtcggaagtgggcaga

gagggaaggctgtccgcggcaattcgcgcctcccagccccgccttctcttccagatcttc gggactggtcatagctccttggaatcaccaacaaacatgccttctccttctcctgattat tttacatggaatctcacctggataatgaaagactccttccctttcctgtctcatcgcagc cgatatggtctggagtgcagctttgacttcccctgtgagctggagtattcccctccactg catgacctcaggaaccagagctggtcctggcgccgcatcccctccgaggaggcctcccag atggacttgctggatgggcctggggcagagcgttctaaggagatgcccagaggctccttt ctccttctcaacacctcagctgactccaagcacaccatcctgagtccgtggatgaggagc agcagtgagcactgcacactggccgtctcggtgcacaggcacctgcagccctctggaagg tacattgcccagctgctgccccacaacgaggctgcaagagagatcctcctgatgcccact ccagggaagcatggttggacagtgctccagggaagaatcgggcgtccagacaacccattt cgagtggccctggaatacatctccagtggaaaccgcagcttgtctgcagtggacttcttt gccctgaagaactgcagtgaaggaacatccccaggctccaagatggccctgcagagctcc ttcacttgttggaatgggacagtcctccagcttgggcaggcctgtgacttccaccaggac tgtgcccagggagaagatgagagccagatgtgccggaaactgcctgtgggtttttactgc aactttgaagatggcttctgtggctggacccaaggcacactgtcaccccacactcctcaa tggcaggtcaggaccctaaaggatgcccggttccaggaccaccaagaccatgctctattg ctcagtaccactgatgtccccgcttctgaaagtgctacagtgaccagtgctacgtttcct gcaccgatcaagagctctccatgtgagctccgaatgtcctggctcattcgtggagtcttg aggggaaacgtgtccttggtgctagtggagaacaaaaccgggaaggagcaaggcaggatg gtctggcatgtcgccgcctatgaaggcttgagcctgtggcagtggatggtgttgcctctc ctcgatgtgtctgacaggttctggctgcagatggtcgcatggtggggacaaggatccaga gccatcgtggcttttgacaatatctccatcagcctggactgctacctcaccattagcgga gaggacaagatcctgcagaatacagcacccaaatcaagaaacctgtttgagagaaaccca aacaaggagctgaaacccggggaaaattcaccaagacagacccccatctttgaccctaca gttcattggctgttcaecacatgtggggccagcgggccccatggccccacccaggcacag tgcaacaacgcctaccagaactccaacctgagcgtggaggtggggagcgagggccccctg aaaggcatccagatctggaaggtgccagccaccgacacctacagcatctcgggctacgga gctgctggcgggaaaggcgggaagaacaccatgatgcggtcccacggcgtgtctgtgctg ggcatcttcaacctggagaaggatgacatgctgtacatcctggttgggcagcagggagag gacgcctgccccagtacaaaccagttaatccagaaagtctgcattggagagaacaatgtg atagaagaagaaatccgtgtgaacagaagcgtgcatgagtgggcaggaggcggaggagga gggggtggagccacctacgtatttaagatgaaggatggagtgccggtgcccctgatcatt gcagccggaggtggtggcagggcctacggggccaagacagacacgttccacccagagaga ctggagaataactcctcggttctagggctaaacggcaattccggagccgcaggtggtgga ggtggctggaatgataacacttccttgctctgggccggaaaatctttgcaggagggtgcc accggaggacattcctgcccccaggccatgaagaagtgggggtgggagacaagagggggt ttcggagggggtggaggggggtgctcctcaggtggaggaggcggaggatatataggcggc aatgcagcctcaaacaatgaccccgaaatggatggggaagatggggtttccttcatcagt ccactgggcatcctgtacaccccagctttaaaagtgatggaaggccacggggaagtgaat attaagcattatctaaactgcagtcactgtgaggtagacgaatgtcacatggaccctgaa agccacaaggtcatctgcttctgtgaccacgggacggtgctggctgaggatggcgtctcc tgcattgtgtcacccaccccggagccacacctgccactctcgctgatcctctctgtggtg acctctgccctcgtggccgccctggtcctggctttctccggcatcatgattgtgtaccgc cggaagcaccaggagctgcaagccatgcagatggagctgcagagccctgagtacaagctg agcaagctccgcacctcgaccatcatgaccgactacaaccccaactactgctttgctggc aagacctcctccatcagtgacctgaaggaggtgccgcggaaaaacatcaccctcattcgg ggtctgggccatggcgcctttggggaggtgtatgaaggccaggtgtccggaatgcccaac gacccaagccccctgcaagtggctgtgaagacgctgcctgaagtgtgctctgaacaggac gaactggatttcctcatggaagccctgatcatcagcaaattcaaccaccagaacattgtt cgctgcattggggtgagcctgcaatccctgccccggttcatcctgctggagctcatggcg gggggagacctcaagtccttcctccgagagacccgccctcgcccgagccagccctcctcc ctggccatgctggaccttctgcacgtggctcgggacattgcctgtggctgtcagtatttg gaggaaaaccacttcatccaccgagacattgctgccagaaactgcctcttgacctgtcca ggccctggaagagtggccaagattggagacttcgggatggcccgagacatctacagggcg agctactatagaaagggaggctgtgccatgctgccagttaagtggatgcccccagaggcc ttcatggaaggaatattcacttctaaaacagacacatggtcctttggagtgctgctatgg gaaatcttttctcttggatatatgccataccccagcaaaagcaaccaggaagttctggag tttgtcaccagtggaggccggatggacccacccaagaactgccctgggcctgtataccgg ataatgactcagtgctggcaacatcagcctgaagacaggcccaactttgccatcattttg gagaggattgaatactgcacccaggacccggatgtaatcaacaccgctttgccgatagaa tatggtccacttgtggaagaggaagagaaagtgcctgtgaggcccaaggaccctgagggg gttcctcctctcctggtctctcaacaggcaaaacgggaggaggagcgcagcccagctgcc atctctgttcgagtccctagagggccggccgtggaagggggacacgtgaatatggcattc tctcagtccaaccctccttcggagttgcacaaggtccacggatccagaaacaagcccacc agcttgtggaacccaacgtacggctcctggtttacagagaaacccaccaaaaagaataat cctatagcaaagaaggagccacacgacaggggtaacctggggctggagggaagctgtact gtcccacctaacgttgcaactgggagacttccgggggcctcactgctcctagagccctct tcgctgactgccaatatgaaggaggtacctctgttcaggctacgtcacttcccttgtggg aatgtcaattacggctaccagcaacagggcttgcccttagaagccgctactgcccctgga gctggtcattacgaggataccattctgaaaagcaagaatagcatgaaccagcctgggccc tga

EGFR cDNA Sequence Reference SEQ ID NO: 7716

ATGCGACCCTCCGGGACGGCCGGGGCAGCGCTCCTGGCGCTGCTGGCTGCGCTCTGCCCG GCGAGTCGGGCTCTGGAGGAAAAGAAAGTTTGCCAAGGCACGAGTAACAAGCTCACGCAG TTGGGCACTTTTGAAGATCATTTTCTCAGCCTCCAGAGGATGTTCAATAACTGTGAGGTG GTCCTTGGGAATTTGGAAATTACCTATGTGCAGAGGAATTATGATCTTTCCTTCTTAAAG ACCATCCAGGAGGTGGCTGGTTATGTCCTCATTGCCCTCAACACAGTGGAGCGAATTCCT TTGGAAAACCTGCAGATCATCAGAGGAAATATGTACTACGAAAATTCCTATGCCTTAGCA GTCTTATCTAACTATGATGCAAATAAAACCGGACTGAAGGAGCTGCCCATGAGAAATTTA CAGGAAATCCTGCATGGCGCCGTGCGGTTCAGCAACAACCCTGCCCTGTGCAACGTGGAG AGCATCCAGTGGCGGGACATAGTCAGCAGTGACTTTCTCAGCAACATGTCGATGGACTTC CAGAACCACCTGGGCAGCTGCCAAAAGTGTGATCCAAGCTGTCCCAATGGGAGCTGCTGG GGTGCAGGAGAGGAGAACTGCCAGAAACTGACCAAAATCATCTGTGCCCAGCAGTGCTCC GGGCGCTGCCGTGGCAAGTCCCCCAGTGACTGCTGCCACAACCAGTGTGCTGCAGGCTGC ACAGGCCCCCGGGAGAGCGACTGCCTGGTCTGCCGCAAATTCCGAGACGAAGCCACGTGC AAGGACACCTGCCCCCCACTCATGCTCTACAACCCCACCACGTACCAGATGGATGTGAAC CCCGAGGGCAAATACAGCTTTGGTGCCACCTGCGTGAAGAAGTGTCCCCGTAATTATGTG GTGACAGATCACGGCTCGTGCGTCCGAGCCTGTGGGGCCGACAGCTATGAGATGGAGGAA GACGGCGTCCGCAAGTGTAAGAAGTGCGAAGGGCCTTGCCGCAAAGTGTGTAACGGAATA GGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAAA AACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCC TTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAA ATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTT GAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTC GTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGAT GTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTG TTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAG GCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCC AGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAAC CTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCA GAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATC CAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATG GGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGC CATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGG CCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTG GCCCTGGGGATCGGCCTCTTCATGCGAAGGCGCCACATCGTTCGGAAGCGCACGCTGCGG AGGCTGCTGCAGGAGAGGGAGCTTGTGGAGCCTCTTACACCCAGTGGAGAAGCTCCCAAC CAAGCTCTCTTGAGGATCTTGAAGGAAACTGAATTCAAAAAGATCAAAGTGCTGGGCTCC GGTGCGTTCGGCACGGTGTATAAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATT CCCGTCGCTATCAAGGAATTAAGAGAAGCAACATCTCCGAAAGCCAACAAGGAAATCCTC GATGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATC TGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGAC TATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGTGTGCAG ATCGCAAAGGGCATGAACTACTTGGAGGACCGTCGCTTGGTGCACCGCGACCTGGCAGCC AGGAACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAA CTGCTGGGTGCGGAAGAGAAAGAATACCATGCAGAAGGAGGCAAAGTGCCTATCAAGTGG ATGGCATTGGAATCAATTTTACACAGAATCTATACCCACCAGAGTGATGTCTGGAGCTAC GGGGTGACTGTTTGGGAGTTGATGACCTTTGGATCCAAGCCATATGACGGAATCCCTGCC AGCGAGATCTCCTCCATCCTGGAGAAAGGAGAACGCCTCCCTCAGCCACCCATATGTACC ATCGATGTCTACATGATCATGGTCAAGTGCTGGATGATAGACGCAGATAGTCGCCCAAAG TTCCGTGAGTTGATCATCGAATTCTCCAAAATGGCCCGAGACCCCCAGCGCTACCTTGTC ATTCAGGGGGATGAAAGAATGCATTTGCCAAGTCCTACAGACTCCAACTTCTACCGTGCC CTGATGGATGAAGAAGACATGGACGACGTGGTGGATGCCGACGAGTACCTCATCCCACAG CAGGGCTTCTTCAGCAGCCCCTCCACGTCACGGACTCCCCTCCTGAGCTCTCTGAGTGCA ACCAGCAACAATTCCACCGTGGCTTGCATTGATAGAAATGGGCTGCAAAGCTGTCCCATC AAGGAAGACAGCTTCTTGCAGCGATACAGCTCAGACCCCACAGGCGCCTTGACTGAGGAC AGCATAGACGACACCTTCCTCCCAGTGCCTGAATACATAAACCAGTCCGTTCCCAAAAGG CCCGCTGGCTCTGTGCAGAATCCTGTCTATCACAATCAGCCTCTGAACCCCGCGCCCAGC AGAGACCCACACTACCAGGACCCCCACAGCACTGCAGTGGGCAACCCCGAGTATCTCAAC ACTGTCCAGCCCACCTGTGTCAACAGCACATTCGACAGCCCTGCCCACTGGGCCCAGAAA GGCAGCCACCAAATTAGCCTGGACAACCCTGACTACCAGCAGGACTTCTTTCCCAAGGAA GCCAAGCCAAATGGCATCTTTAAGGGCTCCACAGCTGAAAATGCAGAATACCTAAGGGTC GCGCCACAAAGCAGTGAATTTATTGGAGCATGA

BRAF cDNA Sequence Reference SEQ ID NO: 7717

atggcggcgctgagcggtggcggtggtggcggcgcggagccgggccaggctctgttcaac ggggacatggagcccgaggccggcgccggcgccggcgccgcggcctcttcggctgcggac cctgccattccggaggaggtgtggaatatcaaacaaatgattaagttgacacaggaacat atagaggccctattggacaaatttggtggggagcataatccaccatcaatatatctggag gcctatgaagaatacaccagcaagctagatgcactccaacaaagagaacaacagttattg gaatctctggggaacggaactgatttttctgtttctagctctgcatcaatggataccgtt acatcttcttcctcttctagcctttcagtgctaccttcatctctttcagtttttcaaaat cccacagatgtggcacggagcaaccccaagtcaccacaaaaacctatcgttagagtcttc ctgcccaacaaacagaggacagtggtacctgcaaggtgtggagttacagtccgagacagt ctaaagaaagcactgatgatgagaggtctaatcccagagtgctgtgctgtttacagaatt caggatggagagaagaaaccaattggttgggacactgatatttcctggcttactggagaa gaattgcatgtggaagtgttggagaatgttccacttacaacacacaactttgtacgaaaa acgtttttcaccttagcattttgtgacttttgtcgaaagctgcttttccagggtttccgc tgtcaaacatgtggttataaatttcaccagcgttgtagtacagaagttccactgatgtgt gttaattatgaccaacttgatttgctgtttgtctccaagttctttgaacaccacccaata ccacaggaagaggcgtccttagcagagactgccctaacatctggatcatccccttccgca cccgcctcggactctattgggccccaaattctcaccagtccgtctccttcaaaatccatt ccaattccacagcccttccgaccagcagatgaagatcatcgaaatcaatttgggcaacga gaccgatcctcatcagctcccaatgtgcatataaacacaatagaacctgtcaatattgat gacttgattagagaccaaggatttcgtggtgatggaggatcaaccacaggtttgtctgct accccccctgcctcattacctggctcactaactaacgtgaaagccttacagaaatctcca ggacctcagcgagaaaggaagtcatcttcatcctcagaagacaggaatcgaatgaaaaca cttggtagacgggactcgagtgatgattgggagattcctgatgggcagattacagtggga caaagaattggatctggatcatttggaacagtctacaagggaaagtggcatggtgatgtg gcagtgaaaatgttgaatgtgacagcacctacacctcagcagttacaagccttcaaaaat gaagtaggagtactcaggaaaacacgacatgtgaatatcctactcttcatgggctattcc acaaagccacaactggctattgttacccagtggtgtgagggctccagcttgtatcaccat ctccatatcattgagaccaaatttgagatgatcaaacttatagatattgcacgacagact gcacagggcatggattacttacacgccaagtcaatcatccacagagacctcaagagtaat aatatatttcttcatgaagacctcacagtaaaaataggtgattttggtctagctacagtg aaatctcgatggagtgggtcccatcagtttgaacagttgtctggatccattttgtggatg gcaccagaagtcatcagaatgcaagataaaaatccatacagctttcagtcagatgtatat gcatttggaattgttctgtatgaattgatgactggacagttaccttattcaaacatcaac aacagggaccagataatttttatggtgggacgaggatacctgtctccagatctcagtaag gtacggagtaactgtccaaaagccatgaagagattaatggcagagtgcctcaaaaagaaa ttgccaaaaattcaccgcagtgcatcagaaccctccttgaatcgggctggtttccaaaca gaggattttagtctatatgcttgtgcttctccaaaaacacccatccaggcagggggatat ggtgcgtttcctgtccactga

KRAS cDNA Sequence Reference SEQ ID NO: 7718

atgactgaatataaacttgtggtagttggagctggtggcgtaggcaagagtgccttgacg atacagctaattcagaatcattttgtggacgaatatgatccaacaatagaggattcctac aggaagcaagtagtaattgatggagaaacctgtctcttggatattctcgacacagcaggt caagaggagtacagtgcaatgagggaccagtacatgaggactggggagggctttctttgt gtatttgccataaataatactaaatcatttgaagatattcaecattatagagaacaaatt aaaagagttaaggactctgaagatgtacctatggtcctagtaggaaataaatgtgatttg ccttctagaacagtagacacaaaacaggctcaggacttagcaagaagttatggaattcct tttattgaaacatcagcaaagacaagacagggtgttgatgatgccttctatacattagtt cgagaaattcgaaaacataaagaaaagatgagcaaagatggtaaaaagaagaaaaagaag tcaaagacaaagtgtgtaattatgtaa

Claims

CLAIMS What is claimed is:

1. A kinase inhibitor resistance panel comprising one or more primer sets from one or more of the genes selected from group of genes comprising KRAS, BRAF, EGFR, ALK, and KIT.

2. The kinase inhibitor resistance panel of claim 1, wherein at least one primer sets hybridizes and amplifies nucleic acid from exon 1 or 2 oiKRAS.

3. The kinase inhibitor panel of claim 2, wherein one or more KRAS hybridizing primers or primer sets comprise one or more of the primers of Tables 10 and/or 14.

4. The kinase inhibitor panel of claim 2, wherein one or more KRAS hybridizing primers or primer sets comprise one or more of the primers of SEQ ID NOs: 4601-5200 and 7181-7610.

5. The kinase inhibitor resistance panel of claim 1, wherein at least one primer sets hybridizes and amplifies nucleic acid from exon 18, 19, 20, 21 or 22 oiEGFR.

6. The kinase inhibitor panel of claim 5, wherein one or more EGFR hybridizing primers or primer sets comprise one or more of the primers of Tables 8 and/or 12.

7. The kinase inhibitor panel of claim 6, wherein one or more EGFR hybridizing primers or primer sets comprise one or more of the primers of SEQ ID NOs: 1641-2440 and 5819-6524.

8. The kinase inhibitor resistance panel of claim 1, wherein at least one primer sets hybridizes and amplifies nucleic acid from exon 21, 22, 23, 24, or 25 oiALK.

9. The kinase inhibitor panel of claim 8, wherein one or more ALK hybridizing primers or primer sets comprise one or more of the primers of Tables 7 and/or 11.

10. The kinase inhibitor panel of claim 9, wherein one or more ALK hybridizing primers or primer sets comprise one or more of the primers of SEQ ID NOs: 1-1640 and 5201-5818.

1 1. The kinase inhibitor resistance panel of claim 1, wherein at least one primer sets hybridizes and amplifies nucleic acid from exon 8, 9, 10, 11, 12, 13, or 17 of KIT.

12. The kinase inhibitor panel of claim 1 1, wherein one or more KIT hybridizing primers or primer sets comprise one or more of the primers of Table 9.

13. The kinase inhibitor panel of claim 12, wherein one or more KIT hybridizing primers or primer sets comprise one or more of the primers of SEQ ID NOs: 2441-4600.

14. The kinase inhibitor resistance panel of claim 1, wherein at least one primer sets hybridizes and amplifies nucleic acid from exon 10, 11, 13, 14, or 15 oiBRAF.

15. The kinase inhibitor panel of claim 14, wherein one or more BRAF hybridizing primers or primer sets comprise one or more of the primers of Table 13.

16. The kinase inhibitor panel of claim 15, wherein one or more BRAF hybridizing primers or primer sets comprise one or more of the primers of SEQ ID NOs: 6525-7180.

17. The kinase inhibitor panel of claim 1, wherein the panel comprises two or more primer sets for one or more of the genes selected from group of genes comprising KRAS, BRAF, EGFR, ALK, and KIT.

18. The kinase inhibitor panel of claim 1, wherein the panel comprises 3, 4, 5, 6, 7, 8, 9, 10, or more primer sets for one or more of the genes selected from group of genes comprising KRAS, BRAF, EGFR, ALK, and KIT

19. The kinase inhibitor panel of claim 1, wherein the panel comprises one or more primer sets for 2, 3, 4, of all 5 of the genes selected from group of genes comprising KRAS, BRAF, EGFR, ALK, and KIT

20. The kinase inhibitor of claim 1, wherein the kinase inhibitor resistance is resistance to an ALK kinase inhibitor.

21. The kinase inhibitor resistance panel of claim 20, wherein the kinase inhibitor is selected from the group consisting of crizotinib, afatinib, Axitinib, bevacizumab, Bosutinib, Cetuximab, Dasatinib, Erlotinib, Fostamati nib, Gefitinib, Imatinib, Lapatinib, Lenvatinib, Nilotinib, Panitumumab, Pazopanib, Pegaptanib, Ranibizumab, Ruxolitinib, Sorafenib, Sunitinib, Trastuzumab, and Vemurafenib.

22. The kinase inhibitor resistance panel of claim 21, wherein the kinase inhibitor comprises crizotinib.

23. The kinase inhibitor panel of claim 1, wherein the one or more primer sets amplify one or more mutations associated with kinase inhibitor resistance, wherein the mutation is identified in Table 2, 3, 4, 5, or 6.

24. A method for the detection of kinase inhibitor resistance comprising obtaining a tissue sample from a subject with a cancer and conducting a high throughput sequencing (also known as next generation sequencing) reaction on the sample using the kinase inhibitor resistant panel of Claim 1, wherein the presence of a mutation in the nucleic acid sequence of a gene associated with kinase inhibitor resistance indicates that that the cancer is resistant or will become resistant to a kinase inhibitor.

25. A method for the detection of kinase inhibitor resistance comprising obtaining a tissue sample from a subject with a cancer and conducting a high throughput sequencing (also known as next generation sequencing) reaction on the sample using one or more primer sets or primer panels with primer sets that specifically hybridizes to one or more of the genes selected from the group consisting oiALK, KRAS, EGFR, KIT, and BRAF, wherein the presence of a mutation in the nucleic acid sequence of a gene associated with kinase inhibitor resistance indicates that that the cancer is resistant or will become resistant to a kinase inhibitor.

26. The method of Claim 25, wherein at least one primer sets hybridizes and amplifies nucleic acid from exon 1 or 2 oiKRAS.

27. The method of claim 26, wherein one or more KRAS hybridizing primers or primer sets comprise one or more of the primers of Tables 10 and/or 14.

28. The method of claim 26, wherein one or more KRAS hybridizing primers or primer sets comprise one or more of the primers of SEQ ID NOs: 4601-5200 and 7181-7610.

29. The method of claim 25, wherein at least one primer sets hybridizes and amplifies nucleic acid from exon 18, 19, 20, 21 or 22 oiEGFR.

30. The method of claim 29, wherein one or more EGFR hybridizing primers or primer sets comprise one or more of the primers of Tables 8 and/or 12.

31. The method of claim 29, wherein one or more EGFR hybridizing primers or primer sets comprise one or more of the primers of SEQ ID NOs: 1641-2440 and 5819-6524.

32. The method of claim 25, wherein at least one primer sets hybridizes and amplifies nucleic acid from exon 21, 22, 23, 24, or 25 oiALK.

33. The method of claim 32, wherein one or more ALK hybridizing primers or primer sets comprise one or more of the primers of Tables 7 and/or 1 1.

34. The method of claim 32, wherein one or more ALK hybridizing primers or primer sets comprise one or more of the primers of SEQ ID NOs: 1-1640 and 5201-5818.

35. The method of claim 25, wherein at least one primer sets hybridizes and amplifies nucleic acid from exon 8, 9, 10, 11, 12, 13, or 17 of KIT.

36. The method of claim 35, wherein one or more KIT hybridizing primers or primer sets comprise one or more of the primers of Table 9.

37. The method of claim 35, wherein one or more KIT hybridizing primers or primer sets comprise one or more of the primers of SEQ ID NOs: 2441-4600.

38. The method of claim 25, wherein at least one primer sets hybridizes and amplifies nucleic acid from exon 10, 11, 13, 14, or 15 oiBRAF.

39. The method of claim 38, wherein one or more BRAF hybridizing primers or primer sets comprise one or more of the primers of Table 13.

40. The method of claim 38, wherein one or more BRAF hybridizing primers or primer sets comprise one or more of the primers of SEQ ID NOs: 6525-7180.

41. The method of claim 25, wherein the panel comprises two or more primer sets for one or more of the genes selected from group of genes comprising KRAS, BRAF, EGFR, ALK, and KIT

42. The method of claim 25, wherein the panel comprises 3, 4, 5, 6, 7, 8, 9, 10, or more primer sets for one or more of the genes selected from group of genes comprising KRAS, BRAF, EGFR, ALK, and KIT

43. The method of claim 25, wherein the panel comprises one or more primer sets for 2, 3, 4, of all 5 of the genes selected from group of genes comprising KRAS, BRAF, EGFR, ALK, and KIT

44. The method of claim 25, wherein the kinase inhibitor resistance is resistance to an ALK kinase inhibitor.

45. The method of claim 25, wherein the kinase inhibitor is selected from the group consisting of crizotinib, afatinib, Axitinib, bevacizumab, Bosutinib, Cetuximab, Dasatinib, Erlotinib, Fostamati nib, Gefitinib, Imatinib, Lapatinib, Lenvatinib, Nilotinib,

Panitumumab, Pazopanib, Pegaptanib, Ranibizumab, Ruxolitinib, Sorafenib, Sunitinib, Trastuzumab, and Vemurafenib.

46. The method of claim 45, wherein the kinase inhibitor comprises crizotinib.

47. The method of claim 25, wherein the one or more primer sets amplify one or more mutations associated with kinase inhibitor resistance, wherein the mutation is identified in Table 2, 3, 4, 5, or 6.