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WO2012000152A1 - Méthode de séquençage par pcr basée sur une technologie d'index moléculaire de l'adn et une stratégie de cassure incomplète d'adn - Google Patents

Méthode de séquençage par pcr basée sur une technologie d'index moléculaire de l'adn et une stratégie de cassure incomplète d'adn Download PDF

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Publication number
WO2012000152A1
WO2012000152A1 PCT/CN2010/001834 CN2010001834W WO2012000152A1 WO 2012000152 A1 WO2012000152 A1 WO 2012000152A1 CN 2010001834 W CN2010001834 W CN 2010001834W WO 2012000152 A1 WO2012000152 A1 WO 2012000152A1
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pairs
primer
pcr
hla
dna
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Chinese (zh)
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李剑
刘涛
赵美茹
张现东
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the invention relates to the field of nucleic acid sequencing technology, in particular to the field of PCR sequencing technology. In another aspect, the invention also relates to the field of PCR-index/barcode technology.
  • the method of the present invention is particularly applicable to second generation sequencing techniques, particularly Pair-end sequencing technology in second generation sequencing technology, and can also be used for HLA genotyping. Background technique
  • the PCR sequencing method is a technique for obtaining a DNA fragment of a target gene by PCR, and then performing DNA sequence detection on the obtained DNA fragment of the target gene, thereby obtaining a DNA sequence information of the target gene, which is intuitive and accurate. Its characteristics have long been widely used in the field of gene mutation detection and genotyping.
  • PCR-index/barcode technology by adding a primer index sequence at the 5' end of the PCR primer, a unique primer label can be introduced into each sample during PCR, allowing the sample to utilize second-generation DNA sequencing technology (In the detection process of large-scale, high-throughput, single-molecule sequencing sequencer represented by Illumina GA, Roche 454, ABI Solid and other sequencers, except for the PCR section, which must be processed one by one, other experimental links can be more The samples are mixed and processed simultaneously, and finally the results of each sample can be returned by its unique primer tag sequence; this method has the characteristics of low cost, large flux and multiple different gene loci that can simultaneously detect a large number of samples.
  • the second generation of DNA sequencing technology has a shorter length of sequential sequencing than the first generation (Sanger method) DNA sequencing technology.
  • Illumina GA Illumina's The Genome Analyzer sequencer (Illumina GA for short) is an example.
  • the current maximum sequencing length of Illumina GA is 200 bp.
  • the length of the PCR product is greater than 200 bp, the entire PCR product cannot be tested by Illumina GA sequencing and directly by PCR sequencing.
  • the entire DNA sequence information of the PCR product to be detected is not obtained.
  • Short sequencing reads limit the application of second-generation sequencing technology, in addition to progressively improving sequencing technology to achieve longer actual sequencing reads, development can overcome the second-generation DNA sequencer's existing sequencing read length in PCR sequencing applications New technologies with insufficient fields have also become a top priority. Summary of the invention
  • Illumina GA has ultra-high sequencing throughput, but its measurement length is only 200bp; although the length of Roche 454 GS-FLX can reach 500bp, its sequencing cost is higher and the throughput is smaller; the first generation of sequencer Although the length measurement can reach more than lOObp, its throughput and cost cannot be compared with the second generation sequencer.
  • the second-generation sequencing technology includes the Pair-end sequencing technology in the second-generation sequencing technology, and the PCR sequencing technology for the PCR template with the DNA Reference Sequence.
  • the present invention provides a method for PCR sequencing, which reduces the limitation caused by the short read length of the self-sequencing and expands the application of the second generation DNA sequencing technology in the field of PCR sequencing applications.
  • the design of the primer label varies according to the experimental platform used. Considering the characteristics of the Illumina GA sequencing platform itself, the present invention mainly considers the following points when designing the primer label: 1: Avoid more than 3 in the primer label sequence (including 3 Single base repeat, 2: the total content of base A and base C in the same site of all primer tags is between 30% and 70% of the total base content, 3: GC of the primer tag sequence itself The content is between 40-60%, 4: the sequence difference between the primer tags is greater than 4 bases, 5: the sequence with high similarity to the Illumina GA sequencing primer is avoided in the primer tag sequence, 6: the primer tag sequence addition is reduced.
  • 1 Avoid more than 3 in the primer label sequence (including 3 Single base repeat, 2: the total content of base A and base C in the same site of all primer tags is between 30% and 70% of the total base content, 3: GC of the primer tag sequence itself The content is between 40-60%, 4: the sequence difference between the primer tags is greater than 4 bases, 5: the sequence with high similarity to the Illumina GA sequencing
  • the present invention introduces a primer tag at each end of the PCR product by PCR reaction (the primer tag sequence may be the same or different), so that the primer tag at either end of the PCR product can specifically label the sample information of the PCR product.
  • the resulting PCR product was subjected to incomplete typing.
  • the so-called incomplete interruption means that the final product contains a complete unbroken PCR product and a partially interrupted PCR product.
  • the breaking method includes, but is not limited to, a chemical breaking method (for example, enzymatic cutting) and a physical breaking method, and the physical breaking method includes an ultrasonic breaking method or a mechanical breaking method.
  • Interrupted DNA is electrophoresed on 2% agarose, and the gel is purified to recover all DNA bands from the maximum read length of the sequencer to the longest DNA length range that the sequencer can use (the longest DNA available for the Illumina GA sequencer) Is 700bp, this length is the original DNA length, no There are lengths of the library linker included). Purification and recovery methods include, but are not limited to, electrophoretic tapping recovery, magnetic bead recovery, and the like. The recovered DNA fragment is then subjected to a second-generation sequencer sequencing library construction process to construct a sequencing library, followed by sequencing, preferably using a PCR-FREE sequencing library construction process to construct a sequencing library, which is preferably sequenced using the Pair-End method.
  • Adapter or “library adapter” tag technology refers to the addition of different library linkers to multiple sequencing libraries (the sequence of the different library linkers is different, and the different parts of the sequence are called adapter indices). ), constructing a tag sequencing library, thereby enabling a plurality of different tag sequencing libraries to be mixed and sequenced, and finally a library tagging technique in which the sequencing results of each tag sequencing library can be distinguished from each other.
  • the PCR-FREE library construction method combining the library linker technology means that the library linker is directly ligated to both ends of the DNA fragment in the sequencing library, and the introduction process of the library linker is called PCR-Free library construction because there is no PCR involved. .
  • the access method can be ligated using DNA ligase. There is no PCR participation in the whole library construction process, which avoids the error caused by PCR and leads to the final conclusion during the construction of PCR product pooling library with high sequence similarity. Inaccuracy.
  • the second generation sequencing technology when the second generation sequencing technology is applied to the field of PCR sequencing, the actual measurable PCR product length exceeds the maximum sequencing of the sequencer. length.
  • a set of primer indices comprising at least 10 pairs, or at least 20 pairs, or at least 30 pairs, or at least 40 pairs of 95 pairs of primer labels shown in Table 1. Or at least 50 pairs, at least 60 pairs, or at least 70 pairs, or at least 80 pairs, or at least 90 pairs, or 95 pairs (or the set of primer labels are 10 - 95 pairs of 95 pairs of primer labels shown in Table 1) (eg 10 - 95 pairs, 20 - 95 pairs, 30 - 95 pairs, 40 - 95 pairs, 50 - 95 pairs, 60 - 95 pairs, 70 - 95 pairs, 80 - 95 pairs, 90 - 95 pairs, or 95 pairs ))), and
  • the set of primer tags preferably comprises at least PI-1 to PI-10, or PI-11 to ⁇ -20, or PI-21 to ⁇ -30, or PI-31 in the 95 pairs of primer labels shown in Table 1.
  • each pair of primer tags is combined with a PCR primer pair for amplifying a sequence of interest to be tested into a pair of tag primers
  • the 5th ends of the forward and reverse PCR primers have (or are optionally joined by a linker sequence) a forward primer tag and a reverse primer tag, respectively.
  • the PCR primer is a PCR primer for amplifying a specific gene of HLA, preferably for amplifying HLA-A/B 2, 3, 4 exon and HLA-DRB1 PCR primers for exon 2, preferably the PCR primers are shown in Table 2.
  • a set of tag primers comprising a set of primer tags described above and a PCR primer pair for amplifying a sequence of interest, wherein each pair of primer tags is combined with a PCR primer pair
  • the 5th ends of the forward and reverse PCR primers have (or are optionally joined by a linker sequence) a forward primer label and a reverse primer label, respectively.
  • the PCR primer in the above-described tag primer is a PCR primer for amplifying a specific gene of HLA, preferably for amplifying HLA-A/B 2, 3, 4 PCR primers for the exon and HLA-DRB1 exon 2, preferably the PCR primers are shown in Table 2.
  • the tag primer is used in a PCR sequencing method.
  • a method of determining a nucleotide sequence of a nucleic acid of interest in a sample comprising:
  • n samples n being an integer greater than or equal to 1, the sample preferably being from a mammal, more preferably a human, in particular a human blood sample; alternatively, dividing the n samples to be analyzed into m groups m is an integer and n > m >l;
  • each pair of label primers are used, and in the presence of a template from the sample, PCR amplification is carried out under conditions suitable for amplifying the nucleic acid of interest, wherein each pair of label primers comprises primers
  • the label's forward label primer and reverse label primer (both may be degenerate primers), wherein the forward label il and the reverse label primer may contain the same or different primer labels; Primer labels are different from each other;
  • the recovered DNA mixture is sequenced using a second generation sequencing technique, preferably a Pair-End technique (e.g., Illumina GA, Illumina Hiseq 2000), to obtain a sequence of the broken DNA;
  • a Pair-End technique e.g., Illumina GA, Illumina Hiseq 2000
  • the sequencing results obtained will be Corresponding to the sample, the alignment sequence (such as Blast, BWA program) is used to locate each sequencing sequence to the corresponding DNA reference sequence of the PCR product, and the complete target nucleic acid is spliced from the sequence of the broken DNA by sequence overlap and linkage. .
  • the alignment sequence such as Blast, BWA program
  • a method of determining a nucleotide sequence of a nucleic acid of interest in a sample comprising:
  • n samples n being an integer greater than or equal to 1, the sample preferably being from a human, in particular a human blood sample; optionally, dividing n samples to be analyzed into m groups, m being an integer and n > m > 1 ;
  • each pair of label primers are used, and in the presence of a template from the sample, PCR is performed under conditions suitable for amplifying the nucleic acid of interest, wherein each pair of label primers comprises a primer label
  • the forward label primer and the reverse label primer both may be degenerate primers, wherein the forward label primer and the reverse label primer may contain the same or different primer labels; the primer label in the label primer pair used for different samples Different from each other;
  • Sequencing The recovered DNA mixture is sequenced using a second-generation sequencing technique, preferably a Pair-End technique (eg, Illumina GA, Illumina Hiseq 2000), to obtain a sequence of the interrupted DNA;
  • a second-generation sequencing technique preferably a Pair-End technique (eg, Illumina GA, Illumina Hiseq 2000)
  • each pair of primer tags and PCR primer pairs are combined into a pair of tag primers, and the 5th ends of the forward and reverse PCR primers respectively have (or optionally pass
  • the ligation sequence is ligated with a forward primer tag and a reverse primer tag.
  • the PCR primer is a PCR primer for amplifying a specific gene of HLA, preferably 2 for amplifying HLA-A/B.
  • PCR primers for exon 3, exon 4 and exon 2 of HLA-DRB1, preferably the PCR primers are shown in Table 2.
  • the primer tag is designed for PCR primers, preferably for PCR primers for amplifying a specific gene of 3 ⁇ 4A, more preferably for use.
  • the set of primer tags preferably comprises at least PI-1 to PI-10, or PI-11 to PI-20, or PI-21 to PI-30, or PI-31 in 95 pairs of primer labels shown in Table 1.
  • PI-40 or PI-41 to PI-50, or PI-51 to PI-60, or PI-61 to PI-70, or PI-71 to PI-80, or PI-81 to PI-90, Or PI-91 to PI-95, or it Any combination of two or more of them.
  • the DNA disruption includes a chemical disruption method and a physical disruption method, wherein the chemical method includes an enzyme digestion method, and the physical The breaking method includes an ultrasonic breaking method or a mechanical breaking method. After the DNA was disrupted, fragments of 450-750 bp in length were isolated.
  • an HLA typing method comprising: sequencing a sample (especially a blood sample) from a patient using the sequencing method described above, and sequencing the result with an HLA database (eg, IMGT HLA)
  • an HLA database eg, IMGT HLA
  • the standard sequence data alignment in the professional database the 100% match of the sequence alignment results is the HLA genotype of the corresponding sample.
  • Figure 1 Sequence primers for primer labeling, DNA disruption, and DNA sequencing.
  • the positive and negative primer tag sequences Index-NF/R (1) were introduced into the PCR product of the No. N sample.
  • the products of the PCR product interrupted by physical methods include: a product with a primer tag sequence at one end, both ends a product without a primer tag sequence, a product that is completely unbroken, and a tapping purification to recover all DNA bands located between the maximum read length of the sequencer and the longest DNA length range applicable to the sequencer for sequencing (2), Using the Index-NF/R to retrieve the sequencing result of the PCR product belonging to the No. N sample in the sequencing result, and using the known reference sequence information of the PCR product to locate the relative reference position of each sequencing sequence, and according to the overlap between the sequencing sequences And the linkage relationship assembled into a complete PCR product (3, 4).
  • FIG. 2 Electrophoresis results of the corresponding exon PCR products of sample No. 1 HLA-A/B/DRB1. From the electropherogram, the PCR product is a series of single bands with a fragment size of 300bp-500bp, wherein lane M is the molecular weight marker. (DL 2000, Takara), Lanes 1-7 are the HLA-A/B/DRB1 exons of sample No. 1 (A2, A3, A4, B2, B3, B4, DRBl-2) PCR amplification products, negative control (N) without amplification bands. The results for the other samples are similar.
  • Figure 3 DNA electrophoresis after HLA-Mix interruption (before and after tapping), the tapping area is 450-750 bp.
  • Lane M is a molecular weight marker (NEB-50bp DNA Ladder)
  • lane 1 is the electrophoresis of HLA-Mix before tapping
  • lane 2 is the gel of HLA-Mix after tapping.
  • Figure 4 Screenshot of the consensus sequence constructor for sample No. 1, which illustrates the complete sequence of the spliced PCR product based on the overlap between the primer tag and the DN A fragment. detailed description
  • the primers to be analyzed are introduced into the primers of the HLA-A/B 2, 3, 4 exons and the HLA-DRB1 exon 2 PCR product by PCR, so that the specific PCR products are labeled.
  • Sample information The PCR amplification products of the three sites of HLA-A/B/DRB1 in each sample were mixed together to obtain a PCR product library; the PCR product library was not completely interrupted by ultrasound, and a PCR-Free sequencing library was constructed.
  • the sequencing library was electrophoresed by 2% low melting agarose, pure gel All DNA bands between 450 bp and 750 bp in length were recovered (the library linker was added to both ends of the DNA fragment during the construction of the PCR-Free sequencing library, so that the length of the DNA fragment on the electropherogram is longer than the actual The length is about 250 bp, so the 450 bp to 700 bp fragment is recovered here, which is equivalent to recovering a DNA fragment of 200 bp to 500 bp in length.
  • the recovered DNA was sequenced by Illumina GA PE-100.
  • the sequence information of all the tested samples can be found by the primer tag sequence, and the sequence of the entire PCR product is assembled by the overlapping and linkage relationship between the reference sequence information of the known DNA fragment and the sequence of the DNA fragment, and then passed through HLA-A/
  • the alignment of the standard database of the corresponding exons of B/DRB1 can assemble the entire sequence of the original PCR product to achieve genotyping of HLA-A/B/DRB1.
  • DNA was extracted from 95 blood samples of known HLA-SBT typing (Chinese Hematopoietic Stem Cell Donor Database (hereinafter referred to as "Zhonghua Marrow Bank") using the KingFisher Automatic Extractor (American Thermo).
  • the main steps are as follows: Take out the deep hole plate and one shallow hole plate of 6 Kingfisher automatic extractor. Add a certain amount of matching reagents according to the instructions and mark them. Place all the well plates with reagents as required. Corresponding position, select the program "Bioeasy a 200ul Blood DNA-KF.msz” program, press “star” to execute the program for nucleic acid extraction. At the end of the program, approximately 100 ⁇ L of the eluted product in the plate Elution was collected as the extracted DNA.
  • Example 2 Example 2
  • the PCR primers are PCR primers for exons 2, 3, 4 of HLA-A/B and exon 2 of HLA-DRB1.
  • a primer tag is then introduced at both ends of the PCR product by a PCR reaction to specifically label PCR products from different samples.
  • each set of PCR-label primers consisting of a pair of bidirectional primer tags (Table 1) and exons 2, 3, 4 for amplifying HLA-A/B and A PCR primer (Table 2) consisting of exon 2 of HLA-DRB1, wherein each forward PCR primer has a forward primer label attached to a pair of primer tags at the 5' end, and the 5' end of the reverse PCR primer is ligated.
  • a reverse primer label for a pair of primer tags The primer tag is added directly to the 5, end of the PCR primer when the primer is synthesized.
  • the 95 DNAs obtained in the sample extraction step of Example 1 were sequentially numbered 1-95, and the PCR reaction was carried out in a 96-well plate, a total of 7 plates, numbered HLA-P-A2, HLA-P-A3, HLA, respectively.
  • a negative control without template was set in the plate, and the primer used in the negative control was the same as the corresponding primer of template 1.
  • D2-F1, D2-F2, D2-F3, D2-F4, D2-F5, D2-F6, D2-F7 are forward primers for amplifying HLA-DRB1 exon 2
  • D2-R is for amplifying HLA- Reverse primer for exon 2 of DRB1.
  • the PCR reaction system of HLA-DRB1 is as follows:
  • PI Ar A/B/D2-F 1/2/3/4/5/6/7 represents primer 5, and HLA-A/B/DRB1 with the nth forward primer tag sequence (Table 1) at the end F primer, PI nr A/B/D2-R 2/3/4 denotes primer 5, and the R primer of HLA-A/B/DRB1 with the nth reverse primer tag sequence at the end (here n ⁇ 95 ), others and so on.
  • each sample corresponds to a specific set of PCR primers ( nr deletion - ⁇ listen l4 / sl6n , PI nr A / B / D2 - R 2 / 3 / 4 ).
  • FIG. 1 shows the electrophoresis results of the corresponding exon PCR products of sample No. 1 HLA-A/B/DRB1.
  • the DNA molecular marker is DL 2000 ( Takara).
  • the gel map has a series of single bands with a fragment size of 300bp-500bp, indicating The HLA-A/B/DRB1 exons (A2, A3, A4, B2, B3, B4, DRB1-2) of sample No. 1 were successfully amplified by PCR, and the negative control (N) had no amplified bands.
  • the results of other samples are similar to this.
  • Example 3 shows the electrophoresis results of the corresponding exon PCR products of sample No. 1 HLA-A/B/DRB1.
  • the DNA molecular marker is DL 2000 ( Takara).
  • the gel map has a series of single bands with a fragment size of 300bp-500bp, indicating The HLA-A/B/DRB1 exons (A2, A3, A4, B2, B
  • HLA-A3-Mix labeled HLA-A3-Mix, HLA-A4-Mix, HLA-B2-Mix, HLA-B3-Mix v HLA-B4-Mix and HLA-D2-Mix, oscillating and mixing, from HLA-A2-Mix 200 ul of HLA-A3-Mix, HLA-A4-Mix, HLA-B2-Mix, HLA-B3-Mix, HLA-B4-Mix and HLA-D2-Mix were mixed in a 3 ml EP tube, labeled For HLA-Mix, 500ul DNA mixture from HLA-Mix was purified by Qiagen DNA Purification kit (QIAGEN). For details, see the instructions), 200 ul of DNA obtained was purified, and the concentration of HLA-Mix DNA was determined to be 48 ng/ul by Nanodrop 8000 (Thermo Fisher Scientific).
  • QIAGEN Qiagen DNA Purification kit
  • T4 DNA Polymerase (T4 DNA Polymerase) 5 ⁇
  • the reaction conditions are: Thermomixer (Thermomixer, Eppendorf)
  • reaction product was recovered by QIAquick PCR Purification Kit and dissolved in 34 ⁇ M EB (QIAGEN Elution Buffer).
  • the DNA was recovered in the previous step, and the end was added with A reaction.
  • the system was as follows (reagents were purchased from Enzymatics):
  • the reaction conditions were: Constant Temperature Mixer (Thermomixer, Eppendorf) 37 Warm bath for 30 min.
  • reaction product was recovered and purified by MiniElute PCR Purification Kit (QIAGEN) and dissolved in 13 ⁇ M EB solution (QIAGEN Elution Buffer).
  • PCR-Free library adaptor refers to a designed set of bases whose primary function is to assist in the immobilization of DNA molecules on a sequencing chip and to provide a binding site for universal sequencing primers.
  • PCR-Free library linkers can pass DNA. The ligase directly ligates it to both ends of the DNA fragment in the sequencing library, and the introduction process of the library linker is called PCR-Free library linker because there is no PCR involved.
  • PCR-free oligonucleotide linker mix (30mM) (PCR-free Adapter ⁇
  • thermomixer Thermomixer, Eppendorf 20X bath for 15 min.
  • the reaction product was purified by Ampure Beads (Beckman Coulter Genomics) and dissolved in 50 ul of deionized water.
  • the DNA concentration was determined by real-time PCR (QPCR) as follows:
  • the sequencing result of Illumina GA is a series of DNA sequences. By searching the sequence of the positive and negative primers and the primer sequences in the sequencing results, the sequencing results of the PCR products of the HLA-A/B/DRB1 exons corresponding to each primer label are established. database. The sequencing results of each exon are mapped to the reference sequence of the corresponding exon by BWA (Burrows-Wheeler Aligner) (reference sequence source: http://www.ebi.ac.uk/imgt/hla/) Build consistency across databases
  • the (pair-End linkage) relationship can be assembled into the corresponding sequence of each exon of HLA-A/B/DRB1.
  • the obtained DNA sequence was aligned with the sequence database of the corresponding exons of HLA-A/B/DRB1 in the IMGT HLA professional database, and the 100% match of the sequence alignment results was the HLA-A/B/DRB1 genotype of the corresponding sample. do not.
  • a screenshot of the exon 2 consensus sequence constructor for the HLA-A site of sample No. 1 illustrated in Figure 4 can be seen. For all 95 samples, the results obtained were completely identical to the results of the original known classification.
  • the specific results of the samples No. 1-32 are as follows:
  • Tiercy J M Molecular basis of HLA polymorphism: implications in clinical transplantation. [J]. Transpl Immunol, 2002, 9: 173-180.

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Abstract

La présente invention concerne une méthode de séquençage pour des acides nucléiques, laquelle méthode comprend : l'amplification de l'acide nucléique cible à l'aide d'amorces indexées, la cassure incomplète des amplicons et la purification et la récupération d'ADN mélangés, le séquençage des ADN mélangés par une méthode de séquençage de nouvelle génération afin d'obtenir les séquences des ADN cassés et l'assemblage des séquences des ADN cassés en une séquence complète pour l'acide nucléique cible. La présente invention concerne en outre des index d'amorces pour la méthode de séquençage.
PCT/CN2010/001834 2010-06-30 2010-11-15 Méthode de séquençage par pcr basée sur une technologie d'index moléculaire de l'adn et une stratégie de cassure incomplète d'adn Ceased WO2012000152A1 (fr)

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CN201010213717.6A CN101921840B (zh) 2010-06-30 2010-06-30 一种基于dna分子标签技术和dna不完全打断策略的pcr测序方法
CN201010213717.6 2010-06-30

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