A capture probe-based amplicon library construction method and its application

Abstract

The present invention discloses a capture probe-based amplicon library construction method and its application, which belongs to the field of medical technology. The present invention realizes a library construction technology that performs probe capture and multiple primer amplification in the same process: using probe hybridization capture to perform non-directional enrichment of DNA / RNA templates and using primers to perform specific enrichment of target fragments to construct libraries, respectively, to achieve DNA and RNA co-construction of libraries. The above method is suitable for detection technologies such as MRD that require trace sample library construction and mutation information amplification, and can simultaneously analyze DNA-level variation SNV / INDEL / fusion variation and RNA-level fusion variation, thereby improving detection sensitivity, while reducing clinical biological background noise caused by the probe method and improving clinical specificity.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a capture probe-based amplicon library construction method and application thereof. Background Art

[0002] Minimal residual disease (MRD) refers to the state in which a cancer patient is in clinical remission after treatment, has no clinical symptoms but still has tumor cells in the body. MRD monitoring can improve prognostic testing and provide guidance for treatment after CR, identify relapse and conduct early intervention. MRD monitoring is currently divided into two main technical routes, one is Tumor-informed and the other is Tumor agnostic; the Tumor-informed technical route is the monitoring of personalized gene panels, including tissue sequencing using WES sequencing, screening a certain number of mutation sites to form a personalized gene panel, and using blood for MRD monitoring. The advantage is strong personalization and high clinical sensitivity; the disadvantage is that the monitoring panel is very personalized and has not undergone a large number of reagent performance verifications, so it needs to be developed, the reagent performance verification is insufficient, and the TAT time is therefore relatively long.

[0003] Tumor agnostic's technical route is the monitoring of fixed gene panels. It uses a fixed panel composed of a group of common and high-frequency mutation genes and sites in tumor patients to monitor the patient's blood. The advantage is that the reagent performance is fully verified, which can reduce the development time. In addition, it can analyze the clonal evolution of patients as the disease progresses, and promptly prompt new clonal information. The performance sensitivity of MRD monitoring reagents usually needs to reach 0.02%. Therefore, both personalized panels and fixed panels have very high requirements on the sensitivity of library construction technology. In addition, MRD monitoring technology also needs to set clinical decision values, that is, MRD positive judgment values. Incorrect mutation detection will result in a decrease in clinical specificity, and too few mutation detections will result in a decrease in clinical sensitivity.

[0004] There are currently two mainstream library construction methods used for immobilized gene panels: one is based on the capture probe method. Since the probe has a 15% mismatch, its technical characteristics reflect that it is conducive to discovering unknown gene mutations; the other is based on multiple primer amplification. The primers are amplified and enriched within a certain length range of the gene. Its technical characteristics reflect that it can fully enrich the target fragments and reduce the amount of sequencing data. The two library constructions have different application scenarios and their steps are also very different.

[0005] The capture method library construction mainly goes through 1) end repair and A addition; 2) adapter ligation; 3) purification of ligation products, 4) library enrichment and PCR product purification; 5) probe hybridization; 6) streptavidin binding; 7) library purification; 8) library enrichment and purification of expected PCR products to obtain a qualified library.

[0006] Amplicon library construction requires 1) one round of PCR, 2) PCR product purification, 3) two rounds of PCR, and 4) library purification.

[0007] The capture probe method is based on the principle of complementary DNA pairing. The probe is cross-linked with the DNA template and can be purified for downstream library construction and analysis. Since the probe has a 15% mismatch, its capture specificity is relatively poor, and it is often necessary to use expensive and complex COTIDNA for blocking. However, nonspecific enrichment will still produce more biological noise and sequencing redundant sequences, which will have a great impact on the specific determination of SNV / INDEL, etc.; and since multiple primers are used to enrich the library in a specific target segment, they have a strong ability to enrich specific fragments and can reduce the generation of biological background noise; at the same time, the capture probe method will cause a considerable amount of invalid and redundant sequencing data due to the mismatch of the probe; and multiple primers have an enrichment effect on the target fragment, which can reduce the generation of redundant data; however, the amplicon-based library construction method has a poor ability to discover unknown variations, while the probe capture library construction method has the ability to discover unknown variations.

[0008] MRD detection is a technology for detecting trace mutations. The sequencing depth is usually very deep, ≥50,000×. Technical means based solely on the capture method will result in a large amount of variation information when detecting based on the fixed gene panel due to the non-specific enrichment of the probe capture method. Sequencing data will produce a large number of gene variations while generating a large number of redundant sequences. These variation information has no suggestive effect on patients and has no clinical significance, which will cause a decrease in the clinical specificity of MRD. The library construction method based solely on amplicon, using primers to enrich specific target fragments, is difficult to discover unknown variations with guiding significance, and is prone to missing real variations, resulting in a decrease in the clinical sensitivity of MRD.

[0009] MRD detection technology is very sensitive to sequencing background noise. Background noise is composed of two parts: one is the chemical background noise introduced by library construction reagents, sequencing reagents, and PCR reactions, and the other is the biological background noise caused by environmental or age factors. Biological background noise includes clonal hematopoietic variation of leukocytes, etc. Since biological background noise is spontaneously generated by the human body, especially at low frequencies, it is difficult to distinguish through technical means; some current technical means will establish a data baseline or filter the sequencing data blacklist through healthy human leukocyte sequencing, but it still cannot be effectively solved. The clinical positive judgment value of MRD, the current mainstream technical methods such as signatera are ≥2SNVs, and do not include fusion; and due to current technical limitations, the detection ability of fusion at the DNA level is inferior to that at the RNA level. However, many fusion variants are also driver mutations, which also have considerable guiding value for the clinical detection of MRD, especially in patients with hematological tumors, where there are a large number of fusion variants. Therefore, using limited blood samples to accurately and comprehensively detect the real variants of tumor patients, including fusion fusion, is a technical difficulty that needs to be solved by MRD monitoring technology. Summary of the invention

[0010] One of the purposes of the present invention is to provide an amplicon library construction method based on capture probes, which includes reverse transcription and fragmentation after DNA / RNA co-extraction, capturing the fragmented DNA / RNA using a set of capture probes, incubating the capture library to extend the artificial sequence (the artificial sequence is a non-human sequence), using a set of multiple primer sets to perform non-specific and specific amplification of the target gene region, library purification, Index universal primer amplification and library purification.

[0011] Preferably, reverse transcription of the co-extracts is performed by M-MLV reverse transcriptase and random primers.

[0012] Preferably, the capture probe is a group of probes with a length of 100-145nt, of which 20nt are artificial sequences and the rest are target region probes, one end of the artificial sequence of the capture probe is biotin-labeled, and the capture probe is divided into a positive chain capture probe and an antisense chain capture probe; the multiple primer set is a group of primers with a length of 35-50nt; the primers include a primer amplification region and a connector connection region, the connector connection region contains a 20nt connector nucleic acid sequence, and the primer amplification region includes a specific sequence and a non-specific sequence; the primers include an upstream primer and a downstream primer; the upstream primer contains a 20nt connector nucleic acid sequence and a specific sequence complementary to the target gene, and has a length of 15-30nt; the downstream primer includes two types: one contains a 20nt connector nucleic acid sequence and a 15-30nt specific sequence complementary to the target gene, and the other contains a 20nt connector nucleic acid sequence and a non-specific sequence complementary to the artificial sequence.

[0013] More preferably, the fragmented DNA / cDNA is prepared by a mechanical method or an enzymatic method.

[0014] More preferably, the main peak of the DNA / cDNA fragmentation length is 80 to 100 nt.

[0015] More preferably, the probe capture process includes capturing the DNA / cDNA mixture with the antisense chain capture probe, incubating and binding with streptavidin magnetic beads, and removing the supernatant by magnetic separation; capturing the supernatant with the sense chain capture probe, incubating and binding with streptavidin magnetic beads, and discarding the supernatant by magnetic separation; mixing the library carrying streptavidin magnetic beads captured by the positive / antisense chain probes, and adding a commercial Klenow enzyme mixture to achieve artificial sequence extension of the captured fragment on the capture probe to fill in the nick.

[0016] More preferably, the capture temperature of the forward / antisense strand is 65°C, the enzyme to be used for the incubation extension is Klenow enzyme, and the incubation extension temperature is 37°C.

[0017] A second object of the present invention is to provide a library constructed by the above-mentioned amplicon library construction method.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The library construction technology process of simultaneous probe capture and multiple primer amplification is realized, which not only avoids the poor specificity of the probe capture method, but also retains the ability of the probe capture method to discover unknown sequences; it not only avoids the poor uniformity of panel amplicons, but also has the ability of high sensitivity in the target region of multiple amplification library construction.

[0020] (2) The MRD panel can simultaneously detect SNV / INDEL / fusion at the DNA level and fusion variation at the RNA level by co-constructing DNA and RNA libraries. The dual detection of fusion variation at the DNA and RNA levels not only improves the fusion detection capability, but also improves the clinical detection sensitivity of MRD.

[0021] (3) It reduces the redundancy of sequencing data and biological background noise caused by nonspecific enrichment of the capture method, simplifies the bioinformatics analysis process, improves the specificity of clinical detection, and reduces the occurrence of false positives; at the same time, it avoids the expensive and complex COTIDNA blocking redundant sequences required by traditional capture methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a flow chart of the library construction of the present invention.

[0023] Figure 2 Schematic diagram of the structure of the sense strand / antisense strand capture probe in Example 1.

[0024] Figure 3 Schematic diagram of the primer structure in Example 1.

[0025] Figure 4 It is a schematic diagram of the present invention. DETAILED DESCRIPTION

[0026] Example 1

[0027] This example is used to illustrate the probe design method for amplification, library construction process, library quality control standards and sequencing analysis provided by the present invention.

[0028] (1) Probe panel design and primer panel design:

[0029] The probes include a sense strand capture probe and an antisense strand capture probe, which are ssDNA, with a probe length of 100 to 145 nt and a 20 nt fixed artificial sequence. For specific structures, see Figure 2 , synthesized at Nanoda Technology Co., Ltd.; the primers include specific primers and non-specific primers, both of which contain 20nt P5 or P7 linker sequences. For specific structures, see Figure 3 , synthesized at Sangon Biotech Co., Ltd.

[0030] (2) DNA and RNA co-extraction

[0031] DNA and RNA were extracted by co-extraction kit and then measured by nanodrop and OD260 / 230 at the same time; the sum of the concentrations needed to be ≥500ng;

[0032] (3) Co-extract reverse transcription

[0033] The RNA in the DNA and RNA co-extracts was reverse transcribed into cDNA using random primers under the action of M-MLV type reverse transcriptase;

[0034] (4) DNA and cDNA fragmentation:

[0035] Fragmentation can be performed by a fragmentation instrument or an enzyme fragmentation kit; the main peak of the fragment size is in the range of 80 to 100 bp, ensuring that the probe completely covers the fragmented fragments and improving the binding efficiency.

[0036] (5) Probe capture phase:

[0037] The method is divided into three main processes: 1) antisense chain capture probe hybridization capture process: the antisense chain capture probe hybridizes the fragmented DNA and cDNA, the streptavidin magnetic beads bind to the biotin probe, the supernatant is removed by magnetic separation, and the streptavidin magnetic beads carrying the antisense chain capture product are collected; 2) supernatant positive chain capture probe hybridization capture process: the positive chain capture probe hybridizes the supernatant produced in step 1) and the fragmented DNA therein, the streptavidin magnetic beads bind to the biotin probe, the supernatant is discarded by magnetic separation, and the streptavidin magnetic beads carrying the positive / antisense chain capture product are collected; 3) the streptavidin magnetic beads carrying the positive / antisense chain probe capture product are re-dissolved with TE buffer and combined into one tube, and incubated at 37 degrees Celsius for 30 minutes in the presence of a commercial Klenow enzyme mixture to extend the artificial sequence on the capture probe, and the artificial sequence is added to the captured DNA sequence through extension.

[0038] (6) Multiple primer amplification stage:

[0039] It is divided into two main processes: 1) Multiplex primer amplification: multiplex primer panel is amplified with the hybridization capture product containing cDNA and forward and reverse strands under certain PCR amplification program conditions with the addition of commercial multiplex amplification mix; 2) Enriched library elution: Magbio magnetic beads are bound, ethanol is washed, and the library is eluted. The formed library contains some P5 and P7 adapters.

[0040] (7) Adaptor amplification stage:

[0041] It is divided into two main processes: 1) Index universal primer amplification: Index universal primers are hybridized with the forward and reverse strands to capture the library, and amplified under certain PCR amplification program conditions with the addition of commercial high-fidelity enzymes; 2) Library elution: Magbio magnetic beads are combined, ethanol is washed, and the library is eluted. The library formed is a complete library. Index universal primers include double-ended index universal sequence primers for the Illumina sequencing platform.

[0042] (8) Library quality control and sequencing:

[0043] The library quality control is carried out by qsep or agarose gel electrophoresis, and the main peak is obvious; the sequencer can be selected from the illumina platform or the BGI platform, and the sequencing strategy can be selected from PE150 or SE150.

[0044] Application Example 1

[0045] This embodiment discloses a method for constructing a library and performing quality control of a library based on a probe-capture-post-amplification library construction technology for a myeloid hematologic malignancy MRD immobilization panel, as well as a method for analyzing sequencing data on a sequencing machine:

[0046] (1) Design of MRD probe panel and primer panel for myeloid hematologic malignancies:

[0047] According to the NCCN guidelines and public databases such as COSMIC and CLINVAR, and combined with the company's own database, 25 hotspot regions and 3 fusion gene regions of 17 genes were screened, with a panel size of approximately 6.6kb; 64 positive chain capture probe panels of 7.7kb, 67 antisense chain capture probe panels of approximately 8.1kb and a set of multiple primer panels were designed.

[0048] Table 1. List of 25 hotspot regions and 3 fusion gene regions of 20 genes

[0049]

[0050]

[0051] Table 2 Probe sequence list

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] Table 3 Multiple primer sequence list

[0064]

[0065]

[0066]

[0067] The probe panel was synthesized by Nanjing Nanoda Biotechnology Co., Ltd., and the primer panel was synthesized by Sangon Biotechnology Co., Ltd.

[0068] (2) DNA and RNA co-extraction

[0069] Peripheral blood samples from 10 patients diagnosed with AML were used for DNA and RNA co-extraction. The extraction kit selected was Zymo DNA / RNA co-extraction kit (Cat: D7005). The extraction volume was 1.5 ml of peripheral blood and the elution volume was 20 μL. After extraction, 1 μL was taken by nanodrop to determine the DNA and RNA concentrations respectively.

[0070] Table 4 Sample information table

[0071]

[0072] (3) Reverse transcription of DNA and RNA co-extracts

[0073] Take 500 ng (the sum of DNA and RNA concentrations) of DNA and RNA co-extracts and select 1st StrandSynthesis Kit (Cat. No.: 12249ES24) was used for reverse transcription, and the reverse transcription product was a mixture of DNA and cDNA.

[0074] The reverse transcription system is:

[0075] Table 5 Reverse transcription system

[0076] Components Dosage (μL) Reverse transcriptase 2 Random Primers 2 Response buffer 2 template 500ng Nuclease-free water Make up to 20 μL

[0077] The reverse transcription procedure is:

[0078] Table 6 Reverse transcription procedure

[0079] Serial number Temperature(℃) time 1 25 5min 2 37 45min 3 85 5s 4 4 Hold

[0080] (3) DNA and cDNA fragmentation

[0081] Take 10ul of the reverse transcribed DNA and cDNA mixture from the co-extracts and fragment them using QIAGEN QIAseq FX in the QIAseq FX DNA Library Kit (Cat. no. 180473) for unbiased fragmentation.

[0082] The fragmentation system is:

[0083] Table 7 Fragmentation system

[0084] Components Dosage (μL) Fragmentase 2 Fragmented buffer 2 DNA and cDNA mixture 10 Nuclease-free water Make up to 20 μL

[0085] The fragmentation procedure is:

[0086] Table 8 Fragmentation procedure

[0087] Serial number Temperature(℃) time 1 95 10min 2 40 60min

[0088] (4) Probe capture and library incubation

[0089] Before capturing the fragmented DNA / cDNA, the fragmented DNA / cDNA was first concentrated using an Eppendorf Concentrator plus vacuum centrifugal concentrator.

[0090] The concentrated product was first captured by the antisense capture probe, and then captured by streptavidin magnetic beads. The capture system is shown in Table 9, and the capture procedure is shown in Table 10. The working concentration of the antisense capture probe is 10 pmol / μL.

[0091] The process of binding the streptavidin magnetic beads to the captured product is as follows: a. After the capture process is completed, immediately take 50 μL of streptavidin magnetic beads and add them to the antisense chain capture probe capture product; b. Add 1 ml of reaction buffer and bind for 30 minutes, shaking and mixing every 5 minutes; c. Use a magnetic stand to magnetically separate and collect the streptavidin magnetic beads. Transfer 4.8 μL of the supernatant for hybridization with the sense chain capture probe.

[0092] The supernatant was captured by the positive chain capture probe, and the captured fragments were captured by streptavidin magnetic beads. The capture system is shown in Table 9, and the capture procedure is shown in Table 10. The working concentration of the positive chain capture probe is 10 pmol / μL. Hyb#1 and Hyb#2 buffers were purchased from Nanjing Nanoda Biotechnology Co., Ltd.

[0093] The process of binding the streptavidin magnetic beads to the captured product is as follows: a. After the capture program is completed, immediately take 50 μL of streptavidin magnetic beads and add them to the positive chain capture probe capture product; b. Add 1 ml of reaction buffer and bind for 30 minutes, shaking and mixing every 5 minutes; c. Use a magnetic stand for magnetic separation, discard the supernatant, and collect the streptavidin magnetic beads.

[0094] Table 9 Capture system

[0095]

[0096]

[0097] The forward / antisense strand hybridization capture procedure is:

[0098] Table 10 Capture procedure

[0099] Serial number Temperature(℃) time 1 95 30s 2 65 60min 3 65 hold

[0100] The two tubes of streptavidin magnetic beads containing the captured products were resuspended with 10 μL TE buffer respectively and combined into one tube of captured products with a total volume of 20 μL.

[0101] Capture product incubation:

[0102] Table 11 Amplification system

[0103] Serial number Components Dosage (μL) 1 Capture product 20 2 10×ERAbuffer (containing Klenow enzyme) 3 3 RNase-free H2O 7

[0104] The incubation program was 37°C for 30 min.

[0105] 10×ERA buffer (containing Klenow enzyme) was purchased from Tiangen Biochemical Technology Co., Ltd., catalog number: NG302.

[0106] (5) Multiple primer amplification

[0107] 30 μL of the incubation product was taken for amplification. The working concentration of each primer in the multiple primer panel was 5 nM, and the total amplification volume was 50 μL. Ringcap enzyme and Ringcap buffer were purchased from Xiamen Feishuo Biotechnology Co., Ltd.

[0108] The amplification system is as follows:

[0109] Table 12 Amplification system

[0110] Serial number Components Dosage (μL) 1 Ringcap enzyme 5 2 Ringcap Buffer 5 3 Multiplex Primer Panel 2 4 Incubation product 30 5 Nuclease-free water 8

[0111] The amplification program is shown in Table 13.

[0112] Table 13 Amplification program

[0113]

[0114] The amplified product was purified using 60 μL of 1.2× magbio magnetic beads, washed twice with 70% ethanol, and eluted with 22 μL of TE buffer to obtain an enriched library. Magbio magnetic beads were purchased from Nanjing Nanoda Biotechnology Co., Ltd.

[0115] (6) Adapter amplification

[0116] Take 20 μL of the enriched library for amplification, and the total amplification volume is 40 μL;

[0117] The amplification system is as follows:

[0118] Table 14 Amplification system

[0119] Serial number Components Dosage (μL) 1 Ringcap enzyme 5 2 Ringcap Buffer 5 3 Index Universal Primer 2 4 Enriched library 20 5 Nuclease-free water 8

[0120] The amplification program is shown in Table 15.

[0121] Table 15 Amplification program

[0122]

[0123] The amplified product was purified using 60 μL of 1.2× magbio magnetic beads, washed twice with 70% ethanol, and eluted with 22 μL TE buffer.

[0124] (7) Library quality control and sequencing

[0125] Take 1 μL of library for qubit detection, the concentration should be greater than 1 ng / μL, take 1 μL and use qsep instrument for agarose gel electrophoresis detection, the main peak should be located around 200 bp to pass the library quality inspection.

[0126] The library quality control results are shown in Table 16.

[0127] Table 16 Library quality control results

[0128]

[0129]

[0130] The sequencing platform selected was the Illumina sequencer Nexstseq550, and the sequencing strategy selected was PE150. The MRD monitoring results of 10 patients with myeloid hematological tumors are shown in Table 17.

[0131] Table 17 MRD monitoring results of patients with myeloid hematological malignancies

[0132]

[0133]

[0134] The expected fusion variant detection results in Table 17 were verified by fluorescence in situ hybridization (FISH).

[0135] From the data analysis of Table 17, it can be seen that the capture probe-based multiple amplicon library construction process of the present application can stably detect all ultra-low frequency mutations and ultra-low copy fusions, and the fusion detection at the RNA level increases the detection capability of fusion.

[0136] In summary, the capture probes and multiplex amplicon library construction methods designed in the present application are suitable for next-generation high-throughput sequencing, and can accurately detect DNA and RNA at the same time. The fusion detection effect is better than the single DNA fusion detection effect, and blood samples are effectively utilized. Blood sample DNA and RNA do not need to be extracted and / or library constructed separately. It has strong operability and a wide range of applications, improves the MRD detection positivity rate, and has good applicability.

[0137] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A capture probe-based amplicon library construction method, characterized in that: The library construction method includes reverse transcription and fragmentation after DNA / RNA co-extraction, capturing the fragmented DNA / cDNA with a set of capture probes, merging the captured libraries, non-specific and specific amplification of the target gene region with a set of multiple primer sets, library purification, index universal primer amplification and library purification; the sequence of the capture probe is shown in SEQ ID NO.1-131; the sequence of the multiple primer set is shown in SEQ ID NO.132-266.

2. The amplicon library construction method according to claim 1, characterized in that: Reverse transcription of the co-extracts was performed using M-MLV reverse transcriptase and random primers.

3. The amplicon library construction method according to claim 2, characterized in that: The fragmented DNA / cDNA is prepared by a mechanical method or an enzymatic method.

4. The amplicon library construction method according to claim 3, characterized in that: The main peak of the DNA / cDNA fragmentation length is 80-100 nt.

5. The method for building an amplicon library according to claim 4, characterized in that: The probe capture process includes capturing with antisense chain capture probes, incubating and binding with streptavidin magnetic beads, and removing the supernatant by magnetic separation; capturing with the supernatant sense chain capture probes, incubating and binding with streptavidin magnetic beads, and discarding the supernatant by magnetic separation; re-dissolving the streptavidin magnetic beads carrying the positive / antisense chain probe capture products and merging them into one tube, and incubating and extending the positive / antisense chain probe capture library.

6. The method for building an amplicon library according to claim 5, characterized in that: The capture temperature of the forward / antisense strands is 65°C, the enzyme required for the incubation extension is Klenow enzyme, and the incubation extension temperature is 37°C.

7. The method for building an amplicon library according to claim 6, characterized in that: The capture procedure of the forward / antisense strand is shown in the following table: 。 8. A library constructed by the amplicon library construction method according to any one of claims 1 to 7.

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