CN105986015A - Method and kit for detecting one or more target sequence of multiple samples based on high-throughput sequencing - Google Patents

Method and kit for detecting one or more target sequence of multiple samples based on high-throughput sequencing Download PDF

Info

Publication number
CN105986015A
CN105986015A CN201510061690.6A CN201510061690A CN105986015A CN 105986015 A CN105986015 A CN 105986015A CN 201510061690 A CN201510061690 A CN 201510061690A CN 105986015 A CN105986015 A CN 105986015A
Authority
CN
China
Prior art keywords
sequence
sample
joint
primer
amplification
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201510061690.6A
Other languages
Chinese (zh)
Other versions
CN105986015B (en
Inventor
刘琦
许立志
胡小许
郑新
杨文辉
其他发明人请求不公开姓名
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dalian Jingtai medical laboratory Co.,Ltd.
Original Assignee
Dalian Gentalker Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dalian Gentalker Biotechnology Co Ltd filed Critical Dalian Gentalker Biotechnology Co Ltd
Priority to CN201510061690.6A priority Critical patent/CN105986015B/en
Publication of CN105986015A publication Critical patent/CN105986015A/en
Application granted granted Critical
Publication of CN105986015B publication Critical patent/CN105986015B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention provides a method or a kit for capturing and labeling one or more target sequences of multiple samples based on high-throughput sequencing. A novel labeling technical scheme is provided by integrating a multiplex amplification technology, a ligation technology and a PCR-index (polymerase chain reaction-index) technology; the target sequences are amplified by virtue of specific capturing primers; by virtue of a ligation reaction, an adaptor, which is provided with a U base and has a known sequence, is introduced to each of two ends of a PCR product; the U bases are cut open by virtue of a USER enzyme; PCR primers are designed in accordance with the known sequences of the adaptors; tag primers are separately introduced to two ends of the PCR product by virtue of a PCR reaction; sample information of the PCR product can be specifically obtained by virtue of the sequences of the tag primers which are introduced to two ends of the PCR product; and then, in accordance with the sequence of the capturing primer of each of the target sequences, a corresponding sequencing result is correspondingly applied to each of the target sequences of the samples.

Description

The detection method of one or more target sequences of a kind of multisample based on high-flux sequence and kit
Technical field
The present invention relates to biological technical field, be specifically related to capture and mark the side of the target sequence of multiple sample Method and kit, particularly relate to a kind of capture based on high-flux sequence and mark one of multisample or The method of multiple target sequences or kit.
Background technology
A new generation high-flux sequence instrument appearance greatly reduce nucleic acid sequencing cost, its have high flux, The features such as low cost, order-checking error rate are low.Application second generation high throughput sequencing technologies, can be to mixing Nucleic acid molecules carries out sequencing, differentiates simultaneously and measure each independent sequence so that this sequencing technologies It is possibly realized at high flux marker development.
With extensive at aspects such as human diseases research, microbial genome order-checkings of high throughput sequencing technologies Carry out, how to play the feature such as high flux and big data quantity of second generation sequencing technologies to greatest extent, reduce Single sample order-checking cost becomes the developing direction of next step sequencing technologies, has very big market prospects and valency Value.
But, conventional PCR-index technology, the index label for each PCR primer is to need Individually to carry out amplification label, then by the mixing order-checking of all of product, when PCR fragment is more, no Only complex operation, and it is difficult to the homogeneity of control fragment mixing, fragment in final sequencing result can be caused Between data volume gap huge.
Content of the invention
It is an object of the invention to utilize high throughput sequencing technologies same to the target sequence correlated series in great amount of samples Shi Jinhang sequencing analysis, solves the problem that conventional method flux is low, order-checking cost is high, expands the second generation and surveys Sequence technology is in the application in PCR order-checking field.
For the purpose of the present invention, the present invention incorporates multiplex amplification technology, interconnection technique and PCR-index Technology, provides a kind of novel markings technical scheme, uses specific capture primer amplification target sequence (the One takes turns amplification), utilize coupled reaction respectively introduce at the two ends of first round PCR primer one band U base and Joint known to sequence, is digested out U base with USER, according to joint known array tag design primer, And utilize PCR reaction (second takes turns amplification) to take turns PCR primer two ends second and introduce Tag primer respectively Sequence, can obtain PCR specifically by the second Tag primer sequence taking turns the introducing of PCR primer two ends The sample information of product, takes turns capture primer sequence contained in amplified production further according to second and ties order-checking Fruit corresponds on each target sequence of sample.
According to an aspect of the present invention, a kind of capture based on high-flux sequence is provided and marks multiple sample The method of this one or more target sequences, comprises the following steps:
The first round expands: uses the capture primer designing according to target sequence, is being suitable to multiplex amplification purpose core Respectively the target area of each sample is expanded under conditions of acid, reclaim designed target area scope Within all DNA bands;
Second takes turns amplification: use the product that first round amplification obtains as template carry out second take turns amplification so that Second takes turns on amplified production with the label that can make a distinction each sample;
Mixing is with recovery: mix the Enrichment Amplification product of each sample equably, reclaims designed Target area within the scope of all DNA bands;
Order-checking: the DNA mixture of recovery is checked order;
Analyze: based on label unique in the amplified production of each sample, by the sequencing result of acquisition first With sample one_to_one corresponding;Capture primer sequence according to each target sequence, then sequencing result is corresponded to sample On this target sequence.
Preferably, according to an aspect of the present invention, a kind of capture based on high-flux sequence and mark are provided Remember the method for one or more target sequences of multiple sample, comprise the following steps:
The first round expands: uses the capture primer designing according to target sequence, is being suitable to multiplex amplification purpose core Respectively the target area of each sample is expanded under conditions of acid, reclaim designed target area scope Within all DNA bands, carry out end reparation to amplified production and add A tail;
Joint designs: described joint comprises two palindromic sequences, U base and 3 ' T ends, wherein, One palindromic sequence is positioned at described joint 5 ' end, is connected with another palindromic sequence by U base, its Can stably form hairpin structure;
Jointing (Adaptor): by coupled reaction at the two ends of first round amplified production plus band U The joint of base;Carry out U base shearing, and digest remaining primer with strand digestive ferment;
Tag primer designs: described Tag primer comprises sequence label and 5 ' the end palindrome with described joint The complementary sequence of sequence, wherein, described sequence label is positioned at 5 ' ends of described Tag primer, wherein, Quantity >=the sample size of the quantity × joint of sequence label;
Second takes turns amplification: use label (index) primer for joint and each sample design, with correspondence Product after sample jointing carries out Enrichment Amplification as template, and remaining with the digestion of strand digestive ferment Primer, to give the amplified production of each sample plus the Tag primer sequence that can distinguish respectively;
Mixing is with recovery: mix the Enrichment Amplification product of each sample equably, reclaims designed Target area within the scope of all DNA bands;
Order-checking: the DNA mixture of recovery is checked order;
Analyze: based on Tag primer sequence unique in the amplified production of each sample, the order-checking that will obtain Result first with sample one_to_one corresponding;Capture primer sequence according to each target sequence, then by sequencing result Correspond on each target sequence of sample.
Preferably, the invention provides a kind of capture based on high-flux sequence and mark multiple sample The method of individual or multiple target sequence, comprises the following steps:
1) primer and joint design:
First round amplimer: according to target sequence design corresponding capture primer to be measured, form the first round Amplimer combines;
Joint sequence: design one or more joint sequence, described joint comprises two palindromic sequences, U Base and 3 ' T ends, wherein, a palindromic sequence is positioned at described joint 5 ' end, by U base Being connected with another palindromic sequence, it can stably form hairpin structure;
Second takes turns amplimer: designs different sequence labels according to sample size, and adds at its 3 ' end Tag primer used by Enrichment Amplification for the upper sequence composition complementary with 5 ' end palindromic sequences of described joint, Composition second takes turns amplimer combination, wherein, due to the joint of the second target sequence two ends connection taking turns amplification Identical, the forward Tag primer for same sample is identical with reverse Tag primer, the Tag primer of design Quantity follow following rule: the quantity >=sample size of the quantity × joint of sequence label, and wherein, Take turns in amplimer second, the sequence that sequence label adds and connecing of being connected in first round amplified production 5 ' end palindromic sequences of head are complementary;
2) first round amplification (PCR capture): will amplification target sequence capture primer mix, be suitable to many Respectively each sample is expanded under conditions of the nucleic acid of re-spread gaining;Reclaim designed target area model All DNA bands within enclosing, carry out end reparation to amplified production, and by the two of amplified production End all adds A tail;
3) jointing (Adaptor): by coupled reaction at amplified production two ends plus with U base Joint, uses USER enzyme to interrupt U base, with the remaining primer of strand digestive ferment digestion;
4) second amplification (product enrichment) is taken turns: according to the Tag primer of different samples and joint design, make Product after processing with corresponding sample jointing carries out Enrichment Amplification as template, to give each sample respectively This amplimer all adds the Tag primer sequence that can distinguish;Draw with the digestion of strand digestive ferment is remaining Thing, and be purified;
5) mixing is with recovery: mix the enriched product of each sample equably;Designed by reclaiming All DNA bands within the scope of target area;
6) check order: DNA mixture is checked order;
7) analyze: based on the unique Tag primer sequence of each sample, by the sequencing result that obtains first with Sample one_to_one corresponding;Capture primer sequence according to each target sequence, then sequencing result is corresponded to sample Each target sequence on.
Preferably, in step 1, the length of described sequence label can be 5-20bp, it is highly preferred that institute The length stating sequence label can be 6-8bp;Described joint sequence comprises the palindromic sequence in two regions, Preferably, each palindromic sequence can be 8-15bp (most preferably 15bp).
Preferably, in step 2, expanding used system is high-fidelity multi-PRC reaction system, with Reduce the brought DNA mutation of amplification, and ensure that each purpose fragment can effectively be expanded;
Preferably, in step 2, PCR amplification carries out 18-20 circulation.
Preferably, in step 4, expanding used enzyme is high-fidelity DNA polymerase, thus reduces The DNA mutation rate that amplification brings.
Preferably, the end reparation in step 2, the coupled reaction institute adding in A end reaction and step 3 Being respectively Neb with reagent recommended for two generations built storehouse kitEnd Repair Module、DA-Tailing Module andQuick Ligation Module.Preferably, Strand digestive ferment used in step 3 and 4 is exonuclease I (Exonuclease I), and this enzyme is single Specific 3 ' → 5 ' exonucleases of chain, do not decompose double-stranded DNA and RNA.
Preferably, step 4 second is taken turns amplification and carried out 15-18 circulation.
Preferably, in step 6, order-checking utilizes two generation sequencing technologies, preferably pair-End technology (example Such as Illumina Hiseq2000, Illumina Hiseq2500 and Illumina Miseq) check order, obtain Obtain the sequence of DNA mixture.
On the other hand, the present invention also provides a kind of capture based on high-flux sequence and marks multiple sample The kit of one or more target sequences, wherein, described kit includes following primer:
First round amplimer: according to target sequence design corresponding capture primer to be measured, form the first round Amplimer combines;
Joint sequence: one or more joint sequences, described joint comprises two palindromic sequences, U base With 3 ' T ends, wherein, palindromic sequence is positioned at described joint 5 ' end, by U base with another One palindromic sequence connects, and it can stably form hairpin structure;And/or
Second takes turns amplimer: designs different sequence labels according to sample size, and adds at its 3 ' end Tag primer used by Enrichment Amplification for the upper sequence composition complementary with 5 ' end palindromic sequences of described joint, Composition second takes turns amplimer combination, wherein, due to the joint of the second target sequence two ends connection taking turns amplification Identical, the forward Tag primer for same sample is identical with reverse Tag primer, the Tag primer of design Quantity follow following rule: the quantity >=sample size of the quantity × joint of sequence label, and wherein, Take turns in amplimer second, the sequence that sequence label adds and connecing of being connected in first round amplified production 5 ' end palindromic sequences of head are complementary.
Preferably, in the present invention, the quantity of described sample can be: sample size≤programmable label The quantity of the quantity of sequence × programmable joint.
It is highly preferred that the present invention provides a kind of mark based on high-flux sequence and captures multiple (10) The examination of one or more (21) extron of the related ATP7B gene of the Wilson syndrome of sample Agent box, wherein said extron for selected from EXON1, EXON2, EXON3, EXON4, EXON5, EXON6、EXON7、EXON8、EXON9、EXON10、EXON11、EXON12、EXON13、 EXON14、EXON15、EXON16、EXON17、EXON18、EXON19、EXON20、 One or more in EXON21;Described kit includes following primer and joint:
It is one or more pairs of that first round amplimer includes in following:
For being connected to the joint with following sequence at first round amplified production two ends:
ADP1:5'P-AGGACAGAAGCTCGA-U-TCGAGCTTCTGTCCT-s-T-3'
ADP2:5'P-ACCTTGAGCGATCGA-U-TCGATCGCTCAAGGT-s-T-3';
Second takes turns one or more Tag primers composition that amplimer includes in following Tag primer Primer pair, it is mutual with the 5 ' of the joint being connected in first round product end palindromic regions for wherein drawing horizontal line part The sequence mended:
ADP1A:GCTCATTCGAGCTTCTGTCCT
ADP1B:TAGCCTTCGAGCTTCTGTCCT
ADP1C:AGAGGCTCGAGCTTCTGTCCT
ADP1D:TATCCTTCGAGCTTCTGTCCT
ADP1E:CTCTGATCGAGCTTCTGTCCT
ADP2A:GCTCATTCGATCGCTCAAGGT
ADP2B:TAGCCTTCGATCGCTCAAGGT
ADP2C:AGAGGCTCGATCGCTCAAGGT
ADP2D:TATCCTTCGATCGCTCAAGGT
ADP2E:CTCTGATCGATCGCTCAAGGT
Wherein, due to second take turns amplification when, target sequence two ends connect joint identical, be used for same sample Forward Tag primer identical with reverse Tag primer.
There is advantages that
The present invention uses specific capture primer (first round amplimer) amplification target sequence, by even Connect reaction and the joint (Adaptor) with U base is connected to the two ends of target sequence amplification product, use After USER is digested out U base, drawn by using the second of sequence label+joint sequence composition to take turns amplification Thing expands, and finally the 5` end in PCR primer with the addition of Tag primer sequence.By to each sample This all introduces the Tag primer sequence of uniqueness, when second generation high throughput sequencing technologies detects, each sample Sequencing result can be given for change by the Tag primer sequence of its uniqueness, catching further according to each target sequence Obtaining primer sequence and corresponding to sequencing result on each target sequence of sample, therefore, the present invention can apply In the multiple different genes sites detecting great amount of samples simultaneously, greatly reduce order-checking cost.
Brief description
Fig. 1 is the capture of the present invention and the schematic diagram of method of target sequence marking multiple sample.
Detailed description of the invention
Now will be described in further details the present invention, embodiment is only limitted to the present invention is described in conjunction with the embodiments, Rather than limitation of the invention.
Equipment used in following example and reagent are as follows:End Repair Module、DA-Tailing Module andQuick Ligation Module, Exonucleolytic Enzyme Takara (Exonuclease I (E.coli)).
Embodiment 1
Wilson syndrome is related to 21 extrons of ATP7B gene (NCBI Gene ID:540) (EXON1、EXON2、EXON3、EXON4、EXON5、EXON6、EXON7、EXON8、 EXON9、EXON10、EXON11、EXON12、EXON13、EXON14、EXON15、 EXON16, EXON17, EXON18, EXON19, EXON20 and EXON21) order-checking, altogether 10 case clinical samples:
1) primer and joint design:
21 extrons for ATP7B gene separately design corresponding capture primer, relevant parameter: Tm value 58.0 DEG C-65.0 DEG C, GC value 40.0%-60.0%, primer size 23 ± 3bp, purpose piece Duan great little is 150-300bp, and designed primer is as follows:
According to 2 joints of 10 sample designs, joint is single stranded DNA, comprises two regions in sequence The palindromic sequence of (each zone length is 15bp), a U base and 3 ' T ends, can form steady Fixed hairpin structure, designed joint sequence is as follows:
ADP1:5'P-AGGACAGAAGCTCGA-U-TCGAGCTTCTGTCCT-s-T-3'
ADP2:5'P-ACCTTGAGCGATCGA-U-TCGATCGCTCAAGGT-s-T-3'
According to following rule tag design primer: Tag primer comprises sequence label and the 5 ' ends with joint The complementary sequence of palindromic sequence, wherein, the quantity >=sample size of the quantity × joint sequence of sequence label, In the present embodiment, according to 10 samples and 2 joints design 10 strip label primers, i.e. with connect 5 ' ends of the sequence of 5 ' end palindromic sequences complementations of head are plus sequence label (6bp) structure that can distinguish Become Tag primer, wherein, owing to second takes turns the target sequence of amplification (after first round amplified production jointing Product) palindromic sequence of joint that connects of two ends is identical, for same sample forward Tag primer and Reverse Tag primer is identical.Designed Tag primer is as follows, wherein draws horizontal line part for producing with the first round The sequence of 5 ' end palindromic regions complementations of the joint connecting in thing:
ADP1A:GCTCATTCGAGCTTCTGTCCT
ADP1B:TAGCCTTCGAGCTTCTGTCCT
ADP1C:AGAGGCTCGAGCTTCTGTCCT
ADP1D:TATCCTTCGAGCTTCTGTCCT
ADP1E:CTCTGATCGAGCTTCTGTCCT
ADP2A:GCTCATTCGATCGCTCAAGGT
ADP2B:TAGCCTTCGATCGCTCAAGGT
ADP2C:AGAGGCTCGATCGCTCAAGGT
ADP2D:TATCCTTCGATCGCTCAAGGT
ADP2E:CTCTGATCGATCGCTCAAGGT
2) first round amplification (PCR capture):
The every pair of capture primer individually debug qualified after, be respectively directed to each clinical sample, by the 21 pairs of primers respectively It is diluted to 100 μM, then mixed in equal amounts, expand by following system and condition:
Reactive component 50μL
Multiplex amplification PCR premixed liquid (Multiplex PCR Mix) 25
GC strengthens buffer solution (GC Enhance) 3
Primer mixed liquor (Primer Mix) 1.25
Template (10ng/ul) 2
H2O 18.75
Reaction condition:
95 DEG C 10 minutes;
95 DEG C 30 seconds;58 DEG C 5 minutes (20 circulations);
72 DEG C 7 minutes.
Product reclaims: cut all DNA bands within the scope of glue reclaims designed target area (150-300bp)。
End is repaired: carry out end reparation to above-mentioned recovery product according to following reaction system and condition:
Reaction condition:
20℃30min
65℃20min
Products therefrom is carried out post purifying, final 50 μ L wash-outs.
Add A tail: add A tail to above-mentioned product fragment 3 ' end according to following reaction system and condition:
Reactive component 50μL
NEBNext adds A reaction buffer (NEBNext dA-Tailing Reaction Buffer) (10 ×) 5
Klenow Fragment enzyme (Klenow Fragment (3 ' → 5 ' exo)) 3
Purified product 42
Reaction condition: 37 DEG C of 30min, 65 DEG C of 20min.
Products therefrom is carried out post purifying, final 30 μ L wash-outs.
3) jointing
Coupled reaction: add joint at above-mentioned product fragment two ends according to following reaction system and condition:
Reaction condition: 20 DEG C of 15min, 12 DEG C of 30min, 65 DEG C of 10min.
Wherein, take turns in amplification Tag primer used second, hold, with sequence label 3 ', the sequence being connected The sequence of 5 ' end palindromic sequences complementations of the joint being and being connected in first round amplified production, each sample The joint that connects in first round product and take turns amplification Tag primer used as shown in following table one second.
USER enzyme and Exonuclease I enzymic digestion: in above-mentioned system, add 1 μ L USER enzyme, 37 DEG C Reaction 30min, cutting the U base in joint, is subsequently adding 1 μ L Exonuclease I enzyme, 37 DEG C Reaction 30min is with the primer strand of residual in digestion system.Carry out post purifying to products therefrom, finally 50 μ L wash-outs.
4) second take turns amplification (product enrichment) and use the above-mentioned Tag primer designing according to sample and joint, Product after processing using corresponding sample jointing carries out Enrichment Amplification as template, to give each sample respectively This amplified production adds the Tag primer sequence that can distinguish, and reaction system and condition are as follows:
Reactive component 50μL
2 × PCR premixed liquid (2 × PCR Mix) 25
Index primer (100 μM) 1
Taq polymerase (Taq polymerse) 1
Template 23
Reaction condition: 95 DEG C of 10min;95 DEG C of 1min, 50 DEG C of 5min, (15-18 is individual for 72 DEG C of 1min Circulation);
Wherein, the joint and the second Tag primer taken turns used in product that connect in first round product are as follows Shown in table one:
Table one
Sample number Joint Tag primer
1# ADP1 ADP1A
2# ADP1 ADP1B
3# ADP1 ADP1C
4# ADP1 ADP1D
5# ADP1 ADP1E
6# ADP2 ADP2A
7# ADP2 ADP2B
8# ADP2 ADP2C
9# ADP2 ADP2D
10# ADP2 ADP2E
Primer digestion and product reclaim: the remaining primer of amplified production strand digestive ferment digestion, and carry out Purify, all DNA bands within the scope of the designed target area of recovery;
5) mixing with reclaim: by for each clinical sample with the second of flag sequence take turns PCR amplification Product is according to homogeneous the mixing of concentration, owning within the scope of the designed target area of recovery DNA band;
6) checking order: the DNA mixture that will reclaim, using Illumina Miseq, PE300 checks order, Obtain the sequence of DNA mixture.
7) analyze: the sequencing result of Illumina Miseq product is a series of DNA sequence dnas, by looking into Look for the Tag primer sequence (group of joint sequence and sequence label that in sequencing result, 10 samples are each unique Close), by obtain sequencing result first with sample one_to_one corresponding, the then capture according to 21 extrons Primer sequence, then sequencing result is corresponded on each target sequence of sample.All of 10 samples 21 extrons can find corresponding data, each sample corresponding reads number in sequencing result As shown in following table two, between 10 samples, reads number difference is maximum at about 3 times, 21 extron sequences Arrange corresponding reads difference maximum about 10 times (data see table three, as a example by 1-2# sample), Show that our multisample labeling method can effectively distinguish the corresponding DNA sequence dna of each label.
Each sample of table two corresponding reads number and GC number
The corresponding reads number of 3 21 exon sequences of table (as a example by 1-2# sample)
1# 2#
Exon 1 14231 10272
Exon 2 9831 6098
Exon3 17246 11428
Exon 4 13888 9649
Exon 5 25132 16094
Exon 6 8032 10857
Exon 7 16989 8565
Exon 8 7564 5826
Exon 9 15789 20667
Exon 10 13476 10758
Exon 11 17428 12633
Exon 12 13085 9964
Exon 13 15714 7234
Exon 14 10857 8574
Exon 15 11428 9771
Exon 16 13308 10034
Exon 17 18571 11241
Exon 18 14330 12571
Exon 19 10094 8143
Exon 20 22455 13215
Exon 21 11500 10428
Amount to 300,948 224,022

Claims (10)

1. the capture based on high-flux sequence and the side of one or more target sequences marking multiple sample Method, comprises the following steps:
The first round expands: uses the capture primer designing according to target sequence, is being suitable to multiplex amplification purpose core Respectively the target area of each sample is expanded under conditions of acid, reclaim designed target area scope Within all DNA bands;
Second takes turns amplification: use the product that first round amplification obtains as template carry out second take turns amplification so that Second takes turns on amplified production with the label that can make a distinction each sample;
Mixing is with recovery: mix the Enrichment Amplification product of each sample equably, reclaims designed Target area within the scope of all DNA bands;
Order-checking: the DNA mixture of recovery is checked order;
Analyze: based on label unique in the amplified production of each sample, by the sequencing result of acquisition first With sample one_to_one corresponding;Capture primer sequence according to each target sequence, then sequencing result is corresponded to sample On this each target sequence.
2. the capture based on high-flux sequence and the side of one or more target sequences marking multiple sample Method, comprises the following steps:
The first round expands: uses the capture primer designing according to target sequence, is being suitable to multiplex amplification purpose core Respectively the target area of each sample is expanded under conditions of acid, reclaim designed target area scope Within all DNA bands, carry out end reparation to amplified production and add A tail;
Joint designs: described joint comprises two palindromic sequences, U base and 3 ' T ends, wherein, One palindromic sequence is positioned at described joint 5 ' end, is connected with another palindromic sequence by U base, its Can stably form hairpin structure;
Jointing: by coupled reaction at the two ends of first round amplified production plus the joint with U base; Carry out U base shearing, and digest remaining primer with strand digestive ferment;
Tag primer designs: described Tag primer comprises sequence label and 5 ' the end palindrome with described joint The complementary sequence of sequence, wherein, described sequence label is positioned at 5 ' ends of described Tag primer, wherein, Quantity >=the sample size of the quantity × joint of sequence label;
Second takes turns amplification: use the Tag primer for joint and each sample design, connects with corresponding sample Product after joint carries out Enrichment Amplification as template, and digests remaining primer with strand digestive ferment, with Give the amplified production of each sample plus the Tag primer sequence that can distinguish respectively;
Mixing is with recovery: mix the Enrichment Amplification product of each sample equably, reclaims designed Target area within the scope of all DNA bands;
Order-checking: the DNA mixture of recovery is checked order;
Analyze: based on Tag primer sequence unique in the amplified production of each sample, the order-checking that will obtain Result first with sample one_to_one corresponding;Capture primer sequence according to each target sequence, then by sequencing result Correspond on the target sequence of sample.
3. the capture based on high-flux sequence and the side of one or more target sequences marking multiple sample Method, comprises the following steps:
1) primer and joint design:
First round amplimer: according to target sequence design corresponding capture primer to be measured, form the first round Amplimer combines;
Joint sequence: design one or more joint sequence, described joint comprises two palindromic sequences, U Base and 3 ' T ends, wherein, a palindromic sequence is positioned at described joint 5 ' end, by U base Being connected with another palindromic sequence, it can stably form hairpin structure;
Second takes turns amplimer: designs different sequence labels according to sample size, and adds at its 3 ' end Tag primer used by Enrichment Amplification for the upper sequence composition complementary with 5 ' end palindromic sequences of described joint, Composition second takes turns amplimer combination, wherein, due to the joint of the second target sequence two ends connection taking turns amplification Identical, the forward Tag primer for same sample is identical with reverse Tag primer, the Tag primer of design Quantity follow following rule: the quantity >=sample size of the quantity × joint of sequence label, and wherein, Take turns in amplimer second, the sequence that sequence label adds and connecing of being connected in first round amplified production 5 ' end palindromic sequences of head are complementary;
2) first round amplification: by the capture primer mixing of amplification target sequence, be suitable to multiplex amplification purpose core Respectively each sample is expanded under conditions of acid;Owning within the scope of the target area designed by reclaiming DNA band, carries out end reparation, and the two of amplified production ends is all added A to amplified production Tail;
3) jointing (Adaptor): by coupled reaction at amplified production two ends plus with U base Joint, uses USER enzyme to interrupt U base, with the remaining primer of strand digestive ferment digestion;
4) second amplification is taken turns: according to the Tag primer of different samples and joint design, use corresponding sample even Product after joint is processed carries out Enrichment Amplification as template, to give the amplimer of each sample respectively All add the Tag primer sequence that can distinguish;Digest remaining primer with strand digestive ferment, and carry out pure Change;
5) mixing is with recovery: mix the enriched product of each sample equably;Designed by reclaiming All DNA bands within the scope of target area;
6) check order: DNA mixture is checked order;
7) analyze: based on the unique Tag primer sequence of each sample, by the sequencing result that obtains first with Sample one_to_one corresponding;Capture primer sequence according to each target sequence, then sequencing result is corresponded to sample Target sequence on.
4. according to the method in claim 2 or 3, wherein, a length of 5-20bp of described sequence label, Preferably, a length of 6-8bp of described sequence label;Described joint comprises the palindrome sequence in two regions Row, each palindromic sequence is 8-15bp, most preferably 15bp.
5. according to the method in claim 2 or 3, wherein, the used system of first round amplification is High-fidelity multi-PRC reaction system;Second to take turns the enzyme that amplification used be high-fidelity DNA polymerase, Described strand digestive ferment is exonuclease I.
6. according to the method in claim 2 or 3, wherein, first round amplification carries out 18-20 and follows Ring, second takes turns amplification carries out 15-18 circulation.
7. according to the method in claim 2 or 3, wherein, end reparation, add A end reaction with And coupled reaction agents useful for same is respectively Neb and recommended for two generations built storehouse kitEnd Repair Module、DA-Tailing Module andQuick Ligation Module。
8. according to the method in claim 2 or 3, wherein, order-checking utilizes two generation sequencing technologies, excellent Choosing is pair-End technology (such as Illumina Hiseq2000, Illumina Hiseq2500 and Illumina Miseq) check order, it is thus achieved that the sequence of DNA mixture.
9. the capture based on high-flux sequence and the examination of one or more target sequences marking multiple sample Agent box, wherein, described kit includes following primer and joint:
First round amplimer: according to target sequence design corresponding capture primer to be measured, form the first round Amplimer combines;
Joint sequence: one or more joint sequences, described joint comprises two palindromic sequences, U base With 3 ' T ends, wherein, palindromic sequence is positioned at described joint 5 ' end, by U base with another One palindromic sequence connects, and it can stably form hairpin structure;And/or
Second takes turns amplimer: designs different sequence labels according to sample size, and adds at its 3 ' end Tag primer used by Enrichment Amplification for the upper sequence composition complementary with 5 ' end palindromic sequences of described joint, Composition second takes turns amplimer combination, wherein, due to the joint of the second target sequence two ends connection taking turns amplification Identical, the forward Tag primer for same sample is identical with reverse Tag primer, the Tag primer of design Quantity follow following rule: the quantity >=sample size of the quantity × joint of sequence label, and wherein, Take turns in amplimer second, the sequence that sequence label adds and connecing of being connected in first round amplified production 5 ' end palindromic sequences of head are complementary.
10. one kind related with the Wilson syndrome capturing multiple sample based on the mark of high-flux sequence The kit of one or more extrons of ATP7B gene, wherein said extron for selected from EXON1, EXON2、EXON3、EXON4、EXON5、EXON6、EXON7、EXON8、EXON9、 EXON10、EXON11、EXON12、EXON13、EXON14、EXON15、EXON16、 One or more in EXON17, EXON18, EXON19, EXON20, EXON21;Described Kit includes following primer and joint:
It is one or more pairs of that first round amplimer includes in following:
E1-F CTTCCCCGGTCCCAAATGAAG
E1-R CCTCCTGGTGGGAGTGAGCAC
E2-F TGCCAGAGAAGCTGGGATGTTG
E2-R GCAAACCTGTTGCAGGCACAC
E3-F GAGCCCTGAAACCTCTTGTTCTG
E3-R CGAGGTCTATACGCAGCATTCCT
E4-F GCCCTGCCCACCCAGAGTG
E4-R CAAAGATGGATGTGTCCAAAATGC
E5-F CTGGCTTTCACAGGCTTTCCT
E5-R TTACTTACCTCAATAATTTTGATAATATCC
E6-F CTGCCAATGCATATTTTAACCAAGT
E6-R GAAGGGACTTAGATGAGAGCTGGAG
E7-F AATCCAGGTGACAAGCAGCATC
E7-R GCATGGAAGGGAGAGGTCTGC
E8-F ACTTGCTGGCAGCCTTCACTG
E8-R GATTTGTTTACTGAAGGAGCAGCTC
E9-F CGATAGCTCTCATTTCACATTCTGG
E9-R CACACAGATTGATAGATACCAACCAC
E10-F TGACCCGGTGACCGAATGAGT
E10-R ATGATATCCTCCTGAGGGAACATG
E11-F CAAGTGACAGTTGTCTCTTTCCTACG
E11-R TTCCCAGAACTCTTCACATAATTTCT
E12-F CCATGGTCTTGGTGTTTTATTTTC
E12-R TGAAAGAACAGGATCAATGTCAGTAG
E13-F TGGGAGCTTCCTTATTGAACTCTC
E13-R CATCTCTCAGGATGGGGAAAGC
E14-F CCTCCATCTGTATTGTGGTCAGTG
E14-R GGTGAGGAATAAAAGAGCATTGGC
E15-F TCTTGGCTTACAGTTTCCTCTTCC
E15-R CGTGGTGCTCTCTGTGGTTTGA
E16-F GGACCATTTAGAAATAACCACAGCC
E16-R TTTGCCTGATATCTGCAGAAAACTG
E17-F CCAACTTGTGTAGCTGCTGATGC
E17-R TGGTGCTTACTTTTGTCTCTAACTGC
E1819-F TGATACCTTTTGCCAACACTAGGC
E1819-R TGGGAGACAGAAGCCTTTCTGG
E20F TGGCTCCTCTCCCCAGACCTA
E20-R ACTGTGCTAAGCATGCAGAATGAC
E21-F GAGAGGCCTTCACCAGGCTTAG
E21-R GCCTGCCTGAAGTCATCAGATG
For being connected to the joint with following sequence at first round amplified production two ends:
ADP1:5'P-AGGACAGAAGCTCGA-U-TCGAGCTTCTGTCCT-s-T-3'
ADP2:5'P-ACCTTGAGCGATCGA-U-TCGATCGCTCAAGGT-s-T-3';
Second takes turns one or more Tag primers composition that amplimer includes in following Tag primer Primer pair, it is mutual with the 5 ' of the joint being connected in first round product end palindromic regions for wherein drawing horizontal line part The sequence mended:
ADP1A:GCTCATTCGAGCTTCTGTCCT
ADP1B:TAGCCTTCGAGCTTCTGTCCT
ADP1C:AGAGGCTCGAGCTTCTGTCCT
ADP1D:TATCCTTCGAGCTTCTGTCCT
ADP1E:CTCTGATCGAGCTTCTGTCCT
ADP2A:GCTCATTCGATCGCTCAAGGT
ADP2B:TAGCCTTCGATCGCTCAAGGT
ADP2C:AGAGGCTCGATCGCTCAAGGT
ADP2D:TATCCTTCGATCGCTCAAGGT
ADP2E:CTCTGATCGATCGCTCAAGGT
Wherein, due to second take turns amplification when, target sequence two ends connect joint identical, be used for same sample Forward Tag primer identical with reverse Tag primer.
CN201510061690.6A 2015-02-05 2015-02-05 Method and kit for detecting one or more target sequences of multiple samples based on high-throughput sequencing Active CN105986015B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510061690.6A CN105986015B (en) 2015-02-05 2015-02-05 Method and kit for detecting one or more target sequences of multiple samples based on high-throughput sequencing

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510061690.6A CN105986015B (en) 2015-02-05 2015-02-05 Method and kit for detecting one or more target sequences of multiple samples based on high-throughput sequencing

Publications (2)

Publication Number Publication Date
CN105986015A true CN105986015A (en) 2016-10-05
CN105986015B CN105986015B (en) 2020-10-23

Family

ID=57037841

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510061690.6A Active CN105986015B (en) 2015-02-05 2015-02-05 Method and kit for detecting one or more target sequences of multiple samples based on high-throughput sequencing

Country Status (1)

Country Link
CN (1) CN105986015B (en)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106554955A (en) * 2016-10-25 2017-04-05 大连晶泰生物技术有限公司 Build method and kit of the sequencing library of PKHD1 gene mutations and application thereof
CN106554999A (en) * 2016-10-25 2017-04-05 大连晶泰生物技术有限公司 High flux detects the sequencing library construction method in neonatal diabetes mellitus Disease-causing gene mutational site, kit and application thereof
WO2019114146A1 (en) * 2017-12-15 2019-06-20 格诺思博生物科技南通有限公司 Method for enriching gene target regions and library construction kit
CN110468188A (en) * 2019-08-22 2019-11-19 广州微远基因科技有限公司 For the sequence label collection and its design method of the sequencing of two generations and application
CN110468179A (en) * 2018-05-10 2019-11-19 北京大学 The method of selective amplification nucleic acid sequence
CN110603334A (en) * 2017-06-20 2019-12-20 深圳华大智造科技有限公司 PCR primer pair and application thereof
CN111118127A (en) * 2019-12-28 2020-05-08 郑州大学 Specific label error-proofing kit for second-generation DNA sequencing sample
CN111748611A (en) * 2019-03-28 2020-10-09 深圳华大基因科技服务有限公司 PCR primer and application thereof in DNA fragment connection
CN112458195A (en) * 2020-12-28 2021-03-09 广州迈景基因医学科技有限公司 Multiplex PCR primer set, kit and method for detecting sexually transmitted pathogens based on high-throughput sequencing
EP3752614A4 (en) * 2018-02-14 2021-11-10 Deep Genomics Incorporated Oligonucleotide therapy for wilson disease

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120244525A1 (en) * 2010-07-19 2012-09-27 New England Biolabs, Inc. Oligonucleotide Adapters: Compositions and Methods of Use
CN104153004A (en) * 2014-08-11 2014-11-19 上海美吉生物医药科技有限公司 Database-building method for amplicon sequencing

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120244525A1 (en) * 2010-07-19 2012-09-27 New England Biolabs, Inc. Oligonucleotide Adapters: Compositions and Methods of Use
CN104153004A (en) * 2014-08-11 2014-11-19 上海美吉生物医药科技有限公司 Database-building method for amplicon sequencing

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ANNU AGGARWAL1 ET AL.: "Wilson Disease Mutation Pattern with Genotype-Phenotype Correlations from Western India: Confirmation of p.C271* as a Common Indian Mutation and Identification of 14 Novel Mutations", 《ANNALS OF HUMAN GENETICS》 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106554955B (en) * 2016-10-25 2020-07-14 大连晶泰生物技术有限公司 Method and kit for constructing sequencing library of PKHD1 gene mutation and application thereof
CN106554999A (en) * 2016-10-25 2017-04-05 大连晶泰生物技术有限公司 High flux detects the sequencing library construction method in neonatal diabetes mellitus Disease-causing gene mutational site, kit and application thereof
CN106554955A (en) * 2016-10-25 2017-04-05 大连晶泰生物技术有限公司 Build method and kit of the sequencing library of PKHD1 gene mutations and application thereof
CN106554999B (en) * 2016-10-25 2020-03-17 大连晶泰生物技术有限公司 Sequencing library construction method for high-throughput detection of neonatal diabetes pathogenic gene mutation site, kit and application thereof
CN110603334B (en) * 2017-06-20 2024-01-16 深圳华大智造科技股份有限公司 PCR primer pair and application thereof
CN110603334A (en) * 2017-06-20 2019-12-20 深圳华大智造科技有限公司 PCR primer pair and application thereof
WO2019114146A1 (en) * 2017-12-15 2019-06-20 格诺思博生物科技南通有限公司 Method for enriching gene target regions and library construction kit
EP3752614A4 (en) * 2018-02-14 2021-11-10 Deep Genomics Incorporated Oligonucleotide therapy for wilson disease
US11578327B2 (en) * 2018-02-14 2023-02-14 Deep Genomics Incorporated Oligonucleotide therapy for Wilson disease
CN110468179B (en) * 2018-05-10 2021-03-05 北京大学 Method for selectively amplifying nucleic acid sequences
CN110468179A (en) * 2018-05-10 2019-11-19 北京大学 The method of selective amplification nucleic acid sequence
CN111748611A (en) * 2019-03-28 2020-10-09 深圳华大基因科技服务有限公司 PCR primer and application thereof in DNA fragment connection
CN110468188B (en) * 2019-08-22 2023-08-22 广州微远医疗器械有限公司 Tag sequence set for second generation sequencing and design method and application thereof
CN110468188A (en) * 2019-08-22 2019-11-19 广州微远基因科技有限公司 For the sequence label collection and its design method of the sequencing of two generations and application
CN111118127A (en) * 2019-12-28 2020-05-08 郑州大学 Specific label error-proofing kit for second-generation DNA sequencing sample
CN111118127B (en) * 2019-12-28 2023-05-02 郑州大学 Specific tag error-proofing kit for second-generation DNA sequencing sample
CN112458195A (en) * 2020-12-28 2021-03-09 广州迈景基因医学科技有限公司 Multiplex PCR primer set, kit and method for detecting sexually transmitted pathogens based on high-throughput sequencing

Also Published As

Publication number Publication date
CN105986015B (en) 2020-10-23

Similar Documents

Publication Publication Date Title
CN105986015A (en) Method and kit for detecting one or more target sequence of multiple samples based on high-throughput sequencing
CN106555226B (en) A kind of method and kit constructing high-throughput sequencing library
CN109468384B (en) Composite amplification detection kit for simultaneously detecting 45Y loci
CN105524983B (en) The method and kit of one or more specific genes of label and the multiple samples of capture based on high-flux sequence
CN104562213A (en) Amplification sublibrary and construction method thereof
CN110129415B (en) NGS library-building molecular joint and preparation method and application thereof
CN104263726A (en) Primer applied to amplicon sequencing library construction and method for constructing amplicon sequencing library
CN108611398A (en) Genotyping is carried out by new-generation sequencing
CN103173441A (en) Amplification method, primer, sequencing method and mutation detection method of mitochondria whole genome DNA (Deoxyribonucleic Acid)
CN110117574B (en) Method and kit for enriching circulating tumor DNA based on multiple PCR
CN109593757B (en) Probe and method for enriching target region by using same and applicable to high-throughput sequencing
CN102839168A (en) Nucleic acid probe, and preparation method and application thereof
CN105039322B (en) DNA sequence labels and sequencing library construction method and kit
CN102604934B (en) Method for amplifying and sequencing nucleic acid based on solid phase carrier
CN106995836A (en) The primer and method and kit of sample pre-treatments was sequenced in two generations
CN108138175A (en) For reagent, kit and the method for molecular barcode coding
WO2023284768A1 (en) Fusion primer direct amplification method-based human mitochondrial whole genome high-throughput sequencing kit
US20220056519A1 (en) Method and system for constructing sequencing library on the basis of methylated dna target region, and use thereof
CN110603327A (en) PCR primer pair and application thereof
CN108330546A (en) A kind of library constructing method and reagent of simplification
WO2024104130A1 (en) Whole genome molecular marker development method utilizing degenerate primer amplification
CN106065417B (en) A kind of STR classification systems and kit
CN108359723A (en) A method of reducing deep sequencing mistake
CN115715323A (en) High-compatibility PCR-free library building and sequencing method
US20180100180A1 (en) Methods of single dna/rna molecule counting

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
TR01 Transfer of patent right

Effective date of registration: 20220111

Address after: 116600 no.9-2 Jinqi Road, Dalian Economic and Technological Development Zone, Liaoning Province

Patentee after: Dalian Jingtai medical laboratory Co.,Ltd.

Address before: 116635 No. 9-2, Jinqi Road, advanced equipment manufacturing park, Dalian Economic and Technological Development Zone, Dalian City, Liaoning Province

Patentee before: DALIAN GENTALKER BIOTECHNOLOGY Co.,Ltd.

TR01 Transfer of patent right