WO2025240460A2 - Assay for detection of mutations conferring resistance to treatment with an immunotherapeutic agent - Google Patents

Abstract

The present disclosure is directed to compositions, kits, and methods for identifying one or more immunotherapy resistant mutations in a nucleic acid sample obtained from a subject, such as a subject in need of treatment with one or more targeted immunotherapeutic agents. In some embodiments, the present disclosure is directed to compositions, kits, and methods of target enrichment by unidirectional primer extension, whereby the compositions, kits, and methods include one or more target capture primers designed for the selective enrichment of one or more genes encoding one or more proteins targeted or capable of being targeted with an immunotherapeutic agent.

Description

ASSAY FOR DETECTION OF MUTATIONS CONFERRING RESISTANCE TO TREATMENT WITH AN IMMUNOTHERAPEUTIC AGENT

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of the filing date of United States Provisional Patent Application No. 63/647,109, filed on May 14, 2024, the disclosure of which is hereby incorporated by reference herein in its entirety.

REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on May 12, 2025, is named "Ventana-0278WO.xml" and is 2,209,620 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.

FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to compositions and assays, such as compositions and assays for identifying immunotherapy resistant mutations.

BACKGROUND OF THE DISCLOSURE

[0004] Cancer is a leading cause of death worldwide. Current treatments for cancer include surgery, chemotherapy, targeted therapy, radiation therapy, endocrine therapy, and immunotherapy. Therapeutic antibodies which recognize specific oncogenic proteins expressed in cancer cells have become a key component of cancer treatment due to their specificity and sensitivity. For instance, monoclonal antibody (MAbs) drugs such as Rituximab (anti-CD20) and Trastuzumab (anti-HER2) have been approved for the treatment of B-cell malignancies and breast cancer, respectively, with encouraging results.

[0005] Bispecific antibodies have been developed to address drug resistance and improve efficacy. It is believed that bispecific antibodies have improved efficacy and safety as compared with monoclonal antibody treatments by simultaneously recognizing and binding two different antigens or antigenic epitopes. (See Sun Y, et. al., "Bispecific antibodies in cancer therapy: Target selection and regulatory requirements." Acta Pharm Sin B. 2023 Sep;13(9):3583-3597. doi: 10.1016/j.apsb.2023.05.023. Epub 2023 May 23. PMID: 37719370; PMCID: PMC 10501874).

[0006] To-date, bispecific monoclonal antibodies (BsMAbs) have been applied in multiple disease types including non-small cell lung cancer, macular degeneration, melanoma, and hemophilia. Looking at the FDA approval history, it is apparent that these therapeutics have had a particular impact in relapsed or refractory Non-Hodgkin's Lymphoma (R/R NHL) (US Food and Drug Administration. (2024) "Bispecific Antibodies: An Area of Research and Clinical Applications." https://www.fda.gov/drugs/spotlight-cder-science/bispecific-antibodies-area- research-and-clinical-applications. Accessed March 29th, 2024.). The first FDA BsMAb approval was in 2014 for the use of blinatumobab for the treatment of relapsed/refractory acute lymphoblastic leukemia. Blinatumomab co-targets CD3 and CD 19 and is manufactured by Amgen under the trade name Blincyto. Since 2022, four BsMAbs have been approved by the FDA for the treatment of various NHL subtypes. For NHL, recently approved BsMAbs co-target CD3 and CD20, with the exception of Teclistamab (trade name Tecvayli - Janssen), which co-targets CD3 and BCMA. In either case, by targeting CD3 BsMAbs facilitate the recruitment of T cells to tumor cells, thus facilitating a major histocompatibility complex (MHC) independent cytotoxic response. Some examples of CD3xCD20 bi specifics include mosunetuzumab (Lunsumio - Genentech/Roche), glofitamab (Columvi - Roche), epcoritamab (Epkinly -Abbvie), and odronextamab (REGN1979 - Regeneron) (Ayyappan S (2023). "Final Analysis of the Phase 2 ELM-2 Study: Odronextamab in Patients with Relapsed/Refractory (R/R) Diffuse Large B-cell Lymphoma (DLBCL)." Blood. 142 (Supplement 1): 436). All of these examples are FDA approved treatments with the exception of odronextamab, which is currently in clinical trials. Each therapeutic employs unique strategies in the design and configuration of the antibody. For example, mosunetuzumab targets CD20 and CD3 in a monovalent fashion and fuses the heavy chains through a knob-into-hole approach, whereas glofitamab targets CD20 in a bivalent fashion and achieves fusion via a head-to-tail approach (Schuster SJ (2021) "Bispecific antibodies for the treatment of lymphomas: Promises and Challenges." Hematological Oncology. https://doi.org/10.1002/hon.2858, Falchi F (2023) "Bispecific antibodies for the treatment of B- cell lymphoma: promises, unknowns, and opportunities." Blood. 10.1182/blood.2021011994), Sun LL, Ellerman D, Mathieu M, et al. Anti-CD20/CD3 T cell-dependent bispecific antibody for the treatment of B cell malignancies. Sci Transl Med. 2015.; 7(287):287ra70., Bacac M, Colombetti S, Herter S, et al. CD20-TCB with obinutuzumab pretreatment as next-generation treatment of hematologic malignancies. Clin Cancer Res. 2018. ;24(19):4785-4797., Engelberts PJ, Hiemstra IH, de Jong B, et al. DuoBody-CD3xCD20 induces potent T-cell-mediated killing of malignant B cells in preclinical models and provides opportunities for subcutaneous dosing. EBioMedicine. 2020. ;52: 102625.)

[0007] The relatively significant number of FDA approved CD3xCD20 BsMAbs in the NHL space speaks to the efficacy of these drugs. Results of phase 1 and 2 clinical trials showed that among 197 patients with R/RNHL, mosunetuzumab therapy resulted in an objective response rate (ORR) of 35% and a complete response (CR) of 19% for aggressive NHL and an ORR of 66% and CR of 48% for indolent NHL. Glofitamab showed strong performance in phase 1 and 2 trials among highly pre-treated patients with aggressive NHL, reporting an ORR of 61% and CR of 49% for phase 1 (n=171) and an ORR of 52% and CR of 39% for phase 2 (n=155). Glofitimab is given after a pre-treatment course with the monoclonal antibody obinutuzumab. It is notable that a percentage of these cohorts across all trials were pre-treated with CAR T cells. It is also notable that adverse events across trials have been manageable, with cytokine release syndrome (CRS) being the most common presentation. ORR and CR for epcoritamab was 63% and 39% respectively, by phase 2.

[0008] Overall, BsMAbs, particularly those co-targeting CD3xCD20 are a promising therapeutic strategy for cases of resistant NHL. They exhibit good efficacy and safety across multiple NHL subtypes. Relative to CAR-T cells, they are easier to manufacture and indeed can serve as an effective therapeutic among patients where CAR-T cells have failed.

[0009] Despite the achievements made in treating cancers, resistance to novel targeted therapeutic agents, such as MAbs or BsMAbs, poses problem in cancer treatment. It is believed that resistance to targeted therapeutic agents can occur either through loss of expression or alteration of monoclonal antibody binding sites, the latter being a rare occurrence (see Foran et al 2001. "Loss of CD20 expression following treatment with rituximab (chimaeric monoclonal anti- CD20): a retrospective cohort analysis." British Journal of Haematology. doi: https://doi.Org/10.1046/j.1365-2141.2001.03019.x, Johnson et al 2009 "CD20 mutations involving the rituximab epitope are rare in diffuse large B-cell lymphomas and are a significant cause of R-CHOP failure." Haematalogica. 94(3). doi: https://doi.org/10.3324/haematol.2008.001024, Mishima et al 2011 "The identification of irreversible rituximab-resistant lymphoma caused by CD20 gene mutations." Blood Cancer Journal. I(el5). doi: 10.1038/bcj .2011.11, Rushton et al 2020 ) "Genetic and evolutionary patterns of treatment resistance in relapsed B-cell lymphoma." Blood Advances. doi: 10.1182/bloodadvances.2020001696).

[0010] Emerging research also reports this mechanism in resistance to bispecific monoclonal antibody (BsMAb) treatment (Brouwer- Visser et al 2020 "Baseline Biomarkers of T- cell Function Correlate with Clinical Responses to Odronextamab (REGEN1979), and Loss of CD20 Target Antigen Expression Identified as a Mechanism of Treatment Resistance." Blood. 136(S1). doi: https://doi.org/10.1182/blood-2020-137499, Schuster et al 2022 "Characterization of CD20 expression loss as a mechanism of resistance to mosunetuzumab in patients with relap se/refractory B-cell non-Hodgkin’s lymphomas." Journal of Clinical Oncology. 140(S16). doi: 10.1200/JC0.2022.40.16_suppl.7526, Parrondo et a. 2022 "Plamotamab (XmAb® 13676) for Ibruntinib refractory CXCR4-mutated extramedullary Waldenstrom macroglobulinemia." Leukemia and Lymphoma. 63:3, 738-742, doi: 10.1080/10428194.2021.2005045).

BRIEF SUMMARY OF THE DISCLOSURE

[0011] It is desirable to screen patients in need of treatment with targeted therapeutic agents, e.g., BsMAbs, to identify treatment resistant mutations in one or more genes encoding one or more proteins targeted by the therapeutic agents and tailor treatment accordingly. In view of the foregoing, Applicant has developed compositions, kits, and methods which facilitate the identification of one or more immunotherapy resistant mutations, thereby permitting the selection of one or more therapeutic agents most suitable for treating a subject's diagnosed form of cancer. More particularly, the present disclosure provides compositions, kits, and methods which utilize Primer Extension Target Enrichment (PETE) technology to selectively enrich a sample for target nucleic acid molecules corresponding to one or more genes potentially harboring mutations which could confer resistance to treatment with a targeted therapeutic agent, and then sequencing the target nucleic acid molecules to identify gene variants.

[0012] Applicant has discovered that a PETE-based enrichment strategy combined with next-generation sequencing (NGS) facilitates an approach which (1) is relatively rapid compared to other enrichment strategies, and (2) provides high throughput. While existing methodologies can generate similar results; they fall short in specific aspects. For example, digital polymerase chain reaction (dPCR) offers a sensitive and rapid solution for the quantification of rare variants in cell free DNA (cfDNA), it suffers from the requirement to design primers and probes against a known region of interest with a small amplicon size, making adaptions to screening for unknown mutations unwieldy. Sanger sequencing is an established method for variant confirmation in a single gene. This method, however, cannot offer the same high throughput and sensitivity that NGS does.

[0013] A first aspect of the present disclosure is a method of identifying immunotherapy resistant mutations, comprising: obtaining a sample comprising a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules comprises one or more target nucleic acid molecules and one or more non-target nucleic acid molecules; hybridizing one of a plurality of different target capture primers to each one of the one or more target nucleic acid molecules in the obtained sample, wherein each target capture primer of the plurality of different target capture primers comprises a capture moiety, and wherein each target capture primer targets a different region within one or more coding regions, non-coding regions, and/or intronic regions one or more genes encoding one or more proteins capable of being targeted with a immunotherapy agent, and wherein each target capture primer of the plurality of target capture primers does not target a region within the one or more coding exons of the one or more genes that overlap with one or more regions corresponding to one or more known variants; extending each of the hybridized one of the plurality of different target capture primers to provide one or more extended target primer extension complexes, wherein each extended target capture primer complex of the one or more extended target capture primer complexes comprises one of the one or more target nucleic acid molecules and the one of the plurality of different target capture primers; and enriching the sample for the one or more target nucleic acid molecules. In some embodiments, the immunotherapy agent is a bispecific monoclonal antibody. In some embodiments, the one or more genes are selected from one or more of CD19, CD22, CD58, CD79B, MS4A1, TNFSF13B, TNFRSF13C, and TNFRSF17.

[0014] In some embodiments, the one or more genes is the MS4A1 gene, and wherein at least three different coding exons of the MS4A1 gene are targeted by the plurality of different target capture primers. In some embodiments, at least four different coding exons of the MS4A1 gene are targeted by the plurality of different target capture primers. In some embodiments, at least five different coding exons of the MS4A1 gene are targeted by the plurality of different target capture primers. In some embodiments, the coding exons of the MS4A1 gene are selected from the group consisting of coding exons 1, 2, 3, 4, 5, and 6. In some embodiments, the coding exons of the MS4A1 gene are selected from the group consisting of coding exons 1, 2, 4, 5, and 6. In some embodiments, the coding exons of the MS4A1 gene are selected from the group consisting of coding exons 3, 4, 5, and 6. In some embodiments, the plurality of different target capture primers comprises at least eight different target capture primers targeting different regions within the MS4A1 gene. In some embodiments, the plurality of different target capture primers comprises at least twelve different target capture primers targeting different regions within the MS4A1 gene. In some embodiments, the plurality of different target capture primers comprises at least sixteen different target capture primers targeting different regions within the MS4A1 gene.

[0015] In some embodiments, the plurality of different target capture primers further comprises at least four different target capture primers targeting different regions within one or more coding regions, non-coding regions, and/or intronic regions of any one of the CD 19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes.

[0016] In some embodiments, the plurality of different target capture primers further comprises at least eight different target capture primers targeting different regions within one or more coding regions, non-coding regions, and/or intronic regions of any one of the CD 19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes.

[0017] In some embodiments, the at least eight different target capture primers targeting different regions within the MS4A1 gene have at least 90% identity to any one of SEQ ID NOS: 41 to 122. In some embodiments, the at least eight different target capture primers targeting different regions within the MS4A1 gene have any one of SEQ ID NOS: 41 to 122. In some embodiments, the at least four different target capture primers targeting different regions within the one or more coding regions, non-coding regions, and/or intronic regions of the CD 19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes have at least 90% identity to any one of SEQ ID NOS: 1 to 40 and 123 to 462. In some embodiments, the at least four different target capture primers targeting different regions within the one or more coding regions, noncoding regions, and/or intronic regions of the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes have any one of SEQ ID NOS: 1 to 40 and 123 to 462.

[0018] In some embodiments, the different regions targeted correspond to one or more locations within Chromosome 11 ranging from between about 60463002 to about 60463121, from between about 60464288 to about 60464344, from between about 60465921 to about 604661 7, and/or from between about 60466959 to about 60467060, based on genome build HG38 or an equivalent position in a genome build other than HG38.

[0019] In some embodiments, the plurality of different target capture primers hybridize to at least 15 consecutive nucleotides of complementary nucleic acid sequences located at positions within Chromosome 11 ranging from between about 60463002 to about 60463121, from between about 60464288 to about 60464344, from between about 60465921 to about 60466157, and/or from between about 60466959 to about 60467060, based on genome build HG38 or an equivalent position in a genome build other than HG38.

[0020] In some embodiments, the different regions targeted correspond to one or more locations within Chromosome 11 ranging from between about 60462374 to about 60462533, from between about 60463001 to about 60463121, from between about 60464287 to about 60464344, from between about 60465920 to about 60466157, from between about 60466958 to about 60467060, and/or from between about 60468249 to about 60468468, based on genome build HG38 or an equivalent position in a genome build other than HG38.

[0021] In some embodiments, the plurality of different target capture primers hybridize to at least 15 consecutive nucleotides of complementary nucleic acid sequences located at positions within Chromosome 11 ranging from between about 60462374 to about 60462533, from between about 60463001 to about 60463121, from between about 60464287 to about 60464344, from between about 60465920 to about 60466157, from between about 60466958 to about 60467060, and/or from between about 60468249 to about 60468468, based on genome build HG38 or an equivalent position in a genome build other than HG38.

[0022] In some embodiments, the different regions targeted correspond to one or more locations within Chromosome 11 ranging from between about 60455815 to about 60456010, from between about 60457995 to about 60458316, from between about 60461002 to about 60461269, from between about 60462140 to about 60462600, from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, from between about 60466902 to about 60467140, and/or from between about 60468195 to about 60470813, based on genome build HG38 or an equivalent position in a genome build other than HG38. [0023] In some embodiments, the plurality of different target capture primers hybridize to at least 15 consecutive nucleotides of complementary nucleic acid sequences located at positions within Chromosome 11 ranging from between about 60455815 to about 60456010, from between about 60457995 to about 60458316, from between about 60461002 to about 60461269, from between about 60462140 to about 60462600, from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, from between about 60466902 to about 60467140, and/or from between about 60468195 to about 60470813, based on genome build HG38 or an equivalent position in a genome build other than HG38.

[0024] In some embodiments, the plurality of target capture primers includes 12 or more different target capture primers, wherein each different target capture primer has at least at least 90% identity to any one of SEQ ID NOS: 1 - 462, SEQ ID NOS: 925 - 942, or SEQ IDS NO: 961 - 1436. In some embodiments, the plurality of target capture primers includes 12 or more different target capture primers, wherein each different target capture primer has any one of SEQ ID NOS: 1 - 462, SEQ ID NOS: 925 - 942, or SEQ IDS NO: 961 - 1436.

[0025] In some embodiments, the enriching of the sample for the one or more target nucleic acid molecules comprises (i) capturing the one or more extended target capture primer complexes; (ii) removing the one or more non-target nucleic acid molecules; and (iii) releasing the one or more target nucleic acid molecules from the one or more extended target capture primer complexes. In some embodiments, the capturing of the one or more extended target capture primer complexes comprises contacting the one or more extended target capture primer complexes with a functionalized substrate.

[0026] In some embodiments, the capture moiety of the plurality of different target capture primers comprises a first member of a pair of specific binding entities, and wherein the functionalized substrate comprises a second member of the pair of specific binding entities. In some embodiments, the first member of the pair of specific binding entities is selected from the group consisting of biotin, an antigenic molecule, an enzyme substrate, a receptor ligand, a polysaccharide, a thiolated molecule, and an amine-terminated molecule. In some embodiments, the second member of the pair of specific binding entities is selected from the group consisting of streptavidin, an antibody, an enzyme, a receptor, a lectin, a gold p article, and an NHS-activated moiety. In some embodiments, the plurality of different target capture primers is coupled to a substrate through the capture moiety prior to the step (b), and wherein the step (b) captures the one or more target nucleic acid molecules to the substrate.

[0027] In some embodiments, the capturing of the one or more extended target capture primer complexes comprises: (i) hybridizing a universal capture oligonucleotide to the capture moiety of the one or more extended target capture primer complexes to form one or more universal capture oligonucleotide complexes, wherein the universal capture oligonucleotide comprises (a) a first member of a pair of specific binding entities, and (b) a nucleotide sequence complementary to at least a portion of a capture sequence of the capture moiety; (ii) contacting the one or more universal capture oligonucleotide complexes with a functionalized substrate, wherein the functionalized substrate comprises a second member of the pair of specific binding entities. In some embodiments, the removing of the non-target nucleic acid molecules comprises washing the sample one or more times.

[0028] In some embodiments, the releasing of the one or more target nucleic acid molecules from the one or more extended target capture primer complexes comprises: (i) hybridizing a release primer to the one or more extended target capture primer complexes; and (b) extending the hybridized release primer. In some embodiments, the hybridized one of the plurality of different target capture primers is extended with a first polymerase; and wherein the hybridized release primer is extended with a second polymerase.

[0029] In some embodiments, the one or more target nucleic acid molecules are in low abundance as compared with the one or more non-target nucleic acid molecules. In some embodiments, the one or more target nucleic acid molecules in the enriched sample are amplified. In some embodiments, the one or more target nucleic acid molecules in the enriched sample are sequenced.

[0030] A second aspect of the present disclosure is a method of identifying immunotherapy resistant mutations, comprising: obtaining a sample comprising a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules comprises one or more target nucleic acid molecules and one or more non-target nucleic acid molecules; hybridizing one target capture primer from a first set of different target capture primers to one of the one or more target nucleic acid molecules in the obtained sample, wherein each target capture primer of the first set of different target capture primers comprises a capture moiety, and wherein each target capture primer of the first set of different target capture primers targets a different region within one or more coding exons of MS4A1 , and wherein each target capture primer of the first set of target capture primers does not target a region within the one or more coding exons that overlap with one or more regions corresponding to one or more known variants; extending each of the hybridized one of the first set of different target capture primers to provide one or more extended target capture primer complexes, wherein each target capture primer extension complex of the one or more extended target capture primer complexes comprises one of the one or more target nucleic acid molecules and the one of the plurality of different target capture primers; and enriching the sample for the one or more target nucleic acid molecules.

[0031] In some embodiments, the different regions targeted by the different target capture primers within the first set of different target capture primers correspond to one or more sequences within each of at least three different coding exons of the MS4A1 gene. In some embodiments, the different regions targeted by the different target capture primers within the first set of different target capture primers correspond to one or more sequences within each of at least four different coding exons of the MS4A1 gene. In some embodiments, the different regions targeted by the different target capture primers within the first set of different target capture primers correspond to one or more sequences within each of at least five different coding exons of the MS4A1 gene. In some embodiments, the different regions targeted by the different target capture primers within the first set of different target capture primers correspond to one or more sequences within each of coding exons 3, 4, 5, and 6 of the MS4A1 gene. In some embodiments, the different regions targeted by the different target capture primers within the first set of different target capture primers correspond to one or more sequences within each of coding exons 1, 2, 3, 4, 5, and 6 of the MS4A1 gene. In some embodiments, the different regions targeted by the different target capture primers within the first set of different target capture primers correspond to one or more sequences within each of coding exons 1, 2, 4, 5, and 6 of the MS4A1 gene.

[0032] In some embodiments, the method further comprises hybridizing one target capture primer from a second set of different target capture primers to another one of the one or more target nucleic acid molecules in the obtained sample wherein each target capture primer of the second set of different target capture primers comprises a capture moiety, and wherein each target capture primer of the second set of different target capture primers targets a different region within one or more coding regions, non-coding regions, and/or intronic regions of one or more genes selected from the group consisting of a CD19 gene, a CD22 gene, a CD58 gene, a CD79B gene, a TNFSF13B gene, a TNFRSF13C gene, and a TNFRSF17 gene.

[0033] In some embodiments, the one of a first set of different target capture primers has a nucleic acid sequence having at least 90% identity to any one SEQ ID NOS: 943 - 960.

[0034] In some embodiments, the method further comprises sequencing the one or more target nucleic acid molecules.

[0035] A third aspect of the present disclosure is a method of identifying immunotherapy resistant mutations, comprising: obtaining a sample comprising a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules comprises one or more target nucleic acid molecules and one or more non-target nucleic acid molecules; hybridizing one of a plurality of different target capture primers to each one of the one or more target nucleic acid molecules in the obtained sample, wherein each target capture primer of the plurality of different target capture primers comprises a capture moiety, and wherein each target capture primer targets a different region within one or more coding regions, non-coding regions, and/or intronic regions of one or more genes selected from the group consisting of a CD19 gene, a CD22 gene, a CD58 gene, a CD79B gene, a MS4A1 gene, a TNFSF13B gene, a TNFRSF13C gene, and a TNFRSF17 gene, and wherein each target capture primer of the plurality of target capture primers does not target a region within the one or more coding exons that overlap with one or more regions corresponding to one or more known variants; extending each of the hybridized one of the plurality of different target capture primers to provide one or more extended target capture primer complexes, wherein each target capture primer extension complex of the one or more extended target capture primer complexes comprises one of the one or more target nucleic acid molecules and the one of the plurality of different target capture primers; and enriching the sample for the one or more target nucleic acid molecules.

[0036] In some embodiments, the plurality of target capture primers includes 12 or more different target capture primers, wherein each different target capture primer has at least at least 90% identity to any one of SEQ ID NOS: 1 - 462, SEQ ID NOS: 925 - 942, or SEQ IDS NO: 961 - 1436. In some embodiments, the plurality of target capture primers includes 12 or more different target capture primers, wherein each different target capture primer has any one of SEQ ID NOS: 1 - 462, SEQ ID NOS: 925 - 942, or SEQ IDS NO: 961 - 1436. In some embodiments, the plurality of target capture primers includes 16 or more different target capture primers, wherein each different target capture primer has at least at least 90% identity to any one of SEQ ID NOS: 1 - 462, SEQ ID NOS: 925 - 942, or SEQ IDS NO: 961 - 1436. In some embodiments, the plurality of target capture primers includes 16 or more different target capture primers, wherein each different target capture primer has any one of SEQ ID NOS: 1 - 462, SEQ ID NOS: 925 - 942, or SEQ IDS NO: 961 - 1436.

[0037] In some embodiments, each target capture primer of the plurality of different target capture primers hybridize to at least 15 consecutive nucleotides of complementary nucleic acid sequences located at any of the positions set forth within any one of Tables 1 - 10, based on genome build HG38 or an equivalent position in a genome build other than HG38.

[0038] In some embodiments, the plurality of different target capture primers hybridize to at least 15 consecutive nucleotides of complementary nucleic acid sequences located at positions within chromosome 11 ranging from between about 60455815 to about 60456010, from between about 60457995 to about 60458316, from between about 60461002 to about 60461269, from between about 60462140 to about 60462600, from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, from between about 60466902 to about 60467140, and/or from between about 60468195 to about 60470813, based on genome build HG38 or an equivalent position in a genome build other than HG38. In some embodiments, at least 12 different target capture primers of the plurality of different target capture primers target regions within the MS4A1 gene; and wherein at least 6 different target capture primers of the plurality of different target capture primers target regions within the CD 19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes. In some embodiments, at least 12 different target capture primers of the plurality of different target capture primers target regions within the MS4A1 gene; and wherein at least 12 different target capture primers of the plurality of different target capture primers target regions within the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes.

[0039] In some embodiments, the method further comprises sequencing the one or more target nucleic acid molecules.

[0040] A fourth aspect of the present disclosure is composition comprising at least 12 different target capture primers, wherein each target capture primer targets a different region within a MS4A1 gene, provided that each of the different target capture primers within the composition do not target a region within the MS4A1 gene that overlaps with one or more regions corresponding to one or more known variants.

[0041] In some embodiments, the different regions correspond to locations within Chromosome 11 ranging from between about 60455815 to about 60456010, from between about 60457995 to about 60458316, from between about 60461002 to about 60461269, from between about 60462140 to about 60462600, from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, from between about 60466902 to about 60467140, and/or from between about 60468195 to about 60470813, based on genome build HG38 or an equivalent position in a genome build other than HG38. In some embodiments, the different regions correspond to locations within Chromosome 11 ranging from between about 60463002 to about 60463121, from between about 60464288 to about 60464344, from between about 60465921 to about 60466157, and/or from between about 60466959 to about 60467060, based on genome build HG38 or an equivalent position in a genome build other than HG38. In some embodiments, the different regions correspond to locations within Chromosome 11 ranging from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, and/or from between about 60466902 to about 60467140, based on genome build HG38 or an equivalent position in a genome build other than HG38.

[0042] In some embodiments, the at least 12 different target capture primers have at least 85% identity to any one of SEQ ID NOS: 41 - 122 or 925 - 942. In some embodiments, the at least 12 different target capture primers have at least 90% identity to any one of SEQ ID NOS: 41 - 122 or 925 - 942. In some embodiments, the at least 12 different target capture primers have any one of SEQ ID NOS: 41 - 122 or 925 - 942. In some embodiments, the at least 12 different target capture primers have at least 85% identity to any one of SEQ ID NOS: 925 - 942. In some embodiments, the at least 12 different target capture primers have at least 90% identity to any one of SEQ ID NOS: 925 - 942. In some embodiments, the at least 12 different target capture primers have any one of SEQ ID NOS: 925 - 942.

[0043] A fifth aspect of the present disclosure is a composition comprising (i) at least 12 different target capture primers targeting different regions within a MS4A1 gene, wherein the different regions correspond to locations within Chromosome 11 ranging from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, and/or from between about 60466902 to about 60467140, based on genome build HG38 or an equivalent position in a genome build other than HG38; (ii) at least six additional target capture primers targeting one or more regions within one or more genes selected from the group consisting of a CD19 gene, a CD22 gene, a CD58 gene, a CD79B gene, a TNFSF13B gene, a TNFRSF13C gene, and a TNFRSF17 gene.

[0044] A sixth aspect of the present disclosure is a composition comprising (i) at least 12 different target capture primers, wherein each of the at least 12 different target capture primers have different nucleic acid sequences, wherein the different nucleic acid sequences have at least 90% identity to any one of SEQ ID NOS: 925 - 942; and (ii) at least six additional target capture primers targeting one or more regions within one or more genes selected from the group consisting of a CD19 gene, a CD22 gene, a CD58 gene, a CD79B gene, a TNFSF13B gene, a TNFRSF13C gene, and a TNFRSF17 gene.

[0045] A seventh aspect of the present disclosure is a composition comprising (i) at least 12 different target capture primers, wherein each of the at least 12 different target capture primers have different nucleic acid sequences, wherein the different nucleic acid sequences have any one of SEQ ID NOS: 925 - 942; and (ii) at least six additional target capture primers targeting one or more regions within one or more genes selected from the group consisting of a CD 19 gene, a CD22 gene, a CD58 gene, a CD79B gene, a TNFSF13B gene, a TNFRSF13C gene, and a TNFRSF17 gene.

[0046] An eighth aspect of the present disclosure is a composition comprising at least 12 different target capture primers, wherein the at least 12 different target capture primers have at least 90% identity to any one of SEQ ID NOS: 1 - 462, SEQ ID NOS: 925 - 942, or SEQ IDS NO: 961 - 1436. In some embodiments, the composition comprises at least 18 different target capture primers.

[0047] A ninth aspect of the present disclosure is a composition comprising at least 12 different target capture primers, wherein the at least 12 different target capture primers have any one of SEQ ID NOS: 1 - 462, SEQ ID NOS: 925 - 942, or SEQ IDS NO: 961 - 1436. In some embodiments, the composition comprises at least 18 different target capture primers. In some embodiments, the composition includes at least one corresponding release primer. [0048] A tenth aspect of the present disclosure is a kit comprising at least 12 target capture primers and at least 12 corresponding release primers, wherein the at least 12 target capture primers and the at least 12 corresponding release primers have the sequences identified in Table 29, herein. [0049] An eleventh aspect of the present disclosure is a kit comprising at least 12 target capture primers and at least 12 corresponding release primers, wherein the at least 12 target capture primers and the at least 12 corresponding release primers have any one of the sequences identified in Table 30, herein.

[0050] A twelfth aspect of the present disclosure is a composition comprising at least 12 different target capture primers, wherein the at least 12 different target capture primers have any one of SEQ ID NOS: 961 - 1436. In some embodiments, the composition comprises at least 18 different target capture primers. In some embodiments, the composition includes at least one corresponding release primer.

BRIEF DESCRIPTION OF THE FIGURES

[0051] For a general understanding of the features of the disclosure, reference is made to the drawings. In the drawings, like reference numerals have been used throughout to identify identical elements.

[0052] FIG. 1A illustrates a method of sequencing an enriched population of nucleic acid molecules corresponding to one or more genes potentially harboring mutations which could confer resistance to treatment with a targeted therapeutic agent.

[0053] FIG. IB provides a flowchart illustrating the methods of a primer extension target enrichment protocol in accordance with the present disclosure.

[0054] FIG. 2 provides the mean barcoded deduplicated base coverage obtained from pure cfDNA (n=3 technical replicates per condition). The barcode deduplicated base coverage indicates the true read support across the ROIs when removing PCR duplicates and base call errors. More coverage indicates better sensitivity, with a minimum average coverage of 3000 reads expected at 5 Ong of input.

[0055] FIG. 3 depicts a minimum dilution of gBlock containing MS4A1 c.256T>C mutation in sheared K562 DNA passing pipeline (n=3 technical replicates per condition). These results support sufficient analytical sensitivity for mutation screening. [0056] FIGS. 4A - 4C illustrate clinical characteristics and mutation kinetics from patient A. FIG. 4A provides overall clinical characteristics, including CD20 IHC status, time of biopsy, and response as measured by PET/CT. FIG. 4A also indicates the mutations identified as well as their VAF (variant allele frequency) and total MMPM (mutant molecules per m ) at the pre and post treatment time point. MMPM is listed in parentheses in the table. VAF and MMPM was directly reported by the pipeline. Total circulating tumor DNA (ctDNA) VAF and MMPM was obtained from previously available study sequencing data, and total tumor MMPM was calculated by multiplying ctDNA VAF by total MMPM. The variant MMPM was calculated as VAF multiplied by tumor MMPM. The final two columns indicate the overall kinetics of the ctDNA across treatment. FIG. 4B provides the location of mutations in CD20. Mutations identified at pre and post treatment timepoints are circled in red, and mutations identified only at the post treatment timepoint are circled in blue. Diagram adapted from Schuster et al (Schuster et al. 2022). FIG. 4C provides kinetics of VAF across treatment timepoints for mutations identified in the case.

[0057] FIGS. 5A - 5C illustrate clinical characteristics and mutation kinetics from patientB. FIG. 5A provides overall clinical characteristics, including response as measured by PET/CT. The table of FIG. 5A also indicates the mutations identified as well as their VAF and total MMPM in parentheses at the pre, mid, and post treatment time point. VAF and MMPM calculated as previously described. The final two columns indicate the overall kinetics of the ctDNA across treatment. FIG. 5B provides the location of mutations in CD20 is circled in red. Diagram adapted from Schuster et al (Schuster et al. 2022). FIG. 5C provides the kinetics of mutation MMPM (red) versus total tumor MMPM (blue) across treatment timepoints.

[0058] FIG. 6 is a table illustrating the clinical relevance of the various targets included in the expanded panel.

[0059] FIG. 7 compares the Fold80Base penalty obtained from 50ng input K562 DNA in the original CD20 panel to the expanded panel, indicating that coverage uniformity improves with the expanded panel (Error bars represent 95% confidence interval, n=3 technical replicates per condition). Fold 80 Base penalty is the ratio of the median base coverage to the base coverage at the 20^th percentile. As this metric approaches one, this indicates a decreasing difference between the worst performing regions and the median coverage, overall indicating better coverage uniformity. [0060] FIGS. 8 A - F depict barcode deduplicated per positional base coverage across exon 2 obtained from 5 Ong input K562 DNA comparing the original panel to the expanded panel. FIG. 8A indicates per positional coverage of exon 2 for the expanded panel. FIG. 8B indicates per positional coverage for the original panel. FIG. 8C is an annotation for the exon where the thick blue bar with arrows indicates the coding region and ROI, and the thin line with arrows indicates the intron. FIG. 8D indicates the position of known variants derived from the COSMIC and gnomad databases. FIG. 8E indicates the capture primer placement in this region for the original panel. FIG. 8F indicates the capture primer placement in this region for the expanded panel. This schematic illustrates how the addition of a capture primer downstream of the locations of the known variants improves the coverage uniformity over the ROI. Image generated using IGV version 2.14.1.

[0061] FIG. 9A depicts the mean barcode deduplicated base coverage from sheared K562 (n=2 technical replicates) for the expanded panel, across a typical range of total cfDNA inputs. This shows that we are meeting the minimum expected mean coverage of 3000 across all ROIs at 50ng of input. Lower total DNA inputs yield a deduplicated coverage commensurate with the input.

[0062] FIG. 9B is a simple random binomial simulation illustrating the implications for assay sensitivity at 3000 reads. If we were to perform a sequencing run 10,000 times at this depth (read. depth) and expected to detect a variant at 0.1% allele frequency (alt_obs), we would get 2-3 reads supporting that allele, most of the time.

[0063] FIG. 10 depicts barcode deduplicated per positional base coverage for an ROI encompassing exons 2 and 3 of CD 19 obtained from 50ng input K562 DNA. FIG 10, row A, illustrates the ROI enriched by the capture and release primers. FIG. 10, row B, illustrates the capture primer placement, and FIG. 10, row C, indicates the coordinates of known variants derived from the COSMIC and gnomad databases. Circled areas indicate detection and supporting coverage for two such variants matching these coordinates. Note that capture primers are positioned such that the 3’ end avoids the indicated variant coordinates. DETAILED DESCRIPTION

[0064] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0065] Definitions

[0066] As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "includes" is defined inclusively, such that "includes A or B" means including A, B, or A and B.

[0067] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, e g., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (e.g., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0068] The terms "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Similarly, "comprises," "includes," "has," and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of "comprising" and is therefore interpreted to be an open term meaning "at least the following," and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase: "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Moreover, while the steps and processes may be outlined herein in a particular order, the skilled artisan will recognize that the ordering steps and processes may vary.

[0069] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A orB," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0070] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0071] As used herein, the term "adapter" refers a nucleotide sequence that may be added to another sequence so as to import additional properties to that sequence. An adapter can be single- or double-stranded or may have both a single-stranded portion and a double-stranded portion.

[0072] As used herein "amplification" refers to a process in which a copy number increases. Amplification may be a process in which replication occurs repeatedly over time to form multiple copies of a template. Amplification can produce an exponential or linear increase in the number of copies as amplification proceeds. Exemplary amplification strategies include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), rolling circle replication (RCA), cascade-RCA, nucleic acid-based amplification (NASBA), and the like. Also, amplification can utilize a linear or circular template. Amplification can be performed under any suitable temperature conditions, such as with thermal cycling or isothermally. Furthermore, amplification can be performed in an amplification mixture (or reagent mixture), which is any composition capable of amplifying a nucleic acid target, if any, in the mixture. PCR amplification relies on repeated cycles of heating and cooling (i.e., thermal cycling) to achieve successive rounds of replication. PCR can be performed by thermal cycling between two or more temperature setpoints, such as a higher denaturation temperature and a lower annealing/extension temperature, or among three or more temperature setpoints, such as a higher denaturation temperature, a lower annealing temperature, and an intermediate extension temperature, among others. PCR can be performed with a thermostable polymerase, such as Taq DNA polymerase. PCR generally produces an exponential increase in the amount of a product amplicon over successive cycles. [0073] As used herein, the term "biological sample," "tissue sample," "specimen," "sample," or the like refers to any sample including a biomolecule (such as a protein, a peptide, a nucleic acid, a lipid, a carbohydrate, or a combination thereof) that is obtained from any organism including viruses. Other examples of organisms include mammals (such as humans; veterinary animals like cats, dogs, horses, cattle, and swine; and laboratory animals like mice, rats, and primates), insects, annelids, arachnids, marsupials, reptiles, amphibians, bacteria, and fungi. Biological samples include tissue samples (such as tissue sections and needle biopsies of tissue), cell samples (such as cytological smears such as Pap smears or blood smears or samples of cells obtained by microdissection), or cell fractions, fragments, or organelles (such as obtained by lysing cells and separating their components by centrifugation or otherwise). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus, biopsi ed tissue (for example, obtained by a surgical biopsy or a needle biopsy), nipple aspirates, cerumen, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from a first biological sample. In certain embodiments, the term "biological sample" as used herein refers to a sample (such as a homogenized or liquefied sample) prepared from a tumor or a portion thereof obtained from a subject.

[0074] As used herein, the term "complementary" generally refers to the capability for precise pairing between two nucleotides. The term "complementary" refers to the ability to form favorable thermodynamic stability and specific pairing between the bases of two nucleotides at an appropriate temperature and ionic buffer conditions. Complementarity is achieved by distinct interactions between the nucleobases adenine, thymine (uracil in RNA), guanine and cytosine, where adenine pairs with thymine or uracil, and guanine pairs with cytosine. For example, if a nucleotide at a given position of a nucleic acid is capable of hydrogen bonding with a nucleotide of another nucleic acid, then the two nucleic acids are considered to be complementary to one another at that position. Complementarity between two single-stranded nucleic acid molecules may be "partial," in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single- stranded molecules. A first nucleotide sequence can be said to be the "complement" of a second sequence if the first nucleotide sequence is complementary to the second nucleotide sequence. A first nucleotide sequence can be said to be the "reverse complement" of a second sequence, if the first nucleotide sequence is complementary to a sequence that is the reverse (i.e., the order of the nucleotides is reversed) of the second sequence.

[0075] As used herein, the term "conjugate" refers to two or more molecules (and/or materials such as nanoparticles) that are covalently linked into a larger construct. In some embodiments, a conjugate includes one or more biomolecules (such as peptides, proteins, enzymes, sugars, polysaccharides, lipids, glycoproteins, and lipoproteins) covalently linked to one or more other molecules, such as one or more other biomolecules.

[0076] As used herein, the term "enrichment" refers to the process of increasing the relative abundance of a population of molecules, e.g., nucleic acid molecules, in a sample relative to the total amount of the molecules initially present in the sample before treatment. Thus, an enrichment step provides a percentage or fractional increase rather than directly increasing for example, the copy number of the nucleic acid sequences of interest as amplification methods, such as a polymerase chain reaction, would.

[0077] As used herein, the term "fluid" refers to any liquid or liquid composition, including water, solvents, buffers, solutions (e.g., polar solvents, non-polar solvents), washes or washing solutions, and/or mixtures. The fluid may be aqueous or non-aqueous. In some embodiments, washing solutions include a surfactant to facilitate spreading of the washing liquids over the specimen-bearing surfaces of the slides. In some embodiments, acid solutions include deionized water, an acid (e.g., acetic acid), and a solvent. In some embodiments, alkaline solutions include deionized water, a base, and a solvent. In some embodiments, transfer solutions include one or more glycol ethers, such as one or more propylene-based glycol ethers (e.g., propylene glycol ethers, di(propylene glycol) ethers, and tri(propylene glycol) ethers, ethylene-based glycol ethers (e g., ethylene glycol ethers, di(ethylene glycol) ethers, and tri(ethylene glycol) ethers), and functional analogs thereof.

[0078] Non-liming examples of buffers include citric acid, potassium dihydrogen phosphate, boric acid, diethyl barbituric acid, piperazine-N,N'-bis(2-ethanesulfonic acid), dimethylarsinic acid, 2-(N-morpholino)ethanesulfonic acid, tris(hydroxymethyl)methylamine (TRIS), 2-(N-morpholino)ethanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine(Bicine), N-tris(hydroxymethyl)methylglycine (Tri cine), 4-2-hydroxy ethyl -1 -piperazineethanesulfonic acid (HEPES), 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), and combinations thereof. In other embodiments, the buffer may be comprised of tris(hydroxymethyl)methylamine (TRIS), 2-(N-morpholino)ethanesulfonic acid (TAPS), N,N- bis(2-hydroxyethyl)glycine(Bicine), N -tris(hydroxymethyl)methylglycine (Tricine), 4-2- hydroxy ethyl- 1 -piperazineethanesulfonic acid (HEPES), 2-

{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), or a combination thereof. Additional wash solutions, transfer solutions, acid solutions, and alkaline solutions are described in United States Patent Application Publication No. 2016/0282374, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0079] As used herein, the term "hybridize" refers to the base-pairing between different nucleic acid molecules consistent with their nucleotide sequences.

[0080] As used herein, the term "mutant molecules per m " is calculated as follows: Total extracted DNA in ng is converted to DNA copies via the conversion 330 DNA copies per ng DNA. This number is multiplied by the variant allele frequency to obtain mutant molecules, and this is subsequently divided by the total volume in mL of plasma to obtain mutant molecules per mL. When total DNA yield in ng and total plasma volume is included as input into the analysis pipeline, mutant molecules per mL will be calculated and reported by the pipeline.

[0081] As used herein, the term "next generation sequencing" refers to sequencing technologies having high-throughput sequencing as compared to traditional Sanger- and capillary electrophoresis-based approaches, wherein the sequencing process is performed in parallel, for example producing thousands or millions of relatively small sequence reads at a time. Some examples of next generation sequencing techniques include, but are not limited to, sequencing by synthesis, sequencing by ligation, and sequencing by hybridization. These technologies produce shorter reads (anywhere from about 25 - about 500 bp) but many hundreds of thousands or millions of reads in a relatively short time. Examples of such sequencing devices available from Illumina (San Diego, CA) include, but are not limited to iSEQ, MiniSEQ, MiSEQ, NextSEQ, NoveSEQ.

[0082] It is believed that the Illumina next-generation sequencing technology uses clonal amplification and sequencing by synthesis (SBS) chemistry to enable rapid sequencing. The process simultaneously identifies DNA bases while incorporating them into a nucleic acid chain. Each base emits a unique fluorescent signal as it is added to the growing strand, which is used to determine the order of the DNA sequence. A non-limiting example of a sequencing device available from ThermoFisher Scientific (Waltham, MA) includes the Ion Personal Genome Machine™ (PGM™) System.

[0083] It is believed that Ion Torrent sequencing measures the direct release of H+ (protons) from the incorporation of individual bases by DNA polymerase. A non -limiting example of a sequencing device available from Pacific Biosciences (Menlo Park, CA) includes the PacBio Sequel Systems. A non-limiting example of a sequencing device available from Roche (Pleasanton, CA) is the Roche 454. Next-generation sequencing methods may also include nanopore sequencing methods. In general, three nanopore sequencing approaches have been pursued: strand sequencing in which the bases of DNA are identified as they pass sequentially through a nanopore, exonuclease-based nanopore sequencing in which nucleotides are enzymatically cleaved one-by-one from a DNA molecule and monitored as they are captured by and pass through the nanopore, and a nanopore sequencing by synthesis (SBS) approach in which identifiable polymer tags are attached to nucleotides and registered in nanopores during enzyme- catalyzed DNA synthesis. Common to all these methods is the need for precise control of the reaction rates so that each base is determined in order.

[0084] Strand sequencing requires a method for slowing down the passage of the DNA through the nanopore and decoding a plurality of bases within the channel; ratcheting approaches, taking advantage of molecular motors, have been developed for this purpose. Exonuclease-based sequencing requires the release of each nucleotide close enough to the pore to guarantee its capture and its transit through the pore at a rate slow enough to obtain a valid ionic current signal. In addition, both methods rely on distinctions among the four natural bases, two relatively similar purines and two similar pyrimidines. [0085] The nanopore SBS approach utilizes synthetic polymer tags attached to the nucleotides that are designed specifically to produce unique and readily distinguishable ionic current blockade signatures for sequence determination. In some embodiments, sequencing of nucleic acid molecules includes via nanopore sequencing includes preparing nanopore sequencing complexes and determining polynucleotide sequences. Methods of preparing nanopores and nanopore sequencing are described in U.S. Patent Application Publication No. 2017/0268052, and PCT Publication Nos. WO2014/074727, W02006/028508, WO2012/083249, and

WO/2014/074727, the disclosures of which are hereby incorporated by reference herein in their entireties. In some embodiments, tagged nucleotides may be used in the determination of the polynucleotide sequences (see, e.g., PCT Publication No. WO/2020/131759, WO/2013/191793, and WO/2015/148402, the disclosures of which are hereby incorporated by reference herein in their entireties).

[0086] The "Sequencing by Expansion" (SBX) protocol, developed by Stratos Genomics (see, e.g., Kokoris et al., U.S. Pat. No. 7,939,259, "High Throughput Nucleic Acid Sequencing by Expansion," the disclosure of which is hereby incorporated by reference herein in its entirety) is based on the polymerization of highly modified, non-natural nucleotide analogs referred to as "XNTPs." In general, SBX uses biochemical polymerization to transcribe the sequence of a DNA template onto a measurable polymer called an "Xpandomer." The transcribed sequence is encoded along the Xpandomer backbone in high signal-to-noise reporters that are separated by ~10 nm and which are designed for high-signal-to-noise, well -differentiated responses. Xpandomers can facilitate several next generation DNA sequencing detection technologies and are well suited to nanopore sequencing.

[0087] Analysis of the data generated by sequencing is performed using software and/or statistical algorithms that perform various data conversions, e.g., conversion of signal emissions into base calls, conversion of base calls into consensus sequences for a nucleic acid template, etc. Such software, statistical algorithms, and the use of such are described in detail, in U.S. Patent Application Publication Nos. 2009/0024331 2017/0044606 and in PCT Publication No. WO/2018/034745, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0088] As used herein, the terms "nucleic acid" or "polynucleotide" (used interchangeably herein) refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Unless specifically limited, the terms encompass nucleic acids or polynucleotides including known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, synthetic polynucleotides, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified, such as by conjugation with a labeling component. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologues, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91 - 98 (1994)).

[0089] As used herein, the term "nucleic acid molecule" refers to a molecule whose presence is to be enriched, detected, measured, amplified, and/or subject to further assays and analyses. A nucleic acid molecule may comprise any single and/or double-stranded nucleic acid. Nucleic acid s can exist as isolated nucleic acid fragments or be a part of a larger nucleic acid fragment. Nucleic acid molecules can be derived or isolated from any source, such as cultured microorganisms, uncultured microorganisms, complex biological mixtures, biological samples, tissues, sera, ancient or preserved tissues or samples, environmental isolates, or the like. Further, nucleic acid molecules include or are derived from cDNA, RNA, genomic DNA, cloned genomic DNA, genomic DNA libraries, enzymatically fragmented DNA or RNA, chemically fragmented DNA or RNA, physically fragmented DNA or RNA, or the like. In some embodiments, a nucleic acid molecule may include a whole genome. In exemplary embodiments, a nucleic acid molecule may include the entire nucleic acid content of a sample and/or biological sample. In exemplary embodiments, a nucleic acid molecule may include circulating or cell-free DNA's, e.g., circulating tumor DNA ("ctDNA") present in individuals with cancer or circulating fetal or circulating maternal DNA ("cfDNA") fragments present in plasma or serum of pregnant women. Nucleic acid s can come in a variety of different forms including, for example, simple or complex mixtures, or in substantially purified forms. For example, a nucleic acid molecule can be part of a sample that includes other components or can be the sole or major component of the sample. Also, a nucleic acid molecule can have either a known or unknown sequence.

[0090] As used herein, the term "nucleotide" refers to a nucleoside-5 '-oligophosphate compound, or structural analog of a nucleoside-5 '-oligophosphate, which can act as a substrate or inhibitor of a nucleic acid polymerase. Exemplary nucleotides include, but are not limited to, nucleoside-5 '-triphosphates (e.g., dATP, dCTP, dGTP, dTTP, and dUTP); nucleosides (e.g., dA, dC, dG, dT, and dU) with 5 '-oligophosphate chains of 4 or more phosphates in length (e.g., 5'- tetraphosphosphate, 5'-pentaphosphosphate, 5'-hexaphosphosphate, 5'-heptaphosphosphate, 5'- octaphosphosphate); and structural analogs of nucleoside-5 '-triphosphates that can have a modified base moiety (e.g., a substituted purine or pyrimidine base), a modified sugar moiety (e.g., an O-alkylated sugar), and/or a modified oligophosphate moiety (e.g., an oligophosphate comprising a thio-phosphate, a methylene, and/or other bridges between phosphates).

[0091] As used herein, the term "oligonucleotide," refers to an oligomer of nucleotide or nucleoside monomer units wherein the oligomer optionally includes non-nucleotide monomer units, and/or other chemical groups attached at internal and/or external positions of the oligomer. The oligomer can be natural or synthetic and can include naturally-occurring oligonucleotides, or oligomers that include nucleosides with non-naturally-occurring (or modified) bases, sugar moi eties, phosphodiester-analog linkages, and/or alternative monomer unit chiralities and isomeric structures (e.g., 5'- to 2'-linkage, L-nucleosides, a-anomer nucleosides, P-anomer nucleosides, locked nucleic acids (LNA), peptide nucleic acids (PNA)).

[0092] As used herein, a "reaction" between any two different reactive groups (such as any two reactive groups of a reagent and a particle) may mean that a covalent linkage is formed between the two reactive groups (or two reactive functional groups); or may mean that the two reactive groups (or two reactive functional groups) associate with each other, interact with each other, hybridize to each other, hydrogen bond with each other, etc. Tn some embodiments, the "reaction" includes binding events, e.g., binding events between reactive function groups or binding events between first and second members of a pair of specific binding entities.

[0093] As used herein, the term "polymerase" refers to an enzyme that performs template- directed synthesis of polynucleotides. A DNA polymerase can add free nucleotides only to the 3' end of the newly forming strand. This results in elongation of the newly forming strand in a 5 '-3' direction. No known DNA polymerase is able to begin a new chain (de novo). DNA polymerase can add a nucleotide only on to a pre-existing 3 '-OH group, and, therefore, needs a primer at which it can add the first nucleotide. Non-limiting examples of polymerases include prokaryotic DNA polymerases (e.g. Pol I, Pol II, Pol III, Pol IV, and Pol V), eukaryotic DNA polymerase, archaeal DNA polymerase, telomerase, reverse transcriptase, and RNA polymerase. Reverse transcriptase is an RNA-dependent DNA polymerase which synthesizes DNA from an RNA template. The reverse transcriptase family contain both DNA polymerase functionality and RNase H functionality, which degrades RNA base-paired to DNA. RNA polymerase is an enzyme that synthesizes RNA using DNA as a template during the process of gene transcription. RNA polymerase polymerizes ribonucleotides at the 3' end of an RNA transcript.

[0094] In some embodiments, a polymerase from the following may be used in a polymerase-mediated primer extension, end-modification (e.g., terminal transferase, degradation, or polishing), or amplification reaction: archaea (e.g., Thermococcus litoralis (Vent, GenBank: AAA72101), Pyrococcus furiosus (Pfu, GenBank: D12983, BAA02362), Pyrococcus woesii, Pyrococcus GB-D (Deep Vent, GenBank: AAA67131), Thermococcus kodakaraensis KODI (KOD, GenBank: BD175553, BAA06142; Thermococcus sp. strain KOD (Pfx, GenBank: AAE68738)), Thermococcus gorgonarius (Tgo, Pdb: 4699806), Sulfolobus solataricus (GenBank: NC002754, P26811), Aeropyrum pemix (GenBank: BAA81109), Archaeglobus fulgidus (GenBank: 029753), Pyrobaculum aerophilum (GenBank: AAL63952), Pyrodictium occultum (GenBank: BAA07579, BAA07580), Thermococcus 9 degree Nm (GenBank: AAA88769, Q56366), Thermococcus fumicolans (GenBank: CAA93738, P74918), Thermococcus hydrothermalis (GenBank: CAC 18555), Thermococcus sp. GE8 (GenBank: CAC 12850), Thermococcus sp. JDF-3 (GenBank: AX135456; WO0132887), Thermococcus sp. TY (GenBank: CAA73475), Pyrococcus abyssi (GenBank: P77916), Pyrococcus glycovorans (GenBank: CAC12849), Pyrococcus horikoshii (GenBank: NP 143776), Pyrococcus sp. GE23 (GenBank: CAA90887), Pyrococcus sp. ST700 (GenBank: CAC 12847), Thermococcus pacificus (GenBank: AX411312.1), Thermococcus zilligii (GenBank: DQ3366890), Thermococcus aggregans, Thermococcus barossii, Thermococcus cel er (GenBank: DD259850.1), Thermococcus profundus (GenBank: E14137), Thermococcus siculi (GenBank: DD259857.1), Thermococcus thioreducens, Thermococcus onnurineus NA1, Sulfol obus acidocaldarium, Sulfol obus tokodaii, Pyrobaculum calidifontis, Pyrobaculum islandicum (GenBank: AAF27815), Methanococcus jannaschii (GenBank: Q58295), Desulforococcus species TOK, Desulforococcus, Pyrolobus, Pyrodi ctium, Staphylothermus, Vulcanisaetta, Methanococcus (GenBank: P52025) and other archaeal B polymerases, such as GenBank AAC62712, P956901, BAAA07579)), thermophilic bacteria Thermus species (e.g., flavus, ruber, thermophilus, lacteus, rubens, aquaticus), Bacillus stearothermophilus, Thermotoga maritima, Methanothermus fervidus, KOD polymerase, TNA1 polymerase, Thermococcus sp. 9 degrees N-7, T4, T7, phi29, Pyrococcus furiosus, P. abyssi, T. gorgonarius, T. litoralis, T. zilligii, T. sp. GT, P. sp. GB-D, KOD, Pfu, T. gorgonarius, T. zilligii, T. litoralis and Thermococcus sp. 9N-7 polymerases.

[0095] As used herein, the term "thermostable polymerase" refers to an enzyme that is stable to heat, is heat resistant, and retains sufficient activity to effect subsequent polynucleotide extension reactions and does not become irreversibly denatured (inactivated) when subjected to the elevated temperatures for the time necessary to effect denaturation of double-stranded nucleic acids. The heating conditions necessary for nucleic acid denaturation are well known in the art and are exemplified in, e g., U.S. Pat. Nos. 4,683,202, 4,683,195, and 4,965,188, which are incorporated herein by reference. As used herein, a thermostable polymerase is suitable for use in a temperature cycling reaction such as the polymerase chain reaction ("PCR"), a primer extension reaction, or an end-modification (e.g., terminal transferase, degradation, or polishing) reaction. Irreversible denaturation for purposes herein refers to permanent and complete loss of enzymatic activity. For a thermostable polymerase, enzymatic activity refers to the catalysis of the combination of the nucleotides in the proper manner to form polynucleotide extension products that are complementary to a template nucleic acid strand. Thermostable DNA polymerases from thermophilic bacteria include, e.g., DNA polymerases from Thermotoga maritima, Thermus aquaticus, Thermus thermophilus, Thermus flavus, Thermus filiformis, Thermus species spsl7, Thermus species Z05, Thermus caldophilus, Bacillus caldotenax, Thermotoga neopolitana, Thermosipho africanus, and other thermostable DNA polymerases disclosed above. [0096] In some cases, the nucleic acid (e.g., DNA or RNA) polymerase may be a modified naturally occurring Type A polymerase. A further embodiment of the disclosure generally relates to a method wherein a modified Type A polymerase, e.g., in a primer extension, end-modification (e.g., terminal transferase, degradation, or polishing), or amplification reaction, may be selected from any species of the genus Meiothermus, Thermotoga, or Thermomicrobium. Another embodiment of the disclosure generally pertains to a method wherein the polymerase, e.g., in a primer extension, end-modification (e.g., terminal transferase, degradation or polishing), or amplification reaction, may be isolated from any of Thermus aquaticus (Taq), Thermus thermophilus, Thermus caldophilus, or Thermus filiformis. A further embodiment of the disclosure generally encompasses a method wherein the modified Type A polymerase, e.g., in a primer extension, end-modification (e.g., terminal transferase, degradation, or polishing), or amplification reaction, may be isolated from Bacillus stearothermophilus, Sphaerobacter therm ophilus, Dictoglomus therm ophilum, or Escherichia coli. In another embodiment, the disclosure generally relates to a method wherein the modified Type A polymerase, e.g., in a primer extension, end-modification (e.g., terminal transferase, degradation, or polishing), or amplification reaction, may be a mutant Taq-E507K polymerase. Another embodiment of the disclosure generally pertains to a method wherein a thermostable polymerase may be used to effect amplification of the target nucleic acid.

[0097] As used herein, the term "primer" refers to an oligonucleotide which binds to a specific region of a single-stranded template nucleic acid molecule and initiates nucleic acid synthesis via a polymerase-mediated enzymatic reaction, extending from the 3' end of the primer and complementary to the sequence of the template molecule. PCR amplification primers can be referred to as 'forward' and 'reverse' primers, one of which is complementary to a nucleic acid strand and the other of which is complementary to the complement of that strand. Typically, a primer comprises fewer than about 100 nucleotides and preferably comprises fewer than about 30 nucleotides. Exemplary primers range from about 5 to about 25 nucleotides. Primers can comprise, for example, RNA and/or DNA bases, as well as non-naturally occurring bases. The directionality of the newly forming strand (the daughter strand) is opposite to the direction in which DNA polymerase moves along the template strand. In some cases, a target capture primer specifically hybridizes to a target polynucleotide under hybridization conditions. Such hybridization conditions can include, but are not limited to, hybridization in isothermal amplification buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4), 50 mM KC1, 2 mM MgSO4, 0.1% TWEEN® 20, pH 8.8 at 25° C) at a temperature of about 40° C, 45° C, 50° C, 55° C, 60° C, 65° C, or 70° C.

[0098] As used herein, the term "sequence," when used in reference to a nucleic acid molecule, refers to the order of nucleotides (or bases) in the nucleic acid molecules. In cases, where different species of nucleotides are present in the nucleic acid molecule, the sequence includes an identification of the species of nucleotide (or base) at respective positions in the nucleic acid molecule. A sequence is a property of all or part of a nucleic acid molecule. The term can be used similarly to describe the order and positional identity of monomeric units in other polymers such as amino acid monomeric units of protein polymers.

[0099] As used herein, the term "sequencing" refers to the determination of the order and position of bases in a nucleic acid molecule. More particularly, the term "sequencing" refers to biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. Sequencing, as the term is used herein, can include without limitation parallel sequencing or any other sequencing method known of those skilled in the art, for example, chain-termination methods, rapid DNA sequencing methods, wandering-spot analysis, Maxam-Gilbert sequencing, dye- terminator sequencing, or using any other modern automated DNA sequencing instruments.

[0100] As used herein, the term "substrate" refers to any material capable of interacting with a capture moiety. In some embodiments, the substrate is a solid support. In some embodiments, a solid support may encompass any type of solid, porous, or hollow sphere, ball, bearing, cylinder, capillary, channel, or other similar configuration composed of plastic, ceramic, metal, or polymeric material (e.g., hydrogel) onto which a nucleic acid molecule may be immobilized (e.g., covalently or non-covalently). In some embodiments, a solid support may comprise a discrete particle that may be spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical, oblong, or disc-shaped, and the like. In some embodiments, a solid support cmay include silica chips, microparticles, nanoparticles, plates, arrays, capillaries, flat supports such as glass fiber filters, glass surfaces, metal surfaces (steel, gold silver, aluminum, silicon and copper), glass supports, plastic supports, silicon supports, chips, filters, membranes, microwell plates, slides, plastic materials including multiwell plates or membranes (e.g., formed of polyethylene, polypropylene, polyamide, polyvinylidenedifluoride), and/or wafers, combs, pins or needles (e.g., arrays of pins suitable for combinatorial synthesis or analysis) or beads in an array of pits or nanoliter wells of flat surfaces such as wafers (e.g., silicon wafers), wafers with pits with or without fdter bottoms. In some embodiments, a solid support may be a solution-phase support capable of suspension in a solution (e. g., a glass bead, a magnetic bead, or another like particle), or a solid-phase support (e.g., a silicon wafer, a glass slide, or the like). Non-limiting examples of solution-phase supports include superparamagnetic spherical polymer particles such as DYNABEADS magnetic beads from INVITROGEN or magnetic glass particles such as described in U.S. Pat. Nos. 656,568, 6,274,386, 7,371,830, 6,870,047, 6,255,477, 6,746,874 and 6,258,531, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0101] As used herein, the term "specific binding entity" refers to a member of a specificbinding pair. Specific binding pairs are pairs of molecules that are characterized in that they bind each other to the substantial exclusion of binding to other molecules (for example, specific binding pairs can have a binding constant that is at least 10³ M^-1 greater, 10⁴ M’¹ greater or 10⁵ M'¹ greater than a binding constant for either of the two members of the binding pair with other molecules in a biological sample). Examples of specific binding moieties include specific binding proteins (for example, antibodies, lectins, avidins such as streptavidins, and protein A). Specific binding moieties can also include the molecules (or portions thereof) that are specifically bound by such specific binding proteins.

[0102] As used herein, the term "subject" or "individual" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0103] As used herein, the term "substantially" means the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. In some embodiments, "substantially" means within about 5%. In some embodiments, "substantially" means within about 10%. In some embodiments, "substantially" means within about 15%. In some embodiments, "substantially" means within about 20%.

[0104] As used herein, the terms "target" or "target sequence" refer to nucleic acid molecule sequences of interest. [0105] As used herein, the term "universal primer" refers to a primer that can hybridize to and support amplification of target polynucleotides having a shared complementary universal primer binding site. Similar, the term "universal primer pair" refers to a forward and reverse primer pair that can hybridize to and support PCR amplification of target polynucleotides having shared complementary forward and reverse universal primer binding sites. Such universal primer(s) and universal primer binding site(s) can allow single or double primer mediated universal amplification (e.g., universal PCR) of target polynucleotide regions of interest. The headings provided herein are for convenience only and do not interpret the scope or meaning of the disclosed embodiments. [0106] As used herein, "variant allele frequency" is the percentage of sequence reads observed matching a specific DNA variant divided by the overall coverage at that locus.

[0107] OVERVIEW

[0108] The present disclosure is directed to compositions, kits, and methods for identifying one or more immunotherapy resistant mutations in a nucleic acid sample (e.g., a cell free DNA (cfDNA) sample) obtained from a subject, such as a subject in need of treatment with one or more targeted immunotherapeutic agents (e.g., BsMAbs). In some embodiments, the identification of the one or more immunotherapy resistant mutations in the obtained nucleic acid sample facilitates the personalized treatment of the subject with one or more targeted immunotherapeutic agents.

[0109] More particularly, the present disclosure is directed to compositions, kits, and methods of target enrichment by unidirectional primer extension, whereby the compositions, kits, and methods include one or more target capture primers designed for the selective enrichment of one or more genes (e.g., MS4A1) encoding one or more proteins (e.g., CD20) targeted or capable of being targeted with an immunotherapeutic agent, e.g., a BsMAb, such as a BsMAb for the treatment of NHL.

[0110] In some embodiments, the present disclosure is directed to the longitudinal screening of NHL patients undergoing treatment with BsMAbs. In some embodiments, if a mutation is identified that confers loss of CD20 expression (such as by utilizing the methods, compositions, and/or kits disclosed herein), BsMAbs are available that target additional B cell antigens (e.g. CD 19 - Robinson et al 2018) or alternative treatment strategies may be pursued, such as the use of CAR-T cells (Denlinger et al 2022, Sworder et al), if they have not already been used as a first-line therapeutic. Therefore, by allowing clinicians to respond quickly and effectively to emerging BsMAb resistance, the present disclosure has the potential to improve outcomes for NHL patients that are refractory to treatment.

[0111] The present disclosure is also directed to methods of amplifying a target enriched sample, such as a target enriched sample prepared according to any one of the methods described herein. Additionally, the present disclosure is directed to methods of sequencing a target enriched sample (e.g., with next generation sequencing), such as a target enriched sample prepared according to any one of the methods described herein. The methods of the present disclosure can be used as a part of a sequencing protocol, including a high throughput single molecule sequencing protocol. In some embodiments, the method of the present disclosure generates a library of enriched target nucleic acid molecules to be sequenced. The enriched target nucleic acid molecules in the library may optionally incorporate barcodes for molecular identification and sample identification, such as described in U.S. Publication No. 2020/0032244, and in U.S. Patent Nos. 7,393,665, 8,168,385, 8,481,292, 8,685,678, and 8,722,368, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0112] METHODS OF IDENTIFYING IMMUNOTHERAPY RESISTANT MUTATIONS IN A SAMPLE

[0113] In some embodiments, the present disclosure is directed to methods of identifying one or more immunotherapy resistant mutations in a nucleic acid sample which could confer treatment resistance, such as resistance to treatment with a BsMAb.

[0114] In general, and with reference to FIG. 1 A, the method comprises obtaining a library of nucleic acid molecules, the library of nucleic acid molecules including both target and nontarget nucleic acid molecules (step 10). Next, the library is enriched for the presence of target nucleic acid molecules, such as by using a primer extension target enrichment protocol (step 20). In some embodiments, the enrichment comprises contacting the library of nucleic acid molecules with a plurality of different target capture primers targeting one or more regions within one or more genes, such as one or more genes harboring immunotherapy resistant mutations. The enriched library is then sequenced, such as with a NGS technique, to identify one or more immunotherapy resistant mutations (step 30). Each of these steps are described in further detail herein.

[0115] Library Preparation [0116] With reference to FIG. IB, a library of nucleic acid molecules is prepared for downstream processing, such as downstream target enrichment and/or sequencing. As described herein, the library of nucleic acid molecules includes, in some embodiments, both target and nontarget nucleic acid molecules derived from a sample.

[0117] In some embodiments, the methods disclosed herein first comprise obtaining a sample comprising a plurality of nucleic acid molecules (step 101). In some embodiments, the plurality of nucleic acid molecules in the obtained sample may include one or more target nucleic acid molecules and one or more non-target nucleic acid molecules. In some embodiments, the non-target nucleic acid molecules are in high abundance as compared with the target nucleic acid molecules within the obtained sampled. In some embodiments, the non-target nucleic acid molecules represent at least about 70% of the nucleic acid molecules in the obtained sample.

[0118] In some embodiments, samples may be obtained from any source e.g., tissue (including tumor tissue or FFPE tissue), blood, serum, plasma, skin, swab (e.g., buccal, vaginal), urine, saliva, etc. In some embodiments, the biological sample is derived from a subject or a patient. In some embodiments the biological sample may include a fragment of a solid tissue, or a tumor sample derived from the subject or the patient, e.g., by biopsy. As used herein, the term "tumor sample" encompasses samples prepared from a tumor or from a sample potentially including or suspected of comprising cancer cells, or to be tested for the potential presence of cancer cells, such as a lymph node. As used herein, the term "tumor" refers to a mass or a neoplasm, which itself is defined as an abnormal new growth of cells that usually grow more rapidly than normal cells and will continue to grow if not treated, sometimes resulting in damage to adjacent structures. Tumor sizes can vary widely. In some embodiments, a tumor may be solid, or fluid filled. A tumor can refer to benign (not malignant, generally harmless), or malignant (capable of metastasis) growths. In some embodiments, tumors can include neoplastic cells that are benign (such as carcinoma in situ) and, simultaneously, contain malignant cancer cells (such as adenocarcinoma). In some embodiments, this should be understood to include neoplasms found in multiple locations throughout the body. Therefore, for purposes of the disclosure, tumors include primary tumors, lymph nodes, lymphatic tissue, and metastatic tumors.

[0119] In some embodiments, the plurality of nucleic acid molecules is derived from a tissue sample, such as a tissue sample derived from a mammalian subject. In other embodiments, the plurality of nucleic acid molecules is derived from a cytology sample, such as a cytology sample derived from a mammalian subject. In other embodiments, the plurality of nucleic acid molecules is derived from a plasma sample, such as a plasma sample derived from a mammalian subject.

[0120] Methods for isolating nucleic acid molecules from biological samples and/or purifying the samples are known, e.g., as described in Sambrook, and several kits are commercially available (e.g., High Pure RNA Isolation Kit, High Pure Viral Nucleic Acid Kit, and MagNA Pure LC Total Nucleic Acid Isolation Kit, DNA Isolation Kit for Cells and Tissues, DNA Isolation Kit for Mammalian Blood, High Pure FFPET DNA Isolation Kit, available from Roche). In the context of the presently disclosed methods, genomic DNA can be collected, purified, and/or isolated.

[0121] It will be appreciated that nucleic acid molecules may be isolated from biological samples using any of a variety of procedures known in the art, for example, MagMAX™ DNA Multi-Sample Ultra Kit (Applied Biosystems, Thermo Fisher Scientific), the MagMAX™ Express-96 Magnetic Particle Processor and the KingFisher™ Flex Magnetic Particle Processor (Thermo Fisher Scientific), a RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE and PureLink™ FFPE RNA Isolation Kit (Ambion™, Thermo Fisher Scientific), the ABI Prism™ 6100 Nucleic Acid PrepStation and the ABI Prism™ 6700 Automated Nucleic Acid Workstation (Applied Biosystems, Thermo Fisher Scientific), and the like.

[0122] In some embodiments, the nucleic acid molecules within the obtained sample are selected from DNA molecules, genomic DNA molecules, cfDNA molecules, cDNA molecules, RNA molecules, mRNA molecules, rRNA molecules, mtDNA, siRNA molecules, or any combination thereof. In some embodiments, the plurality of nucleic acid molecules comprises single stranded polynucleotides.

[0123] In some embodiments, the nucleic acid molecules within the obtained sample may be prepared for downstream target enrichment by fragmenting, cutting, or shearing the nucleic acids. In some embodiments, the fragmenting, cutting, and/or shearing of the plurality of nucleic acid molecules in the obtained sample may be carried out using such procedures as mechanical force, sonication, restriction endonuclease cleavage, or any method known in the art. In other embodiments, no fragmentation is necessary (some genomic samples may already consist of appropriately sized fragments and will not require additional fragmentation). [0124] In some embodiments, the nucleic acid molecules within any obtained biological sample have a size ranging from between about 10 mer to about 300 mer. In some embodiments, the nucleic acid molecules within any obtained biological sample have a size ranging from between about 10 mer to about 250 mer. In some embodiments, the nucleic acid molecules within any obtained biological sample have a size ranging from between about 10 mer to about 200 mer. In some embodiments, the nucleic acid molecules within any obtained biological sample have a size ranging from between about 10 mer to about 180 mer. In other embodiments, the nucleic acid molecules within any obtained biological sample have a size ranging from between about 15 mer to about 150 mer. In other embodiments, the nucleic acid molecules within any obtained biological sample have a size ranging from between about 15 mer to about 100 mer. In other embodiments, the nucleic acid molecules within any obtained biological sample have a size ranging from between about 15 mer to about 75 mer.

[0125] In some embodiments, adapters are added via a ligation reaction to the plurality of nucleic acid molecules within the obtained sample. In some embodiments, the adapters include one or more unique molecular identifiers (UMIs). A UMI is a barcode that identifies a nucleic acid molecule to which it is attached. As used herein, the term "barcode" refers to a nucleic acid sequence that can be detected and identified. In some embodiments, the barcodes include between about 5 and about 20 nucleotides, such that in a sample, the nucleic acids incorporating the barcodes can be distinguished or grouped according to the barcodes. In some embodiments, the barcodes include between about 5 and about 15 nucleotides. In some embodiments, the barcodes include between about 5 and about 10 nucleotides. In some embodiments, the barcodes include between about 10 and about 15 nucleotides. In some embodiments, the barcodes include about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 nucleotides. Non-limiting examples of barcodes and/or unique molecular identifiers (UMIs) are described in U. S. Publication No. 2020/0032244, and in U.S. Patent Nos. 7,393,665, 8,168,385, 8,481,292, 8,685,678, and 8,722,368, and in PCT Publication No. WO/2018/138237, the disclosures of which are hereby incorporated by reference herein in their entireties. In some embodiments, UMIs may be incorporated as part of an overall DNA amplification and sequencing workflow to perform error correction.

[0126] Methods of ligating adapters to a nucleic acid molecule are described in U.S. Patent

Publication Nos. 2017/0037459, 2018/0334709, 2018/0016630 and in PCT Publication No. WO2017021449, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0127] Primer Extension Target Enrichment

[0128] In some embodiments, a primer extension target enrichment (PETE) workflow may be used to enrich for one or more target nucleic acid molecules in the prepared nucleic acid library. PETE workflows are described in United Patent Application Publication Nos. 2021/0207211 and 2020/0392483; in United States Patent Nos. 10,907,204 and 11,499,180; and in International Publication Nos. WO/2018/013710 and WO/2022/008578, the disclosures of which are each incorporated by reference herein in their entireties. By way of non-limiting example only, a PETE workflow may be utilized to enrich an obtained nuclei acid library for one or more target nucleic acid molecules by:

[0129] (a) providing a reaction mixture comprising the sample including the prepared nucleic acid library and one or more target capture primers (e.g., 6 or more different capture target primers, 9 or more different capture target primers, 12 or more, 16 or more different capture target primers, 20 or more different capture target primers, 24 or more different capture target primers, etc.);

[0130] (b) hybridizing the one or more target capture primers to one or more singlestranded nucleic acid molecules in the reaction mixture, where the one or more different target capture primers comprises a capture moiety (e.g., an affinity ligand);

[0131] (c) extending the one or more hybridized target capture primers with a DNA polymerase to form one or more first double-stranded products including the one or more nucleic acid molecules hybridized to the one or more extended target capture primers;

[0132] (d) removing single- stranded target and non-nucleic acid molecules from the reaction mixture by capturing the capture moiety (e.g., affinity ligand) of the one or more first double-stranded products;

[0133] (e) hybridizing one or more release primers to the one or more captured first double stranded products (e.g., a different release primer for each of the introduced different target capture primers); and

[0134] (f) extending the one or more hybridized release primers with a DNA polymerase, such as where the DNA polymerase comprises strand displacement activity, 5 '-3' double stranded DNA exonuclease activity, or a combination thereof, thereby displacing or degrading the one or more extended target capture primers and forming one or more second double-stranded products, wherein the one or more second double-stranded products comprise the nucleic acid molecule hybridized to one or more extended release primers, wherein the one or more extended release primers comprises a complement of at least a portion of the nucleic acid molecule.

[0135] The steps of a PETE workflow are further described herein.

[0136] Hybridization of a Different Target Capture Primers

[0137] Following the preparation of the nucleic acid library, a plurality of different target capture primers (e.g., about 6 different target capture primers, about 8 different target capture primers, about 10 different target capture primers, about 12 different target capture primers, about 16 different target capture primers, about 20 different target capture primers, about 24 different target capture primers, about 30 different target capture primers, about 35 different target capture primers, about 40 different target capture primers, about 45 different target capture primers) are introduced to the nucleic acid library (or to a reaction mixture including the nucleic acid library and other components) to effectuate hybridization (e.g., buffers, etc.).

[0138] In some embodiments, the plurality of different target capture primers are targetspecific and, thus, designed to at least partially hybridize to a subset of nucleic acid molecules within the nucleic acid library having at least partially complementary sequences, such as a subset of nucleic acid molecules within the nucleic acid library which include desired target genes, exons, introns, and/or other genomic regions of interest, including but not limited to subsets and/or portions of the preceding list. As such, following their introduction, in some embodiments one of the different target capture primers of the plurality of different target capture primers at least partially hybridizes to each one of the one or more target nucleic acid molecules within the nucleic acid library to which it is at least partially complementary (step 110).

[0139] In some embodiments, each of the different target capture primers of the plurality of different target capture primers may be comprised of ribonucleic acids, deoxyribonucleic acids, and/or other nucleic acid analogs known in the art. In some embodiments, each different target capture primer of the plurality of different target capture primers may include one or more nonnatural nucleotides, e.g., locked nucleic acid (LNA), peptide nucleic acid (PNA), gamma-PNA, as glycol nucleic acid (GNA), and threose nucleic acid (TNA). In some embodiments, each different target capture primer of the plurality of different target capture primers has a length ranging from between about 15 nucleotides to about 50 nucleotides. In other embodiments, each different target capture primer of the plurality of different target capture primers has a length ranging from between about 15 nucleotides to about 40 nucleotides. In yet other embodiments, each different target capture primer of the plurality of different target capture primers has a length ranging from between about 15 nucleotides to about 30 nucleotides. In yet other embodiments, each different target capture primer of the plurality of different target capture primers has a length ranging from between about 15 nucleotides to about 26 nucleotides. In yet other embodiments, each different target capture primer of the plurality of different target capture primers has a length of at least 15 nucleotides. In yet other embodiments, each different target capture primer of the plurality of different target capture primers has a length of at least 18 nucleotides. In yet other embodiments, each different target capture primer of the plurality of different target capture primers has a length of at least 21 nucleotides. In yet other embodiments, each different target capture primer of the plurality of different target capture primers has a length of at least 24 nucleotides.

[0140] In some embodiments, each different target capture primer of the plurality of different target capture primers is designed to at least partially hybridize to a complementary nucleic acid sequence within a gene encoding a protein capable of being targeted with an immunotherapeutic agent, such as a BsMAb, provided that each of the different target capture primers do not target a region within a gene that overlaps with one or more regions corresponding to one or more known variants. As described herein, in some embodiments the PETE workflow enriches for targets of interest through a capture primer extension and a subsequent release primer extension, designed with sensitivity and specificity in mind, respectively. Among many parameters incorporated in into capture primer design is that of variant avoidance, where a capture primer is designed not to overlap a region with a known variant reported in COSMIC or gnomad databases. This is, it is believed, is due to data indicating that the measured alternative allele frequency deviates from the expected value when the capture primer overlaps that variant due to enrichment of the reference allele.

[0141] The skilled artisan will appreciate the plurality of different target capture primers may target different regions within the same gene or different regions within different genes, provided that each of the different target capture primers of the plurality of different target capture primers do not target regions within the genes that overlap with one or more regions corresponding to one or more known variants. By way of example, in some embodiments, each different target capture primer of a plurality of different target capture primers targets a different region within the same gene of a gene encoding a protein capable of being targeted with an immunotherapeutic agent, provided that each of the different target capture primers of the plurality of different target capture primers do not target regions within the gene that overlaps with one or more regions corresponding to one or more known variants. By way of another example, the plurality of different target capture primers target one or more regions within one or more different genes encoding one or more proteins capable of being targeted with an immunotherapeutic agent, e.g., one or more regions within a first gene and one or more regions within a second gene, provided that each of the different target capture primers of the plurality of different target capture primers do not target regions within the one or more genes that overlap with one or more regions corresponding to one or more known variants. By way of yet another example, the plurality of different target capture primers target one or more regions within at least two different genes (e.g., at least three different genes, at least four different genes, at least 6 different genes, etc.) encoding one or more proteins capable of being targeted with an immunotherapeutic agent, provided that each of the different target capture primers of the plurality of different target capture primers do not target regions within the two or more genes that overlap with one or more regions corresponding to one or more known variants.

[0142] In some embodiments, the plurality of different target capture primers are designed to at least partially hybridize to complementary nucleic acid sequences within one or more of (e.g., 2 or more of, 3 or more of, 4 or more of, 6 or more of, etc.) the CD19, CD22, CD58, CD79B, MS4A1, TNFSF13B, TNFRSF13C, and TNFRSF17 genes, provided that each of the different target capture primers of the plurality of different target capture primers do not target regions within the CD19, CD22, CD58, CD79B, MS4A1, TNFSF13B, TNFRSF13C, and TNFRSF17 genes that overlap with one or more regions corresponding to one or more known variants.

[0143] In some embodiments, each of the different target capture primers of the plurality of different target capture primers target one or more regions within one or more coding exons within the MS4A1 gene. In some embodiments, each of different target capture primers of the plurality of different target capture primers target one or more regions within at least two coding exons within the MS4A1 gene, such as within three or more coding exons within the MS4A1 gene, such as within four or more coding exons within the MS4A1 gene.

[0144] In some embodiments, a plurality of different target capture primers comprises at least 6 different target capture primers targeting one or more regions within at least two coding exons within the MS4A1 gene, such as within three or more coding exons within the MS4A1 gene, such as within four or more coding exons within the MS4A1 gene. In some embodiments, a plurality of different target capture primers comprises at least 9 different target capture primers targeting one or more regions within at least three coding exons within the MS4A1 gene, such as within three or more coding exons within the MS4A1 gene, or such as within four or more coding exons within the MS4A1 gene. In some embodiments, a plurality of different target capture primers comprises at least 12 different target capture primers targeting one or more regions within at least three coding exons within the MS4A1 gene, such as within three or more coding exons within the MS4A1 gene, or such as within four or more coding exons within the MS4A1 gene. In some embodiments, a plurality of different target capture primers comprises at least 16 different target capture primers targeting one or more regions within at least three coding exons within the MS4A1 gene, such as within three or more coding exons within the MS4A1 gene, or such as within four or more coding exons within the MS4A1 gene.

[0145] In some embodiments, a plurality of different target capture primers comprises at least 6 different target capture primers (e.g., about 8 or more, about 12 or more, about 16 or more, about 20 or more, etc. different target capture primers) targeting one or more regions within at least two coding exons (such as 3 or more, 4 or more, 5 or more, etc. coding exons) within the MS4A1 gene; and wherein the plurality of different target capture primers further comprises one or more different target capture primers (e.g., about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 12 or more, about 16 or more, about 20 or more, etc. different target capture primers) targeting one or more different regions within at least one of the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes. In some embodiments, a plurality of different target capture primers comprises at least 6 different target capture primers (e.g., about 8 or more, about 12 or more, about 16 or more, about 20 or more, etc. different target capture primers) targeting one or more regions within at least two coding exons within the MS4A1 gene (such as about 3 or more, about 4 or more, about 5 or more, etc. coding exons); and wherein the plurality of different target capture primers further comprises one or more different target capture primers (e.g., about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 12 or more, about 16 or more, about 20 or more, etc. different target capture primers) targeting one or more different regions within at least two of the CD 19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes. In some embodiments, a plurality of different target capture primers comprises at least 6 different target capture primers (e.g., about 8 or more, about 12 or more, about 16 or more, about 20 or more, etc. different target capture primers) targeting one or more regions within at least two coding exons within the MS4A1 gene (such as about 3 or more, about 4 or more, about 5 or more, etc. coding exons); and wherein the plurality of different target capture primers further comprises one or more different target capture primers (e.g., about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 12 or more, about 16 or more, about 20 or more, etc. different target capture primers) targeting one or more different regions within at least three of the CD 19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes. In some embodiments, a plurality of different target capture primers comprises at least 6 different target capture primers (e.g., about 8 or more, about 12 or more, about 16 or more, about 20 or more, etc. different target capture primers) targeting one or more regions within at least two coding exons within the MS4A1 gene (such as about 3 or more, about 4 or more, about 5 or more, etc. coding exons); and wherein the plurality of different target capture primers further comprises one or more different target capture primers (e.g., about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 12 or more, about 16 or more, about 20 or more, etc. different target capture primers) targeting one or more different regions within at least four of the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes.

[0146] In some embodiments, the plurality of different target capture primers are designed to at least partially hybridize to complementary nucleic acid sequences located in one or more genes encoding one or more proteins capable of being targeted with an immunotherapeutic agent, such as those nucleic acid sequences identified in any of Tables 1 to 10 (based on genome build HG38 or the equivalent position in a genome build other than HG38 (e.g., a previously known genome build or a future genome build), provided that each of the different target capture primers of the plurality of different target capture primers do not target a region within the one or more genes that overlap with one or more regions corresponding to one or more known variants. In some embodiments, the different target capture primers are designed to hybridize to about 10 or more consecutive nucleotides, such as about 15 or more consecutive nucleotides, such as about 20 or more consecutive nucleotides of complementary nucleic acid sequences located in one or more genes encoding one or more proteins capable of being targeted with an immunotherapeutic agent, such as those nucleic acid sequences identified in any of Tables 1 to 10 (based on genome build HG38 or the equivalent position in a genome build other than HG38 (e.g., a previously known genome build or a future genome build), provided that each of the different target capture primers of the plurality of different target capture primers do not target a region within the one or more genes that overlap with one or more regions corresponding to one or more known variants. By way of example only, a target capture primer having a length of about 20 nucleotides may be at least partially complementary to a nucleic acid sequence located within chromosome 1 at a location ranging from 116514454 to 116514905. By way of another example only, a mixture comprising two different target capture primers each having a length of about 20 nucleotides may be at least partially complementary to: (i) two different nucleic acid sequences located within chromosome 1 at a location ranging from 116514454 to 116514905; (ii) two different nucleic acid sequences located within chromosome 1 at a location ranging from 116518611 to 116519319; or (iii) one nucleic acid sequence within chromosome 1 at a location ranging from 116514454 to 116514905 and one nucleic acid sequence located within chromosome 1 at a location ranging from 116518611 to 116519319.

Table 1: Locations in various chromosomes which may be targeted by one or more target capture primers.

Table 2: Alternative locations within the MS4A1 gene which may be targeted by one or more target capture primers.

Table 3: Alternative locations within the CD58 gene which may be targeted by one or more target capture primers (EC = extracellular; TM = transmembrane). Table 4: Alternative locations within the MS4A1 gene which may be targeted by one or more target capture primers (EC = extracellular; TM = transmembrane).

Table 5: Alternative locations within the TNFSF13B gene which may be targeted by one or more target capture primers (EC = extracellular; TM = transmembrane).

Table 6: Alternative locations within the TNFRSF17 gene which may be targeted by one or more target capture primers (EC = extracellular; TM = transmembrane).

Table 7: Alternative locations within the TNFRSF17 gene which may be targeted by one or more target capture primers (EC = extracellular; TM = transmembrane). Table 8: Alternative locations within the CD19 gene which may be targeted by one or more target capture primers (EC = extracellular; TM = transmembrane).

Table 9: Alternative locations within the CD79B gene which may be targeted by one or more target capture primers (EC = extracellular; TM = transmembrane).

Table 10: Alternative locations within the CD22 gene which may be targeted by one or more target capture primers (EC = extracellular; TM = transmembrane).

[0147] In some embodiments, the plurality of target capture primers includes about 6 or more, about 8 or more, about 9 or more, about 12 or more, about 16 or more, about 20 or more, about 24 or more, etc. different target capture primers, wherein each different target capture primer has a different nucleic acid sequence, wherein the different nucleic acid sequences have at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOS: 1 - 462, SEQ ID NOS: 925 - 942, or SEQ IDS NO: 961 - 1436. In some embodiments, the plurality of target capture primers includes about 6 or more, about 8 or more, about 9 or more, about 12 or more, about 16 or more, about 20 or more, about 24 or more, etc. different target capture primers, wherein each different target capture primer has a different nucleic acid, wherein the different nucleic acid sequences have any one of SEQ ID NOS: 1 - 462, SEQ ID NOS: 925 - 942, or SEQ IDS NO: 961 - 1436. In some embodiments, the plurality of target capture primers includes: (i) 6 or more different target capture primers (e.g., about 8 or more, about 12 or more, about 16 or more, about 20 or more, etc. different target capture primers), wherein each of the 6 or more target capture primers have a different nucleic acid sequence, where the different nucleic acid sequences have at least 80%, at least 85%, 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one SEQ ID NOS: 943 - 960; and (ii) 6 or more different target capture primers (e.g., 8 or more, 12 or more, 16 or more, 20 or more, etc. different target capture primers) targeting one or more different regions within one or more of the CD 19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes; provided that each of the different target capture primers of the plurality of different target capture primers do not target a region within the one or more genes that overlap with one or more regions corresponding to one or more known variants.

[0148] In some embodiments, the plurality of different target capture primers includes: (i) 6 or more different target capture primers (e.g., about 8 or more, about 12 or more, about 16 or more, about 20 or more, etc. different target capture primers), wherein each of the about 6 or more target capture primers have a different nucleic acid sequence, where the different nucleic acid sequences have at least 80%, at least 85%, 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one SEQ ID NOS: 943 - 960; and (ii) 6 or more different target capture primers (e.g., about 8 or more, about 12 or more, about 16 or more, about 20 or more, etc. different target capture primers) each targeting a different region within one or more of the CD 19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes, wherein the different regions different region within the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes correspond to locations set forth in Tables 3 and 5 - 10; provided that each of the different target capture primers of the plurality of different target capture primers do not target a region within the one or more genes that overlap with one or more regions corresponding to one or more known variants. [0149] In some embodiments, the plurality of different target capture primers introduced to the nucleic acid library include any of the compositions and/or target capture primer panels described herein, e.g., any of the panels set forth in Tables 11 - 28.

[0150] Capture Moiety

[0151] In some embodiments, each of the one or more target capture primers are conjugates comprising (i) a capture moiety; and (ii) a sequence that is substantially complementary to a sequence of the one or more target nucleic acid molecules in the nucleic acid library. In some embodiments, each of the one or more target capture primers are capable of binding to a functionalized substrate through the capture moiety after hybridization and/or extension and as described herein. In other embodiments, the target capture primers are bound to a functionalized substrate through the capture moiety prior to hybridization as described herein.

[0152] In some embodiments, the capture moiety of each the one or more target capture primers comprises a first moiety (e.g., a first reactive functional group) which is reactive with a second moiety (e.g., a second reactive functional group) of another entity (e.g., a second moiety conjugated to a functionalized substrate). In some embodiments, the first moiety is a first member of a pair of specific binding entities; and the second moiety is a second member of the same pair of specific binding entities. In some embodiments, a "reaction" between a first moiety and a second moiety may mean that a covalent linkage is formed between two reactive groups or two reactive functional groups of the two moieties; or may mean that the two reactive groups or two reactive functional groups of the two moieties associate with each other, interact with each other, hybridize to each other, hydrogen bond with each other, etc. In some embodiments, the "reaction" thus includes binding events, such as the binding of a hapten with an anti-hapten antibody, or the binding of biotin with streptavidin. In some embodiments, each of the target capture primers includes the same capture moiety, e.g. biotin. In other embodiments, different subsets of target capture primers include different capture moieties.

[0153] In some embodiments, the capture moiety may include a biotin moiety to bind to a functionalized substrate including an avidin moiety or a streptavidin moiety. In other embodiments, the capture moiety may include a thiolated molecule to bind to a functionalized substrate which includes gold particles. In yet other embodiments, the capture moiety may include an amine-terminated molecule to bind to an NHS-activated substrate. [0154] In some embodiments, the capture moiety includes immobilized antibodies, which may be used to bind to molecules including or conjugated to specific antigenic molecules, such as an antigenic molecule bound to a functionalized substrate. In other embodiments, the capture moiety includes an antigenic molecule which may be used to bind to an immobilized antibodies, such as an antibody bound to a functionalized substrate.

[0155] In some embodiments, the capture moiety includes enzymes, which may be used to bind to molecules including or conjugated to specific enzyme substrates. In other embodiments, the capture moiety includes a substrate for an enzyme, which may be used to bind to an enzyme, such as an enzyme coupled to a functionalized substrate.

[0156] In some embodiments, the capture moiety includes receptors, which may be used to bind to molecules including or conjugated to specific receptor ligands, such as a receptor ligand bound to a functionalized substrate. In other embodiments, the capture moiety includes one or more receptor ligands, which may be used to bind to molecules including receptors, such as receptors bound to a functionalized substrate.

[0157] In some embodiments, the capture moiety includes lectins, which may be used to bind to molecules including or conjugated to specific polysaccharides, such as a polysaccharide bound to a functionalized substrate. In other embodiments, the capture moiety includes one or more polysaccharides, which may be used to bind to molecules including or conjugated to one or more lectins, such as one or more lectins bound to a functionalized substrate.

[0158] In even further embodiments, the capture moiety includes one or more nucleic acid sequences which may be used to bind to molecules including or conjugated to complementary base sequences. In other embodiments, the capture moiety may include tethered DNA/RNA aptamers which may specifically bind to target analytes such as small molecules, peptides, proteins, cells.

[0159] Extension of the Hybridized Different Target Capture Primers

[0160] Following the at least partial hybridization of the different target capture primers to the target nucleic acid molecules present within the nucleic acid library (step 110), each of the hybridized target capture primers are extended (step 120) to form one or more extended target capture primer complexes. In some embodiments, the extended target capture primer complexes are double-stranded products, each including an extended target capture primer (which itself includes the hybridized target capture primer including the capture moiety) and the target nucleic acid sequence to which the target capture primer hybridized to. In some embodiments, the extended target capture primer includes the reverse complement of at least a portion of the target nucleic acid molecule to which the target capture primer hybridized to.

[0161] In some embodiments, the hybridized target capture primers are extended with a first polymerase, thus forming the one or more extended target capture primer complexes. Depending on the type of nucleic acid molecule being analyzed, the polymerase may be a DNA- dependent DNA polymerase ("DNA polymerase") or an RNA-dependent DNA polymerase ("reverse transcriptase"). Non-limiting examples of suitable polymerases are selected from a Taq or Taq-derived polymerase (e.g., KAPA 2G polymerase from KAPA BIOSYSTEMS); or a B- family DNA polymerase (e.g., KAPA HIFI polymerase from KAPA BIOSYSTEMS).

[0162] In some embodiments, the hybridization and extension processes (steps 110 and 120 are performed simultaneously. In other embodiments, the hybridization and extension processes (steps 110 and 120) are performed sequentially. In some embodiments, the length of the extensions of the one or more hybridized target capture primers may be controlled actively through techniques such as inactivating the first and/or second polymerases added, or passively by enabling the reaction to go to completion such as through the consumption of limiting reactants. Such methods are further described in U.S. Patent Publication No. 2020/0032244, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0163] Removal of Non-Target Nucleic Acid Molecules

[0164] Following the extending of the hybridized target capture primers (step 120), the nucleic acid library is enriched for the presence of the one or more target nucleic acid molecules (step 130). In some embodiments, enrichment involves increasing the concentration of the one or more target nucleic acid molecules through depletion (i.e., removal) of other members of the library of nucleic acid molecules that are not target nucleic acid molecules.

[0165] In some embodiments, target nucleic acid molecule enrichment may be performed by capturing the one or more extended target capture primer complexes. In some embodiments, capture of the one or more extended target capture primer complexes may be achieved in a variety of ways as disclosed herein and can be achieved prior to, concurrent with, or subsequent to either of the hybridization and/or extension steps described above.

[0166] In some embodiments, capturing comprises contacting the one or more extended target capture primer complexes with an appropriately functionalized substrate (e.g., functionalized beads) after the step of hybridization and/or extension such that the capture moiety of each of one or more extended target capture primer complexes reacts with a corresponding moiety of the functionalized substrate, thereby binding the one or more extended target capture primer complexes to the functionalized substrate. For example, an extended target capture primer complex may include a capture moiety comprising biotin which would bind to a functionalized substrate including streptavidin. In those embodiments where the capture moiety is coupled to a functionalized substrate (e.g., beads) prior to the step of hybridization, capture is effectuated through the hybridization of each target capture primer to its corresponding target nucleic acid. Other methods of capturing the one or more extended target capture primer complexes are described further herein and described in U.S. Publication No. 2020/0032244, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0167] In some embodiments, the functionalized substrate comprises beads having an appropriately functionalized surface (where the substrate may be functionalized with any of the moieties described above). In some embodiments, the beads are included within a column or a microfluidic device. In some embodiments, the beads are magnetic beads. In other embodiments, the beads are non-magnetic beads.

[0168] Following the capture of the one or more extended target capture primer complexes, one or more purification process is performed such that non-target nucleic acid molecules, as well as any other unused reaction components (e.g., nucleotides, primer molecules, enzymes, buffers, etc.), are removed. For instance, the non-target nucleic acid molecules may be removed by flowing a wash fluid and/or buffer through a column including the functionalized substrate (e.g., a column including a plurality of beads having an appropriately functionalized surface). In some embodiments, washing is performed at least one. In other embodiments, washing is performed at least twice. In yet other embodiments, washing is performed at least three times. The skilled artisan will appreciate that the one or more extended target capture primer complexes bound to the functionalized substrates (e.g. beads) will remain bound to the functionalized substrates as the wash fluid and/or buffer is flowed through the column housing the functionalized substrate, while those unbound non-target nucleic acid molecules will be washed away, resulting in a reaction mixture enriched with the one or more target nucleic acid molecules.

[0169] In some embodiments, the target nucleic acid molecules may be optionally amplified following enrichment. In some embodiments, the captured target nucleic acid molecules are directly amplified while coupled to the functionalized surface. Such methods are described in U.S. Patent Nos. 10,240,192, 10,160995, and 7,842,457, the disclosures of which are hereby incorporated by reference herein in their entireties. In other embodiments, the one or more target nucleic acid molecules are released from the captured one or more extended target capture primer complexes (step 203) prior to amplification step.

[0170] Release of the Target Nucleic Acid Molecules

[0171] In some embodiments, the one or more target nucleic acid molecules are then released from the extended target capture primer complexes. In some embodiments, one or more release primers are hybridized to the one or more extended target capture primer complexes to effectuate release (step 150). In some embodiments, the one or more release primers are designed to hybridize to a portion of the one or more target nucleic acid molecules (of the one or more extended target capture primer complexes) and between the first and second adapters. In other embodiments, the one or more release primers are designed to hybridize to portions of the target nucleic acid molecules which are upstream relative to the location in which the target capture primer is hybridized to the target nucleic acid molecule within the extended target capture primer complex. As such, the design of any release primer may be dependent upon the regions of the one or more genes to which target capture primers were designed to hybridize.

[0172] For instance, if the target capture primers were designed to hybridize to complementary nucleic acid sequences located in one or more genes encoding one or more proteins capable of being targeted with an immunotherapeutic agent, such as those nucleic acid sequences identified in any of Tables 1 to 10 (based on genome build HG38 or the equivalent position in a genome build other than HG38 (e.g., a previously known genome build or a future genome build), then, in some embodiments, release primers would be designed to hybridize to complementary nucleic acid sequences upstream relative to the location in the one or more genes to which the respective target capture primer is designed to hybridize to.

[0173] Non-limiting examples of suitable target capture primers and corresponding release primers are set forth herein (see Tables 29 and 30).

[0174] Next, the one or more hybridized release primers are extended using a polymerase, thereby forming one or more extended hybridized release primers (step 150). In some embodiments, the extension of the one or more hybridized release primers with the polymerase liberates the target capture primer from each of the one or more extended target capture primer complexes. In some cases, the polymerase exhibits strand displacement activity. In some cases, the polymerase exhibits 5' - 3' exonuclease activity that digests a single strand of a double-stranded nucleic acid molecule (referred to herein as 5' - 3' double-stranded exonuclease activity) or doublestranded exonuclease activity. In some cases, the polymerase exhibits both strand displacement and 5' - 3' double-stranded exonuclease activity. In some cases, the strand displacement, 5' - 3' double-stranded exonuclease activity, or combination thereof, can displace a target nucleic acid molecule (e.g., original target nucleic acid molecule from a provided sample) into solution.

[0175] For example, in some embodiments, an extended target capture primer complex including a target nucleic acid molecule is immobilized on a functionalized substrate, e.g., by affinity capture of a ligand (e.g., biotin or a derivative thereof) through the capture moiety of the extended target capture primer. In such embodiments, the strand displacement activity of the polymerase that extends the hybridized release primer displaces the target nucleic acid molecule from the sample into solution. Alternatively, 5' - 3' exonuclease activity can degrade an extended target capture primer that is immobilized by affinity capture and hybridized to target nucleic acid molecule, wherein the 5' - 3' exonuclease activity thereby releases the target nucleotide into solution. Additional methods of liberating a target nucleic acid molecule from a formed target capture primer extension complex are described in US. Patent Publication No. 2018/0016630, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0176] The released one or more target nucleic acid molecules may then be used in one or more downstream processes, e.g., sequencing, amplification, further coupling, etc. in some embodiments, the nucleic acid library enriched with the one or more target nucleic acid molecules is optionally amplified. In some embodiments, the step of optional amplification comprises one of a linear or exponential amplification (e.g., polymerase chain reaction (PCR)). As used herein, "PCR" refers to a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA. As used herein, PCR encompass derivative forms of the reaction, including but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, digital PCR, digital droplet PCR, and assembly PCR. Amplification of the target nucleic acid molecules following enrichment may comprise non- PCR based methods. Non-limiting examples of non-PCR based methods include nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), whole transcriptome amplification (WTA), whole genome amplification (WGA), multiple displacement amplification (MDA), strand displacement amplification (SDA), real-time SDA, rolling circle amplification, and/or circle-to-circle amplification.

[0177] In general, amplification comprises amplifying the target nucleic acid molecule with a polymerase, a first amplification primer, and a second amplification primer. In some embodiments, the first and second amplification primers are designed to be complementary to the sequences of the adapters incorporated into the one or more target nucleic acid molecules in the library of nucleic acid molecules. For example, the first amplification primer may have a 3' end complementary to the first adapter and the second amplification primer may have a 3' end complementary to the second adapter. In some embodiments, the amplification primers may include any sequences that are present within the target nucleic acid molecule being amplified (e.g., gene/target specific primers, universal primers, or the like) and can support synthesis of one or both strands (i.e., both the top and bottom strands of a double-stranded nucleic acid molecules corresponding to the template of the amplification reaction). In some embodiments, the first and second amplification primers are universal primers. Additional methods of amplification are described in U.S. Publication Nos. 2020/0032244 and 2018/0016630, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0178] Sequencing

[0179] In some embodiments, sequencing of the target enriched library of nucleic acids may be performed according to any method known to those of ordinary skill in the art (see, step 30 of FIG. 1 A). In some embodiments, sequencing methods include Sanger sequencing and dyeterminator sequencing, as well as next-generation sequencing technologies such as pyrosequencing, nanopore sequencing, micropore-based sequencing, nanoball sequencing, MPSS, SOLiD, Illumina, Ion Torrent, Starlite, SMRT, tSMS, sequencing by synthesis, sequencing by expansion, sequencing by ligation, mass spectrometry sequencing, polymerase sequencing, RNA polymerase (RNAP) sequencing, microscopy-based sequencing, microfluidic Sanger sequencing, microscopy-based sequencing, RNAP sequencing, etc. Instruments and methods of sequencing are disclosed, for example, in PCT Publication Nos. WO2014144478, W02015058093, W02014106076 and WO2013068528, the disclosures of which are hereby incorporated by reference in their entireties.

[0180] In some embodiments, sequencing can be performed by a number of different methods, such as by employing sequencing by synthesis technology. Sequencing by synthesis according to the prior art is defined as any sequencing method which monitors the generation of side products upon incorporation of a specific deoxynucleoside-triphosphate during the sequencing reaction (Hyman, 1988, Anal. Biochem. 174:423-436; Rhonaghi et al., 1998, Science 281 :363-365). One prominent embodiment of the sequencing by synthesis reaction is the pyrophosphate sequencing method. In this case, generation of pyrophosphate during nucleotide incorporation is monitored by an enzymatic cascade which results in the generation of a chemoluminescent signal. The 454 Genome Sequencer System (Roche Applied Science cat. No. 04 760 085 001), an example of sequence by synthesis, is based on the pyrophosphate sequencing technology. For sequencing on a 454 GS20 or 454 FLX instrument, the average genomic DNA fragment size is in the range of 200 or 600 bp, respectively, as described in the product literature. [0181] In some embodiments, a sequencing by synthesis reaction can alternatively be based on a terminator dye type of sequencing reaction. In this case, the incorporated dye deoxynucleotriphosphates (ddNTPs) building blocks comprise a detectable label, which is preferably a fluorescent label that prevents further extension of the nascent DNA strand. The label is then removed and detected upon incorporation of the ddNTP building block into the template/primer extension hybrid for example by using a DNA polymerase comprising a 3 '-5' exonuclease or proofreading activity.

[0182] In some embodiments, and in the case of the Genome Sequencer workflow (Roche Applied Science Catalog No. 04 896 548 001), in a first step, (clonal) amplification is performed by emulsion PCR. Thus, it is also within the scope of the present disclosure, that the step of amplification is performed by emulsion PCR methods. The beads carrying the clonally amplified target nucleic acid molecules may then become arbitrarily transferred into a picotiter plate according to the manufacturer's protocol and subjected to a pyrophosphate sequencing reaction for sequence determination.

[0183] In some embodiments, sequencing is performed using a next-generation sequencing method such as that provided by Illumina, Inc. (the "Illumina Sequencing Method"). Without wishing to be bound by any particular theory, the Illumina next-generation sequencing technology uses clonal amplification and sequencing by synthesis (SBS) chemistry to enable rapid, accurate sequencing. The process simultaneously identifies DNA bases while incorporating them into a nucleic acid chain. Each base emits a unique fluorescent signal as it is added to the growing strand, which is used to determine the order of the DNA sequence. [0184] COMPOSITIONS / TARGET CAPTURE PRIMER COMPOSITIONS

[0185] The present disclosure is also directed to compositions comprising a plurality of different target capture primers, wherein each target capture primer of the plurality of different target capture primers targets a different region within one or more genes of interest. In some embodiments, each target capture primer targets a different region within one or more genes of interest, provided that each of the different target capture primers within the pool of different target capture primers do not target a region within a gene that overlaps with one or more regions corresponding to one or more known variants.

[0186] In some embodiments, the present disclosure is directed to a composition comprising a plurality of different target capture primers. In some embodiments, the composition includes at least 6 different target capture primers, each targeting a different region within one or more coding exons of MS4A1. In some embodiments, the composition includes at least 9 different target capture primers, each targeting a different region within one or more coding exons of MS4A1. In some embodiments, the composition includes at least 12 different target capture primers, each targeting a different region within one or more coding exons of MS4A1. In some embodiments, the composition includes at least 16 different target capture primers, each targeting a different region within one or more coding exons of MS4A1.

[0187] In some embodiments, the composition includes at least 6 different target capture primers, each targeting a different region within one or more coding exons of MS4A1, provided that each of the different target capture primers within the panel do not target a region within the MS4A1 that overlaps with one or more regions corresponding to one or more known variants. In some embodiments, the composition includes at least 9 different target capture primers, each targeting a different region within one or more coding exons of MS4A1, each targeting a different region within MS4A1, provided that each of the different target capture primers within the panel do not target a region within the MS4A1 that overlaps with one or more regions corresponding to one or more known variants. In some embodiments, the composition includes at least 12 different target capture primers, each targeting a different region within MS4A1, each targeting a different region within one or more coding exons of MS4A1, provided that each of the different target capture primers within the panel do not target a region within the MS4A1 that overlaps with one or more regions corresponding to one or more known variants. In some embodiments, the composition includes at least 16 different target capture primers, each targeting a different region within MS4A1, each targeting a different region within one or more coding exons of MS4A1 , provided that each of the different target capture primers within the panel do not target a region within the MS4A1 that overlaps with one or more regions corresponding to one or more known variants.

[0188] In some embodiments, the composition includes: (i) at least 6 different target capture primers (e.g., 8 or more, 12 or more, 16 or more, 20 or more, etc. different target capture primers) targeting one or more regions within at least two coding exons (such as 3 or more, 4 or more, 5 or more, etc. coding exons) within the MS4A1 gene; and (ii) one or more different target capture primers (e.g., 2 or more, 3 or more, 4 or more, 6 or more, 8 or more, 12 or more, 16 or more, 20 or more, etc. different target capture primers) targeting one or more different regions within at least one of the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes, such as within at least two of the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes, such as within at least three of the CD 19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes, or such as within at least four of the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes.

[0189] In some embodiments, the composition includes at least 6 different target capture primers (e.g., at least 8, at least 12, at least 16, at least 20, etc. different target capture primers), each targeting a different region within MS4A1, wherein the different regions correspond to locations within Chromosome 11 ranging from between about 60455815 to about 60456010, from between about 60457995 to about 60458316, from between about 60461002 to about 60461269, from between about 60462140 to about 60462600, from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, from between about 60466902 to about 60467140, and/or from between about 60468195 to about 60470813, based on genome build HG38 or an equivalent position in a genome build other than HG38. In some embodiments, the at least 6 different target capture primers are designed to hybridize to 10 or more consecutive nucleotides, such as 15 or more consecutive nucleotides, such as 20 or more consecutive nucleotides of complementary nucleic acid sequences corresponding to locations within Chromosome 11 ranging from between about 60455815 to about 60456010, from between about 60457995 to about 60458316, from between about 60461002 to about 60461269, from between about 60462140 to about 60462600, from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, from between about 60466902 to about 60467140, and/or from between about 60468195 to about 60470813, based on genome build HG38 or an equivalent position in a genome build other than HG38.

[0190] In other embodiments, the composition includes (i) at least 6 different target capture primers (e.g., at least 8, at least 12, at least 16, at least 20, etc. different target capture primers), each targeting a different region within MS4A1, wherein the different regions correspond to locations within Chromosome 11 ranging from between about 60455815 to about 60456010, from between about 60457995 to about 60458316, from between about 60461002 to about 60461269, from between about 60462140 to about 60462600, from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, from between about 60466902 to about 60467140, and/or from between about 60468195 to about 60470813, based on genome build HG38 or an equivalent position in a genome build other than HG38; and (ii) at least three additional target capture primers targeting one or more regions within one of the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes, such as at least 6 additional target capture primers targeting one or more regions within one of the CD 19, CD22, CD58, CD79B, TNFSF13B, TNFRSF 13 C, and TNFRSF 17 genes, such as at least nine additional target capture primers targeting one or more regions within one of the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes, such as at least nine additional target capture primers targeting one or more regions within one of the CD 19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF 17 genes, etc.

[0191] In yet other embodiments, the composition includes at least 6 different target capture primers (e.g., at least 8, at least 12, at least 16, at least 20, etc. different target capture primers), each targeting a different region within MS4A1, wherein the different regions correspond to locations within Chromosome 11 ranging from between about 60463002 to about 60463121, from between about 60464288 to about 60464344, from between about 60465921 to about 60466157, and/or from between about 60466959 to about 60467060, based on genome build HG38 or an equivalent position in a genome build other than HG38.

[0192] In further embodiments, the composition includes (i) at least 6 different target capture primers (e.g., at least 8, at least 12, at least 16, at least 20, etc. different target capture primers), each targeting a different region within MS4A1, wherein the different regions correspond to locations within Chromosome 11 ranging from between about 60463002 to about 60463121, from between about 60464288 to about 60464344, from between about 60465921 to about 60466157, and/or from between about 60466959 to about 60467060, based on genome build HG38 or an equivalent position in a genome build other than HG38, (ii) at least three additional target capture primers (e.g., at least 6, at least 9, at least 12, at least 16, at least 20, etc. additional target capture primers) targeting one or more regions within one or more of the CD 19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes.

[0193] In yet further embodiments, the composition includes at least 6 different target capture primers (e.g., at least 8, at least 12, at least 16, at least 20, etc. different target capture primers), each targeting a different region within MS4A1, wherein the different regions correspond to locations within Chromosome 11 ranging from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, and/or from between about 60466902 to about 60467140, based on genome build HG38 or an equivalent position in a genome build other than HG38.

[0194] In yet even further embodiments, the composition includes (i) at least 6 different target capture primers (e.g., at least 8, at least 12, at least 16, at least 20, etc. different target capture primers), each targeting a different region within MS4A1, wherein the different regions correspond to locations within Chromosome 11 ranging from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, and/or from between about 60466902 to about 60467140, based on genome build HG38 or an equivalent position in a genome build other than HG38; (ii) at least three additional target capture primers (e.g., at least 6, at least 9, at least 12, at least 16, at least 20, etc. additional target capture primers) targeting one or more regions within one or more of the CD 19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes.

[0195] In some embodiments, the composition of comprises at least 6 different target capture primers (e.g., at least 9, at least 12, at least 16, etc. different target capture primers), wherein each of the at least 6 different target capture primers have different nucleic acid sequences, wherein the different nucleic acid sequences have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOS: 41 - 122 or 925 - 942. In some embodiments, the composition comprises (i) at least 6 different target capture primers (e.g., at least 9, at least 12, at least 16, etc. different target capture primers), wherein each of the at least 6 different target capture primers have different nucleic acid sequences, wherein the different nucleic acid sequences have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOS: 41 - 122 or 925 - 942; and (ii) at least three additional target capture primers (e.g., at least 6, at least 9, at least 12, at least 16, at least 20, etc. additional target capture primers) targeting one or more regions within one or more of the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes.

[0196] In some embodiments, the composition of comprises at least 6 different target capture primers (e.g., at least 9, at least 12, at least 16, etc. different target capture primers), wherein each of the at least 6 different target capture primers have different nucleic acid sequences, wherein the different nucleic acid sequences have any one of SEQ ID NOS: 41 - 122 or 925 - 942. In other embodiments, the composition of comprises (i) at least 6 different target capture primers (e.g., at least 9, at least 12, at least 16, etc. different target capture primers), wherein each of the at least 6 different target capture primers have different nucleic acid sequences, wherein the different nucleic acid sequences have any one of SEQ ID NOS: 41 - 122 or 925 - 942; and (ii) at least three additional target capture primers (e.g., at least 6, at least 9, at least 12, at least 16, at least 20, etc. additional target capture primers) targeting one or more regions within one or more of the CD 19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes.

[0197] In some embodiments, the composition of comprises at least 6 different target capture primers (e.g., at least 9, at least 12, at least 16, etc. different target capture primers), wherein each of the at least 6 different target capture primers have different nucleic acid sequences, wherein the different nucleic acid sequences have any one of SEQ ID NOS: 925 - 942. In some embodiments, the composition of comprises (i) at least 6 different target capture primers (e.g., at least 9, at least 12, at least 16, etc. different target capture primers), wherein each of the at least 6 different target capture primers have different nucleic acid sequences, wherein the different nucleic acid sequences have any one of SEQ ID NOS: 925 - 942; and (ii) at least three additional target capture primers (e.g., at least 6, at least 9, at least 12, at least 16, at least 20, etc. additional target capture primers) targeting one or more regions within one or more of the CD 19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes.

[0198] In some embodiment, the composition comprises a plurality of different target capture primers (e.g., at least 6, at least 9, at least 12, at least 16, at least 20, at least 24, etc. different capture primers) each designed to at least partially hybridize to a complementary nucleic acid sequence in a gene within any one of chromosomes 1, 13, 16, 17, 19, and 22 and as set forth in any of Tables 2 - 10.

[0199] In some embodiments, the composition of comprises at least 6 different target capture primers (e.g., at least 9, at least 12, at least 16, etc. different target capture primers), wherein each of the at least 6 different target capture primers have different nucleic acid sequences, wherein the different nucleic acid sequences have any one of SEQ ID NOS: 925 - 942. In some embodiments, the composition of comprises (i) at least 6 different target capture primers (e.g., at least 9, at least 12, at least 16, etc. different target capture primers), wherein each of the at least 6 different target capture primers have different nucleic acid sequences, wherein the different nucleic acid sequences have any one of SEQ ID NOS: 925 - 942; and (ii) at least three additional target capture primers (e.g., at least 6, at least 9, at least 12, at least 16, at least 20, etc. additional target capture primers) each designed to at least partially hybridize to a complementary nucleic acid sequence in a gene within any one of chromosomes 1, 13, 16, 17, 19, and 22 and as set forth in any of Tables 3 and 5 - 10.

[0200] In some embodiments, the present disclosure provides for a panel targeting the BAFF-R/BAFF-L/BCMA axis, where the panel targeting the BAFF-R/BAFF-L/BCMA axis comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have at least 90% identity to the sequences set forth in Table 1 1. In some embodiments, the present disclosure provides for a panel targeting the BAFF-R/BAFF-L/BCMA axis, where the panel targeting the BAFF-R/BAFF-L/BCMA axis comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have the sequences set forth in Table 11. In some embodiments, the panel targeting the BAFF-R/BAFF- L/BCMA axis comprises at least 8 different target capture primers targeting one or more regions within the TNFSF13B gene; and at least 8 different target capture primers targeting one or more regions within the TNFRSF17 gene. Table 11: A panel targeting the BAFF-R/BAFF-L/BCMA axis. A kit comprising this panel may include an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0201] In some embodiments, the present disclosure provides for a panel targeting the BAFF-R/BAFF-L/BCMA axis, where the panel targeting the BAFF-R/BAFF-L/BCMA axis comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have at least 90% identity to the sequences set forth in Table 12. In some embodiments, the present disclosure provides for a panel targeting the BAFF-R/BAFF-L/BCMA axis, where the panel targeting the BAFF-R/BAFF-L/BCMA axis comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have the sequences set forth in Table 12. In some embodiments, the panel targeting the BAFF-R/BAFF- L/BCMA axis comprises at least 6 different target capture primers targeting one or more regions within the TNFSF13B gene; and at least 6 different target capture primers targeting one or more regions within the TNFRSF17 gene. In some embodiments, the panel targeting the BAFF- R/BAFF-L/BCMA axis comprises at least 8 different target capture primers targeting one or more regions within the TNFSF13B gene; and at least 8 different target capture primers targeting one or more regions within the TNFRSF17 gene. Table 12: A panel targeting the BAFF-R/BAFF-L/BCMA axis. A kit comprising this panel may include an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0202] In some embodiments, the present disclosure provides for a panel targeting CD20 and CD58, where the panel targeting CD20 and CD58 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have at least 90% identity to the sequences set forth in Table 13. In some embodiments, the present disclosure provides for a panel targeting CD20 and CD58, where the panel targeting CD20 and CD58 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have the sequences set forth in Table 13. In some embodiments, the panel targeting CD20 and CD58 comprises at least 6 different target capture primers targeting one or more regions within the CD20 gene; and at least 6 different target capture primers targeting one or more regions within the CD58 gene. In some embodiments, the panel targeting CD20 and CD58 comprises at least 8 different target capture primers targeting one or more regions within the CD20 gene; and at least 8 different target capture primers targeting one or more regions within the CD58 gene.

Table 13: A panel targeting CD20 and CD58. A kit comprising this panel may include an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0203] In some embodiments, the present disclosure provides for a panel targeting CD20 and CD58, where the panel targeting CD20 and CD58 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have at least 90% identity to the sequences set forth in Table 14. In some embodiments, the present disclosure provides for a panel targeting CD20 and CD58, where the panel targeting CD20 and CD58 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have the sequences set forth in Table 14. In some embodiments, the panel targeting CD20 and CD58 comprises at least 6 different target capture primers targeting one or more regions within the CD20 gene; and at least 6 different target capture primers targeting one or more regions within the CD58 gene. In some embodiments, the panel targeting CD20 and CD58 comprises at least 8 different target capture primers targeting one or more regions within the CD20 gene; and at least 8 different target capture primers targeting one or more regions within the CD58 gene.

Table 14: A panel targeting CD20 and CD58. A kit comprising this panel may include an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0204] In some embodiments, the present disclosure provides for a panel targeting CD20 and CD 19, where the panel targeting CD20 and CD 19 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have at least 90% identity to the sequences set forth in Table 15. In some embodiments, the present disclosure provides for a panel targeting CD20 and CD 19, where the panel targeting CD20 and CD 19 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have the sequences set forth in Table 15. In some embodiments, the panel targeting CD20 and CD19 comprises at least 6 different target capture primers targeting one or more regions within the CD20 gene; and at least 6 different target capture primers targeting one or more regions within the CD 19 gene. In some embodiments, the panel targeting CD20 and CD 19 comprises at least 8 different target capture primers targeting one or more regions within the CD20 gene; and at least 8 different target capture primers targeting one or more regions within the CD 19 gene.

Table 15: A panel targeting CD20 and CD19. A kit comprising this panel may include an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0205] In some embodiments, the present disclosure provides for a panel targeting CD20 and CD 19, where the panel targeting CD20 and CD 19 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have at least 90% identity to the sequences set forth in Table 16. In some embodiments, the present disclosure provides for a panel targeting CD20 and CD19, where the panel targeting CD20 and CD19 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have the sequences set forth in Table 16. In some embodiments, the panel targeting CD20 and CD 19 comprises at least 6 different target capture primers targeting one or more regions within the CD20 gene; and at least 6 different target capture primers targeting one or more regions within the CD 19 gene. In some embodiments, the panel targeting CD20 and CD 19 comprises at least 8 different target capture primers targeting one or more regions within the CD20 gene; and at least 8 different target capture primers targeting one or more regions within the CD 19 gene.

Table 16: A panel targeting CD20 and CD19. A kit comprising this panel may include an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0206] In some embodiments, the present disclosure provides for a panel targeting CD 19, CD79b, CD58, and CD20, where the panel targeting CD19, CD79b, CD58, and CD20 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have at least 90% identity to the sequences set forth in Table 17. In some embodiments, the present disclosure provides for a panel targeting CD 19, CD79b, CD58, and CD20, where the panel targeting CD 19, CD79b, CD58, and CD20 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have the sequences set forth in Table 17. In some embodiments, the panel targeting CD19, CD79b, CD58, and CD20 comprises at least 4 different target capture primers targeting one or more regions within the CD 19 gene; at least 4 different target capture primers targeting one or more regions within the CD79b gene; at least 4 different target capture primers targeting one or more regions within the CD58 gene; and at least 4 different target capture primers targeting one or more regions within the CD20 gene.

Table 17: A panel targeting CD19, CD79b, CD58, and CD20. A kit comprising this panel may include an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0207] In some embodiments, the present disclosure provides for a panel targeting CD19, CD79b, CD58, and CD20, where the panel targeting CD19, CD79b, CD58, and CD20 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have at least 90% identity to the sequences set forth in Table 18. In some embodiments, the present disclosure provides for a panel targeting CD 19, CD79b, CD58, and CD20, where the panel targeting CD 19, CD79b, CD58, and CD20 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have the sequences set forth in Table 18. In some embodiments, the panel targeting CD19, CD79b, CD58, and CD20 comprises at least 4 different target capture primers targeting one or more regions within the CD 19 gene; at least 4 different target capture primers targeting one or more regions within the CD79b gene; at least 4 different target capture primers targeting one or more regions within the CD58 gene; and at least 4 different target capture primers targeting one or more regions within the CD20 gene.

Table 18: A panel targeting CD19, CD79b, CD58, and CD20. A kit comprising this panel may include an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0208] In some embodiments, the present disclosure provides for a panel targeting CD 19, CD79b, CD58, CD22, and CD20, where the panel targeting CD19, CD79b, CD58, CD22, and CD20 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have at least 90% identity to the sequences set forth in Table 19. In some embodiments, the present disclosure provides for a panel targeting CD19, CD79b, CD58, and CD20, where the panel targeting CD19, CD79b, CD58, CD22, and CD20 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have the sequences set forth in Table 19. In some embodiments, the panel targeting CD19, CD79b, CD58, CD22, and CD20 comprises at least 4 different target capture primers targeting one or more regions within the CD 19 gene; at least 4 different target capture primers targeting one or more regions within the CD79b gene; at least 4 different target capture primers targeting one or more regions within the CD58 gene; at least 4 different target capture primers targeting one or more regions within the CD22 gene; and at least 4 different target capture primers targeting one or more regions within the CD20 gene.

Table 19: A panel targeting CD19, CD79b, CD58, CD22, and CD20. A kit comprising this panel may include an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0209] In some embodiments, the present disclosure provides for a panel targeting CD 19, CD79b, CD58, CD22, and CD20, where the panel targeting CD 19, CD79b, CD58, CD22, and CD20 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have at least 90% identity to the sequences set forth in Table 20. In some embodiments, the present disclosure provides for a panel targeting CD19, CD79b, CD58, and CD20, where the panel targeting CD19, CD79b, CD58, CD22, and CD20 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have the sequences set forth in Table 20. In some embodiments, the panel targeting CD 19, CD79b, CD58, CD22, and CD20 comprises at least 4 different target capture primers targeting one or more regions within the CD 19 gene; at least 4 different target capture primers targeting one or more regions within the CD79b gene; at least 4 different target capture primers targeting one or more regions within the CD58 gene; at least 4 different target capture primers targeting one or more regions within the CD22 gene; and at least 4 different target capture primers targeting one or more regions within the CD20 gene.

Table 20: A panel targeting CD19, CD79b, CD58, CD22, and CD20. A kit comprising this panel may include an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0210] In some embodiments, the present disclosure provides for a panel targeting CD 19, where the panel targeting CD 19 at least 8 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have at least 90% identity to the sequences set forth in Table 21. In some embodiments, the present disclosure provides for a panel targeting CD19, where the panel targeting CD 19 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have the sequences set forth in Table 21. Table 21 : A panel targeting CD19. A kit comprising this panel may include an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0211] In some embodiments, the present disclosure provides for a panel targeting CD22, where the panel targeting CD22 at least 8 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have at least 90% identity to the sequences set forth in Table 22. Tn some embodiments, the present disclosure provides for a panel targeting CD22, where the panel targeting CD22 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have the sequences set forth in Table 22.

Table 22: A panel targeting CD22. A kit comprising this panel may include an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0212] In some embodiments, the present disclosure provides for a panel targeting CD58, where the panel targeting CD58 at least 8 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have at least 90% identity to the sequences set forth in Table 23. In some embodiments, the present disclosure provides for a panel targeting CD58, where the panel targeting CD58 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have the sequences set forth in Table 23.

Table 23: A panel targeting CD58. A kit comprising this panel may include an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0213] In some embodiments, the present disclosure provides for a panel targeting CD79B, where the panel targeting CD79B at least 8 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have at least 90% identity to the sequences set forth in Table 24. In some embodiments, the present disclosure provides for a panel targeting CD79B, where the panel targeting CD79B comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have the sequences set forth in Table 24.

Table 24: A panel targeting CD79B. A kit comprising this panel may include an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0214] In some embodiments, the present disclosure provides for a panel targeting MS4A1, where the panel targeting MS4A1 at least 8 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have at least 90% identity to the sequences set forth in Table 25. In some embodiments, the present disclosure provides for a panel targeting MS4A1, where the panel targeting MS4A1 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have the sequences set forth in Table 25.

Table 25: A panel targeting CD20. A kit comprising this panel may include an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0215] In some embodiments, the present disclosure provides for a panel targeting TNFRSF13C, where the panel targeting TNFRSF13C at least 8 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have at least 90% identity to the sequences set forth in Table 26. In some embodiments, the present disclosure provides for a panel targeting TNFRSF13C, where the panel targeting TNFRSF13C comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have the sequences set forth in Table 26. Table 26: A panel targeting TNFRSF13C. A kit comprising this panel may include an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0216] In some embodiments, the present disclosure provides for a panel targeting TNFRSF17, where the panel targeting TNFRSF17 at least 8 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have at least 90% identity to the sequences set forth in Table 27. In some embodiments, the present disclosure provides for a panel targeting TNFRSF17, where the panel targeting TNFRSF17 comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have the sequences set forth in Table 27.

Table 27: A panel targeting TNFRSF17. A kit comprising this panel may include an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0217] In some embodiments, the present disclosure provides for a panel targeting TNFSF13B, where the panel targeting TNFSF13B at least 8 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have at least 90% identity to the sequences set forth in Table 28. In some embodiments, the present disclosure provides for a panel targeting TNFSF13B, where the panel targeting TNFSF13B comprises at least 10 different target capture primers, such as at least 12 different target capture primers, such as at least 16 different target capture primers, such as at least 20 different target capture primers, such as at least 24 different target capture primers, wherein the at least 10 different target capture primers have the sequences set forth in Table 28.

Table 28: A panel targeting TNFSF13B. A kit comprising this panel may include an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0218] KITS

[0219] The present disclosure is also directed to one or more kits, such as one or more kits including a plurality of different target capture primers and release primers.

[0220] In some embodiments, the present disclosure provides for a kit comprising: (i) 10 or more, 12 or more, 14 or more, 16 or more, 18 or more, 20 or more, 24 or more, 30 or more, 36 or more, etc. different target capture primers; and (ii) an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0221] In some embodiments, the present disclosure provides for a kit comprising: (i) 10 or more, 12 or more, 14 or more, 16 or more, 18 or more, 20 or more, 24 or more, 30 or more, 36 or more, etc. different target capture primers, wherein each different target capture primer has a different nucleic acid sequence, wherein the different nucleic acid sequences have at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOS: 1 - 462, SEQ ID NOS: 925 - 942, or SEQ IDS NO: 961 - 1436; and (ii) an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0222] In some embodiments, the present disclosure provides for a kit comprising: (i) 10 or more, 12 or more, 14 or more, 16 or more, 18 or more, 20 or more, 24 or more, 30 or more, 36 or more, etc. different target capture primers, wherein each different target capture primer has a different nucleic acid sequence, wherein the different nucleic acid sequences have any one of SEQ ID NOS: 1 - 462, SEQ ID NOS: 925 - 942, or SEQ IDS NO: 961 - 1436; and (ii) an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0223] In some embodiments, the present disclosure provides for a kit comprising: (i) 10 or more, 12 or more, 14 or more, 16 or more, 18 or more, 20 or more, 24 or more, 30 or more, 36 or more, etc. different target capture primers, wherein each different target capture primer has a different nucleic acid sequence, wherein the different nucleic acid sequences have at least 80%, at least 85%, 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one SEQ ID NOS: 925 - 942; and (ii) an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0224] In some embodiments, the present disclosure provides for a kit comprising: (i) 10 or more, 12 or more, 14 or more, 16 or more, 18 or more, 20 or more, 24 or more, 30 or more, 36 or more, etc. different target capture primers, wherein each different target capture primer have any one SEQ ID NOS: 925 - 942; and (ii) an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0225] In some embodiments, the present disclosure provides for a kit comprising: (i) 10 or more, 12 or more, 14 or more, 16 or more, 18 or more, 20 or more, 24 or more, 30 or more, 36 or more, etc. different target capture primers, wherein each different target capture primer has a different nucleic acid sequence, wherein the different nucleic acid sequences have at least 80%, at least 85%, 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one the sequences set forth in any one of Tables 11 - 28; and (ii) an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize. [0226] In some embodiments, the present disclosure provides for a kit comprising: (i) 10 or more, 12 or more, 14 or more, 16 or more, 18 or more, 20 or more, 24 or more, 30 or more, 36 or more, etc. different target capture primers, wherein each different target capture primer have any one the sequences set forth in any one of Tables 11 - 28; and (ii) an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

[0227] In some embodiments, the kit includes 10 or more, 12 or more, 14 or more, 16 or more, 18 or more, 20 or more, 24 or more, 30 or more, 36 or more, etc. pairs of target capture primers and their corresponding release primers, such as any of those pairs of target capture primers and corresponding release primers identified in Tables 29, 30, and 31.

Table 29: Target capture primer capable of at least partially hybridizing to a region within a gene encoding a protein capable of being targeted with an immunotherapeutic agent, and a corresponding release primer capable of at least partially hybridizing to a region within the gene upstream from the region in which the target capture primer hybridizes.

Ill

Table 30: Target capture primer capable of at least partially hybridizing to a region within a gene encoding a protein capable of being targeted with an immunotherapeutic agent, and a corresponding release primer capable of at least partially hybridizing to a region within the gene upstream from the region in which the target capture primer hybridizes.

Table 31: Target capture primer capable of at least partially hybridizing to a region within a gene encoding a protein capable of being targeted with an immunotherapeutic agent, and a corresponding release primer capable of at least partially hybridizing to a region within the gene upstream from the region in which the target capture primer hybridizes.

[0228] In some embodiments, the kit may include reagents for amplification (a master mix), e.g., polymerase, dNTPs, buffers, and/or other elements (e.g., cofactors or aptamers) appropriate for amplification. Typically, the reagent mixture(s) is concentrated, so that an aliquot is added to the final reaction volume, along with sample (e g., RNA or DNA), enzymes, and/or water. In some embodiments, the kit further includes at least one polymerase. In some embodiments, the kit further includes at least two different polymerases. In some embodiments, the kit further includes a plurality of nucleotides. In some embodiments, the kit further includes one or more buffer solutions and/or wash solutions. In some embodiments, the kit further includes beads having a functionalized surface. In some embodiments, the kit further includes one or more release primers. In some embodiments, the kit further comprises first and second amplification primers.

[0229] EXAMPLES

[0230] EXAMPLE 1 - LONGITUDINAL ASSESSMENT FROM LIQUID BIOPSY

OF MUTATIONS IN CD20 ASSOCIATED WITH RESISTANCE TO BISPECIFIC MONOCLONAL ANTIBODY THERAPY: A PILOT STUDY USING A PETE ENRICHMENT STRATEGY

[0231] Background

[0232] CD20 is a membrane protein encoded by MS4A1, expressed during the differentiation and development of B cells. The protein is expressed in a ubiquitous and stable manner across B cell neoplasms. As such, it is a parsimonious diagnostic and therapeutic target for Non-Hodgkin’s Lymphoma (NHL). For example, the monoclonal antibody (MAb) rituximab in combination with chemotherapy (R-CHOP) is now a standard treatment for DLBCL, the most common NHL subtype. In recent years, a potent new class of therapeutics called bispecific monoclonal antibodies (BsMAbs) have emerged. BsMAbs facilitate MHC independent cytotoxicity by co-targeting T cells and tumor antigen, in this case CD20 (Falchi et al 2023).

[0233] Although targeted treatment with monoclonal antibodies has improved clinical outcomes, a subset of patients experiences relapsed or refractory disease. Continuing to use DLBCL as an example, patients not responding to R-CHOP have a 2-year survival rate ranging from 20%-40% (Rushton et al, 2020). One such mechanism of resistance is via the selective pressure from treatment resulting in the expansion of clones harboring mutations in MS4A1 preventing MAb binding. This can occur either through loss of expression or alteration of MAb binding sites, the latter being a rare occurrence. This has been well documented in R-CHOP resistance (Foran et al 2001, Johnson et al 2009, Mishima et al 2011, Rushton et al 2020,), however emerging research also reports this mechanism in resistance to BsMAb treatment (Brouwer- Visser et al 2020, Schuster et al 2022, Parrondo et a. 2022). Patients undergoing treatment with BsMAb could benefit from routine screening of the MS4A1 gene to identify resistance mutations and tailor treatment accordingly. Furthermore, given that such a screening is likely to be longitudinal in nature, it is preferable that it be optimized for cfDNA (cell free DNA) samples obtained by liquid biopsy. Here, we describe a pilot study assessing the performance of a single gene PETE-based enrichment strategy, designed for this purpose, targeting the coding exons of MS4A1. This strategy is advantageous due to its rapid turnaround, potential for high throughput at a low per sample cost, and ability to screen for novel mutations.

[0234] Methods

[0235] Proof of concept studies tested 1) total barcode deduplicated coverage from 10 ng and 50ng of plasma derived and 2) minimum detectable alternative allele frequency of gblocks with clinically significant MS4A1 mutations diluted into a background of K562 DNA at progressively lower concentrations. gBlocks were uniformly 180bp and K562 DNA was sheared by sonicator to an identical mean fragment length. In both cases samples were sequenced on the MiSeq platform at 2xl50bp, targeting a mean depth of IxlO⁶ reads. For the clinical pilot study, libraries from the plasma and paired blood (where available) of 93 patients with DLBCL, enrolled in a clinical trial for a bispecific antibody were enriched for MS4A1 DNA and sequenced at 2xl50bp to a mean depth of IxlO⁶ reads on the MiSeq platform. Sequencing results were demultiplexed, aligned to the hg38 reference genome, and variant calls generated using an inhouse custom analysis pipeline. The pipeline reported both variant allele frequency (VAF) as well as mutant molecules per mL (MMPM). To assess whether a variant was likely to be associated with treatment resistance several criteria were applied, here listed in order of increasing weight. 1) variant was not synonymous, present in paired blood, or present in most samples. 2) variant passes the caller support threshold at a VAF >0.1%, 3) mutation is predicted to be pathogenic in silica using publicly available tools such as FATHMM indel and Polyphen. 4) Mutations were previously reported in the literature to be associated with resistance to treatment. 5) Clinical data were congruent with reported mutation, such as CD20 status by IHC at pre-treatment timepoint (if available), and final treatment response as measured by PET/CT.

[0236] Results

[0237] Proof of concept studies demonstrated sufficient coverage from 50ng input of total cfDNA to detect alternative allele frequencies as low as 0.1% with good coverage (FIG 2-3). Six cases from the screened cohort met our criteria, summarized in Table 1. Notable for subject A was the detection of a missense mutation at position 98, within the intracellular domain, which exhibited a 10-fold expansion in MMPM measured by the post treatment timepoint (FIG. 4A - C). This mutation impacted the binding site for the bispecific monoclonal antibody of interest as reported in Rushton et al (Rushton et al 2020) and PET/CT status was congruent with resistance to treatment. In two additional cases (5272 and 5209), we also observed mutations previously reported at the same position within the intracellular domain in the literature (Schuster et al. 2022), also impacting the BsMAb binding site. Of particular interest was a novel mutation in subject B located at position 148 within the extracellular domain, resulting in a truncated protein (FIG. 5 A - C). No IHC data was available for this case to determine CD20 status, however PET/CT status at the post treatment timepoint revealed progressive metabolic disease. As a percentage of total circulating tumor DNA (ctDNA) the mutation had a VAF of 7.6% at pre-treatment (9001). By cycle 3 of treatment (9003), although the absolute number of tumor derived DNA molecules had contracted from 2776 MMPM to 11 MMPM, this mutation comprised nearly 77% of the residual disease signature. By the post treatment timepoint, the total ctDNA had expanded to 12485 MMPM and the mutation of interest had a VAF of 100%, indicating that this was potentially a case of a resistant clone selected during treatment and expanding as a result.

[0238] Discussion

[0239] This work demonstrates the utility of a single gene screening assay to monitor the variant allele frequency of mutation in CD20 conferring resistance to treatment with BsMAbs. Furthermore, our screening approach both confirmed previous mutations reported in the literature and revealed potentially novel mutations. The use of longitudinal sampling allowed for the assessment of mutation kinetics across the treatment time course. By identifying a dominant clone mid treatment with the potential to inhibit response, this affords the opportunity to identify a more suitable therapeutic approach and reduce the rate of non-responders and refractory cases.

[0240] EXAMPLE 2 - PROOF-OF-CONCEPT FOR A KAPA HYPERPETE- BASED LIQUID BIOPSY ASSAY FOR SCREENING MUTATIONS CONFERRING RESISTANCE TO NHL IMMUNOTHERAPY TARGETS

[0241] Introduction/Background

[0242] Non-Hodgkin’s lymphoma (NHL) is the most common hematologic malignancy in the world (Wang, M.L., et al. "Breakthrough Therapies in B-Cell Non-Hodgkin Lymphoma." Annals of Oncology, 6 Jan. 2020, www. sciencedirect.com/sci ence/article/pii/S0923753419374137#ab0015) and one of the most prevalent cancers in the United States, accounting for approximately 4% ("Key Statistics for Non-Hodgkin’s Lymphoma." American Cancer Society. https://www.cancer.org/cancer/types/non-hodgkin-lymphoma/about/key-statistics.html.) of all cancer cases. Immunotherapy is a common treatment option for NHL. For example, monoclonal antibodies, which promote immune toxicity toward cancer cells by binding to specific antigens on their surface, are often used as a frontline NHL treatment. Bispecific antibodies (BsMAb) are another emerging treatment used to combat NHL. These antibodies have one region that selectively targets the lymphoma cancer cell and another region that selectively targets T cells, bringing the two cells closer together to modulate an immune attack against the cancer. Although these treatments can relieve the disease burden for many patients, there are a subset of patients who acquire resistance to these treatments and undergo relapse. Patients who relapse following treatment often have poor survival outcomes. As such, there is a need to overcome the challenges associated with treating patients who acquire resistance to frontline therapies.

[0243] A subset of NHL relapse cases is caused by expansion of mutations due to selective pressure from treatment which impact the ability of a BsMAb to bind to its intended target. These mutations can range in impact from loss of expression to a functional protein with a conformational change preventing binding. Therefore, if clinicians were able to longitudinally sample cfDNA to track the kinetics of such mutations, they could quickly identify patients at risk of undergoing relapse and pivot their treatment accordingly before the resistant cancer spreads. Doing so would likely increase the chances of survival for these patients. Thus, it is desirable to develop a robust, sensitive, and specific liquid biopsy assay to rapidly sequence a set of genes relevant to NHL immunotherapy, particularly BsMAb therapy (FIG 6, Cao, Yang et al. "Mutations or copy number losses of CD58 and TP53 genes in diffuse large B cell lymphoma are independent unfavorable prognostic factors." Oncotarget vol. 7,50 (2016): 83294-83307. doi: 10.18632/oncotarget.13065, Robinson, Hannah R et al. "A CD19/CD3 bispecific antibody for effective immunotherapy of chronic lymphocytic leukemia in the ibrutinib era." Blood vol. 132,5 (2018): 521-532. doi : 10.1182/blood-2018-02-830992, Yin, Z., Zhang, Y. & Wang, X. Advances in chimeric antigen receptor T-cell therapy for B-cell non-Hodgkin lymphoma. Biomark Res 9, 58 (2021). https://doi.org/10.1186/s40364-021-00309-5, Sun LL et al (2015). Anti-CD20/CD3 T celldependent bispecific antibody for the treatment of B cell malignancies. Sci Transl Med. May 13;7(287):287ra70. doi: 10 1126/scitranslmed.aaa4802. PMID: 259720 02, Center for Drug Evaluation and Research. "FDA Approves Polatuzumab Vedotin-Piiq for Previously Untreated Diffuse Large B-Cell Lymphoma, Not Otherwise Specified, and High-Grade B-Cell Lymphoma." U.S. Food and Drug Administration, www.fda.gov/drugs/resources-information-approved- drugs/fda-approves-polatuzumab-vedotin-piiq-previously-untreated-diffuse-large-b-cell- lymphoma-not, Zheng WW et al (2023). Anti-CD79b/CD3 bispecific antibody combined with CAR19-T cells for B-cell lymphoma treatment. Cancer Immunol Immunother. 2023 Nov;72(l l):3739-3753. doi: 10.1007/s00262-023-03526-z. Epub Sep 14. PMID: 37707586. Cassanello G, et al (2024) Trial watch: bi specific antibodies for the treatment of relapsed or refractory large B-cell lymphoma. Oncoimmunology. 3; 13( 1):2321648. doi: 10.1080/2162402X.2024.2321648. PMID: 38445082; PMCID: PMC1091371 L Yan X, et al (2022) CD58 loss in tumor cells confers functional impairment of CAR T cells. Blood Adv. Nov 22;6(22):5844-5856. doi: 10.1 182/bioodadvances.2022007891. PMII): 35728062; PMCID: PMC9649996. Wong, D P , el al. (2022) A BAFF ligand-based CAR-T cell targeting three receptors and multiple B cell cancers Nat Comimwi 13, 217. https://doi.org/10.1038/ s41467-021- 27853-w).

[0244] Here we demonstrate proof-of-concept for such an assay showing that the barcode deduplicated depth at 50ng total DNA input is sufficient for detection of low allele frequency variants. We also review specific expansions and improvements in coverage as compared to an assay targeting only the coding regions ofMS4SAl .

[0245] Methods

[0246] K562 cell line DNA was sheared by sonication to a mean fragment length of 180bp and libraries prepared at inputs of lOng, 20ng, 30ng, 40ng, and 50ng. Libraries were enriched for the exons (coding and non-coding) as well as the intronic splicing acceptor, donor, and branch point of the MS4A1, CD 19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes. These enriched libraries were sequenced on the NextSeq High Output flow cell at targeted mean read depth of 30xl0⁶ reads. Sequencing results were demultiplexed, aligned to the hg38 reference genome, and mean barcode deduplicated depth reported using an in-house custom analysis pipeline. The .bam alignment and .bai index files were generated in the same fashion. Per positional barcode deduplicated base coverage was visualized by importing a representative .bam and .bai info file IGV version 2.14.1. These were visualized against imported .bed files containing target ROIs, capture primer coordinates, and coordinates of variants of interest. Random binomial simulations were performed in R using rbinom, results were stored as a dataframe using data.table and visualized using ggplot2.

[0247] Results

[0248] When comparing to a problematic region of worse coverage uniformity in an assay only enriching for coding exons of MS4A1, the revised primer design of the expanded panel gave a mean Fold 80 base penalty approaching 1, indicating much improved coverage uniformity for the target ROI (FIG 7). This was accomplished by placing a capture primer within the downstream intronic region to the ROI, while also avoiding placement at the 3’ end overlapping with a known variant (FIG 8).

[0249] The mean barcode deduplicated coverage across all targets at 50ng met the minimum cutoff of 3000 reads, and lower inputs are generally yielded commensurate coverage (FIG 9A). At this coverage, an alternative allele at an expected allele frequency of 0.1% should be supported by 2-3 reads, most of the time (FIG 9B).

[0250] Discussion

[0251] This work demonstrates the proof-of-concept for sufficient coverage on an expanded panel targeting regions of the MS4A1, CD 19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes. It also demonstrates improvements in coverage for MS4A1 compared to a single gene assay targeting only the coding regions of the gene. This panel includes both current and future BsMAb targets as well as targets for additional therapies such as CAR T- cells and antibody drug conjugates. Therefore, although it is intended to be paired with BsMAb treatments, it can potentially serve as an end-to-end solution for tracking resistance to treatment with a broad range of immunotherapies.

[0252] ADDITIONAL EMBODIMENTS

[0253] A first additional embodiment is a method of identifying immunotherapy resistant mutations, comprising: obtaining a sample comprising a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules comprises one or more target nucleic acid molecules and one or more non-target nucleic acid molecules; hybridizing one of a plurality of different target capture primers to each one of the one or more target nucleic acid molecules in the obtained sample, wherein each target capture primer of the plurality of different target capture primers comprises a capture moiety, and wherein each target capture primer targets a different region within one or more coding regions, non-coding regions, and/or intronic regions one or more genes encoding one or more proteins capable of being targeted with a immunotherapy agent, and wherein each target capture primer of the plurality of target capture primers does not target a region within the one or more coding exons of the one or more genes that overlap with one or more regions corresponding to one or more known variants; extending each of the hybridized one of the plurality of different target capture primers to provide one or more extended target primer extension complexes, wherein each extended target capture primer complex of the one or more extended target capture primer complexes comprises one of the one or more target nucleic acid molecules and the one of the plurality of different target capture primers; and enriching the sample for the one or more target nucleic acid molecules. In some embodiments, the immunotherapy agent is a bispecific monoclonal antibody. In some embodiments, the one or more genes are selected from one or more of CD19, CD22, CD58, CD79B, MS4A1, TNFSF13B, TNFRSF13C, and TNFRSF17.

[0254] In some embodiments, the different regions targeted correspond to one or more sequences within each of at least three different coding exons of the MS4A1 gene. In some embodiments, the different regions targeted correspond to one or more sequences within each of at least four different coding exons of the MS4A1 gene. In some embodiments, the different regions targeted correspond to one or more sequences within each of at least five different coding exons of the MS4A1 gene. In some embodiments, the different regions targeted correspond to one or more sequences within each of coding exons 3, 4, 5, and 6 of the MS4A1 gene. In some embodiments, the different regions targeted correspond to one or more sequences within each of coding exons 1, 2, 3, 4, 5, and 6 of the MS4A1 gene. In some embodiments, the different regions targeted correspond to one or more sequences within each of coding exons 1, 2, 4, 5, and 6 of the MS4A1 gene. In some embodiments, the different regions targeted further comprise one or more sequences within one or more coding exons of CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17.

[0255] In some embodiments, the plurality of different target capture primers comprises at least 8 different target capture primers targeting different regions within the MS4A1 gene. In some embodiments, the plurality of different target capture primers comprises at least 12 different target capture primers targeting different regions within the MS4A1 gene. In some embodiments, the plurality of different target capture primers comprises at least 16 different target capture primers targeting different regions within the MS4A1 gene.

[0256] In some embodiments, the plurality of different target capture primers targeting different regions within the MS4A1 gene have at least 90% sequence homology to any one of SEQ ID NOS: 41 to 122. In some embodiments, the plurality of different target capture primers targeting different regions within the MS4A1 gene have any one of SEQ ID NOS: 41 to 122. In some embodiments, the plurality of different capture primers further comprises at least one target capture primer targeting a region within any one of the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes. In some embodiments, the plurality of different capture primers further comprises at least two target capture primers targeting a region within any one of the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes. In some embodiments, the plurality of different capture primers further comprises at least two target capture primers targeting a region within any two of the CD 19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes. In some embodiments, the plurality of different capture primers further comprises at least one target capture primer having at least 90% sequence identity to any one of SEQ ID NOS: 1 to 40 and 123 to 462. In some embodiments, the plurality of different capture primers further comprises at least one target capture primer having any one of SEQ ID NOS: 1 to 40 and 123 to 462. In some embodiments, the plurality of different capture primers further comprises at least two target capture primer having at least 90% sequence identity to any one of SEQ ID NOS: 1 to 40 and 123 to 462. In some embodiments, the plurality of different capture primers further comprises at least two target capture primers having any one of SEQ ID NOS: 1 to 40 and 123 to 462. In some embodiments, the plurality of different capture primers further comprises at least four target capture primers having at least 90% sequence identity to any one of SEQ ID NOS: 1 to 40 and 123 to 462. In some embodiments, the plurality of different capture primers further comprises at least four target capture primers having any one of SEQ ID NOS: 1 to 40 and 123 to 462.

[0257] In some embodiments, the different regions targeted correspond to one or more locations within Chromosome 11 ranging from between about 60463002 to about 60463121, from between about 60464288 to about 60464344, from between about 60465921 to about 60466157, and/or from between about 60466959 to about 60467060, based on genome build HG38 or an equivalent position in a genome build other than HG38. In some embodiments, the plurality of different target capture primers hybridize to at least 15 consecutive nucleotides complementary nucleic acid sequences corresponding to locations within Chromosome 11 ranging from between about 60463002 to about 60463121, from between about 60464288 to about 60464344, from between about 60465921 to about 60466157, and/or from between about 60466959 to about 60467060, based on genome build HG38 or an equivalent position in a genome build other than HG38. In some embodiments, the different regions targeted correspond to one or more locations within Chromosome 11 ranging from between about 60462374 to about 60462533, from between about 60463001 to about 60463121, from between about 60464287 to about 60464344, from between about 60465920 to about 60466157, from between about 60466958 to about 60467060, and/or from between about 60468249 to about 60468468, based on genome build HG38 or an equivalent position in a genome build other than HG38. In some embodiments, the plurality of different target capture primers hybridize to at least 15 consecutive nucleotides complementary nucleic acid sequences corresponding to locations within Chromosome 1 1 ranging from between about 60462374 to about 60462533, from between about 60463001 to about 60463121, from between about 60464287 to about 60464344, from between about 60465920 to about 60466157, from between about 60466958 to about 60467060, and/or from between about 60468249 to about 60468468, based on genome build HG38 or an equivalent position in a genome build other than HG38. In some embodiments, the different regions targeted correspond to one or more locations within Chromosome 11 ranging from between about 60455815 to about 60456010, from between about 60457995 to about 60458316, from between about 60461002 to about 60461269, from between about 60462140 to about 60462600, from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, from between about 60466902 to about 60467140, and/or from between about 60468195 to about 60470813, based on genome build HG38 or an equivalent position in a genome build other than HG38. In some embodiments, the plurality of different target capture primers hybridize to at least 15 consecutive nucleotides complementary nucleic acid sequences corresponding to locations within Chromosome 11 ranging from between about 60455815 to about 60456010, from between about 60457995 to about 60458316, from between about 60461002 to about 60461269, from between about 60462140 to about 60462600, from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, from between about 60466902 to about 60467140, and/or from between about 60468195 to about 60470813, based on genome build HG38 or an equivalent position in a genome build other than HG38.

[0258] In some embodiments, the enriching of the sample for the one or more target nucleic acid molecules comprises (i) capturing the one or more extended target capture primer complexes; (ii) removing the one or more non-target nucleic acid molecules; and (iii) releasing the one or more target nucleic acid molecules from the one or more extended target capture primer complexes. In some embodiments, the capturing of the one or more extended target capture primer complexes comprises contacting the one or more extended target capture primer complexes with a functionalized substrate. In some embodiments, the capture moiety of the plurality of different target capture primers comprises a first member of a pair of specific binding entities, and wherein the functionalized substrate comprises a second member of the pair of specific binding entities. In some embodiments, the first member of the pair of specific binding entities is selected from the group consisting of biotin, an antigenic molecule, an enzyme substrate, a receptor ligand, a polysaccharide, a thiolated molecule, and an amine-terminated molecule. In some embodiments, the second member of the pair of specific binding entities is selected from the group consisting of streptavidin, an antibody, an enzyme, a receptor, a lectin, a gold p article, and an NHS-activated moiety.

[0259] In some embodiments, the plurality of different target capture primers is coupled to a substrate through the capture moiety prior to the step (b), and wherein the step (b) captures the one or more target nucleic acid molecules to the substrate. In some embodiments, the capturing of the one or more extended target capture primer complexes comprises: (i) hybridizing a universal capture oligonucleotide to the capture moiety of the one or more extended target capture primer complexes to form one or more universal capture oligonucleotide complexes, wherein the universal capture oligonucleotide comprises (a) a first member of a pair of specific binding entities, and (b) a nucleotide sequence complementary to at least a portion of a capture sequence of the capture moiety; (ii) contacting the one or more universal capture oligonucleotide complexes with a functionalized substrate, wherein the functionalized substrate comprises a second member of the pair of specific binding entities. In some embodiments, the removing of the non-target nucleic acid molecules comprises washing the sample one or more times. In some embodiments, the releasing of the one or more target nucleic acid molecules from the one or more extended target capture primer complexes comprises: (i) hybridizing a release primer to the one or more extended target capture primer complexes; and (b) extending the hybridized release primer. In some embodiments, the hybridized one of the plurality of different target capture primers is extended with a first polymerase; and wherein the hybridized release primer is extended with a second polymerase. In some embodiments, the one or more target nucleic acid molecules are in low abundance as compared with the one or more non-target nucleic acid molecules. In some embodiments, the one or more target nucleic acid molecules in the enriched sample are amplified. In some embodiments, the one or more target nucleic acid molecules in the enriched sample are sequenced.

[0260] All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary, to employ concepts of the various patents, applications, and publications to provide yet further embodiments. [0261] Although the present disclosure has been described with reference to a number of illustrative embodiments, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure. More particularly, reasonable variations and modifications are possible in the component parts and/or arrangements of the subject combination arrangement within the scope of the foregoing disclosure, the drawings, and the appended claims without departing from the spirit of the disclosure. In addition to variations and modifications in the component parts and/or arrangements, alternative uses will also be apparent to those skilled in the art.

Claims

1. A method of identifying immunotherapy resistant mutations, comprising:

(a) obtaining a sample comprising a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules comprises one or more target nucleic acid molecules and one or more non-target nucleic acid molecules;

(b) hybridizing one of a plurality of different target capture primers to each one of the one or more target nucleic acid molecules in the obtained sample, wherein each target capture primer of the plurality of different target capture primers comprises a capture moiety, and wherein each target capture primer targets a different region within one or more coding regions, non-coding regions, and/or intronic regions one or more genes encoding one or more proteins capable of being targeted with a immunotherapy agent, and wherein each target capture primer of the plurality of target capture primers does not target a region within the one or more coding exons of the one or more genes that overlap with one or more regions corresponding to one or more known variants;

(c) extending each of the hybridized one of the plurality of different target capture primers to provide one or more extended target primer extension complexes, wherein each extended target capture primer complex of the one or more extended target capture primer complexes comprises one of the one or more target nucleic acid molecules and the one of the plurality of different target capture primers; and

(d) enriching the sample for the one or more target nucleic acid molecules.

2. The method of claim 1, wherein the immunotherapy agent is a bispecific monoclonal antibody.

3. The method of claim 1, wherein the one or more genes are selected from one or more of CD19, CD22, CD58, CD79B, MS4A1, TNFSF13B, TNFRSF13C, and TNFRSF17.

4. The method of claim 3, wherein the one or more genes is the MS4A1 gene, and wherein at least three different coding exons of the MS4A1 gene are targeted by the plurality of different target capture primers.

5. The method of claim 4, wherein at least four different coding exons of the MS4A1 gene are targeted by the plurality of different target capture primers.

6. The method of claim 4, wherein at least five different coding exons of the MS4A1 gene are targeted by the plurality of different target capture primers.

7. The method of any one of claims 4 to 6, wherein the coding exons of the MS4A1 gene are selected from the group consisting of coding exons 1, 2, 3, 4, 5, and 6.

8. The method of any one of claims 4 to 6, wherein the coding exons of the MS4A1 gene are selected from the group consisting of coding exons 1, 2, 4, 5, and 6.

9. The method of any one of claims 4 and 5, wherein the coding exons of the MS4A1 gene are selected from the group consisting of coding exons 3, 4, 5, and 6.

10. The method of claim 3, wherein the plurality of different target capture primers comprises at least eight different target capture primers targeting different regions within the MS4A1 gene.

11. The method of claim 3, wherein the plurality of different target capture primers comprises at least twelve different target capture primers targeting different regions within the MS4A1 gene.

12. The method of claim 3, wherein the plurality of different target capture primers comprises at least sixteen different target capture primers targeting different regions within the MS4A1 gene.

13. The method of any one of claims 10 to 12, wherein the plurality of different target capture primers further comprises at least four different target capture primers targeting different regions within one or more coding regions, non-coding regions, and/or intronic regions of any one of the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes.

14. The method of any one of claims 10 to 12, wherein the plurality of different target capture primers further comprises at least eight different target capture primers targeting different regions within one or more coding regions, non-coding regions, and/or intronic regions of any one of the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes.

15. The method of any one of claims 10 to 14, wherein the at least eight different target capture primers targeting different regions within the MS4A1 gene have at least 90% identity to any one of SEQ ID NOS: 41 to 122.

16. The method of any one of claims 10 to 14, wherein the at least eight different target capture primers targeting different regions within the MS4A1 gene have any one of SEQ ID NOS: 41 to 122.

17. The method of any one of claims 13 to 14, wherein the at least four different target capture primers targeting different regions within the one or more coding regions, non-coding regions, and/or intronic regions of the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes have at least 90% identity to any one of SEQ ID NOS: 1 to 40 and 123 to 462.

18. The method of any one of claims 13 to 14, wherein the at least four different target capture primers targeting different regions within the one or more coding regions, non-coding regions, and/or intronic regions of the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes have any one of SEQ ID NOS: 1 to 40 and 123 to 462.

19. The method of claim 1, wherein the different regions targeted correspond to one or more locations within Chromosome 11 ranging from between about 60463002 to about 60463121, from between about 60464288 to about 60464344, from between about 60465921 to about 60466157, and/or from between about 60466959 to about 60467060, based on genome build HG38 or an equivalent position in a genome build other than HG38.

20. The method of claim 1, wherein the plurality of different target capture primers hybridize to at least 15 consecutive nucleotides of complementary nucleic acid sequences located at positions within Chromosome 11 ranging from between about 60463002 to about 60463121, from between about 60464288 to about 60464344, from between about 60465921 to about 60466157, and/or from between about 60466959 to about 60467060, based on genome build HG38 or an equivalent position in a genome build other than HG38.

21. The method of claim 1, wherein the different regions targeted correspond to one or more locations within Chromosome 11 ranging from between about 60462374 to about 60462533, from between about 60463001 to about 60463121, from between about 60464287 to about 60464344, from between about 60465920 to about 60466157, from between about 60466958 to about 60467060, and/or from between about 60468249 to about 60468468, based on genome build HG38 or an equivalent position in a genome build other than HG38.

22. The method of claim 1, wherein the plurality of different target capture primers hybridize to at least 15 consecutive nucleotides of complementary nucleic acid sequences located at positions within Chromosome 11 ranging from between about 60462374 to about 60462533, from between about 60463001 to about 60463121, from between about 60464287 to about 60464344, from between about 60465920 to about 60466157, from between about 60466958 to about 60467060, and/or from between about 60468249 to about 60468468, based on genome build HG38 or an equivalent position in a genome build other than HG38.

23. The method of claim 1, wherein the different regions targeted correspond to one or more locations within Chromosome 11 ranging from between about 60455815 to about 60456010, from between about 60457995 to about 60458316, from between about 60461002 to about 60461269, from between about 60462140 to about 60462600, from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, from between about 60466902 to about 60467140, and/or from between about 60468195 to about 60470813, based on genome build HG38 or an equivalent position in a genome build other than HG38.

24. The method of claim 1, wherein the plurality of different target capture primers hybridize to at least 15 consecutive nucleotides of complementary nucleic acid sequences located at positions within Chromosome 11 ranging from between about 60455815 to about 60456010, from between about 60457995 to about 60458316, from between about 60461002 to about 60461269, from between about 60462140 to about 60462600, from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, from between about 60466902 to about 60467140, and/or from between about 60468195 to about 60470813, based on genome build HG38 or an equivalent position in a genome build other than HG38.

25. The method of claim 1, wherein the plurality of target capture primers includes 12 or more different target capture primers, wherein each different target capture primer has at least at least 90% identity to any one of SEQ ID NOS: 1 - 462 or SEQ ID NOS: 925 - 942.

26. The method of claim 1, wherein the plurality of target capture primers includes 12 or more different target capture primers, wherein each different target capture primer has any one of SEQ ID NOS: 1 - 462 or SEQ ID NOS: 925 - 942.

27. The method of any one of the preceding claims, wherein the enriching of the sample for the one or more target nucleic acid molecules comprises (i) capturing the one or more extended target capture primer complexes; (ii) removing the one or more non-target nucleic acid molecules; and (iii) releasing the one or more target nucleic acid molecules from the one or more extended target capture primer complexes.

28. The method of claim 27, wherein the capturing of the one or more extended target capture primer complexes comprises contacting the one or more extended target capture primer complexes with a functionalized substrate.

29. The method of claim 28, wherein the capture moiety of the plurality of different target capture primers comprises a first member of a pair of specific binding entities, and wherein the functionalized substrate comprises a second member of the pair of specific binding entities.

30. The method of claim 29, wherein the first member of the pair of specific binding entities is selected from the group consisting of biotin, an antigenic molecule, an enzyme substrate, a receptor ligand, a polysaccharide, a thiolated molecule, and an amine-terminated molecule.

31. The method of claim 30, wherein the second member of the pair of specific binding entities is selected from the group consisting of streptavidin, an antibody, an enzyme, a receptor, a lectin, a gold p article, and an NHS-activated moiety.

32. The method of claim 27, wherein the plurality of different target capture primers is coupled to a substrate through the capture moiety prior to the step (b), and wherein the step (b) captures the one or more target nucleic acid molecules to the substrate.

33. The method of claim 27, wherein the capturing of the one or more extended target capture primer complexes comprises: (i) hybridizing a universal capture oligonucleotide to the capture moiety of the one or more extended target capture primer complexes to form one or more universal capture oligonucleotide complexes, wherein the universal capture oligonucleotide comprises (a) a first member of a pair of specific binding entities, and (b) a nucleotide sequence complementary to at least a portion of a capture sequence of the capture moiety; (ii) contacting the one or more universal capture oligonucleotide complexes with a functionalized substrate, wherein the functionalized substrate comprises a second member of the pair of specific binding entities.

34. The method of claim 27, wherein the removing of the non-target nucleic acid molecules comprises washing the sample one or more times.

35. The method of claim 27, wherein the releasing of the one or more target nucleic acid molecules from the one or more extended target capture primer complexes comprises: (i) hybridizing a release primer to the one or more extended target capture primer complexes; and (b) extending the hybridized release primer.

36. The method of claim 35, wherein the hybridized one of the plurality of different target capture primers is extended with a first polymerase; and wherein the hybridized release primer is extended with a second polymerase.

37. The method of any one of the preceding claims, wherein the one or more target nucleic acid molecules are in low abundance as compared with the one or more non-target nucleic acid molecules.

38. The method of any one of the preceding claims, wherein the one or more target nucleic acid molecules in the enriched sample are amplified.

39. The method of any one of the preceding claims, wherein the one or more target nucleic acid molecules in the enriched sample are sequenced.

40. A method of identifying immunotherapy resistant mutations, comprising:

(a) obtaining a sample comprising a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules comprises one or more target nucleic acid molecules and one or more non-target nucleic acid molecules;

(b) hybridizing one target capture primer from a first set of different target capture primers to one of the one or more target nucleic acid molecules in the obtained sample, wherein each target capture primer of the first set of different target capture primers comprises a capture moiety, and wherein each target capture primer of the first set of different target capture primers targets a different region within one or more coding exons of MS4A1, and wherein each target capture primer of the first set of target capture primers does not target a region within the one or more coding exons that overlap with one or more regions corresponding to one or more known variants;

(c) extending each of the hybridized one of the first set of different target capture primers to provide one or more extended target capture primer complexes, wherein each target capture primer extension complex of the one or more extended target capture primer complexes comprises one of the one or more target nucleic acid molecules and the one of the plurality of different target capture primers; and (d) enriching the sample for the one or more target nucleic acid molecules.

41. The method of claim 40, wherein the different regions targeted by the different target capture primers within the first set of different target capture primers correspond to one or more sequences within each of at least three different coding exons of the MS4A1 gene.

42. The method of claim 40, wherein the different regions targeted by the different target capture primers within the first set of different target capture primers correspond to one or more sequences within each of at least four different coding exons of the MS4A1 gene.

43. The method of claim 40, wherein the different regions targeted by the different target capture primers within the first set of different target capture primers correspond to one or more sequences within each of at least five different coding exons of the MS4A1 gene.

44. The method of claim 40, wherein the different regions targeted by the different target capture primers within the first set of different target capture primers correspond to one or more sequences within each of coding exons 3, 4, 5, and 6 of the MS4A1 gene.

45. The method of claim 40, wherein the different regions targeted by the different target capture primers within the first set of different target capture primers correspond to one or more sequences within each of coding exons 1, 2, 3, 4, 5, and 6 of the MS4A1 gene.

46. The method of claim 40, wherein the different regions targeted by the different target capture primers within the first set of different target capture primers correspond to one or more sequences within each of coding exons 1, 2, 4, 5, and 6 of the MS4A1 gene.

47. The method of any one of claims 40 - 46, further comprising hybridizing one target capture primer from a second set of different target capture primers to another one of the one or more target nucleic acid molecules in the obtained sample wherein each target capture primer of the second set of different target capture primers comprises a capture moiety, and wherein each target capture primer of the second set of different target capture primers targets a different region within one or more coding regions, non-coding regions, and/or intronic regions of one or more genes selected from the group consisting of a CD 19 gene, a CD22 gene, a CD58 gene, a CD79B gene, a TNFSF13B gene, a TNFRSF13C gene, and a TNFRSF17 gene.

48. The method of any one of claims 40 - 47, wherein the one of a first set of different target capture primers has a nucleic acid sequence having at least 90% identity to any one SEQ ID NOS: 943 - 960.

49. The method of claim 40, further comprising sequencing the one or more target nucleic acid molecules.

50. A method of identifying immunotherapy resistant mutations, comprising:

(a) obtaining a sample comprising a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules comprises one or more target nucleic acid molecules and one or more non-target nucleic acid molecules;

(b) hybridizing one of a plurality of different target capture primers to each one of the one or more target nucleic acid molecules in the obtained sample, wherein each target capture primer of the plurality of different target capture primers comprises a capture moiety, and wherein each target capture primer targets a different region within one or more coding regions, non-coding regions, and/or intronic regions of one or more genes selected from the group consisting of a CD19 gene, a CD22 gene, a CD58 gene, a CD79B gene, a MS4A1 gene, a TNFSF13B gene, a TNFRSF13C gene, and a TNFRSF17 gene, and wherein each target capture primer of the plurality of target capture primers does not target a region within the one or more coding exons that overlap with one or more regions corresponding to one or more known variants;

(c) extending each of the hybridized one of the plurality of different target capture primers to provide one or more extended target capture primer complexes, wherein each target capture primer extension complex of the one or more extended target capture primer complexes comprises one of the one or more target nucleic acid molecules and the one of the plurality of different target capture primers; and

(d) enriching the sample for the one or more target nucleic acid molecules.

51. The method of claim 50, wherein the plurality of target capture primers includes 12 or more different target capture primers, wherein each different target capture primer has at least at least 90% identity to any one of SEQ ID NOS: 1 - 462, SEQ ID NOS: 925 - 942, or SEQ IDS NO: 961 - 1436.

52. The method of claim 50, wherein the plurality of target capture primers includes 12 or more different target capture primers, wherein each different target capture primer has any one of SEQ ID NOS: 1 - 462, SEQ ID NOS: 925 - 942, or SEQ IDS NO: 961 - 1436.

53. The method of claim 50, wherein the plurality of target capture primers includes 16 or more different target capture primers, wherein each different target capture primer has at least at least 90% identity to any one of SEQ ID NOS: 1 - 462, SEQ ID NOS: 925 - 942, or SEQ IDS NO: 961 - 1436.

54. The method of claim 50, wherein the plurality of target capture primers includes 16 or more different target capture primers, wherein each different target capture primer has any one of SEQ ID NOS: 1 - 462, SEQ ID NOS: 925 - 942, or SEQ IDS NO: 961 - 1436.

55. The method of claim 50, wherein each target capture primer of the plurality of different target capture primers hybridize to at least 15 consecutive nucleotides of complementary nucleic acid sequences located at any of the positions set forth within any one of Tables 1 - 10, based on genome build HG38 or an equivalent position in a genome build other than HG38.

56. The method of claim 50, wherein the plurality of different target capture primers hybridize to at least 15 consecutive nucleotides of complementary nucleic acid sequences located at positions within chromosome 11 ranging from between about 60455815 to about 60456010, from between about 60457995 to about 60458316, from between about 60461002 to about 60461269, from between about 60462140 to about 60462600, from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, from between about 60466902 to about 60467140, and/or from between about 60468195 to about 60470813, based on genome build HG38 or an equivalent position in a genome build other than HG38.

57. The method of claim 50, wherein at least 12 different target capture primers of the plurality of different target capture primers target regions within the MS4A1 gene; and wherein at least 6 different target capture primers of the plurality of different target capture primers target regions within the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes.

58. The method of claim 50, wherein at least 12 different target capture primers of the plurality of different target capture primers target regions within the MS4A1 gene; and wherein at least 12 different target capture primers of the plurality of different target capture primers target regions within the CD19, CD22, CD58, CD79B, TNFSF13B, TNFRSF13C, and TNFRSF17 genes.

59. The method of claim 50, further comprising sequencing the one or more target nucleic acid molecules.

60. A composition comprising at least 12 different target capture primers, wherein each target capture primer targets a different region within a MS4A1 gene, provided that each of the different target capture primers within the composition do not target a region within the MS4A1 gene that overlaps with one or more regions corresponding to one or more known variants.

61. The composition of claim 60, wherein the different regions correspond to locations within Chromosome 11 ranging from between about 60455815 to about 60456010, from between about 60457995 to about 60458316, from between about 60461002 to about 60461269, from between about 60462140 to about 60462600, from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, from between about 60466902 to about 60467140, and/or from between about 60468195 to about 60470813, based on genome build HG38 or an equivalent position in a genome build other than HG38.

62. The composition of claim 60, wherein the different regions correspond to locations within Chromosome 11 ranging from between about 60463002 to about 60463121, from between about 60464288 to about 60464344, from between about 60465921 to about 60466157, and/or from between about 60466959 to about 60467060, based on genome build HG38 or an equivalent position in a genome build other than HG38.

63. The composition of claim 60, wherein the different regions correspond to locations within Chromosome 11 ranging from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, and/or from between about 60466902 to about 60467140, based on genome build HG38 or an equivalent position in a genome build other than HG38.

64. The composition of claim 60, wherein the at least 12 different target capture primers have at least 85% identity to any one of SEQ ID NOS: 41 - 122 or 925 - 942.

65. The composition of claim 60, wherein the at least 12 different target capture primers have at least 90% identity to any one of SEQ ID NOS: 41 - 122 or 925 - 942.

66. The composition of claim 60, wherein the at least 12 different target capture primers have any one of SEQ ID NOS: 41 - 122 or 925 - 942.

67. The composition of claim 60, wherein the at least 12 different target capture primers have at least 85% identity to any one of SEQ ID NOS: 925 - 942.

68. The composition of claim 60, wherein the at least 12 different target capture primers have at least 90% identity to any one of SEQ ID NOS: 925 - 942.

69. The composition of claim 60, wherein the at least 12 different target capture primers have any one of SEQ ID NOS: 925 - 942.

70. A composition comprising (i) at least 12 different target capture primers targeting different regions within a MS4A1 gene, wherein the different regions correspond to locations within Chromosome 11 ranging from between about 60462961 to about 60463200, from between about 60463696 to about 60464418, from between about 60465859 to about 60466235, and/or from between about 60466902 to about 60467140, based on genome build HG38 or an equivalent position in a genome build other than HG38; (ii) at least six additional target capture primers targeting one or more regions within one or more genes selected from the group consisting of a CD19 gene, a CD22 gene, a CD58 gene, a CD79B gene, a TNFSF13B gene, a TNFRSF13C gene, and a TNFRSF17 gene.

71. A composition comprising (i) at least 12 different target capture primers, wherein each of the at least 12 different target capture primers have different nucleic acid sequences, wherein the different nucleic acid sequences have at least 90% identity to any one of SEQ ID NOS: 925 - 942; and (ii) at least six additional target capture primers targeting one or more regions within one or more genes selected from the group consisting of a CD 19 gene, a CD22 gene, a CD58 gene, a CD79B gene, a TNFSF13B gene, a TNFRSF13C gene, and a TNFRSF17 gene.

72. A composition comprising (i) at least 12 different target capture primers, wherein each of the at least 12 different target capture primers have different nucleic acid sequences, wherein the different nucleic acid sequences have any one of SEQ ID NOS: 925 - 942; and (ii) at least six additional target capture primers targeting one or more regions within one or more genes selected from the group consisting of a CD19 gene, a CD22 gene, a CD58 gene, a CD79B gene, a TNFSF13B gene, a TNFRSF13C gene, and a TNFRSF17 gene.

73. A composition comprising at least 12 different target capture primers, wherein the at least 12 different target capture primers have at least 90% identity to any one of SEQ ID NOS: 1 - 462, SEQ ID NOS: 925 - 942, or SEQ IDS NO: 961 - 1436.

74. The composition of claim 73, wherein the composition comprises at least 18 different target capture primers.

75. A composition comprising at least 12 different target capture primers, wherein the at least 12 different target capture primers have any one of SEQ ID NOS: 1 - 462, SEQ ID NOS: 925 - 942, or SEQ IDS NO: 961 - 1436.

76. The composition of claim 75, wherein the composition comprises at least 18 different target capture primers.

77. A kit comprising the compositions of any one of claims 73 - 76, and an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

78. The kit of claim 77, further comprising a polymerase, dNTPs, and a buffer.

79. A kit comprising at least 12 target capture primers and at least 12 corresponding release primers, wherein the at least 12 target capture primers and the at least 12 corresponding release primers have the sequences identified in Table 29.

80. A kit comprising at least 12 target capture primers and at least 12 corresponding release primers, wherein the at least 12 target capture primers and the at least 12 corresponding release primers have any one of the sequences identified in Table 30.

81. A composition comprising at least 10 different capture target primers, wherein the at least 10 different target capture primers have any one of the sequences identified in Table 11.

82. A composition comprising at least 10 different capture target primers, wherein the at least 10 different target capture primers have any one of the sequences identified in Table 12.

83. A composition comprising at least 10 different capture target primers, wherein the at least 10 different target capture primers have any one of the sequences identified in Table 13.

84. A composition comprising at least 10 different capture target primers, wherein the at least 10 different target capture primers have any one of the sequences identified in Table 14.

85. A composition comprising at least 10 different capture target primers, wherein the at least 10 different target capture primers have any one of the sequences identified in Table 15.

86. A composition comprising at least 10 different capture target primers, wherein the at least 10 different target capture primers have any one of the sequences identified in Table 16.

87. A composition comprising at least 10 different capture target primers, wherein the at least 10 different target capture primers have any one of the sequences identified in Table 17.

88. A composition comprising at least 10 different capture target primers, wherein the at least 10 different target capture primers have any one of the sequences identified in Table 18.

89. A composition comprising at least 10 different capture target primers, wherein the at least 10 different target capture primers have any one of the sequences identified in Table 19.

90. A composition comprising at least 10 different capture target primers, wherein the at least 10 different target capture primers have any one of the sequences identified in Table 20.

91. A composition comprising at least 10 different capture target primers, wherein the at least 10 different target capture primers have any one of the sequences identified in Table 21.

92. A composition comprising at least 10 different capture target primers, wherein the at least 10 different target capture primers have any one of the sequences identified in Table 22.

93. A composition comprising at least 10 different capture target primers, wherein the at least 10 different target capture primers have any one of the sequences identified in Table 23.

94. A composition comprising at least 10 different capture target primers, wherein the at least 10 different target capture primers have any one of the sequences identified in Table 24.

95. A composition comprising at least 10 different capture target primers, wherein the at least 10 different target capture primers have any one of the sequences identified in Table 25.

96. A composition comprising at least 10 different capture target primers, wherein the at least 10 different target capture primers have any one of the sequences identified in Table 26.

97. A composition comprising at least 10 different capture target primers, wherein the at least 10 different target capture primers have any one of the sequences identified in Table 27.

98. A composition comprising at least 10 different capture target primers, wherein the at least 10 different target capture primers have any one of the sequences identified in Table 28.

99. A kit comprising the compositions of any one of claims 80 - 98, and further comprising an equal number of different release primers, wherein the different release primers are designed to hybridize to portions of target nucleic acid molecules which are upstream relative to the locations in which the different target capture primers are designed to hybridize.

100. A method of identifying immunotherapy resistant mutations in one or more samples derived from a patient diagnosed with NHL, comprising:

(a) obtaining a first sample from the patient, wherein the obtained sample comprises a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules comprises one or more target nucleic acid molecules and one or more non-target nucleic acid molecules;

(b) hybridizing one of a plurality of different target capture primers to each one of the one or more target nucleic acid molecules in the obtained sample, wherein each target capture primer of the plurality of different target capture primers comprises a capture moiety, and wherein each target capture primer targets a different region within one or more coding regions, non-coding regions, and/or intronic regions of one or more genes selected from the group consisting of a CD 19 gene, a CD22 gene, a CD58 gene, a CD79B gene, a MS4A1 gene, a TNFSF13B gene, a TNFRSF13C gene, and a TNFRSF17 gene, and wherein each target capture primer of the plurality of target capture primers does not target a region within the one or more coding exons that overlap with one or more regions corresponding to one or more known variants;

(c) extending each of the hybridized one of the plurality of different target capture primers to provide one or more extended target capture primer complexes, wherein each target capture primer extension complex of the one or more extended target capture primer complexes comprises one of the one or more target nucleic acid molecules and the one of the plurality of different target capture primers; and

(d) enriching the sample for the one or more target nucleic acid molecules;

(e) sequencing the one or more target nucleic acid molecules to provide a first sequencing data set; and

(f) deriving a variant allele frequency (VAF) and a mutant molecules per mb (MMPM) from the first sequencing data set for each of the one or more genes.

101. The method of claim 100, wherein a first VAF and a first MMPM are derived from the first sample, wherein the first sample is obtained prior to the patient receiving an immunotherapeutic agent.

102. The method of claim 101, further comprising obtaining two or more additional samples from the patient diagnosed with NHL, wherein each additional sample of the two or more additional samples is obtained after the patient has received one or more doses of the immunotherapeutic agent.

103. The method of claim 101, further comprising obtaining two or more additional samples from the patient diagnosed with NHL, wherein each additional sample of the two or more additional samples is obtained at different time points during treatment with the immunotherapeutic agent.

104. The method of any one of claims 102 to 103, wherein the two or more additional samples are each sequenced to provide two or more additional sequencing data sets, and wherein additional VAF and MMPM are derived from each of the two or more additional sequencing data sets.

105. The method of claim 104, further comprising deriving one or more kinetics parameters based on the first VAF, the first MMPM, and the additional VAF and MMPM.

106. The method of claim 105, further comprising deriving a clinical decision based on the one or more derived kinetics parameters.

107 . A composition comprising at least 12 different target capture primers, wherein the at least 12 different target capture primers have any one of SEQ IDS NO: 961 - 1436.

108. The composition of claim 107, further comprising at least 12 different release primers corresponding to the at least 12 different target capture primers, wherein the at least 12 different release primers having any one of SEQ ID NOS: 1437 - 1912.

109. A composition comprising at least 12 different target capture primers, wherein the at least 12 different target capture primers have any one of SEQ IDS NO: 1 - 462.

110. The composition of claim 107, further comprising at least 12 different release primers corresponding to the at least 12 different target capture primers, wherein the at least 12 different release primers having any one of SEQ ID NOS: 463 - 924.