CN102140523B - Sequencing method for in-situ copying high-flux sequencing template and increasing reading length thereof - Google Patents
Sequencing method for in-situ copying high-flux sequencing template and increasing reading length thereof Download PDFInfo
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Abstract
The invention relates to a sequencing method for in-situ copying high-flux sequencing template and increasing reading length thereof, which comprises the following steps of: after obtaining a sequence fragment by sequencing a prepared DNA (Deoxyribonucleic Acid) sequencing template, denaturing the DNA sequencing template to DNA single chains--old templates; copying the template by activating a previously introduced extending primer and then fully cutting off the old template to obtain DNA single chains--new templates which completely complement the original DNA sequencing template; carrying out sequence measurement by using the DNA single chains as DNA sequencing templates to obtain new measured sequences complementary with the other ends of the old templates. The sequence fragments obtained by measuring the new templates and the old templates are spliced so that the reading length of the sequencing template is increased, the difficulty of splicing short fragment sequences is reduced, and the accuracy of sequences is improved.
Description
Technical field
The invention belongs to biological technical field, specifically is a kind of method that realizes in the dna sequence analysis increasing the order-checking reading length, relates in particular to a kind of high-flux sequence method that copies to increase reading length by the original position of sequencing template.
Background technology
Along with the carrying out and finish of the Human Genome Project and various model animals genome plans, make the mankind step into back era gene, biological study and the medical research in the present age produced tremendous influence, the molecular biology related discipline has obtained swift and violent development.From the difference of gene level understanding life, disease takes place, the rule of development, and the interaction of medicine and life entity will become possibility.With regard to gene sequencing, the emphasis of back era gene has been measured by whole genome sequence and has been transferred to the comparison of hereditary difference between idiogenetics difference and species in the genome.At present, no matter look for new or confirm known SNP site, traditional Sanger dna sequencing method still is in the status that can not be substituted.But there is the low and problem of ultra-high price of flux in this method.The expense that first human genomic sequence is measured is approximately 1,000,000,000 dollars, and this expense has been reduced to about 2,000 ten thousand dollars at present.But the progress of functional genome still is subject to the dna sequencing technology.For this reason, U.S. Venter foundation proposed the goal in research of the human genome sequencings of 1000 U.S. dollars in 2003.Based on traditional Sanger dna sequencing method, will finish a complete genomic order-checking of Mammals at present in the world needs up to ten million dollars.Be example with current the most advanced ABI Prism 3730 dna sequencing instrument, finish the order-checking of 3,000,000,000 bases in the human genome, need 150 ABI Prism 3730 dna sequencing instrument runnings 1 year, its order-checking cost reaches 24,000,000 dollars.Now based on Sanger dna sequencing method, at the highdensity capillary array of development, improving the parallelism of order-checking, so this research thinking of order-checking speed that improves DNA improve dna sequencing speed and reduce cost aspect room for improvement also very limited.
At present, the complete genome DNA sequencing technologies has become research field that competition is very fierce in the world.Most of research synthetic order-checking tactful aspect, the marketization at present comparatively successful example be that 454 Life Sciences companies of the U.S. are based on the burnt sequencing technologies of the high-flux parallel of emulsion PCR product; Bridge-type amplification-DNA the chip of Illumina (Solexa) company extends sequencing technologies; And Applied Biosestems(SOLiD) company is based on the hybridization-enzyme connection-enzyme cutting high throughput sequencing technologies of emulsion PCR product.In these synthetic sequence measurements, no matter be burnt order-checking or the extension of labeled monomer order-checking, or connection sequence measurement, along with the increase of extending (perhaps connecting) reaction times, because the influences such as loss of its extension (perhaps connecting) efficient, cutting efficiency, sequencing primer, the mistake of order-checking can constantly increase, and causes the reduction of sequence reading length, and sequence reading length remarkably influenced splicing packaging efficiency.Existing document shows, when the sequence reading length is 20 bases, need carry out the sequencing more than 50 times, and when the sequence reading length is 80 bases, only need about 5-6 time sequencing just human genomic sequence can be carried out effective completed assembled.Therefore, the reading length that improves order-checking not only can improve the accuracy of sequence, and can reduce the cost of sequencing greatly.
Summary of the invention
Goal of the invention:Purpose of the present invention is exactly that original position by a kind of high-flux sequence template that has checked order copies, and is that dna sequence analysis increases the order-checking reading length, set up fast, accurately, cheap genome sequence measuring method.
Technical scheme:A kind of original position of high-flux sequence template copies and increases the sequence measurement of reading length, the dna sequencing template that has prepared, after order-checking obtains one section sequence fragment, be dna single chain-old template with its sex change, by the extension primer that activates previous introducing it is copied again, and with behind the old template complete resection, obtain the dna single chain-new template complementary fully with original dna sequencing template, these dna single chains are carried out sequencing as the dna sequencing template, just obtain and the old template the other end, and complementary new mensuration sequence, will be new, the sequence fragment splicing that old template is measured, increased the reading length of sequencing template, reduce the difficulty of short-movie section sequence assembly, improved the accuracy of sequence.
The old template of high-flux sequence contains the site that can cut, the cleavage site directly universal primer by being included in cleavage site is connected with the order-checking fragment and obtains, and the amplimer that maybe will be included in cleavage site acquires by methods such as emulsion PCR, rolling circle amplification or bridge-type PCR.
The chemical bond that the old template cleavage site of described high-flux sequence is chemical chop, described chemical bond are the disulfide linkage of reductive agent fracture or the o-dihydroxy of oxygenant fracture,
The old template cleavage site of described high-flux sequence is enzyme identification cleavage site, and described cleavage site is xanthoglobulin (I) base of endonuclease identification or uridylic (U) base of ura DNA glycosidase identification.
Amplification and sequencing reaction can not take place in the previous extension primer of introducing in the order-checking process of the amplification procedure that obtains old template and the old template of use, before amplification, this 3 ' end that extends primer can be by mode non-hydroxylated such as phosphorylations; And in the sequencing reaction process, when adopt extending order-checking, this extends 3 ' of primer and holds also that right and wrong are hydroxylated, is adopting when connecting order-checking, and then this 3 ' end that extends primer is hydroxylated.
After the order-checking of old template is finished and sex change becomes strand, after the extension primer 3 ' terminal hydroxy groupization to previous introducing, under the effect of polysaccharase, A, G, four bases of C, T are added in the lump carry out extension, the acquisition of new template is finished by an extension that extends primer or is carried out repeatedly extension by annealing repeatedly and realize.
Described sequencing is for extending sequence measurement or connecting sequence measurement; Described sequencing template is single-molecule sequencing template or polymolecular sequencing template.
Beneficial effect:
The present invention compared with prior art has following advantage:
1. great advantage of the present invention is that realized checking order the original position of section of DNA template copies, and carry out sequencing again to copy template, this is equivalent to the other end with original dna profiling has been carried out sequencing, increased the sequence reading length, improved the exactness of splicing, reduce the number of times of replication, greatly reduce the expense of sequencing.
2. the high-throughput of the section of DNA template that checks order that the present invention relates to copies, the excision of old template, fixing and the activation of primer is all carried out according to the chemistry of tradition maturation and popular molecular biology method, there are not technological difficulties, easily in existing technical enforcement.
Description of drawings
Fig. 1 is the sequence measurement synoptic diagram that the original position of a kind of high-throughput single-molecule sequencing of the present invention template copied and increased reading length.Have among the figure: genome (1); Genomic fragment (2); Connexon (3,4), 5 ' end of connexon (3) have can with the active group of substrate generation bonding reaction, contain chemistry or enzyme cleavage site in the middle of the sequence; The genomic fragment (5) that connects connexon (3), connexon (4); The sealing primer (6), 5 ' end have can with the active group of substrate generation bonding reaction, sequence and connexon 4 all or part of complementations; Activation substrate (7) is as modifying the sheet glass of avidin etc.; Sequencing primer (8), all or part of complementation of sequence and connexon (4); Primer (6); Extend template (9); Sequencing primer (10), all or part of complementation of sequence and connexon (3).It is the fragment of 50-1000 base that genome (1) (a) becomes size with enzyme cutting (perhaps ultrasonication), and under the effect of ligase enzyme with these fragmentation nucleotide sequences (2) with the known general connexon (3 of pair of sequences, 4) carry out ligation (b) and become the fragmentation sequence (5) that contains connecting arm, the active group generation chemical bonding (c) that contains the fragmentation sequence (5) of connecting arm and the active group of sealing primer (6) (as vitamin H etc.) and substrate (7) makes it fixing, add sequencing primer (8) unit molecule template (5) is carried out a series of sequencing reaction (d) (Harris, T.D.
Et al. Single-molecule DNA sequencing of a viral genome. Science, 2008,320,106 – 109), realize one section sequencing to template 5; After the sequencing reaction of template 5 is finished, remove the synthetic product of sequencing primer (8), and activation (e) sealing primer 6, under the polysaccharase effect, extension (polymerization of monomer A, G, C, T is synthetic) (f, g) takes place in primer 6, obtains the single stranded DNA template 9 complementary fully with template 5, template 4 is cut (h), and with after its sealing (i), add sequencing primer (10) unit molecule template (9) is carried out a series of sequencing reaction (j) (Braslavsky, I.
Et al. Sequence information can be obtained from single DNA molecules. Proc. Natl. Acad. Sci. USA.2003,100,3960 – 3964), realize one section sequencing to template 9.Because template (5) and template (9) are complementary fully sequences, can be transformed into the sequence information of template (5) to the mensuration of new template (9) sequence, thereby obtain the information of each one section sequence fragment of template (5) two ends.
Fig. 2 is the sequence measurement synoptic diagram that the original position of a kind of microballoon emulsion amplification of the present invention high-flux sequence template copied and increased reading length.Have among the figure: genome (1); Genomic fragment (2); Connexon (3,4); The genomic fragment (5) that connects connexon (3), connexon (4); Microballoon (6); Primer (7), 5 ' end have can with the active group of microballoon generation bonding reaction; Primer (8), 5 ' end have can with the active group of microballoon generation bonding reaction, contain chemistry or enzyme cleavage site in the middle of the sequence; The microballoon (9) of immobilized primer (7) and primer (8); Amplified production (10); The substrate (11) that microballoon is fixing; The order-checking product (12) of template (10).It is the fragment of 50-1000 base that genome (1) (a) becomes size with enzyme cutting (perhaps ultrasonication), and under the effect of ligase enzyme, these fragmentation nucleotide sequences (2) are carried out ligation (b) with the known general connexon of pair of sequences (3,4) and become the fragmentation sequence (5) that contains connecting arm, fragmentation sequence (5) is by pre-amplification reaction, and behind electrophoresis, the fragment of getting length 100-200bp is used for the amplification sequencing template; Simultaneously, primer (7,8) fixing (c) is fixed primer 7,8 microballoon (9) on microballoon (6).The fragmentation sequence (5) of microballoon (9) and length 100-200bp is carried out pcr amplification (d) (Williams, R. in microemulsion system
Et al. Amplification of complex gene libraries by emulsion PCR. Nature Methods, 2006,3(7), 545-550; Dieh, F.
Et al. BEAMing:single-molecule PCR on microparticles in water-in-oil emulsions. Nature Methods, 2006,3(7), 551-559), obtain the dna profiling that can be used for checking order, utilize 3 ' the end group group and substrate generation bonding reaction (e) of primer on the microballoon (7) then, make it to be fixed on the substrate, template (10) is carried out a series of sequencing reaction (f), realize one section sequencing of template 10; After the sequencing reaction of template (10) is finished, order-checking product (12) is removed in sex change, and activation (g) sealing primer (7), under the polysaccharase effect, extension (polymerization of monomer A, G, C, T is synthetic) (h, i) takes place in primer (7), obtains the single stranded DNA template (13) complementary fully with template (10), template 10 is cut (j), and with after its sealing (k), template (13) is carried out a series of sequencing reaction (l), realize one section sequencing to template (13).Because template (13) and template (10) are complementary fully sequences, can be transformed into the sequence information of template (10) to the mensuration of new template (13) sequence, thereby obtain the information of each one section sequence fragment of template (10) two ends.
The agarose gel electrophoresis figure that the original DNA sample ultrasonic of Fig. 3 different concns is smashed (the sign length DNA of M1, M2: gradient 100bp, A:DNA concentration is 100ng/mL, B:DNA concentration is 150ng/m L)
Fig. 4 template magnetic bead is at the fixing displaing micro picture of sheet glass substrate;
Fig. 5 connects sequencing and once connects the four look fluorograms (part) that obtain, wherein (1) Cyanine 3; (2) Cyanine 5; (3) Texas Red; (4) Fuorescein Isothiocyannate.
Embodiment
The invention will be further described below in conjunction with example:
It is to have finished the preparation of high-flux sequence template that the original position of high-flux sequence template copies, and finish after the high-throughput dna profiling one terminal sequence mensuration, its sex change is become single strand dna, and the extension primer that will before be fixed on around the template activates, be to extend template then with the single strand dna, extend and obtain a chain (new template) complementary fully with original single strand dna after primer is finished extension, behind old template complete resection, new template can continue on for the mensuration of dna sequence dna, this section sequence of measuring and one section sequence of the old template the other end are complementary fully, like this can be with the sequence fragment splicing of this section sequence and old template mensuration, increased the reading length of sequencing template, reduce the difficulty of short-movie section sequence assembly, improved the accuracy of sequence.
Embodiment 1: the regeneration of high-flux sequence template and connection sequence measurement thereof are measured the bacillus coli gene group
(1) general～100ng/mL bacillus coli gene group sample 100mL selects 20 minutes low frequency ultrasound time in ultrasonic apparatus, and ultrasonic experiments has been carried out electrophoresis detection (as Fig. 3), reclaims the dna fragmentation of 100 ± 30bp length from gel.
(2) connexon 1, the concrete sequence of 2(are seen the following form) under the effect of ligase enzyme, be connected (identical sequence, i.e. connexon are all contained in the two ends of all different templates molecules) with above-mentioned 100 ± 30bp fragmentation nucleotide sequence.
(3) with amplimer and the extension primer (concrete sequence sees the following form) of biotin modification, the magnetic microsphere of modifying with avidin fully reacts, and it is fixed on the magnetic microsphere.
| Primer | Sequence |
| The template amplification primer | 5’-Bio-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT |
| Extend primer | 5’-Bio-AACTGCCCCGGGTTCCTCATTCTCT- PO 4 3- |
(4) will connect the fragmentation nucleotide sequence of connexon and fixedly amplimer and the magnetic microsphere that extends primer carry out pcr amplification under microemulsion system, and utilize to extend fixedly magnetic bead (Fig. 4) of 3 end phosphate groups of primer and on-chip amino bonded, obtain magnetic microsphere bacillus coli gene group sequencing template chip.
(5) 3 terminal hydroxy groupization that will extend primer with the T4 kinases are to avoid interference follow-up connection sequencing reaction.
The fixing sheet glass of microballoon is installed in the high-flux sequence instrument makes up reaction tank, according to the program (Shendure, the J. that connect sequence measurement
Et al. Accurate multiplex polony sequencing of an evolved bacterial genome. Science, 2005,309,1728 –, 1732. documents), obtain the fluorescent signal of the each reaction of each magnetic bead, and signal is converted into base information (Fig. 5), measure 30 base sequence information of template when carrying out connecting order-checking for 30 times.
(6) 0.1M NaOH solution is joined make order-checking product sex change become single stranded DNA in the reaction tank.
(7) polysaccharase and monomer A, G, C, T are joined in the reaction tank, through the annealing of order-checking product and single stranded DNA template, extend, copy the dna profiling of order-checking for the first time.
(8) handle the said chip cutting dna profiling of order-checking for the first time with endonuclease, and under alkaline condition with its removing.
(9) add sequencing primer new template is carried out sequencing, and the sequence of measuring is transformed in the sequence of corresponding template for the first time.
(10) all sequences fragment is compared in bacillus coli gene group reference sequences, splicing is (on the identical microspheres, first base of sequence fragment of measuring at a distance of 0～95bp), is finished the order-checking again of bacillus coli gene group apart from the 30th base of the sequence fragment of measuring for the first time for the second time.
Embodiment 2: the regeneration of high-flux sequence template and connection sequence measurement thereof are measured the people's gene group
(1) method according to embodiment 1 prepares magnetic microsphere people's gene group sequencing template chip (seeing (1) among the embodiment 1～(4) step).
(2) fixedly the sheet glass of microballoon people's gene group sequencing template is installed in the high-flux sequence instrument and makes up reaction tank, according to extending sequence measurement (Bentley, D. R.
Et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature, 2008,456, program 53-59), obtain the fluorescent signal of the each reaction of each magnetic bead, and signal is converted into base information (Fig. 5), measure 35 base sequence information of template when carrying out extending order-checking for 35 times.
(3) 0.1M NaOH solution is joined make order-checking product sex change become single stranded DNA in the reaction tank.
(4) use the T4 kinases to handle 3 end phosphate groups of the extension primer of predetermined fixed, with its hydroxylation.
(5) polysaccharase and monomer A, G, C, T are joined in the reaction tank, through the annealing of order-checking product and single stranded DNA template, extend, copy the dna profiling of order-checking for the first time.
(6) handle the said chip cutting dna profiling of order-checking for the first time with ura DNA glycosidase, and under alkaline condition with its removing.
(7) add sequencing primer new template is carried out sequencing, and the sequence of measuring is transformed in the sequence of corresponding template for the first time.
(8) all sequences fragment is compared in people's gene group reference sequences, splicing is (on the identical microspheres, first base of sequence fragment of measuring at a distance of 0～95bp), is finished the order-checking again of people's gene group apart from the 35th base of the sequence fragment of measuring for the first time for the second time.
Sequence table
＜110〉Southeast China University
＜120〉a kind of original position of high-flux sequence template copies and increases the sequence measurement of reading length
<130>
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 43
<212> DNA
＜213〉artificial sequence
<400> 1
aaccactacg cctccgcttt cctctctatg ggcagtcggt gat 43
<210> 2
<211> 43
<212> DNA
＜213〉artificial sequence
<400> 2
ttggtgatgc ggaggcgaaa ggagagatac ccgtcagcca cta 43
<210> 3
<211> 25
<212> DNA
＜213〉artificial sequence
<400> 3
aactgccccg ggttcctcat tctct 25
<210> 4
<211> 25
<212> DNA
＜213〉artificial sequence
<400> 4
ttgacggggc ccaaggagta agaga 25
<210> 5
<211> 41
<212> DNA
＜213〉artificial sequence
<400> 5
ccactacgcc tccgctttcc tctctatggg cagtcggtga t 41
<210> 6
<211> 25
<212> DNA
＜213〉artificial sequence
<400> 6
aactgccccg ggttcctcat tctct 25
Sequence table
＜110〉Southeast China University
＜120〉a kind of original position of high-flux sequence template copies and increases the sequence measurement of reading length
<130>
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 43
<212> DNA
＜213〉artificial sequence
<400> 1
aaccactacg cctccgcttt cctctctatg ggcagtcggt gat 43
<210> 2
<211> 43
<212> DNA
＜213〉artificial sequence
<400> 2
ttggtgatgc ggaggcgaaa ggagagatac ccgtcagcca cta 43
<210> 3
<211> 25
<212> DNA
＜213〉artificial sequence
<400> 3
aactgccccg ggttcctcat tctct 25
<210> 4
<211> 25
<212> DNA
＜213〉artificial sequence
<400> 4
ttgacggggc ccaaggagta agaga 25
<210> 5
<211> 41
<212> DNA
＜213〉artificial sequence
<400> 5
ccactacgcc tccgctttcc tctctatggg cagtcggtga t 41
<210> 6
<211> 25
<212> DNA
＜213〉artificial sequence
<400> 6
aactgccccg ggttcctcat tctct 25
Claims (1)
1. the original position of a high-flux sequence template copies and increases the sequence measurement of reading length, it is characterized in that 100ng/ μ L bacillus coli gene group sample 100 μ L are selected 20 minutes low frequency ultrasound time in ultrasonic apparatus, ultrasonic experiments has been carried out electrophoresis detection, reclaims the dna fragmentation of 100 ± 30bp length from gel; Connexon 1,2 is connected described connexon 1:5 '-PO with above-mentioned 100 ± 30bp fragmentation nucleotide sequence under the effect of ligase enzyme
4 3--AACCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT; Connexon 1 complementary sequence: 3 '-TTGGTGATGCGGAGGCGAAAGGAGAGATACCCGTCAGCCACTA; Connexon 2:5 '-PO
4 3--AACTGCCCCGGGTTCCTCATTCTCT; Connexon 2 complementary sequences: 3 '-TTGACGGGGCCCAAGGAGTAAGAGA; With amplimer and the extension primer of biotin modification, the magnetic microsphere of modifying with avidin fully reacts, and it is fixed on the magnetic microsphere, and wherein amplimer is: 5 '-Bio-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT; Extending primer is: 5 '-Bio-AACTGCCCCGGGTTCCTCATTCTCT-PO
4 3-Under microemulsion system, carry out pcr amplification with connecting the fragmentation nucleotide sequence of connexon with the fixing magnetic microsphere of amplimer and extension primer, and utilize to extend fixedly magnetic bead of 3 end phosphate groups of primer and on-chip amino bonded, obtain magnetic microsphere bacillus coli gene group sequencing template chip; To extend 3 terminal hydroxy groupization of primer with the T4 kinases, to avoid interference follow-up connection sequencing reaction: fixedly the sheet glass of microballoon is installed in the high-flux sequence instrument and makes up reaction tank, according to the program that connects sequence measurement, obtain the fluorescent signal of the each reaction of each magnetic bead, and signal is converted into base information, measure 30 base sequence information of template when carrying out connecting order-checking for 30 times; 0.1M NaOH solution joined make order-checking product sex change become single stranded DNA in the reaction tank; Polysaccharase and monomer A, G, C, T are joined in the reaction tank, through the annealing of order-checking product and single stranded DNA template, extend, copy the dna profiling of order-checking for the first time; Handle the said chip cutting dna profiling of order-checking for the first time with endonuclease, and under alkaline condition with its removing; Add sequencing primer new template is carried out sequencing, and the sequence of measuring is transformed in the sequence of corresponding template for the first time; The all sequences fragment is compared in bacillus coli gene group reference sequences, splicing, on the identical microspheres, first base of sequence fragment of measuring at a distance of 0～95bp, is finished the order-checking again of bacillus coli gene group apart from the 30th base of the sequence fragment of measuring for the first time for the second time.
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| US7425431B2 (en) * | 2004-02-27 | 2008-09-16 | President And Fellows Of Harvard College | Polony fluorescent in situ sequencing beads |
| CN101307361A (en) * | 2008-07-15 | 2008-11-19 | 南京大学 | Method for identifying miRNA in blood serum of patient with lung cancer by Solexa technology |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7425431B2 (en) * | 2004-02-27 | 2008-09-16 | President And Fellows Of Harvard College | Polony fluorescent in situ sequencing beads |
| CN101307361A (en) * | 2008-07-15 | 2008-11-19 | 南京大学 | Method for identifying miRNA in blood serum of patient with lung cancer by Solexa technology |
Non-Patent Citations (6)
| Title |
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| Accurate whole human genome sequencing using reversible terminator chemistry;David R. Bentley et al.;《Nature》;20081106;第456卷(第7218期);参见第54页图1及其注释，摘要，第53页正文部分左栏第18行至右栏第5行，补充数据第3-4页，图17a * |
| David R. Bentley et al..Accurate whole human genome sequencing using reversible terminator chemistry.《Nature》.2008,第456卷(第7218期),参见第54页图1及其注释，摘要，第53页正文部分左栏第18行至右栏第5行，补充数据第3-4页，图17a. |
| Michael L. Metzker.Sequencing technologies —the next generation.《NATuRe RevIewS》.2010,第11卷(第1期),第33页图1及注释. |
| Sequencing technologies —the next generation;Michael L. Metzker;《NATuRe RevIewS》;20100131;第11卷(第1期);第33页图1及注释 * |
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| 王升跃.新一代高通量测序技术及其临床应用前景.《广东医学》.2010,第31卷(第3期),269-272. |
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