CN108546739A - A method of the nucleic acid target sequence enrichment for NGS sequencings - Google Patents
A method of the nucleic acid target sequence enrichment for NGS sequencings Download PDFInfo
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- CN108546739A CN108546739A CN201810357468.4A CN201810357468A CN108546739A CN 108546739 A CN108546739 A CN 108546739A CN 201810357468 A CN201810357468 A CN 201810357468A CN 108546739 A CN108546739 A CN 108546739A
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Abstract
The present invention provides it is a kind of for NGS sequencing nucleic acid target sequence enrichment method, the method includes：Each target sequence probe is carried out isothermal duplication to form target sequence probes complementary chain, connects each target sequence probe, bound fraction is added on probe, obtains the single-stranded target sequence probes pond containing bound fraction by the design of target sequence probe.
Description
Technical field
The present invention relates in biotechnology, more particularly, it relates to which NGS sequencing amplifying nucleic acid target sequences are rich
The method of collection.
Background technology
With the fast development of NGS sequencing technologies (Next-generation sequencing technology), science
Boundary and clinical medicine circle start more and more NGS sequencing technologies to be applied in scientific research and clinical diagnosis；However for big
The gene order-checking of the species of type or need carry out superelevation deep sequencing when, sequencing data amount is big, give data analysis and storage tape
It greatly bothers, and cost superelevation is sequenced.At this point, the appearance of nucleic acid enriching capture technique solves, data volume is huge to ask
Topic.
Nucleic acid enriching capture technique refers to the target nucleic acid sequence using capture researcher's research of nucleic acid hybridization technique orientation
Row, are then sequenced, and to improve the depth of target nucleic acid sequence sequencing, accuracy and sensitivity, and make sequencing cost big
It is big to reduce.Multiple PCR technique is a kind of means of target sequence enrichment relatively common at present, for capturing multiple tracts
Section；Multiplex PCR technology has data volume few, the high advantage of depth, but this technical costs is higher, complicated for operation, to personnel's technology
It is required that high, sequencing data is uneven, and is mainly used for Hot spots detection, can not carry out the parallel sequencing of lots of genes.
Invention content
To solve the above-mentioned problems, the present invention provides a kind of nucleic acid enriching capture techniques for NGS sequencings.
In a first aspect, the present invention provides it is a kind of for NGS sequencing nucleic acid target sequence enrichment method, the side
Method includes：
A) design object sequence probes：To target sequence assessment, whether there is or not hairpin structures, if so, target sequence is extended outwardly
To no hairpin structure；
B) target sequence probe is added into universal sequence；
C) T of every probe is assessedmValue；
D) copy number adjustment is carried out to target sequence probe；
Each target sequence probe is carried out isothermal duplication to form target sequence probe by the e) preparation of target sequence probe
Complementary strand connects each target sequence probe, and bound fraction is added on probe, obtains the single-stranded target sequence containing bound fraction
Row probe cell.
In this embodiment, it is DNA single-stranded probes pond containing bound fraction target sequence probe cell.
In this embodiment, the target sequence probe has the characteristics that：1) hairpin structure itself is not generated, 2) copy
Several melting temperature T according to target sequence probemValue and space structure are determined.
In this embodiment, the target sequence probe on a solid carrier, such as on microarray slide.
In this embodiment, wherein the bound fraction is biotin binding species.
In this embodiment, wherein the target sequence probe length is 70-150bp, preferably 90-130bp, more preferably
105bp。
In this embodiment, it with reference to known group sequence, by stacked tile type design object sequence probes group, and calculates every
Target sequence probes Tm values, Tm≤ 80 DEG C, preferably Tm≤75℃；It is preferred that Tm values are joined based on 2007 thermodynamics of santalucia
The k-nearest neighbor of number table.
In this embodiment, wherein the no hairpin structure generation refers to that any one target sequence probe itself will not
Form hairpin structure.
In this embodiment, copy number compensation scheme is：With the identical spy of Tm values in stacked tile type design object sequence probes group
Tm values on the basis of the most Tm values of needle item number, other target sequence probes compared to benchmark Tm values often differ 0.02 DEG C (containing with
It is interior), probe copy number just more increases by 0.08 times, for example, Tm values on the basis of the Tm values of probe A, the Tm values of probe B are than benchmark Tm
It is worth 0.09 DEG C high, then the copy number of probe B is 1.36 times of probe A；It is preferred that Tm values are joined based on 2007 thermodynamics of santalucia
The k-nearest neighbor of number table.
In this embodiment, the region for target sequence less than an aim sequence probe length can use the target
Complementary design probe area, the region within preferably 50bp are used as within the 150bp of sequence both sides.
In this embodiment, for there is the aim sequence region of hairpin structure, can with the target sequence both sides 150bp with
It is interior to be used as complementary design probe area, the region within preferably 50bp.
In this embodiment, aim sequence probe increases a universal sequence at 3 ' ends, and probe structure is as follows：
5 '-probe sequence-CGTGGATGAGGAGCC-3 '
In this embodiment, target sequence probe is synthesized by oligonucleotides pond (Oligo Pools) technology, and passes through ammonia
Water elutes target sequence probe, is utilizing ultrapure water dissolution.The concentration of the ammonium hydroxide can be 50%.
In this embodiment, the complementary strand for preparing target sequence probe utilizes the Bst X DNA of Enzymatics companies
Polymerase kits, and the complementary series of universal primer in probe is added, carry out the complementary strand of synthesising probing needle；Specific reaction
System can be：10 μ l of target sequence probe cell, 2 μ l, 10x Bst X Reaction Buffer of universal primer, 5 μ l, 10mM
dNTP 1μl、Bst X DNA Polymerase 2μl、H2O 30μl.Target sequence probe cell described in the reaction system
Concentration can be that 10x Bst X Reaction Buffer and Bst X DNA Polymerase described in 10ng/ μ l. are
Reagent in the Bst X DNA Polymerase kits of Enzymatics companies, and 10mM dNTP buyings are public from Thermo
Department.The reaction condition is 65 DEG C of 30min, 80 DEG C of 10min.
In this embodiment, the T4DNA Ligase reagents of Enzymatics companies can be used by connecting each target sequence probe
Box carries out.The linked system is：Synthesize 20 μ l of target sequence probe, 25 μ of 2X T4DNA Ligase Buffer of complementary strand
l、 T4DNA Ligase 5μl.The reaction condition of the connection is 20 DEG C of 5h.
In this embodiment, the phi29DNA that Thermo companies are utilized containing bound fraction target sequence probe cell is prepared
The complementary primer sequences of universal sequence, specific reaction system can be in Polymerase kits and aim sequence probe：Even
10 μ l of aim sequence probe, 5 μ l of universal sequence complementary primer, 5 μ l of 10X Reaction Buffer, 10mM dATP after connecing
1μl、10mM dCTP 1μl、10mM dGTP 1μl、10mM dTTP 0.5μl、10mM Bio-16-dUTP 0.5μl、H2O 16
μl.Wherein universal sequence complementary primer concentration can be 2uM, and reaction condition is 95 DEG C of 10min, is placed in ice bath after reaction
Then the Phi29DNA Polymerase, 37 DEG C of 8h of 5 μ l is added in 5min.
In second aspect, the present invention also provides a kind of kit, the kit includes described in invention first aspect
The method of design object sequence probes and preparation containing bound fraction target sequence probe cell.The kit further includes using
In the hybridization solution, cleaning solution, eluent and the magnetic bead that can be combined with probe cell bound fraction that carry out nucleic acid target sequence enrichment.
Advantageous effect：
The method of amplifying nucleic acid target sequence enrichment of the present invention, has experimental implementation simple, greatly reduces sequencing and reality
Cost is tested, the parallel sequencing of lots of genes can be carried out, and sequencing data uniformity is good, coverage is high.
Specific implementation mode
The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The method of nucleic acid target sequence enrichment provided by the present invention, including：The design of target sequence probe, by each mesh
Mark sequence probes are prepared as the target sequence probe containing complementary strand, connect each target sequence probe, plus knot on probe
Part is closed, the single-stranded target sequence probes pond containing bound fraction is obtained.
Embodiment 1, target sequence probe design
It looks for 20 genes at random on human genome below, designs its exon region probe, it is how sharp to be specifically described
The method that can be enriched with target sequence is prepared with the method for the nucleic acid target sequence enrichment of the present invention.
Table 1：Randomly selected 20 target gene lists
Target sequence probe design procedure is as follows：
1. first finding this 20 objective gene sequences by reference to genome sequence, arranged, is designed using stacked tile type
Go out preliminary probe sequence；
2. whether there is or not hairpin structures for assessment sequence, if there is hairpin structure, then need to extend outwardly target sequence 50bp,
For design object sequence probes, hairpin structure is reappraised after the completion of design；
3. target sequence probe is added universal sequence；
4. the Tm values of every probe will be assessed plus the target sequence probe of universal sequence；It is preferred that Tm values are based on
The k-nearest neighbor of 2007 thermodynamic parameter tables of santalucia.
5. according to the Tm values of target sequence probe, the copy number of every probe is calculated, the method that copy number calculates is as follows：With
Tm values on the basis of the most Tm values of Tm values same probe item number in stacked tile type design object sequence probes group, other target sequences are visited
Needle often differs 0.02 DEG C (within containing) compared to benchmark Tm values, and probe copy number just more increases by 0.08 times, for example, the Tm of probe A
The Tm values of Tm values on the basis of value, probe B are 0.09 DEG C higher than benchmark Tm values, then the copy number of probe B is 1.36 times of probe A；It is excellent
Select Tm values based on the k-nearest neighbor of 2007 thermodynamic parameter tables of santalucia.
6. the target sequence probe conditions of final design are as follows：
1) Preliminary design goes out 321 target sequence probes；
2) wherein there are 8 probes to have hairpin structure, redesigned, the probe item number after redesign is 333；
3) the copy number such as following table of target sequence probe is calculated according to Tm values：
Table 2：The copy number of target sequence probe
| Tm values | Copy number | Number of probes |
| 70.14 | 1.18 | 1 |
| 70.16 | 1.16 | 1 |
| 70.18 | 1.14 | 1 |
| 70.20 | 1.12 | 7 |
| 70.22 | 1.1 | 5 |
| 70.24 | 1.08 | 10 |
| 70.26 | 1.06 | 28 |
| 70.28 | 1.04 | 37 |
| 70.30 | 1.02 | 45 |
| 70.32 | 1 | 63 |
| 70.34 | 1.02 | 45 |
| 70.36 | 1.04 | 42 |
| 70.39 | 1.06 | 35 |
| 70.41 | 1.08 | 22 |
| 70.43 | 1.1 | 5 |
| 70.45 | 1.12 | 3 |
| 70.47 | 1.14 | 2 |
| 70.49 | 1.16 | 2 |
| 70.51 | 1.18 | 1 |
K-nearest neighbor of the Tm values based on 2007 thermodynamic parameter tables of santalucia.
Embodiment 2 prepares and contains bound fraction target sequence probe cell
1, sequence preparation is carried out according to the target sequence that embodiment 1 designs, method prepared by sequence is as follows：
2, the 1-333 articles respectively above-mentioned by oligonucleotides pond well known in the art (Oligo Pools) synthetic technology
Target sequence in chip synthetic oligonucleotide probe, and by concentration can for 50% ammonium hydroxide by 333 target sequence probes (see
Table 4) it elutes, by being dissolved in ultra-pure water after purification, target sequence probe cell, measured concentration are formed, and be diluted to
10ng/μl。
3, using target sequence probe cell as masterplate, the primer with the complementation of 3 ' end-specificity utilizes Enzymatics companies
Bst X DNA Polymerase kits, and the complementary series of universal primer in probe is added, carry out the complementation of synthesising probing needle
Chain, concrete operation step are as follows：
1) reaction system is as follows：
| Component | Volume (μ l) |
| Target sequence probe cell (10ng/ μ l) | 10 |
| 10X Bst X Reaction Buffer | 5 |
| Universal primer (10uM) | 2 |
| 10mM dNTP | 1 |
| Bst X DNA Polymerase | 2 |
| H20 | 30 |
2) reaction condition is as follows：
| Temperature | Time |
| 65℃ | 30min |
| 80℃ | 10min |
3) QIAGEN PCR purification kits are used, product purification is carried out according to its operational manual；
4) connection reaction：The target sequence probe product containing complementary strand that step 3) is obtained, utilizes Enzymatics
The T4 DNA Ligase kits of company are attached, and coupled reaction system is as follows：
| Component | Volume (μ l) |
| Target sequence probe containing complementary strand | 20μl |
| 2X T4 DNA Ligase Buffer | 25μl |
| T4 DNA Ligase | 5μl |
5) reaction condition is as follows：
| Temperature | Time |
| 20℃ | 5h |
| 70℃ | 10min |
6) QIAGEN PCR purification kits are used, product purification is carried out according to its operational manual；
7) the target sequence probe cell containing biotin is prepared：The ring target sequence containing complementary strand that step 6) is obtained
Probe product utilizes general sequence in the phi29 DNA Polymerase kits and aim sequence probe of Thermo companies
The complementary primer sequences of row, specific reaction system can be：
8) by above-mentioned system in PCR instrument 95 DEG C of 10min, be immediately placed in ice bath after 10min, cooling 5min, Zhi Houjia
Enter Phi29 the DNA Polymerase, 37 DEG C of 8h of 5 μ l.
9) QIAGEN PCR purification kits are used, product purification is carried out according to its operational manual；It obtains containing biotin
Target sequence probe cell, be diluted to 50ng/ μ l, be placed in -20 DEG C of preservations.
Embodiment 3, the enrichment of target sequence library
1. test experiments scheme：The full-length genome library sample of 5 people is selected, confirmatory experiment is carried out.
2. being enriched with the target sequence in above-mentioned 5 samples using the target sequence probe cell containing biotin：
1) HUMAN COT-1DNA are mixed with Herring Sperm DNA equal proportions, is labeled as Block Mix；
2) 2ml 20xSSPE, 1.5ml 50x Denhards, 0.5ml 10%SDS mixing are taken, is labeled as
Hybridization Buffer；
3) H2O of 5ml 1M Tris-HCl (pH 7.5), 1ml 0.5M EDTA, 5M NaCl 2.5ml, 16.5ml are taken
It is mixed, is labeled as BW Buffer；
4) it takes 5ml 20X SSC, 1ml 20%SDS, 14ml H2O to be mixed, is labeled as Wash Buffer；
5) 1ug full-length genomes library (25 μ l, 40ng/ μ l), 5 μ l Block Mix, the mesh containing biotin of 5 μ l are taken respectively
Mark sequence probes pond, 20 μ l Hybridization Buffer；
6) mixed system of step 5) is placed in PCR instrument：95 DEG C, 10min, 65 DEG C, 10h, 65 DEG C of constant temperature, PCR instrument heat
Lid is set as 105 DEG C.
7) it takes the Dynabeads MyOne C1 (INVITROGEN) of 20ul to be placed in the centrifuge tube of 1.5ml, 200ul is added
BW Buffer, shake 5s, be put into after of short duration centrifugation on magnetic frame static one minute, remove supernatant.
8) centrifuge tube is removed, the BW Buffer of 50 μ l are added, shakes 5s, room temperature on reversion blending instrument is placed in and combines
20min。
9) after 20min, centrifuge tube is placed in 2min on magnetic frame, supernatant is abandoned in suction；
10) by centrifuge tube under magnetic frame gets on, the Wash Buffer of 65 DEG C of preheatings of 500ul are added, are placed in constant temperature gold
Belong to 10min in bath, is taken out per 2min and overturn mixing；
11) 10min is placed on 1min on magnetic frame, and supernatant is abandoned in suction；
12) step 10-11 is repeated twice, Wash Buffer are washed three times altogether；
13) 20ul ultra-pure waters are added, and slowly for several times, suspend magnetic bead again for piping and druming；
14) the 2X HIFI PCR Mix of Enzymatics is used to carry out PCR amplification：
15) amplification system is as follows：
| Component | Volume (μ l) |
| Enriched product | 20μl |
| 2X HIFI PCR Mix | 25μl |
| Primer Mix(10uM) | 5μl |
16) K amplification programs are as follows：
17) Beckman AMPure XP Kit is used to carry out the purifying of PCR product；
18) Illumina microarray datasets is used to carry out the sequencing of target sequence enriched library, sequencing reading length PE150.
3. data analysis
1) 1) with cutadapt softwares remove fastq files in primer from connect, low quality, more N and sequence it is too short
Reads is compared with BWA softwares by the data after QC to (hg19) in the reference gene group of the mankind, then to the data of sequencing
Quality control is carried out, data are shown in Table 3.
Table 3, sequencing data table
The explanation of table 3：
A. sequencing data amount refers to completing sequencing data total caused by target area enrichment sample；
B. target sequence coverage：Refer to after enrichment is sequenced, base number/purpose base number of sequencing data covering；；
C. average sequencing depth：Refer to target sequence in sequencing, average read number；
D. 10X ratios are averagely covered：Feeling the pulse with the finger-tip mark series account for all alkali of target sequence by the base for having read 10 times or more
The ratio of base；
E. 50X ratios are averagely covered：Feeling the pulse with the finger-tip mark series account for all alkali of target sequence by the base for having read 50 times or more
The ratio of base；
F. 100X ratios are averagely covered：Feeling the pulse with the finger-tip mark series account for target sequence by the base for having read 100 times or more to be owned
The ratio of base；
From table 3 it can be seen that by taking sample 1 as an example, average sequencing depth 1063.85 ×, 10 × coverage be
100%,
50 × coverage be 100%, 100 × coverage be 99.91%, have fabulous coverage and homogeneity,
And sequencing data is only 49.75M.In terms of data result, nucleic acid target sequence enrichment is carried out using the method for the present invention, is had：
1) the advantages that sequencing data amount is few, and sequencing coverage is high, and sequencing homogeneity is good.
Although combining embodiment, invention has been described, it should be understood that protection scope of the present invention is not limited to
The embodiment of this description.Embodiment is exemplary only, and true scope of the invention and purport are defined in the claims.
Table 4
。
Claims (10)
1. a kind of method of nucleic acid target sequence enrichment for NGS sequencings, which is characterized in that
A) design object sequence probes：To target sequence assessment, whether there is or not hairpin structures, if so, target sequence is extended out to nothing
Hairpin structure；
B) target sequence probe is added into universal sequence；
C) T of every probe is assessedmValue；
D) copy number adjustment is carried out to target sequence probe；
E) each target sequence probe is subjected to isothermal duplication to form target sequence probes complementary chain, and connects each target sequence
Row probe adds bound fraction on target sequence probe, obtains the single-stranded target sequence probes pond containing bound fraction.
2. the method for the nucleic acid target sequence enrichment of sequencing, which is characterized in that target sequence probe cell is DNA single-stranded probes pond.
3. according to the method for going the nucleic acid target sequence enrichment described in claim 1 for NGS sequencings, which is characterized in that right
In the aim sequence region for having hairpin structure, use within the target sequence both sides 150bp as complementary design probe area, it is excellent
Select the region within 50bp.
4. according to the method for going the nucleic acid target sequence enrichment described in claim 1 for NGS sequencings, which is characterized in that logical
With sequence, probe structure is as follows：5 '-probe sequence-CGTGGATGAGGAGCC-3 '.
5. the method for the nucleic acid target sequence enrichment according to claim 1 or 2 for NGS sequencings, which is characterized in that mesh
Mark sequence probes have the characteristics that：1) itself do not generate hairpin structure, 2) copy number is according to the unwinding temperature of target sequence probe
Spend TmValue and space structure are determined.
6. the method for the nucleic acid target sequence enrichment according to claim 1 for NGS sequencings, which is characterized in that described
Target sequence probe length is 70-150bp, preferably 90-130bp, more preferable 105bp.
7. the method for the nucleic acid target sequence enrichment according to claim 1 for NGS sequencings, which is characterized in that pass through
With reference to known group sequence, by stacked tile type design object sequence probes group, and every target sequence probes Tm value is calculated,
Tm≤ 80 DEG C, preferably Tm≤75℃；It is preferred that k-nearest neighbor of the Tm values based on 2007 thermodynamic parameter tables of santalucia.
8. the method for the nucleic acid target sequence enrichment according to claim 1 for NGS sequencings, which is characterized in that in institute
It states in scheme, the number compensation scheme of copy is：Most with Tm value same probe items number in stacked tile type design object sequence probes group
Tm values on the basis of more Tm values, other target sequence probes often differ 0.02 DEG C (within containing) compared to benchmark Tm values, and probe is copied
Shellfish number just more increases by 0.08 times.
9. the method for the nucleic acid target sequence enrichment according to claim 1 for NGS sequencings, it is characterised in that：To containing
There are the ring target sequence probes of complementary strand to carry out single stranded amplification, and amplified production is marked using biotin.
10. a kind of target area sequence enrichment kit, which is characterized in that comprising containing engaging portion described in claim 1-9
The single-stranded target sequence probes pond divided and target sequence probe cell are applied to agent prescription used in the enrichment of target area.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114300052A (en) * | 2021-12-15 | 2022-04-08 | 纳昂达(南京)生物科技有限公司 | Method and device for evaluating capture specificity of nucleic acid probe |
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| CN105925671A (en) * | 2016-04-22 | 2016-09-07 | 艾吉泰康生物科技(北京)有限公司 | Method for enriching nucleic acids with target sequence from nucleic acid sample |
| CN106191256A (en) * | 2016-07-15 | 2016-12-07 | 艾吉泰康生物科技(北京)有限公司 | A kind of method carrying out DNA methylation order-checking for target area |
| CN106520978A (en) * | 2016-11-29 | 2017-03-22 | 北京迈博恒业科技有限责任公司 | Preparation method for target-DNA-enriching probe |
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- 2018-04-20 CN CN201810357468.4A patent/CN108546739A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105925671A (en) * | 2016-04-22 | 2016-09-07 | 艾吉泰康生物科技(北京)有限公司 | Method for enriching nucleic acids with target sequence from nucleic acid sample |
| CN106191256A (en) * | 2016-07-15 | 2016-12-07 | 艾吉泰康生物科技(北京)有限公司 | A kind of method carrying out DNA methylation order-checking for target area |
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| CN114300052A (en) * | 2021-12-15 | 2022-04-08 | 纳昂达(南京)生物科技有限公司 | Method and device for evaluating capture specificity of nucleic acid probe |
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