CN117965709A

CN117965709A - Joint for detecting mutation frequency of gene by single-ended multiple amplification and using method

Info

Publication number: CN117965709A
Application number: CN202410390449.7A
Authority: CN
Inventors: 叶邦全
Original assignee: BOE Technology Group Co Ltd; Beijing BOE Technology Development Co Ltd
Current assignee: BOE Technology Group Co Ltd; Beijing BOE Technology Development Co Ltd
Priority date: 2024-04-02
Filing date: 2024-04-02
Publication date: 2024-05-03

Abstract

A connector for detecting mutation frequency of gene by single-end multiple amplification and a use method thereof. The linker comprises a long single strand comprising a unique molecular tag sequence adjacent to the 3' end of said long single strand and the unique molecular tag sequence and its upstream and downstream partial sequences are binding sites for a unique molecular tag blocking sequence comprising at least one modified blocking base; a short single strand; the short single strand is complementarily paired with the downstream sequence of the unique molecular tag sequence to form an incomplete double-stranded adaptor. The adaptor is applied to constructing and sequencing libraries compatible with genome DNA and free DNA samples and constructing and sequencing methylation libraries. The joint and the use method improve the detection efficiency, correct the PCR amplification and sequencing amplification errors, ensure the accuracy and reduce the cost compared with multiplex PCR and hybrid capture, and have the mutation frequency detection sensitivity as low as 0.1 percent and the genome comparison rate of 99 percent.

Description

Joint for detecting mutation frequency of gene by single-ended multiple amplification and using method

Technical Field

The disclosure relates to the technical field of targeted sequencing, in particular to the field of single-ended multiple-specific primer extension and UMI closed amplification sequencing.

Background

The target sequencing is also called target region sequencing, is a technical means for amplifying or capturing and enriching a genomic region of interest and then carrying out high-throughput sequencing, can detect genetic variation, somatic cell point mutation, indel mutation, fusion mutation and the like aiming at the target genomic region, can obtain high-depth, high-coverage and high-accuracy detection performance, can reduce cost compared with whole genome sequencing, is suitable for researching a large number of samples, is beneficial to finding and verifying disease-related gene loci, and has great application prospects in clinical diagnosis and drug development.

Disclosure of Invention

The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the claims.

The present disclosure provides a linker for single-ended multiplex amplification detection of gene mutation frequencies and methods of use thereof.

In one aspect, the present disclosure provides a linker for single-ended multiplex amplification detection of mutation frequencies of genes, the linker comprising:

A long single strand comprising a unique molecular tag sequence adjacent to the 3' end of the long single strand, and the unique molecular tag sequence and its upstream and downstream partial sequences being binding sites for a unique molecular tag blocking sequence comprising at least one modified blocking base; and

A short single strand;

Wherein the short single strand is complementarily paired with a sequence downstream of the unique molecular tag sequence to form an incomplete double-stranded adaptor.

In some exemplary embodiments, the long single stranded unique molecular tag sequence is upstream of a sequencing primer sequence and the long single stranded unique molecular tag sequence is downstream of a specific sequence for the genome to be tested; the short single strand is the complement of the specific sequence.

In some exemplary embodiments, the sequencing primer sequence may include an Illumina sequencing platform Read1 sequencing primer sequence.

In some exemplary embodiments, the specific sequence is not fully complementary to the aligned human genome.

In some exemplary embodiments, the unique molecular tag comprises 2 to 12 random bases; the number of modified blocked bases is the same as the number of bases of the unique molecular tag sequence; the modified blocked base is selected from at least one of C3 spacer, C6 spacer, C12 spacer, spacer, spacer 18, dspacer.

In some exemplary embodiments, the unique molecular tag comprises 8 random bases.

In some exemplary embodiments, the long single stranded sequence is shown in SEQ ID NO. 1; the sequence of the short single strand is shown in SEQ ID NO. 2.

In some exemplary embodiments, the unique molecular tag blocking sequence is shown in SEQ ID NO. 3 or SEQ ID NO. 4.

In some exemplary embodiments, the single-stranded sequence upstream and the C bases in the double-stranded sequence downstream of the unique molecular tag sequence of the incomplete double-stranded adaptor may also contain methylation modifications.

The present disclosure provides the use of the above-described linkers in library construction and sequencing of compatible genomic DNA, episomal DNA samples, and methylation library construction and sequencing.

In some exemplary embodiments, the application comprises the steps of:

fragmenting the genomic DNA and purifying to recover fragmented DNA fragments;

performing end repair on the fragmented DNA fragments;

ligating the repaired DNA fragment to the adaptor; purification is then performed to recover the ligated DNA fragments;

Denaturing the connected DNA fragments to form single-stranded DNA with the linker, then adding the single-stranded DNA into an amplification system containing a unique molecular tag blocking sequence and single-ended specific primers, performing linear amplification, and purifying an amplification product; amplifying and enriching the purified amplified product by using a universal primer, and purifying the enriched product; and finally, carrying out on-machine sequencing on the purified enriched product.

In some exemplary embodiments, the application further comprises:

fragmenting the genomic DNA and purifying to recover fragmented DNA fragments;

performing end repair on the fragmented DNA fragments;

Ligating the repaired DNA fragment to the adaptor containing the methylation modification; purification is then performed to recover the ligated DNA fragments;

Denaturing the connected DNA fragments to form single-stranded DNA with the linker, performing oxidation protection, deamination reaction and purification on the single-stranded DNA with the linker, adding the purified product into an amplification system containing a unique molecular tag blocking sequence and single-ended specific primers, performing linear amplification, and purifying the amplified product; amplifying and enriching the purified amplified product by using a universal primer, and purifying the enriched product; and finally, carrying out on-machine sequencing on the purified enriched product.

The present disclosure provides an apparatus for executing the above application.

The present disclosure provides a kit for constructing a high throughput sequencing library, the kit comprising a linker as described above or a linker involved in the use as described above, a unique molecular tag blocking sequence, a specific primer for single ended linear multiplex PCR amplification; and the kit further comprises a DNA fragmentation system, a terminal repair system, a ligation system, and an amplification system.

In some exemplary embodiments, the specific primer for single-ended linear multiplex PCR amplification comprises a specific sequence capable of base complementary pairing with a target sequence of a gene to be sequenced.

In some exemplary embodiments, the specific primer for single ended linear multiplex PCR amplification is selected from any one or more of SEQ ID NO. 5 through SEQ ID NO. 118.

In some exemplary embodiments, the fragmented DNA fragments are 200-500bp in length.

Compared with the prior art, the method has the following advantages:

1) The spacer is modified on the closed structure of UMI, so that the subsequent amplification of UMI is not influenced, and the closed effect is better;

2) Single primer amplification: since multiplex amplification (e.g., 2 primers) is extremely prone to primer dimer generation and amplification is biased, the generation of primer dimer can be avoided with a single primer

3) Linear amplification: amplification bias can be avoided.

In addition, the single-ended multiplex primer extension technology adopted by the disclosure has the advantages that the reaction time is 5 hours (calculated according to the embodiment of the disclosure), compared with the hybridization capture technology for 2 days (calculated according to the flow of the prior art), the detection efficiency is greatly improved, compared with the multiplex PCR amplification technology, UMI sequences are introduced, PCR amplification and sequencing amplification errors can be corrected, the mutation frequency detection sensitivity is as low as 0.1%, the genome comparison rate is 99%, and the effective data amount is improved from 50% to 99%, so that the accuracy is ensured, and meanwhile, the cost is greatly reduced compared with the traditional multiplex PCR technology and the hybridization capture technology.

Other aspects will become apparent upon reading and understanding the accompanying drawings and detailed description.

Drawings

The accompanying drawings are included to provide a further understanding of the disclosed embodiments and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain, without limitation, the embodiments of the disclosure. The shape and size of one or more modules in the drawings are not to scale, and are intended to be illustrative of the present disclosure.

Fig. 1 shows a flow chart of an exemplary embodiment of the present disclosure.

FIG. 2 is a diagram showing the sequence composition and structure of an exemplary embodiment linker of the present disclosure.

Fig. 3 shows a diagram of the sequence and structure of a long single strand containing a UMI linker and one unique molecular tag blocking sequence of a linker of an exemplary embodiment of the present disclosure.

Fig. 4 shows a diagram of the sequence and structure of a long single strand containing a UMI linker and another unique molecular tag blocking sequence of a linker of one exemplary embodiment of the present disclosure.

FIG. 5 illustrates a flow chart of an exemplary embodiment of the present disclosure of an application of a linker to methylation rate detection.

FIG. 6 is a diagram showing the sequence and structure of a C base in a single-stranded sequence upstream and a double-stranded sequence downstream of a random base of a linker according to an exemplary embodiment of the present disclosure when the linker is applied to methylation rate detection.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present disclosure more apparent, embodiments of the present disclosure will be described in detail hereinafter. It should be noted that, without conflict, the embodiments of the present disclosure and features of the embodiments may be arbitrarily combined with each other.

Embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings. Embodiments may be implemented in a number of different forms. One of ordinary skill in the art will readily recognize the fact that the patterns and matters may be changed into one or more forms without departing from the spirit and scope of the present disclosure. Accordingly, the present disclosure should not be construed as being limited to the following description of the embodiments. Embodiments of the present disclosure and features of embodiments may be combined with each other arbitrarily without conflict.

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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described. For the purposes of this disclosure, the following terms are defined below.

The term "about" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation according to practice in the art. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a particular value. In other examples, an amount of "about 10" includes 10 and any amount from 9 to 11.

In still other examples, the term "about" referring to a reference value may also include a range of values of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that value. Alternatively, particularly in relation to biological systems or processes, the term "about" may mean within an order of magnitude of a value. When a particular value is described in the present disclosure and claims, unless otherwise specified, the term "about" shall be assumed to mean within an acceptable error range for the particular value.

As used in this specification and one or more claims, the words "comprise/include", "have" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any of the embodiments discussed in this specification can be implemented with reference to any method or combination of the present disclosure, and vice versa. Furthermore, combinations of the present disclosure may be used to implement the methods of the present disclosure.

"Genome" when used in eukaryotic cells encompasses not only chromosomal DNA present in the nucleus, but also organelle DNA present in subcellular components of the cell (e.g., mitochondria, plastids).

"Polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "nucleic acid fragment" are used interchangeably and are single-or double-stranded RNA or DNA polymers, optionally containing synthetic, unnatural or modified nucleotide bases. Nucleotides are referred to by their single letter designations as follows: "A" is adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), "C" represents cytidine or deoxycytidine, "G" represents guanosine or deoxyguanosine, "U" represents uridine, "T" represents deoxythymidine, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, "H" represents A or C or T, "I" represents inosine, and "N" represents any nucleotide.

DdC: dideoxycytosine.

UMI: unique Molecular Identifier, unique molecular tags.

Primer: and (5) a primer.

Index: a sample tag for labeling each sequenced sample.

OligoA: in the present disclosure, refers to a single stranded oligonucleotide comprising one strand of the adaptors used in the present disclosure, the single stranded oligonucleotide having from 1 to 100, preferably from 1 to 90, more preferably from 1 to 80, still more preferably from 1 to 70, 1 to 60, 1 to 50, or 1 to any number or range of nucleotides between 50 and 100; and the single stranded oligonucleotide comprises UMI having a random sequence.

OligoB: in the present disclosure, single stranded oligonucleotides comprising one strand of a linker as used in the present disclosure, having from 1 to 30, preferably from 1 to 20, more preferably from 1 to 10, or from 1 to 10 and 30 nucleotides of any number or range.

Dspacer, including THF tetrahydrofuran modification, in the present disclosure refers to introducing a null deoxynucleotide without a base at the nucleotide site, which does not contain a base, cannot form hydrogen bonds, and can play a role in increasing the spatial distance, so as to cover the UMI sequence, and prevent the primer from combining with the UMI sequence in space, so as to play a role in blocking.

At present, two main technical means of targeted sequencing exist, namely, a multiplex PCR amplification technology, namely, amplicon sequencing, and the principle is that a series of specific primers aiming at a target region are designed in advance, the tail ends of the specific primers are provided with universal sequences, and then the target region is amplified and enriched by utilizing the universal primer sequences, but the multiplex PCR amplification has the following defects: 1, the difficulty of primer design is high, so that a larger area cannot be effectively amplified, 2, in the process of primer combination, SNP loci can have a certain influence on primer combination efficiency, 3, no UMI correction function is provided, in the amplification process, error bases can be introduced, and particularly in the case of more amplification cycles, the risk of false positive results is increased, and low-frequency mutation cannot be detected efficiently; the 4 th is that the primer amplification efficiency has serious bias, primer dimers are extremely easy to form, a large amount of invalid sequencing data is generated, the depth of a target area is extremely unbalanced, and site variation information cannot be effectively and accurately detected; the second is a probe hybridization capture method, which is based on the principle of base complementation pairing of nucleic acid molecules, designing a probe set with biotin marks, constructing a whole genome library in advance, hybridizing the target area library molecules by using probes, combining probes with biotin marks and target area library molecule complexes by using avidin magnetic beads, and amplifying and enriching the target area after a series of elution steps. Compared with the multiplex PCR method, the probe design of the hybridization capture method is more flexible, the size of the target area is not limited, the amplification process can be reduced as much as possible, and the higher library complexity is ensured. For detection of fusion genes, the method of hybrid capture is more advantageous and some unknown fusion subtypes can be obtained. At the same time, the library is more homogeneous and CNV can be detected based on sequencing depth. However, hybrid capture technology suffers from several drawbacks: 1) relatively complex operation, high personnel requirements, 2) long hybridization process, generally 2-3 days, correspondingly prolonged detection period, 3) high reagent cost, especially probe reagent cost.

In addition, because PCR amplification is exponential amplification, i.e. the product can reach the power N of amplification efficiency (N represents the amplification cycle number), different DNA templates have different amplification efficiencies (such as different fragment lengths, different GC contents and the like) during PCR amplification, the more the amplification cycle number is, the higher the amplification efficiency is, the lower the amplification efficiency is, and the further amplification of the difference of the amplification efficiencies is, so that the difference of the PCR amplification yields is larger and larger, and the result that the PCR has amplification bias is presented.

Because single-end amplification adopts only one primer for amplification, linear amplification is realized, the amplification efficiency is still kept in the initial state no matter how many amplification cycles are carried out, and the bias of PCR amplification can be effectively overcome. Therefore, the low-frequency mutation detection effect with low cost, high efficiency and high accuracy is realized through single-ended primer extension and UMI closed amplification technology. In the experimental procedure, after the end of the fragmented DNA is repaired, a UMI adapter is used for a ligation reaction (see fig. 1, wherein, on the display of the penultimate sequence, a horizontal black arrow indicates a specific sequence of a target sequence of a gene to be sequenced, and a downward inclined line segment behind the horizontal black arrow indicates a universal sequencing sequence), so that the problem that false positives are caused by the failure in PCR amplification correction is solved, and the subsequent extension is performed by adopting single-ended multiple specific primers, which are halved compared with the multiple PCR amplification technology, so that the problems of primer dimer generation and small target region amplification are solved, and the linear amplification of single-ended primer extension is realized, thereby solving the problem of bias in PCR amplification. And a blocking sequence capable of blocking the UMI sequence is added in the single-end primer extension process, so that the single-end multiplex primer is prevented from being combined with the UMI sequence to amplify to form a joint product, the problem that an invalid amplified product occupies the sequencing data amount due to the UMI sequence is solved, and the high capture efficiency, the high uniformity and the high accuracy of the subsequent sequencing data are ensured.

The present disclosure uses single-ended primer extension amplification techniques, specifically including genome fragmentation, fragmented DNA end repair, UMI adapter ligation, amplification of target region with single-ended multi-specific primers & blocking sequences, amplification of target region sequences with universal primers, and specific flow is shown in fig. 1.

Example 1

1. And (3) preparation of a standard substance: pan tumor 800 gDNA standard 1 (cat# GW-OGTM 800), tumor SNV wild type gDNA standard 2 was purchased from cyanine gene, wherein pan tumor 800 gDNA standard 1 contained mutations and corresponding mutation frequencies as follows: EGFR L858R mutation frequency was 1%, KRAS A146T mutation frequency was 1%, EGFR T790M mutation frequency was 2%, and BRAF V600E mutation frequency was 7%. 10 ng pan blood tumor 800 gDNA standard 1 was mixed with 90 ng tumor SNV wild gDNA standard 2 to form standard 3.

2. Primer design: designing primers aiming at lung cancer related hot spot mutation sites in EGFR, ALK, ROS genes, BRAF and KRAS, wherein the sequences of the primers are as follows:

Name of the name	Sequence (5 '-3')
		Oligo1(SEQ ID NO:5)	CAGACGTGTGCTCTTCCGATCTAGAAAACAAGATTTTGACTGTACC
Oligo2(SEQ ID NO:6)	CAGACGTGTGCTCTTCCGATCTAAGAATTGCTAAAGTTTGTCGACA
		Oligo3(SEQ ID NO:7)	CAGACGTGTGCTCTTCCGATCTAGCCTTAGAAAACAAATGGAGTTT
Oligo4(SEQ ID NO8:)	CAGACGTGTGCTCTTCCGATCTTCCAGAGAATTTTTCTTAAGGGGA
		Oligo5(SEQ ID NO:9)	CAGACGTGTGCTCTTCCGATCTGGCCCATCCTTTCCAAAAGG
Oligo6(SEQ ID NO:10)	CAGACGTGTGCTCTTCCGATCTAGGTTTACATTGGCAAGTGCT
		Oligo7(SEQ ID NO:11)	CAGACGTGTGCTCTTCCGATCTAGTCATTACTGCTAGGTTTTTAAT
Oligo8(SEQ ID NO:12)	CAGACGTGTGCTCTTCCGATCTATTTTGGTATATGAAGCTTCTGGG
		Oligo9(SEQ ID NO:13)	CAGACGTGTGCTCTTCCGATCTCAAGAGAGTAGATACGTCAGTTTC
Oligo10(SEQ ID NO:14)	CAGACGTGTGCTCTTCCGATCTACGATGGAATATTAGGGAGCCA
		Oligo11(SEQ ID NO:15)	CAGACGTGTGCTCTTCCGATCTGTCTGCCAGTCAACTTTGCC
Oligo12(SEQ ID NO:16)	CAGACGTGTGCTCTTCCGATCTTCCCTTTAGTATTAAGTTAGCTGT
		Oligo13(SEQ ID NO:17)	CAGACGTGTGCTCTTCCGATCTGGGGAACGGAACTGATTTTTCTG
Oligo14(SEQ ID NO:18)	CAGACGTGTGCTCTTCCGATCTTCTGGCACTTATTTTGTAATTTGA
		Oligo15(SEQ ID NO:19)	CAGACGTGTGCTCTTCCGATCTACCACTGTACTAAATGATCTCTAA
Oligo16(SEQ ID NO:20)	CAGACGTGTGCTCTTCCGATCTGTACCTGAGGTGGCCCAG
		Oligo17(SEQ ID NO:21)	CAGACGTGTGCTCTTCCGATCTTCAAAACACAGTTCTGTTCCTCA
Oligo18(SEQ ID NO:22)	CAGACGTGTGCTCTTCCGATCTGGAGAGTAGCAACAAAAGGAAAA
		Oligo19(SEQ ID NO:23)	CAGACGTGTGCTCTTCCGATCTACCACCCAGATTTTCATTCTTCT
Oligo20(SEQ ID NO:24)	CAGACGTGTGCTCTTCCGATCTGGTGTAATACATACATCGTAAGCT
		Oligo21(SEQ ID NO:25)	CAGACGTGTGCTCTTCCGATCTAGACAGGTGTTTTAATGGTAAAAG
Oligo22(SEQ ID NO:26)	CAGACGTGTGCTCTTCCGATCTTTGACTCTAAGAGGAAAGATGAAG
		Oligo23(SEQ ID NO:27)	CAGACGTGTGCTCTTCCGATCTCCAATGAAGAGCCTTTACTGCT
Oligo24(SEQ ID NO:28)	CAGACGTGTGCTCTTCCGATCTCACGCCAAGTCAATCATCCAC
		Oligo25(SEQ ID NO:29)	CAGACGTGTGCTCTTCCGATCTCCCAAGCCTGGACTTAACGT
Oligo26(SEQ ID NO:30)	CAGACGTGTGCTCTTCCGATCTGCGCTGAGAAGTCTGTGGTT
		Oligo27(SEQ ID NO:31)	CAGACGTGTGCTCTTCCGATCTGGACCCATTAGAACCAACTCCA
Oligo28(SEQ ID NO:32)	CAGACGTGTGCTCTTCCGATCTCCCAAAACCTCCAAAAGCCA
		Oligo29(SEQ ID NO:33)	CAGACGTGTGCTCTTCCGATCTTAATGTCACCGACACCCTGC
Oligo30(SEQ ID NO:34)	CAGACGTGTGCTCTTCCGATCTTTGCCGGAAAACTTGGGAGA
		Oligo31(SEQ ID NO:35)	CAGACGTGTGCTCTTCCGATCTGGAGAAGACACAGGTGGCAA
Oligo32(SEQ ID NO:36)	CAGACGTGTGCTCTTCCGATCTAGACACGTGAAGGCATGAGG
		Oligo33(SEQ ID NO:37)	CAGACGTGTGCTCTTCCGATCTTGAACAATCATCTGGCAGCG
Oligo34(SEQ ID NO:38)	CAGACGTGTGCTCTTCCGATCTGGACAGCCTTCAAGACCTGG
		Oligo35(SEQ ID NO:39)	CAGACGTGTGCTCTTCCGATCTGAAACAGGAAAGGACGGGCG
Oligo36(SEQ ID NO:40)	CAGACGTGTGCTCTTCCGATCTTGAGGACAAAGTTGGCACGT
		Oligo37(SEQ ID NO:41)	CAGACGTGTGCTCTTCCGATCTACTGCTAATGGCCCGTTCTC
Oligo38(SEQ ID NO:42)	CAGACGTGTGCTCTTCCGATCTTGGAGTTTCCCAAACACTCAGT
		Oligo39(SEQ ID NO:43)	CAGACGTGTGCTCTTCCGATCTGCTGTCTCTAAGGGGAGGGA
Oligo40(SEQ ID NO:44)	CAGACGTGTGCTCTTCCGATCTATGTGAGGATCCTGGCTCCT
		Oligo41(SEQ ID NO:45)	CAGACGTGTGCTCTTCCGATCTTCCACCACTATCACCTGGGT
Oligo42(SEQ ID NO:46)	CAGACGTGTGCTCTTCCGATCTCATCCTCCCCTGCATGTGTT
		Oligo43(SEQ ID NO:47)	CAGACGTGTGCTCTTCCGATCTGCAGCTGGACTCGATTTCCT
Oligo44(SEQ ID NO:48)	CAGACGTGTGCTCTTCCGATCTCAAAGGCCTCAGCTGTTTGG
		Oligo45(SEQ ID NO:49)	CAGACGTGTGCTCTTCCGATCTCTCTAATGCGCTGGAGGGTT
Oligo46(SEQ ID NO:50)	CAGACGTGTGCTCTTCCGATCTAAGTGGAATTCTGCCCAGGC
		Oligo47(SEQ ID NO:51)	CAGACGTGTGCTCTTCCGATCTTGACTTCATATCCATGTGAGTTTC
Oligo48(SEQ ID NO:52)	CAGACGTGTGCTCTTCCGATCTGGCGATGCTACTACTGGTCC
		Oligo49(SEQ ID NO:53)	CAGACGTGTGCTCTTCCGATCTCCTTACGCCCTTCACTGTGT
Oligo50(SEQ ID NO:54)	CAGACGTGTGCTCTTCCGATCTTGGTGTATGTGTGGTGTGTGT
		Oligo51(SEQ ID NO:55)	CAGACGTGTGCTCTTCCGATCTGTAGTCAGGGTTGTCCAGGC
Oligo52(SEQ ID NO:56)	CAGACGTGTGCTCTTCCGATCTCTGTTGGGATGGAGGACCTG
		Oligo53(SEQ ID NO:57)	CAGACGTGTGCTCTTCCGATCTTCCAATGTGGGCTGGAATCC
Oligo54(SEQ ID NO:58)	CAGACGTGTGCTCTTCCGATCTGGACTTTGGGCAGCTCCTC
		Oligo55(SEQ ID NO:59)	CAGACGTGTGCTCTTCCGATCTTGGGCTGCACCTGAGAAAAT
Oligo56(SEQ ID NO:60)	CAGACGTGTGCTCTTCCGATCTTGCATTTATGAACCCCCAGC
		Oligo57(SEQ ID NO:61)	CAGACGTGTGCTCTTCCGATCTTGCAGTCTCTCTAAAGTAAACTGT
Oligo58(SEQ ID NO:62)	CAGACGTGTGCTCTTCCGATCTACAGGGTTACACTGGCAGATG
		Oligo59(SEQ ID NO:63)	CAGACGTGTGCTCTTCCGATCTTCCACCCTTAATAAAGAGAGAAAC
Oligo60(SEQ ID NO:64)	CAGACGTGTGCTCTTCCGATCTAGGTACTCAGGAGTAAAGAACAAT
		Oligo61(SEQ ID NO:65)	CAGACGTGTGCTCTTCCGATCTACTAAACATGGCTTCTCTCCC
Oligo62(SEQ ID NO:66)	CAGACGTGTGCTCTTCCGATCTGCATGCGGTGAGATTTGCAA
		Oligo63(SEQ ID NO:67)	CAGACGTGTGCTCTTCCGATCTGCTAATTGACAGCTCCCCCA
Oligo64(SEQ ID NO:78)	CAGACGTGTGCTCTTCCGATCTAGGCAAACACATCCACCCAA
		Oligo65(SEQ ID NO:69)	CAGACGTGTGCTCTTCCGATCTTCCCATTGCCTAACCTAGCT
Oligo66(SEQ ID NO:70)	CAGACGTGTGCTCTTCCGATCTTGTGGATCCAGAGGAGGAGT
		Oligo67(SEQ ID NO:71)	CAGACGTGTGCTCTTCCGATCTTGACAAAAGTTGTGGACAGGT
Oligo68(SEQ ID NO:72)	CAGACGTGTGCTCTTCCGATCTTGTTGTTGAGTTGTATATAACACC
		Oligo69(SEQ ID NO:73)	CAGACGTGTGCTCTTCCGATCTACATGTGCTTGTTTTTCTAAGAGA
Oligo70(SEQ ID NO:74)	CAGACGTGTGCTCTTCCGATCTGGTTATCTTATCTTGAATGAGTTA
		Oligo71(SEQ ID NO:75)	CAGACGTGTGCTCTTCCGATCTTGGGAATGAACTCATCAACCTCT
Oligo72(SEQ ID NO:76)	CAGACGTGTGCTCTTCCGATCTCACGTCTGCAGTCAACTGGA
		Oligo73(SEQ ID NO:77)	CAGACGTGTGCTCTTCCGATCTACCCAGCAGTTACCTTAAAGCT
Oligo74(SEQ ID NO:78)	CAGACGTGTGCTCTTCCGATCTTCAAGCTACTTTATGTAAATCACT
		Oligo75(SEQ ID NO:79)	CAGACGTGTGCTCTTCCGATCTCACAAGGCACTGGGTATATGGT
Oligo76(SEQ ID NO:80)	CAGACGTGTGCTCTTCCGATCTCTTGTCACCCAGGCTGGAAT
		Oligo77(SEQ ID NO:81)	CAGACGTGTGCTCTTCCGATCTTGACCTAATCACTAATTTTCAGGT
Oligo78(SEQ ID NO:82)	CAGACGTGTGCTCTTCCGATCTCCACGGTCATCCAGTGTTGT
		Oligo79(SEQ ID NO:83)	CAGACGTGTGCTCTTCCGATCTCCTGTACACATGAAGCCATCG
Oligo80(SEQ ID NO:84)	CAGACGTGTGCTCTTCCGATCTTGTGCCATTGGTTATCCTTGTC
		Oligo81(SEQ ID NO:85)	CAGACGTGTGCTCTTCCGATCTACAAACCAGGATTCTAGCCCA
Oligo82(SEQ ID NO:86)	CAGACGTGTGCTCTTCCGATCTAGTCACACCTTGCACCGAAT
		Oligo83(SEQ ID NO:87)	CAGACGTGTGCTCTTCCGATCTCCACTCTCTTCTTGGCTGTGT
Oligo84(SEQ ID NO:88)	CAGACGTGTGCTCTTCCGATCTAGCTAAAGTCAGTTTTCTCATGCA
		Oligo85(SEQ ID NO:89)	CAGACGTGTGCTCTTCCGATCTGCACCAGAGAACCCCAGAAT
Oligo86(SEQ ID NO:90)	CAGACGTGTGCTCTTCCGATCTAGCACTTCCAAAATTGCAAATTTTGA
		Oligo87(SEQ ID NO:91)	CAGACGTGTGCTCTTCCGATCTACTACTTGCATGGACCAGCC
Oligo88(SEQ ID NO:92)	CAGACGTGTGCTCTTCCGATCTTGTCACTTGAAAGCCATTAACTG
		Oligo89(SEQ ID NO:93)	CAGACGTGTGCTCTTCCGATCTTGTTGAGGTGCAGATGTGAT
Oligo90(SEQ ID NO:94)	CAGACGTGTGCTCTTCCGATCTGCCCTTGCAGATGTCATCCT
		Oligo91(SEQ ID NO:95)	CAGACGTGTGCTCTTCCGATCTTGTTGGGAATGCTGTCTCCT
Oligo92(SEQ ID NO:96)	CAGACGTGTGCTCTTCCGATCTAGGCTGTTTCTCTCACACTGA
		Oligo93(SEQ ID NO:97)	CAGACGTGTGCTCTTCCGATCTTTCTAATGTGCAGCCAGGGC
Oligo94(SEQ ID NO:98)	CAGACGTGTGCTCTTCCGATCTTGCTTCACCTTCTGCCTGAG
		Oligo95(SEQ ID NO:99)	CAGACGTGTGCTCTTCCGATCTTTGGCCCATGTGGAATTCCT
Oligo96(SEQ ID NO:100)	CAGACGTGTGCTCTTCCGATCTGGAGGTGTATGAAGGCCAGG
		Oligo97(SEQ ID NO:101)	CAGACGTGTGCTCTTCCGATCTGTGAAGAACTGGAAGCCCGA
Oligo98(SEQ ID NO:102)	CAGACGTGTGCTCTTCCGATCTTGTAGTGCTTCAAGGGCCAG
		Oligo99(SEQ ID NO:103)	CAGACGTGTGCTCTTCCGATCTAGTGTGACAAGGTCTCCAGC
Oligo100(SEQ ID NO:104)	CAGACGTGTGCTCTTCCGATCTTGTGCTTCCAGTGGCTATGG
		Oligo101(SEQ ID NO:105)	CAGACGTGTGCTCTTCCGATCTTCCAAATTCCCATAGCACACCA
Oligo102(SEQ ID NO:106)	CAGACGTGTGCTCTTCCGATCTTGAGCCAAATCCCACATGCA

Primers were synthesized in Shanghai engineering, TE Buffer was added to dissolve the primers according to the indicated volume on the synthesis tube, 102 Oligo primers in the above table were diluted to 10. Mu.M, and the 102 primers after dilution were mixed in equal proportions to give primer Mix1.

3. Genome fragmentation: 100 ng standard (standard 1, standard 3 respectively) was added to the 0.6 mL PCR tube, the TE buffer was made up to 50. Mu.L, well mixed and centrifuged, the samples were crushed with a Biorupter ultrasonic crusher for 20 cycles, and the ultrasonic crusher was turned on for 30s and off for 30s in each cycle.

4. And (3) terminal repair: the reaction system was formulated as follows:

Component (A)	Volume (mu L)
		The product of the last step	50
T4 DNA polymerase (5U/. Mu.L)	0.2
		Klenow fragment (10U/. Mu.L)	0.1
T4 PNK(10U/μL)	1
		Taq DNA polymerase (1U/. Mu.L)	1
10X T4 DNA ligase buffer (10 mM ATP)	6.5
		dNTP (10mM)	1.63
dATP (100mM)	0.16
		PEG4000 (50%)	3.25
Nuclease-free water	1.16
		Totals to	65

Well mixed, immediately incubate 15 min at 20 ℃, then incubate 15 min at 65 ℃.

5. And (3) joint connection: after the completion of the previous reaction, a ligation reaction system was prepared immediately for linker ligation according to the following table:

Component (A)	Volume (mu L)
		The product of the last step	65
10X T4 DNA ligation buffer	10
		T4 DNA ligase (10U/. Mu.L)	5
Joint	5
		PEG4000 (50%)	10
Nuclease-free water	5
		Totals to	100

Note that: wherein the linker (Adapter) was annealed by OligoA (SEQ ID NO:1, 5 '-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNGCTCGATTCT-3') and OligoB (SEQ ID NO:2, 5 '-AGAATCGAGC-3'), the annealing procedure was 95℃5min, followed by slow cooling to room temperature. OligoA and OligoB are synthesized by Shanghai technology, and specific sequences and linker (Adapter) structures are shown in FIG. 2.

6. Purification of the ligation product: adding 80 mu L of nuuzuan purified magnetic beads into the connection product, fully mixing, standing at room temperature for 5 min, placing on a magnetic rack for about 5 min to enable the magnetic beads to be completely adsorbed and the solution to be clear, and carefully removing the supernatant; adding 200 μl of newly prepared 80% ethanol for rinsing, incubating at room temperature for 30-60 s, carefully removing supernatant, repeating for one time, adding 22 μl of ultrapure water for eluting after magnetic beads are dried, standing at room temperature for 3 min, placing in a magnetic rack, and sucking 20 μl of supernatant for use after the solution is clarified.

7. Single-ended primer extension: the reaction system was formulated as follows:

Component (A)	Volume (mu L)
		Purified ligation products	20
10X Taq polymerase buffer	10
		Taq DNA polymerase (10U/. Mu.L)	5
Primer Mix1	5
		Blocking sequences	5
dNTP	5
		Totals to	50

Note that: the blocking sequence (5 ^' -CTCGc3spacer (6) AGAT-3', SEQ ID NO:3: CTCGCCCCCCCCCCCCAGAT, wherein c3spacer (6) instead of cytosine is represented by c at positions 5 to 22 in the 5' -3' direction) was synthesized by Shanghai engineering at a concentration of 100. Mu.M, the specific sequence and structure is shown in FIG. 3.

Gently beating, mixing, centrifuging briefly, and placing into a PCR instrument to perform the following reaction system:

8. Single end extension product purification: adding 60 mu L of nuuzuan purified magnetic beads into the extension product, fully mixing, standing at room temperature for 5min, placing on a magnetic rack for about 5min to enable the magnetic beads to be completely adsorbed and the solution to be clarified, and carefully removing the supernatant; adding 200 μl of newly prepared 80% ethanol for rinsing, incubating at room temperature for 30-60 s, carefully removing supernatant, repeating for one time, adding 22 μl of ultrapure water for eluting after magnetic beads are dried, standing at room temperature for 3 min, placing in a magnetic rack, and sucking 20 μl of supernatant for use after the solution is clarified.

9. Amplification and enrichment of universal primers: using VAHTS HIFI Universal Amplification Mix (cat No. N618) and VAHTS Unique Dual Index Primer (cat No. 34401-01) of Northenan, the reaction system was prepared as follows:

Component (A)	Volume (mu L)
		The product of the last step	20
VAHTS HiFi Universal Amplification Mix	25
		VAHTS Unique Dual Index Primer	5
Totals to	50

10. purification of amplification products: adding 75 mu L of nuuzuan purified magnetic beads into the amplification product, fully mixing, standing at room temperature for 5 min, placing on a magnetic rack for about 5 min to enable the magnetic beads to be completely adsorbed, clarifying the solution, and carefully removing the supernatant; adding 200 μl of newly prepared 80% ethanol for rinsing, incubating at room temperature for 30-60 s, carefully removing supernatant, repeating for one time, adding 22 μl of ultrapure water for eluting after magnetic beads are dried, standing at room temperature for 3 min, placing in a magnetic rack, and sucking 20 μl of supernatant for use after the solution is clarified.

11. Sequencing on a machine: sequencing was performed using Illumina Novaseq6000 platform, with standard 1 and 3 data volumes of 5G, respectively.

Example 2

The experimental procedure is the same as in example 1, step 7 without addition of a blocking sequence, and the reaction system is shown in the following table:

Component (A)	Volume (mu L)
		Purification of the ligation product	20
10X Taq polymerase buffer	10
		Taq DNA polymerase (10U/. Mu.L)	5
Primer Mix1	5
		dNTP	5
Nuclease-free water	5
		Totals to	50

The remaining steps were the same as in example 1.

Example 3

Comparing and testing multiplex PCR amplification technology, the multiplex amplification technology kit adopts a vendor Ai Jitai (EGFR, ALK, ROS1, BRAF, KRAS, HER 2) customized kit, samples adopt standard products 1 and 3, 100 ng are respectively taken for library construction and sequencing, and the data volume of each sample is 5G.

Example 4

Comparing the hybridization capture, customizing the kit by using a Twist Bioscience (EGFR, ALK, ROS, BRAF, KRAS, HER 2), adopting the standard products 1 and 3 as samples, respectively taking 100 ng for library construction and sequencing, and taking 5G of data amount on each sample.

Example 5

The experimental procedure is as in example 1, wherein step 7 replaces the blocking sequence, and the modification group in the middle of the blocking sequence employs 4C 6 spacers, and the specific results and sequences (5 ^' -CTCGc6 spacers (4) AGAT-3', SEQ ID NO:4: CTCCCCCCCCCCCCCCCCCCCCCCCAGAT, wherein C from position 5' -3' represents C6Spacer (4) instead of cytosine) are shown in FIG. 4.

Example 6

The technical method can be compatible with peripheral blood free DNA library construction, and the specific test flow is as follows:

1. Peripheral blood 5 mL was collected using a roche cfDTM free nucleic acid collection tube (07785674001), and the following plasma separation was performed within 24 hours.

2. Plasma separation procedure: first centrifugation, 1350 g, low temperature centrifugation at 4℃12 min, carefully remove the pale yellow supernatant (avoiding contaminating leukocyte layers) and transfer to a 2mL DNase-free sterile centrifuge tube. The second centrifugation, 13500 g, low temperature centrifugation at 4℃5min, carefully remove the supernatant (complete removal of leukocytes) and transfer to 2-3 tube 2mL DNase-free sterile centrifuge tube.

3. Extracting cfDNA from the separated blood plasma by using a nucleic acid extraction or purification kit (QIAGEN, QIAamp Circulating Nucleic Acid, cat# 55114), marking the cfDNA as a standard substance 4, measuring the concentration by using Qubit, and performing fragment analysis quality inspection by Agilent Tapestation

4. Standard 4 the subsequent experimental procedure was performed as described in example 1, steps 4-11, and a comparative example was designed, which differs from this example in that step 7 did not add a blocking sequence.

Example 7

The technical method can be applied to methylation sequencing at the same time, and the methylation conversion flow is carried out after the connection of the connectors is completed, and the whole flow is shown in figure 5.

1. And (3) preparation of a standard substance: human methylation standards (cat# D5014) were purchased from ZYMO Research, and included a total methylation standard (methylation modification of all C bases in the sequence) and a total unmethylation standard (non-methylation modification of all C bases in the sequence), mixed in a ratio to 1 μg of 50% methylation standard, labeled as standard 5, and then added to a 0.6 mL PCR tube.

2. Methylation amplification primer: at the mutation site covered in example 1, the first 12 sites (Oligo 1-12 covered region) were selected for methylation primer design, the methylation primer sequences were as follows:

Name of the name	Sequence (5 '-3')
		Oligo1’ (SEQ ID NO:107)	CAGACGTGTGCTCTTCCGATCTAAAATCTTTTAATAAAAAACAAAAT
Oligo2’ (SEQ ID NO:108)	CAGACGTGTGCTCTTCCGATCTTTAATTCTCTTTTATAAAAATTACT
		Oligo3’ (SEQ ID NO:109)	CAGACGTGTGCTCTTCCGATCTATAAAATTTAAATAACTAAAATATA
Oligo4’ (SEQ ID NO:110)	CAGACGTGTGCTCTTCCGATCTTTAACTACTATTCTTCCAAAAAATT
		Oligo5’ (SEQ ID NO:111)	CAGACGTGTGCTCTTCCGATCTATCTATATTTAAACATACACTACATT
Oligo6’ (SEQ ID NO:112)	CAGACGTGTGCTCTTCCGATCTAATAATTATTTCATTATAAATTTAC
		Oligo7’ (SEQ ID NO:113)	CAGACGTGTGCTCTTCCGATCTAATACTTTAATAATTTTAAAAATAT
Oligo8’ (SEQ ID NO:114)	CAGACGTGTGCTCTTCCGATCTCTAAAAATAAAATTTAATCTCAATT
		Oligo9’ (SEQ ID NO:115)	CAGACGTGTGCTCTTCCGATCTTCAATTTCTAAAAAATTTTCTTATA
Oligo10’ (SEQ ID NO:116)	CAGACGTGTGCTCTTCCGATCTACCATTTAAATTTCTAACAATAAAAT
		Oligo11’ (SEQ ID NO:117)	CAGACGTGTGCTCTTCCGATCTATCTACCAATCAACTTTACCCTTCT
Oligo12’ (SEQ ID NO:118)	CAGACGTGTGCTCTTCCGATCTTTAATATTAAATTAACTATAAAAAT

Primer synthesis in Shanghai engineering, dissolving primer by adding TE Buffer according to the prompt volume on the synthesis tube, diluting the concentration of Oligo1'-12' in the table to 10 mu M, synthesizing primer Mix 2 by the diluted Oligo1'-12' according to an equal proportion, and mixing the mixture with Oligo1-12 in example 1 to 10 mu M according to an equal proportion to obtain Mix 3 according to a ratio of 1:1.

3. Genome fragmentation: standard 5 was made up to 50 μl using TE buffer, thoroughly mixed and centrifuged, and the samples were crushed with a bioreroller sonicator for 20 cycles with 30 s on and 30 s off for each cycle.

4. And (3) terminal repair: the reaction system was formulated as follows:

Component (A)	Volume (mu L)
		The product of the last step	50
T4 DNA polymerase (5U/. Mu.L)	0.2
		Klenow fragment (10U/. Mu.L)	0.1
T4 PNK(10U/μL)	1
		Taq DNA polymerase (1U/. Mu.L)	1
10X T4 DNA ligase buffer (10 mM ATP)	6.5
		dNTP(10mM)	1.63
dATP(100mM)	0.16
		PEG4000(50%)	3.25
Nuclease-free water	1.16
		Totals to	65

Mixing well, incubating at 20deg.C for 15 min, and incubating at 65deg.C for 15 min.

5. After the previous step of reaction is completed, a connection reaction system is prepared immediately according to the following table:

Component (A)	Volume (mu L)
		The product of the last step	65
10X T4 DNA ligation buffer	10
		T4 DNA ligase (10U/. Mu.L)	5
Joint	5
		PEG4000(50%)	10
Nuclease-free water	5
		Totals to	100

Note that: wherein the linker (Adapter) is annealed by Oligo C (SEQ ID NO: 1) and Oligo D (SEQ ID NO: 2) with an annealing procedure of 95℃of 5 min, followed by a slow cooling to room temperature. Oligo C (containing methylation modification) and Oligo D (containing methylation modification) were synthesized by Shanghai engineering, the linker structure is shown in FIG. 6, and the specific sequence is as follows:

Oligo name	Sequence (5 '-3')	Remarks
			Oligo C (SEQ ID NO: 1)	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNGCTCGATTCT	Methylation modification of all C bases except N bases
Oligo D (SEQ ID NO: 2)	AGAATCGAGC	Methylation of all C bases

7. Oxidation reaction: reagents in the following table were thawed and mixed upside down and then immediately separated, and then sequentially added to the previous PCR tube (ensuring that the operation was performed on ice).

Reagent name	Volume (mu L)
		The product of the last step	20
TET2 reaction buffer	10
		Oxidation Supplement	1
DTT	1
		Oxidation Enhancer	1
TET2	4
		Nuclease-free water	8
Totals to	45

Gently stirring, mixing, centrifuging briefly, adding 5 μl of Fe (II) Solution, mixing thoroughly again, centrifuging briefly, and placing into a PCR instrument for the following reaction: (Note: table reagent is NEB company enzymatic methylation conversion Module kit (product number: E7125).

Temperature (. Degree. C.)	Time (min)
		Thermal cover 75	On
37	60

After the reaction was completed, 1. Mu.L of Stop Reagent was added on ice and incubated at 37℃for 30 min.

8. And (3) purifying a product: adding 80 mu L of nuuzuan purified magnetic beads into the product obtained in the previous step, fully mixing, standing at room temperature for 5 min, placing on a magnetic rack for about 5 min to enable the magnetic beads to be completely adsorbed and the solution to be clear, and carefully removing the supernatant; adding 200 μl of newly prepared 80% ethanol for rinsing, incubating at room temperature for 30-60 s, carefully removing supernatant, and repeating for one time; after the magnetic beads are dried, adding 18 mu L of ultrapure water for eluting, placing the magnetic beads at room temperature for 3 min, placing the magnetic beads on a magnetic rack, and sucking 16 mu L of supernatant for standby after the solution is clarified.

9. Deamination reaction: adding 4 mu L of formamide to the product of the previous step, fully mixing to obtain a mixture A, briefly centrifuging, incubating for 10min at 85 ℃, immediately placing the mixture on ice after the reaction is completed, and preparing a reaction system as follows:

Reagent name	Volume (mu L)
		Mixture A	20
Nuclease-free water	68
		APOBEC reaction buffer	10
BSA	1
		APOBEC	1
Totals to	100

Gently beating, mixing, centrifuging briefly, and placing into a PCR instrument to perform the following reaction: (Note: table reagent is NEB company enzymatic methylation conversion Module kit (product number: E7125).

Temperature (. Degree. C.)	Time (min)
		Thermal cover 75	On
37	180

10. And (3) purifying a product: adding 100 mu L of nuuzuan purified magnetic beads into the product obtained in the previous step, fully mixing, standing at room temperature for 5 min, placing on a magnetic rack for about 5 min to enable the magnetic beads to be completely adsorbed and the solution to be clear, and carefully removing the supernatant; adding 200 μl of newly prepared 80% ethanol for rinsing, incubating at room temperature for 30-60 s, carefully removing supernatant, and repeating for one time; after the magnetic beads are dried, 22 mu L of ultrapure water is added for elution, the mixture is placed at room temperature for 3 min and then placed on a magnetic rack, and 20 mu L of supernatant is sucked for standby after the solution is clarified.

11. Single-ended primer extension: the reaction system was formulated as follows:

Component (A)	Volume of
		The product of the last step	20
10X Taq polymerase buffer	10
		Taq DNA polymerase (10U/. Mu.L)	5
Primer Mix 3	5
		Blocking sequences	5
dNTP	5
		Totals to	50

(Note: the blocking sequence was synthesized by Shanghai Biotechnology at a concentration of 100. Mu.M, and the specific sequence and structure are shown in FIG. 3).

12. Single end extension product purification: adding 60 mu L of nuuzuan purified magnetic beads into the extension product, fully mixing, standing at room temperature for 5min, placing on a magnetic rack for about 5min to enable the magnetic beads to be completely adsorbed and the solution to be clarified, and carefully removing the supernatant; adding 200 μl of newly prepared 80% ethanol for rinsing, incubating at room temperature for 30-60 s, carefully removing supernatant, repeating for one time, adding 22 μl of ultrapure water for eluting after magnetic beads are dried, standing at room temperature for 3 min, placing in a magnetic rack, and sucking 20 μl of supernatant for use after the solution is clarified.

13. Amplification and enrichment of universal primers: using KAPA HiFi HotStart Uracil + ReadyMix (product number KK 2801) from Roche corporation and VAHTS Unique Dual Index Primer (product number 34401-01) from Northenan, the reaction system was prepared as follows:

Component (A)	Volume (mu L)
		The product of the last step	20
KAPA HiFi HotStart Uracil+ ReadyMix	25
		VAHTS Unique Dual Index Primer	5
Totals to	50

14. Purification of amplification products: adding 75 mu L of nuuzuan purified magnetic beads into the amplification product, fully mixing, standing at room temperature for 5 min, placing on a magnetic rack for about 5 min to enable the magnetic beads to be completely adsorbed, clarifying the solution, and carefully removing the supernatant; adding 200 μl of newly prepared 80% ethanol for rinsing, incubating at room temperature for 30-60 s, carefully removing supernatant, repeating for one time, adding 22 μl of ultrapure water for eluting after magnetic beads are dried, standing at room temperature for 3 min, placing in a magnetic rack, and sucking 20 μl of supernatant for use after the solution is clarified.

15. Sequencing on a machine: sequencing was performed using Illumina Novaseq6000 platforms, the data volumes were 5G each.

Comparison of experimental data:

As can be seen from the above table, the comparison of example 1 and example 2 shows a significant decrease (55%) in the genome alignment (MAPPING RATE%) in example 2, due to the unblocked UMI sequence, resulting in the binding of UMI sequence upon amplification of the specific primer, resulting in a large number of sequencable adaptor products.

Example 1 is inferior to example 3 in the ultra low frequency mutation detection result (0.1% -0.7%), and since the absence of UMI sequence cannot correct the mutation introduced by PCR amplification error, the ultra low frequency mutation cannot be detected effectively.

The results were substantially consistent compared to example 1 and example 4, but the cost of hybridization capture (example 4) was far higher than example 1.

The results are substantially identical compared to example 1 and example 5, indicating that C6 spacer has the same efficacy as C3 spacer.

The genome alignment of example 6 was expected to indicate that it was applicable to cfDNA pooling detection and that it was effective in increasing the amount of effective data compared to the comparative example.

Example 7 methylation rate detection values are consistent with theoretical values, indicating that the disclosed technique is applicable to methylation library sequencing.

Therefore, compared with the existing multiplex PCR amplification technology, the single-end multiplex primer extension technology adopted by the method has the advantages that the detection efficiency is greatly improved, errors introduced in PCR amplification and sequencing amplification can be corrected, mutation frequencies as low as 0.1% can be detected, the detection sensitivity is better than that of multiplex PCR 1%, the genome comparison rate is 99%, the effective data size is improved from 50% to 99%, and compared with the traditional multiplex PCR technology and hybrid capture technology, the accuracy is ensured, and meanwhile, the cost is greatly reduced. And the technical method is compatible with cfDNA, and can be applied to the field of methylation sequencing.

Claims

1. A linker for single-ended multiplex amplification to detect a mutation frequency of a gene, the linker comprising:

A short single strand;

2. The adaptor of claim 1, wherein the long single stranded unique molecular tag sequence is upstream of a sequencing primer sequence and downstream of the long single stranded unique molecular tag sequence is a specific sequence for the genome to be tested; the short single strand is the complement of the specific sequence.

3. The linker of claim 2 wherein said unique molecular tag comprises 2 to 12 random bases; the number of modified blocked bases is the same as the number of bases of the unique molecular tag sequence; the modified blocked base is selected from at least one of C3 spacer, C6 spacer, C12 spacer, spacer, spacer 18, dspacer.

4. A linker according to claim 3 wherein the unique molecular tag comprises 8 random bases.

5. A linker according to any one of claims 1 to 3, wherein the sequence of the long single strand is shown in SEQ ID No. 1; the sequence of the short single strand is shown in SEQ ID NO. 2.

6. The adaptor of claim 5, wherein the single-stranded sequence upstream and the C bases in the double-stranded sequence downstream of the unique molecular tag sequence of the incomplete double-stranded adaptor are further capable of containing methylation modifications.

7. Use of the linker of any one of claims 1 to 6 for library construction and sequencing of compatible genomic DNA, episomal DNA samples and methylation library construction and sequencing.

8. The use according to claim 7, comprising the steps of:

fragmenting the genomic DNA and purifying to recover fragmented DNA fragments;

performing end repair on the fragmented DNA fragments;

ligating the repaired DNA fragment with the adaptor of any one of claims 1 to 5; purification is then performed to recover the ligated DNA fragments;

Denaturing the connected DNA fragments to form single-stranded DNA with the linker, then adding an amplification system comprising a unique molecular tag blocking sequence and single-ended specific primers, performing linear amplification, and purifying an amplification product; amplifying and enriching the purified amplified product by using a universal primer, and purifying the enriched product; finally, carrying out on-machine sequencing on the purified enriched product; or alternatively

The application also includes in the construction and sequencing of methylation library:

fragmenting the genomic DNA and purifying to recover fragmented DNA fragments;

performing end repair on the fragmented DNA fragments;

ligating the repaired DNA fragment to the adaptor of claim 6; purification is then performed to recover the ligated DNA fragments;

9. An apparatus for executing the application of claims 7 to 8.

10. A kit for constructing a high throughput sequencing library, the kit comprising a linker according to any one of claims 1 to 6 or a linker involved in the use of claims 7 to 8, a unique molecular tag blocking sequence, a specific primer for single ended linear multiplex PCR amplification; and the kit further comprises a DNA fragmentation system, a terminal repair system, a ligation system, and an amplification system.