CN116179710A - Primer group for BRCA1/2 gene mutation detection based on high-throughput sequencing platform, library construction and cyclization method - Google Patents
Primer group for BRCA1/2 gene mutation detection based on high-throughput sequencing platform, library construction and cyclization method Download PDFInfo
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Abstract
The invention belongs to the technical field of nucleic acid detection, and particularly relates to a primer group and library construction and cyclization method for BRCA1/2 gene mutation detection based on a high-throughput sequencing platform. And provides a library construction and library cyclization method, wherein the data volume required by a single sample is only 2.5M, and the sequencing data comparison rate can be 100%, the capture rate is more than 99%, the uniformity is more than 99%, and the coverage under the depth of 500 times sequencing is 100%.
Description
Technical Field
The invention belongs to the technical field of nucleic acid detection, and particularly relates to a primer group for BRCA1/2 gene mutation detection based on a high-throughput sequencing platform, a library construction method and a cyclization method.
Background
Breast cancer is the most common malignancy in chinese females, and is the 5 th cause of cancer-related death. Studies have shown that 5% -10% of breast cancer patients have a definite genetic mutation, called hereditary breast cancer (hereditary breast cancer, HBC), wherein BRCA1/2 gene mutation accounts for 15%, and other main susceptibility genes are TP53, CDH1, LKB1, PTEN, CHEK2, ATM, PALB2 and the like. Breast cancer susceptibility genes including breast cancer susceptibility gene 1 (breast cancer susceptibility gene 1, brca 1) and breast cancer susceptibility gene 2 (breast cancer susceptibility gene, brca 2) are important cancer suppressing genes, and the coding products thereof are involved in DNA damage homology recombination repair group (homologous recombination), gene transcription regulation, cell cycle regulation and the like.
The traditional Sanger sequencing is a main detection means for detecting BRCA1/2 gene mutation in China at present, but the method has the defects of small detection range, large sample use amount, large primer reagent consumption and the like. For the detection of all the coding sequences and adjacent intron sequences of BRCA1/2, the detection flux is low, the period is long, and the single sample detection cost is high (the cost is different from 600-1000 yuan). In addition, detection kits based on high resolution melting point curve analysis (High Resolution Melting Analysis, HRM) techniques are also available on the market, but can only detect a few known sites, which have very limited effect. The high-throughput sequencing technology (Next-Gene Sequneng, NGS) can realize the amplification detection of germ line mutation of all coding regions of two genes BRCA1 and BRCA2 by one experiment, and has high throughput and accurate detection.
There are a number of kits or inventive patents (including multiplex PCR targeting library building techniques and specific probe hybridization capture) for detecting human breast cancer susceptibility genes BRCA1 and BRCA2 based on the NGS technology. However, there are problems such as high initial sample input (20 ng-50ng is not equal), expensive probe, complicated operation due to excessive initial amplification tube number, large sample use (40 ng-400ng is not equal), high reagent consumption cost, and increased cost due to large primer pair number (97-216 pair primer is not equal) or large primer input (200. Mu.M-400. Mu.M).
Disclosure of Invention
In order to further optimize the content in the prior art, the invention provides a group of primer groups which can be used for amplifying exon coding regions of all transcripts of BRCA1 genes and BRCA2 genes and +/-20 bp at the junction of exons and introns, the primer groups are low in use amount, the total input amount is 107.7 mu M at the minimum, and the input molar amount of the primers is between 0.1 mu M and 3.0 mu M.
And providing a library construction and library cyclization method for MGI platform, adapting to various sample types, completing the whole library construction and cyclization process within 7h by only 6ng samples, wherein the PCR components are simple, the number of amplification tubes is small, the operation is simple and convenient, in the library cyclization step, template denaturation and primer annealing are performed in the same space and time, the whole reaction can be performed at room temperature, single-chain renaturation is not required, the yield of library loops is greatly improved, the required data size of a single sample is 2.5M, the sequencing data comparison rate is 100%, the capture rate is more than 99%, the uniformity is more than 99%, and the coverage under 500×sequencing depth is 100%.
The aim of the invention is realized by the following technical scheme:
the primer group for BRCA1/2 gene mutation detection based on the high-throughput sequencing platform comprises 133 pairs of primer pairs divided into three groups, wherein the 5 'end to the 3' end of each forward primer and each reverse primer of each primer pair sequentially comprise a general sequence and a specific sequence;
the specific sequences of the forward primers of the first primer pair are shown as SEQ ID NO.1,2,3,4,5 and … …, and the specific sequences of the corresponding reverse primers are shown as SEQ ID NO.134,135,136,137,138 … … and 177;
the specific sequences of the forward primers of the second primer pair are shown as SEQ ID NO.45,46,47,48,49 and … … 89, and the specific sequences of the corresponding reverse primers are shown as SEQ ID NO.178,179,180,181,182 … … and 222;
the specific sequences of the forward primers of the third primer pair are shown as SEQ ID NO.90,91,92,93,94 and … …, and the specific sequences of the corresponding reverse primers are shown as SEQ ID NO.223,224,225,226,227 … … and 266.
Further, the molar amount of the 133 primer pairs is in the following range:
further, the universal adaptor sequences of the forward primer and the reverse primer of each primer pair are adaptor sequences that are compatible with the genetic sequencing platform.
Further, the universal adaptor sequence of the forward primer of each primer pair adapted to the MGI high throughput sequencing platform is shown as SEQ ID NO.267, and the universal adaptor sequence of the reverse primer is shown as SEQ ID NO. 268.
SEQ ID NO.267：TTGTCTTCCTAAGACCGCTTGGCCTCCGACTT；
SEQ ID NO.268：GAACGACATGGCTACGATCCGACTT。
The invention also provides a library construction method for BRCA1/2 gene mutation detection, which comprises the following steps:
s1, amplifying sample genes by using the first primer pair, the second primer pair and the third primer pair respectively;
the method comprises the following steps: diluting the extracted sample gDNA to 2ng, preparing a 3-tube PCR reaction system for PCR amplification, wherein a first group of primer pairs are required to be added in the reaction system 1, a second group of primer pairs are required to be added in the reaction system 2, and a third group of primers are required to be added in the reaction system 3.
S2, mixing the first primer pair amplification products, the second primer pair amplification products and the third primer pair amplification products in the step S1 according to the ratio of (1-3): 3-7): 5-11, and purifying to obtain purified first-round PCR amplification products; preferably, the first, second and third sets of primer pair amplification products are added in amounts of 4. Mu.L, 12. Mu.L, 20. Mu.L, respectively.
S3, performing a second round of PCR amplification reaction by taking the first round of PCR amplification product as a template, so as to further increase the target product quantity and add a sample identification tag;
s4, purifying the PCR amplification product of the second round to obtain the library for detecting the BRCA1/2 gene mutation.
Further, the library construction method for BRCA1/2 gene mutation detection specifically comprises the following steps:
further, in step S1, the types of the sample include peripheral blood, oral swab, and cervical exfoliated cells.
Further, in step S1, the total amount of the primer set is 107.7 to 134.3. Mu.M, preferably 121. Mu.M.
Further, in step S1, the PCR reaction system comprises 5. Mu.L of 5 XPCR Mix, 18. Mu.L of primer set 1, 1. Mu.L of gDNA (2 ng/. Mu.L), 1. Mu.L of ddH 2 O; reaction system 2 included 5. Mu.L of 5 XmPCRMix, 17. Mu.L of primer set 2, 1. Mu.L of gDNA (2 ng/. Mu.L), 2. Mu.L of ddH 2 O; the reaction system 3 included 5. Mu.L of 5 XmPCRMix, 10. Mu.L of primer set 3, 1. Mu.L of gDNA (2 ng/. Mu.L), 9. Mu.L of ddH 2 O。
Further, in step S1, the 5 XmPCR Mix (1 mL) comprises 500. Mu.L of 10 XMultiPCR Buffer, 125. Mu.L of dNTPs (10 mM), 50. Mu. LHotStart Taq Polymerase (5U/. Mu.L), 325. Mu.L of ddH 2 O。
Further, in step S1, the 10×MultiPCR Buffer fraction comprises 8-12mM Tris-HCl (pH 8.3), 240-270mM KCl, 0.8-1.2mM MgCl 2 10mL of 50% glycerol, 2.5mL of 20% triton 100, 0.75mg of BSA, ddH 2 O was constant to 50mL.
Further, in step S1, the PCR reaction procedure is a drop PCR, and the procedure is divided into pre-denaturation at 94℃for 3min, denaturation at 94℃for 20S, annealing at 66℃for 20S (-1 ℃/cycles), elongation at 72℃for 25S,14 cycles, denaturation at 94℃for 20S, annealing at 53℃for 20S, elongation at 72℃for 25S, and elongation at 21 cycles, and elongation at 72℃for 1min.
Furthermore, the purification in the above steps can adopt beaver biological DNA fragments to sort magnetic beads, and the volume ratio of the magnetic beads is 0.8 times.
Further, in step S3, the PCR reaction system of the second round of PCR amplification reaction comprises 5. Mu.L of 5 XmPCR Mix, 4. Mu.L of upstream universal primer, 4. Mu.L of downstream universal primer, 8. Mu.L of purified first round PCR amplification product, 4. Mu.L of ddH 2 O. The 5' end of the upstream general primer GAACGACATGGCTACGATCCGA needs to be labeled with 1 base by adopting phosphorylation; downstream universal primer
TGTGAGCCAAGGAGTTGNNNNNNNNNNTTGTCTTCCTAAGACCGCTTGGC where NNNNNNNNNN represents the Barcode sequence of the MGI platform, it is required that each sample is unique.
Further, in step S3, the PCR reaction procedure was 94℃for 3min, 94℃for 20S,62℃for 20S,72℃for 30S,11 cycles, and 72℃for 1m 30S.
The invention also provides a library cyclization method for BRCA1/2 gene mutation detection, which is characterized by comprising the library construction method for BRCA1/2 gene mutation detection, and further comprising the following steps:
s5, carrying out Qubit quantification on the library for detecting BRCA1/2 gene mutation obtained in the step S4, mixing the quantitative and equal mass, and adding ddH into the mixed library with the total amount of 100-400ng 2 O is fixed to a volume of 27 mu L;
s6, adding terminal repair enzyme and a corresponding buffer1, and performing terminal repair reaction to obtain a mixed library after terminal repair; preferably, the end repair enzyme is 1UNEB company DNA polymerase I large fragment (Klenow);
s7, carrying out single-chain treatment on the mixed library with the repaired tail end, adding a cyclization primer, and carrying out denaturation and annealing reaction to obtain a single-chain mixed library;
s8, cyclizing the single-chain mixed library, adding ligase and corresponding buffer2, and carrying out cyclization reaction to obtain a single-chain library ring; preferably, the ligase is NEB company T4 DNA ligase;
s9, digesting the single-chain library ring, and adding a digestive enzyme Mix, wherein the digestive enzyme Mix is a mixture of exonuclease I and exonuclease III of NEB company, and the mixing ratio is 1:1;
s10, purifying the single-chain library loop, and sequencing the single-chain library loop on a machine after quantitative Qubit.
Further, in step S5, 10-40ng of each library is mixed by equal mass, and ddH is supplemented after mixing 2 O to library quantity N X2 volumes (. Mu.L).
Further, in step S6, the buffer1 comprises 10-100mM Tris-HCl (pH 7.9), 50-500mM NaCl, 10-100mM MgCl 2 、1-10mM DTT。
Further, in step S6, the condition of the end repair reaction is that the reaction is carried out at 25 ℃ for 15min and at 80 ℃ for 10min.
Further, in step S7, the circularized primer sequence is shown as SEQ ID NO. 269; SEQ ID NO.269: GCCATGTCGTTCTGTGAGCCAAGG.
In step S7, the denaturation and annealing reaction is carried out at 95 ℃ for 3min, and the temperature is reduced to 4 ℃ in a PCR instrument.
Further, in step S8, the buffer2 comprises 60-300mM Tris-HCl (pH 7.6), 10-50mM MgCl 2 、1-5mMATP、1-5mM DTT、76-380mg/mLPEG6000。
Further, in step S8, the cyclization reaction conditions are 25℃for 30min.
Further, in step S9, the single-stranded library loop was digested at 37℃for 30min and at 80℃for 20min, followed by cooling to 4 ℃.
Further, the library cyclization method can be accomplished at room temperature without the need for configuration on ice.
Furthermore, the on-machine sequencing method is MGISEQ-200RS high-throughput sequencing reagent kit, and PE150 read length, FCS or FCL can be selected.
The beneficial effects of the invention are as follows:
1. the initial sample of the invention has small usage amount. The total usage amount of samples required by the prior similar technology is varied from 40ng to 400ng, and the invention only needs 6ng, and the minimum detection limit can reach 2ng.
2. The primer pair number is small. The primer pairs in the prior art are different from 97 to 216 pairs of primers, and only 133 pairs of primers are needed in the invention. The invention patent with 97 pairs of primer pairs is divided into 8 tubes for amplification, so that the amplification specificity and uniformity are ensured, and the 133 pairs of primers can ensure the amplification specificity and uniformity by optimizing the molar ratio and only 3 tubes.
3. The primer of the invention has less molar usage. The molar quantity of the primer in the prior similar technology is 200-400 mu M, and the minimum quantity of the primer is only 107.7 mu M.
4. The PCR amplification tube has a small number. The number of amplification tubes in the prior art is 2-8, and only 3 tubes are needed in the invention. In the prior art with 2 amplification tubes, the primer pair number is more, the molar amount is large, and the non-specific amplification is more and the uniformity is poor. The 133 pairs of primers are amplified by 3-tube PCR, and sequencing results show that the specificity and the uniformity are good and reach 99%.
5. The PCR reaction component of the invention is simple. Compared with the similar technology with few amplification tubes, the PCR component of the invention needs to be added with chemical substances such as TMAC and the like for improving the reaction specificity, and the invention can realize high-specificity amplification only by using the common PCR component without adding chemical substances with extra high specificity.
6. The invention requires a small amount of data for a single sample. The data size of a single sample of the existing high-throughput sequencing technology for BRCA gene mutation detection is different from 2M to 25M. The data volume required for a single sample is 2M, the coverage is more than 99% at a depth of 100×sequencing, and the capture rate and uniformity are poor. The 2.5M data volume of a single sample can reach 100% of coverage under 500 Xsequencing depth, and the capture rate and uniformity are good.
7. The library construction, cyclization method, etc. of the present invention do not need to be performed on ice. The MGI sequencing platform needs a library cyclization process, and a template is denatured into a single chain and then kept on ice.
8. The invention has short library construction time. The time length of manual library establishment is only 3 hours, and the manual library establishment and the library cyclization are carried out for about 7 hours.
9. The invention has rich types of adaptation samples. The kit comprises peripheral blood, an oral swab, cervical exfoliated cells and the like, and can meet the warehouse building requirement of low-concentration samples caused by poor sampling or poor extraction due to the fact that the minimum detection limit is as low as 2ng.
Drawings
FIG. 1 is an electrophoresis quality assay of 4 libraries randomly selected in example 1;
FIG. 2 is a diagram showing forward primer peaks for Sanger sequencing verification of heterozygous pathogenic mutation 13-32954030 (c.9097dup, p.Thr3033 fs) in example 2;
FIG. 3 is a diagram showing the reverse primer peaks for Sanger sequencing verification of heterozygous pathogenic mutation 13-32954030 (c.9097dup, p.Thr3033 fs) in example 2.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to the accompanying drawings, but the scope of the present invention is not limited to the following description.
Example 1
gDNA of the cell sample of the peripheral blood, the oral swab and the cervical exfoliated to be detected is extracted by a common commercial kit, and the extracted gDNA is subjected to ddH 2 O was diluted to 2 ng/. Mu.L.
1) Carrying out a first round of multiplex PCR targeting specific amplification reaction, wherein each sample needs to be respectively prepared into a 3-tube PCR reaction system, and the molar usage of the primers is as follows:
in the examples, the total molar input amount of the primer pool 1 was 50. Mu.M, the total molar input amount of the primer pool 2 was 43.4. Mu.M, the total molar input amount of the primer pool 3 was 27.4. Mu.M, and the total use amount was 121. Mu.M, and the reaction system was as follows:
2) After the reaction system is prepared, the reaction is carried out in a PCR instrument, and the PCR amplification procedure comprises pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 20s, annealing at 66 ℃ for 20s (-1 ℃/cycles), extension at 72 ℃ for 25s,14 cycles, denaturation at 94 ℃ for 20s, annealing at 53 ℃ for 20s, extension at 72 ℃ for 25s, and extension at 21 cycles and 72 ℃ for 1min.
3) After the completion of the reaction, the mixture was taken out, and the PCR reaction tube 1 was taken out as 4. Mu. L, PCR reaction tube 2, the reaction tube 3 was taken out as 12. Mu. L, PCR reaction tube 3, and mixed into one tube, followed by purification of the product.
4) Purifying by separating beaver biological DNA fragment with magnetic beads of 28.8 μl, and finally using 20 μl ddH 2 O is eluted, and the rest operation steps are according to instructions.
5) Taking the PCR product purified in the step 4) as a template, and carrying out a second round of PCR reaction, wherein the reaction system is as follows:
| component (A) | Volume (mu L) |
| 5×mPCRMix | 5 |
| Upstream general primer (10 mu M) | 4 |
| Downstream general primer (10. Mu.M) | 4 |
| Purified first round PCR product | 8 |
| ddH 2 O | 4 |
The sequence of Barcode in the downstream universal primer used in this example is as follows:
6) The second round of PCR reaction was performed at 94℃for 3min, 94℃for 20s,62℃for 20s,72℃for 30s,11 cycles, and 72℃for 1m 30s.
7) Purifying by separating magnetic beads with the amount of 16 μl and 20 μl ddH 2 O is eluted, and the rest operation steps are according to instructions.
8) And (3) carrying out Qubit quantification on the purified library, wherein a test kit is a Qubit 1 x dsDNA HS detection kit, and the specific operation steps are the same as the instruction book.
9) The purified library is subjected to fragment size quality control, and the result is shown in figure 1, the bands are distributed between 300bp and 400bp, the size meets the purpose, and no obvious nonspecific bands exist.
10 40ng of each library after quantification was mixed and then supplemented with ddH 2 O to library quantity N X2 volumes (μL), library concentrations in this example were as follows, library total volume 27.3 μL, supplemented with ddH 2 O 20.7μL。
| Sequence number | Barcode | Barcode library concentration (ng/. Mu.L) | Volume (mu L) |
| 1 | 1 | 53.0 | 0.8 |
| 2 | 2 | 56.0 | 0.7 |
| 3 | 3 | 53.0 | 0.8 |
| 4 | 4 | 20.8 | 1.9 |
| 5 | 13 | 32.1 | 1.2 |
| 6 | 14 | 30.2 | 1.3 |
| 7 | 15 | 48.3 | 0.8 |
| 8 | 16 | 58.0 | 0.7 |
| 9 | 25 | 53.0 | 0.8 |
| 10 | 26 | 52.0 | 0.8 |
| 11 | 28 | 45.5 | 0.9 |
| 12 | 29 | 23.5 | 1.7 |
| 13 | 30 | 30.2 | 1.3 |
| 14 | 32 | 24.7 | 1.6 |
| 15 | 33 | 37.2 | 1.1 |
| 16 | 34 | 52.0 | 0.8 |
| 17 | 35 | 41.7 | 1.0 |
| 18 | 36 | 38.7 | 1.0 |
| 19 | 37 | 35.8 | 1.1 |
| 20 | 38 | 33.3 | 1.2 |
| 21 | 39 | 36.1 | 1.1 |
| 22 | 41 | 33.2 | 1.2 |
| 23 | 42 | 15.5 | 2.6 |
11 20. Mu.L of the above mixed library was taken and 7. Mu.L of ddH was added 2 O, 1. Mu.L of 1U/. Mu.L of large DNA polymerase I fragment, 3. Mu.L of buffer1, and performing reaction on a PCR instrument after mixing uniformly, wherein the reaction procedure is 25 DEG CThe reaction was carried out for 15min at 80℃for 10min and cooled to 4 ℃.
12 1. Mu.L of circularized primer (1. Mu.M) was added to the above-mentioned system, and after mixing, the mixture was reacted on a PCR apparatus with a reaction procedure of 95℃for 3min, and the temperature was lowered to 4℃in the PCR apparatus.
13 0.5 mu L T DNA ligase and 8 mu L buffer2 were added to the above system, and the mixture was reacted on a PCR apparatus after mixing, the reaction procedure was 25℃for 30min.
14 After the reaction, 1. Mu.L of digestive enzyme Mix is added, the mixture is uniformly mixed and then reacted with a PCR instrument, the reaction procedure is that the reaction is carried out for 30min at 37 ℃, the reaction is carried out for 20min at 80 ℃, and the reaction is cooled to 4 ℃.
15 Purifying the magnetic beads after the reaction is finished, selecting the magnetic beads for selecting beaver biological DNA fragments, wherein the volume of the magnetic beads is 60 mu L, and finally 20 mu L of ddH is adopted 2 O is eluted, and the rest steps are the same as instruction book.
16 The above cyclized library was quantitated using a kit of Qubit ssDNA detection kit followed by on-machine sequencing, in this example the sequencing reagent was MGISEQ-200RS high throughput rapid sequencing kit (FCS PE 150), and the sequencer was MGI200.
The sequencing results of each sample in this example are as follows:
2 cases of peripheral blood, oral swab and cervical exfoliated cell samples are randomly selected respectively, all targeting sequences of BRCA1 gene and BRCA2 gene are verified in a yin-yang manner by adopting Sanger sequencing, and in the embodiment, sanger sequencing is completed by a Optimago, and the comparison result is as follows:
example 2
Several cell lines were pooled, library circularized and sequenced on-press, all from ATCC, the molar amounts of primers used in example 2 are as follows:
the procedure of example 1 was repeated except that the total molar amount of primer pool 1 in example 2 was 45.8. Mu.M, the total molar amount of primer pool 2 was 38.9. Mu.M, the total molar amount of primer pool 3 was 23. Mu.M, and the total amount of primer pool was 107.7. Mu.M.
Wherein the 22RV1 cell line contains 1 pathogenic mutation on BRCA2 gene, and other BRCA1 and BRCA2 gene mutations are detected in the coverage area of the primer set of the invention, the peak patterns of Sanger sequencing are shown in fig. 2 and 3, the peak patterns of the BRCA1 and BRCA2 gene are consistent with the peak patterns of heterozygous insertion or deletion Sanger, the mutation is detected in the 22RV1 cell line as shown in the following table, and the mutation is verified by Sanger, and the coincidence rate is 100%:
| Chr | Start | End | Sanger | REF | ALT | InterVar | Otherinfo |
| 13 | 32907536 | 32907537 | √ | TT | - | Uncertain significance | het |
| 13 | 32911888 | 32911888 | √ | A | G | Benign | het |
| 13 | 32913055 | 32913055 | √ | A | G | Benign | hom |
| 13 | 32913920 | 32913920 | √ | G | A | Likely benign | het |
| 13 | 32915005 | 32915005 | √ | G | C | Benign | hom |
| 13 | 32915411 | 32915414 | √ | AATT | - | Benign | het |
| 13 | 32929232 | 32929232 | √ | A | G | Benign | het |
| 13 | 32929387 | 32929387 | √ | T | C | Benign | hom |
| 13 | 32936646 | 32936646 | √ | T | C | Benign | hom |
| 13 | 32953388 | 32953388 | √ | T | C | Benign | hom |
| 13 | 32954030 | 32954030 | √ | - | A | Pathogenic | het |
| 17 | 41223094 | 41223094 | √ | T | C | Benign | het |
| 17 | 41234470 | 41234470 | √ | A | G | Benign | het |
| 17 | 41244000 | 41244000 | √ | T | C | Benign | het |
| 17 | 41244435 | 41244435 | √ | T | C | Benign | het |
| 17 | 41244936 | 41244936 | √ | G | A | Benign | het |
| 17 | 41245237 | 41245237 | √ | A | G | Benign | het |
| 17 | 41245466 | 41245466 | √ | G | A | Benign | het |
| 17 | 41249364 | 41249364 | √ | A | - | Benign | het |
| 17 | 41251931 | 41251931 | √ | G | A | Benign | het |
example 3
Library cyclization tests were performed on the sample libraries of example 1 at different input amounts of 10ng, 20ng, 30ng and 40ng for each library, and the total amounts of 100ng, 200ng, 300ng and 400ng were sequentially taken for library cyclization in the same mixing manner as in example 1, and the final cyclization library concentrations were 1.23 ng/. Mu.L, 1.58 ng/. Mu.L, 1.69 ng/. Mu.L and 2.02 ng/. Mu.L, respectively, and after sequencing on a machine, all the library cyclized solutions met 100% coverage, and the detected mutations were completely consistent with Sanger sequencing results. The provided library cyclization method has high efficiency and good sensitivity, and can meet the cyclization requirements of libraries with different concentrations in a wide range.
The foregoing is merely a preferred embodiment of the invention, and it is to be understood that the invention is not limited to the form disclosed herein but is not to be construed as excluding other embodiments, but is capable of numerous other combinations, modifications and environments and is capable of modifications within the scope of the inventive concept, either as taught or as a matter of routine skill or knowledge in the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
Claims (10)
1. The primer group for BRCA1/2 gene mutation detection based on the high-throughput sequencing platform is characterized by comprising 133 pairs of primer pairs divided into three groups, wherein the forward primer and the reverse primer of each primer pair sequentially comprise a general sequence and a specific sequence from the 5 'end to the 3' end;
the specific sequences of the forward primers of the first primer pair are shown as SEQ ID NO.1,2,3,4,5 and … …, and the specific sequences of the corresponding reverse primers are shown as SEQ ID NO.134,135,136,137,138 … … and 177;
the specific sequences of the forward primers of the second primer pair are shown as SEQ ID NO.45,46,47,48,49 and … … 89, and the specific sequences of the corresponding reverse primers are shown as SEQ ID NO.178,179,180,181,182 … … and 222;
the specific sequences of the forward primers of the third primer pair are shown as SEQ ID NO.90,91,92,93,94 and … …, and the specific sequences of the corresponding reverse primers are shown as SEQ ID NO.223,224,225,226,227 … … and 266.
2. The primer set for BRCA1/2 gene mutation detection based on high throughput sequencing platform according to claim 1, wherein the ratio of the usage ranges of the 133 pairs of primers is as shown in the following table:
3. the primer set for BRCA1/2 gene mutation detection based on high throughput sequencing platform according to claim 1, wherein the universal adaptor sequence of the forward primer and the reverse primer of each primer pair is an adaptor sequence adapted to the gene sequencing platform.
4. The primer set for BRCA1/2 gene mutation detection based on a high-throughput sequencing platform according to claim 1, wherein the high-throughput sequencing platform is an MGI sequencing platform, the universal adaptor sequence of the forward primer of each primer pair is shown as SEQ ID No.267, and the universal adaptor sequence of the reverse primer is shown as SEQ ID No. 268.
The library construction method for BRCA1/2 gene mutation detection is characterized by comprising the following steps:
s1, amplifying sample genes by using the first primer pair, the second primer pair and the third primer pair according to any one of claims 1-4;
s2, mixing and purifying the first primer pair amplification products, the second primer pair amplification products and the third primer pair amplification products in the step S1 according to the proportion of 1-3:3-7:5-11 to obtain purified first-round PCR amplification products;
s3, performing a second PCR amplification reaction by taking the first PCR amplification product as a template;
s4, purifying the PCR amplification product of the second round to obtain the library for detecting the BRCA1/2 gene mutation.
6. The method for constructing a library for BRCA1/2 gene mutation detection according to claim 5, wherein in step S1, the types of the sample include peripheral blood, oral swab and cervical exfoliated cells.
7. The method for constructing a library for detecting BRCA1/2 gene mutation according to claim 5, wherein the total amount of the primer set used in step S1 is 107.7 to 134.4. Mu.M.
Library cyclization method for BRCA1/2 gene mutation detection, characterized by comprising the library construction method for BRCA1/2 gene mutation detection according to any one of claims 5-7, further comprising the steps of:
s5, carrying out Qubit quantification on the library for detecting the BRCA1/2 gene mutation obtained in the step S4, mixing the library with the equal mass ratio, and carrying out volume fixation on the library with the total amount of 100-400ng after mixing to 27 mu L;
s6, adding terminal repair enzyme and a corresponding buffer1, and performing terminal repair reaction to obtain a mixed library after terminal repair;
s7, carrying out single-chain treatment on the mixed library with the repaired tail end, adding a cyclization primer, and carrying out denaturation and annealing reaction to obtain a single-chain mixed library;
s8, cyclizing the single-chain mixed library, adding ligase and corresponding buffer2, and carrying out cyclization reaction to obtain a single-chain library ring;
s9, digesting the single-chain library ring, and adding a digestive enzyme Mix, wherein the digestive enzyme Mix is a mixture of exonuclease I and exonuclease III, and the mixing ratio is 1:1;
s10, purifying the single-chain library loop, and sequencing the single-chain library loop on a machine after quantitative Qubit.
9. The method for library cyclization of BRCA1/2 gene mutation detection according to claim 8, wherein in step S7, the cyclization primer sequence is shown as SEQ ID No. 269.
10. The library cyclization method of BRCA1/2 gene mutation detection of claim 8, wherein said library cyclization method is not configured on ice.
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