Multi-position double-tag connector set for detecting gene mutation and preparation method therefor and application thereof

Abstract

A multi-position double-tag adapter set for detecting gene mutation and preparation method therefor and application thereof, the multi-position double-tag adapter set comprising a double-tag adapter A, a double-tag adapter B and a double-tag adapter C. The double-tag adapter A, the double-tag adapter B and the double-tag adapter C are obtained respectively by hybridizing an adapter primer P5 with an adapter primer P7-A, an adapter primer P7-B and an adapter primer P7-C 5′ ends of which are all modified with biotin. Using the multi-position double-tag adapter set, the mutation rate of 1×10.sup.−5 genes may be accurately detected and the sensitivity of gene mutation detection may be effectively improved. A plurality of mutation sites of a plurality of genes may be detected by one-time sequencing in combination with throughput of high-throughput sequencing.

Claims

1. A multi-position double-tag adapter set for detecting gene mutation, comprising: a first double-tag adapter, a second double-tag adapter, and a third double-tag adapter, wherein: the first double-tag adapter, the second double-tag adapter, and the third double-tag adapter are respectively obtained by hybridizing an adapter primer P5 to a first adapter primer P7, a second adapter primer P7, and a third adapter primer P7, all 5′ ends of the first adapter primer P7, the second adapter primer P7, and the third adapter primer P7 are modified with biotin, the adapter primer P5 is obtained by connecting SEQ ID NO:01 with a sequence shown in SEQ ID NO:02 through an I5 index sequence, the first adapter primer P7 is obtained by serially connecting FFFFFEEEEEJJJJJNNNNNNNNNNNN with 5′ end of SEQ ID NO:03 and connecting SEQ ID NO:03 with SEQ ID NO:04 through an I7 index sequence, the second adapter primer P7 is obtained by serially connecting FFFFFEEEEEKKKKKNNNNNNNNNNNN with the 5′ end of SEQ ID NO:03 and connecting SEQ ID NO:03 with SEQ ID NO:04 through the I7 index sequence, the third adapter primer P7 is obtained by serially connecting FFFFFEEEEELLLLLNNNNNNNNNNNN with the 5′ end of SEQ ID NO:03 and connecting SEQ ID NO:03 with SEQ ID NO:04 through the I7 index sequence, the FFFFF defines protective bases for restriction sites, EEEEE defines the restriction sites, JJJJJ, KKKKK, and LLLLL define position-tag sequences, the JJJJJ, the KKKKK, and the LLLLL are different, NNNNNNNNNNNN defines a random tag sequence, a sequence of the I7 index sequence comprises 6 bases, and the NNNNNNNNNNNN comprises 4 to 12 random bases, in which no more than four continuous bases are identical.

2. The multi-position double-tag adapter set for detecting gene mutation according to claim 1, wherein: the NNNNNNNNNNNN is represented as BDHVBDHV, B represents a base other than A, D represents a base other than C, H represents a base other than G, and V represents a base other than T.

3. The multi-position double-tag adapter set for detecting gene mutation according to claim 1, wherein: the I5 index sequence is selected from SEQ ID NO:05-12, the I7 index sequence is selected from SEQ ID NO: 13-24, a sequence of the JJJJJ, the KKKKK, or the LLLLL is configured to be partially or completely overlapped with a sequence of the EEEEE, and when the sequence of the JJJJJ, the KKKKK, or the LLLLL is partially or completely overlapped with the sequence of the EEEEE, one or more bases of an overlapped part appear only once.

4. The multi-position double-tag adapter set for detecting gene mutation according to claim 1, wherein the FFFFF, the JJJJJ, the KKKKK, the LLLLL, and the EEEEE respectively comprise five identical bases.

5. A method for preparing the multi-position double-tag adapter set according to claim 1, comprising: (1) after mixing the adapter primer P5, the first adapter primer P7, the second adapter primer P7, the third adapter primer P7, a buffer, and deionized water, annealing to obtain a first annealed adapter, a second annealed adapter, and a third annealed adapter; (2) elongating the first annealed adapter, the second annealed adapter, and the third annealed adapter to obtain a first elongated adapter, a second elongated adapter, and a third elongated adapter by polymerase elongation; (3) purifying the first elongated adapter, the second elongated adapter, and the third elongated adapter to obtain a first purified elongated adapter, a second purified elongated adapter, and a third purified elongated adapter due to ethanol precipitation or isopropanol precipitation; (4) adding restrictive endonuclease capable of producing 3′T protruding ends into the first purified elongated adapter, the second purified elongated adapter, and the third purified elongated adapter, and performing enzyme digestion to obtain a first enzymatic digested adapter, a second enzymatic digested adapter, and a third enzymatic digested adapter; (5) purifying the first enzymatic digested adapter, the second enzymatic digested adapter, and the third enzymatic digested adapter to obtain the first double-tag adapter, the second double-tag adapter, and the third double-tag adapter due to ethanol precipitation or isopropanol precipitation; (6) purifying the first double-tag adapter, the second double-tag adapter, and the third double-tag adapter to obtain a product due to a biotin-avidin system; and (7) after purifying the product to obtain the multi-position double-tag adapter set by ethanol precipitation or isopropanol precipitation.

6. A method for preparing the multi-position double-tag adapter set according to claim 2, comprising: (1) after mixing the adapter primer P5, the first adapter primer P7, the second adapter primer P7, the third adapter primer P7, a buffer, and deionized water, annealing to obtain a first annealed adapter, a second annealed adapter, and a third annealed adapter; (2) elongating the first annealed adapter, the second annealed adapter, and the third annealed adapter to obtain a first elongated adapter, a second elongated adapter, and a third elongated adapter by polymerase elongation; (3) purifying the first elongated adapter, the second elongated adapter, and the third elongated adapter to obtain a first purified elongated adapter, a second purified elongated adapter, and a third purified elongated adapter due to ethanol precipitation or isopropanol precipitation; (4) adding restrictive endonuclease capable of producing 3′T protruding ends into the first purified elongated adapter, the second purified elongated adapter, and the third purified elongated adapter, and processing enzyme digestion to obtain a first enzymatic digested adapter, a second enzymatic digested adapter, and a third enzymatic digested adapter; (5) purifying the first enzymatic digested adapter, the second enzymatic digested adapter, and the third enzymatic digested adapter to obtain the first double-tag adapter, the second double-tag adapter, and the third double-tag adapter due to ethanol precipitation or isopropanol precipitation; (6) purifying the first double-tag adapter, the second double-tag adapter, and the third double-tag adapter to obtain a product due to a biotin-avidin system; and (7) after purifying the product to obtain the multi-position double-tag adapter set by ethanol precipitation or isopropanol precipitation.

7. A method for preparing the multi-position double-tag adapter set according to claim 3, comprising: (1) after mixing the adapter primer P5, the first adapter primer P7, the second adapter primer P7, the third adapter primer P7, a buffer, and deionized water, annealing to obtain a first annealed adapter, a second annealed adapter, and a third annealed adapter; (2) elongating the first annealed adapter, the second annealed adapter, and the third annealed adapter to obtain a first elongated adapter, a second elongated adapter, and a third elongated adapter by polymerase elongation; (3) purifying the first elongated adapter, the second elongated adapter, and the third elongated adapter to obtain a first purified elongated adapter, a second purified elongated adapter, and a third purified elongated adapter due to ethanol precipitation or isopropanol precipitation; (4) adding restrictive endonuclease capable of producing 3′T protruding ends into the first purified elongated adapter, the second purified elongated adapter, and the third purified elongated adapter, and processing enzyme digestion to obtain a first enzymatic digested adapter, a second enzymatic digested adapter, and a third enzymatic digested adapter; (5) purifying the first enzymatic digested adapter, the second enzymatic digested adapter, and the third enzymatic digested adapter to obtain the first double-tag adapter, the second double-tag adapter, and the third double-tag adapter due to ethanol precipitation or isopropanol precipitation; (6) purifying the first double-tag adapter, the second double-tag adapter, and the third double-tag adapter to obtain a product due to a biotin-avidin system; and (7) after purifying the product to obtain the multi-position double-tag adapter set by ethanol precipitation or isopropanol precipitation.

8. A method for constructing a library, comprising: after breaking 10 ng-1 μg of deoxyribonucleic acid (DNA) into 200-500 bp of DNA fragments: adding terminal repair enzymes into the DNA fragments to repair terminals of the DNA fragments and adding A-tails, adding the multi-position double-tag adapter set according to claim 1 for a connection with the DNA fragments, and selecting 340-660 bp of fragments using Ampure magnetic beads or gel cutting after the connection is complete.

9. A method for sequencing a library, comprising: (1) constructing the library by the method for constructing the library according to claim 8; and (2) sequencing the library.

10. A method for analyzing a nucleic acid sequence, comprising: (1) constructing the library by the method for constructing the library according to claim 8; (2) sequencing a sequence of the library; and (3) analyzing results of the sequencing, wherein a method for the analyzing the results comprises: a. selecting a unique matching sequence for sequencing with a base Q value greater than 30 according to preset parameters; b. judging duplication according to the random tag sequence, and processing base rectifying; c. detecting to obtain single nucleotide polymorphisms (SNP) sites by a software; and d. comparing the SNP sites with mutant sites of a control group and a population genome mutation database, filtering out identical mutant sites, and defining finally remaining mutant site information as final detecting mutant site information.

11. A method for constructing a library, comprising: after breaking 10 ng-1 μg of deoxyribonucleic acid (DNA) into 200-500 bp of DNA fragments: adding terminal repair enzymes into the DNA fragments to repair terminals of the DNA fragments, adding A-tails, adding the multi-position double-tag adapter set according to claim 2 for a connection with the DNA fragments, and selecting 340-660 bp of fragments using Ampure magnetic beads or gel cutting after the connection is complete.

12. A method for sequencing a library, comprising: (1) constructing the library by the method for constructing the library according to claim 11; and (2) sequencing the library.

13. A method for analyzing a nucleic acid sequence, comprising: (1) constructing the library by the method for constructing the library according to claim 11; (2) sequencing a sequence of the library; and (3) analyzing results of the sequencing, wherein a method for the analyzing the results comprises: a. selecting a unique matching sequence for sequencing with a base Q value greater than 30 according to preset parameters; b. judging duplication according to the random tag sequence, and processing base rectifying; c. detecting to obtain single nucleotide polymorphisms (SNP sites) by a software; and d. comparing the SNP sites with mutant sites of a control group and a population genome mutation database, filtering out identical mutant sites, and defining final remaining mutant site information as final detecting mutant site information.

14. A method for constructing a library, comprising: after breaking 10 ng-1 μg of deoxyribonucleic acid (DNA) into 200-500 bp of DNA fragments: adding terminal repair enzymes into the DNA fragments to repair terminals of the DNA fragments, adding A-tails, adding the multi-position double-tag adapter set according to claim 3 for a connection with the DNA fragments, and selecting 340-660 bp of fragments using Ampure magnetic beads or gel cutting after the connection is complete.

15. A method for sequencing a library, comprising: (1) constructing the library by the method for constructing the library according to claim 14; and (2) sequencing the library.

16. A method for analyzing a nucleic acid sequence, comprising: (1) constructing the library by the method for constructing the library according to claim 14; (2) sequencing a sequence of the library; and (3) analyzing results of the sequencing, wherein a method for the analyzing the results comprises: a. selecting a unique matching sequence for sequencing with a base Q value greater than 30 according to preset parameters; b. judging duplication according to the random tag sequence, and processing base rectifying; c. detecting to obtain single nucleotide polymorphisms (SNP) sites by a software; and d. comparing the SNP sites with mutant sites of a control group and a population genome mutation database, filtering out identical mutant sites, and defining final remaining mutant site information as final detecting mutant site information.

Description

DRAWINGS

(1) FIG. 1 is a schematic diagram of a single position double-tag adapter prepared by embodiment 1 of the present invention.

(2) FIG. 2 is a schematic diagram of the effect of flat-end adapter (residual elongated adapter) on sequencing libraries in the present invention.

(3) FIG. 3 is a schematic diagram of a double-tag adapter of introducing Index by PCR in the present invention.

(4) FIG. 4 is a flow chart for the construction of a single position and double-tag adapter Library in embodiment 2 of the present invention.

(5) FIG. 5 is a flow chart for identifying cell mutations by single position double-tag adapter in embodiment 3 of the present invention.

(6) FIG. 6 is a schematic diagram of the preparation process of the multi-position double-tag adapter set in embodiment 4 of the present invention.

DETAILED DESCRIPTION

(7) The following is a further description and description of the technical scheme of the present invention through specific embodiments in conjunction with the accompanying drawings.

Embodiment 1: Preparation of Single Position Double-Tag Adapter

(8) Two primers, adapter primer P5 and adapter primer P7 (the adapter primer P5 was obtained by SEQ ID NO:01 ligating the sequence of SEQ ID NO: 02 with 15 index sequence, the adapter primer P7 was obtained by SEQ ID NO:03 ligating the sequence of SEQ ID NO: 04 with 17 index sequence, wherein, FFFFFEEEEEJJJJJNNNNNNNNNNNN, in turn connecting to 5′ end of SEQ ID NO:03; synthetic manufacturers: Bioengineering (Shanghai) Co., Ltd.) were diluted with ddH.sub.2O (or TE buffer) to 100 μM.

(9) Wherein FFFFF is the protective base of restriction site, EEEEE is the restriction site, DDDDD is the position tag sequence, NNNNNNNNNNNN is random molecule maker, the 15 index sequence is selected from SEQ ID NO:5-12. The 17 index sequence is selected from SEQ ID NO:13-24.

(10) Meanwhile, FFFFF/DDDDD/EEEEE/includes but is not limited to five identical bases. NNNNNNNNNNNNNNN is 4 to 12 random bases, and there are no four consecutive identical bases.

(11) The preparation method of the single position double-tag adapter is as follows (as shown in FIG. 3):

(12) (1) Annealing: The following system was prepared in 0.2 mL EP pipe: adapter primer P5:10 μL, adapter primer P7: 10 μL, NEB buffer2: 3 μL, ddH2O: 7 μL; in total 30 μL. The system was annealed on a polymerase chain reaction (PCR) instrument: 95° C., 5 min; gradient cooling of 95° C.˜24° C., 0.2-0.5° C./s; maintenance at 24° C.;

(13) (2) Amplified annealed fragments: In the original PCR tube, add: 10×NEB buffer: 2 μL, 10 mM dNTP mix: 5 μL, ddH2O: 8 μL, Klenow exo-(5U/μL): 5 μL, 50 μL in total. After mixing, it was placed at 37° C. for 1 hour.

(14) (3) First precipitation: 1/10 volume of NaAC (3M) and 2.5 times volume of absolute ethanol were added to the product of the step (2), and then mixed and placed at −20° C. for 2 h; centrifuged at 13000 g for 30 min; the supernatant was removed, 600 mu L 70% ethanol was added for rinsing and precipitating, centrifuged at 13000 g and 4° C. for 30 min; after the supernatant was removed, the DNA was dried at room temperature for 5-10 minutes, and the DNA was re-suspended with 30 μL ddH.sub.2O.

(15) (4) Enzymatic hydrolysis (for example, HpyCH4III endonuclease, restriction site: ACNGT, the corresponding primer P7 sequence EEEEE is ACAGT): The product obtained by step (3) was 30 μL and 5 μL 10×NEB CutSmart buffer was added:ddH.sub.2O: 10 μL, HpyCH4III(5 U/μL): 5 μL, 50 μL in total. After mixing, enzymatic hydrolysis was carried out at 37° C. for 16 hours.

(16) (5) Second precipitation: 1/10 volume of NaAC (3M) and 2.5 times volume of absolute ethanol were added to the product of the step (4), and then mixed and placed at −20° C. for 2 hours; centrifuged for 30 min at 14 000 g and 4° C.; the supernatant was removed, 600 μL 70% ethanol was added for rinsing and precipitating, centrifuged at 13000 g and 4° C. for 30 min; The supernatant was removed, the DNA was dried at room temperature for 5-10 minutes, and the DNA was re-suspended with 26 μL TE low buffer. The final single position double-tag adapter (25 μM, the structure as shown in FIG. 2) was obtained and sub-packed at 5 μL and frozen at −80° C. for reserve.

Embodiment 2: Detection of Plasma DNA Mutation Rate by Single Position Double-Tag Adapter

(17) In this embodiment: The protective base of the single position double-tag adapter prepared by embodiment 1 is TCTTCT. The sequence of restriction sites was (position base in box, partial overlap of restriction site and position base). The molecular tag is BDHVBDHV.

(18) The combination of I5 index sequence and I7 index sequence may be:I501-I701, I502-I702, I503-I703, I504-I704, I505-I705, I506-I706, I507-I707, I508-I708, I501-I707, I502-I708, I503-I709, I504-I710. (The base sequence corresponding to the serial number is shown in Table 1)

(19) Sample selection and quality control: Five plasma samples from patients with lung cancer were collected, and plasma DNA was extracted by QIAGEN plasma DNA extraction kit. The purity of DNA samples was determined by spectrophotometer (A260/280 was required to be between 1.8 and 20). Then the DNA concentration was determined by Qubit 2.0 (the total amount was between 5-15 ng), the DNA fragment distribution was detected by D1000 chip (Agilent) and the mutation rate of EGFR gene T790M sites (1.9%, 0.8%, 0.18%, 0.12% and 1.44%) was determined by digital PCR (Bio-rad).

(20) Library Construction: The KAPA DNA library kit was used to construct the library, and all DNA samples were used to construct the library.

(21) KAPA HTP Library Preparation Kit Illumina® Platforms, end-repair enzymes and end-repair buffers used in the following experiments were all derived from the kit.

(22) End repair of DNA samples (adding 7 μL 10× end repair buffer, 5 μL end repair enzyme, 20° C., 30 min); after purification, the product was added A-tail with A-taling enzyme (5 μL 10× end repair buffer, 3 μL end repair enzyme 30° C., 30 min), the product was divided into two parts after purification, and the single position double-tag adapter prepared in Embodiment 2 was used in the connecting step (a single position double tag adapter was added to the fragment with the A tail according to a 10:1 molar ratio) to build the library (As shown in FIG. 4) or ordinary building adapter (the sequence was as SEQ ID NO: 24 and 25), 10 μL of 5× ligation buffer+5 μL of T4 DNA ligase was added and connect at 20° C. for 20 min, the product was purified by two steps of 1× Ampure magnetic beads, and the purified product was amplified by KAPA high fidelity enzyme mix (25 μL) and upstream and downstream amplification primers (25 μM) with 1 μL each.

(23) Wherein the common library building adapter was added as a control. In the experimental set, the single position double-tag adapter which was prepared in embodiment 2 was added. The steps of the control group were the same as those of the experimental set, but the sequence of adapters used were different.

(24) The upstream and downstream amplification primers used in the common Library building adapter sample set were general primers (SEQ ID NO:5) and index primers (SEQ ID NO:6). The upstream and downstream primers used in the single position and double-tag adapter sample set prepared by embodiment 2 were PCR-P5 primers+PCR-P7 primers.

(25) Common Library building adapter sequence information:

(26) TABLE-US-00003 SEQ ID NO: 24 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3′ SEQ ID NO: 25 3′-CTGACCTCAAGTCTGCACACGAGAAGGCTAG-p-5′

(27) The sequence of upstream and downstream primers corresponding to common Library building adapters:

(28) TABLE-US-00004 Universal primers: SEQ ID NO: 26 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCT CTTCCGATC-s-T-3′ (-s-denotes thio, the same as the following) Index primers: obtained by SEQ ID NO: 27 connecting SEQ ID NO: 28 through I7 index sequence: SEQ ID NO: 27 5′-CAAGCAGAAGACGGCATACGAGAT SEQ ID NO: 28 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3′

(29) Wherein I7 Index sequence is selected from SEQ ID NO:13-24.

(30) The sequence of P5 and P7 primers corresponding to the single position double-tag junction prepared by embodiment 1: When the adapters were added to the single position double-tag adapters prepared by embodiment 1, the following primer sequences were used:

(31) TABLE-US-00005 PCR-P5: SEQ ID NO: 29 AATGATACGGCGACCACCG-s-A PCR-P7: SEQ ID NO: 30 CAAGCAGAAGACGGCATACG-s-A

(32) Capture: According to Roche SeqCap EZ custom kit (250k) the library target capture is carried out, the capture library was qualified and subjected to sequencing (Agilent 2100/2200 judges the size distribution of the library fragment, for example, the size of the insert (template) is 200-350 bp when the library is constructed, after two end adapters P5,P7 were added, it increase 140 bp, the size of the library should be 340 bp-490 bp; QPCR was used to judge the capture effect—if the average enrichment factor is less than 10, the capture failed and needed to be recaptured).

(33) Result: The sequencing depth of each sample was 20000× and the raw data of the sample of sequence was 8.20 G, clean data Q20 was 94.25%, Q30 was 0.3%, mapping rate was 99.9% and coverage was 99.89%. In terms of detection results, two of 1.9% and 1.44% of the sample mutation sites can be accurately detected in the common adapter sample set, while all sample mutation sites of 1.9%, 0.8%, 0.18%, 0.12% and 1.44% can be detected in the single position and double-tag adapter sample (based on the mutation sites and mutation rate information detected by digital PCR before the establishment of the database, high-throughput sequencing data were analyzed by software (FastQC, samtools, BWA/bowtie2, GATK, Freebayes/picard, etc.), to analyze whether these sites have mutations and mutation rates, compare them with the results of digital PCR, and determine the detection rate). The detection rate was 100% (compared with the results of digital PCR, if digital PCR detects 10 low-frequency mutation sites in these 5 samples, if high-throughput sequencing can detect all 10 sites, the detection rate is 100%, if five sites were detected, the detection rate would be 50%).

Embodiment 3: Detection of Mutation Rate of Single Position Double-Tag Adapter Cell Lines

(34) NCI-H1650 and HCT cell lines were selected as experimental materials. NCI-H1650 cell DNA was incorporated into HCT cell DNA in 10%, 1% and 0.1% mass ratio, respectively. In addition, 100% DNA of NCI-H1650 and HCT cell were used as two samples, respectively, corresponding to 10%, 1%, 0.1%, NCI-H1650 and HCT sets. (NCI-H1650 and HCT sets are only used to determine the genetic background of DNA used for mixing proportions, i.e. allele sites information, such as heterozygosity and homozygosity. Through the sequencing information of these two samples, some homozygous base sites can be found, and then the sites with different bases at the same site can be selected as statistical analysis sites for other sample sets.)

(35) DNA samples were fully blended and then taken 2 μg for DNA library preparation (KAPA DNA library kit). Wherein 10%, 1% and 0.1% samples were equally divided into two sets after adding A-tail step, adding common adapters (such as SEQ ID NO:3 and SEQ ID NO:4) and single position double-tag adapter (as shown in FIG. 5) prepared in embodiment 1, and then subsequent library preparation and capture steps were carried out. Roche SeqCap EZ custom kit (250k) is used in capture step, and finally sequenced on the computer. The depth of the sequence is 2000×. The sequencing results are detected by filtered Q30 unique mapping reads.

(36) the single position double-tag adapter FFFFF is TCTTCT and EEEEE is ACAGT;DDDDD is AGT; it overlaps with the EEEEE sequence mentioned above.

(37) The NNNNNNNNNNNN is showed as BDHVBDHV, wherein B indicates that the position is a base other than A, D indicates that the position is a base other than C, H indicates that the position is a base other than G, V indicates that the position is a base other than T.

(38) Result: Firstly, the data of NCI-H1650 and HCT samples were analyzed, and the base MAF (secondary allele frequency) in the 250 Kbp capture region of Roche capture chip was found according to the SNP detection information. The base sites with 0% of MAF (SNP homozygous negative) and 100% of base sites (SNP homozygous positive) were screened out (the actual criterion is to set a threshold, such as 0.1%. If the MAF value of a site is less than 0.1%, it is considered that the sites is 0% base site, that is, SNP homozygous negative sites; 100% sites is analogous in turn). Screening the corresponding sites in two cell lines (the same position in the genome), one sites was homozygous positive and the other was homozygous negative. These sites were used as the statistical detection rate and false-positive and false-negative information of the analysis site samples of other sample sets.

(39) In NCI-H1650 and HCT sets (100%) 178 homozygous allele SNP sites were detected (i.e., each site was homozygous negative in one cell line and homozygous positive in another cell line). Then, 10%, 1% and 0.1% samples of different adapters were analyzed for these 178 sites, and the mutation rate of 178 sites in different proportion samples (heterozygosity) were 10%, 1% and 0.1% respectively. The positive detection rate of common adapter was 100% in 10% sample set, 98.86% in 1% sample set and 81.29% in 0.1% sample set. The detection rates of single position and double-tag adapters prepared by embodiment 1 were 100% in 10%, 1% and 0.1% sets. False positive rate: under 1% sensitivity, the false positive rate of common adapters was 0.01%, and under 0.1% sensitivity, the false positive rate of common adapters was more than 5%. The false positive rate of single position double-tag adapter prepared by embodiment 1 was 0.001% at the sensitivity of 0.1%. (The site whose sensitivity value exceeded a certain threshold was considered to be the detected mutation site. For example, 1% sensitivity was defined as the mutation site whose threshold value of base mutation frequency was 1%, and the site whose sensitivity value exceeded 1% was considered to be the detected mutation site.)

Embodiment 4: Preparation of Multi-Position Double-Tag Adapter Set

(40) adapter primers P7-A, P7-B and P7-C (synthesizer: Biotechnology and Bioengineering (Shanghai) Co., Ltd.) were diluted with ddH.sub.2O to 100 μM, respectively.

(41) The adapter primer P5 is obtained by SEQ ID NO:01 ligating the sequence shown in SEQ ID NO: 02 with I5 index sequence;

(42) FFFFFEEEEEJJJJJNNNNNNNNNNNN, in turn connect to 5′ end of SEQ ID NO:03, and SEQ ID NO:03 connect SEQ ID NO:04 through I7 index sequence, then the adapter primers P7-A is obtained;

(43) FFFFFEEEEEKKKKKNNNNNNNNNNNN, in turn connect to 5′ end of SEQ ID NO:03, and SEQ ID NO:03 connect SEQ ID NO:04 through I7 index sequence, then the adapter primers P7-B is obtained;

(44) FFFFFEEEEELLLLLNNNNNNNNNNNN, in turn connect to 5′ end of SEQ ID NO:03, and SEQ ID NO:03 connect SEQ ID NO:04 through I7 index sequence, then the adapter primers P7-C is obtained;

(45) the FFFFF is the protective base of the restriction site, EEEEE is the restriction site, JJJJJ, KKKKK and LLLLL are the position-tag sequences, and JJJJJ, KKKKK and LLLLL are different, NNNNNNNNNNNN is the random molecular tag sequence. FFFFF, JJJJJ, KKKKK, LLLLL and EEEEE contain but are not limited to five identical bases. The sequence of I7 index is 6-8 bases. NNNNNNNNNNNN is 4 to 12 random bases, and there are no four consecutive identical bases.

(46) Preferably, the NNNNNNNNNNNN is showed as BDHVBDHV, wherein B indicates that the position is a base other than A, D indicates that the position is a base other than C, H indicates that the position is a base other than G, V indicates that the position is a base other than T. The 15 index sequence is selected from SEQ ID NO:05˜12; The I7 index sequence is selected from SEQ ID NO:13˜23.

(47) Preferably, the sequences of JJJJJ, KKKKK and LLLLL can overlap partially or completely with the sequences of EEEEE. When the sequences are partially or completely overlapped, the base of the overlapped part appears only once.

(48) The preparation process of the multi-position double-tag adapter set is as follows (FIG. 6):

(49) (1) Annealing: after mixing the adapter primers P5, P7-A, P7-B, P7-C, buffer and proper deionized water, annealing treatment was carried out to obtain annealed adapter A, annealed adapter B and annealed adapter C; specifically: the following systems are prepared in a 15 mL centrifugal tube: adapter primer P5:1 mL, P7-A:334 μL, P7-B:334 μL, P7-C:334 μL, NEB buffer2: 300 μL, ddH.sub.2O: 700 μL; 3 mL in total. After mixing the system, the following reactions were carried out: In the water bath pot at 95° C. for 5 minutes. Then immediately it is putted into a beaker filled with hot water at 95° C., slowly cooling to 24-27° C. at room temperature.

(50) (2) Elongating annealed adapters: the obtained annealed adapter A, annealed adapter B and annealed adapter C were elongated by polymerase chain reaction to obtain elongated adapter A, elongated adapter B and elongated adapter C. In the original 15 mL centrifugal tube, add: 10×NEB buffer: 200 μL, 25 mM dNTP mix: 200 μL, 500 mM DTT: 6 μL, Klenow exo-(5 U/μL): 100 μL, supplemented with ddH.sub.2O to 5 mL. After blending, the mixture was rotated in a 37° C. thermostat and incubated for 1 hour.

(51) (3) First precipitation: the purified elongated adapter A, B and C were obtained by precipitation and purification with ethanol or isopropanol, respectively, specifically 1/10 volume of NaAC (3M) and 2.5 times volume of absolute ethanol were added to the product of the step (2), and then mixed and placed at −20° C. for 2 h; centrifuged at 13000 g for 30 min; the supernatant is removed, 5 ml 70 volume % ethanol was added to rinse and precipitate, centrifuged at 13000 g and 4° C. for 30 min; The supernatant was removed, the DNA was dried at room temperature for 20-30 minutes, the DNA was suspended with 3 mL ddH.sub.2O, and the concentration was measured by Quantus.

(52) (4) Enzyme digestion: the purified elongated adapter A, B and C were added to restrictive endonuclease capable of producing 3′T protruding ends, respectively, for enzyme digestion, and the enzymatic digested adapter A, B and C were obtained; specifically (Take HpyCH4III endonuclease as an example, the restriction site: ACNGT, corresponding primer P7 sequence EEEEEEE was changed to ACAGT):the product obtained from the above step (3) is added 10×NEB CutSmart buffer according to its mass x(μg): 2×μL, HpyCH4III(5 U/μL): 2×μL, supplemented with ddH.sub.2O to 20×μL, mixed, incubated in a 37° C. incubator, enzymatic hydrolysis for 16 h;

(53) (5) Second precipitation: the obtained digested adapter A, the digested adapter B and the digested adapter C are subjected to ethanol or isopropanol precipitation to obtain a double-tag adapter A, a double-tag adapter B and a double-tag adapter C; specifically: 1/10 volume of NaAC and 2.5 times volume of absolute ethanol were added to the product of the step (4), and then mixed and placed at −20° C. for 2 hours; centrifuged for 30 min at 14 000 g and 4° C.; the supernatant was removed, 10 m L 70% ethanol was added or rinsing and precipitating, centrifuged at 13000 g and 4° C. for 30 min; the supernatant was removed, the DNA were dried at room temperature for 20-30 minutes and suspended with 2 mL ddH.sub.2O.

(54) (6) Biotin purification: the affinity purification of biotin was carried out on the double-tag adapter A, the double-tag adapter B and the double-tag adapter C obtained in step (5); specifically:2 mL Dynabeads MyOne Streptavidin C1 magnetic beads were rinsed with 1×B&W buffer magnetic beads and then re-suspended with 2 mL 2×B&W buffer magnetic beads. 2 mL the products obtained from step (5) were added to the magnetic beads, incubated at 4° C. for 30 minutes, and placed on the magnetic rack, and the supernatant was taken to a new 50 mL centrifugal tube.

(55) (7) Third precipitation: after precipitating and purifying the product obtained in step (6) with ethanol or isopropanol, the multi-position double-tag adapter set is obtained, specifically: 1/10 volume of NaAC and 2.5 times volume of absolute ethanol were added to the product of the step (6), and then mixed and placed at −20° C. for 2 hours; centrifuged for 30 min at 14 000 g and 4° C.; the supernatant was removed, 10 m L 70% ethanol was added or rinsing and precipitating, centrifuged at 13000 g and 4° C. for 30 min; the supernatant is removed, the DNA is dried at room temperature for 20-30 minutes and re-suspended with 1.5 mL TE low buffer, that is, the multi-position double-tag adapter set, which is subpacked after qualified, is frozen at −20° C. for reserve.

Embodiment 5: Improvement of Sequencing PF Value of the Multi-Position Double-Tag Adapter Set

(56) A library was constructed after 30 ng interrupted Leukocyte DNA (average length 220 bp). The experiment was divided into two sets. One set constructed the library with single position double-tag adapter prepared by embodiment 1 and the other set constructed the library with multi-position double-tag adapter set prepared by embodiment 4. The library kit uses NEBNext Ultra II DNA Library Prep Kit. The library construction steps are as follows: (1) 30 ng interrupted DNA was added into 7 μL NEBNext ULtra II End Prep Reaction Buffer and 3 μL NEBNext ULtra II End Prep Enzyme Mix, and the volume was supplemented with deionized water to 60 μL. The DNA was at 20° C., 30 min.fwdarw.65° C., 30 min.fwdarw.4° C. maintained on the PCR. (2) In the system, 1 μL adapter (the single position double-tag adapter or the multiple-position double-tag adapter set) was added, then a 30 μL NEBNext ULtra II Ligation Master Mix and 1 μL NEBNext Ligation Enhancer were added, and the reaction time was 15 minutes after mixing at 20° C. The conjugated product was purified by 0.9× Ampure magnetic beads and eluted by 23 μL purified water. (3) 23 μL of the above conjugated product, 1 μL (25 μM) of each of the I5 and I7 index primers, NEBNext ULtra III Q5 Master Mix respectively were added to the PCR tube. After mixing, the following reactions were performed on the PCR apparatus:

(57) 98° C., 30s;

(58) 98° C., 10s.fwdarw.65° C., 75s (8 cycles);

(59) 65° C., 5 min;

(60) Maintenance at 4° C.

(61) After the completion of the PCR, the purified product was purified with 0.9× magnetic beads, and then the quality control was carried out with Qubit 2.0 (or Quantus) and Agilent 2100 Bioanalyzer (or Agilent 2200 TapeStation).

(62) After the quality control of the library is qualified, the NextSeq500 platform is used for sequencing. The sequencing reagent is Mid Output Kit (300 cycles). Phix incorporation ratio is 1%. Each library is sequenced separately on the computer. The experiment was repeated three times and sequenced on computer respectively. The sequencing platform was NextSeq500, the sequencing reagent was Mid Output kit (300 cycles), and Phix incorporation rate was 1%. The quality control of the sequencing results was as follows:

(63) TABLE-US-00006 Cluster Phix PF Experience set density ratio (%) value Q30 Single Position Double-tag 190 K/mm.sup.2 1.2% 33.80% 90.4% connector Library-1 Single Position Double-tag 186 K/mm.sup.2 0.8% 31.50% 85.8% connector Library-2 Single Position Double-tag 200 K/mm.sup.2 1.5% 33.20% 82.0% connector Library-3 Multi-Position and 200 K/mm.sup.2 0.9%   87% 88.6% Double-tag connector set Library-1 Multi-Position and 181 K/mm.sup.2 1.2%   90% 87.3% Double-tag connector set Library-2 Multi-Position and 210 K/mm.sup.2 1.3%   91% 90.1% Double-tag connector set Library-3

Embodiment 6: After Purification of Biotin, Residue of the Sequence of Adapter Library Building

(64) The library was constructed with 30 ng interrupted Leukocyte DNA (average length 220 bp), the experiment was divided into two sets: one set constructed the library with single position double-tag adapter prepared by embodiment 1, the other set constructed the library with multi-position double-tag adapter set prepared by embodiment 4, and the library kit was NEBNext ULtra II DNA Library Prep Kit. The steps of library construction are as follows: (1) 30 ng interrupted DNA was added into 7 μL NEBNext ULtra II End Prep Reaction Buffer and 3 μL NEBNext ULtra II End Prep Enzyme Mix, and the volume was supplemented with deionized water to 60 μL. The DNA was at 20° C., 30 min.fwdarw.65° C., 30 min.fwdarw.4° C. maintained on the PCR. (2) In the system, 1 μL adapter (the single position double-tag adapter or the multiple-position double-tag adapter set) was added, then a 30 μL NEBNext ULtra II Ligation Master Mix and 1 μL NEBNext Ligation Enhancer were added, and the reaction time was 15 minutes after mixing at 20° C. The conjugated product was purified by 0.9× Ampure magnetic beads and eluted by 23 μL purified water. (3) 23 μL of the above conjugated product, 1 μL (25 μM) of each of the I5 and I7 index primers, NEBNext ULtra III Q5 Master Mix respectively were added to the PCR tube. After mixing, the following reactions were performed on the PCR apparatus:

(65) 98° C., 30s;

(66) 98° C., 10s.fwdarw.65° C., 75s (8 cycles);

(67) 65° C., 5 min;

(68) Maintenance at 4° C.

(69) After the completion of the PCR, the purified product was purified with 0.9× magnetic beads, and then the quality control was carried out with Qubit 2.0 (or Quantus) and Agilent 2100 Bioanalyzer (or Agilent 2200 TapeStation).

(70) After the quality control of the library is qualified, the NextSeq500 platform is used for sequencing. The sequencing reagent is Mid Output Kit (300 cycles). Phix incorporation ratio is 1%. The amount of data in each library was 1 GB. The sequencing results were as follows:

(71) TABLE-US-00007 Read1 Read2 Data unMapped connector connector Experience set amount Q30 % residue residue Single Position Double-tag 955.13M 85.03% 1.50% 4.66% 0.15% connector Library-1 Single Position Double-tag 1.09 G 84.86% 1.39% 4.64% 0.11% connector Library-2 Single Position Double-tag 1.13 G 84.89% 1.44% 4.12% 0.16% connector Library-3 Multi-Position and 956.17M 84.68% 0.99% 0.07% 0.07% Double-tag connector set Library-1 Multi-Position and 1.14 G 84.82% 0.57% 0.08% 0.06% Double-tag connector set Library-2 Multi-Position and 1.08 G 85.50% 0.51% 0.11% 0.07% Double-tag connector set Library-3

(72) As mentioned above, it is only a better embodiment of the present invention, so the scope of implementation of the present invention can not be limited accordingly. That is, the equivalent changes and modifications made according to the patent scope and description content of the present invention should still be within the scope of the present invention.

INDUSTRIAL APPLICABILITY

(73) The invention provides a multi-position double-tag adapter set for detecting gene mutation, a preparation method and a specific application thereof. The gene mutation rate of 1×10.sup.−5 may be accurately detected, the sensitivity of gene mutation detection may be effectively improved. Combined with the throughput of high-throughput sequencing, one-time sequencing can detect multiple mutation sites of multiple genes.