CN106498504A - Two generations sequencing database technology based on multiplex PCR - Google Patents
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Abstract
The present invention relates to biological technical field, more particularly to two generation sequencing library constructing technologies.Storehouse is built using two-wheeled multiplex PCR in the program.For each targeting regions or target site, two specific primers are designed, the primer OP of upstream is used for first round multiplex PCR, and the primer I P in downstream is used for the second wheel multiplex PCR.By the second wheel PCR primer after purification, Q PCR are carried out quantitative, is diluted to suitable concentration and is sequenced for two generations.The present invention can effectively shorten the problem of Ampliseq kits presence, for the panel design production cycles of customization only need to two weeks, and reduce the cost that single sample builds storehouse, and Ion Torrent and Illumina microarray datasets are all general.
Description
Technical field
The present invention relates to biological technical field, more particularly to two generation sequencing library constructing technologies.
Background technology
In two generations, were sequenced and there is particularly important application due to the sequencing ability of its superelevation in scientific research and clinic.In two generations, were sequenced
Technology is also referred to as deep sequencing, large-scale parallel sequencing, and core concept is that (Sequencing by are sequenced in synthesis
Synthesis), i.e., determining the sequence of DNA, existing technology platform by the mark of the end of the new synthesis of seizure mainly has
Roche/454FLX, Illumina/Solexa Genome Analyzer and Applied Biosystems SOLID
system.These three technology platforms respectively have advantage, and the sequencing fragment of 454FLX is long, and high-quality reading length can reach 400bp；
Solexa is sequenced cost performance highest, and not only the price of machine is lower than other two kinds, and operating cost is also low, identical in data volume
In the case of, cost only has the 1/10 of 454 sequencings；The degree of accuracy of SOLID sequencings is high, and the degree of accuracy of original base data is more than
99.94%, and the degree of accuracy in 15 × coverage rate can reach 99.999%.
The general principle of Illumina/Solexa Genome Analyzer sequencings is that side synthesis becomes sequencing.In Sanger
On the basis of Deng sequence measurement, by technological innovation, with four kinds of different dNTP of fluorescence labeling of different colours, when DNA is polymerized
During enzymatic synthesis complementary strand, often add a kind of dNTP and will discharge different fluorescence, according to the fluorescence signal for catching and through special
Fixed software processing, so that obtain the sequence information of DNA to be measured.
The two generations general flow of sequencing is as follows：1) prepared by library, DNA is atomized or the chemical conversion of ultrasonic wave random fragment is hundreds of
Base or shorter small fragment.DNA fragmentation is cut into flat end with polymerase and exonuclease, and then phosphorylation is increased
One nucleotides cohesive terminus,cohesive termini.Then sequence measuring joints are connected with fragment；2) template molecule is added chip to use by the establishment of cluster
Circulate in producing clone's cluster and being sequenced.Chip has the silicon chip of 8 longitudinal swimming lanes.In each swimming lane, chip surface has countless quilts
Fixed single-stranded joint.The DNA fragmentation denaturation of the belt lacing that above-mentioned steps are obtained is drawn into the single-stranded rear joint with sequencing passage
Thing combines to form bridge-like structure, so that follow-up pre- amplification is used.The double of up to a million cluster distributions are obtained by constantly circulation
Chain fragment to be measured；3) it is sequenced, the reversible terminator of archaeal dna polymerase combined with fluorescent, fluorescence labeling cluster is imaged, starts in next circulation
Front combining nucleotide excision simultaneously decomposes；4) data analysis, it is tens bases that the initial data that sequencing is obtained is length
Sequence, the frame of the Contigs even whole gene groups that these short sequence assemblings are grown up by bioinformatics tools to be passed through
Frame, or on these sequence alignments to existing genome or close species gene group sequence, further analysis is had
The result of biological significance.
Polymerase chain reaction (i.e. PCR) is a kind of technology for being widely used in molecular genetic and diagnosis, for amplifying amplification
Specific DNA fragmentation, is considered as the special DNA replication dna of in vitro, can analyze any short DNA sequence dna, and the maximum of PCR is special
Point, is micro DNA to be significantly increased, even if comprising only low-level DNA in sample.Round pcr can be used for Amplification Analysis
DNA or RNA selectes fragment.
Most successful commercialization multiplex PCR database technology is the Ampliseq of Thermo Fisher companies in the market
Technology, the technology can complete thousands of amplifications to primer in the reaction of same hole so that the enrichment of multiple targeting regions can be with
Completed by a PCR reaction, subsequently the universal joint plus Ion Torrent microarray datasets is built up library and surveyed for two generations
Sequence.However, this product exist maximum defect be customization the panel design production cycles longer, need two to three
Individual month, serious increased time cost；And each sample build storehouse costly up to 1000-2000 unit, cause its economy into
This is high；In addition the kit is only applicable to Ion Torrent microarray datasets, is affected greatly, to be also unfavorable for being sequenced by platform factor
The reduction of expense.
Content of the invention
In view of problem above, the invention provides a kind of two generations sequencing database technology based on multiplex PCR, the technology can be with
Ampliseq kits are solved the problems, such as effectively, for the panel design production cycles of customization only need to two
Week, and can greatly reduce the cost that single sample builds storehouse, and all lead in IonTorrent and Illumina microarray datasets
With.
In order to realize that foregoing invention purpose, the present invention provide technical scheme below：
Use two-wheeled multiplex PCR to build storehouse in the present invention.
First against each targeting regions or target site, need to design two specific primers, a primer is another
The position of 3 ' downstream 100-150bp of one primer, the primer in downstream is at 5 ' ends plus the general survey of two generation microarray datasets
Sequence joint.The primer OP of upstream is used for first round multiplex PCR, and the primer I P in downstream is used for the second wheel multiplex PCR.
Specifically building storehouse process is：
Sample DNA is carried out fragmentation process by step 1, and subsequently each fragment two ends adds special joint；
Step 2, adds the upstream primer mixing pit of first round amplification, and complementary universal primer, is configured to special joint
PCR total systems, carry out first round amplification；
Step 3, by first round PCR primer after purification, adds the downstream primer mixing pit of the second wheel amplification, in second step
The complementary universal primer of used special joint is configured to PCR total systems, carries out the second wheel amplification；
Step 4, by the second wheel PCR primer after purification, configuration reaction total system carries out Q-PCR quantitatively, and it is suitable to be diluted to
Concentration, you can be sequenced for two generations.
In above-mentioned technical proposal, preferably, in step 2, first round specific primer is common pcr amplification primer thing,
The base of totally 20 or so, Tm values are set in 60 DEG C, and sequence is obtained according to target area design, and multiple primers blend together an OP and draw
Upstream primer of the thing pond as first round PCR.PCR total systems are：2 times of PCR Mix 35uL, 10umol/L P7 1uL,
1umol/L OP 1uL or 0.5umol/L OP 2uL, DNA 25uL.
In above-mentioned technical proposal, preferably, in step 3, the second wheel specific primer is common pcr amplification primer thing,
5 '-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCG of sequence measuring joints is added at its 5 ' end
ATCT-3 ' the bases of totally 80 or so, the sequence length for wherein being obtained according to target area design is 20 bases or so, Tm values
60 DEG C are set in, multiple primers blend together upstream primer of the IP primers pond as the second wheel PCR.The sequence of P7：5’-
CAAGCAGAAGACGGCATACGAG 3’.PCR total systems are：2 times of PCR Mix 15uL, 10umol/L P7 1uL, 1umol/L
OP 1uL or 0.5umol/L OP 2uL, DNA 5uL, NFW 5uL.
In above-mentioned technical proposal, preferably, PCR total systems 10uL in the step 4 are specially：VAHTS SYBR
QPCR Master Mix 5uL, qPCR Primer Mix 1uL, DNA 2uL, ROXReference Dye 2 0.2uL, NFW
5uL.
In above-mentioned technical proposal, preferably, described first round PCR amplification cycles number is 10-14.
In above-mentioned technical proposal, preferably, the second described wheel PCR amplification cycles number is 10-14.
The advantages of the present invention are：A kind of two generations sequencing database technology based on multiplex PCR is provided.Two
Storehouse kit is built in sequencing procedure using what the technology then need not buy business again, all reaction reagents individually can be purchased
Combination is bought, experimental cost is greatly reduced.Additionally, due to the universal primer for securing one end during PCR, greatly drop
Primer type and quantity in low multi-PRC reaction system, effectively can suppress between non-specific amplification and primer
Interfere, also reduce the difference of the amplification efficiency between different primers.
Description of the drawings
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
Accompanying drawing to be used needed for having technology description is briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, without having to pay creative labor, may be used also
To obtain other accompanying drawings according to these accompanying drawings.
Fig. 1 builds storehouse process for the two generations sequencing of two-wheeled multiplex PCR.
Fig. 2 is the average overburden depth of 10 100 SNPs of sample.
Specific embodiment
With reference to the accompanying drawings and examples, the specific embodiment of the present invention is further described.Following examples are only
For technical scheme is clearly described, and can not be limited the scope of the invention with this.
1. genomic DNA fragment of embodiment
A. Qsonica Ultrasonic Cell Disruptors are opened, allows circulator bath to be lowered the temperature in advance, operating temperature is 4 DEG C.
B. transfer total amount for 1ug genomic DNA in the centrifuge tube of 0.2mL, with Nuclease Free Water (with
Call NFW in the following text) 100uL is supplied, concussion is mixed, is centrifuged in short-term.
C., the centrifuge tube of 0.2mL is loaded the adapter of Ultrasonic Cell Disruptor, axis is screwed, top cover is inserted, machine is closed, if
Program is interrupted surely：Amplitude -25%, on＆off be the 20s-30 seconds, duration 15 minutes.Start to interrupt.
D. after interrupting end, 0.2mL centrifuge tubes are taken out, marshalling, uncaps in order.
2. 3in of embodiment, 1 joints connect
A. reaction system 22uL is configured, and the reagent being first configured in addition to DNA is matched somebody with somebody according to the 125% of theoretical value, and concussion is mixed
Even, it is centrifuged in short-term, is dispensed in 96 orifice plates, often hole adds 7uL, shifts the DNA of 15uL afterwards to often from above 0.2mL centrifuge tubes
Kong Zhong, closes the lid.Concussion is mixed, and is centrifuged in short-term.
B.96 orifice plate or eight unions are put into PCR instrument.Setting program：25 DEG C keep for 30 minutes, 72 DEG C for 15 minutes, 4 DEG C.
C. the Adapter of the T4 Ligase (single rifle) and 1uL of 0.5uL after terminating, is added in every hole, covers new lid.
Concussion is mixed, and is centrifuged in short-term.20-30 minutes are incubated under room temperature.
D. magnetic bead shakes 30 seconds, after fully mixing, adds the magnetic bead of 25uL in each hole, covers new lid.Concussion is mixed,
It is centrifuged in short-term.It is incubated 5 minutes under room temperature.
E. 96 orifice plates or eight unions are put magnetic frame, is waited 2 minutes, after solution clarification, inhaled and abandon supernatant.
F. 80% ethanol of 100uL is added per hole, stand 30 seconds, inhale and abandon supernatant.Come again.It is centrifuged in short-term, inhales and abandon
Clearly.
G. dry 3-5 minutes, observation magnetic bead surfaces are no longer moist reflective, and mist formation planar can leave magnetic frame, add per hole
30uL NFW, piping and druming mix resuspended magnetic bead, incubated at room 1 minute.
H. orifice plate puts magnetic frame again, waits 2 minutes, and after solution clarification, transfer supernatant is in new orifice plate.
3. multiplex PCR of embodiment
First, first round multiplex PCR
A. according to Qubit dsDNA HS Assay Kit operation manual detection sample concentration, according to the sample of Qubit detections
This concentration, each sample take same amount (10ng), mix per 5～10 samples and are transferred in new orifice plate.
B. first round specific primer is common pcr amplification primer thing, the base of totally 20 or so, and Tm values are set in 60
DEG C, sequence is obtained according to target area design, and multiple primers blend together upstream primer of the OP primers pond as first round PCR.
Configuration PCR total systems, match somebody with somebody according to the 125% of theoretical value, and concussion is mixed, and is centrifuged in short-term.
C. orifice plate is put in PCR instrument, selects MAPlex-1st programs to run 2 hours.
2nd, PCR primer purifying
After a.PCR terminates, orifice plate is removed, open lid, added per hole the magnetic bead of 60uL, piping and druming to mix, under room temperature, be incubated 5
Minute.
B. orifice plate or eight unions are put magnetic frame, is waited 2 minutes, after solution clarification, inhaled and abandon supernatant.
C. 80% ethanol of 100uL is added per hole, stand 30 seconds, inhale and abandon supernatant.Come again.
D. dry 3-5 minutes, observation magnetic bead surfaces are no longer moist reflective, and mist formation planar can leave magnetic frame, add per hole
20uL NFW, piping and druming mix resuspended magnetic bead, incubated at room 1 minute.
E. orifice plate puts magnetic frame again, waits 2 minutes, and after solution clarification, transfer supernatant is in new orifice plate.
3rd, the second wheel multiplex PCR
A. the second wheel specific primer is common pcr amplification primer thing, its 5 ' end plus sequence measuring joints 5 '-
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT CT-3 ' the alkali of totally 80 or so
Base, is 20 bases or so wherein according to the target area sequence length that obtains of design, and Tm values are set in 60 DEG C, and multiple primers are mixed
Into an IP primers pond as the second upstream primer for taking turns PCR.The sequence of P7：5’-CAAGCAGAAGACGGCATACGAG-3’.
Configuration PCR total systems, prepare according to the 125% of theoretical value, and concussion is mixed, and is centrifuged in short-term.
B. orifice plate is put in PCR instrument, selects MAPlex-2nd programs to run 2 hours.
4th, PCR primer purifying
Step is done and is purified twice with two, thoroughly removes primer dimer.For the first time using the magnetic beads for purifying of 28uL, use
The NFW wash-outs of 20uL；Second magnetic beads for purifying with 20uL, is eluted with the NFW of 25uL.
5th, electroresis appraisal
Each sample takes 5uL, carries out 2.5% agarose electrophoresis, has seen whether band, and whether band length exists
Between 300-400bp.
5. library of embodiment quantitatively with mix
A. standard items, fluorescent quantitation reagent, primer are positioned over thawed on ice from -20 DEG C of taking-ups.
B. 10000 times of Sample Dilution, in two times gradient dilution.Take library stoste 2uL for the first time, add the NFW of 198uL,
Vibration is mixed or is mixed with rifle piping and druming, is centrifuged in short-term.It is taken out 10uL again, adds 990uL NFW, vibration to mix or use
Rifle piping and druming is mixed, and is centrifuged in short-term.
C. reaction system 10uL is configured, to match somebody with somebody according to the 125% of theoretical value, concussion is mixed, and is centrifuged in short-term.
D. ABI7500 quantitative real time PCR Instruments are opened, is put into sample, starts to detect.Detection terminates, will according to testing result
All samples are diluted to same concentration, make final concentration not less than 2.5nmol/L, and volume is sent not less than after 30uL, then mixed in equal amounts
Go out sequencing.
E. experimental result is as shown in Fig. 2 include 100 SNPs, genomic DNA is initial in MAPlex custom panel
Amount is 250ng, builds storehouse by MAPlex methods, and HiSeq2500 microarray datasets PE 2*150 is sequenced, and single sample takes 100Mb numbers
According to, upper target rate 93%, mean depth is 321 ×, 92% site sequencing depth 20 × more than, repeatable 100%.
Presently preferred embodiments of the present invention is the foregoing is only, not in order to limit the present invention, all in essence of the invention
Within god and principle, any modification, equivalent substitution and improvements that is made etc. should be included within the scope of the present invention.
SEQUENCE LISTING
<110>Shanghai Mei Diweikang bio tech ltd
<120>Two generations sequencing database technology based on multiplex PCR
<130> 123
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 58
<212> DNA
<213>Artificial synthesized
<400> 1
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatct 58
<210> 2
<211> 22
<212> DNA
<213>Artificial synthesized
<400> 2
caagcagaag acggcatacg ag 22
Claims (10)
1. the construction method of two generation sequencing libraries, it is characterised in that comprise the steps：
Sample DNA is carried out fragmentation process by step 1, and subsequently each fragment two ends adds special joint；
Step 2, adds the upstream primer mixing pit of first round amplification, is configured to PCR with the universal primer of special joint complementation total
System, carries out first round amplification；
Step 3, by first round PCR primer after purification, adds the downstream primer mixing pit of the second wheel amplification, used in second step
The universal primer of the special joint complementation that crosses is configured to PCR total systems, carries out the second wheel amplification；
Step 4, by the second wheel PCR primer after purification, prepares reaction total system, carries out Q-PCR quantitatively, be diluted to suitably dense
Degree, you can be sequenced for two generations.
2. the construction method of two generations sequencing library according to claim 1, it is characterised in that the PCR in the step 2 is total
System is specially：2 times of PCR Mix 35uL, 10umol/L P7 1uL, 1umol/L OP 1uL, DNA 25uL.
3. the construction method of two generations sequencing library according to claim 1, it is characterised in that the PCR in the step 2 is total
System is specially：2 times of PCR Mix 35uL, 10umol/L P7 1uL, 0.5umol/L OP 2uL, DNA 25uL.
4. the construction method of two generations sequencing library according to claim 1, it is characterised in that the PCR in the step 3 is total
System is specially：2 times of PCR Mix 15uL, 10umol/L P7 1uL, 1umol/L OP 1uL, DNA 5uL, NFW 5uL.
5. the construction method of two generations sequencing library according to claim 1, it is characterised in that the PCR in the step 3 is total
System is specially：2 times of PCR Mix 15uL, 10umol/L P7 1uL, 0.5umol/L OP 2uL, DNA 5uL, NFW 5uL.
6. the construction method of two generations sequencing library according to claim 1, it is characterised in that the PCR in the step 4 is total
System 10uL is specially：VAHTS SYBR qPCR Master Mix 5uL, qPCR Primer Mix 1uL, DNA 2uL, ROX
2 0.2uL of Reference Dye, NFW 5uL.
7. the construction method of two generations sequencing library according to claim 1, it is characterised in that described first round PCR expands
Increasing number of cycles is 10-14.
8. the construction method of two generations sequencing library according to claim 1, it is characterised in that the second described wheel PCR expands
Increasing number of cycles is 10-14.
9. the construction method of two generation sequencing libraries according to Claims 2 or 3, it is characterised in that in described step 2,
First round specific primer is common pcr amplification primer thing, the base of totally 20 or so, and Tm values are set in 60 DEG C, sequence according to
Target area design is obtained, and multiple primers blend together upstream primer of the OP primers pond as first round PCR.
10. the construction method of two generation sequencing libraries according to claim 4 or 5, it is characterised in that in described step 3,
Second wheel specific primer is common pcr amplification primer thing, adds sequence measuring joints at its 5 ' end
5 '-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC TCTTCCGATCT-3 ' totally 80 left sides
Right base, the sequence length for wherein being obtained according to target area design is 20 bases or so, and Tm values are set in 60 DEG C, multiple
Primer blendes together upstream primer of the IP primers pond as the second wheel PCR；The sequence of P7：5’-
CAAGCAGAAGACGGCATACGAG 3’.
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| CN107475429A (en) * | 2017-09-28 | 2017-12-15 | 上海思路迪生物医学科技有限公司 | The primer and kit of more target library constructions are uniformed based on high-flux sequence |
| CN107513570A (en) * | 2017-09-28 | 2017-12-26 | 上海思路迪生物医学科技有限公司 | The method and kit of more target library constructions are uniformed based on high-flux sequence |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104946629A (en) * | 2015-07-14 | 2015-09-30 | 天津诺禾医学检验所有限公司 | Method for fragmenting trace DNA sample and method for establishing DNA library by utilizing trace DNA sample |
| CN106192018A (en) * | 2015-05-07 | 2016-12-07 | 深圳华大基因研究院 | A kind of method of grappling Nest multiplex PCR enrichment DNA target area and test kit |
-
2016
- 2016-12-13 CN CN201611145969.3A patent/CN106498504A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106192018A (en) * | 2015-05-07 | 2016-12-07 | 深圳华大基因研究院 | A kind of method of grappling Nest multiplex PCR enrichment DNA target area and test kit |
| CN104946629A (en) * | 2015-07-14 | 2015-09-30 | 天津诺禾医学检验所有限公司 | Method for fragmenting trace DNA sample and method for establishing DNA library by utilizing trace DNA sample |
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| CN110144387A (en) * | 2019-05-08 | 2019-08-20 | 中国科学院植物研究所 | A multiplex PCR method |
| CN110144383A (en) * | 2019-05-08 | 2019-08-20 | 中国科学院植物研究所 | Method for Enriching Target DNA Fragments Using Multiplex PCR |
| CN110453000A (en) * | 2019-08-15 | 2019-11-15 | 深圳谱元科技有限公司 | A kind of two generations sequencing banking process for identifying the typing of bacteria and resistant type of helicobacter pylori in sample |
| CN111118001B (en) * | 2019-12-31 | 2022-03-18 | 苏州贝康医疗器械有限公司 | Universal joint for multiple sequencing platforms, library construction method suitable for multiple sequencing platforms and kit |
| CN111118001A (en) * | 2019-12-31 | 2020-05-08 | 苏州贝康医疗器械有限公司 | Universal joint for multiple sequencing platforms, library construction method suitable for multiple sequencing platforms and kit |
| CN111621599A (en) * | 2020-06-12 | 2020-09-04 | 武汉菲沙基因信息有限公司 | Third-generation database construction sequencing method based on whole genome full-length amplification of new coronavirus |
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| CN114657239A (en) * | 2021-12-30 | 2022-06-24 | 迈基诺（重庆）基因科技有限责任公司 | Primer combination, kit and library building method for multiplex PCR (polymerase chain reaction) second-generation sequencing |
| CN114657239B (en) * | 2021-12-30 | 2024-05-10 | 迈基诺（重庆）基因科技有限责任公司 | Primer combination, kit and library construction method for multiplex PCR second generation sequencing |
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