CN110114472A - The method that linear sequencing library is converted into cyclic annular sequencing library - Google Patents
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Abstract
Provide the method that linear sequencing library is converted into cyclic annular sequencing library.Wherein, method includes the following steps: linear sequencing library is carried out PCR amplification using positive adapter-primer and reversed adapter-primer, to obtain amplified production；The amplified production is subjected to thermal denaturation processing, to obtain single stranded DNA mixture；The single stranded DNA mixture is subjected to cyclisation processing, to obtain cyclic DNA product, the cyclic DNA product constitutes the cyclic annular sequencing library, wherein, the at least partly sequence complementary pairing of the positive connector of positive adapter-primer and the linear sequencing library, 5 ' terminal nucleotides of at least one of at least partly sequence complementary pairing of the reversed connector of reversed adapter-primer and the linear sequencing library, positive adapter-primer and the reversed adapter-primer pass through phosphorylation modification.
Description
Priority information
Nothing
The present invention relates to library constructions and sequencing technologies field, and in particular, to the method that linear sequencing library is converted to cyclic annular sequencing library.
For some items in commerce, sample is rare, sample size is few, the library sample only having had been built up, when to convert microarray dataset because of project demands, new sequencing library directly can not be constructed using remaining sample since sample size is few, and the library sample built also cannot be used directly for the purpose microarray dataset entirely different with it.
And how the linear sequencing library of Illumina, Proton is converted into library and is converted to cyclic annular sequencing library and is applicable in other microarray datasets, such as BGISEQ-500 there is no mature solution at present.
Summary of the invention
The present invention is directed at least solve one of the technical problems existing in the prior art.For this purpose, it is an advantage of the invention to provide a kind of means that existing linear libraries are effectively converted to cyclic annular sequencing library.
It should be noted that the present invention is following discovery based on inventor and work and completes:
Currently, having more business or research institution starts generally to replace Hiseq 2000/Hiseq 4000 using BGISEQ-500.And common preciousness rare sample or unicellular sample in practicing, its existing library Illumina or Proton, but sample surplus be not enough to carry out it is secondary build library, the library BGISEQ-500 (existing micro and conventional build library scheme not applicable) of standard can not be obtained using BGISEQ-500 routine database technology；The unicellular library Illumina of the high-throughput unicellular library apparatus for preparation preparation of another aspect 10X Genomics company can not be compatible with BGISEQ-500, therefore, is badly in need of the technical solution that corresponding library carries out microarray dataset conversion at present.
For above-mentioned two situations, inventor have passed through a series of researchs and exploration, develop a set of specific phosphorylation modification primer and reaction system, the linear sequencing library of dsDNA (such as library Illumina or Proton) can be made to be quickly converted circlewise sequencing library (such as library BGISEQ-500) using the system, library can be directly sequenced in cyclic annular library microarray dataset after conversion.Also, the present invention not only can be adapted for precious rare sample library conversion, apply also for the high-throughput library Illumina conversion.
In turn, in one aspect of the invention, the present invention provides a kind of methods that linear sequencing library is converted to cyclic annular sequencing library.According to an embodiment of the invention, method includes the following steps: linear sequencing library is carried out PCR amplification using positive adapter-primer and reversed adapter-primer, to obtain amplified production；The amplified production is subjected to thermal denaturation processing, to obtain single stranded DNA mixture；The single stranded DNA mixture is subjected to cyclisation processing, to obtain cyclic DNA product, the cyclic DNA product constitutes the cyclic annular sequencing library, wherein the forward direction adapter-primer and the linear sequencing
At least partly sequence complementary pairing of the positive connector in library, the at least partly sequence complementary pairing of the reversed connector of the reversed adapter-primer and the linear sequencing library, and, 5 ' terminal nucleotides of the forward direction adapter-primer and at least one of the reversed adapter-primer pass through phosphorylation modification, the cyclisation processing uses cyclisation primer to carry out, and 3 ' the end sub-sequence complementary pairings with the positive end of adapter-primer 5 ', the reversed adapter-primer respectively ' are held in end and 3 ' in the 5 of the cyclisation primer.
It is surprisingly found by the inventors that dsDNA linear libraries can be made to be quickly converted circlewise sequencing library, library is used directly for being sequenced after conversion using this method.And, present invention is particularly suitable for the conversions in the library Illumina and the library Proton, wherein, for different linear sequencing libraries, only need the method according to the invention, 5 ' terminal nucleotides of the positive adapter-primer and at least one of reversed adapter-primer that make pass through phosphorylation modification, and make be cyclized primer 5 ' to hold 3 ' the end sub-sequence complementary pairings with the forward direction adapter-primer 5 ' end, reversed adapter-primer respectively in end and 3 '.Also, for the cyclic annular sequencing library (such as library BGISEQ-500) that conversion obtains, as long as sequencing can be effectively realized using the linear sequencing primer identical or complementary with junction portion sequence.
In another aspect of this invention, the present invention also provides a kind of methods of DNA sequence dna for determining linear sequencing library.According to an embodiment of the invention, method includes the following steps: the linear sequencing library is converted to cyclic annular sequencing library according to the mentioned-above method that linear sequencing library is converted to cyclic annular sequencing library；And the cyclic annular sequencing library is sequenced using the corresponding microarray dataset of the ring-type sequencing library, to determine the DNA sequence dna of the linear sequencing library.Thereby, it is possible to effectively realize the purpose that linear sequencing library is used for cyclic annular library microarray dataset (such as BGISEQ-500 microarray dataset).Also, sequencing result is accurate, reliable, reproducible.
In addition, it should be noted that, the present invention has at least one of following advantages:
(1) double-stranded linear DNA the library especially library Illumina, the library Ion Proton fast and efficiently can be converted circlewise sequencing library (such as library BGISEQ-500) by technical solution of the present invention, and it is easy to operate, sequencing result is stablized, entire library conversion process can be completed in 5 hours, being suitble to initial amount is nanogram level (1-100ng) and the library conversion of Gamma Magnitude, is greatly promoted the application and whole sequencing speed of cyclic annular sequencing library class sequenator such as BGISEQ500 gene sequencer.
(2) present invention can promote high-throughput library preparation system 10X Genomics company and GemCodeTMThe combination of instrument and BGISEQ-500 microarray dataset.
Additional aspect and advantage of the invention will be set forth in part in the description, and partially will become apparent from the description below, or practice through the invention is recognized.
Above-mentioned and/or additional aspect of the invention and advantage will be apparent and are readily appreciated that from the description of the embodiment in conjunction with the following figures, in which:
Fig. 1 shows the H1975 cell line gene expression quantity testing result according to an embodiment of the invention, BGISEQ-500 platform and the sequencing of Illumina platform；
Fig. 2 is shown according to an embodiment of the invention, the library the H1975Tn5 large fragment CNV testing result based on the sequencing of BGISEQ500 platform；
Fig. 3 is shown according to an embodiment of the invention, the library Illumina is converted to the technical principle schematic diagram of BGISEQ-500 microarray dataset by method according to the present invention.
Detailed description of the Invention
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and for explaining only the invention, and is not considered as limiting the invention.
For convenience of understanding, first BGISEQ-500 sequencing technologies are simply introduced below.
BGISEQ-500 is the gene sequencer of new generation of Hua Da gene research and development, it passes through rolling circle amplification (Rolling circle amplification, RCA single stranded circle DNA library) is expanded into the 2-3 order of magnitude, form DNA nanosphere (DNA nanoball, DNB), final nanosphere loads technology by DNB and is fixed on the silicon chip of array, and joint probe is anchored polymerization technique (cPAS) sequencing.Compared with other two generations sequencing technologies, which has many advantages, such as to can reduce amplification error rate, improves chip utilization rate.
Current BGISEQ-500 microarray dataset can mainly obtain the library BGISEQ-500 of standard using two kinds of conventional database technologies:
The conventional BGISEQ-500 library construction techniques of (1) 1 microgram starting
The technology interrupts technology using conventional Ultrasound and realizes DNA fragmentation, the DNA of fragmentation is after magnetic bead screens, carry out end reparation using end repair enzyme and " A " added to handle, after joint sequence is added at two sections of DNA of fragmentation, a side connector uses phosphorylation modification, after denatured double stranded, phosphorylation modification that it is single-stranded can be with the help of external source single-stranded DNA templates, itself is cyclized, formed ssDNA, product after cyclisation passes through Exonucleolytic enzymatic treatment, removes the dsDNA in ssDNA.The product of digestion obtains the more clean library ssDNA by magnetic beads for purifying.
(2) the BGISEQ-500 library construction techniques of micro starting
The technology realizes nanogram level DNA fragmentation using TN5 transposase, the DNA of fragmentation directly passes through PCR plus joint sequence, one side connector uses phosphorylation modification, after denatured double stranded, phosphorylation modification that it is single-stranded can be with the help of external source single-stranded DNA templates, itself is cyclized, formed ssDNA, product after cyclisation passes through Exonucleolytic enzymatic treatment, removes the dsDNA in ssDNA.The product of digestion obtains the more clean library ssDNA by magnetic beads for purifying.
The gene sequencer of Illumina is as gene sequencer most widely used at present, it is that DNA clusters is formed by bridge-type PCR amplification, each cluster combines microarray technology and proprietary reversible terminator technology extensive parallel be sequenced in synthesis to DNA clusters there are about the identical DNA profiling that 1000-6000 is copied.
For the technological disparity and library construction feature of two class sequenators, inventor studies discovery, for the library dsDNA such as the library Illumina or the library Proton, using specific positive adapter-primer and reversed adapter-primer (at least partly sequence complementary pairing of the positive connector of the forward direction adapter-primer and the linear sequencing library, the at least partly sequence complementary pairing of the reversed connector of the reversed adapter-primer and the linear sequencing library, and 5 ' the terminal nucleotides of at least one of the positive adapter-primer and described reversed adapter-primer pass through phosphorylation modification) carry out PCR amplification, and amplified production is subjected to thermal denaturation processing, then using specific cyclisation primer, (' end and 3 ' ends are held with the positive adapter-primer 5 ' respectively for the 5 of the cyclisation primer, the 3 of the reversed adapter-primer ' end sub-sequence complementary pairing) single stranded DNA is subjected to cyclisation processing, the library dsDNA can be successfully set to be quickly converted into the library BGISEQ-500ssDNA, and then convert the BGISEQ-500 sequencing library obtained to be sequenced effective for BGISEQ-500 platform.In short, the present invention carries out PCR amplification by the adapter-primers of 5 ' phosphorylation modifications, make the 5 ' of Illumina double-stranded DNA library that bases be held to obtain phosphorylation modifications, after by thermal denaturation,
The just single stranded DNA of phosphorylation modification makes recirculation at ssDNA by external source single-stranded DNA templates, to be converted to BGISEQ-500 sequencing library, in turn, by designing special sequencing primer, the library that can obtain the conversion is used for BGISEQ-500 sequencing.
In turn, in one aspect of the invention, the present invention provides a kind of methods that linear sequencing library is converted to cyclic annular sequencing library.According to an embodiment of the invention, method includes the following steps: linear sequencing library is carried out PCR amplification using positive adapter-primer and reversed adapter-primer, to obtain amplified production；The amplified production is subjected to thermal denaturation processing, to obtain single stranded DNA mixture；The single stranded DNA mixture is subjected to cyclisation processing, to obtain cyclic DNA product, the cyclic DNA product constitutes the cyclic annular sequencing library, wherein, the at least partly sequence complementary pairing of the positive connector of the forward direction adapter-primer and the linear sequencing library, the at least partly sequence complementary pairing of the reversed connector of the reversed adapter-primer and the linear sequencing library, and, 5 ' terminal nucleotides of the forward direction adapter-primer and at least one of the reversed adapter-primer pass through phosphorylation modification, the cyclisation processing is carried out using cyclisation primer, ' end and 3 ' ends are held with the positive adapter-primer 5 ' respectively for the 5 of the cyclisation primer, 3 ' end sub-sequence complementary pairings of the reversed adapter-primer.
It is surprisingly found by the inventors that dsDNA linear libraries can be made to be quickly converted into ssDNA ring-type library (such as BGISEQ-500 sequencing library) using this method, library can be directly sequenced in the microarray dataset of corresponding cyclic annular sequencing library after conversion.And, present invention is particularly suitable for the conversions in the library Illumina and the library Proton, wherein, for different linear sequencing libraries, only need the method according to the invention, 5 ' terminal nucleotides of at least one of positive adapter-primer and reversed adapter-primer are subjected to phosphorylation modification, and make be cyclized primer 5 ' to hold 3 ' the end sub-sequence complementary pairings with the forward direction adapter-primer 5 ' end, reversed adapter-primer respectively in end and 3 '.Also, for the cyclic annular sequencing library that conversion obtains, as long as using the identical sequencing primer with known linear sequencing library, the microarray dataset that can effectively utilize cyclic annular sequencing library is sequenced.
As previously mentioned, the method for the invention that linear sequencing library is converted to cyclic annular sequencing library, is suitable for double-stranded DNA library, and be particularly suitable for the conversion of Illumina sequencing library, Ion Protona sequencing library.Thus, according to an embodiment of the invention, the linear sequencing library is Illumina sequencing library or Ion Protona sequencing library.And according to an embodiment of the invention, the ring-type sequencing library is the library BGISEQ-500.
According to an embodiment of the invention, before thermal denaturation processing, further comprising after PCR amplification: the step of amplified production is carried out Piece Selection and purifying.The cyclic annular sequencing library finally obtained as a result, is high-quality.
It is handled 3 minutes according to an embodiment of the invention, carrying out the thermal denaturation under 95 degrees Celsius.Thermal denaturation high treating effect as a result, is conducive to the progress of subsequent step.
According to an embodiment of the invention, carrying out the cyclisation processing using T4DNA ligase.It is cyclized high treating effect as a result,.
According to an embodiment of the invention, the ratio of the single stranded DNA mixture and the cyclisation primer is 1:10 in cyclisation processing.It is cyclized high treating effect as a result, the cyclic annular sequencing library quality of acquisition is high.
According to an embodiment of the invention, further comprising: the step of cyclic DNA product is successively carried out digestion process and purification process.The BGISEQ-500 sequencing library obtained as a result, is with high purity, high-quality, accurate and reliable for result after being sequenced.
Some specific examples according to the present invention, the linear sequencing library are Illumina sequencing library, and the sequence of the forward direction adapter-primer is as shown in SEQ ID NO:1: 5 '-PhosAATGATACGGCGACCACCGA-3 ', the reversed connector
The sequence of primer is as shown in SEQ ID NO:2: 5 '-CAAGCAGAAGACGGCATACGA-3 ', and the sequence of the cyclisation primer is as shown in SEQ ID NO:3: 5 '-GCCGTATCATTCAAGCAGAAGACG-3 '.Thus, Illumina sequencing library effectively can be converted into cyclic annular sequencing library (such as BGISEQ-500 sequencing library), in turn, cyclic annular sequencing library is used for corresponding microarray dataset (such as BGISEQ-500 microarray dataset), the DNA sequence dna that can effectively realize sequencing, determine original Illumina sequencing library.And, when carrying out library transformation for the library Proton, those skilled in the art only need to carry out according to method of the invention above-mentioned, and according to Ion Proton library sequence and forward and reverse connector, positive adapter-primer, reversed adapter-primer, cyclisation primer and sequencing primer are adjusted accordingly.
In another aspect of this invention, the present invention also provides a kind of methods of DNA sequence dna for determining linear sequencing library.According to an embodiment of the invention, method includes the following steps: the linear sequencing library is converted to cyclic annular sequencing library according to the mentioned-above method that linear sequencing library is converted to cyclic annular sequencing library；And the cyclic annular sequencing library is sequenced using the corresponding microarray dataset of the ring-type sequencing library, to determine the DNA sequence dna of the linear sequencing library.Thereby, it is possible to effectively realize the purpose that linear sequencing library is used for the microarray dataset (such as BGISEQ-500 microarray dataset) of cyclic annular sequencing library.Also, sequencing result is accurate, reliable, reproducible.
According to an embodiment of the invention, the sequencing is carried out using sequencing primer, the sequencing primer is identical as junction portion sequence or complementary pairing.
Embodiment according to the present invention, when utilizing adapter-primer shown in SEQ ID NO:1-3 and cyclisation primer, Illumina sequencing library is converted into cyclic annular sequencing library according to preceding method --- BGISEQ-500 sequencing library, and when the BGISEQ-500 sequencing library is sequenced applied to BGISEQ-500, sequencing primer can be consistent with the sequencing primer of the Illumina sequencing library.Specifically, according to an embodiment of the invention, the sequence of the sequencing primer is as follows:
Forward primer is as shown in SEQ ID NO:4: 5 '-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC,
Reverse primer is as shown in SEQ ID NO:5: 5 '-TCGTCGGCAGCGTCAGATGTGTATAAGAGACA.
Thereby, it is possible to which sequencing is effectively performed, to realize the purpose for determining the DNA sequence dna of linear sequencing library using BGISEQ-500 microarray dataset.Also, sequencing result is accurate, reliable, reproducible.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that the following examples are merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or condition are not specified in embodiment, described technology or conditions (such as write with reference to J. Pehanorm Brooker etc. according to the literature in the art, " Molecular Cloning:A Laboratory guide " that Huang Peitang etc. is translated, the third edition, Science Press) or carry out according to product description.Reagents or instruments used without specified manufacturer, being can be with conventional products that are commercially available.
Conventional method:
According to an embodiment of the invention, the method for the invention that linear sequencing library is converted to cyclic annular sequencing library, can generally be carried out using following steps:
Linear sequencing library is subjected to PCR amplification using positive adapter-primer and reversed adapter-primer, to obtain amplified production；
The amplified production is subjected to thermal denaturation processing, to obtain single stranded DNA mixture；
The single stranded DNA mixture is subjected to cyclisation processing, to obtain cyclic DNA product, the cyclic DNA is produced
Object constitutes the cyclic annular sequencing library,
Wherein,
At least partly sequence complementary pairing of the positive connector of the forward direction adapter-primer and the linear sequencing library, the at least partly sequence complementary pairing of the reversed connector of the reversed adapter-primer and the linear sequencing library, and, 5 ' terminal nucleotides of the forward direction adapter-primer and at least one of the reversed adapter-primer pass through phosphorylation modification
The cyclisation processing uses cyclisation primer to carry out, and 3 ' the end sub-sequence complementary pairings with the positive end of adapter-primer 5 ', the reversed adapter-primer respectively ' are held in end and 3 ' in the 5 of the cyclisation primer.
Embodiment 1:
The present embodiment is by taking the library Illumina (library Illumina that sample is a lung cancer H1975 cell line) as an example, linear sequencing library is converted into cyclic annular sequencing library (BGISEQ-500 sequencing library) using method of the invention and is sequenced, the specific steps are as follows:
1, connector converts PCR amplification
A) sterilizing PCR pipe is placed in ice bath, successively adds each reactive component:
The sequence of positive adapter-primer: 5 '-PhosAATGATACGGCGACCACCGA-3 ' (SEQ ID NO:1),
The sequence of reversed adapter-primer: 5 '-CAAGCAGAAGACGGCATACGA-3 ' (SEQ ID NO:2).
B) 10 times are gently blown and beaten using pipettor to mix well.
C) PCR pipe is placed in PCR instrument, following response procedures (Hot lid:105 DEG C) is set:
2, amplified production Piece Selection
Each sample uses 0.8X+0.2X Agencourt AMPure XP beads, carries out selective purification.
A) starting PCR product volume should be 50ul.Because sample volatilization will lead to bulk product less than 50ul during PCR,
Sterile purified water must be used volume polishing to 50ul before carrying out operation below, otherwise separation length can be inconsistent with expection.
B) concussion that is vortexed mixes AMPure XP beads and draws 40ul volume into 50ul PCR product, and 10 times are gently blown and beaten using pipettor and is mixed well.Incubation at room temperature 10 minutes.
C) the of short duration centrifugation of reaction tube is placed in magnetic frame and separates magnetic bead and liquid.To solution clarification (about 5 minutes), carefully transfer supernatant abandons magnetic bead into clean EP pipe.
D) concussion that is vortexed mixes AMPure XP beads and draws 10ul volume into supernatant, and 10 times are gently blown and beaten using pipettor and is mixed well.Incubation at room temperature 5 minutes.
E) the of short duration centrifugation of reaction tube is placed in magnetic frame and separates magnetic bead and liquid.Supernatant is carefully removed to solution clarification (about 5 minutes).
F) keep EP pipe always in magnetic frame, 80% ethyl alcohol that 200ul Fresh is added rinses magnetic bead.Incubation at room temperature carefully removed supernatant after 30 seconds.
G) it repeats to walk, amounts to rinsing twice.
H) EP pipe is kept in magnetic frame, to uncap always and be air-dried magnetic bead 10 minutes.
I) EP pipe is taken out from magnetic frame, the ultrapure water elution of 25ul sterilizing is added.Vortex oscillation or gently blown and beaten using pipettor is mixed well.The of short duration centrifugation of reaction tube is placed in magnetic frame and separates magnetic bead and liquid.Supernatant is carefully drawn to solution clarification (about 5 minutes) into sterilizing EP pipe, is saved in -20 DEG C.
Note: plastic recovery kit can be used to carry out fragment length sorting and purifying for the library more concentrated if you need to obtain distribution of lengths, amplified production.If production concentration is relatively low after PCR, the elution volume in step i) can be suitably reduced.
3, amplified production QC quality standard and assessment
| Quality index title | Quality index | Measurement method |
| Clip size | 300bp±100bp | Agilent 2100 |
| Mass concentration | >3.5ng/μl | QPCR |
4、Pooling
A) the pooling volume of each sample is determined according to sample pooling radix according to DNA mix total amount 390ng pooling；
B) current pooling radix can only be 8 multiple, 8*N sample surveys 1 lane, and then pooling radix is 8*N, INDEX only has 48 kinds (when building library Pooling at present, the same library must be one group 8 set of (or 8 multiple) Barcode Pooling together, guarantee base balance.If being that the non-set of sequencing of Barcode when will cause sequencing number of base is uneven, sequencing quality is poor, influences Barcode fractionation.)
C) conversion method: for example, 16 sample pool, at 1 library, it is 390/16=24.375ng that each sample, which should obtain total amount, the concentration of sample 1 is 5ng/ul, then the corresponding volume of sample 1 is 24.375ng/ (5ng/ul)=4.875ul；Sample 2 calculates in this way；
D) Pool good DNA mix can freeze -20 DEG C it is spare.
Points for attention: if concentration is in 1.0ng/ul~2.0ng/ul after the double choosings of previous step PCR product, according to 390ng total amount DNA
DNA mix volume after pooling is greater than 96ul, and the sample after pooling should use 1.6*XP magnetic beads for purifying, using 40ul NF H2O back dissolving solution, then continue to test by subsequent system.
5, the processing of ssDNA thermal denaturation, cyclisation
A) prepare reagent according to formula as below
It is cyclized the sequence of primer: 5 '-GCCGTATCATTCAAGCAGAAGACG-3 ' (SEQ ID NO:3).
B) parameter is set according to following procedure, carries out thermal denaturation processing:
C) ssDNA is cyclized, and above-mentioned PCR pipe is placed in ice bath, each reactive component is successively added:
D) after preparing above-mentioned reaction solution, vortex gently gets rid of 5s；37 DEG C of incubations 1h, 4 DEG C of hold；Product takes 1ul to survey dsDNA concentration after connection；
6, EXO digests
A) step 5 reaction solution is placed in ice bath, successively adds each reactive component:
B) after preparing above-mentioned reaction solution, vortex gently gets rid of 5s；37 DEG C of incubations 30min, 4 DEG C of hold；Digestion
Product takes 1ul to survey dsDNA concentration and ssDNA concentration afterwards, determines dsDNA digestion effect.
7, magnetic beads for purifying
A) 170ul PEG32beads is taken, above-mentioned reaction solution is added to, the concussion that is vortexed mixes, and is incubated at room temperature 10 minutes；
B) the of short duration centrifugation of reaction tube is placed in magnetic frame and separates magnetic bead and liquid.(about 5 minutes) are clarified to solution, carefully remove supernatant；
C) keep EP pipe always in magnetic frame, 80% ethyl alcohol that 200ul Fresh is added rinses magnetic bead.Incubation at room temperature carefully removed supernatant after 30 seconds；
D) it repeats to walk, amounts to rinsing twice；
E) EP pipe is kept in magnetic frame, to uncap always and be air-dried magnetic bead 10 minutes；
F) EP pipe is taken out from magnetic frame, the ultrapure water elution of 25ul sterilizing is added.Vortex oscillation or gently blown and beaten using pipettor is mixed well.The of short duration centrifugation of reaction tube is placed in magnetic frame and separates magnetic bead and liquid.Supernatant is carefully drawn to solution clarification (about 5 minutes) into sterilizing EP pipe, is saved in -20 DEG C；
G) product after purification takes 1ul to survey ssDNA concentration.
Purified product is BGISEQ500 sequencing library.
8, quality standard and assessment
| Quality index title | Quality index | Measurement method |
| Clip size | 300bp±100bp | Agilent 2100 |
| Concentration | >1.5ng/ul | QPCR |
9, sequencing and interpretation of result
For the BGISEQ500 sequencing library of above-mentioned acquisition, 10M reads is surveyed using BGISEQ500 microarray dataset.
Wherein, it is sequenced using following sequencing primer:
Forward primer: 5 '-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC (SEQ ID NO:4),
Reverse primer: 5 '-TCGTCGGCAGCGTCAGATGTGTATAAGAGACA (SEQ ID NO:5).
Under data after machine, using onhouse pipleline by lower machine data filtering, and then it is compared using tophat and fq is switched to bam, and it compares reads is compared onto gene, with using edgeR calculation expression amount (the result is shown in Figure 1, wherein, type is sequenced in BGISEQ-500: type: PE100 is sequenced in SE50, Illumina platform)；It is compared using bwa and fq is switched to bam, samtools duplicate removal, onhouse pipleline chooses unique, and calculates CNV (result is shown in Fig. 2).Wherein, experiment is using the Illumina sequencing result in the library Illumina before conversion as control.
For Fig. 1, above histogram in, abscissa: H1975-1 indicates the sequencing result of unicellular starting；H1975-5 indicates the sequencing result of 5 cells starting；BGI indicates the sequencing of BGISEQ-500 platform, and Hiseq indicates the sequencing of Illumina platform.Ordinate indicates transcript profile sequencing result after downstream information is analyzed, the gene dosage detected.
In table below Fig. 1, abscissa indicates that the gene dosage detected, ordinate indicate the abundance of gene.
Wherein, RPKM is the abbreviation of Reads Per Kilobases per Millionreads, represents and comes from every million reads
The reads number of the every kilobase length of Mr. Yu's gene, by the read number of map to gene divided by the length of all read numbers (as unit of million) and RNA on map to genome (as unit of KB).RPKM is used to indicate gene expression amount or index abundant in two generation sequencing technologies.It has been generally acknowledged that the gene expression amount of RPKM > 1 is relatively abundanter, detect that the quantity of the gene of RPKM > 1 is more believable；RPKM < 1 or < 0.1 gene abundance it is lower, the gene of 0 < RPKM < 0.1 detected has been generally acknowledged that confidence level is slightly worse.
For Fig. 2, abscissa indicates that chromosome sequence number, ordinate indicate to detect the large fragment copy number variation (multiplication or missing) above DNA
Based on known to Fig. 2:
1, large fragment CNV can be detected using the sequencing result that BGISEQ-500 platform is sequenced；
2, unicellular largely consistent with the CNV result of 5 cell mix, the single celled result of CNV (on 1p, 3p, 11q) and cell mix have differences on a small number of chromosomes, just illustrate the heterogeneity of tumour cell.
Further, be that template prepares BGISEQ500 sequencing library with the unicellular library H1975DNA and RNA 10ng (library Illumina) according to above technical scheme, final product ssDNA after purification, 20ul NF H2O back dissolving.Then, the same Illumina sequencing result using the library Illumina before conversion is as control, the BGISEQ500 sequencing library of acquisition is surveyed into 10M reads using BGISEQ500 microarray dataset, under data after machine, using onhouse pipleline by lower machine data filtering, it is compared using tophat and fq is switched to bam, and compared a reads is compared onto gene, with using edgeR calculation expression amount；It is compared using bwa and fq is switched to bam, samtools duplicate removal, onhouse pipleline chooses unique, and calculates CNV.The results show that the sequencing result for the BGISEQ500 sequencing library that conversion obtains is reliable.
The method that linear sequencing library is converted into cyclic annular sequencing library of the invention, dsDNA the linear libraries especially library Illumina and the library Proton can be made to be quickly converted into ssDNA ring-type sequencing library (such as library BGISEQ-500), library can directly be sequenced in the corresponding microarray dataset of cyclic annular sequencing library after conversion.
Although a specific embodiment of the invention has obtained detailed description, it will be understood to those of skill in the art that.According to all introductions having disclosed, those details can be carry out various modifications and be replaced, these changes are within the scope of the present invention.Full scope of the invention is given by the appended claims and any equivalents thereof.
In the description of this specification, the description of reference term " one embodiment ", " some embodiments ", " illustrative examples ", " example ", " specific example " or " some examples " etc. means that particular features, structures, materials, or characteristics described in conjunction with this embodiment or example are included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms may not refer to the same embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be combined in any suitable manner in any one or more of the embodiments or examples.
Claims (11)
- A method of linear sequencing library is converted into cyclic annular sequencing library, which comprises the following steps:Linear sequencing library is subjected to PCR amplification using positive adapter-primer and reversed adapter-primer, to obtain amplified production；The amplified production is subjected to thermal denaturation processing, to obtain single stranded DNA mixture；The single stranded DNA mixture is subjected to cyclisation processing, to obtain cyclic DNA product, the cyclic DNA product constitutes the cyclic annular sequencing library,Wherein,At least partly sequence complementary pairing of the positive connector of the forward direction adapter-primer and the linear sequencing library, the at least partly sequence complementary pairing of the reversed connector of the reversed adapter-primer and the linear sequencing library, and, 5 ' terminal nucleotides of the forward direction adapter-primer and at least one of the reversed adapter-primer pass through phosphorylation modificationThe cyclisation processing uses cyclisation primer to carry out, and 3 ' the end sub-sequence complementary pairings with the positive end of adapter-primer 5 ', the reversed adapter-primer respectively ' are held in end and 3 ' in the 5 of the cyclisation primer.
- The method according to claim 1, wherein the linear sequencing library is Illumina sequencing library or Ion Protona sequencing library, the ring-type sequencing library is the library BGISEQ-500.
- The method according to claim 1, wherein before thermal denaturation processing, further comprising after PCR amplification:The step of amplified production is subjected to Piece Selection and purifying.
- It is handled 3 minutes the method according to claim 1, wherein carrying out the thermal denaturation under 95 degrees Celsius.
- The method according to claim 1, wherein carrying out the cyclisation processing using T4 DNA ligase.
- The method according to claim 1, wherein the ratio of the single stranded DNA mixture and the cyclisation primer is 1:10 in cyclisation processing.
- The method according to claim 1, wherein further comprising:The step of cyclic DNA product is successively subjected to digestion process and purification process.
- The method according to claim 1, wherein the linear sequencing library is Illumina sequencing library, the sequence of the forward direction adapter-primer is as shown in SEQ ID NO:1: 5 '-PhosAATGATACGGCGACCACCGA-3 ', the sequence of the reversed adapter-primer is as shown in SEQ ID NO:2: 5 '-CAAGCAGAAGACGGCATACGA-3 ', and the sequence of the cyclisation primer is as shown in SEQ ID NO:3: 5 '-GCCGTATCATTCAAGCAGAAGACG-3 '.
- A method of determining the DNA sequence dna of linear sequencing library, which comprises the following steps:The linear sequencing library is converted to cyclic annular sequencing library by method according to claim 1-8；AndThe cyclic annular sequencing library is sequenced using the corresponding microarray dataset of the ring-type sequencing library, to determine the DNA sequence dna of the linear sequencing library.
- According to the method described in claim 9, the sequencing primer is identical as junction portion sequence or complementary pairing it is characterized in that, the sequencing is carried out using sequencing primer.
- According to the method described in claim 10, it is characterized in that, the sequence of the sequencing primer is as follows:Forward primer is as shown in SEQ ID NO:4: 5 '-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC,Reverse primer is as shown in SEQ ID NO:5: 5 '-TCGTCGGCAGCGTCAGATGTGTATAAGAGACA.
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