CN107217052A - The preparation method and its matched reagent box of a kind of quantitative high-throughput sequencing library - Google Patents
The preparation method and its matched reagent box of a kind of quantitative high-throughput sequencing library Download PDFInfo
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- CN107217052A CN107217052A CN201710552185.0A CN201710552185A CN107217052A CN 107217052 A CN107217052 A CN 107217052A CN 201710552185 A CN201710552185 A CN 201710552185A CN 107217052 A CN107217052 A CN 107217052A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
Abstract
The invention provides a kind of preparation method of quantitative high-throughput sequencing library and its matched reagent box, specifically, the invention provides a kind of adapter, the adapter has Formulas I or the structure shown in II：In various, S1 is optionally guide portion (N)m, N is any base in A, T, G, C, and m is 1 150 positive integer；PS1 is the S1 of phosphorylation；S2 is stem 1, with SEQ ID NO.:Nucleotide sequence shown in 1；S3 is ring portion, wherein containing at least one base U；S4 is stem 2, with SEQ ID NO.:Nucleotide sequence shown in 2；S2 and S4 complete complementaries or partial complementarity；S5 is the extension without or with guide portion S1 complementations；S6 is nothing or the joint portion containing at least one base T；If S5 is nothing, S6 is nothing；"-" is chemical bond.5'‑S1─S2─S3─S4─S5─S6‑3' (I)5'‑pS1─S2─S3─S4─S5─S6‑3' (II).
Description
Technical field
The present invention relates to biological technical field, high-throughput sequencing library is prepared more particularly, to a kind of accurate quantification
Method and its matched reagent box.
Background technology
With the popularization of high throughput sequencing technologies, for the high-throughput sequencing library preparation method of various separate sources samples
And commercial reagents box is also widely applied.The high flux library reagent preparation box such as Illumina companies of in the market main flow
Truseq series DNA/RNA libraries reagent preparation box, the Hyper series of KAPA companies builds storehouse kit, and NEB companies were used for for two generations
NEBNext DNA/RNA libraries reagent preparation of sequencing etc., these species are various, Various Functions kits can tackle different surveys
The various demands such as sequence platform, different samples sources, farthest meet the practical applications such as scientific research and clinic.But, at present also
There is no a kind of library preparation method of complete quantification, it is impossible to meet being actually needed for accurate medical science and quantitative biology.
Therefore, this area still lacks a kind of high-throughput sequencing library preparation method specifically designed for accurate quantification demand.
The content of the invention
It is an object of the invention to provide a kind of high-throughput sequencing library preparation side for DNA sample of accurate quantification
Method.
The first aspect of the present invention provides a kind of adapter, and the adapter has Formulas I or the structure shown in II：
5'-S1─S2─S3─S4─S5─S6-3' (I)
5'-pS1─S2─S3─S4─S5─S6-3' (II)
In various,
S1 is optionally guide portion (N)m, N is any base in A, T, G, C, and m is 1-150 positive integer；
PS1 is the S1 of phosphorylation；
S2 is stem 1, with SEQ ID NO.:Nucleotide sequence shown in 1；
S3 is ring portion, wherein containing at least one base U；
S4 is stem 2, with SEQ ID NO.:Nucleotide sequence shown in 2；
S2 and S4 complete complementaries or partial complementarity；
S5 is the extension without or with guide portion S1 complementations；
S6 is nothing or the joint portion containing at least one base T；
If S5 is nothing, S6 is nothing；
"-" is chemical bond.
In another preference, described adapter is linear structure or loop-stem structure.
In another preference, described adapter has structure shown in formula III or IV：
In another preference, m is 1-100, more preferably 1-50, most preferably 5-20 any positive integer.
In another preference, m be 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,
23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、
48th, 49 or 50.
In another preference, the ring portion S3 has SEQ ID NO.:Nucleotide sequence shown in 3.
In another preference, the S6 contains 1 base T.
In another preference, the S2 of the adapter has SEQ ID NO.:Nucleotide sequence shown in 1；S3 has
SEQ ID NO.:Nucleotide sequence shown in 3；S4 has SEQ ID NO.:Nucleotide sequence shown in 2.
In another preference, S2 and S4 are complete complementaries, or in addition to 1 or 2 not complementary base are to be
Complementary.
The second aspect of the present invention provides a kind of method that sequencing library is built based on nucleic acid samples, including step：
(a) nucleic acid samples for being used to build sequencing library are provided；
(b) end is carried out to described nucleic acid samples and repairs reaction, obtain the repaired nucleic acid samples in end；
(c) the nucleic acid samples progress 3' end repaired to described end adds dA end reactions, obtains the nucleic acid through tailing
Sample；
(d) nucleic acid samples through tailing are mixed with any described adapter in claim 1-5, and connected
It is reversed to answer, so as to form the first mixture containing " adapter-nucleic acid-adapter " compound；
(e) " adapter-nucleic acid-adapter " compound obtained in step (d) is mixed with USER enzymes so that adapter
In U be degraded, formed not complementary " adapter-nucleic acid-adapter " compound containing end furcations the second mixture；
(f) the second mixture containing the compound obtained in step (e) is mixed with fragment sorting magnetic bead so that institute
State fragment sorting magnetic bead and adsorb unnecessary adapter, and the adsorbed adapter is removed, obtain the 3rd mixture；
(g) compound using step (f) acquisition is template, with the first primer and spy for specifically binding to adapter
The second primer that the opposite sex is incorporated into adapter enters performing PCR reaction, obtains amplified production；
(h) using the amplified production that will obtain in step (g) as raw material, obtained sequencing library.
In another preference, in step (h), in addition to the step of purified to the amplified production.
In another preference, described purifying includes sorting the unnecessary primer of magnetic bead removal with fragment.
In another preference, step is also included between the step (c) and (d)：
(i) 5' terminal phosphateizations are carried out to the adapter shown in Formulas I to react, forms it into structure shown in Formula II；
Or the reaction of 5' terminal phosphateizations is carried out to the adapter shown in formula III, form it into structure shown in formula IV.
In another preference, step is also included between the step (c) and (d)：
(j) Formulas I or shown adapter are annealed, forms it into structure shown in formula III；
Or Formula II or shown adapter are annealed, form it into structure shown in formula IV.
In another preference, step is also included between the step (c) and (d)：
(k) when S5 be without when, it is any to Formulas I-IV shown in adapter carry out chain extension reaction, polishing degeneracy base portion
Form complementary double-strand.
In another preference, step is also included between the step (c) and (d)：
(l) when S5 be not without when, it is any to I-IV shown in adapter carry out 3' ends and add dT end reactions；Or
When S5 be without when, 3' ends are carried out to the adapter obtained in step (k) and add dT end reactions.
In another preference, the fragment sorting magnetic bead is selected from the group：DNA points of AMPure XP magnetic beads, Hieff NGS
Select magnetic bead, Imag fragments sorting magnetic bead.
In another preference, the sequencing library can be used for Illunima high-flux sequence platform direct Sequencings.
In another preference, the sequencing library can be used for accurate quantification.
In another preference, step (a) the amplifying nucleic acid sample total amount is the μ g of 500pg~1.
The third aspect of the present invention provides a kind of kit that sequencing library is built based on nucleic acid samples, the kit
Including：
Repair/add dA tails complex enzyme and its buffer solution in one end；
One T4DNA ligases；
One adapter, described adapter is as described in any in the first aspect of the present invention；
And specification, described specification describes application method.
In another preference, the kit also includes：The first primer and second for specifically binding to adapter draw
Thing.
In another preference, second primer is with sequencing mark (barcode).
In another preference, first primer is universal primer P5, its sequence such as SEQ ID NO.:Shown in 5.
In another preference, second primer is special primer P7.
In another preference, second primer is special primer P7, its sequence such as SEQ ID NO.:Shown in 6.
In another preference, the kit also includes enzyme buffer liquid.
In another preference, the kit also includes PCR reaction premixed liquids.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, so as to constitute new or preferred technical scheme.As space is limited, exist
This no longer tires out one by one states.
Brief description of the drawings
Fig. 1 is the schematic diagram of adapter preparation process.
Embodiment
The present inventor's in-depth study by long-term, surprisingly have developed a kind of with any shown knot in Formulas I-IV
The adapter of structure, it can be used in preparing DNA high-throughput sequencing libraries；And measured using the constructed dna high pass of the adapter
The method and corresponding reagent box in preface storehouse, using methods described, can prepare the high-throughput sequencing library of accurate quantification.Based on upper
Discovery is stated, the present invention is completed.
The high-throughput sequencing library method of accurate quantification is prepared for DNA sample
The invention provides a kind of method that quantification high-throughput sequencing library is prepared based on nucleic acid samples, described method
Including step：
(a) providing one is used to preparing the nucleic acid samples of sequencing library, and total amount is the μ g of 500pg~1 (or 1-200ng, or 0.5-
10ng)；
(b) end is carried out to described nucleic acid samples and repairs reaction；
(c) 3' ends are carried out to described nucleic acid samples and adds dA end reactions；
(d) a single-chain nucleic acid sample for being used to prepare adapter (adaptor) is provided, sequence is its 5' ends shown in annex
End includes 5~20 degeneracy bases, and 3' ends the 34th base reciprocal is U；
(e) 5' terminal phosphateizations are carried out to above-mentioned single-chain nucleic acid sample to react；
(f) nucleic acid samples that previous step is obtained are annealed, forms it into loop-stem structure；
(g) chain extension reaction is carried out to the nucleic acid samples that previous step is obtained, polishing degeneracy base portion forms complementary double-strand；
(h) 3' ends are carried out to the nucleic acid samples that previous step is obtained and adds dT end reactions, so far loop-stem structure adapter
(adaptor) prepare and complete, structure is as shown in formula IV, and wherein S6 is base T；
(i) nucleic acid samples of dA tails are added to mix progress coupled reaction with adapter after end is repaired, so as to form " linking
The compound of son-nucleic acid-adapter "；
(j) " adapter-nucleic acid-adapter " compound that previous step is obtained is mixed with USER enzymes so that in adapter
U be degraded, form not complementary " adapter-nucleic acid-adapter " compound of end furcations；
(k) compound for obtaining previous step is mixed with appropriate fragment sorting magnetic bead, the unnecessary adapter of Adsorption；
(l) compound for obtaining previous step and the universal primer (P5) and the specific binding that specifically bind to adapter
Mixed in the special primer (P7) of adapter, enter performing PCR reaction, obtain amplified production；
(m) amplified production for obtaining previous step is mixed with appropriate fragment sorting magnetic bead, and to remove unnecessary primer, this is
The sequencing library completed is prepared, Illunima high-flux sequence platforms are can be used to after length range detection and concentration quantitative
Direct Sequencing.
(n) each obtained after being sequenced reads the long individual mark for carrying 5~20 degeneracy base in (reads) two ends
Label, the complexity of this label depends on the quantity of degeneracy base, covers all nucleic acid samples enough in theory, and in amplification
By loyalty reservation in amplification process, therefore it can be used for accurate quantification.
The kit of sequencing library is built based on Method for Microarray Applications
Present invention also offers a kind of kit that high-throughput sequencing library is prepared based on nucleic acid samples, the kit bag
Include：
Repair/add A tails complex enzyme and its buffer solution in one end；
One T4DNA ligases；
One adapter, described adapter is loop-stem structure, and single stranded circle part is with a base U, complementary double-strand end
End carries 5~20 degeneracy bases,
And specification, described specification describes application method.
Typically, kit of the present invention also includes：One universal primer and/or a special primer.
Typically, in kit of the present invention, the special primer is with sequencing mark (barcode), the sequencing mark
The listed sequence in table 1.
Typically, kit of the present invention also includes：One PCR reaction premixed liquids.
Main advantages of the present invention include：
(1) present invention is by the adapter (adaptor) of particular design, and it is 5~20 degeneracy bases to introduce a segment length,
As the exclusive label of each nucleic acid fragment, and it will not obscure during amplification, sequencing and later stage bioinformatics analysis, energy
Enough review the value volume and range of product of each nucleic acid fragment in primary sample, it is possible to achieve complete accurate quantification.
(2) compared with prior art, the present invention realizes the preparation of high-throughput sequencing library using operation simple and easy to apply.It is right
As little as 500pg nucleic acid samples also can smoothly build storehouse, and the purifying of magnetic bead is sorted by conventional fragment, and Library Quality is high, and impurity is few,
Can directly upper machine sequencing.
(3) present invention is suitable for various DNA and RNA sample prepares high-throughput sequencing library, and required is all normal experiment
Technology and the reagent and medicine easily bought, condition are easy to get, and easy to operate, and general experiment technical personnel all can be grasped independently
Complete.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor
Laboratory Press, 1989) described in condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, it is no
Then percentage and number are calculated by weight.
The structure of the DNA high-throughput sequencing libraries of embodiment 1
Use exemplified by adapter shown in Formulas I (for nothing, S6 is nothing to wherein S5) (referring to Fig. 1), constructed dna high-flux sequence text
Storehouse, adapter pretreatment process is as follows：
1st, adapter phosphorylation
The single stranded DNA sample being commercially synthesized is dissolved in pure water and obtains the solution that concentration is 100 μM, 5 μ L solution are taken to one
200 μ L PCR light-wall pipes, add 1 μ 10 × T4 of L ligase buffer solutions (final concentration of 50mM Tris-HCl, 10mM MgCl2,
1mM ATP, 10mM DTT), 1 μ L T4 polynueleotide kinases (PNK) (10U/ μ L), moisturizing to cumulative volume is 10 μ L.
37 DEG C react 60 minutes, then 70 DEG C reaction 25min inactivate enzyme.
2nd, anneal
1 10 × NEBuffer of μ L 2 (final concentration of 50mM NaCl, 10mM Tris-HCl, 10mM are added in above-mentioned reaction
MgCl2, 1mM DTT), moisturizing to cumulative volume is 20 μ L.
95 DEG C are heated 10 minutes, are then preserved on ice to 4 DEG C with 0.1 DEG C/sec of cooling rate slow cooling.
3rd, filling-in
2 10 × NEBuffer of μ L 2 (final concentration of 50mM NaCl, 10mM Tris-HCl, 10mM are added in above-mentioned system
MgCl2, 1mM DTT), 2 μ L concentration are 10mM dNTP, and 3 μ L concentration are 5U/ μ L Klenow enzymes, and moisturizing to cumulative volume is 40 μ
L。
25 DEG C are reacted 30 minutes, are then reclaimed with the QIAquick nucleotide removal kit of QIAGEN companies
Reaction product, cumulative volume is 60 μ L.
4th, dT tails are added
Added in above-mentioned solution 10 10 × NEBuffer of μ L 2 (final concentration of 50mM NaCl, 10mM Tris-HCl,
10mM MgCl2, 1mM DTT), 3 μ L concentration are 10mM dTTP, and 5 μ L concentration are 5U/ μ L Klenow (exo-) enzyme, and moisturizing is extremely
Cumulative volume is 100 μ L.
37 DEG C are reacted 30 minutes, are then reclaimed with the QIAquick nucleotide removal kit of QIAGEN companies
Reaction product, cumulative volume is 30 μ L.So far adapter pretreatment is completed, -20 DEG C of preservations after can dispensing.
Banking process flow is as follows, and initial sample DNA bound is the μ g of 500pg~1：
1st, A tails are repaired/added in end
Take 200 μ LPCR to manage, add initial sample DNA, the μ L of dA tails complex enzyme 3 are repaired/added in end, and 10 × end is repaired slow
The μ L of fliud flushing 6.5, moisturizing to 65 μ L.
20 DEG C are reacted 30 minutes, and then 65 DEG C are reacted 30 minutes, 4 DEG C of preservations.
2nd, connect
The μ L of T4DNA ligases 5 are added in above-mentioned reaction, the μ L of adapter 1 prepared are added (if initial sample DNA is total
Amount<100ng, adapter is used after must diluting 10 times with water), moisturizing to cumulative volume is 80 μ L.
25 DEG C are reacted 2 hours.
3rd, 3 μ L USER enzymes are added in above-mentioned system, then 37 DEG C are reacted 15 minutes.
80 μ L AMPure XP magnetic beads are added in above-mentioned system, is incubated at room temperature after mixing 5 minutes, then puts quiet on magnet stand
Put 5 minutes, inhaled after solution becomes clear and abandon supernatant.Magnetic bead is washed three times with 80% ethanol solution newly matched somebody with somebody, supernatant is abandoned in suction,
Put standing and drying 10 minutes on magnet stand.Then 28 μ L 10mM Tris-HCl, pH8.0 are added, mixes and 5 points is stood on rearmounted magnet stand
Clock, then draws 23 μ L of supernatant into another 200 μ L PCR pipes, continuously adds following solution：
The μ L of 2 × PCR premixed liquids 25, the concentration with sequencing mark (barcode) is 25 μM of the μ L of special primer 1, concentration
For 25 μM of the μ L of universal primer 1, cumulative volume is 50 μ L.
Following response procedures are performed in PCR instrument：
98 DEG C of denaturations 30 seconds, then 98 DEG C are denatured 10 seconds, and 65 DEG C are annealed 30 seconds, and 72 DEG C extend 30 seconds, and this program is held
5~16 circulations of row, last 72 DEG C extend 5 minutes, 4 DEG C of preservations.
If originating DNA sample close to 1 μ g, then PCR cycle number can be controlled in 5~7；If initial sample is 50ng,
PCR cycle number is 10 or so；If initial sample number is 500pg, then PCR cycle needs 13~16.
Product is cleared out with AMPure XP magnetic beads
45 μ L AMPure XP magnetic beads are added in above-mentioned system, is incubated at room temperature after mixing 5 minutes, then puts quiet on magnet stand
Put 5 minutes, inhaled after solution becomes clear and abandon supernatant.Magnetic bead is washed three times with 80% ethanol solution newly matched somebody with somebody, supernatant is abandoned in suction,
Put standing and drying 10 minutes on magnet stand.Then 33 μ L 10mM Tris-HCl, pH8.0 are added, mixes and 5 points is stood on rearmounted magnet stand
Clock, then draws 28 μ L of supernatant into another new pipe, and this is the library that can be used for upper machine to be sequenced prepared.
In the present invention, each part S1-S6 sequence is as follows in adapter：
S1 is N5-20(degeneracy base (N) length is 5~20)；
S2 sequence：5'-AGATCGGAAGAGC-3'(SEQ ID NO.:1)；
S4 sequence：5'-GCTCTTCCGATCT-3'(SEQ ID NO.:2)；
S3 sequence：5'-ACACGTCTGAACTCCAGTCUACACTCTTTCCCTACACGAC-3'(SEQ ID NO.:
3)。
In the specific embodiment of the present invention, adapter sequential structure is Z1-Z2,
Wherein, Z1 is N5-20；
Z2 is 5'-AGATCGGAAGAGCACACGTCTGAACTCCAGTCUACACTCTTTCCCTACACGAC GCTCTTCCG
ATCT-3'(SEQ ID NO.:4)。
In the specific embodiment of the present invention, primer sequence is as follows：
Universal primer P5 is SEQ ID NO.:5：
5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC GCTCTTCCGATCT-3'；
Special primer P7 is
P1-B-P2
In formula,
P1 is CAAGCAGAAGACGGCATACGAGATTG (SEQ ID NO.:1-26 in 6)；
B is sequencing mark (barcode) sequence, such as TTGACT；
P2 is GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO.:33-66 in 6).
In another embodiment, the length of the B is 6 nt.
In another embodiment, the B is selected from table 1 below：
The Illumina high-flux sequences of table 1 mark Barcode numberings and correspondence sequence
In another embodiment, special primer P7 sequence is SEQ ID NO.:6：
5'-CAAGCAGAAGACGGCATACGAGATTGTTGACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
-3'。
All documents referred in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Sequence table
<110>Shanghai Communications University
<120>The preparation method and its matched reagent box of a kind of quantitative high-throughput sequencing library
<130> P2017-0449
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 13
<212> DNA
<213>Artificial sequence
<400> 1
agatcggaag agc 13
<210> 2
<211> 13
<212> DNA
<213>Artificial sequence
<400> 2
gctcttccga tct 13
<210> 3
<211> 40
<212> DNA
<213>Artificial sequence
<400> 3
acacgtctga actccagtcu acactctttc cctacacgac 40
<210> 4
<211> 66
<212> DNA
<213>Artificial sequence
<400> 4
agatcggaag agcacacgtc tgaactccag tcuacactct ttccctacac gacgctcttc 60
cgatct 66
<210> 5
<211> 58
<212> DNA
<213>Artificial sequence
<400> 5
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatct 58
<210> 6
<211> 66
<212> DNA
<213>Artificial sequence
<400> 6
caagcagaag acggcatacg agattgttga ctgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
Claims (10)
1. a kind of adapter, it is characterised in that the adapter has Formulas I or the structure shown in II：
5'-S1─S2─S3─S4─S5─S6-3' (I)
5'-pS1─S2─S3─S4─S5─S6-3' (II)
In various,
S1 is optionally guide portion (N)m, N is any base in A, T, G, C, and m is 1-150 positive integer；
PS1 is the S1 of phosphorylation；
S2 is stem 1, with SEQ ID NO.:Nucleotide sequence shown in 1；
S3 is ring portion, wherein containing at least one base U；
S4 is stem 2, with SEQ ID NO.:Nucleotide sequence shown in 2；
S2 and S4 complete complementaries or partial complementarity；
S5 is the extension without or with guide portion S1 complementations；
S6 is nothing or the joint portion containing at least one base T；
If S5 is nothing, S6 is nothing；
"-" is chemical bond.
2. adapter as claimed in claim 1, it is characterised in that m is 1-100, more preferably 1-50, most preferably 5-20 any
Positive integer.
3. adapter as claimed in claim 1, it is characterised in that the ring portion S3 has SEQ ID NO.:Nucleosides shown in 3
Acid sequence.
4. adapter as claimed in claim 1, it is characterised in that the S6 contains 1 base T.
5. adapter as claimed in claim 1, it is characterised in that the S2 of the adapter has SEQ ID NO.:Shown in 1
Nucleotide sequence；S3 has SEQ ID NO.:Nucleotide sequence shown in 3；S4 has SEQ ID NO.:Nucleotides shown in 2
Sequence.
6. a kind of method that sequencing library is built based on nucleic acid samples, it is characterised in that including step：
(a) nucleic acid samples for being used to build sequencing library are provided；
(b) end is carried out to described nucleic acid samples and repairs reaction, obtain the repaired nucleic acid samples in end；
(c) the nucleic acid samples progress 3' end repaired to described end adds dA end reactions, obtains the nucleic acid samples through tailing；
(d) nucleic acid samples through tailing are mixed with any described adapter in claim 1-5, and be attached anti-
Should, so as to form the first mixture containing " adapter-nucleic acid-adapter " compound；
(e) " adapter-nucleic acid-adapter " compound obtained in step (d) is mixed with USER enzymes so that in adapter
U is degraded, and forms second mixture of not complementary " adapter-nucleic acid-adapter " compound containing end furcations；
(f) the second mixture containing the compound obtained in step (e) is mixed with fragment sorting magnetic bead so that described
Section sorting magnetic bead adsorbs unnecessary adapter, and the adsorbed adapter is removed, and obtains the 3rd mixture；
(g) compound using step (f) acquisition is template, with the first primer and specificity for specifically binding to adapter
The second primer for being incorporated into adapter enters performing PCR reaction, obtains amplified production；
(h) using the amplified production that will obtain in step (g) as raw material, obtained sequencing library.
7. method as claimed in claim 6, it is characterised in that step (a) the amplifying nucleic acid sample total amount is the μ g of 500pg~1.
8. a kind of kit that sequencing library is built based on nucleic acid samples, it is characterised in that the kit includes：
Repair/add dA tails complex enzyme and its buffer solution in one end；
One T4DNA ligases；
One adapter, described adapter is as described in any in claim 1-5；
And specification, described specification describes application method.
9. kit as claimed in claim 8, it is characterised in that the kit also includes：Specifically bind to adapter
The first primer and the second primer.
10. kit as claimed in claim 9, it is characterised in that second primer is marked with sequencing.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710552185.0A CN107217052A (en) | 2017-07-07 | 2017-07-07 | The preparation method and its matched reagent box of a kind of quantitative high-throughput sequencing library |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988320A (en) * | 2017-11-10 | 2018-05-04 | 至本医疗科技（上海）有限公司 | A kind of molecular label connector and its preparation method and application |
| CN109797196A (en) * | 2019-01-30 | 2019-05-24 | 艾吉泰康生物科技(北京)有限公司 | A kind of extensible application of the probe in cfDNA library construction of zip mode |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102296065A (en) * | 2011-08-04 | 2011-12-28 | 盛司潼 | System and method for constructing sequencing library |
| CN102586472A (en) * | 2011-12-27 | 2012-07-18 | 盛司潼 | Method and kit for assaying hepatitis B virus DNA (deoxyribonucleic acid) sequence |
| CN103668471A (en) * | 2013-12-19 | 2014-03-26 | 上海交通大学 | Method for constructing high-throughput DNA sequencing library and matching kit thereof |
| CN103748236A (en) * | 2011-04-15 | 2014-04-23 | 约翰·霍普金斯大学 | Safe sequencing system |
| CN105143525A (en) * | 2013-03-15 | 2015-12-09 | 纽亘技术公司 | Methods, compositions and kits for generation of stranded RNA or DNA libraries |
| CN106047998A (en) * | 2016-05-27 | 2016-10-26 | 深圳市海普洛斯生物科技有限公司 | Detection method and application of lung cancer genes |
| CN106048009A (en) * | 2016-06-03 | 2016-10-26 | 人和未来生物科技（长沙）有限公司 | Label joint for detection of ultra-low-frequency gene mutation and application of label joint |
| CN106811460A (en) * | 2015-11-30 | 2017-06-09 | 安诺优达基因科技(北京)有限公司 | For the construction method and kit of two generation sequencing libraries of low frequency abrupt climatic change |
| CN106834275A (en) * | 2017-02-22 | 2017-06-13 | 天津诺禾医学检验所有限公司 | The analysis method of the construction method, kit and library detection data in ctDNA ultralow frequency abrupt climatic changes library |
| CN106906211A (en) * | 2017-04-13 | 2017-06-30 | 江苏为真生物医药技术股份有限公司 | A kind of molecular adaptor and its application |
| CN107058310A (en) * | 2016-08-12 | 2017-08-18 | 艾吉泰康生物科技(北京)有限公司 | A kind of amplicon library constructing method for improving gene low frequency abrupt climatic change sensitivity |
-
2017
- 2017-07-07 CN CN201710552185.0A patent/CN107217052A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103748236A (en) * | 2011-04-15 | 2014-04-23 | 约翰·霍普金斯大学 | Safe sequencing system |
| CN102296065A (en) * | 2011-08-04 | 2011-12-28 | 盛司潼 | System and method for constructing sequencing library |
| CN102586472A (en) * | 2011-12-27 | 2012-07-18 | 盛司潼 | Method and kit for assaying hepatitis B virus DNA (deoxyribonucleic acid) sequence |
| CN105143525A (en) * | 2013-03-15 | 2015-12-09 | 纽亘技术公司 | Methods, compositions and kits for generation of stranded RNA or DNA libraries |
| CN103668471A (en) * | 2013-12-19 | 2014-03-26 | 上海交通大学 | Method for constructing high-throughput DNA sequencing library and matching kit thereof |
| CN106811460A (en) * | 2015-11-30 | 2017-06-09 | 安诺优达基因科技(北京)有限公司 | For the construction method and kit of two generation sequencing libraries of low frequency abrupt climatic change |
| CN106047998A (en) * | 2016-05-27 | 2016-10-26 | 深圳市海普洛斯生物科技有限公司 | Detection method and application of lung cancer genes |
| CN106048009A (en) * | 2016-06-03 | 2016-10-26 | 人和未来生物科技（长沙）有限公司 | Label joint for detection of ultra-low-frequency gene mutation and application of label joint |
| CN107058310A (en) * | 2016-08-12 | 2017-08-18 | 艾吉泰康生物科技(北京)有限公司 | A kind of amplicon library constructing method for improving gene low frequency abrupt climatic change sensitivity |
| CN106834275A (en) * | 2017-02-22 | 2017-06-13 | 天津诺禾医学检验所有限公司 | The analysis method of the construction method, kit and library detection data in ctDNA ultralow frequency abrupt climatic changes library |
| CN106906211A (en) * | 2017-04-13 | 2017-06-30 | 江苏为真生物医药技术股份有限公司 | A kind of molecular adaptor and its application |
Non-Patent Citations (1)
| Title |
|---|
| SICHAI: "二代测序：碱基平衡与barcode选择", 《百度文库》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988320A (en) * | 2017-11-10 | 2018-05-04 | 至本医疗科技（上海）有限公司 | A kind of molecular label connector and its preparation method and application |
| CN109797196A (en) * | 2019-01-30 | 2019-05-24 | 艾吉泰康生物科技(北京)有限公司 | A kind of extensible application of the probe in cfDNA library construction of zip mode |
| CN109797196B (en) * | 2019-01-30 | 2022-06-07 | 艾吉泰康生物科技(北京)有限公司 | Application of zipper type extensible probe in cfDNA library construction |
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