CN108456713A - The construction method of tab closure sequence, library construction Kit and sequencing library - Google Patents
The construction method of tab closure sequence, library construction Kit and sequencing library Download PDFInfo
- Publication number
- CN108456713A CN108456713A CN201711209208.4A CN201711209208A CN108456713A CN 108456713 A CN108456713 A CN 108456713A CN 201711209208 A CN201711209208 A CN 201711209208A CN 108456713 A CN108456713 A CN 108456713A
- Authority
- CN
- China
- Prior art keywords
- sequence
- tab closure
- library
- capture
- dna
- 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.)
- Pending
Links
Classifications
-
- 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
-
- 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
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of tab closure sequence, the construction methods of library construction Kit and sequencing library.Tab closure sequence includes：P5 tab closure sequences, P5 tab closure sequences have SEQ ID NO:Sequence shown in 1：AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT C, and SEQ ID NO:There are reversed dT to modify for 3 ' ends of sequence shown in 1, partition modification, the modification of dideoxycytidine acid and any one of phosphorylation modification.By being modified plus closing in 3 ' ends of closing sequence, the joint efficiency of closing sequence is increased, improves capture rate by reducing the nontarget area capture caused by connector connects, and reduce the influence of the closing follow-up PCR processes of sequence pair.
Description
Technical field
The present invention relates to sequencing libraries to build field, in particular to a kind of tab closure sequence, library construction reagent
The construction method of box and sequencing library.
Background technology
Using Illumina TrueSeq connectors carry out library construction general flow be：DNA is interrupted as 300- first
400bp carries out end reparation to the DNA of fragmentation, and adds A base tails, then by library and Illumina TrueSeq connectors
It is attached, finally carries out PCR amplification and enrichment by primer of P5 the and P7 sequences of the known general-purpose at connector both ends, build
Library can be used to hybrid capture.
The closing in progress library is needed before hybrid capture process, is on the one hand the repetitive sequence closed in Insert Fragment,
On the other hand it is closing library joint sequence.Library capture probe after closing captures target area, and then side is closed
Be sequenced at side, by being absorbed in the sequencing of target area, sequencing depth improved under same quantity of data, to DNA more as possible into
The sequencing of the high sequencing depth of row.
But closed process before the capturing is low in the presence of closing efficiency at present, so that the other capture rate ratio in target area
It is relatively low.For this reason, it is necessary to be improved to existing method, to improve the capture rate of target area.
Invention content
The main purpose of the present invention is to provide a kind of tab closure sequence, the structures of library construction Kit and sequencing library
Construction method, the capture rate to solve the problems, such as target fragment in sequencing library in the prior art are low.
To achieve the goals above, according to an aspect of the invention, there is provided a kind of tab closure sequence, tab closure
Sequence includes：P5 tab closure sequences, P5 tab closure sequences have SEQ ID NO:Sequence shown in 1：
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT C, and SEQ ID NO:Sequence shown in 1
Row there are reversed dT modify for 3 ' ends, partition modification, dideoxycytidine acid are modified and any one of phosphorylation modification.
Further, tab closure sequence further includes：P7 tab closure sequences, P7 tab closure sequences have SEQ ID
NO:Sequence shown in 2：CAAGCAGAAGACGGCATACGAGATNGTGACTGGAGTTCAGACGTGTGCTCTTCCGA TC, wherein N
Represent the reverse complementary sequence of index, and SEQ ID NO:There are reversed dT to modify for 3 ' ends of sequence shown in 2, partition is modified,
Any one of the modification of dideoxycytidine acid and phosphorylation modification.
To achieve the goals above, it according to an aspect of the invention, there is provided a kind of library construction Kit, including connects
Head sealer, contains any of the above-described kind of tab closure sequence in tab closure agent.
Further, in tab closure reagent, the working concentration of tab closure sequence is 0.1.~0.25nM.
Further, kit further includes repetitive sequence sealer in Insert Fragment；It is preferably inserted into repetitive sequence in segment
Sealer is Cot-1 DNA.
Further, kit further includes hybrid capture reagent.
Further, hybrid capture reagent is DNA hybridization capture agent, and preferably DNA hybridization capture agent is Integrated Device Technology, Inc.
DNA hybridization capture agent.
Further, hybrid capture reagent is RNA hybrid capture reagents, and preferably RNA hybrid captures reagent is Agilent public
The RNA hybrid capture reagents of department.
According to another aspect of the present invention, a kind of construction method of sequencing library is provided, which includes pre-expansion
It is used in the step of the step of increasing the capture of structure, tab closure, solution hybridization and the capture amplified library in library, tab closure
Any of the above-described kind of tab closure sequence is closed, or is closed using the closed reagent in any of the above-described kit.
It applies the technical scheme of the present invention, the present invention is increased by being modified plus closing in 3 ' ends of closing sequence
The joint efficiency for closing sequence improves capture rate by reducing the nontarget area capture caused by connector connects, and drops
The influence of the low closing follow-up PCR processes of sequence pair.
Description of the drawings
The accompanying drawings which form a part of this application are used to provide further understanding of the present invention, and of the invention shows
Meaning property embodiment and its explanation are not constituted improper limitations of the present invention for explaining the present invention.In the accompanying drawings：
Fig. 1 shows the principle schematic of the tab closure in the application.
Specific implementation mode
It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase
Mutually combination.Below in conjunction with embodiment, the present invention will be described in detail.
3 '-Spacer (partition) are modified, and Spacer can provide necessary interval to reduce label base for oligonucleotide marker
Interaction between group and oligonucleotides is mainly used in DNA hairpin structures and duplex structure research.C3spacer is mainly used for
Imitate three carbon intervals between 3' the and 5' hydroxyls of ribose, or base unknown in " replacement " sequence.3'-Spacer C3 are used
Arm is to prevent the ends 3' excision enzyme and the ends 3' polymerase from acting between introducing one 3 '.
3 '-Dideoxy-C (dideoxycytidine acid) modification, 3 ' phosphorylations (Phosphorylation) modification and 3 '
Inverted dT (reversed dT) are modified in the related experiment that can be used for resisting 3 ' circumscribed enzymic digestions, it can also be used to prevent archaeal dna polymerase
The DNA chain extension of catalysis.
In order to improve in existing solution hybridization capture library construction process, the capture rate of destination region is relatively low
Existing hybrid capture library constructing method is studied in detail in defect, inventor, finds the connector at current library both ends
Sequence of the sequence using the sequence reverse complemental with connector is closed, by completing text with the base pair complementarity of joint sequence
The closing of library connector.Specifically, tab closure sequence is divided into two parts, the P5 and sequencing primer 1 of a part and sequence measuring joints
(SP1) region sequence reverse complemental, another part and sequencing primer 2 (SP2), label (index) and sequence measuring joints P7 region sequences are anti-
To complementation, tab closure is carried out by the complementary pairing of corresponding part.However, the sequence using reverse complemental carries out library
Closing combine when, during hybrid capture, close sequence combination be easy influenced by system temperature, close sequence
Between easily form dimer and cause close efficiency reduce so that the capture rate of target area also reduces.
In addition, if the sequence of reverse complemental does not completely remove before PCR processes, subsequent captured library also can influenced just
Often amplification.Here it is always 65 DEG C of cleanings that influence, which refers to after capture, and theoretically at 65 DEG C, (Tm is big for library and closed reagent
General 76 DEG C) it is possible without and unlocks double-strand, and the double-strand that closed reagent is formed at both ends at this time just always exists.If index
It is not corresponding and if 3 ' ends are not decorated, PCR after capture during very likely will appear closed reagent as primer after
The situation of continuous amplification, and be likely to cause index chaotic in this way, sample can not be differentiated.
On the basis of the studies above result, inventor has carried out various improvement to existing tab closure sequence, finds
It is modified by the 3 ' ends to existing tab closure sequence, helps to improve the sealing effect of docking header sequence, and then made
The capture rate for obtaining target area improves.Moreover, inventor also found, by carrying out 3 '-Spacer to existing joint sequence
(partition) modification, 3 '-Dideoxy-C (dideoxycytidine acid) modifications, 3 '-Phosphorylation (phosphorylation) modifications and
3 '-Inverted dT modifications, can improve the sealing effect of docking header sequence to some extent, and wherein, 3 '-Inverted
The sealing effect of docking header sequence is best after dT modifications, and capture rate improves most notable.
Applicant provides a kind of tab closure sequence based on above-mentioned discovery in a kind of typical embodiment of the application
Row, the tab closure sequence include：P5 tab closure sequences, P5 tab closure sequences have SEQ ID NO：Sequence shown in 1：
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT C, and SEQ ID NO：Shown in 1
There are reversed dT to modify for 3 ' ends of sequence, partition modification, the modification of dideoxycytidine acid and any one of phosphorylation modification.
Above-mentioned tab closure sequence provided by the present application is mainly used in and is built using Illumina TrueSeq connectors
Library uses before this library carries out hybrid capture as tab closure reagent.Tab closure in compared with prior art
Sequence, the tab closure sequence provided by the present application that closing modification is carried out in 3 ' ends are capable of the envelope of reinforced joint closing sequence
Effect is closed, and then improves the capture rate of solution hybridization acquisition procedure, the tab closure sequence of especially reversed dT modifications, docking
The sealing effect of header sequence is best, is improved to the capture rate of solution hybridization acquisition procedure most notable.
Above-mentioned P5 tab closures sequence has improved closing efficiency compared to existing tab closure sequence, and then improves
Capture rate.In order to further increase the hybrid capture efficiency in existing library construction process, as shown in Figure 1, another in the application
In a kind of preferred embodiment, above-mentioned tab closure sequence further includes：P7 tab closure sequences, P7 tab closure sequences have
SEQ ID NO:Sequence shown in 2：CAAGCAGAAGACGGCATACGAGATNGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C, wherein N represent the reverse complementary sequence of index, and SEQ ID NO:There are reversed dT to modify for 3 ' ends of sequence shown in 2,
Any one of wall modification, the modification of dideoxycytidine acid and phosphorylation modification.
By after the 3 ' ends to P5 tab closure sequences carry out closing modification (especially reversed dT modification), then into one
Step carries out closing modification (especially reversed dT is modified) to 3 ' ends of P7 tab closure sequences, while improving P5 tab closures
The closing efficiency of sequence and P7 tab closures sequence in tab closure step, so that the capture of solution hybridization acquisition procedure
It is more efficient.Wherein, in P7 tab closures sequence, length and the index sequences of the reverse complementary sequence of index representated by N
Length is identical, usually 6~12bp.
Tab closure sequence provided by the present application be suitable for Illumina TrueSeq connectors structure all pre- libraries into
Joint sequence closing before the capture of row target area, and follow-up available capture system include but not limited to Agilent,
The business and customization capture agent and chip of Nimblegen and Illumina.
In second of typical embodiment of the application, a kind of library construction Kit is additionally provided, the kit packet
Include tab closure agent, wherein contain any of the above-described kind of tab closure sequence in tab closure agent.
Include the library construction Kit of any of the above-described kind of tab closure sequence, builds the tab closure effect during library
Fruit is good so as to the capture rate higher in target fragment/region when hybrid capture.
To the specifically used concentration of the tab closure reagent in mentioned reagent box, can be carried out according to the amount in pre- amplification library
Reasonable set.In a kind of preferred embodiment of the application, in tab closure reagent, the working concentration of tab closure sequence is
0.1~0.25nM.In the range by the working concentration setting of tab closure sequence, have closing efficient, and tab closure
Sequence is not easy excess and leads to the normal structure for PCR processes being interfered and being influenced when subsequent captured amplified library library.
Can also include that other library constructions are common other than comprising above-mentioned tab closure agent in mentioned reagent box
Reagent, to make library construction more convenient and quicker.In a kind of preferred embodiment of the application, mentioned reagent box further includes being inserted into
Repetitive sequence sealer in segment.In the application another kind preferred embodiment, mentioned reagent box further includes hybrid capture examination
Agent.
The kit of the repetitive sequence sealer of purpose Insert Fragment in library is further included, convenient in closing step
It docks header sequence simultaneously and the repetitive sequence of purpose Insert Fragment is closed, to improve subsequent capture rate.And into
One step includes the kit of hybrid capture reagent, convenient for so that closing step is more convenient with hybrid capture step operation.
Repetitive sequence sealer in Insert Fragment in mentioned reagent box uses existing repetitive sequence sealer.
In a kind of preferred embodiment of the application, repetitive sequence sealer is Cot-1 DNA in Insert Fragment.People Cot-1 DNA are
Placenta dna, length are mainly 50~300bp, and are rich in reiterated DNA sequences, so for closing repetitive sequence, DNA with
It is required for using in the hybrid capture system of DNA, DNA and RNA.Salmon sperm DNA are salmon sperm dna, are used in DNA
With (solution hybridization of such as Agilent captures system) in RNA hybrid system, effect is sample in closing DNA and RNA hybrid processes
Non-specific signals.And without the hybrid capture body of salmon sperm dna, such as IDT in the hybridization system of general dna and DNA
System.
Hybrid capture reagent in the kit of the prior art is typically to match with closing related reagent, is caught with improving
Obtain efficiency.And in the mentioned reagent box of the application, hybrid capture reagent can select DNA hybridization capture examination according to actual needs
Agent or RNA hybrid capture reagents, regardless of being DNA hybridization capture agent or RNA hybrid capture reagents, upper with the application
Closed reagent cooperation where applicable is stated, all has and closes efficient and high capture rate advantage.
Therefore, in a kind of preferred embodiment of the application, above-mentioned hybrid capture reagent is DNA hybridization capture agent, excellent
It is the DNA hybridization capture agent of Integrated Device Technology, Inc. to select DNA hybridization capture agent.In the application another kind preferred embodiment, hybridization
Capture agent is RNA hybrid capture reagents, and preferably RNA hybrid captures reagent is the RNA hybrid capture reagents of Agilent companies.
In the application in the third typical embodiment, a kind of construction method of sequencing library, the structure are additionally provided
Method includes the steps that structure, tab closure, solution hybridization capture and the capture amplified library in pre- amplification library, tab closure
The step of in closed using any of the above-described kind of tab closure sequence, or using the closing examination in any of the above-described kind of kit
Agent is closed.
By the closing modification in 3 ' ends of closing sequence plus reversed dT, the joint efficiency of closing sequence is increased,
By reduce because connector connection caused by nontarget area capture due to improve capture rate, and reduce closing sequence pair after
The influence of continuous PCR processes, thus, there are the higher advantages of purpose target fragment accounting in constructed library.
Further illustrate the advantageous effect of the application below in conjunction with specific embodiments.
Embodiment 1
Reagent used in embodiment 1 is shown in Table 1, and hybrid capture reagent therein is the RNA hybrid captures of Agilent companies
System.
Table 1：
One, sample extraction
Tissue samples use the paraffin-embedded tissue DNA extraction kit (commodity article No. Dp331) of Tiangeng company, extract
DNA be stored in it is spare in -20 DEG C.Sample names and extracted amount are shown in Table 2.
Table 2：
Two, the fragmentation of DNA.
2.1---DNA interrupting
It takes 6 parts of sample (520ng/ parts) to interrupt instrument (instrument model S220) using Covaris, is arranged according to the following table 3
Parameter carry out the broken of DNA.The main peak of breakdown products takes 20ng to carry out electrophoresis detection in 200-350bp.
Table 3：
| Highest incident power | Service factor | Energy transmission is counted | Processing time |
| 450W | 30% | 200 | 350-380s |
2.2---DNA is detected after interrupting
2.2.1 preparing in advance needs reagent to be used：
Prepare 2% Ago-Gel (11 hole glue)：Claim 0.6g agaroses, be dissolved in 1 × TAE of 30mL Buffer,
Micro-wave oven dissolves by heating, and at least to boil 5 times or so, slightly cooling in room temperature after dissolving completely, is poured into after being cooled to 50 degree or so
In ready glue support, its solidification is allowed to be placed in 4 DEG C of preservations after forty minutes.
It is for use that 1 μ L SYBR Green I are added into 6 × Loading of 1mL Buffer.
2.2.2 it takes 5 μ L of the sample after interrupting to be added in 0.2mL centrifuge tubes, adds 1 μ L 6 × loading buffer,
With vortex oscillator oscillation mixing 5s, palm centrifuge 5s.
2.2.3 it takes 5 μ L of 100bp marker to be put into 0.2mL centrifuge tubes, adds 1 μ L 6 × loading buffer,
With vortex oscillator oscillation mixing 5s, palm centrifuge 5s.
2.2.4 sample and prepared marker are subjected to dispensing, electrophoresis detection (120V, 30min).
2.2.5 after electrophoresis, gel imaging system is observed as a result, electrophoretic band carries out sample after concentrating on 150-300bp
This purification process.If the main band of electrophoresis concentrates on 300bp or more, sample DNA is subjected to supplement and interrupts operation.
2.3---1.8X AMPure XP magnetic beads for purifying DNA samples
2.3.1 AMPure XP bead suspensions are mixed well, until suspension color is uniform.
2.3.2 DNA is taken out from 0.2mL centrifuge tubes, is put into the new pipes of 1.5mL, the AMPure XP of 90 μ L mixings are added
Bead suspension.High speed vortex mixing 5s, is placed at room temperature for 5min.
2.3.3 of short duration centrifugation, pipe is placed on magnetic frame, stands about 5min to solution change clarification.
2.3.4 the supernatant in pipe is carefully absorbed on magnetic frame, pipette tips not encounter magnetic bead.
2.3.5 on magnetic frame, add 200 μ L, 80% ethyl alcohol respectively in each pipe.
2.3.6 after standing 1min waiting magnetic bead sedimentations, ethyl alcohol is absorbed.
2.3.7 it is primary that step 2.3.5 and 2.3.6 are repeated.
2.3.8 51 μ L EB are added, in the upper mixing 5s of high speed vortex, are placed at room temperature for 5min.
2.3.9 of short duration centrifugation, pipe is placed on magnetic frame, stands about 2min to solution change clarification.
2.3.10 it draws in 50 μ L supernatants to a new PCR pipe, checks errorless rear discarding magnetic bead.
Three, the structure in pre- library
Repair and add A in the ends 3.1---
The reagent that this step is used：End-Repair＆A-Tailing Buffer, End-Repair＆A-Tailing
Enzyme Mix (are derived from KAPA Hyper Prep Kit Illumina platforms kits, production firm：KAPA, goods
Number：KK8504).Fragmented, double-stranded DNA are the DNA samples that previous step interrupts, totally 4 parts.
3.1.1 according to the form below 4 configures reaction solution in PCR pipe, with the gently upper and lower pressure-vaccum mixing of rifle.
Table 4：
3.1.2 it is put into PCR instrument, according to the form below 5 is arranged program and is reacted (hot lid temperature is set as 70 DEG C), reaction system 60
μL。
Table 5：
3.2--- jointings (Adaptor)
The reagent that this step is used：Connect buffer solution (Ligation Buffer), DNA ligase (DNA Ligase
Enzyme is derived from KAPA Hyper Prep Kit Illumina platforms kits, production firm：KAPA, article No.：
KK8504)；(Truseq Adaptor, are derived from connectorDNA Barcodes -48 kits, production firm：Bioo
Scientific, article No.：NOVA-514104).
3.2.1 according to the form below 6 configures reaction solution in PCR pipe, with the gently upper and lower pressure-vaccum mixing of rifle.
Table 6：
Sample number and connector (Truseq Adaptor) number and corresponding label (index) sequence such as the following table 7.
Table 7：
Above-mentioned system is put in PCR instrument, and 20 DEG C of warm bath 15min, reaction system is set as 100 μ L, not use heat lid.
3.2.2 the AMPure XP magnetic beads for purifying DNA samples of 0.8X.
3.2.2.1 AMPure XP bead suspensions are mixed well, until suspension color is uniform.
3.2.2.2 the AMPure XP bead suspensions and end that 88 μ L mixings are added in the new pipes of 1.5mL add
The DNA library of Adaptor.High speed vortex mixing 5s, is placed at room temperature for 5min.
3.2.2.3 of short duration centrifugation, pipe is placed on magnetic frame, stands about 5min to solution change clarification.
3.2.2.4 the supernatant in pipe is carefully absorbed on magnetic frame, pipette tips not encounter magnetic bead.
3.2.2.5 on magnetic frame, add 200 μ L, 80% ethyl alcohol respectively in each pipe.
3.2.2.6 after standing 1min waiting magnetic bead sedimentations, ethyl alcohol is absorbed.
3.2.2.7 step 5,6 are repeated once.
3.2.2.8 25 μ L EB are added, in the upper mixing 5s of high speed vortex, are placed at room temperature for 5min.
3.2.2.9 of short duration centrifugation, pipe is placed on magnetic frame, stands about 2min to solution change clarification.
3.2.2.10 it draws in 24 μ L supernatants to a new PCR pipe, checks errorless rear discarding magnetic bead.
The library of connector has been gone up in 3.3--- amplification connections
The reagent that this step is used：2X KAPA HiFi Hotstart ReadyMix (are derived from KAPA Hyper Prep
Kit Illumina platforms kits, production firm：KAPA, article No.：KK8504)；(P5+P7) primer (25uM), sequence
Row are as shown in table 8 below.
Table 8：
| SEQ ID NO: | Primer | Sequence (5 ' -3 ') |
| 3 | P5 | AATGATACGGCGACCACCGAGA |
| 4 | P7 | CAAGCAGAAGACGGCATACGAG |
3.1 in PCR pipe according to the form below 9 configure reaction solution, with rifle gently pressure-vaccum mixing up and down.
Table 9：
Above-mentioned reaction solution is put into PCR instrument, according to the form below 10 is arranged program and is reacted.
Table 10：
3.3.2 the AMPure XP magnetic beads for purifying DNA samples of 1.8X
3.3.2.1 AMPure XP bead suspensions are mixed well, until suspension color is uniform.
3.3.2.2 AMPure XP bead suspensions and the PCR product high speed whirlpool of 90 μ L mixings are added in the new pipes of 1.5mL
Mixing 5s is revolved, 5min is placed at room temperature for.
3.3.2.3 of short duration centrifugation, pipe is placed on magnetic frame, stands about 5min to solution change clarification.
3.3.2.4 the supernatant in pipe is carefully absorbed on magnetic frame, pipette tips not encounter magnetic bead.
3.3.2.5 on magnetic frame, add 200 μ L, 80% ethyl alcohol respectively in each pipe.
3.3.2.6 after standing 1min waiting magnetic bead sedimentations, ethyl alcohol is absorbed.
3.3.2.7 step 5,6 are repeated once.
3.3.2.8 26 μ L NF-water are added, in the upper mixing 5s of high speed vortex, are placed at room temperature for 5min.
3.3.2.9 of short duration centrifugation, pipe is placed on magnetic frame, stands about 2min to solution change clarification.
3.3.2.10 it draws in 26 μ L supernatants to a new PCR pipe, checks errorless rear discarding magnetic bead.
It is quantitative that 3.4 pairs of libraries Pre-Capture carry out Qubit
It is quantitative that 1 μ L progress Qubit 2.0 of product in PCR pipe is walked in absorption.The libraries Pre-Capture yield should be greater than at this time
500ng。
Four, Library hybridization
4.1--- configures tab closure agent
The tab closure sequence of synthesis fills for powder, needs addition nuclease-free water to be dissolved, nuclease free water volume
It is calculated according to the volume indicated on tube wall.The tab closure sequence configured is known as tab closure agent, is referred to as P5 sealers
With P7 sealers, wherein P5 sealers have SEQ ID NO:Sequence shown in 1, and P7 sealers have SEQ ID NO:Shown in 2
Sequence.3 ' the ends that differ only in of closing sequence used in following each library whether there is modification, table 11 and table specific as follows
Shown in 12.
Table 11：
| P5 closes sequence -1 | Without modification |
| P7 closes sequence -1 | Without modification |
| P5 closes sequence -2 | 3 ' end Inverted dT are modified |
| P7 closes sequence -2 | 3 ' end Inverted dT are modified |
Table 12：
The closing and hybridization in the libraries 4.2---
4.2.1 take 500ng DNA libraries in a new EP pipe, draining machine with vacuum is concentrated, temperature≤45 DEG C,
Until the volume in library is 5 μ L.
4.2.2 the formula of according to the form below 13 prepares the closing mixed liquor (Block Mix) of sample reactions all enough.
Table 13：
DNA library use after CB030, CB032, CB034-1 concentration is closed without modification sequence, CB031, CB033,
CB034-2 carries the closing sequence of reversed dT modifications using the 3 ' ends added in the application.It is separately added into the P5/P7 envelopes of configuration
Each 1 μ L of agent are closed, pay attention to No. Index corresponding to P7 sealers.With pressure-vaccum above and below rifle 8-10 times, then pipe lid covers tightly, high speed whirlpool
Tube wall liquid is collected into tube bottom by rotation oscillation 5s, of short duration centrifugation, and reaction solution volume is 17 μ L at this time.
4.2.3 it reaffirms that pipe lid covers tightly, is put into PCR instrument, 14 Program of according to the form below is reacted, and is covered using 105 DEG C of heat,
Reaction system is set as 30 μ L.
It is important：PCR programs must be walked in third to be suspended, probe is added.In the first step and second step reaction process
Capture Library hybridization mixed liquor (Capture Library Hybridization Mix) can be prepared in advance.
Table 14：
4.2.4 capture Library hybridization mixed liquor (Capture Library Hybridization are prepared according to the following table 15
Mix), high speed vortex oscillation mixing 5s, of short duration centrifugation carry out next step operation immediately.
Table 15：
4.2.5 when the response procedures in PCR, which proceed to third, walks (65 DEG C of 1min at), suspend response procedures, keep
Library mixed liquor is added to library under the conditions of 65 DEG C, by the Capture Library Hybridization Mix of 13 μ L and mixes
It closes in liquid, with pressure-vaccum mixing 8-10 times above and below rifle, covers pipe lid immediately, pipe inner volume is 30 μ L at this time.
4.2.6 high speed vortex oscillation 5s, of short duration centrifugation, then puts back in PCR instrument, start program.
It is important：It must ensure that pipe lid covers tightly, minimize the evaporation for reducing hybrid mixed liquid product, otherwise will influence to hybridize
Effect.
4.3--- is captured and elution
The reagent that this step is used：Dynabeads My One Streptavidin T1 (manufacturers：Invitogen, goods
Number：65602, hereinafter referred to as T1 magnetic beads), 30min, which takes out, in advance is placed in room temperature；Sure Select Binding Buffer,
Sure Select Wash 1；Sure Select Wash 2 (Agilent SureSelect QXT Reagent Kit are derived from,
Manufacturer：Agilent, article No.：G9681B), after the completion of " combination in library and T1 magnetic beads " step operation, constant-temperature metal bath is used
65 DEG C of preheatings.
4.3.1Sure Select Binding Buffer balance T1 magnetic beads
4.3.1.1 magnetic bead can settle when preserving, and high speed, which is vortexed, acutely shakes, again suspension T1 magnetic beads.
4.3.1.2 it to each hybridization reaction, takes in 50 μ L T1 magnetic beads to PCR pipe.
4.3.1.3 200 μ L Sure Select Binding Buffer 8-10 mixings of pressure-vaccum are added.
4.3.1.4 pipe is placed on magnetic frame, until solution absorbs supernatant after becoming clarification.
4.3.1.5 repeat step 3,4 twice, rinse 3 times in total.
4.3.1.6 last is after the completion of rinsing, and of short duration centrifugation is again placed on magnetic frame, it is ensured that all Sure
Select Binding Buffer are absorbed
4.3.1.7 with 200 μ L Sure Select Binding Buffer again suspension T1 magnetic beads.
4.3.2 the combination in library and T1 magnetic beads
After general hybridization in 2 hours, sample is placed in room temperature, (being estimated with liquid-transfering gun) is estimated and records remaining
The volume of hybrid mixed liquid.
It is important：The volume of hybrid mixed liquid is about 30 μ L at this time, and excessive vaporization will influence subsequent capture effect.
4.3.2.1 after determining that hybrid mixed liquid is reduced to room temperature, by its T1 magnetic bead solution after being applied directly to balance with rifle
In, overturn mixing 3-5 times.
4.3.2.2 mixed liquor is placed on rotary mixer, room temperature (ensureing 24 DEG C or so) mixing 30min.
Note：PCR pipe can be placed in 1.5mL centrifuge tubes and place into rotary mixer.
4.3.3Sure Select Wash 1 rinse magnetic bead
4.3.3.1 brief centrifugation is placed on pipe on magnetic frame, stands to solution and clarifies, and absorbs supernatant.
4.3.3.2 200 μ L Sure Select Wash 1 are added, so that magnetic bead is suspended 15 times with pressure-vaccum above and below rifle, vortex shakes
Swing 8s, brief centrifugation.
4.3.3.3 pipe is placed on magnetic frame, stands to solution and clarifies, absorb supernatant.
4.3.4Sure Select Wash 2 rinse magnetic bead
4.3.4.1 the Sure Select Wash 2 that 200 μ L pass through 65 DEG C of preheatings are added, with 15 flushings of pressure-vaccum above and below rifle
Magnetic bead.
4.3.4.2 65 DEG C of warm bath 5min in PCR instrument, system are set as 100 μ L, and hot lid temperature is set as 105 DEG C.
4.3.4.3 pipe is placed on magnetic frame, stands to solution and clarifies, absorb supernatant.
4.3.4.4 it repeats step 1-3 twice, rinses 3 times in total.
4.3.4.5 last is after the completion of rinsing, and of short duration centrifugation is again placed on magnetic frame, it is ensured that all Sure
Select Wash2 are absorbed.
4.3.4.6 38 μ L NF-water are added, high speed vortex 5s suspends magnetic bead again.
PCR amplification after 4.4--- captures
The reagent that this step is used is：5 × HercuLase II Rxn Buffer, HercuLase II Fusion DNA
Polymerase, 100mM dNTP Mix (are derived from Herculase II Fusion Enzyme with dNTPs Combo
400Rxn Kit, production firm：Agligent, article No.：600679)；(P5+P7)Primer(25uM)
4.4.1 PCR reaction solution is prepared according to the following table 16 system on ice.
Table 16：
4.4.2 confirm after the reaction solution mixing containing magnetic bead, pipe is put into PCR instrument and is expanded, pay attention to replacing
Pipe lid, PCR instrument parameter setting such as the following table 17：
Table 17：
4.4.3 with 1.2 times of AMPure XP magnetic beads for purifying DNA samples
4.4.3.1 AMPure XP bead suspensions are mixed well, until suspension color is uniform.
4.4.3.2 the AMPure XP bead suspensions that 60 μ L mixings are added in the new pipes of 1.5mL and the DNA after PCR amplification
Library.High speed vortex mixing, is placed at room temperature for 5min.
4.4.3.3 of short duration centrifugation, pipe is placed on magnetic frame, stands about 3min to solution change clarification.
4.4.3.4 the supernatant in pipe is carefully absorbed on magnetic frame, pipette tips not encounter magnetic bead.
4.4.3.5 on magnetic frame, add 200 μ L, 80% ethyl alcohol respectively in each pipe.
4.4.3.6 after standing 1min sedimentation magnetic beads, ethyl alcohol is absorbed.
4.4.3.7 step 5,6 are repeated once.
4.4.3.8 room temperature is dried, until remaining ethyl alcohol volatilizees completely in pipe, (the unsuitable overdrying of magnetic bead, otherwise can cause to wash
De- efficiency is remarkably decreased).
4.4.3.9 21 μ L EB are added, in the upper mixing of high speed vortex, are placed at room temperature for 5min.
4.4.3.10 of short duration centrifugation, pipe is placed on magnetic frame, stands about 2min to solution change clarification.
4.4.3.11 it draws in about 20 μ L supernatants to a new 1.5mL pipe, magnetic bead is can drop after review.
4.4.3.12 1 μ L samples are taken to carry out Qubit 2.0 quantitative.
The libraries 4.5--- library is examined and upper machine
Library is diluted to 2ng/ μ L, 1 μ L is taken out and carries out Agilent 2100Bioanalyzer (Agilent company of the U.S.) inspections
It surveys；It is detected for qPCR in addition, further taking out 1ul, determines upper machine concentration according to testing result.It, will according to the concentration obtained by upper step
Library is diluted to confidential (2nmol) after asking, and PE150 sequencings are carried out in the HiseqX microarray datasets of Illumina companies.
Embodiment 2
Specific reagent used in embodiment 2 is shown in Table 18, and hybrid capture reagent therein is that the DNA hybridization of Integrated Device Technology, Inc. is caught
Obtain system.
Table 18：
One, sample extraction
Cell line sample uses paramagnetic particle method poba gene group DNA extraction kit (the commodity article No. DP329- of Tiangeng company
02) DNA, extracted is stored in spare in -20 DEG C.Sample names and extracted amount are shown in Table 19.
Table 19：
Two, the fragmentation of DNA.
2.1---DNA interrupting
It takes 6 parts of sample (120ng/ parts) to interrupt instrument (instrument model S220) using Covaris, is arranged according to the following table 20
Parameter carry out the broken of DNA.The main peak of breakdown products takes 20ng to carry out electrophoresis detection in 200-350bp.
Table 20：
| Highest incident power | Service factor | Energy transmission is counted | Processing time |
| 450W | 30% | 200 | 350-380s |
2.2---DNA is detected after interrupting
2.2.1 preparing in advance needs reagent to be used：
Prepare 2% Ago-Gel (11 hole glue)：Claim 0.6g agaroses, be dissolved in 1 × TAE of 30mL Buffer,
Micro-wave oven dissolves by heating, and at least to boil 5 times or so, slightly cooling in room temperature after dissolving completely, is poured into after being cooled to 50 degree or so
In ready glue support, its solidification is allowed to be placed in 4 DEG C of preservations after forty minutes.
It is for use that 1 μ L SYBR Green I are added into 6 × Loading of 1mL Buffer.
2.2.2 it takes 5 μ L of the sample after interrupting to be added in 0.2mL centrifuge tubes, adds 1 μ L 6 × loading buffer,
With vortex oscillator oscillation mixing 5s, palm centrifuge 5s.
2.2.3 it takes 5 μ L of 100bp marker to be put into 0.2mL centrifuge tubes, adds 1 μ L 6 × loading buffer,
With vortex oscillator oscillation mixing 5s, palm centrifuge 5s.
2.2.4 sample and prepared marker are subjected to dispensing, electrophoresis detection (120V, 30min).
2.2.5 after electrophoresis, gel imaging system is observed as a result, electrophoretic band carries out sample after concentrating on 150-300bp
This purification process.If the main band of electrophoresis concentrates on 300bp or more, sample DNA is subjected to complement operation.
2.3---1.8X AMPure XP magnetic beads for purifying DNA samples
2.3.1 AMPure XP bead suspensions are mixed well, until suspension color is uniform.
2.3.2 DNA is taken out from 0.2mL centrifuge tubes, is put into the new pipes of 1.5mL, the AMPure XP of 90 μ L mixings are added
Bead suspension.High speed vortex mixing 5s, is placed at room temperature for 5min.
2.3.3 of short duration centrifugation, pipe is placed on magnetic frame, stands about 5min to solution change clarification.
2.3.4 the supernatant in pipe is carefully absorbed on magnetic frame, pipette tips not encounter magnetic bead.
2.3.5 on magnetic frame, add 200 μ L, 80% ethyl alcohol respectively in each pipe.
2.3.6 after standing 1min waiting magnetic bead sedimentations, ethyl alcohol is absorbed.
2.3.7 step 5,6 are repeated once.
2.3.8 51 μ L EB are added, in the upper mixing 5s of high speed vortex, are placed at room temperature for 5min.
2.3.9 of short duration centrifugation, pipe is placed on magnetic frame, stands about 2min to solution change clarification.
2.3.10 it draws in 50 μ L supernatants to a new PCR pipe, checks errorless rear discarding magnetic bead.
Three, the structure in pre- library
Repair and add A in the ends 3.1---
The step for by fragmentation DNA end-fillings, and 5 ' end carry out phosphorylations and 3 ' end plus dA tails.
Mixing is overturned after ER＆A mix are thawed, and the reaction system of table 21 is formulated as follows in the PCR pipe that sterilizes：
Table 21：
Mixing (mixing please don't be vibrated) is gently blown and beaten using pipettor, and of short duration centrifugation collects reaction solution to tube bottom.
PCR pipe is placed in PCR instrument, program is reacted shown according to the form below 22.
Table 22：
After reaction, connector Connection Step is carried out immediately.
3.2--- connectors connect
The step for will repair product end jointing in end.Connector is diluted 10 times using nuclease-free water；It will
Rapid ligation buffer overturn mixing after thawing, and are placed in spare on ice.
It is formulated as follows reaction system shown in table 23 in the PCR pipe that step is repaired in end：
Table 23：
* after rapid ligation buffer and rapid DNA ligase premixs, r for 24 hours can be no more than in 4 DEG C of storages,
Connector is added when reaction.Specifically to joint sequence and corresponding library title such as the following table 24 used in sample：
Table 24：
Mixing (mixing please don't be vibrated) is gently blown and beaten using pipettor, and of short duration centrifugation collects reaction solution to tube bottom.It will
PCR pipe is placed in PCR instrument, and program shown in the following table 25 is reacted.
Table 25：
After reaction, connector connection product purification step is carried out immediately.
3.3--- connector connection products purify
Note：Magnetic bead, which shifts to an earlier date 30min, to be taken out, balance to room temperature.
3.31 magnetic beads are balanced to room temperature, and be vortexed concussion mixing VAHTS Particles G.
3.32 draw in 80 μ L VAHTS Particles G to 100 μ L connectors connection reaction products, vortex oscillation or make
It is gently blown and beaten 10 times and is mixed well with pipettor.
3.33 being incubated at room temperature 5min.
The of short duration centrifugation of PCR pipe is placed in separation magnetic bead and liquid in magnetic frame by 3.34, after solution clarification (about 5min),
Carefully remove supernatant (note：Supernatant is removed as possible).
3.35 holding PCR pipes are placed in magnetic frame always, and 80% ethyl alcohol that 200 μ l Fresh are added rinses magnetic bead, room
Temperature is incubated 30s, carefully removes supernatant.
3.36 repeat step 5 once, amount to rinsing twice.
3.37 holding PCR pipes are placed in magnetic frame always, and uncapping is air-dried magnetic bead 5-10min and remains to no ethyl alcohol.
3.38 take out PCR pipe from magnetic frame, 52 μ L elutions are added, vortex oscillation or use pipettor are gently
Piping and druming mixes well, and in being stored at room temperature 2min, the of short duration centrifugation of PCR pipe is placed on magnetic frame and is stood, after solution clarification (about
5min), it carefully draws in 50 μ L supernatants to new PCR pipe, is sure not to touch magnetic bead.
3.4--- amplified library
The step for will to after purification connection product carry out PCR amplification.By PCR primer mix,
Amplification mix2 overturn mixing after thawing, and reaction system shown in table 26 is formulated as follows in the PCR pipe that sterilizes：
Table 26：
Mixing is softly blown and beaten using pipettor, and of short duration centrifugation collects reaction solution to tube bottom.
Reaction tube is placed in PCR instrument, program is reacted shown according to the form below 27：
Table 27：
3.5--- amplified productions purify
Note：Magnetic bead, which shifts to an earlier date 30min, to be taken out, balance to room temperature.
3.51 magnetic beads are balanced to room temperature, and be vortexed concussion mixing VAHTS Particles G.
3.52 draw in 45 μ L VAHTS Particles G to 50 μ L connectors connection reaction products, vortex oscillation or use
Pipettor is gently blown and beaten 10 times and is mixed well.
3.53 being incubated at room temperature 5min.
The of short duration centrifugation of PCR pipe is placed in separation magnetic bead and liquid in magnetic frame by 3.54, after solution clarification (about 5min),
Carefully remove supernatant (note：Supernatant is removed as possible).
3.55 holding PCR pipes are placed in magnetic frame always, and 80% ethyl alcohol that 200 μ l Fresh are added rinses magnetic bead, room
Temperature is incubated 30s, carefully removes supernatant.
3.56 repeat step 5 once, amount to rinsing twice.
3.57 holding PCR pipes are placed in magnetic frame always, and uncapping is air-dried magnetic bead 5-10min and remains to no ethyl alcohol.
3.58 take out PCR pipe from magnetic frame, 22 μ L elutions are added, vortex oscillation or use pipettor are gently
Piping and druming mixes well, and in being stored at room temperature 2min, the of short duration centrifugation of PCR pipe is placed on magnetic frame and is stood, after solution clarification (about
5min), it carefully draws in 20 μ L supernatants to new PCR pipe, is sure not to touch magnetic bead.
3.59 take 1 μ L qubit reagent measured concentrations, and it is spare that remaining library is placed in -20 DEG C of refrigerators.
Four, Library hybridization
Prepare tab closure reagent：
The tab closure sequence of synthesis fills for powder, needs addition nuclease-free water to be dissolved, nuclease free water volume
It is calculated according to the volume indicated on tube wall.The tab closure sequence configured is known as tab closure agent, is referred to as P5Blocker
And P7Blocker.Close sequence such as the following table 28：
Table 28：
Note：Pho is the abbreviation of Phosphorylation.
4.1--- closing
Reagent shown in the following table 29 is added in 0.2mL PCR pipes：
Table 29：
PCR pipe is placed in and is concentrated in vacuo in instrument, rate of drying is tuned into middling speed and is dried, about 1.5h-2h completes drying.
4.2--- hybridization
4.2.1 all reagents are taken out from -20 DEG C of refrigerators, is placed in room temperature and melts.
Remarks：Whether there is crystallization in the pipe of detection 2X Hybridization buffer, if there is crystallization, reagent is set
It is heated to reagent in 65 DEG C completely to melt, during which need to repeatedly shake, this process needs are heated for a period of hours.
4.2.2 reagent is added according to the following table 30 in the pipe of hybrid capture (one) and room temperature is incubated 5-10min：
Table 30：
4.2.3 in the PCR pipe for the 0.2mL for being transferred to a new low adsorption after blowing and beating mixing with liquid-transfering gun.
4.2.4 95 DEG C of incubation 10min in PCR instrument are placed in.
4.2.5 PCR pipe is taken out, the probe of a concentration of 0.75pmol/ μ L of 4 μ L is added immediately.
4.2.6 be vortexed concussion mixing, and PCR pipe, which is placed in PCR instrument 65 DEG C, after brief centrifugation is incubated 4h (temperature of lid is 75
℃)。
4.3--- prepares elution buffer
4.3.1 for each capture reaction, each buffer solution in the following table 31 is diluted to the working solution of 1X.
Remarks：(15-25 DEG C) can at most be placed 4 weeks the working solution of 1X at ambient temperature.
Table 31：
Remarks：If necessary, need to water-bath or metal bath be carried out to be resuspended in reagent at 65 DEG C by the wash buffer of 10X
Particle.
4.3.2 it prepares in the working solution of 1X as shown in following table 32, part elution buffer I and rigorous elution buffer
It needs to be placed on 65 DEG C and at least preheats 2h, remaining 1X working solution is in (15-25 DEG C) preservation of room temperature.
Table 32：
4.4--- prepares M-270 magnetic beads.Points for attention：Magnetic bead should be now with the current, not allow magnetic bead overdrying.
4.4.1 it is taken out from 4 DEG C of refrigeratorsM-270 magnetic beads, vortex 15s place balance 30min at room temperature
Left and right.Remarks：The Streptavidin MagneSphere of replacement is not recommended, because the yield of capture may be reduced by substituting magnetic bead.
4.4.2 the concussion magnetic bead 15s that is vortexed makes it mix well.
4.4.3 each capture reaction needs to use the M-270 magnetic beads of 100 μ L.
4.4.4 the 1.5mL centrifuge tubes equipped with magnetic bead are placed on magnetic frame, are stood, removed supernatant after solution clarification, note
Meaning not be drawn onto pearl.
4.4.5 200 μ 1 × Bead of L Wash Buffer, vortex 10s are added.
4.4.6 centrifuge tube is placed on magnetic frame after solution clarification after brief centrifugation, carefully draws and discards supernatant.
4.4.7 it is primary to repeat step 5-6.
4.4.8 100 μ L 1 × Bead Wash Buffer are added, new low adsorption is transferred to after vortex mixing
In the PCR pipe of 0.2mL.
4.4.9 PCR pipe is placed on magnetic frame and is adsorbed, after solution clarification, carefully drawn and discard supernatant.
Pay attention to：Pearl needs to carry out next step reaction immediately after cleaning.
4.5--- capture
4.5.1 the hybrid product of step (2) is transferred in the PCR pipe containing magnetic bead in step (4), with rifle pressure-vaccum
It is set to mix well 10 times.
4.5.2 PCR pipe is placed in PCR instrument, 65 DEG C are incubated 45min (temperature of lid is 75 DEG C), during which, every 12min
Vortex oscillation 3s makes pearl be maintained at suspended state.
4.6--- elutions are not bonded to the DNA on magnetic bead
4.6.1 65 DEG C of elutions
1) 1 × Wash Buffer I that 100 μ L are preheating to 65 DEG C are added.
2) after vortex oscillation mixing, brief centrifugation centrifuges solution to tube bottom.
3) PCR pipe is placed on magnetic frame, after solution clarification, draws and discard supernatant (containing the DNA on being not associated with).
4) 200 μ L are added and are preheating to 65 DEG C of 1X Stringent Wash Buffer, with the slow pressure-vaccum of liquid-transfering gun 10 times,
It is careful not to generate bubble in this process.
5) PCR pipe is placed in PCR instrument, 65 DEG C of incubation 5min.
6) PCR pipe is placed on magnetic frame to adsorb, after solution is clarified, is carefully drawn with liquid-transfering gun and discard supernatant.
7) step 5 and 6 is repeated once.
4.6.2 room temperature elutes
1) 1 × Wash Buffer I, vortex mixing 2min of 200 μ L room temperatures is added.
2) after brief centrifugation, PCR pipe is placed on magnetic frame and is adsorbed, after solution clarification, carefully inhaled with liquid-transfering gun
It takes and discards supernatant.
3) 1 × Wash Buffer II, vortex mixing 1min of 200 μ L room temperatures is added.
4) after brief centrifugation, PCR pipe is placed on magnetic frame and is adsorbed, after solution clarification, carefully inhaled with liquid-transfering gun
It takes and discards supernatant.
5) 1 × Wash Buffer III, vortex mixing 30s of 200 μ L room temperatures is added.
6) after brief centrifugation, PCR pipe is placed on magnetic frame and is adsorbed, after solution clarification, carefully inhaled with liquid-transfering gun
It takes and discards supernatant.
4.6.3 magnetic bead is resuspended
1) PCR pipe containing the magnetic bead combined with DNA is taken off from magnetic frame and is put on rack for test tube.
2) water that 20 μ L nuclease frees are added ensures that all magnetic beads are resuspended with rifle pressure-vaccum 10 times.
It is expanded after 4.7--- captures
4.7.1 according to preparation pcr amplification reaction liquid shown in the following table 33
Table 33：
4.7.2 by prepared pcr amplification reaction liquid vortex mixing.
4.7.3 PCR pipe is placed in PCR instrument, is reacted according to the program of following table table 34.
Table 34：
Remarks：The pcr amplification product can preserve overnight at 4 DEG C.
4.8---PCR amplified productions purify
4.8.1 75 μ L (1.5 ×) VAHTS Particles G magnetic beads, whirlpool are added in the PCR pipe containing pcr amplification product
After rotation oscillation mixing, room temperature is incubated 5min.
4.8.2 PCR pipe is placed on magnetic frame and is adsorbed, the about 5min after solution clarification is carefully drawn with liquid-transfering gun
And abandon supernatant.
4.8.3 200 μ L, 80% ethyl alcohol is added, after standing 30s on magnetic frame, is drawn with liquid-transfering gun and abandons supernatant.
4.8.4 it is primary to repeat step 3.
4.8.5 brief centrifugation makes the ethyl alcohol on tube wall centrifuge to tube bottom, after residual ethanol is abandoned in careful suction, is stored at room temperature 5min
Ethyl alcohol is set fully to volatilize.
4.8.6 the water of 22 μ L nuclease frees is added, vortex mixing is stored at room temperature 5min.
4.8.7 brief centrifugation is placed on magnetic frame, is stood 2min and is drawn 20 μ L liquid to new EP after solution clarification
Guan Zhong.
4.8.8 1 μ L Qubit reagent measured concentrations are taken.
The result of embodiment one and embodiment two detects：
The sequencing result of embodiment one and embodiment two is compared with reference gene group using bwa softwares, specific matter
Information is controlled as shown in following table 35, shown in the capture rate in the library of different closed reagents following 36.
Table 35：
Table 36：
It is attached：Total bases/covering the full-length genome in=coverage goal the region capture rate (Probe capture ratio)
Total bases * 100%.
It can be seen from the above description that the above embodiments of the present invention realize following technique effect：It is repaiied with not adding
The closing sequence of decorations is compared, and the present invention is by increasing the tab closure sequence of modification (especially reversed dT modify) in 3 ' ends
Row, improve the joint efficiency with connector, improve closing efficiency, reduce the nontarget area caused by connector connection due to capture
Capture rate is improved, and reduces the influence of the closing follow-up PCR processes of sequence pair.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, any made by repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Sequence table
<110>The Tianjin sources Nuo Hezhi biological information Science and Technology Ltd.
<120>The construction method of tab closure sequence, library construction Kit and sequencing library
<130> PN78266NHZY
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 57
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(57)
<223>There are reversed dT to modify for the ends 3', partition modification, dideoxycytidine acid is modified or phosphorylation modification
<400> 1
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatc 57
<210> 2
<211> 58
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(58)
<223>There are reversed dT to modify for 3 ' ends, partition modification, dideoxycytidine acid is modified or phosphorylation modification
<220>
<221> misc_feature
<222> (25)..(25)
<223>N represents the reverse complementary sequence of the index of the 6-12bp of a, t, c or g composition
<400> 2
caagcagaag acggcatacg agatngtgac tggagttcag acgtgtgctc ttccgatc 58
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(22)
<223>P5 joint sequences
<400> 3
aatgatacgg cgaccaccga ga 22
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(22)
<223>P7 joint sequences
<400> 4
caagcagaag acggcatacg ag 22
Claims (9)
1. a kind of tab closure sequence, which is characterized in that the tab closure sequence includes：P5 tab closure sequences, the P5
Tab closure sequence has SEQ ID NO:Sequence shown in 1：
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT C, and the SEQ ID NO:1 institute
Show sequence there are reversed dT to modify for 3 ' ends, partition modification, any one of the modification of dideoxycytidine acid and phosphorylation modification.
2. tab closure sequence according to claim 1, which is characterized in that the tab closure sequence further includes：P7 connects
Head closing sequence, the P7 tab closures sequence have SEQ ID NO:Sequence shown in 2：
CAAGCAGAAGACGGCATACGAGATNGTGACTGGAGTTCAGACGTGTGCTCTTCCGA TC, wherein N represent the anti-of index
To complementary series, and the SEQ ID NO:There are reversed dT to modify for 3 ' ends of sequence shown in 2, partition is modified, double deoxidation born of the same parents
Any one of thuja acid modification and phosphorylation modification.
3. a kind of library construction Kit, including tab closure agent, which is characterized in that contain in the tab closure agent and have the right to want
Seek the tab closure sequence described in 1 or 2.
4. kit according to claim 3, which is characterized in that in the tab closure reagent, the tab closure sequence
The working concentration of row is 0.1.~0.25nM.
5. kit according to claim 3, which is characterized in that the kit further includes repetitive sequence in Insert Fragment
Sealer；It is preferred that repetitive sequence sealer is Cot-1 DNA in the Insert Fragment.
6. kit according to claim 3, which is characterized in that the kit further includes hybrid capture reagent.
7. kit according to claim 6, which is characterized in that the hybrid capture reagent is DNA hybridization capture agent,
It is preferred that the DNA hybridization capture agent is the DNA hybridization capture agent of Integrated Device Technology, Inc..
8. kit according to claim 6, which is characterized in that the hybrid capture reagent is RNA hybrid capture reagents,
It is preferred that the RNA hybrid captures reagent is the RNA hybrid capture reagents of Agilent companies.
9. a kind of construction method of sequencing library, the construction method includes that the structure, tab closure, liquid phase in pre- amplification library are miscellaneous
The step of handing over capture and capture amplified library, which is characterized in that claims 1 or 2 is used in the step of the tab closure
The tab closure sequence is closed, or using the closing examination in the kit described in any one of claim 3 to 9
Agent is closed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711209208.4A CN108456713A (en) | 2017-11-27 | 2017-11-27 | The construction method of tab closure sequence, library construction Kit and sequencing library |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711209208.4A CN108456713A (en) | 2017-11-27 | 2017-11-27 | The construction method of tab closure sequence, library construction Kit and sequencing library |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108456713A true CN108456713A (en) | 2018-08-28 |
Family
ID=63220362
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711209208.4A Pending CN108456713A (en) | 2017-11-27 | 2017-11-27 | The construction method of tab closure sequence, library construction Kit and sequencing library |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108456713A (en) |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109777858A (en) * | 2018-12-20 | 2019-05-21 | 天津诺禾医学检验所有限公司 | The probe and method of hybrid capture are carried out to Duplication region |
| CN109988820A (en) * | 2019-04-19 | 2019-07-09 | 奥明（杭州）基因科技有限公司 | A kind of library constructing method and kit for the detection of breast cancer polygenes |
| CN110564831A (en) * | 2019-08-30 | 2019-12-13 | 北京优迅医学检验实验室有限公司 | Blocking reagent for sequencing library and method for improving targeted capture efficiency |
| CN111005074A (en) * | 2019-12-19 | 2020-04-14 | 江西海普洛斯医学检验实验室有限公司 | DNA library construction kit based on illumina sequencing platform, library construction method and application |
| WO2020073748A1 (en) * | 2018-10-11 | 2020-04-16 | 北京优乐复生科技有限责任公司 | Method for constructing sequencing library |
| CN111394436A (en) * | 2020-03-26 | 2020-07-10 | 上海天昊生物科技有限公司 | DNA (deoxyribonucleic acid) joint as well as preparation method and application thereof |
| CN111534518A (en) * | 2020-05-18 | 2020-08-14 | 纳昂达(南京)生物科技有限公司 | Universal blocking sequence and application thereof |
| CN111748551A (en) * | 2019-03-27 | 2020-10-09 | 纳昂达(南京)生物科技有限公司 | Blocking sequence, capture kit, library hybridization capture method and library construction method |
| CN111961711A (en) * | 2020-08-31 | 2020-11-20 | 伯科生物科技有限公司 | Universal hybridization enhancers and methods for targeted sequencing |
| CN111979307A (en) * | 2020-08-31 | 2020-11-24 | 伯科生物科技有限公司 | Targeted sequencing method for detecting gene fusion |
| CN112375809A (en) * | 2020-11-19 | 2021-02-19 | 天津莱贝生物科技有限公司 | Hybridization capture kit and method for performing hybridization capture by using same |
| CN114134204A (en) * | 2021-12-07 | 2022-03-04 | 上海交通大学 | Sample preparation method for trace labeled nucleic acid sample detection and sequencing |
| CN114657232A (en) * | 2022-03-11 | 2022-06-24 | 上海英基生物科技有限公司 | Universal blocking reagent for improving target capture efficiency and application thereof |
| CN114774515A (en) * | 2022-03-24 | 2022-07-22 | 北京安智因生物技术有限公司 | Capture probe, kit and detection method for detecting polycystic kidney disease gene mutation |
| WO2023179766A1 (en) * | 2022-03-24 | 2023-09-28 | 南京传奇生物科技有限公司 | Method for preparing dna library and detecting retroviral integration site |
| WO2024092562A1 (en) * | 2022-11-02 | 2024-05-10 | 京东方科技集团股份有限公司 | Blocking sequence, kit thereof, and method for using same |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102409047A (en) * | 2010-09-21 | 2012-04-11 | 深圳华大基因科技有限公司 | Method for building sequencing library by hybridization |
| CN104562214A (en) * | 2014-12-26 | 2015-04-29 | 上海派森诺生物科技有限公司 | Reduced-representation genome library building method based on type IIB restriction enzyme digestion |
| CN105925675A (en) * | 2016-04-26 | 2016-09-07 | 序康医疗科技（苏州）有限公司 | Method for amplifying dna |
| WO2017117440A1 (en) * | 2015-12-30 | 2017-07-06 | Bio-Rad Laboratories, Inc. | Droplet partitioned pcr-based library preparation |
-
2017
- 2017-11-27 CN CN201711209208.4A patent/CN108456713A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102409047A (en) * | 2010-09-21 | 2012-04-11 | 深圳华大基因科技有限公司 | Method for building sequencing library by hybridization |
| CN104562214A (en) * | 2014-12-26 | 2015-04-29 | 上海派森诺生物科技有限公司 | Reduced-representation genome library building method based on type IIB restriction enzyme digestion |
| WO2017117440A1 (en) * | 2015-12-30 | 2017-07-06 | Bio-Rad Laboratories, Inc. | Droplet partitioned pcr-based library preparation |
| CN105925675A (en) * | 2016-04-26 | 2016-09-07 | 序康医疗科技（苏州）有限公司 | Method for amplifying dna |
Non-Patent Citations (4)
| Title |
|---|
| B. BEKAERT等: "In-Solution Hybridization for the Targeted Enrichment of the Whole Mitochondrial Genome", 《FORENSIC DNA TYPING PROTOCOLS》 * |
| H. H WON等: "Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing", 《JOURNAL OF VISUALIZED EXPERIMENTS》 * |
| K. R. UPTON等: "Ubiquitous L1 Mosaicism in Hippocampal Neurons", 《CELL》 * |
| 深圳市百迈生命科学有限公司: "修饰引物的常见类型及应用", 《深圳市百迈生命科学有限公司 HTTP://WWW.BM-LS.COM/ZILIAO/2015/77.HTML》 * |
Cited By (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020073748A1 (en) * | 2018-10-11 | 2020-04-16 | 北京优乐复生科技有限责任公司 | Method for constructing sequencing library |
| CN111989406A (en) * | 2018-10-11 | 2020-11-24 | 北京优乐复生科技有限责任公司 | Construction method of sequencing library |
| CN111989406B (en) * | 2018-10-11 | 2023-02-21 | 北京优乐复生科技有限责任公司 | Construction method of sequencing library |
| CN109777858A (en) * | 2018-12-20 | 2019-05-21 | 天津诺禾医学检验所有限公司 | The probe and method of hybrid capture are carried out to Duplication region |
| CN111748551B (en) * | 2019-03-27 | 2021-07-09 | 纳昂达(南京)生物科技有限公司 | Blocking sequence, capture kit, library hybridization capture method and library construction method |
| CN111748551A (en) * | 2019-03-27 | 2020-10-09 | 纳昂达(南京)生物科技有限公司 | Blocking sequence, capture kit, library hybridization capture method and library construction method |
| CN109988820A (en) * | 2019-04-19 | 2019-07-09 | 奥明（杭州）基因科技有限公司 | A kind of library constructing method and kit for the detection of breast cancer polygenes |
| CN110564831A (en) * | 2019-08-30 | 2019-12-13 | 北京优迅医学检验实验室有限公司 | Blocking reagent for sequencing library and method for improving targeted capture efficiency |
| CN111005074A (en) * | 2019-12-19 | 2020-04-14 | 江西海普洛斯医学检验实验室有限公司 | DNA library construction kit based on illumina sequencing platform, library construction method and application |
| CN111394436A (en) * | 2020-03-26 | 2020-07-10 | 上海天昊生物科技有限公司 | DNA (deoxyribonucleic acid) joint as well as preparation method and application thereof |
| CN111394436B (en) * | 2020-03-26 | 2023-12-08 | 天昊基因科技(苏州)有限公司 | DNA (deoxyribonucleic acid) connector as well as preparation method and application thereof |
| CN111534518A (en) * | 2020-05-18 | 2020-08-14 | 纳昂达(南京)生物科技有限公司 | Universal blocking sequence and application thereof |
| CN111534518B (en) * | 2020-05-18 | 2021-07-23 | 纳昂达(南京)生物科技有限公司 | Universal blocking sequence and application thereof |
| WO2021232793A1 (en) * | 2020-05-18 | 2021-11-25 | 纳昂达(南京)生物科技有限公司 | Universal closed sequence and use thereof |
| CN111979307B (en) * | 2020-08-31 | 2022-07-08 | 伯科生物科技有限公司 | Targeted sequencing method for detecting gene fusion |
| CN111979307A (en) * | 2020-08-31 | 2020-11-24 | 伯科生物科技有限公司 | Targeted sequencing method for detecting gene fusion |
| CN111961711A (en) * | 2020-08-31 | 2020-11-20 | 伯科生物科技有限公司 | Universal hybridization enhancers and methods for targeted sequencing |
| CN112375809A (en) * | 2020-11-19 | 2021-02-19 | 天津莱贝生物科技有限公司 | Hybridization capture kit and method for performing hybridization capture by using same |
| CN114134204A (en) * | 2021-12-07 | 2022-03-04 | 上海交通大学 | Sample preparation method for trace labeled nucleic acid sample detection and sequencing |
| CN114657232A (en) * | 2022-03-11 | 2022-06-24 | 上海英基生物科技有限公司 | Universal blocking reagent for improving target capture efficiency and application thereof |
| CN114657232B (en) * | 2022-03-11 | 2024-04-30 | 上海英基生物科技有限公司 | Universal blocking reagent for improving targeted capture efficiency and application thereof |
| CN114774515A (en) * | 2022-03-24 | 2022-07-22 | 北京安智因生物技术有限公司 | Capture probe, kit and detection method for detecting polycystic kidney disease gene mutation |
| WO2023179766A1 (en) * | 2022-03-24 | 2023-09-28 | 南京传奇生物科技有限公司 | Method for preparing dna library and detecting retroviral integration site |
| WO2024092562A1 (en) * | 2022-11-02 | 2024-05-10 | 京东方科技集团股份有限公司 | Blocking sequence, kit thereof, and method for using same |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108456713A (en) | The construction method of tab closure sequence, library construction Kit and sequencing library | |
| CN108676846B (en) | Application of bridge oligonucleotide in library target region capture | |
| WO2013138510A9 (en) | Measurement of nucleic acid variants using highly-multiplexed error-suppressed deep sequencing | |
| CN110564831A (en) | Blocking reagent for sequencing library and method for improving targeted capture efficiency | |
| CN102212612A (en) | Constructing method of double-end library for high throughput 454 sequencing | |
| CN112852802A (en) | Linker for constructing sequencing library and construction method of sequencing library | |
| CN111575343A (en) | Construction method and kit of immune repertoire sequencing library | |
| US11384384B1 (en) | Methods and reagent kits for bisulfite conversion of DNA | |
| CN112662760A (en) | Cancer gene methylation detection system and cancer in-vitro detection method implemented in cancer gene methylation detection system | |
| CN113249457A (en) | Kit and method for constructing DNA nanospheres by one-step method | |
| CN112410331A (en) | Linker with molecular label and sample label and single-chain library building method thereof | |
| CN112662762A (en) | Probe composition for detecting 5 tumors of digestive tract | |
| CN112662765A (en) | Probe composition for detecting 6 Chinese high-incidence cancers | |
| CN109337956B (en) | Design method and kit for enriching multi-gene stem-loop probe based on NGS technology | |
| CN117551771A (en) | Construction method and kit of single-ended anchored multiplex PCR amplicon library for detecting tumor low-frequency mutation | |
| CN116790718A (en) | Construction method and application of multiplex amplicon library | |
| CN111607588B (en) | Kit and method for rapid enrichment of circular RNA | |
| CN112662761A (en) | Probe composition for detecting 3 parenchymal organ tumors | |
| CN113462759B (en) | Method for enrichment sequencing of single-stranded DNA sequence based on combination of multiplex amplification and probe capture and application of method in mutation detection | |
| CN109161580B (en) | HER2 gene fluorescence in situ hybridization probe and preparation method and application thereof | |
| CN113969317B (en) | Noninvasive detection method and noninvasive detection device for venous malformation and related syndromes | |
| CN115992128A (en) | Composition, kit and purification method thereof | |
| CN112391455A (en) | Kit for homologous recombination repair defect detection | |
| CN109777858A (en) | The probe and method of hybrid capture are carried out to Duplication region | |
| CN112375809A (en) | Hybridization capture kit and method for performing hybridization capture by using same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180828 |
|
| RJ01 | Rejection of invention patent application after publication |