CN115110154A

CN115110154A - Method and kit for detecting low-plasma-initiation-amount and low-frequency mutation and application of method and kit

Info

Publication number: CN115110154A
Application number: CN202210685434.4A
Authority: CN
Inventors: 王涛; 胡娟; 马亚茹
Original assignee: Hangzhou Repugene Technology Co ltd
Current assignee: Hangzhou Repugene Technology Co ltd
Priority date: 2022-06-15
Filing date: 2022-06-15
Publication date: 2022-09-27

Abstract

The invention provides a method, a kit and application for detecting low plasma initial amount and low frequency mutation, wherein the method comprises a construction method of a high-throughput sequencing library: 1) performing end repair on the extracted cfDNA; 2) performing joint connection on the end repairing product; 3) performing first magnetic bead purification on the joint connection product; 4) amplifying the first purified product to obtain an amplified product; 5) performing secondary magnetic bead purification on the amplification product to obtain a pre-library; 6) performing mixing treatment by using the single pre-library; 7) preparing a hybridization reaction system by using the mixed pre-library obtained in the step 6), and performing hybridization treatment; 8) performing specific capture on the hybridization product obtained in the step 7) so as to obtain a capture library; 9) and carrying out amplification and third magnetic bead purification treatment on the capture library to obtain the high-throughput sequencing library containing the DNA fragment with the mutation frequency of not less than 0.3% in the sample genome DNA.

Description

Method and kit for detecting low-plasma-initiation-amount and low-frequency mutation and application of method and kit

Technical Field

The invention relates to the technical field of biology, in particular to a method and a kit for detecting low plasma initial amount and low frequency mutation and application thereof.

Background

High-throughput sequencing, also known as "Next-generation" sequencing technology, is used to sequence hundreds of thousands to millions of DNA molecules in parallel at a time. The NGS (high throughput sequencing technology) technology plays an important role in each link of the whole course management of tumor treatment due to the advantages of high detection flux and wide detection range, and plays an indispensable role in the accurate diagnosis and treatment link.

Currently, CFDA approved multiple gene detection kits for NGS tumors in China are mainly used for FFPE samples to detect small Panle gene mutations in tissues, and although tissue biopsy is the gold standard for lung cancer diagnosis, tissue biopsy has the disadvantages of invasiveness, long time consumption and limited use, for example, the detection result depends on the location of tumors and whether tissue samples are sufficient or not. The noninvasive liquid biopsy based on the blood plasma can supplement diagnosis information, has the advantages of small invasiveness and wide applicable population, and provides valuable information for patients with tissue difficulty to a certain extent. Liquid Biopsy (Liquid Biopsy) has many advantages such as rapidness, convenience, and less trauma compared to conventional tissue Biopsy. Among various liquid biopsy technologies, the detection of circulating mu Lating cell-free DNA (ccfDNA) has been rapidly developed due to its unique advantages and the maturity of high throughput sequencing technology. In a human body, circulating tumor DNA (ctDNA) is tumor DNA that is dissociated in the blood circulation system, is a small fragment of DNA released after tumor cells die, and is mainly derived from necrotic tumor cells, apoptotic tumor cells, circulating tumor cells, and exosomes secreted by tumor cells. In clinical application of plasma ctDNA, ctDNA released into blood by tumors may be very trace, and detection sensitivity of NGS may be required to reach a level of 0.01% -1% when detecting low-abundance tumor gene mutation, which is a great challenge for detection sensitivity and detection limit of NGS technology.

Therefore, there is a need to further develop a more widely and accurately applicable method for detecting a low-abundance gene mutation.

Disclosure of Invention

The object of the present invention is to solve at least to some extent one of the related technical problems:

therefore, the inventor optimizes the method and the reagent for obtaining the high-throughput sequencing library, specifically captures and enriches free DNA (cfDNA) mutation in trace blood, can detect various DNA variation with extremely low variation frequency in the blood in a high-sensitivity and high-specificity manner under the condition of no wound, and can be applied to the fields of targeted medication guidance, dynamic monitoring of treatment effect and the like.

Therefore, in a first aspect of the invention, the invention provides a method for constructing a high-throughput sequencing library. According to an embodiment of the invention, the method comprises the following steps: constructing a PCR amplification system by using cfDNA, and carrying out PCR amplification treatment to obtain a PCR amplification library, wherein based on 50 mu L of the PCR amplification system, the using amount of a label primer (10 mu M) is 1-3 mu L, the using amount of an amplification primer 1(10 mu M) is 1-3 mu L, the using amount of a PCR enzyme (main components: MLtraHiFi DNA Polymerase, NaCl, Tris-HCl, MgCl2 and DTT) is 23-27 mu L, and the using amount of the purified cfDNA is 18-24 mu L. The PCR amplification system is obtained by screening and optimizing the DNA fragment, so that a great deal of creative work is paid, the total amount of the prediction sequencing libraries to be hybridized, which contain the DNA fragments with the mutation frequency of not less than 0.3 percent and are obtained by the method of the embodiment of the invention, is higher, and the high-throughput sequencing libraries obtained by hybridizing, capturing and capturing the pre-sequencing libraries and then amplifying can be used for effectively detecting the mutation with the mutation frequency of not less than 0.3 percent.

According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:

according to the embodiment of the invention, the conditions of the PCR amplification treatment are as follows: at 98 deg.C for 2-4 min; (98 ℃, 15-25s, 58-63 ℃, 28-32s, 67-78 ℃, 25-35s, 2-4 steps circulating 8 times); 72 ℃ for 5 min; and preserving at 4 ℃. The PCR amplification treatment conditions are obtained by the inventor through a large number of experimental optimization screening, and have better QC parameters and PCR product yield under the reaction conditions of the application.

According to an embodiment of the present invention, the cfDNA is subjected to a first magnetic bead purification process in advance;

according to an embodiment of the present invention, the cfDNA is subjected to a first magnetic bead purification process using 0.8 × magnetic beads.

According to an embodiment of the invention, the cfDNA is from a sample to be tested.

According to an embodiment of the present invention, the content of the cfDNA obtained in the sample to be tested is 15-30 ng.

According to an embodiment of the invention, the sample to be tested comprises plasma.

According to the embodiment of the invention, when the first magnetic bead purification treatment is performed, the cfDNA obtained from the sample to be tested is subjected to end repairing treatment and adaptor connection treatment in advance in sequence.

According to the embodiment of the invention, based on 50. mu.L of the system for the terminal repairing treatment, the amount of the terminal repairing enzyme is 3 to 8. mu.L, and the amount of the terminal repairing buffer is 8 to 12. mu.L.

According to the embodiment of the invention, the conditions of the end repairing treatment are as follows in sequence: 18-23 ℃ and 27-33 min; 62-68 ℃ for 28-33 min.

According to the embodiment of the invention, based on 100 μ L of the linker ligation system, 45-55 μ L of the end repair product, 2.5-7.5 μ L of linker, 8-13 μ L of ligase and 18-22 μ L of ligation buffer are used.

According to an embodiment of the invention, the method further comprises: and carrying out second magnetic bead purification treatment on the PCR amplification library to obtain a DNA pre-sequencing library.

According to an embodiment of the present invention, the PCR amplification library is subjected to a second magnetic bead purification process using 1 × magnetic beads.

According to an embodiment of the invention, the method further comprises: 1) mixing a plurality of individual DNA prediction prosequences; 2) preparing a hybridization reaction system by using the product obtained in the step 1), and performing hybridization treatment; 3) performing streptavidin magnetic bead (T1 magnetic bead) capture treatment on the hybridization product obtained in the step 2) so as to obtain a capture product; 4) and amplifying the capture product and purifying the capture product by using a third magnetic bead so as to obtain the high-throughput sequencing library, wherein the high-throughput sequencing library comprises DNA fragments with mutation frequency of not less than 0.3% in cfDNA of the sample.

According to an embodiment of the present invention, the average sequencing depth of the library for each sample in the high throughput sequencing library is 18000-25000.

According to an embodiment of the invention, the mutations comprise point mutations, insertions, deletions.

According to an embodiment of the invention, the plurality of sample individual DNA sequencing libraries comprise different tag primers.

According to an embodiment of the invention, the number of DNA sequencing libraries of the sample does not exceed 4.

According to the embodiment of the invention, based on 30 μ L of the hybridization reaction system, 1-3 μ g of template to be hybridized, 3-7 μ L of blocker 1, 1-3 μ L of hybridization probe, 1-3 μ L of blocker diluent and 5-7 μ L of hybridization buffer are used.

According to an embodiment of the invention, the conditions of the hybridization treatment are, in order: 95 deg.C, 5min, 65 deg.C, 10min, 65 deg.C, 1 min; (65 deg.C, 1 min; 37 deg.C, 3s, cycle 60 times), and preserving at 65 deg.C.

According to an embodiment of the present invention, the streptavidin magnetic beads are washed with a binding buffer in advance.

According to the embodiment of the present invention, washing with a washing buffer is performed after 20-40min of capturing.

According to the embodiment of the invention, based on 50. mu.L of the capture amplification system, the amount of the capture product is 22-27. mu.L, the amount of the capture amplification primer is 3-8. mu.L, and the amount of the PCR enzyme reaction solution 2 is 22-27. mu.L.

According to an embodiment of the invention, the conditions for post-capture amplification are, in order: at 98 deg.C for 2-4 min; (98 ℃, 15-25s, 58-63 ℃, 28-32s, 67-78 ℃, 25-35s, 2-4 steps circulating for 14 times); preserving at 72 deg.C for 5min and 4 deg.C.

In a second aspect of the invention, a high throughput sequencing library is provided. According to an embodiment of the invention, constructed according to the method of the first aspect. As described above, the method for constructing the high-throughput sequencing library is obtained by a large number of experimental screening and optimization performed by the inventor, the method according to the embodiment of the present invention can detect a sample to be tested containing a small amount of cfDNA, the total amount of the obtained prediction library to be hybridized is high, and after hybridization capture, the high-throughput sequencing library can be effectively constructed, and the high-throughput sequencing library contains DNA fragments with mutation rate not less than 0.3% in the cfDNA, and the high-throughput sequencing library can be used to effectively detect low-frequency mutation sites in the cfDNA of the sample to be tested.

In a third aspect of the invention, the invention proposes a method of detecting low frequency DNA mutations, according to an embodiment of the invention, comprising: 1) constructing a high throughput sequencing library using the method of the first aspect; 2) and (3) performing machine sequencing on the high-throughput sequencing library to obtain the DNA locus with low-frequency mutation. According to the method provided by the embodiment of the invention, the sample to be detected containing a small amount of cfDNA can be quickly and effectively detected, wherein mutation of as low as 0.3% in the cfDNA can be accurately detected.

according to an embodiment of the present invention, the high throughput sequencing library includes DNA fragments having a mutation frequency of not less than 0.3% in genomic DNA of the sample.

In a fourth aspect of the invention, a kit is provided. According to an embodiment of the invention, the kit comprises: the kit comprises probes, primers, a DNA extraction reagent, a nucleic acid purification reagent, a terminal repair enzyme, a terminal repair buffer solution, a joint, a ligase, a ligation buffer solution, a PCR reaction solution, a blocking agent, a hybridization buffer solution, a binding buffer solution and a washing buffer solution. The kit provided by the embodiment of the invention can be used for quickly and effectively detecting a sample to be detected containing a small amount of cfDNA, wherein mutation of as low as 0.3% in the cfDNA can be accurately detected.

According to an embodiment of the present invention, the kit may further comprise at least one of the following additional technical features:

according to an embodiment of the present invention, the nucleic acid purification reagent includes purified magnetic beads.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a technical roadmap according to an embodiment of the invention;

FIG. 2 is a graph showing the comparative analysis of the detection sensitivity of the kit obtained according to the present invention in the example of the present invention and the commercial kit at the mutation frequencies of 0.5% and 0.2%.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

For ease of understanding, the present invention is briefly described as follows:

the invention provides a library building method for detecting low-initial-amount and low-frequency mutation of plasma, which is obtained by optimizing and screening through a large number of experiments by an inventor, wherein the initial amount of DNA used in the method is 30ng, point mutation, insertion and deletion samples with the mutation proportion as low as 0.3% can be detected under the sequencing depth of 20000X, the specificity reaches 100%, and the low-initial-amount DNA can be used for effectively detecting the low-frequency mutation.

The invention aims to provide a detection kit, wherein all substances in the kit are obtained by screening and optimizing by the inventor, and the detection kit comprises a probe composition, a terminal repair enzyme, a terminal repair buffer solution, a joint, a ligase, a connection buffer solution, a PCR enzyme reaction solution, a primer, a blocking agent, a hybridization buffer solution, a binding buffer solution, a washing buffer solution and the like. The probe type of the kit is an RNA probe, the capture coverage is high, the uniformity is good, the high specificity of the probe is ensured, and the one-time detection of multiple mutations of multiple genes can be realized.

Compared with a commercial kit Agilent SureSelect XTHS, the kit has higher library complexity and higher accuracy.

The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.

Example 1 optimization of kits and methods of use thereof

1.1 optimization of initial volume of Bank construction

In this embodiment, the inventor optimizes the initial library building amount, sets the usage amount of cfDNA to 15ng, 30ng,50ng, and 15ng, respectively sets two mutation frequencies of 2% and 0.5%, and sets two mutation frequencies of 0.5% and 0.3% for 30ng and 50ng, repeats each condition for 3 times, detects pre-PCR yield, QC quality control parameters, mutation detection and other indexes, and screens out the optimal initial library building amount, and the specific experimental operations are as follows:

1.1.1 nucleic acid extraction

1) Sample processing

The sample comprises plasma, and if the sample is fresh plasma, the sample is placed at 16000Xg at 4 ℃ for centrifugation for 10 minutes; if the sample is plasma stored at-80 ℃, the plasma sample is taken out and thawed in a laboratory at room temperature, then the plasma sample is centrifuged for 10 minutes at 16000Xg at 4 ℃, 4mL of centrifuged plasma supernatant is sucked into a 15mL centrifuge tube to prepare for extracting DNA.

2) Extraction of DNA

Plasma DNA was extracted according to the standard procedure of a plasma extraction kit (Hangzhou Ruipu Gen. Tech., Ltd., lot: 20210301), and the sample was stored at-20 ℃ for further use. The extracted cfDNA sample is used for determining the DNA concentration by using the Qubit4.0, and the total amount of the DNA of the cfDNA sample calculated is more than 30 ng.

1.1.2 library construction

1) Tip repair

The end repair enzyme (T4 DNA polymerase, Klenow fragment, T4 polynucleotide kinase) and end repair buffer (KPO) ₄ 、DTT、EDTA、NaCl、Tris-HCl、MgCl ₂ Glycerol) was melted on ice, and the following end-repair reaction mixture was prepared on ice, vortexed, shaken, and mixed uniformly, wherein x1 represents the volume of cfDNA added in this example, that is, 30ng of cfDNA was added, and x1 was 30 ng/concentration, respectively, as shown in table 1.

Table 1: end-repair reaction system

Adding 15 mu L of mixed solution of the end repairing reaction into each DNA sample, and gently sucking and beating for 6-8 times or gently flicking and uniformly mixing; and (3) running a terminal repairing program: 1min at 4 ℃; 30min at 20 ℃; 30min at 65 ℃; hold at 4 ℃, volume 50 μ L, hot lid temperature 70 ℃; after the PCR procedure was completed, the samples were taken out on ice and immediately subjected to the adaptor ligation step.

2) Joint connection

Preparing a joint connection mixed solution shown in the table 2 on ice, and gently blowing, sucking or flicking to mix uniformly; and (3) running a program: 20 ℃ 15min, volume 100. mu.L (PCR instrument without hot lid). After the process is finished, the next process is carried out.

Table 2: linker ligation reaction system

3) Purification after linker attachment

The linker ligation products were purified using purified magnetic Beads AMPure XP Beads (0.8 ×), eluted with 21 μ L nuclease-free water, and 21 μ L purified products were transferred to a new PCR tube.

4) Library amplification

Melting the PCR enzyme reaction solution and the amplification primer 1 on ice, and uniformly mixing by vortex oscillation; preparing PCR reaction mixed liquor for library amplification before capture on ice, and mixing uniformly by vortex oscillation, wherein each human part of library amplification system is shown in table 3, wherein the PCR enzyme reaction liquor 1 mainly comprises the following components: MLtraHiFi DNA Polymerase, NaCl, Tris-HCl, MgCl2, DTT; simultaneously arranging different label primer numbers for each sample, and adding 2 mu L of arranged label primers into each PCR tube; after flick and uniform mixing, the procedure was run, and the PCR amplification reaction procedure is shown in table 4.

Table 3: pre-library amplification reaction system

TABLE 4 Pre-library amplification reaction conditions

5) Purification after library amplification

And (3) purifying the amplified library product by using purified magnetic Beads AMPure XP Beads (BECKMAN CO mu LTER, A63882, lot 1776140) (1X), adding 15 mu L of nuclease-free water for elution after purification, and transferring 15 mu L of the purified product to a new 1.5mL centrifuge tube to obtain the sample DNA pre-library.

6) Library quality control

Diluting the amplified library by 10 times, detecting the concentration by using a QubitdsDNA HS Assay Kit, and calculating to obtain a sample DNA library, wherein the total amount of a positive control library and a negative control library is more than 0.5 mu g. Otherwise, the library sample is not in accordance with the requirements, and the library is to be rebuilt.

1.1.3 hybrid Capture

1) Hybridization of

Respectively selecting single pre-libraries consisting of cfDNA of 4 different label primers to mix, wherein 750ng of each pre-hybridization library and 4 pre-libraries are taken, and the total amount is 3 mu g; if the number of samples is less than 4, the number of samples captured by mixed hybridization can be reduced, and the positive control and the negative control can be captured by mixed hybridization with the library samples. In this experiment, X2 represents the volume of the mixed library used, specifically 3. mu.g/concentration, that is, 3. mu.g of the mixed library was put in.

After the mixed libraries are prepared, 5 mu L of the blocking agent 1 is added into each mixed library, and the mixture is evenly mixed and centrifuged; setting a PCR instrument: hot lid 105 ℃, volume 30 μ Ι _, procedure: 5min at 95 ℃; 10min at 65 ℃; 1min at 65 ℃; (65 ℃ C. for 1min, 37 ℃ C. for 3s, 60 cycles); hold at 65 ℃; hybridization was performed according to the system.

TABLE 5 dilution of 25% blocking agent 2

Components	Volume (μ L)
		Blocking agent 2	0.5
Nuclease-free water	1.5
		Total up to	2

TABLE 6 hybridization reaction System

2) Capture

Taking down the hybridization reaction tube, placing at room temperature, immediately transferring all the liquid into a magnetic bead containing 200 mu L of washed magnetic beads, slowly blowing and sucking for 5-8 times, and uniformly mixing; the tube cover is covered, and the tube is placed in a constant temperature oscillation mixing machine to be mixed for 30min at room temperature of 1500 rpm.

Adding 200 mu L of washing buffer solution 1 into the washing buffer solution 2, and blowing and sucking the heavy suspension magnetic beads for 10-15 times; centrifuging briefly, placing the tube in a magnetic rack, clarifying the solution, and removing the supernatant. Adding 200 μ L of washing buffer solution 2 preheated at 73.5 deg.C, and mixing by blowing and sucking for 10-15 times or mixing by gentle shaking for brief centrifugation; incubating at 73.5 deg.C for 10 min; putting the centrifugal tube into a magnetic frame, clarifying the solution, and removing the supernatant; repeated washing for 3 times, 4 times in total; the last cleaning ensures that the supernatant is discarded; add 20. mu.L of nuclease-free water, suck and mix well 10 times, resuspend the magnetic beads.

3) Amplification of captured products

The post-capture PCR amplification reaction mixture was placed on ice (preparation method shown in Table 7), wherein the PCR enzyme reaction mixture had the following main components: MLtraHiFi DNA Polymerase, NaCl, Tris-HCl, MgCl2, DTT; flicking and uniformly mixing; the PCR tube was placed in a thermal cycler and the PCR program was run as shown in Table 8 below (hot lid temperature 105 ℃ C., reaction volume 50. mu.L); after the PCR program is finished, the PCR tube is instantaneously centrifuged and then placed on a magnetic frame, and after clarification, the supernatant is taken for purification; after purification, 25 mu L of nuclease-free water is added into each tube; mixing, standing at room temperature for 2 min; placing the sample tube into a magnetic frame, and standing for 1-2min until the liquid is clear; transfer 25. mu.L of supernatant to a new 1.5mL centrifuge tube.

TABLE 7 Capture product amplification System

Components	Volume (μ L)
		Trapping the product	20
PCR liquid enzyme reaction mixture 2	25
		Capture amplification primers	5
Total up to	50

TABLE 8 amplification conditions for captured products

4) Quality control of captured libraries

And (3) taking a sample library to perform fragment quality control on a 4150 instrument, wherein main fragments of the library are respectively consistent with the fragments before capture and have no obvious peaks of large fragments or small fragments, and if the requirements are met, the library is judged to be qualified, otherwise, the library is not qualified.

1.1.4 sequencing on machine

It is recommended that the library be selected on NextSeq 550Dx using a sequencing reagent with a sequencing read length of 300cycles (Paired-End Reads, 2X 150cycles), and that 1.5G be used for each sample, and that the sequencing time be about 27-28 hours

The initial amount optimization conditions and QC quality control parameters for library construction are shown in Table 9, and the mutation detection results are shown in Table 10.

TABLE 9 initial library creation optimization conditions and QC quality control

TABLE 10 initial amount optimization of mutation detection in pooling

As the initial amount of the library was increased, the mean concentration of the pre-PCR library was gradually decreased from 113.6 ng/. mu.L at 15ng to 82.5 ng/. mu.L at 50 ng. Under the condition that the sequencing depth is 20000X, when the initial amount of the library is 15ng, the UMI reliability, the complexity and the mean depth are all highest, and the replication is lowest. The mutation detection condition is analyzed by adopting a Vardict analysis method, and all mutations can be detected when the mutation frequency of 15ng library construction initial amount is 2% and 0.5%; 30ng and 50ng of the library was initially constructed at 0.5% and 0.3% mutation frequency, and all mutations were detected.

1.2 optimization of the concentration of each component in the library construction Process

And (3) optimizing the concentration of each component in the library building process on the basis of the experimental result of 1.1, and if no special description is provided, keeping the other operations and parameters consistent with the record of 1.1 except the following optimized parameters.

1.2.1 optimization of linker concentration in ligation reaction System

Keeping the experimental conditions of the tail end and the 3' plus A consistent with 1.1, setting the joint dosage to be 2.5 muL, 5 muL and 7.5 muL, setting the joint connection time to be 15min, keeping the pre-PCR reaction system and the reaction conditions unchanged, purifying by using AMPure magnetic beads after PCR amplification, and detecting the concentration by using Qubit 4.0. The subsequent experiments were performed according to the library building procedure, the on-machine sequencing procedure, and the sequencing analysis procedure described in 1.1, and the obtained sample information is shown in table 11.

Table 11 library construction front end connector usage optimization

From the data, the pre-PCR yield of the library is increased along with the increase of the joint dosage, when the joint dosage is increased to 7.5 mu L, the pre-PCR yield is the highest, but the Mean depth is lower, and the Mean depth is an important parameter influencing the subsequent low-frequency mutation detection, so that the joint dosage is finally selected to be 5.0 mu L by combining the pre-yield, the Mean depth, the UMI utilization rate and the library complexity, and the pre-PCR yield is 2070ng at the moment, so that the smooth operation of the subsequent experiment can be completely ensured.

1.2.2 optimization of primer concentration in the Pre-PCR reaction System (primers contain Index and Forward primers)

The reaction conditions for end repair and ligation were not changed, and the amounts of primers added were set to 1. mu.L, 2. mu.L, and 3. mu.L in pre-PCR amplification, 3 replicates for each condition, and the number of PCR cycles was 9. After PCR amplification, magnetic bead purification was performed, and the concentration was measured by using Qubit4, and the results are shown in Table 12.

Table 12: determination of Index and Forward concentrations in Pre-PCR amplification

As can be seen from the results in Table 12, when the amount of primer added is 1. mu.L, the total yield of pre-PCR does not reach 750ng required for hybridization; when the addition amount of the primer is 2 mu L, the yield is 1862-2058ng, which can meet the dosage of 2 hybridizations and is more suitable; when the amount of the primer added is 3. mu.L, the yield is 2660-2740ng, and when 750ng is adopted for hybridization, 1/4 which is the total yield, some molecules may be lost, affecting the lowest detection limit. Therefore, the optimal amount of primer addition during pre-PCR is 2. mu.L.

1.3 reaction conditions of the System

1.3.1 optimization of ligation time in ligation reactions

The experimental conditions of the tail end and the 3' plus A are unchanged, the joint usage amount is 5 mu L, the joint connection time is set to be 8min, 15min and 1h, the reaction system and the reaction conditions of pre-PCR are unchanged, AMPure magnetic beads are adopted for purification after PCR amplification, and the concentration is detected by Qubit 4.0. The subsequent experiment is carried out according to the established library establishing process, the computer-operated sequencing process and the sequencing analysis process, and finally the obtained sample information is shown in a table 13.

Table 13 library building front end connector connection time optimization

And (4) conclusion: the highest pre-PCR yield of the library is 1803.75ng when the joint connection time is 15min, when the joint connection time is prolonged to 1h, Mean depth and library complexity are reduced to a certain extent, the joint connection time is finally selected to be 15min by comprehensively considering the pre-PCR yield, the Mean depth, the UMI utilization rate and the library complexity, and at the moment, the pre-PCR yield is 1803.75ng, so that the smooth operation of subsequent experiments can be completely ensured.

1.4Pre-PCR reaction System optimization

1.4.1 screening optimization of the number of Pre cycles in the Pre-PCR reaction System

Selecting 2 clinical cfDNA samples, optimizing the cycle number of the pre cycles under the condition that other conditions are not changed, respectively setting 7cycles, 8cycles and 9cycles, repeating each condition for 2 times, and screening out the optimal cycle number of the pre cycles by comparing the pre-PCR yield, post-PCR yield, QC quality control parameters, mutation detection conditions and the like of the samples. The sample information and QC quality control parameters are shown in Table 14.

Table 14: optimization and QC quality control of pre cycles in clinical samples

As seen from the concentration of the pre-PCR library, the average concentration of the pre cycles 8 is the highest, and is about 282 ng/. mu.L, and the yield of the pre-PCR library is as high as 3948 ng; the mean concentration of pre cycles 7 was 172 ng/. mu.L and the mean concentration of pre cycles 9 was 232.5 ng/. mu.L. The duty increases gradually with increasing number of pre cycles, with an average duty of 84.38% for pre cycles 7, 84.47% for pre cycles 8 and 84.8% for pre cycles 9.

Finally, the number of the pre cycles is determined to be 8 according to parameters such as pre-PCR yield and replication.

1.4.2 optimization of annealing temperature during Pre-PCR

The reaction conditions of end repair and ligation were not changed, and in pre-PCR amplification, the annealing temperatures were set to 56 ℃, 60 ℃ and 63 ℃ respectively, 3 replicates were set for each condition, and the number of PCR cycles was 8. After PCR amplification, magnetic bead purification was performed, and the concentration was measured by using Qubit4, and the results are shown in Table 15.

TABLE 15 determination of annealing temperature in pre-PCR amplification

As can be seen from the results in Table 13, the total yield of pre-PCR was between 1456-1694g when the annealing temperature was 56 ℃; when the annealing temperature is 60 ℃, the yield is between 1296.4 and 1554 ng; when the annealing temperature is 63 ℃, the yield is between 1512 and 1736 ng. The three have no obvious difference in PCR product yield, so the annealing temperature on the manufacturer's instruction is finally selected, namely 60 ℃ is selected as the annealing temperature of the pre-PCR process.

As described above, the annealing temperature of 60 ℃ is the optimum condition in the pre-PCR process.

1.5 hybridization Process

1.5.1 hybridization initiation amount optimization

Under the condition that other conditions are not changed, the initial hybridization amount in the hybridization process is optimized, 3 initial hybridization amounts of 500ng, 750ng and 1000ng are respectively set, and the optimal initial hybridization amount is screened out according to the comprehensive consideration of the parameters such as post-PCR library yield, QC quality control parameters, mutation detection and the like. The sample information and QC quality control are shown in Table 16, and the mutation detection is shown in Table 17.

TABLE 16 conditions for optimization of initial hybridization amount and QC quality control

TABLE 17 mutation detection for hybridization initiation optimization

The data in Table 16 show that the post-PCR library concentration gradually increased with increasing initial amount of hybridization. When the initial hybridization amount is 500ng, mean depth, UMI availability and compatibility are all the highest, and replication is the lowest. As can be seen from the data of the detection of mutations in Table 17, all of the three initial hybridization amounts of 500ng, 750ng and 1000ng were detected, and both of the analysis methods of mutect2 and vardict detected no difference in the number of the initial hybridization amounts of 500ng, 750ng and 1000ng and the frequency of the mutations.

1.5.2 hybridization elution temperature optimization

The end of the front end of the library was repaired and the experimental conditions of 3' plus a, linker ligation, pre-PCR were unchanged, the initial total amount of the sample was 750ng during hybridization capture, the hybridization program and capture program were unchanged, the temperature was set at 65 ℃ and 70 ℃ during Wash Buffer2 elution, three technical iterations were performed, the subsequent post-PCR, bead purification and on-machine sequencing experimental conditions were unchanged, and the finally obtained sample information and on-machine sequencing information are shown in table 18.

Table 18: hybridization elution temperature optimization

According to the data shown in Table 18, at a hybridization elution temperature of 70 ℃, the library concentration was in the normal range, and the capture efficiency, Mean depth, UMI utilization rate and library complexity were all higher than 65 ℃, so that it was finally determined that the hybridization elution temperature was 70 ℃.

1.5.3 optimization of the number of cycles in post-PCR

Other reaction conditions were not changed, and only in the post-PCR amplification process, the number of cycles was set to 12, 14 and 16, respectively, 3 replicates were set for each condition, and the PCR annealing temperature was 60 ℃. After PCR amplification, magnetic bead purification was performed, the concentration was determined using the Qubit4.0, and the library was sequenced using a HiseqX sequencer, with the results shown in tables 19 and 20.

TABLE 19 determination of annealing temperature in post-PCR amplification-library quality inspection

TABLE 20 determination of annealing temperature in post-PCR amplification-sequencing QC

TABLE 21 determination of annealing temperature in post-PCR amplification-detection of mutations (theoretical mutation Rate 2% at all sites)

And (4) conclusion: as can be seen from the results in Table 19, the total yield of post-PCR was between 41-51ng when the number of cycles was 12; when the number of the circulation is 14, the yield is between 139.5 and 172 ng; when the number of cycles is 16, the yield is between 505 and 640 ng. The yield at 16 cycles is higher than the yields at 12 and 14 cycles in terms of PCR product yield; as can be seen from the results in tables 20 and 21, the QC results and the detection results of mutations of the library at 14 cycles were superior to those of the QC results and the detection results of mutations of the library at 12 and 16 cycles, and finally 14 cycles were selected as the cycle number of the post-PCR process.

1.5.4 optimization of hybridization sample number

Other reaction conditions were not changed, and only in the hybridization process, the number of hybridization samples was set to 1 (single hybridization), 2, 3 and 4 (all pooling), 2 replicates were set for each condition, and the amount of the probe was 2. mu.L. The library was assayed for concentration using Qubit4.0 and sequenced using a HiseqX sequencer, with results in tables 22, 23 and 24.

Table 22: determination of hybridization sample number-library quality inspection

TABLE 23 determination of hybridization sample number-sequencing QC

TABLE 24 determination of hybridization sample number-detection of mutation (theoretical mutation Rate 2% at all sites)

From the above data, it can be seen that when the number of hybridization samples is 2 and 4, the capture efficiency, UMI utilization ratio, library complexity and redundancy in library QC are all superior to other conditions, and are also the best in mutation detection, and considering that the cost is lower and the experimental operation is more concise when the number of hybridization samples is 4, the number of hybridization samples is determined to be 4.

Example 2 kit and method of use validation

In this example, the kit and the method obtained by the screening in example 1 were used to verify different DNA initial amounts of 15ng, 20ng, 30ng and 50ng and different mutation frequencies of 0.5% and 0.3%, wherein the sequencing depth was 20000X, and the specific experimental procedures were as follows:

1.1 nucleic acid extraction

1) Sample processing

2) Extraction of DNA

1.2 library construction

1) Tip repair

Melting the terminal repair enzyme and the terminal repair buffer solution on ice, preparing the following terminal repair reaction mixed solution on ice, and mixing uniformly by vortex oscillation, wherein each part of reaction system is shown in table 25:

table 25: end-repair reaction system

Components	Add volume (μ L)
		cfDNA samples	X3
End repair enzyme	10
		End repair buffer	5
Nuclease-free water	35-X3
		Total up to	50

2) Joint connection

Preparing a joint connection mixed solution shown in the table 26 on ice, and gently blowing, sucking or flicking to mix uniformly; adding 45 mu L of joint connection mixed liquid into each tube of sample, softly blowing and sucking or softly flicking and uniformly mixing, and operating the program: 20 ℃ 15min, volume 100. mu.L (PCR instrument without hot lid). After the process is finished, the next process is carried out.

Table 26: linker ligation reaction system

3) Purification after linker attachment

And (3) purifying the amplified adaptor connection product by using a purified magnetic bead AMPure XP Beads, adding 21 mu L of nuclease-free water for elution after purification, and transferring 21 mu L of purified product to a new PCR tube.

4) Library amplification

Melting the PCR enzyme reaction solution 1 and the amplification primer 1 on ice, and uniformly mixing by vortex oscillation; preparing PCR reaction mixed solution for library amplification before capture on ice, and mixing uniformly by vortex oscillation, wherein the library amplification system of each person is shown in table 27; simultaneously arranging different label primer numbers for each sample, and adding 2 mu L of arranged label primers into each PCR tube; after flick and mixing, the procedure was run and the PCR amplification reaction procedure is shown in table 28.

Table 27: pre-library amplification reaction system

Components	Volume (μ L)
		PCR enzyme reaction solution 1	25
Amplification primer 1 (10. mu.M)	2
		Label primer (10. mu.M)	2
Linker purified product	21
		Total up to	50

TABLE 28 Pre-library amplification reaction conditions

5) Purification after library amplification

Purifying the amplified library product by using purified magnetic Beads AMPure XP Beads (BECKMAN CO mu LTER, A63882, lot 1776140), adding 15 mu L of nuclease-free water for elution after purification, and transferring 15 mu L of the purified product to a new 1.5mL centrifuge tube to obtain the sample DNA pre-library.

6) Library quality control

1.3 hybrid Capture

1) Hybridization of

Respectively selecting single pre-libraries consisting of cfDNA of 4 different label primers to mix, wherein 750ng of each pre-hybridization library and 4 pre-libraries are taken, and the total amount is 3 mu g; if the number of samples is less than 4, the number of samples captured by mixed hybridization can be reduced, and the positive control and the negative control can be captured by mixed hybridization with the library samples.

TABLE 29 dilution of 25% blocking agent 2

TABLE 30 hybridization reaction System

Components	Volume (μ L)
		Hybridization buffer	6
Nuclease-free water	1
		Probe needle	2
25% blocker 2 diluent	2
		Total up to	11

2) Capture

Taking down the hybridization reaction tube, placing at room temperature, immediately transferring all the liquid into a magnetic bead containing 200 mu L of washed magnetic beads, slowly blowing and sucking for 5-8 times, and uniformly mixing; and covering a tube cover, and placing in a constant-temperature oscillation blending instrument to blend at room temperature of 1500rpm for 30 min.

3) Amplification of captured products

Preparing a captured PCR amplification reaction mixed solution on ice (the preparation method is shown in table 31), and gently and uniformly mixing; the PCR tube was placed in a thermal cycler and the PCR program was run as shown in Table 32 below (hot lid temperature 105 ℃ C., reaction volume 50. mu.L); after the PCR program is finished, the PCR tube is instantaneously centrifuged and then placed on a magnetic frame, and after clarification, the supernatant is taken for purification; after purification, 21 mu L of nuclease-free water is added into each tube; mixing, standing at room temperature for 2 min; placing the sample tube into a magnetic frame, and standing for 1-2min until the liquid is clear; transfer 20. mu.L of supernatant to a new 1.5mL centrifuge tube.

TABLE 31 Capture product amplification System

TABLE 32 amplification conditions for captured products

4) Quality control of captured libraries

Taking a sample library or a reference library, and performing fragment quality control on a 4150 instrument by using Agilent, wherein the main fragments of the library are respectively consistent with the fragments before capture and have no obvious peak of large fragments or small fragments, and the library is judged to be qualified if the requirements are met, otherwise, the library is not qualified.

1.4 sequencing on machine

It is recommended that the library be performed on NextSeq 550Dx using sequencing reagents with a sequencing read length of 300cycles (Paired-End Reads, 2X 150cycles), and that 1.5G be used for each sample, with a sequencing duration of about 27-28 hours.

1.5 analysis of results

The specific experimental results show that as shown in the following table 33, when the mutation frequency is 0.5%, the four input amounts of 15ng, 20ng, 30ng and 50ng can be correctly detected, and the sensitivity reaches 100%; when the mutation frequency is 0.3%, 15ng and 20ng can not be completely detected, the sensitivity is 87.5% and 93.75%, respectively, and the input amount of 30ng and 50ng can be correctly detected, and the sensitivity reaches 100%.

Table 33: comparison of different input quantities

Example 3 assay Rate validation of kits

The experiment was performed on the basis of examples 1 and 2, and this embodiment used the present kit described in example 2 and agilent SureSelect XTHS kit to perform hybrid capture library construction using 0.5% and 0.2% of Horizon HD780 standard (mock cfDNA) as test samples, with the initial library construction amount being 30ng and the initial hybridization amount being 750ng, and the rest of the experimental conditions refer to example 2, comparing the QC 780 quality control and mutation detection of 2 library construction reagents in 0.5% and 0.2% of Horizon HD standard. The results show that as shown in table 34, when the mutation ratio is 0.5%, the QC quality control result shows that the average depth of the kit after de-duplication is significantly lower than agilent SureSelect XTHS, the capture uniformity is higher than agilent SureSelect XTHS, the library complexity is higher than agilent SureSelect XTHS, and the redundancy is lower than agilent SureSelect XTHS. The detection result of the mutation shows that the sensitivity of the kit is 100 percent, and the detection sensitivity of the Agilent SureSelect XTHS is 93.75 percent. When the mutation ratio is 0.2%, QC quality control results show that the average depth of the kit after de-duplication is obviously lower than Agilent SureSelect XTHS, the capture uniformity is higher than Agilent SureSelect XTHS, the library complexity is higher than Agilent SureSelect XTHS, and the redundancy is lower than Agilent SureSelect XTHS. The detection result of the mutation is shown in FIG. 2, the sensitivity of the kit is 81.25%, and the detection sensitivity of Agilent SureSelect XTHS is 56.25%.

Table 34: QC comparison for different kit detection

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. A method for constructing a high-throughput sequencing library is characterized by comprising the following steps: utilizing cfDNA to construct a PCR amplification system, and carrying out PCR amplification treatment to obtain a PCR amplification library, wherein based on 50 mu L of the PCR amplification system, the using amount of a label primer is 1-3 mu L, the using amount of an amplification primer 1 is 1-3 mu L, the using amount of a PCR enzyme is 23-27 mu L, and the using amount of the purified cfDNA is 18-24 mu L.

2. The method according to claim 1, wherein the conditions of the PCR amplification process are, in order: at 98 deg.C for 2-4 min; (98 ℃, 15-25s, 58-63 ℃, 28-32s, 67-78 ℃, 25-35s, 2-4 steps circulating for 8 times); 72 ℃ for 5 min; preserving at 4 ℃;

optionally, the cfDNA is subjected to a first magnetic bead purification treatment in advance;

optionally, subjecting the cfDNA to a first magnetic bead purification process using 0.8 × magnetic beads;

optionally, the cfDNA is from a sample to be tested;

optionally, the content of the cfDNA obtained from the sample to be tested is 15-30 ng;

optionally, the sample to be tested comprises plasma.

3. The method according to claim 1 or 2, wherein when the first magnetic bead purification process is performed, the cfDNA obtained from the sample to be tested is subjected to a terminal repair process and a linker ligation process in advance in this order.

4. The method according to any one of claims 1 to 3, wherein the amount of the terminal repairing enzyme is 3 to 8. mu.L, and the amount of the terminal repairing buffer is 8 to 12. mu.L, based on 50. mu.L of the system for the terminal repairing treatment;

optionally, the conditions of the end repairing treatment are as follows: 18-23 ℃ and 27-33 min; 62-68 ℃; 28-33 min.

5. The method according to any one of claims 1 to 4, wherein the amount of the end repair product is 45 to 55. mu.L, the amount of the linker is 2.5 to 7.5. mu.L, the amount of the ligase is 8 to 13. mu.L, and the amount of the ligation buffer is 18 to 22. mu.L, based on 100. mu.L of the linker-ligated system.

6. The method of claim 1, further comprising: performing second magnetic bead purification treatment on the PCR amplification library to obtain a DNA pre-sequencing library;

optionally, the PCR amplification library is subjected to a second magnetic bead purification process using 1 × magnetic beads.

7. The method of any one of claims 1-6, further comprising:

1) mixing a plurality of individual DNA prediction prosequences;

2) preparing a hybridization reaction system by using the product obtained in the step 1), and performing hybridization treatment;

3) performing streptavidin magnetic bead capture treatment on the hybridization product obtained in the step 2) so as to obtain a capture product;

4) and amplifying the capture product and purifying the capture product by using a third magnetic bead so as to obtain the high-throughput sequencing library, wherein the high-throughput sequencing library comprises DNA fragments with mutation frequency of not less than 0.3% in cfDNA of the sample.

8. The method of claim 7, wherein the average sequencing depth of the library for each sample in the high throughput sequencing library is 18000-25000;

optionally, the mutation comprises a point mutation, insertion, deletion;

optionally, the plurality of sample-individual DNA sequencing libraries comprise different tag primers;

preferably, the number of DNA sequencing libraries of the sample does not exceed 4.

9. The method according to claim 7, wherein the amount of the template to be hybridized is 1 to 3 μ g, the amount of the blocker 1 is 3 to 7 μ L, the amount of the hybridization probe is 1 to 3 μ L, the amount of the diluent for the blocker 2 is 1 to 3 μ L, and the amount of the hybridization buffer is 5 to 7 μ L, based on 30 μ L of the hybridization reaction system.

10. The method according to claim 7, wherein the hybridization treatment is performed under the following conditions: 95 deg.C, 5min, 65 deg.C, 10min, 65 deg.C, 1 min; (65 deg.C, 1 min; 37 deg.C, 3s, cycle 60 times), and preserving at 65 deg.C.

11. The method according to claim 7, wherein the streptavidin magnetic beads are washed with a binding buffer in advance;

optionally, the capture 20-40min later with washing buffer washing.

12. The method according to claim 7, wherein the amount of the capture product is 22 to 27. mu.L, the amount of the capture amplification primer is 3 to 8. mu.L, and the amount of the PCR enzyme reaction solution 2 is 22 to 27. mu.L, based on 50. mu.L of the capture amplification system.

13. The method of claim 7, wherein the conditions for post-capture amplification are, in order: at 98 deg.C for 2-4 min; (98 ℃, 15-25s, 58-63 ℃, 28-32s, 67-78 ℃, 25-35s, 2-4 steps circulating for 14 times); preserving at 72 deg.C for 5min and 4 deg.C.

14. A high throughput sequencing library constructed according to the method of any one of claims 1 to 13.

15. A method for detecting low frequency DNA mutations, comprising:

1) constructing a high throughput sequencing library using the method of any one of claims 1-13;

2) performing on-machine sequencing on the high-throughput sequencing library to obtain low-frequency mutated DNA sites;

optionally, DNA fragments with a mutation frequency of not less than 0.3% in the genomic DNA of the sample are included in the high throughput sequencing library.

16. A kit, comprising at least one of: the kit comprises probes, primers, a DNA extraction reagent, a nucleic acid purification reagent, a terminal repair enzyme, a terminal repair buffer solution, a joint, a ligase, a connection buffer solution, a PCR reaction solution, a blocking agent, a hybridization buffer solution, a binding buffer solution and a washing buffer solution;

optionally, the nucleic acid purification reagent comprises purified magnetic beads.