CN111471746A - NGS library preparation joint for detecting low mutation abundance sample and preparation method thereof - Google Patents
NGS library preparation joint for detecting low mutation abundance sample and preparation method thereof Download PDFInfo
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Abstract
The invention provides a joint for preparing an NGS library for detecting a low mutation abundance sample and a preparation method thereof, relating to the technical field of genetic engineering, wherein the joint contains primer sequences P5 and P7 and also comprises a sequencing primer site SP sequence; sequences of umis; c. sample tag Index sequence. The 3 'end of the P5 end is sequentially connected with a sequencing primer site SP sequence, a UMIS sequence and a sample label Index sequence, and the 5' end of the P7 end is sequentially connected with the sequencing primer site SP sequence, the UMIS sequence and the sample label Index sequence. The adapter sequence uses the uniquely designed sample label and the sequence position of the UMIs, so that the adapter sequence is particularly suitable for but not limited to the construction of a sample library with the insert fragment size of 100-250bp, and in addition, the double-ended UMIs greatly improve the recognition capability of the adapter sequence on false positive mutation, thereby being more suitable for the detection of low mutation abundance samples.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to an NGS library preparation joint for detecting a low mutation abundance sample and a preparation method thereof.
Background
Next Generation Sequencing (NGS) can sequence millions of DNA fragments simultaneously. In order to achieve high throughput parallel sequencing, the target DNA to be tested must have specific NGS linkers at both ends. The basic structure of the NGS linker comprises P5, P7 primer sequences and sequencing primer binding sequences, which must be arranged in a well-designed order in the asymmetric Y-linker. In addition, the advanced NGS adapters may contain other sequences, such as sample tag sequences (indexes) and single Molecule Identification Sequences (UMIs), which may be located at different positions of the adapter in different adapter designs and in different quantities. The basic structure of the NGS linker is an asymmetric structure of the two strands forming a Y-shaped strand, meaning that a portion of the sequences of the two strands (P5 and P7 oligonucleotides) can be complementary to form a double strand, while the other portion of the oligonucleotide chain carries sequences that are not complementary depending on the different functions of each strand. In the basic structure of the joint, different types of joints have been developed to satisfy different functions. For different purposes, the NGS adaptor has different design modes, but the existing NGS adaptor is not particularly suitable for constructing a sample (such as cfDNA) library with the insert fragment size of 100-250bp, and the false positive mutation recognition capability is low, so that the NGS adaptor is not suitable for detecting a low mutation abundance sample.
Disclosure of Invention
In order to solve the problems that the existing NGS adaptor is not particularly suitable for the construction of a sample (such as cfDNA) library with the insert fragment size of 100-250bp, and the false positive mutation identification capability is low, so that the existing NGS adaptor is not suitable for the detection of a low mutation abundance sample, the invention provides a novel NGS library preparation adaptor for detecting the low mutation abundance sample, which is particularly suitable for the construction of a sample (such as cfDNA) library with the insert fragment size of 100-250bp, and a preparation method thereof.
Firstly, the invention provides a NGS library preparation joint for detecting a low mutation abundance sample, which comprises a P5 terminal oligonucleotide chain and a P7 terminal oligonucleotide chain, wherein the oligonucleotide chain contains primer sequences of P5 and P7, and also comprises a sequencing primer site SP sequence; sequences of umis; c. sample tag Index sequence; the oligonucleotide chain at the P5 terminal comprises a P5 primer sequence, the 3 'terminal of the oligonucleotide chain is sequentially connected with a sequencing primer site SP sequence, a connected UMIS sequence and a sample label Index sequence, the oligonucleotide chain at the P7 terminal comprises a P7 primer sequence, and the 5' terminal of the oligonucleotide chain is sequentially connected with a sequencing primer site SP sequence, a connected UMIS sequence and a sample label Index sequence; the P5 and P7 terminal oligonucleotide chain has reverse complementary sequences except the P5 and P7 primer sequences; the joint prepared by the NGS library is of a Y-shaped structure, and the primer sequences of P5 and P7 are respectively two ends above the Y shape.
The primer sequence P5/P7 enables the adaptor to be suitable for an Illumina sequencing platform, the SP sequence of the sequencing primer site enables the adaptor to perform double-end sequencing on the Illumina platform, and the sequence UMIs enables the adaptor to be more suitable for sequencing low mutation abundance samples.
The sequence of the UMIs can be a random oligonucleotide of 1-16 bases.
The sample tag Index sequence may be a 1-16 base oligonucleotide.
Preferably, the sequences of the UMIs and the sequence of the sample tag Index are located between the SP sequence and the insert, and the positions of the sequences of the UMIs and the sequence of the sample tag Index are interchangeable. This structure makes the linker particularly suitable for the construction of a library of samples with insert sizes of 100 and 250 bp.
Preferably, the linker may contain a 3' T cohesive end to facilitate ligation to the insert.
Preferably, the positive and negative strands of the linker may contain a phosphate group at their 5' ends.
Based on the above technical solution, preferably, the base sequences of the positive strand and the negative strand of the linker prepared by the NGS library provided by the present invention may be:
and (3) positive strand: the 5 'end sequence is shown in SEQ ID NO.1, and is sequentially connected with [ UMI ] - [ Index ] and a 3' end sequence ACT, and the sequence is as follows:
5’-/5Phoa/
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-[UMI]-[Index]-ACT-3’
negative chain: the 5 'end sequence GT is sequentially connected with [ Index ] - [ UMI ] and a 3' end sequence shown in SEQ ID NO.2, and the sequence is as follows:
5’-/5Phoa/GT-[Index’]
-[UMI’]-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3’
secondly, the invention also provides a preparation method of the NGS library preparation adaptor for detecting the low mutation abundance sample, which comprises the following steps:
(1) synthesis of the P5-terminal oligonucleotide strand and the P7-terminal oligonucleotide strand:
the oligonucleotide strand comprises a P5/P7 primer sequence and further comprises a. a sequencing primer site SP sequence; sequences of umis; c. sample tag Index sequence.
(2) Synthesizing and preparing a linker:
1) performing chain fusion on the P5 terminal oligonucleotide chain synthesized in the step (1) and the P7 terminal oligonucleotide chain;
2) performing chain extension on the DNA obtained by the chain fusion in the step 1);
3) and (3) carrying out enzyme digestion on the product DNA obtained in the step 2) to prepare a T cohesive end at the double-stranded end.
The sample tag Index sequence of step (1) may be a 1-16 base oligonucleotide which functions to label samples each containing a unique tag. The base length of the sample label Index sequence can be correspondingly adjusted according to the requirements of a sequencing platform, and the base sequence can refer to a bar code sequence provided by the corresponding sequencing platform. The number of random bases can also be selected according to the sequencing depth requirement of the library, and more random bases can be used when the sequencing depth requirement is higher.
The P5 and P7 oligonucleotide chains can be synthesized by selecting from nucleic acid synthesizing companies which are commonly found on the market.
Preferably, the step (1) of synthesizing the NGS library for detecting low mutation abundance samples prepares the adaptor, and in the using process, the sequences of the UMIs and the sequences of the sample tag Index are positioned between the sequences of the SP and the insert, and the positions of the sequences of the UMIs and the sequences of the sample tag Index can be interchanged.
The insertion sequence is the sequence to be detected.
Preferably, the 5' end of the synthesized P5 and P7 terminal oligonucleotide chain in step (1) may contain a phosphate group.
Preferably, the sequences of the synthetic P5 and P7 terminal oligonucleotide chains in step (1) can be:
the sequence of the P5 terminal oligonucleotide chain is shown as SEQ ID NO.3, and the details are as follows:
5’-/5Phoa/
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’
the sequence of the P7 terminal oligonucleotide chain is: the 5 'end sequence is shown in SEQ ID NO.4, and is sequentially connected with [ Index ] - [ UMI ] and a 3' end sequence shown in SEQ ID NO.5, and the sequence is as follows:
5’-/5Phoa/
TCTTCTACAGT-[Index]-[UMI]-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3’
step 1) the chain fusion specifically comprises:
① dissolving the synthesized P5 and P7 terminal oligonucleotide chains, and adjusting the concentration to 1-100uM, wherein the concentrations of the two solutions are equal;
②, mixing the P5 and P7 terminal oligonucleotide chain solutions prepared in the step ① in equal volume, adding equal volume of chain fusion buffer solution, and mixing uniformly;
③ 95 deg.C for 10 min;
④ naturally cooling to room temperature;
⑤ precipitating DNA from the mixed solution;
⑥ washing the DNA precipitate with ethanol solution, air drying, and dissolving with water or TE.
The lysis described in step ① is performed using enzyme-free water or TE buffer or the like.
The chain fusion buffer solution in step ② may be a buffer solution commonly used in biological experiments, such as Tris-buffered saline (TAPS), Bicine-buffered N, N-bis (2-hydroxyethyl) glycine, etc.
The precipitation in step ⑤, using absolute ethanol precipitation or isopropanol precipitation, may be facilitated by the addition of 1/10 volumes of 3M sodium acetate solution.
The ethanol solution of step ⑥ is a 70% wt ethanol solution.
Step 2) the step of performing chain extension on the DNA obtained by the chain fusion in the step 1) comprises the following steps:
① preparing an extension system containing Klenow enzyme;
② incubating the system at 37 deg.C for 10min to 24 h;
③ precipitating DNA from the mixed solution;
④ washing the DNA precipitate with ethanol solution, air drying, and dissolving with water or TE.
The Klenow enzyme-containing extension system described in step ① is shown in the following table:
the precipitation in step ③, using absolute ethanol precipitation or isopropanol precipitation, may be facilitated by the addition of 1/10 volumes of 3M sodium acetate solution.
The ethanol solution of step ④ is a 70% wt ethanol solution.
The step 3) of carrying out enzyme digestion on the product DNA obtained in the step 2) comprises the following steps:
① preparing enzyme digestion system containing HPYCH4III enzyme;
② incubating the system at 37 deg.C for 10min to 24 h;
③ precipitating DNA from the mixed solution;
④ washing the DNA precipitate with ethanol solution, air drying, and dissolving with water or TE.
The cleavage system described in step ① is shown in the following table:
the precipitation in step ③, using absolute ethanol precipitation or isopropanol precipitation, may be facilitated by the addition of 1/10 volumes of 3M sodium acetate solution.
The ethanol solution of step ④ is a 70% wt ethanol solution.
Advantageous effects
The invention adds a sample label sequence index. A pre-designed 6-10 base sequence (namely a sample label) is added to any one/or two oligonucleotide chains (P5 or P7) of the joint, different samples use different sample labels, namely, a plurality of samples can be mixed and detected during high-throughput sequencing, the utilization rate of a sequencing flow cell is improved, and a sequencing result can be split according to the sample label sequence during data analysis. A tag sequence is added to only the P7 oligonucleotide and is referred to as a single-ended tag, while a tag sequence is added to both the P5 and P7 oligonucleotides and is referred to as a double-ended tag.
In addition, the present invention uses single molecule identifiers (UMIs) in the linker. The UMIs are located on the P5 or/and P7 strands and are short random nucleotide sequences. This short random oligonucleotide strand results in each individual linker molecule potentially containing a different sequence. In contrast to the sample tag function, linkers containing different UMIs are ligated to the individual inserts in the library preparation such that the inserts contain different UMI sequences, corresponding to the addition of a molecular tag to each insert. The function of the primer is to identify whether a section of sequencing result is from the same insert, so that the sequencing deviation introduced in experimental operation, such as base mutation introduced in PCR amplification, can be detected, and therefore, the linker containing the double-ended UMIs is extremely useful in detecting low mutation abundance samples (such as plasma free DNA).
Aiming at different purposes, the NGS adaptor has different design modes, the adaptor sequence in the invention uses a uniquely designed sample label and the position of the UMIs sequence, so that the invention is particularly suitable for but not limited to the construction of a sample (such as cfDNA) library with the insert fragment size of 100-250bp, and in addition, the double-ended UMIs greatly improve the identification capability of the double-ended UMIs on false positive mutation, thereby being more suitable for the detection of low mutation abundance samples.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the means particularly pointed out in the written description and claims thereof.
The technical solution of the present invention is further described in detail by the following examples.
Drawings
FIG. 1 is a schematic structural diagram of an NGS library adaptor for detecting low mutation abundance samples provided by the invention; wherein, P5 is P5 primer sequence; p7 is P7 primer sequence; SP is a sequencing primer sequence; UMI is single molecule identification; index sample tag sequence; t is the 3' thymine cohesive end;
FIG. 2 is a schematic view of a joint preparation process; wherein, P5 is P5 primer sequence; p7 is P7 primer sequence; SP is a sequencing primer sequence; UMI is single molecule identification; index sample tag sequence; t is the 3' thymine cohesive end;
FIG. 3 shows the results of qPCR amplification detection of the library, wherein the abscissa represents the number of cycles and the ordinate represents the fluorescence intensity Δ Rn;
FIG. 4 shows the construction of the NGS library in which Insert is the insertion sequence, i.e., the sequence to be tested.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Example 1 preparation of NGS library preparation adaptors for detection of low mutation abundance samples:
(1) synthesis of oligonucleotide chain
The sequence of the P5 terminal oligonucleotide chain is shown as SEQ ID NO.3, and the details are as follows:
5’-/5Phoa/
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’
the sequence of the P7 terminal oligonucleotide chain is: the 5 'end sequence is shown in SEQ ID NO.4, and is sequentially connected with [ Index ] - [ UMI ] and a 3' end sequence shown in SEQ ID NO.5, and the sequence is as follows:
5’-/5Phoa/
TCTTCTACAGT-[Index]-[UMI]-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3’
wherein, the sample label sequence index is shown in the following table, and the UMI sequence is 10 random nucleotide sequences.
The P5 and P7 terminal oligonucleotide chains can be synthesized by selecting from nucleic acid synthesis companies commonly available on the market.
The P5 and P7 terminal oligonucleotide chains contain a phosphorylated group at the 5' terminus.
(2) Linker synthesis and preparation
The method for preparing the linker by using the synthesized oligonucleotide chain comprises the following steps: fusion, extension, and digestion (FIG. 2).
1) Oligonucleotide chain fusion
① the above synthesized oligonucleotide was dissolved sufficiently in enzyme-free water, TE buffer solution or the like, and the concentration was adjusted to 20 uM.
② mixing equal volumes of the well-adjusted concentration of the P5 and P7 terminal oligonucleotide solutions, adding equal volumes of chain fusion buffer solution, and mixing well, wherein the chain fusion buffer solution is trihydroxymethyl methylaminopropane sulfonic acid buffer solution (TAPS).
③ the mixture was incubated at 95 ℃ for 10 minutes.
④ naturally cooling to room temperature.
⑤ the mixture was precipitated by absolute ethanol precipitation during which 1/10 volumes of 3M sodium acetate solution were added to facilitate DNA precipitation.
⑥ DNA was washed 2 times with 70% wt ethanol solution, air dried, and dissolved by adding an appropriate amount of TE.
2) Oligonucleotide chain extension
① the DNA prepared above was subjected to chain extension using Klenow enzyme according to the following table.
② the mixture is incubated at 37 ℃ for 10 minutes to 24 hours.
③ the mixture was precipitated by absolute ethanol precipitation during which 1/10 volumes of 3M sodium acetate solution were added to facilitate DNA precipitation.
④ DNA was washed 3 times with 70% wt ethanol solution, air dried, and added with appropriate amount of enzyme-free water.
3) Digestion by enzyme
① the purpose of the digestion was to create T cohesive ends at the double stranded ends the double stranded linkers that were used to complete the chain extension were digested as described below.
② the mixture is incubated at 37 ℃ for 10 minutes to 24 hours.
③ the DNA in the mixture was precipitated by absolute ethanol precipitation during which 1/10 volumes of 3M sodium acetate solution were added to facilitate precipitation of the DNA
④ DNA was washed 3 times with 70% wt ethanol solution, air dried, and dissolved by adding an appropriate amount of TE.
Example 2 DNA library construction was performed using NGS library preparation adaptors prepared in example 1 to detect low mutation abundance samples:
1. the long-fragment genome DNA is broken by using ultrasonic waves to be fragmented, the length range of the DNA fragment is 100-250bp, and the peak value is about 180 bp.
2. End repair and tailing of the fragmented DNA. This step can be performed using various commercially available DNA end repair and tailing modules.
3. Ligation of the inserts to the linkers was performed as follows. Wherein the dosage ratio of the adaptor to the DNA sample can be 1:1-100: 1. The ligase mixture can be T4DNA ligase or other ligase with similar functions and buffer solution thereof.
4. And (3) fully and uniformly mixing the mixed solution, and quickly centrifuging.
5. Incubate at 4-37 ℃ for 10 minutes until overnight ligation.
6. Add 1 × volumes (60ul) of AmPure XP beads.
7. Mix with shaking for 1 minute.
8. The incubation was carried out at room temperature for 5 minutes to allow the DNA to bind well to the magnetic beads.
9. The centrifuge tube was placed on a magnetic rack and allowed to stand for 3 minutes until the solution became transparent and clear.
10. The supernatant was carefully removed and discarded.
11. The tube was held on a magnetic stand, 200ul of freshly prepared 80% wt ethanol solution was added, and allowed to stand for 30 seconds.
12. Carefully aspirate the ethanol solution.
13. Repeating the steps 11-12 once.
14. The tube was placed on the magnetic stand for about 5 minutes, the lid was kept open, and the beads were dried.
15. The tube was removed from the magnetic stand, 20ul of sterile water or TE buffer was added, and the beads were resuspended by pipetting with a pipette tip.
16. The tube was left to stand at room temperature for 2 minutes. And (4) performing quick centrifugation.
17. The tube was placed back on the magnetic stand and allowed to stand for 3 minutes until the solution was clear.
18. The supernatant was carefully aspirated with a pipette tip.
19. Library qPCR amplification assays were performed according to the following table system.
20. qPCR amplification was performed according to the following table procedure (fig. 2 and 3). FIG. 3 shows a graph of the results of qPCR amplification during library preparation using initial amounts of fragmented genomic DNA of 10ng,50ng,100ng and 200ng, respectively. It can be seen that the linker effect is more stable and increases linearly with the initial amount of DNA.
21. For library preparation and amplification, Platinum from Thermo Fisher can be usedTMSuperFiTMPCRMaster Mix or other similar high fidelity amplificationsThe amplification enzyme replaces Power up SYBR Mix to amplify the library, and the amplification cycle number can be 5-25 cycles according to the required amount of the library. The structure of the NGS library used in the preparation is shown in fig. 4.
The nucleotide sequence table number and corresponding biological sample related to the invention are described as follows:
the 5' terminal sequence of the positive strand of the joint 1 of SEQ ID NO. 1;
3' terminal sequence of negative strand of linker 1 of SEQ ID NO. 2;
the sequence of the oligonucleotide chain at the end of the connector 2P5 of SEQ ID NO. 3;
the 5' terminal sequence of the oligonucleotide chain sequence at the end of the connector 2P7 of SEQ ID NO. 4;
the 3' terminal sequence of the oligonucleotide chain sequence at the end of the connector 2P7 of SEQ ID NO. 5.
It will be apparent to those skilled in the art that various modifications can be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include such modifications.
Sequence listing
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Claims (9)
1. An NGS library preparation adaptor for detecting a low mutation abundance sample, which is characterized in that: the primer comprises a P5 terminal oligonucleotide chain and a P7 terminal oligonucleotide chain, wherein the oligonucleotide chain contains primer sequences of P5 and P7, and also comprises a sequencing primer site SP sequence; sequences of umis; c. sample tag Index sequence; the oligonucleotide chain at the P5 terminal comprises a P5 primer sequence, the 3 'terminal of the oligonucleotide chain is sequentially connected with a sequencing primer site SP sequence, a connected UMIS sequence and a sample label Index sequence, the oligonucleotide chain at the P7 terminal comprises a P7 primer sequence, and the 5' terminal of the oligonucleotide chain is sequentially connected with a sequencing primer site SP sequence, a connected UMIS sequence and a sample label Index sequence; the P5 and P7 terminal oligonucleotide chain has reverse complementary sequences except the P5 and P7 primer sequences; the joint prepared by the NGS library is of a Y-shaped structure, and the primer sequences of P5 and P7 are respectively two ends above the Y shape.
2. The NGS library-generated adaptor for detecting low mutation abundance samples according to claim 1, wherein: the sequence of the UMIs is a random oligonucleotide with 1-16 bases.
3. The NGS library-generated adaptor for detecting low mutation abundance samples according to claim 1, wherein: the sample tag Index sequence is a section of 1-16 base oligonucleotide.
4. The NGS library-generated adaptor for detecting low mutation abundance samples according to claim 1, wherein: the linker contains a 3' T cohesive end.
5. The NGS library-generated adaptor for detecting low mutation abundance samples according to claim 1, wherein: the 5' ends of the positive chain and the negative chain of the joint contain a phosphorylation group.
6. A method for preparing an NGS library preparation adaptor for detecting a low mutation abundance sample according to any one of claims 1-5, wherein the method comprises the following steps: the method comprises the following steps:
(1) synthesis of the P5-terminal oligonucleotide strand and the P7-terminal oligonucleotide strand:
the oligonucleotide strand comprises a P5/P7 primer sequence and further comprises a. a sequencing primer site SP sequence; sequences of umis; c. sample tag Index sequence;
(2) synthesizing and preparing a linker:
1) performing chain fusion on the P5 terminal oligonucleotide chain synthesized in the step (1) and the P7 terminal oligonucleotide chain;
2) performing chain extension on the DNA obtained by the chain fusion in the step 1);
3) and (3) carrying out enzyme digestion on the product DNA obtained in the step 2) to prepare a T cohesive end at the double-stranded end.
7. The method for preparing an NGS library adaptor for detecting low mutation abundance samples according to claim 6, wherein the NGS library adaptor comprises: step 1) the chain fusion specifically comprises:
① dissolving the synthesized P5 and P7 terminal oligonucleotide chains, and adjusting the concentration to 1-100uM, wherein the concentrations of the two solutions are equal;
②, mixing the P5 and P7 terminal oligonucleotide chain solutions prepared in the step ① in equal volume, adding equal volume of chain fusion buffer solution, and mixing uniformly;
③ 95 deg.C for 10 min;
④ naturally cooling to room temperature;
⑤ precipitating DNA from the mixed solution;
⑥ washing the DNA precipitate with ethanol solution, air drying, and dissolving with water or TE.
8. The method for preparing an NGS library adaptor for detecting low mutation abundance samples according to claim 6, wherein the NGS library adaptor comprises: step 2) the step of performing chain extension on the DNA obtained by the chain fusion in the step 1) comprises the following steps:
① preparing an extension system containing Klenow enzyme;
② incubating the system at 37 deg.C for 10min to 24 h;
③ precipitating DNA from the mixed solution;
④ washing the DNA precipitate with ethanol solution, air drying, and dissolving with water or TE.
9. The method for preparing an NGS library adaptor for detecting low mutation abundance samples according to claim 6, wherein the NGS library adaptor comprises: the step 3) of carrying out enzyme digestion on the product DNA obtained in the step 2) comprises the following steps:
① preparing enzyme digestion system containing HPYCH4III enzyme;
② incubating the system at 37 deg.C for 10min to 24 h;
③ precipitating DNA from the mixed solution;
④ washing the DNA precipitate with ethanol solution, air drying, and dissolving with water or TE.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113293205A (en) * | 2021-05-24 | 2021-08-24 | 深圳市真迈生物科技有限公司 | Sequencing method |
| CN113981043A (en) * | 2021-11-22 | 2022-01-28 | 广州迈景基因医学科技有限公司 | Method for preparing second-generation sequencing linker |
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