CN109652513B

CN109652513B - Method and kit for accurately detecting individual mutation of liquid biopsy based on second-generation sequencing technology

Info

Publication number: CN109652513B
Application number: CN201910136422.4A
Authority: CN
Inventors: 郎继东; 田埂
Original assignee: Geneis Beijing Co ltd
Current assignee: Geneis Beijing Co ltd
Priority date: 2019-02-25
Filing date: 2019-02-25
Publication date: 2022-08-23
Anticipated expiration: 2039-02-25
Also published as: CN109652513A

Abstract

The invention discloses a method for accurately detecting individual mutation of liquid biopsy based on a next-generation sequencing technology, which comprises the following steps of (1) classifying cell-free DNA according to the length of the cell-free DNA in a liquid sample collected from a subject to obtain more than two cell-free DNA groups with different length ranges; (2) selecting a desired cell-free DNA group from two or more cell-free DNA groups having different length ranges, and analyzing the gene mutation in the desired cell-free DNA group; (3) and (3) detecting the mutation of the liquid biopsy individual according to the gene mutation situation obtained in the step (2). The method provided by the invention not only improves the accuracy of detecting ctDNA mutation, reduces the false positive rate and false negative rate detected by a conventional experimental method, but also provides a more effective means for early screening of tumors or mutation monitoring and control of early cancer patients.

Description

Method and kit for accurately detecting individual mutation of liquid biopsy based on second-generation sequencing technology

Technical Field

The invention relates to the field of gene detection, in particular to a method and a kit for accurately detecting individual mutation of liquid biopsy based on a second-generation sequencing technology.

Background

Cell free DNA (cfDNA) generally refers to a small segment of double-stranded DNA with a peak length of about 167bp, often found in peripheral blood or other tissue fluids, and is primarily derived from apoptotic degradation of normal cells themselves. Circulating tumor DNA (ctDNA) refers to DNA fragments derived from tumor that constantly flow in the human blood circulation system, and these fragments carry much information about tumors, including gene mutations, deletions, insertions, rearrangements, copy number abnormalities, and methylation. The information can be used for detecting early diagnosis, progression process, prognosis judgment and personalized medication guidance of the tumor, and the importance of the information for detecting ctDNA to clinic can be seen.

The existing ctDNA mutation detection methods are many, but the second-generation sequencing-based method is most applied and the detection means are most abundant. The most important technical implementation means in the method based on next generation sequencing are two: one is a target region capture or amplification method for high depth sequencing, and the other is a library-building sequencing method for adding molecular barcodes or molecular labels. The two methods are experimentally based on the conventional second-generation sequencing library construction method, and not only can effectively detect high-frequency mutation of ctDNA of a sample, but also can effectively detect low-frequency or even ultra-low-frequency mutation (> ═ 0.1%). Nevertheless, there are still major problems in practical applications based on such results. Therefore, there is still a need for more accurate detection methods based on second-generation sequencing.

Disclosure of Invention

In order to solve at least some technical problems in the prior art, the present invention has found that there is a certain rule for ctDNA changes at different stages of a disease, and has been accomplished at least partially based on this rule. Specifically, the present invention includes the following.

In a first aspect of the present invention, there is provided a method for accurately detecting mutations in a liquid biopsy individual based on a next-generation sequencing technique, comprising the steps of:

(1) classifying cell-free DNA in a liquid sample collected from a subject according to the length of the cell-free DNA to obtain two or more groups of cell-free DNA having different length ranges;

(2) selecting a desired cell-free DNA group from the two or more cell-free DNA groups having different length ranges, and analyzing the gene mutation status in the desired cell-free DNA group;

(3) detecting the mutation of the liquid biopsy individual according to the gene mutation situation obtained in the step (2).

In certain embodiments, the liquid sample in step (1) is selected from the group consisting of blood, saliva, urine, interstitial fluid, and isolates thereof.

In certain embodiments, the cell-free DNA is classified in step (1) using gel electrophoresis, or the sequence length is obtained by sequencing and classification is performed according to the obtained length.

In certain embodiments, in step (1), the cell-free DNA in the liquid sample collected from the subject is classified according to its length, resulting in a set of cell-free DNA having a first length range and a set of cell-free DNA having a second length range, and the first length range is less than the second length range.

In certain embodiments, in step (2):

if the liquid sample is from a subject early in the disease, then treating the set of cell-free DNA having the second range of lengths as the desired set of cell-free DNA;

if the liquid sample is from a subject with advanced disease, the set of cell-free DNA having the first length range is designated as the desired set of cell-free DNA.

In certain embodiments, in step (2):

if the assay is aimed at an early screening visit for the subject, then the set of cell-free DNA having the second length range is used as the desired set of cell-free DNA;

if the assay is aimed at providing a medication guide or a drug resistance assay, then the set of cell-free DNA having the first length range is used as the desired set of cell-free DNA;

if the detection is aimed at providing dynamic monitoring of the mutation, both the set of cell-free DNA having the first length range and the set of cell-free DNA having the second length range are used as the desired set of cell-free DNA, and the gene mutation in the two sets of cell-free DNA are analyzed separately.

In certain embodiments, the first length range is 130-160bp and the second length range is 161-230 bp.

In certain embodiments, step (1) comprises:

(1-1) extracting cell-free DNA in the liquid sample;

(1-2) constructing a gene library using the cell-free DNA;

(1-3) classifying cell-free DNA in the obtained gene library by gel electrophoresis.

In a second aspect of the present invention, there is provided a kit for accurately detecting mutation in a liquid biopsy individual based on a second generation sequencing technology, which comprises:

(1) a reagent for extracting cell-free DNA from a liquid sample from a subject;

(2) (ii) an agent that separates the cell-free DNA into two or more sets of cell-free DNA having different ranges of lengths;

(3) reagents for analyzing cell-free DNA for genetic mutations; and

(4) instructions for use in which directions to carry out the method according to any one of claims 1 to 8 are taught.

Preferably, the kit of the present invention further comprises a reagent for constructing a gene library.

The invention establishes a method for accurately detecting ctDNA mutation based on a second-generation sequencing technology, which not only improves the accuracy of detecting ctDNA mutation, reduces the false positive rate and the false negative rate of conventional experimental methods, but also provides a more effective detection method for early screening of tumors or mutation monitoring and control of early cancer patients.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.

The term "Cell free DNA" (cfDNA) as used herein refers to a short piece of double-stranded DNA that exists in a free form in a body fluid (e.g., blood). Typically, cfDNA is released from cells of the body (including normal cells such as leukocytes, diseased cells such as tumor cells, or senescent cells). The length of cfDNA is typically 400bp or less, with a distribution of between 130 and 180bp being predominant, and typically it peaks at about 167 bp.

The term "circulating tumor DNA" (abbreviated ctDNA) as used herein refers to a tumor-derived DNA fragment present in a body fluid such as blood, and the fragment carries much information about tumors, including gene mutation, deletion, insertion, rearrangement, copy number abnormality, methylation, etc. The information can be used for detecting early diagnosis, progress process, prognosis judgment and personalized medication guidance of the tumor. In general, the length of circulating tumor DNA is within a range of 400bp or less.

Research into diagnosing or predicting diseases such as tumors by detecting ctDNA has been receiving increasing attention. However, current research is heavily focused on the improvement of detection sensitivity. For example, mutations with a frequency as low as > -0.1% can be effectively detected by the existing second-generation sequencing technology, and although high sensitivity is favorable for obtaining low-concentration mutations, the content and morphological distribution of ctDNA in organisms are continuously changed along with the progress of diseases, and the change is not favorable for obtaining more practical diagnosis or detection results, and even generates false negative or false positive for disease diagnosis.

The inventors of the present invention found that the total length of the ctDNA fragment of the ultra-low frequency mutation (0.6%) was longer than the corresponding fragment length of the wild-type cfDNA. This is not the same as the previous study results. For example, Jay Shend in 2016 showed that the Fragment Length (132-145 bp) of the mutated ctDNA is shorter than that (165bp) of the wild-type cfDNA (Fragment Length of Circulating Tumor DNA. PLoS Genet.2016Jul 18; 12(7)), and digital microdroplet PCR (digital repeat PCR) was used to verify that different library sizes have certain influence on the mutation frequency, such as 361bp, 345bp, 335bp, 320bp, 307bp and 292bp, and the corresponding mutation frequencies are 0%, 0.03%, 1.6%, 3.0%, 0.09% and 0%, respectively. This is again demonstrated, for example, in an article published in 2018 by Moulire F (Enhanced detection of circulating tumor DNA by fragment size analysis. Sci Transl Med.2018Nov 7; 10 (466)). Based on these conclusions, the inventors propose a solution to enhance the detection of ctDNA based on the analysis of fragment sizes, which can complement or provide some more advanced research methods of cfDNA. Specifically, the present invention includes the following.

[ method for accurately detecting mutation in liquid biopsy individuals based on second-generation sequencing technology ]

In a first aspect of the present invention, a method for accurately detecting a mutation in a liquid biopsy individual based on a second generation sequencing technology is provided, and the method of the present invention selectively detects ctDNA with a specific length according to different situations, thereby improving or enhancing the accuracy of detection. Specifically, the method of the present invention comprises at least the steps of:

(1) classifying cell-free DNA in a fluid sample collected from a subject according to its length, resulting in two or more sets of cell-free DNA having different ranges of lengths.

(2) A desired cell-free DNA group is selected from two or more cell-free DNA groups having different length ranges, and the gene mutation in the desired cell-free DNA group is analyzed.

(3) Detecting the mutation of the liquid biopsy individual according to the gene mutation situation.

Step (1)

The step (1) of the present invention is to classify cell-free DNA in a liquid sample collected from a subject according to the length of the cell-free DNA, resulting in two or more groups of cell-free DNA having different length ranges.

The subject of the present invention can be any subject to be detected, including mammals as well as humans. The subject may be a healthy individual, or may be an individual suspected of having or diagnosed with a disease to be diagnosed, for example, an individual to be screened, an individual diagnosed at an early stage, an individual with a middle or late stage of a disease, or the like.

The liquid sample of the present invention may be any body fluid from an individual, examples of which include, but are not limited to, blood, saliva, urine, and interstitial fluid, as well as isolates from such body fluids. The isolate may be a single substance obtained after separation, or may be a mixture of a plurality of substances. For example, in the case where the body fluid is blood, the isolate includes plasma, serum, or the like.

The classification method of the present invention may employ any method known in the art, examples of which include, but are not limited to, gel electrophoresis, or a classification method based on sequencing results. Examples of gel electrophoresis include, but are not limited to, polyacrylamide gel electrophoresis (PAGE), agarose gel electrophoresis, and the like. The conditions and specific procedures for gel electrophoresis are known in the art and can be found, for example, in the publications of molecular cloning, laboratory Manual, fourth edition, et al, Cold spring harbor. The classification method based on the sequencing result comprises a step of extracting ctDNA in body fluid, a step of constructing a ctDNA library, a step of sequencing the library, a step of classifying fragments based on the length obtained by sequencing and the like.

In the present invention, two or more sets of cell-free DNA having different lengths can be obtained by classification. The number of sets of cell-free DNA having different lengths is not particularly limited, and may be 2 to 50, preferably 2 to 10, and more preferably 2 to 5. In certain embodiments, the two sets of cell-free DNA are obtained by sorting, i.e., a set of cell-free DNA having a first range of lengths and a set of cell-free DNA having a second range of lengths, and the first range of lengths is less than the second range of lengths. In certain embodiments, the three sets of cell-free DNA are obtained by classification, i.e., a set of cell-free DNA having a first length range, a set of cell-free DNA having a second length range, and a set of cell-free DNA having a third length range, and the first length range is less than the second length range, and the second length range is less than the third length range.

In the present invention, the span of the length range (the difference between the upper limit value and the lower limit value of the length range) is not particularly limited, and can be set by a person skilled in the art depending on the type of body fluid, the type of disease, and the type of individual. Typically, the length range spans 1 to 350, preferably 1 to 300, more preferably 1 to 200. The span of the length range may be the same or different in different sets of cell-free DNA. For example, in the case that the span of the first length range is 1, the span of the second length range may be 1, or may be any length other than 1, such as 5, 8, 10, etc. The length range of the present invention is also not particularly limited, but is usually 50 to 400bp, preferably 60 to 350bp, more preferably 80 to 300bp, further preferably 100 and 250bp, etc. In certain embodiments, the length ranges of the invention are 130-160bp or 161-230 bp.

In order to improve the classification effect, the step (1) may include a plurality of sub-steps. For example, the following three substeps may be included:

(1-1) extracting cell-free DNA in the liquid sample;

(1-2) constructing a sequencing library by using cell-free DNA;

(1-3) classifying the cell-free DNA in the library by gel electrophoresis.

The operating conditions for these substeps are known in the art and can be found, for example, in publications such as molecular cloning, a fourth edition of the Experimental guidelines for molecular cloning, from Cold spring harbor.

Step (2)

The step (2) of the present invention is to select a desired cell-free DNA group from two or more cell-free DNA groups having different length ranges and analyze the gene mutation in the desired cell-free DNA group.

In the present invention, the desired cell-free DNA group is a cell-free DNA group selected/screened/selected from the results obtained from the classification for further analysis or detection. That is, a portion of a specific length is selected as necessary after the classification, and a fragment of a specific length which adversely affects the subsequent analysis or detection is excluded from the detection. This classification is not a traditional ctDNA purification. The desired cell-free DNA set may vary depending on the purpose of the assay or depending on the type of the subject to be assayed. In general, in cases where the probability of mutation occurrence is high, a cell-free DNA group having a smaller length is selected. In contrast, in the case where the probability of occurrence of mutation is small, a cell-free DNA group having a large length is selected. For example, in the case where the cell-free DNA group having the first length range and the cell-free DNA group having the second length range are obtained by classification, and the first length range is smaller than the second length range, if the liquid sample is from a subject in an early stage of a disease, the cell-free DNA group having the second length range is regarded as the desired cell-free DNA group; if the liquid sample is from a subject with advanced disease, the set of cell-free DNA having the first length range is designated as the desired set of cell-free DNA. Under the same circumstances, if the detection is aimed at an early screening visit for the subject, the cell-free DNA group having the second length range is taken as the desired cell-free DNA group; if the assay is aimed at providing a medication guide or a drug resistance assay, then the set of cell-free DNA having the first length range is used as the desired set of cell-free DNA; if the detection is aimed at providing dynamic monitoring of the mutation, both the cell-free DNA having the first length range and the set of cell-free DNA having the second length range are taken as the desired set of cell-free DNA.

Step (2) of the present invention further comprises analyzing the gene mutation status within the desired cell-free DNA group. Wherein the genetic mutation conditions include, but are not limited to, the type of genetic mutation (e.g., SNP mutation, deletion mutation Indel, or a combination of both, etc.), the frequency of genetic mutation (e.g., 30%, 1%, etc.). The specific analysis method is not particularly limited, and may include, for example, a step of aligning the sequencing result with a reference sequence. In certain embodiments, the method of analyzing a gene mutation comprises comparing the sequenced sequence to a known sequence, such as a human genome sequence (Hg 19:http://hgdownload.soe.ucsc.edu/ goldenPath/hg19/bigZips/) And (5) carrying out comparison.

Step (3)

Step (3) of the present invention is to detect the mutation of the liquid biopsy individual (also referred to as a subject) according to the gene mutation status obtained in step (2). In the present invention, the gene mutation situation obtained in step (2), for example, the gene mutation frequency, may be directly used as a result of detecting a mutation in the liquid biopsy individual, and the mutation in the liquid biopsy individual may also be predicted or judged based on the gene mutation situation obtained in step (2).

It is understood by those skilled in the art that the order of the above steps is not particularly limited as long as the object of the present invention can be achieved. In addition, other steps or operations may be included before or after steps (1) - (3) above, or between any of these steps, for example, to further optimize and/or improve the methods of the present invention.

In an exemplary embodiment, the method of the present invention comprises the steps of:

1. collecting a liquid biopsy sample, such as peripheral blood, saliva, urine, etc.;

2. extracting free DNA in a sample;

3. constructing a library by using a library construction kit;

4. separating and purifying by agarose gel electrophoresis, selecting but not limited to, selecting libraries of target bands of 130-160bp and 161-230 bp;

5. hybrid capture or direct sequencing according to step 6;

6. performing double-end sequencing by using but not limited to an Illumina sequencer to obtain sequencing data;

7. removing low quality and connector pollution sequences of the original sequencing data to obtain processed data to be analyzed;

8. the data obtained in 7 were aligned to the human reference genome (Hg 19: http:// hgdownload. soe. ucsc. edu/goldenPath/Hg19/bigZips /);

9. extracting the results of the comparison of the two-terminal sequencing sequences, and classifying according to the comparison distance positions 130-160bp and 161-230bp of the two-terminal sequencing sequences to obtain a set A, B;

10. performing mutation detection on the sequence sets of the set A, B respectively;

11. comparing the mutation results of the set A, B, and screening the results according to the type of sample or purpose of detection: (II) selecting the mutation results of set B for prognosis if the sample type is early stage patient (e.g., disease progression stage I or stage II) or the detection is an early screening diagnosis for healthy persons; if the sample type is a late stage patient (for example, the disease progresses to the III stage and above) or the detection purpose is medication guide or drug resistance analysis and the like, selecting the mutation result of the set A for prejudging; if the detection is for dynamic monitoring of mutations or otherwise, the results of set A, B are subjected to a binding prediction.

[ reagent kit for accurately detecting individual mutation of liquid biopsy based on second-generation sequencing technology ]

In a second aspect of the invention, a kit for accurately detecting mutations in a liquid biopsy based on a second generation sequencing technology is provided. The kit at least comprises the following components:

(1) reagents for extracting cell-free DNA from a liquid sample from a subject;

(2) a reagent that separates cell-free DNA into two or more groups of cell-free DNA having different length ranges;

(3) reagents for analyzing cell-free DNA for genetic mutations; and

(4) instructions for carrying out the method of the first aspect of the invention are taught.

Reagent (1)

In the kit of the present invention, the reagent (1) is used for extracting cell-free DNA from a liquid sample from a subject. Reagent (1) is typically a combination of components of a plurality of reagents, each of which may be a single component or a mixture of a plurality of different components. In exemplary embodiments, the reagent (1) of the present invention includes a buffer, proteinase K, and the like. Examples of the buffer include buffer ACL, buffer ACB, buffer ACW, buffer AVE, and the like. These buffers are known in the art.

Reagent (2)

In the kit of the present invention, the reagent (2) is used for separating cell-free DNA into two or more groups of cell-free DNA having different length ranges. In certain embodiments, the agent (2) of the invention comprises an agent for constructing a sequencing library. In certain embodiments, the reagent (2) of the present invention comprises a reagent for gel electrophoresis. Preferably, the gel in gel electrophoresis is a polyacrylamide gel or an agarose gel. The concentration of the gel during electrophoresis is not particularly limited, and is usually 0.5 to 3% by weight. The concentration can be freely selected by those skilled in the art according to the need and various factors (low concentration gel is fragile, and high concentration gel may make DNA bands with similar molecular size not easy to distinguish, causing band deletion phenomenon). For example, low concentrations are used to perform electrophoresis of large fragments of nucleic acids. High concentration gels are typically used for small fragment analysis.

Reagent (3)

In the kit of the present invention, the reagent (3) is used for analyzing gene mutation in cell-free DNA. The reagent (3) may use a reagent known in the art, or a combination of a plurality of reagents. Preferably, the reagent (3) includes a reagent used in the next-generation sequencing, examples of which include a DNA polymerase required for carrying out PCR, various kinds of dNTPs and ions such as Mg ²⁺ And the like.

Instruction manual

The kit of the invention further comprises instructions for use. Wherein instructions, directions or teachings for carrying out the method according to the first aspect of the invention or for using the kit according to the invention are given or taught in the instructions for use.

It is to be noted that the various agents or components of the present invention are known to those skilled in the art and can be readily obtained from publications such as molecular cloning, a laboratory manual, fourth edition, and the like, of cold spring harbor.

Examples

11 blood samples of known positive mutations (see Table 1 below for sample cases) were selected for detection of point mutations by the experimental and data analysis methods of the present invention.

Specifically, 1 example Sample1(KRAS: p.G12C) is exemplified, and the remaining examples repeat steps 1-11 as follows:

1. collecting a Sample of peripheral blood from Sample 1;

2. extracting cell free DNA in the peripheral blood of Sample1 by using a QIAamp Circulating Nucleic Acid Kit;

3. starting with 20ng of DNA, library construction was performed using the Rapid DNA Lib Prep Kit library construction Kit from AB clone;

4. separating and purifying by 2 percent agarose Gel electrophoresis, taking 1.2mg of a library of target bands which are purified by using a QIA quick Gel Extraction Kit of Qiagen, cutting Gel and recovering 130-160bp and 161-230bp target bands;

5. performing hybridization capture on the library obtained in step 4 by using a target region capture probe designed by Integrated DNA technologies;

6. performing double-end read-length 150bp sequencing by using an Illumina Nextseq500 sequencer to obtain a fastq format file of original sequencing data of Sample 1;

7. removing low quality and connector pollution sequences of the original sequencing data obtained in the step 6 by utilizing cutadapt software to obtain a processed data fastq format file to be analyzed;

8. comparing the fastq sequence of the data to be analyzed obtained in the step 7 with a human reference genome by using an aln method of BWA software to obtain a file in a Sam format;

9. sequencing and de-repeating the Sam format by using Samtools software to obtain a Bam file, extracting all sequence results on comparison of double-end sequencing sequences from the Bam file by using the Samtools software, and classifying the sequence results according to the comparison distance positions of the double-end sequencing sequences, namely 160bp and 161 bp respectively to obtain a set A, B;

10. mutation (SNP + Indel) detection is respectively carried out on the sequence set of the set A, B by using Freebayes software;

11. comparing the mutation results of the set A, B, the mutation frequency of p.g12c of KRAS detected in the set a was 13.3%, and the mutation frequency of p.g12c of KRAS detected in the set B was 6.5%. Since Sample1 was known to be a stage IV lung cancer patient, the mutation results in set a were selected for prediction, and the frequency of mutation of p.g12c in KRAS in this Sample was 13.3%. The sample was subjected to absolute quantitative validation by digital microdroplet pcr (digital Droplet pcr) and the mutation frequency at this site was found to be 17.26%, i.e. more closely aligned with the mutation frequency in pool a.

The detailed information of the test results of all 6 samples is shown in Table 1. It can be seen that for Sample4 and Sample5, false negative results can occur if the detection is performed by the conventional method, thereby further reflecting the accuracy of the method of the present invention.

TABLE 1.6 statistics of detection frequency results for samples

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

Claims

1. A method for accurately detecting mutation of a liquid biopsy individual based on a next generation sequencing technology is characterized by comprising the following steps:

(1) classifying cell-free DNA in a fluid sample collected from a subject according to its length, resulting in two sets of cell-free DNA having a first length range and a second length range;

(2) selecting a desired cell-free DNA group from cell-free DNA groups having different length ranges, and analyzing the gene mutation situation in the desired cell-free DNA group;

(3) detecting the mutation of the liquid biopsy individual according to the gene mutation situation obtained in the step (2),

wherein, in step (2):

if the liquid sample is from a subject with advanced disease, then treating the set of cell-free DNA having the first length range as the desired set of cell-free DNA;

wherein the method is a non-diagnostic method, the disease is lung cancer, the first length range is 130-160bp, the second length range is 161-230bp, and the cell-free DNA comprises KRAS, EFGR and/or PIK3CA genes.

2. The method for accurately detecting mutation in liquid biopsy individuals based on the next-generation sequencing technology according to claim 1, wherein the liquid sample in step (1) is selected from blood, saliva, urine, tissue fluid and their isolates.

3. The method for accurately detecting the mutation in the liquid biopsy individual based on the next-generation sequencing technology according to claim 1, wherein the step (1) is to classify the cell-free DNA by gel electrophoresis or obtain the sequence length by sequencing and classify the cell-free DNA according to the obtained length.

4. The method for accurately detecting mutations in liquid biopsy individuals based on the next-generation sequencing technology according to claim 1, wherein in step (1), the cell-free DNA is classified according to the length of the cell-free DNA in the liquid sample collected from the subject, so as to obtain the cell-free DNA group with the first length range and the cell-free DNA group with the second length range, and the first length range is smaller than the second length range.

5. The method for accurately detecting the mutation in the liquid biopsy individual based on the next-generation sequencing technology according to claim 1, wherein the step (1) comprises the following steps:

(1-1) extracting cell-free DNA in the liquid sample;

(1-2) constructing a gene library using the cell-free DNA;

6. Use of a reagent for the preparation of a kit for the accurate detection of mutations in a liquid biopsy individual based on a second generation sequencing technique, wherein the reagent comprises:

(1) a reagent for extracting cell-free DNA from a liquid sample from a subject;

(2) (ii) an agent that separates the cell-free DNA into two sets of cell-free DNA having a first length range and a second length range; and

(3) reagents for analyzing cell-free DNA comprising KRAS, EFGR and/or PIK3CA for genetic mutations;

the method for accurately detecting the mutation of the liquid biopsy individual based on the next generation sequencing technology comprises the following steps:

wherein, in step (2):

if the assay is aimed at early screening for disease for the subject, then the set of cell-free DNA having the second length range is used as the desired set of cell-free DNA;

wherein the disease is lung cancer, the first length range is 160bp along 130-.

7. The use of claim 6, wherein the agent further comprises an agent for constructing a gene library.