CN106497920A - A kind of library constructing method and test kit for nonsmall-cell lung cancer detection in Gene Mutation - Google Patents

Abstract

The invention discloses an optimized sequencing library construction method and a kit for detecting non-small cell lung cancer gene mutations. The method includes: a one-tube reaction to complete genomic DNA fragmentation and adapter ligation, after amplification of the ligated product, hybridization and capture with probes for target regions of non-small cell lung cancer-related genes, followed by BGISEQ-500/1000 platform sequencing and data analysis to obtain mutations Happened. Based on the one-tube reaction, the experimental process is greatly optimized, the operation complexity and time are reduced, and the requirements for the initial volume of clinical samples are reduced; multi-gene multi-locus detection can be performed at one time, covering point mutations, indels, and structural variations And copy number variation, the detection results are accurate and make up for the inability of PCR capture method to detect structural variation at one time, greatly improving the effectiveness of high-throughput sequencing in the detection of non-small cell lung cancer gene mutations. The invention has wide coverage and high cost performance, can provide references for doctors to diagnose, treat and administer medicine, and is suitable for large-scale popularization and application.

Description

A library construction method and test for detecting gene mutations in non-small cell lung cancer Kit

technical field

The invention relates to the technical field of molecular biology, in particular to the technical field of sequencing library construction, in particular to a library construction method and kit for detecting non-small cell lung cancer gene mutations.

Background technique

At present, lung cancer is one of the most common malignant tumors in the world, and has become the first cause of death of malignant tumors in urban population in my country, and about 85% of lung cancers are non-small cell lung cancers. Conventional chemotherapy or radiotherapy will seriously affect the quality of life and physical fitness of patients, so more and more attention has been paid to targeted therapy. For non-small cell lung cancer, existing scientific research and clinical trials have identified a series of targeted treatment sites and drugs. Different from traditional chemotherapy, targeted therapy does not interfere with all continuously dividing cells, but uses drugs to interfere with specific molecules required for canceration or tumor proliferation, thereby specifically preventing the growth of cancer cells. When the drug enters the body, it will specifically select the cancer-causing sites to combine and take effect, so that the tumor cells will die specifically, without affecting the normal tissue cells around the tumor. Compared with traditional chemotherapy and radiotherapy, targeted therapy can not only reduce toxic and side effects, but also significantly improve the effectiveness of treatment, significantly prolong the survival period of patients and improve the quality of life. In the 2016 "Non-Small Cell Lung Cancer Targeted Drug Therapy Related Genetic Recommendations", there are relevant instructions for targeted drug detection genes, which can be used or recommended for detection in non-small cell lung cancer (NSCLC) include: EGFR , ALK and ROS1 fusion genes, c-MET, RET, BRAF and KRAS. Through genetic testing, the molecular classification of non-small cell lung cancer can be clarified, and possible target drug sites or drug resistance sites can be found, which can provide patients with more accurate medication guidance.

At present, high-throughput sequencing technology has been widely used in the fields of medical research and clinical diagnosis, providing a new detection method for the detection of carcinogenic sites. High-throughput sequencing technology is becoming more and more cost-effective in medical research and clinical applications due to its comprehensive coverage of results and decreasing sequencing costs. The limited sample size and the timeliness of test report output put forward higher requirements for this technology. It is imperative to simplify the library construction process and improve the success rate of library construction for low-input samples. By optimizing and improving the library construction method, shortening the enzyme reaction time and library construction steps, and improving the efficiency of sample conversion into library has become a key point for the expansion of application fields of high-throughput sequencing technology.

The classic high-throughput library construction steps include: 1. Genome fragmentation (including physical fragmentation and chemical fragmentation, such as covaris ultrasonic fragmentation, fragmentase fragmentation fragmentation); 2. Double-stranded DNA (Deoxyribonucleic acid, deoxyribonucleic acid, deoxygenated 3. Add dATP (deoxyadenosine triphosphate, deoxyadenosine triphosphate) to the 3' end; 4. Add adapters matching the sequencing platform; 5. PCR (Polymerase Chain Reaction, polymerase chain reaction) amplification. A purification reaction is required between each step, and sometimes a fragment selection (one of steps 1, 4, and 5) is required after the enzyme reaction. The entire process takes about 8-9 hours to complete. In order to simplify the process, some biological companies have introduced new library construction technologies. For example, illumina proposed a rapid library construction method based on transposase interruption and adapter addition in one step, but this method has the problem of base bias in sequencing data.

As mentioned above, the classic high-throughput library construction method is currently the most widely accepted method with the most stable data performance and the least data bias. High, increasingly unable to meet the needs of the fast-growing market for low-initial micro-library construction and fast reporting cycles, which is more prominent in the clinical application of high-throughput sequencing technology. Although the transposase library construction technology can meet the requirements of low initial amount and rapid library construction, its data bias limits the large-scale promotion of this method to a certain extent. Therefore, if the classic high-throughput library construction method can be improved, the required sample input volume can be reduced and the library construction time can be shortened on the basis of maintaining its existing advantages, it will greatly promote the application of high-throughput sequencing methods in the future. In the market, especially medical research and clinical applications, it provides more favorable methods and technologies for research and clinical applications in related fields.

The method in the present invention optimizes and improves the classic high-throughput library construction method, making it compatible with different sample types (including low-quality formalin-fixed paraffin-embedded samples) while maintaining the advantages of existing data performance ), can carry out micro sample library construction, which greatly simplifies the operation process, reduces the loss of samples in the process of library construction, greatly shortens the time of library construction, and reduces the cost of library construction, which is more conducive to the detection of non-small cell lung cancer genes Large-scale promotion and application.

At present, most of the major genetic testing products on the market can only target a few genes and loci at a time, and different mutation types require different starting sample types or testing methods. For example, the detection kit based on the Arms PCR method uses DNA samples when detecting SNV and InDel, and detects one or several genes at a time. Multiple detections are required to detect more comprehensive genes or loci, resulting in increased time and cost; When detecting fusion gene variants, RNA samples are required and only known fusion types can be detected. In addition, the FISH method is commonly used clinically for the detection of fusion and copy number variation. This method generally targets a single gene at a time, and the sensitivity and accuracy of this method are relatively low, requiring high requirements and experience for operators. expensive. Then, using the kit in the present invention, hundreds of lung cancer-related genes and thousands of hotspots can be detected through a DNA library construction sequence, and all mutation types can be detected, including point mutations, indels, gene fusions, and copy number variations. Covering all non-small cell lung cancer targeted drug action sites, greatly saving sample requirements, avoiding complicated operations caused by extraction of different nucleic acid types, and saving valuable clinical samples. The optimized experimental process in this kit greatly improves the utilization rate of nucleic acid, the detection result is accurate, and it has very good sensitivity, specificity and accuracy for the detection of four types of mutations. The optimized experimental operation steps of this kit make the detection process simpler and more convenient, further reduce the detection cost and labor cost, and the lower cost brings high cost performance, which can ultimately benefit patients better.

Contents of the invention

The invention provides an optimized sequencing library construction method and kit for detecting non-small cell lung cancer gene mutations. This method can realize DNA fragmentation, end repair, A addition, and adapter ligation reactions in the same reaction tube, and then carry out hybridization capture and sequencing of target regions of non-small cell lung cancer-related genes on the amplified products of the ligated products, and through information analysis Ascertain the gene mutation status of non-small cell lung cancer. The invention provides an optimized library construction kit that can be used for gene mutation detection of non-small cell lung cancer, and provides a safer, more accurate, sensitive, convenient and cost-effective gene mutation detection means for clinical practice. It solves the problem that the current clinical method cannot rely on a small amount of DNA samples to comprehensively detect point mutations, indels, fusion genes and copy number variations at one time, and greatly saves the demand for precious clinical samples. The detection results are accurate, which can provide doctors with comprehensive and accurate gene mutation information in a relatively short period of time, and also save patients the precious time and money wasted by multiple detections. The simple and optimized operation steps of the kit reduce the complexity of NGS library construction operation to a certain extent, reduce the probability of error, and further improve the safety of the product in clinical application.

In the present invention, the end repair and the addition of A are realized in the same reaction system, which simplifies the reaction process, reduces the purification steps, improves the efficiency of starting DNA conversion into ligated linker DNA, and reduces the impact on the total amount of starting DNA. Requirements, can also reduce the number of cycles of PCR amplification, increase the available data ratio of the library on the machine and reduce the data base bias. The method of the present invention is suitable for rapid library construction of different types of trace starting DNA. On the basis of the library construction method for detecting non-small cell lung cancer gene mutations of the present invention, the present invention also provides a library construction kit for non-small cell lung cancer gene mutation detection.

According to the first aspect of the present invention, the present invention provides a method for constructing a library for non-small cell lung cancer gene mutation detection, comprising:

Add genomic DNA, DNA breaking enzyme and breaking reaction buffer into the reaction tube; under the action of DNA breaking enzyme, genomic DNA is randomly fragmented to generate DNA molecular fragments;

In the reaction tube, directly add polymerase, polynucleotide kinase, polymerase without 3'-5' exolytic activity, dNTP, dATP and polynucleotide kinase buffer for end repair and Add A reaction; in the same reaction system, in the presence of dNTP, use polymerase to fill in or cut the end of the fragmented DNA, for example, use the 5'-3' polymerization activity of the polymerase to fill in the 5' protruding end and/or Or the 3'-5' exo-cleavage activity of the polymerase blunts the 3' protruding end, using polynucleotide kinase to convert the 5' hydroxyl group to a 5' phosphate group and the 3' phosphate group to a 3' hydroxyl group ; In the presence of excess dATP, add dATP to the 3' end of double-stranded DNA by using a polymerase that does not have 3'-5' exonuclease activity;

In the above reaction system, directly add bubble adapter, DNA ligase and ligation reaction buffer, one end of the bubble adapter is a single-base protruding end; the single-base protruding end is T, which is located at The 3' end of the strand; the 3'A overhanging double-stranded DNA produced in the previous step is ligated to the bubble adapter;

After purifying the adapter-ligated product in the previous step, add nucleic acid single strands complementary to both ends of the adapter sequence as primers for PCR amplification. One of the primers has phosphorylation modification at the 5' end to obtain a large amount of double-stranded DNA, and 5' of one strand is There is a phosphate group at the 'end;

Use the non-small cell lung cancer probe to capture the target region of non-small cell lung cancer on the product obtained by PCR amplification in the previous step;

For the product captured in the target region of non-small cell lung cancer in the previous step, a nucleic acid single strand complementary to both ends of the adapter sequence was added as a primer for PCR amplification, and one of the primers was phosphorylated at the 5' end to obtain a large amount of double-stranded DNA, among which One strand has a phosphate group at the 5' end;

After purifying the product amplified by PCR in the previous step, use heat denaturation to convert the above double-stranded DNA into single-stranded deoxynucleic acid, and add a single-stranded nucleic acid complementary to the head and tail sequence of the adapter as a mediating bridge sequence, and the denatured single-stranded deoxygenated nucleic acid Nucleic acid hybridization and ligation reactions to circularize the target single-stranded nucleic acid to obtain a library.

As a further improved solution of the present invention, the aforementioned genomic DNA is surgically frozen tissue DNA, formalin-fixed paraffin-embedded (FFPE) DNA, or punctured tissue DNA.

As a further improved solution of the present invention, the above-mentioned polymerase is T4 DNA polymerase and/or Klenow large fragment; the above-mentioned polynucleotide kinase is T4 polynucleotide kinase; the above-mentioned does not have 3'-5'exo-cutting activity The polymerase used is Taq polymerase and/or Klenow fragment (3'→5'exo-).

As a further improved solution of the present invention, the fragmented DNA is a DNA fragment molecule obtained by enzymatic digestion and fragmentation. When the end is repaired and A is added to the reaction, each 50 μL system contains 1-500 ng of enzyme fragmentation DNA, T4 DNA polymerase 0-40U, Klenow Large Fragment 0-20U, T4 polynucleotide kinase 0-24U, Taq polymerase 1-10U, each dNTP 0.02-1mM, additional dATP 0.02-1mM, and Mg ion 8- 10mM, the condition is that the content of T4 DNA polymerase and Klenow large fragments are not equal to 0 at the same time; more preferably, the reaction conditions are 15-37°C for 10-30min, and then 65-75°C for 10-30min.

As a further improved solution of the present invention, when there are multiple samples to be sequenced, the steps of DNA fragmentation, end repair/addition of A, and adapter ligation are performed on different samples; Contains a tag sequence; the tag sequence can be used to distinguish samples to be sequenced.

As a further improved solution of the present invention, prior to the hybridization capture reaction, the ligation products or PCR amplification products of different samples to be tested can be mixed for hybridization capture.

According to the second aspect of the present invention, the present invention provides a library construction kit for detecting non-small cell lung cancer gene mutations. First, the kit includes a DNA shearing enzyme and a shearing reaction buffer.

Secondly, the kit also includes an end repair-add A enzyme mixture and an end repair-add A reaction buffer; the end repair-add A enzyme mix includes polymerase, polynucleotide kinase, '-5' exo-active polymerase; the end repair-add A reaction buffer includes dNTP, dATP and polynucleotide kinase buffer; the end repair-add A enzyme mixture and end repair-add A reaction buffer is the kit unit used to prepare the following 50μL reaction system: each 50μL system contains 1-500ng of enzyme-cut DNA, 0-40U of T4 DNA polymerase, 0-20U of Klenow large fragment, and more T4 Polynucleotide kinase 0-24U, Taq polymerase 1-10U, each dNTP 0.02-1mM, additional dATP 0.02-1mM, and Mg ion 8-10mM, provided the content of T4 DNA polymerase and Klenow large fragment It is not 0 at the same time.

Third, the kit also includes a bubble adapter, ligase and ligation reaction buffer; the bubble adapter sequence includes SEQ ID NO: 1-2; SEQ ID NO: 1 contains a tag sequence and is in 5 There is phosphorylation modification at the 'end, and the 3' end of SEQ ID NO: 2 is a T base, which can be connected with a double-stranded DNA molecule with a protruding A base at the 3' end.

Fourth, the kit also includes primer sequences for amplifying the ligation product, the primers include SEQ ID NO: 3 and SEQ ID NO: 4, wherein SEQ ID NO: 3 has a phosphorylation modification at the 5' end, so that One strand of amplified double-stranded DNA has a phosphate group at its 5' end.

The advantages of the library construction method for non-small cell lung cancer gene mutation detection of the present invention are reflected in: first, the four steps of DNA fragmentation, end repair, A addition and linker connection of the present invention are carried out in the same reaction tube, wherein DNA end repair and A addition are carried out in the same reaction system, which greatly shortens the time of library construction, saves purification steps, reduces the cost of library construction, and improves the application of high-throughput library construction technology to medical clinical testing projects, especially the detection of non-small cell lung cancer gene mutations effectiveness. Secondly, the present invention carries out the reaction from DNA end repair to adding A to adding linker in the same reaction system, which can reduce the loss of starting DNA in the process of building a library and improve the efficiency of DNA recovery and utilization.

In addition, by optimizing the reaction conditions, the present invention improves the efficiency of converting the starting DNA into the linker DNA, that is, increases the effective utilization rate of the starting DNA sample, reduces the number of cycles of PCR amplification, thereby reducing the number of duplicate data caused by too many PCR cycles. output, increase the ratio of available data, and reduce data base bias.

Furthermore, the present invention captures the target region of non-small cell lung cancer based on the probe hybridization capture method, and can detect all mutation types such as point mutations, indels, fusion genes, and copy number variations at one time.

In addition, the method of the present invention can widely realize the rapid library construction of different types of trace starting DNA, such as surgically frozen tissue DNA, FFPE sample DNA, and puncture tissue DNA. The method of the present invention can be applied to the BGISEQ-500/1000 sequencing platform.

Description of drawings

Fig. 1 is a schematic diagram of the library construction process of the non-small cell lung cancer gene mutation detection library based on the BGISEQ-500/1000 sequencing platform of the present invention; wherein, 1: starting DNA; 2: genomic DNA enzyme cutting and breaking; 3: end repair It is carried out in the same reaction system as adding A, among which 3.1: end repair reaction; 3.2: adding A reaction; 4: linker ligation reaction (steps 2, 3, and 4 are carried out in the same reaction tube, as shown in the figure " One-tube method"); 5: Purify the reaction system to remove adapter contamination; 6: Perform PCR amplification of ligation products; 7: Purify the PCR reaction system to remove primer-dimer contamination; 8: Use non-small cell lung cancer probes for non-small cell lung cancer Small cell lung cancer target region capture; 9: PCR amplification of non-small cell lung cancer target region capture products; 10: purification of PCR reaction system; 11: single-strand circularization.

detailed description

The present invention will be further described in detail below through specific embodiments in conjunction with the accompanying drawings.

The invention discloses an optimized sequencing library construction method that can be used for detecting non-small cell lung cancer gene mutation. In one reaction tube, the four-step reaction (one-tube reaction) of genomic DNA fragmentation, end repair, A addition and adapter ligation can be quickly and optimized. And it can realize DNA end repair and A addition in the same reaction system.

The library construction method of the present invention can be applied to many sequencing research fields, especially in the clinical direction such as the library construction of non-small cell lung cancer gene mutation detection in the present invention. Using the method of the present invention to construct a non-small cell lung cancer gene mutation detection library can realize rapid library construction of clinical samples of non-small cell lung cancer with a low initial amount, shorten the time for library preparation, reduce experimental costs, and increase the effective utilization rate of DNA. , improve the available data output of the library, and the library is fully compatible with the BGISEQ-500/1000 sequencing platform.

The following will take the BGISEQ-500 sequencing platform as an example and describe the technical solution of the present invention in detail in conjunction with the accompanying drawings. It should be noted that the technical solutions of the present invention are not limited to the details of the technical solutions described below with reference to the accompanying drawings.

Please refer to Figure 1. Taking the construction of a non-small cell lung cancer-related gene mutation detection library based on the BGISEQ-500/1000 sequencing platform as an example, the specific steps of the library construction process in this protocol are as follows:

1. Preparation of template DNA: Use the prepared template DNA for library construction reaction. The template DNA includes but is not limited to genomic DNA extracted from frozen tissues, FFPE samples and puncture tissues after non-small cell lung cancer surgery (number 1 in Figure 1 shown).

2. DNA fragmentation treatment: Add any of the above-mentioned genomic DNA, DNA fragmentation enzyme and fragmentation reaction buffer into the reaction tube. Under the action of DNA breaking enzyme, genomic DNA is randomly fragmented to generate DNA molecular fragments (as shown in number 2 in Figure 1).

3. DNA end repair and A addition reaction: In the same reaction tube as above, add polymerase, polynucleotide kinase, polymerase without 3'-5' exolytic activity, dNTP, dATP and polynucleoside Acid kinase buffer. In the same reaction system, in the presence of dNTPs, use a polymerase to fill in or cut the ends of the fragmented DNA, for example, use the 5'-3' polymerization activity of the polymerase to fill in the 5' protruding ends and/or the polymerase's The 3'-5' exonucleating activity blunts the 3' protruding end, using polynucleotide kinase to convert the 5' hydroxyl group to a 5' phosphate group and the 3' phosphate group to a 3' hydroxyl group; in excess dATP In the presence of dATP (as indicated by numbers 3.1 and 3.2 in Figure 1 ) is added to the 3' end of the double-stranded DNA using a polymerase that does not have 3'-5' exo-cutting activity.

The integrity of the ends of the interrupted genomic DNA fragments will be damaged to varying degrees, forming configurations such as 5' protruding ends, 3' protruding ends, 5' hydroxyl groups, and 3' phosphate groups, which are not conducive to subsequent ligation with adapters reaction. The end repair reaction mainly uses the 5'-3' polymerization activity of polymerase (such as T4 DNA polymerase, Klenow large fragment, etc.) in the presence of dNTP (deoxyribonucleosidetriphosphate, deoxyribonucleoside triphosphate) to fill in the 5' protruding end, The 3'-5' exonuclease activity cuts the 3' protruding end flat; the 5' hydroxyl group is converted into a 5' phosphate by the 5' hydroxyl kinase activity of polynucleotide kinase (such as T4 polynucleotide kinase, etc.) group, the 3' phosphatase activity of polynucleotide kinase converts a 3' phosphate group into a 3' hydroxyl group. The reaction of adding A to the 3' end is to use a polymerase without 3'-5' exolytic activity when dATP exists in the reaction system (such as the Klenow large fragment with 3'-5' exolytic activity removed (also known as " Klenow fragment (3'→5'exo-)"), Taq DNA polymerase without 3'-5' correction activity, etc.), add dATP at the 3' end of double-stranded DNA to facilitate the subsequent integration of double-stranded DNA with 5 The linker sequence in the 'T protruding configuration makes an A-T connection.

4. Add adapter: directly add bubble joint, ligase and ligation reaction buffer into the reaction tube in step 3, one end of the bubble joint is a single-base protruding end; the single-base protruding end is T, which is located at the 3' end of the strand; ligate the double-stranded DNA with the 3'A protruding from the previous step to the adapter. One strand of the bubble adapter contains a tag sequence.

The linker is a specially designed deoxyribonucleic acid sequence, fixed at both ends of the DNA fragment by ligation and other methods, and can be recognized during sequencing and used as the starting site for sequencing, allowing the instrument to read subsequent sequence information. Due to the different library structures of different platforms, the adapter structures used are also different. Using different adapters in this step can meet the library requirements of different sequencing platforms. What is used in the present invention is a bubbling joint suitable for the BGISEQ-500 platform (shown as No. 4 in FIG. 1 ).

5. Purifying the adapter ligation product: Purifying the reaction system of end repair-adding A-adding adapter to remove adapter contamination (as shown in number 5 in Figure 1).

6. PCR amplification of the ligation product: add nucleic acid single strands complementary to both ends of the target adapter sequence as primers for PCR amplification, and obtain a large number of DNA products. Since one of the primers has phosphorylation modification at the 5' end, the amplified double There is a phosphate group at the 5' end of one of the strands of DNA (shown as number 6 in Figure 1).

7. PCR product purification: purify the PCR reaction system to remove primer-dimer contamination (as shown in number 7 in FIG. 1 ).

8. Capture of the target region of non-small cell lung cancer: the purified PCR product was used to capture the target region of non-small cell lung cancer (as shown in number 8 in FIG. 1 ). When there are multiple samples to be tested, different samples to be tested can be mixed together for hybridization capture by using bubble adapters with different tag sequences.

At present, most of the cancer detection probes based on sequencing methods pursue a comprehensive coverage area, hoping to include all drug-related genes. In fact, the number of molecular targeting and immune drugs approved for clinical use is limited, and most of them have clear sites, while the number of sites related to chemotherapy is not large. Therefore, in order to improve the detection accuracy, it is possible to design specific probes for the detection of non-small cell lung cancer, thereby greatly reducing the area covered by the probes. While the coverage area of the probe is relatively concentrated, it does not affect the detection effect of the probe on the overall non-small cell lung cancer population, so as to achieve the purpose of low cost, high coverage and accurate detection. The non-small cell lung cancer probe used in the present invention has been consulted with clinical experts to design a clinically oriented non-small cell lung cancer-related gene detection probe, which reduces the chip size and increases the number of people covered under the premise of including all important sites .

The above non-small cell lung cancer-related gene detection probes include the following: a. Mutation sites recommended by FDA and NSCLC-NCCN; b. Mutation sites that have achieved significant results in large-scale clinical studies; c. Polymorphic sites related to chemotherapy of cell lung cancer; d. Full exons of target genes of NSCLC-NCCN and FDA-recommended drugs; e. Full exons of other non-small cell lung cancer (NSCLC) research hotspot genes sub and hot spots; f. Relying on the data mining and screening of existing non-small cell lung cancer patients, use as few regions as possible to ensure that more than 2 single nucleotide variations (SNVs) are included in more than 95% of NSCLC patients, so that The probe is guaranteed to detect mutations in the vast majority of NSCLC patients.

9. Amplification of the capture product: perform PCR amplification on the capture product of the target region of non-small cell lung cancer (shown as number 9 in FIG. 1 ).

10. Purification of amplification products after capture: Purify the PCR amplification products of the capture target region (as shown by number 10 in FIG. 1 ).

11. Single-strand circularization: prepare the double-stranded linear DNA of the purified post-capture amplified product into a single-stranded circular molecule (shown as number 11 in FIG. 1 ).

The single-stranded circularization reaction uses heat denaturation to convert double-stranded DNA into single-stranded deoxynucleic acid, and adds a single-stranded nucleic acid complementary to the head and tail sequence of the linker (which can be called a mediating bridge sequence) to hybridize with the denatured single-stranded deoxynucleic acid. The ligation reaction circularizes the target single-stranded nucleic acid. The reacted single-strand circularization system is directly used for the subsequent rolling circle replication to form the template product DNB (DNA nanoball, DNA nanoball) on the sequencing machine, which is used for the sequencing reaction.

The database construction method of the present invention greatly simplifies the operation steps, shortens the database construction time, saves the database construction cost, reduces the initial quantity, and improves the application of high-throughput database construction technology to medical clinical detection items—especially non-small cell lung cancer gene mutation detection Project - effectiveness.

The technical solutions of the present invention will be described in detail below through the examples. It should be understood that the examples are only exemplary and should not be construed as limiting the protection scope of the present invention. Unless otherwise specified, the reagents used in the present invention can be purchased from the market through open channels, for example, from BGI.

Example 1: DNA library construction and mutation detection of negative and positive non-small cell lung cancer samples

The positive sample DNA is DNA positive standard 1 containing multiple non-small cell lung cancer-related hotspot mutation sites (which contains point mutations and insertion-deletion mutations that occur frequently in non-small cell lung cancer), and contains multiple non-small cell lung cancer DNA positive standard 2 for fusion gene mutation, and DNA positive standard 3 (HorioznDx) containing non-small cell lung cancer-related MET gene amplification, the mutations in the standard have a stable mutation frequency. Negative samples come from cultured Yanhuang cell DNA (YH DNA), which does not contain gene mutations related to non-small cell lung cancer.

1. DNA sample interruption and end repair: Take 100ng of genomic DNA from negative and positive samples, and dilute to 20μL with water.

Configure the system according to Table 1.

Table 1

Reagent name a reaction dose Breaking and End Repair Buffer 6.4μL breaking and end repair enzyme 3.6μL total capacity 10μL

Add 10 μL of the reaction solution to the diluted DNA, mix well, and keep at 37°C for 20 minutes-65°C for 15 minutes-4°C.

2. Linker connection and purification of connection products:

The linker sequence used in this scheme is as follows (the sequence in this example is from 5' end to 3' end from left to right, "//" indicates the modification group, "phos" indicates phosphorylation, B10 indicates 10 bases label sequence).

long chain:

/5Phos/AGTCGGAGGCCAAGCGGTCTTAGGAAGACAA (B10)CAACTCCTTGGCTCACA (SEQ ID NO: 1);

Short chain:

TTGTCTTCCTAAGGAACGACATGGCTACGATCCGACTT (SEQ ID NO: 2).

Add 10 μL adapter to the reaction solution of the previous step, and mix well. Negative and positive standards use adapters with different tag sequences.

Configure the system according to Table 2.

Table 2

Mix the ligation reaction system with the adapter and product mixture, and incubate at 23°C for 1 hour. After the reaction, 40 μL of magnetic beads were added for purification, and 20 μL of nuclease-free water was used to dissolve the recovered product.

Need to explain: There are many ways to purify the reaction product, such as magnetic bead method, column purification method, gel recovery method, etc., all of which can be used for replacement.

3. PCR (polymerase chain reaction) and purification of amplified products

The primer sequences are as follows: (the sequence in this example is from 5' end to 3' end from left to right, "//" indicates a modification group, and "phos" indicates phosphorylation).

Primer 1 sequence: /5Phos/GAACGACATGGCTACGA (SEQ ID NO: 3);

Primer 2 sequence: TGTGAGCCAAGGAGTTG (SEQ ID NO: 4).

Prepare the system according to Table 3.

table 3

Reagent a reaction dose PCR reaction solution 25 μL PCR primers 5μL total capacity 30μL

Add 20 μL of the product recovered from the previous step to the above system, mix well and then react. The conditions are shown in Table 4.

Table 4

After the reaction was completed, 60 μL of magnetic beads were used for purification, and 22 μL of nuclease-free water was used to dissolve the recovered product. Take 1 μL of the recovered product, use the QubitdsDNA HS Analysis Kit (Invitrogen) to quantify the concentration of the negative and positive standard products, take a sample of 1 μg and mix it, then select the magnetic beads again, and finally dissolve the recovered product with 20 μL of nuclease-free water, and proceed to the next step reaction.

4. Hybridization

Use the non-small cell lung cancer detection probe to hybridize and capture the target region of the non-small cell lung cancer mutant gene on the PCR product obtained in the previous step. Add 5.6 μL of hybridization reaction solution 1 to the upward step reaction tube, mix well, and run the reaction program in Table 5.

table 5

temperature time number of cycles 95°C 5min 1 65°C Keep 1

Take a new PCR tube, add 20 μL hybridization reaction solution 2, and run the reaction program in Table 6.

Table 6

temperature time number of cycles 65°C 5min 1 65°C Keep 1

Take a new PCR tube, add 5 μL hybridization reaction solution 3, and run the PCR reaction program in Table 7.

Table 7

temperature time number of cycles 65°C 2min 1 65°C Keep 1

Take out 13 μL of hybridization reaction solution 2 and add it to hybridization reaction solution 1 and mix evenly. After maintaining a constant temperature at 65°C, transfer all the reaction solution to hybridization reaction solution 3, mix gently, place in a PCR instrument, and react at 65°C for 16 hours.

When the above hybridization reaction is almost completed, take 50 μL of streptavidin-labeled magnetic beads, wash the magnetic beads with 200 μL of magnetic bead binding solution for three times, and finally add 200 μL of magnetic bead binding solution to resuspend the magnetic beads; After the reaction was completed, all the hybridization reaction solution was transferred to the washed magnetic beads, and mixed by inversion at room temperature for 30 min. After the reaction is complete, use a magnetic stand to absorb the magnetic beads, discard the supernatant, add 500 μL of washing solution 1 to wash the magnetic beads, and let it stand at room temperature for 15 minutes (during this period, vortex and oscillate for 3 to 4 times), then use the magnetic stand to absorb the magnetic beads , discard the supernatant; then add 500 μL of Wash Solution 2 preheated to 65°C to wash the magnetic beads, incubate at 65°C for 10 minutes (during this period, vortex and oscillate 2 to 3 times), use the magnetic stand to absorb the magnetic beads, discard Wash the supernatant and repeat using Wash Solution 2 two times. After the reaction is complete, add 45 μL of enzyme-free water to resuspend to obtain magnetic beads containing the DNA fragment of the target region.

5. PCR enrichment and product purification

The primer sequences are as follows: (the sequence in this example is from 5' end to 3' end from left to right, "//" indicates a modification group, and "phos" indicates phosphorylation).

Primer 1 sequence: /5Phos/GAACGACATGGCTACGA (SEQ ID NO: 3);

Primer 2 sequence: TGTGAGCCAAGGAGTTG (SEQ ID NO: 4).

Prepare the system according to Table 8.

Table 8

Reagent 1 reaction volume PCR reaction solution 50μL PCR primers 5μL total capacity 55μL

Add 45 μL of the product recovered from the previous step (including magnetic beads) to the above system, mix well and then react. The conditions are shown in Table 9.

Table 9

After the reaction was completed, 120 μL of magnetic beads were used for purification, and 40 μL of elution buffer was used to dissolve the recovered product. Take 1 μL of the recovered product, and quantify the concentration of the product with QubitdsDNA HS Analysis Kit (Invitrogen).

6. Single chain cyclization:

Take 160ng of the amplified product of the target region and configure it with elution buffer to form a 48μL system, place it in a PCR instrument and incubate at 95°C for 5min, and immediately place it on ice for 5-10min.

Prepare the system as shown in Table 10:

Table 10

Reagent a reaction dose Ligase 0.2 μL 20 μM mediator fragment 5μL Cyclization reaction buffer 6.8μL total capacity 12μL

Add the prepared cyclization reaction solution to the single-chain product, mix well, and incubate at 37° C. for 30 min to carry out the single-chain cyclization reaction.

Wherein the mediating fragment has the corresponding complementary sequence for connecting the two ends of the single strand, and its sequence is as follows: (the sequence in this embodiment is from 5' end to 3' end from left to right).

GCCATGTCGTTCTGTGAGCCAAGG (SEQ ID NO: 5).

7. After the library constructed in the previous step is qualified by electrophoresis, it is sequenced by BGISEQ-500/1000 sequencer, and the sequencing results are analyzed to obtain the detection results of non-small cell lung cancer gene mutations.

1) Sequencing

The constructed single-stranded circular DNA library was used for DNA nanosphere preparation and BGISEQ-500/1000 sequencing. The sequencing process was carried out on the machine in strict accordance with the standard operating procedure of BGISEQ-500/1000.

2) Data analysis

By comparing and deduplicating the sequence obtained by sequencing, the variation of the sample is analyzed, and the steps are as follows:

For point mutations and insertion-deletion mutations, the first step is to output potential mutation sites in the target region according to the alignment; the second step is to extract the base quality value, alignment quality value and For the position information in the sequencing sequence, take the site with a p-value value less than or equal to 0.05; the third step counts the number of indel mutations 20 bp before and after the mutation site, filters out the sites with the number of indel mutations greater than 10, and filters Remove the polymorphic sites in the population, and finally output the information of the mutation sites.

For fusion mutations, the first step is to obtain the abnormal double-ended sequencing sequences of the inserted fragments according to the alignment situation, and obtain potential fusions through their alignment positions; the second step is to intercept the truncated sequencing sequence part in the alignment result B1, Re-align these sequencing sequences to the genome to obtain a comparison result B2, combine B1 and B2 to obtain potential fusion mutations; the third step combines the potential fusion mutations obtained in the first step and the second step, and finally determine the fusion mutation results and output Information on the occurrence of fusion mutations.

Table 11 shows the detection results of point mutations and insertion-deletion mutations of negative and positive samples (DNA positive standard 1 and YH DNA) analyzed after library construction and sequencing.

Table 11

Table 12 shows the fusion gene mutation detection results analyzed after the negative and positive samples (DNA positive standard 2 and YH DNA) were sequenced on the machine.

Table 12

Table 13 shows the copy number variation mutation detection results of negative and positive samples (DNA positive standard 3 and YH DNA) analyzed after library construction and sequencing.

Table 13

SEQUENCE LISTING

<110> Shenzhen BGI Research Institute

<120> A library construction method and kit for detecting gene mutations in non-small cell lung cancer

<130> 15I22173

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 58

<212>DNA

<213> Artificial sequence

<220>

<221> tag sequence

<222> (32)..(41)

<220>

<221> misc_feature

<222> (32)..(41)

<223> n is a, c, g, or t

<400> 1

agtcggaggc caagcggtct taggaagaca annnnnnnnn ncaactcctt ggctcaca 58

<210> 2

<211> 43

<212>DNA

<213> Artificial sequence

<400> 2

ttgtcttcct aagaccgcga acgacatggc tacgatccga ctt 43

<210> 3

<211> 17

<212>DNA

<213> Artificial sequence

<400> 3

gaacgacatg gctacga 17

<210> 4

<211> 17

<212>DNA

<213> Artificial sequence

<400> 4

tgtgagccaa ggagttg 17

<210> 5

<211> 24

<212>DNA

<213> Artificial sequence

<400> 5

gccatgtcgt tctgtgagcc aagg 24

Claims (10)

1. A library construction method for non-small cell lung cancer-related gene mutation detection, characterized in that the method comprises: i. Add genomic DNA, DNA breaking enzyme and breaking reaction buffer into the reaction tube; under the action of DNA breaking enzyme, genomic DNA is randomly fragmented to generate DNA molecular fragments; ii. In the reaction tube, directly add polymerase, polynucleotide kinase, polymerase without 3'-5' exolytic activity, dNTP, dATP and polynucleotide kinase buffer for terminal Repair and add A reaction; in the same reaction system, in the presence of dNTP, use polymerase to fill or cut the ends of fragmented DNA, and use polynucleotide kinase to convert 5' hydroxyl to 5' phosphate group And convert the 3' phosphate group into a 3' hydroxyl group; in the presence of excess dATP, add dATP to the 3' end of double-stranded DNA by using a polymerase that does not have 3'-5' excision activity; iii. In the reaction system, directly add bubbling adapter, DNA ligase and ligation reaction buffer, one end of the bubbling adapter is a single-base protruding end; the single-base protruding end is T, It is located at the 3' end of the strand; the 3'A protruding double-stranded DNA generated in the previous step is ligated to the bubble adapter; iv. After purifying the product of the adapter ligation in the previous step, add nucleic acid single strands complementary to both ends of the adapter sequence as primers for PCR amplification, and one of the primers has phosphorylation modification at the 5' end to obtain a large amount of double-stranded DNA, one of which There is a phosphate group at the 5' end of the chain; v. Use the non-small cell lung cancer probe to capture the target region of non-small cell lung cancer on the product obtained by PCR amplification in the previous step; vi. For the product captured in the target region of non-small cell lung cancer in the previous step, add nucleic acid single strands complementary to both ends of the linker sequence as primers for PCR amplification, and one of the primers has phosphorylation modification at the 5' end to obtain a large number of double strands DNA, one strand has a phosphate group at the 5' end; vii. After purifying the product amplified by PCR in the previous step, use thermal denaturation to convert the double-stranded DNA into single-stranded deoxynucleic acid, and add a single-stranded nucleic acid complementary to the head and tail sequence of the adapter as a mediating bridge sequence, and the denatured Single-stranded deoxynucleic acid hybridization and ligation reaction to circularize the target single-stranded nucleic acid to obtain the library. 2. The method according to claim 1, wherein the genomic DNA is surgically frozen tissue DNA, formalin-fixed paraffin-embedded DNA, or punctured tissue DNA. 3. method according to claim 1, is characterized in that, described polymerase is T4 DNA polymerase and/or Klenow large fragment; Described polynucleotide kinase is T4 polynucleotide kinase; The polymerase with 3'-5' exo-cutting activity is Taq polymerase and/or Klenow fragment (3'→5'exo-). 4. The method according to claim 1, characterized in that each 50 μL system of the end repair and adding A step contains 1-500 ng of enzyme-cut DNA fragments, 0-40 U of T4 DNA polymerase, and 0-40 U of Klenow large fragments. 20U, T4 polynucleotide kinase 0-24U, Taq polymerase 1-10U, each dNTP 0.02-1mM, additional dATP 0.02-1mM, and Mg ion 8-10mM, provided T4 DNA polymerase and Klenow The content of large fragments is not 0 at the same time; Preferably, the reaction conditions are 15-37°C for 10-30 minutes, and then 65-75°C for 10-30 minutes. 5. The method according to claim 1, wherein when there are multiple samples to be sequenced, different samples are respectively carried out in steps i, ii and iii; the bubble-type connector used in step iii contains a label Sequence; the tag sequence can be used to distinguish the samples to be sequenced. 6. The method according to claim 1, characterized in that, before performing the hybridization capture reaction, the ligation products or PCR amplification products of different samples to be tested can be mixed for hybridization capture. 7. A kit for constructing a gene mutation detection library for non-small cell lung cancer, characterized in that it comprises a DNA breaking enzyme and a breaking reaction buffer. 8. The non-small cell lung cancer gene mutation detection library construction kit according to claim 7, further comprising end repair-add A enzyme mixture and end repair-add A reaction buffer; said end repair-add A reaction buffer; Adding A enzyme mixture includes polymerase, polynucleotide kinase, and polymerase without 3'-5' exolytic activity; the end repair-adding A reaction buffer includes dNTP, dATP and polynucleotide Kinase buffer; the end repair-add A enzyme mixture and the end repair-add A reaction buffer are kit units for preparing the 50 μL reaction system described in claim 4 . 9. The non-small cell lung cancer gene mutation detection library construction kit according to claim 8, characterized in that, it also includes a bubbling adapter, a ligase and a ligation buffer; the bubbling adapter sequence includes SEQ ID NO: 1-2; SEQ ID NO: 1 contains a tag sequence and has a phosphorylation modification at the 5' end, and the 3' end of SEQ ID NO: 2 is a T base, which can be combined with a double with a protruding A base at the 3' end Strand DNA molecule connection. 10. The non-small cell lung cancer gene mutation detection library construction kit according to claim 9, further comprising primer sequences for amplifying the ligated product, said primers comprising SEQ ID NO: 3 and SEQ ID NO : 4, wherein SEQ ID NO: 3 has a phosphorylation modification at the 5' end, so that there is a phosphate group at the 5' end of one strand in a large amount of double-stranded DNA amplified.