CN112592968B - Molecular tag joint for high-throughput sequencing and synthesis method and application thereof - Google Patents
Molecular tag joint for high-throughput sequencing and synthesis method and application thereof Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C—CHEMISTRY; METALLURGY
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The invention discloses a molecular label joint for high-throughput sequencing and a synthesis method and application thereof, wherein a sequence 1 and a sequence 2 are heated in a hybridization working buffer solution to synthesize a joint A; heating the sequence 3 and the sequence 4 in a hybridization working buffer solution to synthesize a joint B; and mixing the joint A and the joint B to obtain the molecular tag joint for high-throughput sequencing. Aiming at the problems of complex synthesis, detection result accuracy needing to be improved and the like of the existing molecular tag of the Illumina sequencing platform, the invention discloses a novel molecular tag joint for high-throughput sequencing and a synthesis method and application thereof.
Description
Technical Field
The invention belongs to biotechnology, and particularly relates to a molecular tag joint for high-throughput sequencing, and a synthesis method and application thereof, which are used for an Illumina sequencing platform.
Background
Circulating tumor DNA (ctDNA) exists in peripheral blood of a tumor patient, is free DNA (cfDNA) released by tumor cells after necrosis and apoptosis, has short half-life in blood, and can reflect dynamic changes of tumors in real time. The number of tumor cells in a tumor patient body is far lower than that of normal cells, the content of cfDNA in plasma is very low, ctDNA only accounts for 0.1% -5% of the cfDNA, and the content difference of the ctDNA in the plasma of the tumor patients with different disease courses is large in different cancer types, so that the detection of the ctDNA needs higher sensitivity and specificity compared with tissue detection.
Currently, the second generation sequencing technology (NGS) for ctDNA liquid biopsy detects multiple different variants of multiple genes simultaneously, and is the most widely used gene detection technology. However, because the NGS experiment process is complicated, some amplification and sequencing errors, called background noise, are inevitably introduced during the library construction, target region capture and sequencing process, and ctDNA detection is often low in mutation frequency and greatly interfered by the background noise, and low-frequency mutations from the ctDNA sample are often submerged in the background noise to cause false negative or false positive results, which limits the sensitivity and specificity of ctDNA detection.
The Molecular barcode is also called a Molecular marker (UMI) and is based on the principle that a specific marker sequence is added to each original DNA fragment, and sequencing is performed together after library construction and PCR amplification. According to different label sequences, DNA templates from different sources can be distinguished, and false positive mutations caused by random errors in the PCR amplification and sequencing process and true mutations carried by a patient are distinguished, so that the detection sensitivity and specificity are improved. The existing molecular label has high synthesis cost, complex process, easy error generation and unstable detection result accuracy.
Disclosure of Invention
Aiming at the problems of complex synthesis, detection result accuracy needing to be improved and the like of the existing molecular tag of the Illumina sequencing platform, the invention discloses a novel molecular tag joint for high-throughput sequencing and a synthesis method and application thereof, and the molecular tag joint has the advantages of low cost, simple and convenient annealing operation, poor base balance and high production success rate.
The invention adopts the following technical scheme:
a molecular tag joint for high-throughput sequencing consists of a joint A and a joint B; the joint A is synthesized by a sequence 1 and a sequence 2; linker B was synthesized from SEQ ID Nos. 3 and 4.
A gene high-throughput sequencing method comprises the following steps:
(1) synthesizing a linker A from the sequence 1 and the sequence 2; synthesizing a linker B from the sequence 3 and the sequence 4; mixing the joint A and the joint B to obtain a molecular label joint;
(2) fragmenting nucleic acid, repairing tail end, adding A, and connecting with the molecular tag to obtain a joint connection product;
(3) and after the joint connection product is subjected to PCR amplification, sequencing the final amplification product to complete gene high-throughput sequencing.
In the invention, the sequence 1 and the sequence 2 are heated in a hybridization working buffer solution to synthesize a linker A; heating the sequence 3 and the sequence 4 in a hybridization working buffer solution to synthesize the linker B. Hybridization working buffers include KCl, Tris-HCl, Triton X-100, and water. Heating at 55-58 ℃ for 25-35 minutes, then heating at 35-40 ℃ for 13-16 hours, preferably heating at 56 ℃ for 30 minutes, and then heating at 37 ℃ for 15 hours; further, the temperature is reduced to 4 ℃ after heating and is kept.
In the invention, the sequence 1 is AATGATACGGCGACCACCGAGATCTACAC [ i5] ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNT; the sequence 2 is NNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC [ i7] ATCTCGTATGCCGTCTTCTGCTTG; the sequence 3 is AATGATACGGCGACCACCGAGATCTACAC [ i5] ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNVT; the sequence 4 is BNNNNNAGATCGGAAGAGACACGTCTGAACTCCAGTCAC [ i7] ATCTCGTATGCCGTCTTCTGCTTG.
In the invention, N is selected from A, T, C, G, V is selected from A, C, G, and B is selected from T, G, C. Specifically, N is selected from A, T, C, G, the feeding proportion of A, T, C, G bases at the N position is 25% respectively when the sequences are synthesized, and the synthesized sequences are random mixtures; v is selected from A, C, G, the feeding proportion of A, C, G bases at the N position is 1/3 when the sequence is synthesized, and the synthesized sequence is a random mixture; b is selected from T, G, C, the feeding proportion of T, C, G bases at B position is 1/3 when synthesizing the sequence, and the synthesized sequence is a random mixture.
Preferably, in sequence 1, the 2 nd nucleotide at the 3 terminal is a locked nucleic acid; in the sequence 2, the 1 st nucleotide at the 5 end is locked nucleic acid; in the sequence 3, the 2 nd nucleotide at the 3 end is locked nucleic acid; in the sequence 4, the 1 st nucleotide at the 5 end is a locked nucleic acid. The use of locked nucleic acids ensures that base pairing is strong and aligned annealing is performed at the ends, avoiding the problem of weak binding due to individual base mismatches at random N-pairing.
The invention adopts the joint A and the joint B to form the molecular label joint for high-throughput sequencing, realizes the effect of base balance at specific fixed positions, avoids the unbalance of some fixed-position bases and improves the sequencing accuracy.
In the present invention, [ i5] and [ i7] are index sequences consisting of 8-base sequences, such as: AATGACGT, ATTGAGGT, etc., is a single sequence determined, is a conventional technique, and does not influence the realization of the technical effect of the invention.
In the invention, the conventional technology of fragmenting nucleic acid, repairing tail end and adding A is adopted for a sample, and the sample is a gene for high-throughput sequencing; reacting the sample added with the A with a molecular label joint in the presence of a connection buffer solution and DNA ligase to obtain a joint connection product; the reaction is carried out at 20 ℃ for 15 minutes and then maintained at 4 ℃, preferably, after the reaction is finished, the reaction product is purified by magnetic beads and eluted by water to obtain supernatant which is the adaptor-ligated product.
In the present invention, a 50. mu.L system was used for PCR amplification, and the reaction procedure was heating at 95 ℃ for 3 minutes, then cycling at 98 ℃ for 20 seconds +60 ℃ for 15 seconds +72 ℃ for 15 seconds for 4 times, followed by heating at 72 ℃ for 1 minute, and finally holding at 4 ℃.
In the present invention, the reaction is completed when the temperature is lowered to 4 ℃ and maintained, which is a conventional method in the art. Sequencing of the final amplification product is prior art. The specific formula of the PCR system is selected according to a sample to be detected, and for example, a multiplex primer, an amplification reagent, an i7 primer, an i5 primer, product purification and the like are all selected conventionally.
The invention discloses a new four groups of base sequences, wherein a connector A is synthesized from a sequence 1 and a sequence 2, a connector B is synthesized from a sequence 3 and a sequence 4, and a molecular label connector for high-throughput sequencing is composed of the connector A and the connector B; on the basis of low cost, the annealing operation is simple and convenient, and the base balance is good, so that the detection result accuracy is very high when the method is used for high-throughput sequencing.
Drawings
FIG. 1 shows the distribution of 4N combinations of UMI joints according to the present invention;
FIG. 2 is a 4N combinatorial distribution of non-locked nucleic acid UMI linkers;
FIG. 3 is the detection result of the molecular tag linker for high throughput sequencing according to the present invention;
FIG. 4 shows the detection results of the molecular tag linker for high throughput sequencing according to the present invention.
Detailed Description
In the invention, the synthesis of the sequence 1, the sequence 2, the sequence 3 and the sequence 4 adopts the prior art, the sequence 1 and the sequence 2 are annealed to synthesize the linker A, the sequence 3 and the sequence 4 are annealed to synthesize the linker B, and then the linker A and the linker B are mixed to obtain the molecular tag linker for high-throughput sequencing. The synthesis of the specific sequences in the prior art obtains dry powder, namely four groups of dry powder, corresponding to the sequence 1, the sequence 2, the sequence 3 and the sequence 4, each group of dry powder comprises a plurality of sequences, and the synthesis of each sequence only needs one charging, thereby avoiding the problem that the prior art needs multiple charging.
Except for the sequence 1 to the sequence 4, the other reagents (raw materials) involved in the invention are all commercial products, and the specific operation method and the test method are all conventional methods in the field.
Example one
1. Sequence Synthesis
First group of joints
Sequence 1:
5-AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNT
sequence 2:
----3-GTTCGTCTTCTGCCGTATGCTCTA[i7]CACTGACCTCAAGTCTGCACACGAGAAGGCTAGANNNNN
second group of joints
And (3) sequence:
5-AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNVT
and (4) sequence:
----3-GTTCGTCTTCTGCCGTATGCTCTA[i7]CACTGACCTCAAGTCTGCACACGAGAAGGCTAGANNNNNB
n (ATCG): during synthesis, the feeding proportion of ATCG basic groups at the N position is respectively 25%, and the synthesized product is a random mixture;
v (ACG): the feeding proportion of ACG base at the V position is 33 percent respectively during synthesis, and the synthesized product is a random mixture;
b (TGC): during synthesis, the feeding proportion of TCG base at the position B is 33 percent respectively, and the synthesized product is a random mixture;
[i5] and [ i7] is an index sequence consisting of 8-position bases, which are respectively ACATCAGC and GCTTGTGA;
in the sequence 1, the 2 nd nucleotide at the 3 end is locked nucleic acid; in the sequence 2, the 1 st nucleotide at the 5 end is locked nucleic acid; in the sequence 3, the 2 nd nucleotide at the 3 end is locked nucleic acid; in the sequence 4, the 1 st nucleotide at the 5 end is a locked nucleic acid.
The synthesis method of the above sequence of the present invention is a conventional method.
2. UMI joint annealing
The dry powders synthesized in the sequences 1, 2, 3 and 4 are respectively melted by pure water into stock solutions with the concentration of 100uM in 4 tubes.
Configuring annealing buffer
Take the double-labeled linker with annealing number 40ul 15uM as an example:
2.1, diluting the concentration of the hybridization buffer solution by using pure water to be 1X hybridization working buffer solution;
2.2 numbering the first set (100 uM sequence 1, 100uM sequence 2) of linkers to anneal, the second set (100 uM sequence 3, 100uM sequence 4) of linkers;
and 2.3, taking out 2 PCR tubes, marking the numbers of corresponding annealing joints by tube caps, adding corresponding components according to the following table, carrying out vortex mixing, centrifuging, and sealing the centrifugal tube caps.
2.4, place PCR tube in PCR, set hot lid temperature to 40 ℃, set program as follows:
56 ℃/30min +37 ℃/15h, cooling to 4 ℃ for storage; the joint structure formed after annealing is shown below:
during sequence synthesis, the marked position is synthesized by adopting locked nucleic acid; the invention creatively provides random N pairing, and the combination of the random N pairing and the locked nucleic acid avoids the infirm combination caused by the mismatching of individual bases, thereby ensuring the firmness and the alignment annealing at the tail end and improving the detection preparation.
2.5 the annealed 1 st and 2 nd joints were mixed in equal amounts to obtain 80ul of 15uM joints, called UMI joints, which were used in the following experiments.
In the UMI joint, 4N combinations are distributed as shown in figure 1; for comparison, the locked nucleic acids in SEQ ID Nos. 1 to 4 were replaced with conventional nucleic acids, and the rest were not changed, and the distribution of 4N combinations in the F-UMI linker (non-locked nucleic acid UMI linker) was shown in FIG. 2. The results of routine sequencing were as follows:
application examples
And (3) verifying the detection effect of the UMI joint of the invention by taking 8 mutation types of EGFR mutation genes as detection samples and setting 2 negative control samples, and comparing the effect with that of a comparison joint.
Preparation of the experiment:
preparation before routine experiments: and (3) taking out Serapure Beads 30min before the experiment, shaking and centrifuging, vertically placing, and shaking and centrifuging before actual use.
Experimental procedure
4.1 Rapid DNA fragmentation/end-filling-in-and-adding A
A investment of 100ng for constructing a library
A-1, putting each reagent on ice, melting, reversing, mixing uniformly, centrifuging, and preparing the following reaction in a sterilized PCR tube:
and A-2, covering an eight-connecting-tube cover, reversing, mixing uniformly, and centrifuging for a short time.
A-3, placing into a PCR instrument (Langgy A100/T30), and operating according to the following table, wherein the program name of the PCR instrument is < TG1 >.
And A-4, immediately carrying out a joint connection reaction.
Joint connection
4.2.1 thawing 5 × Ligase buffer, mixing, and placing on ice for use.
4.2.2 the components in the following table were added to the reaction tube after the end of the reaction in the above step.
4.2.3 mix by turning over the eight-piece tube cap and centrifuging briefly.
4.2.4 into a PCR machine (Langgy A100/T30) having the program name < TG 2.
4.2.5 immediate purification.
Sample purification operation steps:
1) the PCR tube was briefly centrifuged. Adding 130 ul of uniformly mixed Serapure magnetic beads (1:1.3) into every 100 ul of reaction solution, fully and uniformly mixing, and standing for 2 min at room temperature;
2) placing the PCR tube on a magnetic frame, standing for 5 min, and discarding the supernatant;
3) adding 200 mul of 75% ethanol into each tube on a magnetic frame, and removing supernatant;
4) repeating the step 3) once, discarding the supernatant as much as possible, and standing at room temperature until the residual ethanol is completely volatilized;
5) adding 23ul of eluent (10 mM Tris-HCl, pH 8.0-pH 8.5) for elution, fully mixing, standing at room temperature for 5 min, placing a magnetic frame for standing for 2 min, and taking 23ul of supernatant to be stored in a new PCR to be used as a purified Adapter Ligation product.
Library amplification (using KAPA reagent)
4.3.1 prepare the amplification reaction according to the following table:
4.3.2 mix well and centrifuge briefly.
4.3.3 the reaction program in the following Table, program name < KAPAKZ > of PCR apparatus (BioRad T100) was performed.
4.3.4 after the reaction, the quantity of the Qubit HS is determined, the electrophoresis is carried out, the bands are concentrated between 200-400bp, and the first step of initial denaturation is omitted when circulation is carried out, so that the circulation is directly carried out.
Purification after amplification
And (3) a purification step:
1) the PCR tube was centrifuged briefly. Adding 55 ul of uniformly mixed Serapure magnetic beads (1:1.1) into every 50 ul of reaction solution, fully mixing, and standing for 2 min at room temperature;
2) placing the PCR tube on a magnetic frame, standing for 5 min, and discarding the supernatant;
3) adding 200 mul of 75% ethanol into each tube on a magnetic frame, standing for half a minute, and removing supernatant;
4) repeating the step 3 once, discarding the supernatant as much as possible, and standing at room temperature for 5 min until the residual ethanol is completely volatilized;
5) adding 27ul of ultrapure water for elution, fully and uniformly mixing, standing at room temperature for 5 min, then placing a magnetic frame for standing for 2 min, storing the supernatant into a new PCR for probe capture, and performing QPCR quantification on tissues, whole blood and a leucocyte layer.
5. Sequencing computer
All the libraries after sample purification are mixed and are subjected to machine sequencing (illumina Nextseq 500), the UMI joint connection effect of the invention is shown in attached figures 1 and 2, and the pictures are used as reference to illustrate the actual operation result of the detection of the invention, and the specific mutation frequency is shown in the table below.
And (3) taking 8 mutation types of EGFR mutation genes as detection samples, setting 2 negative control samples, verifying the detection effect of the UMI joint disclosed by the invention, and comparing the effect with that of a comparison joint. The samples were as follows:
the detection results of the UMI joint and the common joint are as follows:
the above-mentioned conventional linker was prepared from the sequences 1-1 and 2-1 in the following table according to the procedure of example-first 2 nd step UMI linker annealing.
[i5] And [ i7] is an index sequence consisting of 8-base, which is ACATCAGC, GCTTGTGA, respectively.
When the detection result of the F-UMI linker (non-locked nucleic acid UMI linker) is examined by taking 23699-KTD024 as a sample, the mutation frequency is 1.06%, and the deviation is slightly larger.
The invention discloses a joint with locked nucleic acid for the first time, four groups of sequences can be obtained by feeding once, and the UMI joint is obtained by conventional annealing.
Sequence listing
<110> Suzhou Kono medical laboratory Co., Ltd
Molecular tag joint for high-throughput sequencing and synthesis method and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 76
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aatgatacgg cgaccaccga gatctacaca catcagcaca ctctttccct acacgacgct 60
cttccgatct nnnnnt 76
<210> 2
<211> 71
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
nnnnnagatc ggaagagcac acgtctgaac tccagtcacg cttgtgaatc tcgtatgccg 60
tcttctgctt g 71
<210> 3
<211> 77
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aatgatacgg cgaccaccga gatctacaca catcagcaca ctctttccct acacgacgct 60
cttccgatct nnnnnvt 77
<210> 4
<211> 72
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
bnnnnnagat cggaagagca cacgtctgaa ctccagtcac gcttgtgaat ctcgtatgcc 60
gtcttctgct tg 72
<210> 5
<211> 70
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
aatgatacgg cgaccaccga gatctacaca catcagcaca ctctttccct acacgacgct 60
cttccgatct 70
<210> 6
<211> 65
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gatcggaaga gcacacgtct gaactccagt cacgcttgtg aatctcgtat gccgtcttct 60
gcttg 65
<210> 7
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
aatgatacgg cgaccaccga gat 23
<210> 8
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
caagcagaag acggcatacg agat 24
Claims (6)
1. A molecular label joint for high-throughput sequencing is formed by mixing a joint A and a joint B, and is characterized in that the joint A is synthesized by a sequence 1 and a sequence 2; the joint B is synthesized by a sequence 3 and a sequence 4; the sequence 1 is AATGATACGGCGACCACCGAGATCTACAC [ i5] ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNT; the sequence 2 is NNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC [ i7] ATCTCGTATGCCGTCTTCTGCTTG; the sequence 3 is AATGATACGGCGACCACCGAGATCTACAC [ i5] ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNVT; the sequence 4 is BNNNNNAGATCGGAAGAGACACGTCTGAACTCCAGTCAC [ i7] ATCTCGTATGCCGTCTTCTGCTTG; n is selected from A, T, C, G, V is selected from A, C, G, B is selected from T, G, C; in the sequence 1, the 2 nd nucleotide at the 3 end is a locked nucleic acid; in the sequence 2, the 1 st nucleotide at the 5 end is locked nucleic acid; in the sequence 3, the 2 nd nucleotide at the 3 end is a locked nucleic acid; in the sequence 4, the 1 st nucleotide at the 5 end is locked nucleic acid; during synthesis, the feeding proportion of ATCG base at the N position is respectively 25%; the ACG base feeding proportion at the V position during synthesis is 1/3 respectively; the feeding proportion of TCG base at the position B during synthesis is 1/3 respectively; [i5] and [ i7] is an index sequence consisting of the base at position 8.
2. The method for preparing the molecular tag joint for high throughput sequencing according to claim 1, which is characterized by comprising the following steps of heating sequence 1 and sequence 2 in a hybridization working buffer solution to synthesize a joint A; heating the sequence 3 and the sequence 4 in a hybridization working buffer solution to synthesize a joint B; and mixing the joint A and the joint B to obtain the molecular tag joint for high-throughput sequencing.
3. The method for preparing the molecular tag linker for high throughput sequencing of claim 2, wherein the hybridization buffer comprises KCl, Tris-HCl, Triton X-100 and water.
4. The method for preparing the molecular tag joint for high throughput sequencing according to claim 3, wherein the heating is performed at 55-58 ℃ for 25-35 minutes and then at 35-40 ℃ for 13-16 hours.
5. The use of the molecular tag linker for high throughput sequencing of claim 1 in the preparation of a gene high throughput sequencing linker reagent.
6. The use of claim 5, wherein the gene is a lung cancer gene.
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