CN110317856A - It is assembled based on apparent group information low cost and parses biological core gene group information - Google Patents
It is assembled based on apparent group information low cost and parses biological core gene group information Download PDFInfo
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Abstract
The present invention relates to the method and its application based on apparent group information low cost assembling core gene group and then parsing biological gene group information.A kind of method that the present invention proposes new acquisition core gene group sequence information, the combined nucleic acid sequence of one or more apparent modifications of the capture active list of controlling gene first, the sequence of acquisition is carried out later to build library, sequencing, splicing, assembling etc., obtains sequence information.Method of the invention can without reference to genome species and the bigger kind of hereditary difference in obtain the gene region array or Regulatory Sequence of long segment, and independent of any existing sequence information.
Description
Technical field
The invention belongs to biological assembling parsing fields, more particularly it relates to be based on apparent group information low cost group
The method and its application of dress parsing biological gene group information.
Background technique
In the past decade, genome sequencing and resurvey sequence be widely used in excavate different lines crop between something lost
Difference is passed, has pushed genetic breeding field that major transformation all has occurred from research method to Economic Application.Sequence is resurveyed to refer to
In the case where knowing species gene group, genome is carried out to the different tissues of Different Individual or some individual in species and resurveys sequence,
The difference between Different Individual or histocyte can be found in full-length genome level.
However, many economic species experienced long-term domestication, genome is complicated and huge.For example, generally planting at present
Wheat be 6 times of bodies, full-length genome has a 17Gb, even coverage require it is lower resurvey sequence experiment require it is high at
This.And much study and do not need to know all base sequences of genome, so people develop for big genome species
The substitution sequencing technologies of various low costs.Its basic principle is usually to carry out selective sequencing to whole genome sequence, be detailed in
Lower two summaries (Genome-wide genetic marker discovery and genotyping using next-
generation sequencing.Nature reviews.Genetics,12,499-510；Using next-
generation sequencing to isolate mutant genes from forward genetic
screens.Nature reviews.Genetics,15,662-676).For be not sequenced or not genetic marker species come
Say, the more classical method such as RAD-seq of the sequencing technologies based on restriction enzyme site, can between kind more identical restriction enzyme site
Neighbouring sequence.However the gene functional research of this unsuitable big genome species of method, because of the sequencing fragment generated
Noncoding region is greatly derived from, it is unrealistic that functional analysis is directly carried out according to its result, it is also necessary to is a large amount of subsequent fine
Heredity work.More importantly if restriction enzyme site changes between kind, sequence can not mutually compare, thus can not be right
Between species and kind that polymorphism is high is compared.Another method is sought using the transcription group information of RNA-seq acquisition
Look for genetic marker, for example, by with mixed pond sequencing (Bulked segregant analysis, BSA) in conjunction with carrying out figure position gram
It is grand.However, montage mode is more due to big by gene expression dose difference, rna editing etc. influences, and speculates base using RNA-seq
There is because of group information and in actual operation bigger difficulty in further gene location.For kind, a kind of conventional means is sequenced
It is that direct enrichment exon sequence is sequenced, i.e. exon trapping technology.It is widely used in the research of people, and advantage is
It is directly enriched with coding region sequence, and follow-up function analysis is relatively easy, but high, production is required existing Genomic sequence information
The early investment cost for capturing chip is big, and the chip made is only used for the less big group of hereditary variation, for heredity
Make a variation big group, resurveys sequence dependent on the technology of reference genome, including exon sequencing or even full-length genome, can all show
Work underestimates polymorphism (Comprehensive comparison of three commercial human whole-exome
capture platforms.Genome Biology,12,R95).And the genetic diversity between industrial crops different lines is remote
Much higher than people's interspecific difference, these alternatives, which are difficult to meet, carries out group's something lost for the relatively high big genome species of polymorphism
Pass the demand of analysis.
Thus, exploitation can be captured directly to gene and the simplification gene order-checking method of Regulatory Sequence is more for studying
The high group of state property has important value.
Summary of the invention
The purpose of the present invention is to provide the methods for assembling parsing biological gene group information based on apparent group information low cost
And its application.
In the first aspect of the present invention, a kind of method for obtaining core gene group sequence information, the method packet are provided
It includes:
(1) nucleic acid sequence of apparent modified regions in genome is captured；
(2) library, sequencing are constructed using the nucleic acid sequence that step (1) obtains；
(3) sequencing result is obtained according to step (2), carries out sequence assembly, assembles, fills up and extend, obtain core gene
Group sequence information.
In a preferred embodiment, in step (1), apparent modified region in genome is captured using the method for co-immunoprecipitation
The nucleic acid sequence in domain.
In another preferred example, in step (1), described is apparently modified to single apparent modification or a variety of apparent modifications.
In another preferred example, it is described be apparently modified in genome be stabilized or conservative existing for apparently repair
Decorations；Preferably, including the apparent modification of chromatin and DNA, transcription factor is combined, and enrichment is incorporated in gene nearby with RNA
Polymerase shears closely related protein complexes with transcription initiation, extension, RNA for representative.
In another preferred example, described to be apparently modified to histone modification；Preferably, the apparent modification includes: sharp
The apparent modification or the apparent modification of suppressive of type living.
In another preferred example, the activated form it is apparent modification include it is below one or more: the 3rd subunit of histone
The one/bis- of No. 4 lysine/tri-methylated (H3K4me1/2/3)；The acylation of the 3rd No. 27 lysine of subunit of histone
(H3K27ac)；One/bis-/tri-methylated (H3K36me1/2/3) of the 3rd No. 36 lysine of subunit of histone；The 3rd subunit of histone
The acylation (H3K9ac) of No. 9 lysine or other genome have stable bond activated form apparently modify.
In another preferred example, the apparent modification of the suppressive includes: histone No. 27 lysines of the 3rd subunit
One/bis-/tri-methylated (H3K27me1/2/3)；The one/bis- of the 3rd subunit 9 lysine of histone/tri-methylated (H3K9me1/
2/3)；Or other in genome there is the suppressive of stable bond apparently to modify.
In another preferred example, the apparent modification includes the apparent modification and at least one suppression of at least one activated form
The apparent modification of type processed.
In another preferred example, the apparent modification includes H3K4me3.
In another preferred example, the apparent modification includes H3K4me3 and H3K27me3.
In another preferred example, the described apparent modification include H3K4me3, H3K4me1, H3K27me3, H3K27ac,
H3K36me3。
In another preferred example, in step (1), using specific antibody of the specific enrichment near gene to capture
The apparent modified regions stated.
In another preferred example, in step (2), double-stranded specific nuclease (Duplex-SpecificNuclease is utilized
The methods of (DSN)) the low abundance nucleic acid sequence of enrichment carries out the building in library.
In another preferred example, in step (3), before carrying out the splicing, further includes: removal low quality sequence and
Short sequence；Preferably, removal base of the sequencing quality score less than 25, removes sequence measuring joints, the segment that above-mentioned steps obtain is such as
Shorter than 20 remove.
In another preferred example, for long segment sequence (the core gene group sequence that such as the method is used to obtain wheat
Information), sequencing fragment can be 120~180 (such as 150) bases longs, utilize Abyss (http://www.bcgsc.ca/
Platform/bioinfo/software/abyss) spliced.
In another preferred example, for short-movie section sequence (the core gene group sequence that such as the method is used to obtain rice
Information), sequencing fragment be single-ended 30~80 (such as 50) bases longs, using Velvet (https: //www.ebi.ac.uk/~
Zerbino/velvet/) spliced；Preferably, splicing parameter is the coverage for being at least 4 times；Using gradient Kmer into
Row splicing, selects 27 and 31 the two best results；By merging two as a result, taking longer sequence.
In another preferred example, the assembling includes, and after completing splicing, for the segment of both-end sequencing, will survey again
Sequence segment is compared into the sequence spliced, and by connecting both-end sequencing fragment, obtains longer splicing result.
In another preferred example, for the sequence fragment of acquisition, (the sequencing fragment covering of special low cover degree is further removed
Degree<the 1/3 of mean coverage) and especially high coverage (5 times of sequencing fragment coverage>mean coverage) false positive
As a result.
In another preferred example, described fill up includes: to compare primitive sequencer segment onto the long segment of splicing, is passed through
The information of the original series compared fills neutral gear present in above-mentioned splice segment.
In another preferred example, the extension includes: to compare primitive sequencer segment again to the institute after filling up step
The splice segment of acquisition extends splice segment using the primitive sequencer segment compared to splice segment end.
In another preferred example, the threshold value of the extension is that primitive sequencer segment base ratio identical as splice segment is big
In the 95% of original segments length, the sequencing fragment number of least support is 5.
In another preferred example, after step (4), further includes: (5) divide the core gene group sequence information of acquisition
Analysis, comprising: species population genetics research is not sequenced for the genetic polymorphism site for determining gene and control region, compare species into
The heredity variation of gene and control region during changing or taming.
In another aspect of this invention, the application of the method for any description above is provided, is used for: determining gene and control region
Genetic polymorphism site, species population genetics research is not sequenced, compare spore or domestication during gene and regulation
The heredity variation in area.
Other aspects of the invention are apparent to those skilled in the art due to this disclosure
's.
Detailed description of the invention
Fig. 1, technical solution of the present invention a kind of embodiment schematic diagram.
Fig. 2, Circos figure illustrate that splicing result and the gene location goodness of fit are high.
Fig. 3, single-gene illustrate splicing result and the gene location goodness of fit is high.
Fig. 4, the present invention obtain sequencing fragment covering gene and promoter region ratio.
Fig. 5, splice segment of the present invention are distributed around in gene.
Fig. 6, thermal map show that the active important ChIP-seq sequencing fragment apparently modified of rice controlling gene is attached in gene
Close distribution situation and the relationship with gene expression, every row represent a gene, and gene is arranged from high to low by expression quantity.
The protein structure domain that Fig. 7, the promoter region for capturing new gene and gene regions are enriched with respectively.
Fig. 8, the ability that the present invention captures new gene and control region in rice is illustrated.
Fig. 9 A~B, method of the invention find more sites (A) in target area compared with weight sequencing technologies, these
Site is predominantly located at the high position of polymorphism (B).
Figure 10 A~C, thermal map show that arabidopsis (A), controlling gene is active in corn (B) and tree cotton (C) important apparently repairs
Distribution situation of the ChIP-seq sequencing fragment near gene of decorations and the relationship with gene expression, every row represent a gene,
Gene is arranged from high to low by expression quantity.
Specific embodiment
The present inventor passes through in-depth study, it is found that the characteristic distributions of the epigenetic modification of genome, abundance are suitable for
It is matched with sequencing technologies, obtains more comprehensive genomic information.Further, it is described apparent to optimize capture by the present inventor
The method of genetic modification, i.e. Immunoprecipitation.Using method of the invention, it is possible to obtain long segment gene region array or
Regulatory Sequence independent of any existing sequence information, and can greatly reduce testing cost.
Method of the present invention includes: the nucleic acid sequence of apparent modified regions in (1) capture genome；(2) step is utilized
Suddenly the nucleic acid sequence that (1) obtains constructs library, sequencing；(3) sequencing result is obtained according to step (2), carries out sequence assembly, group
It fills, fill up and extends, obtain core gene group sequence information.In some embodiments, it may also include that (4) after step (3)
The core gene group sequence information of acquisition is analyzed, comprising: object is not sequenced for the polymorphic site for determining gene and control region
Kind of groups genetics research compares the evolution of gene and control region during spore or domestication.
The high big genome group of needle genetic polymorphism of the present invention, design capture gene nearby activate apparent with suppressive
Modification is to be enriched with the method comprising the core gene group sequence including gene regions and promoter region, to greatly reduce group's core
The cost of heart genome splicing effectively improves the molecular genetic and population genetic study efficiency of big genome species.
It is described be apparently modified in genome be stabilized or conservative existing for apparently modify, such as, but not limited to contaminate
The apparent modification of chromaticness and DNA, transcription factor combine, and enrichment is incorporated in gene nearby with RNA polymerase (e.g., RNA polymerization
Enzyme I/II/III) it is that representative with transcription initiation, extension, RNA shears closely related protein complexes.The apparent modification
It can apparently be modified for single apparent modification or a variety of (such as 2,3,4,5,6 kind).
As preferred embodiment of the invention, the apparent modification includes histone modification.Such as but may be not limited to:
H3K4me1/2/3, H3K27ac, H3K9ac, H3K36me1/2/3, H3K27me1/2/3, H3K9me1/2/3.
For present invention relates generally to apparent modification, analyzed by primary-stage survey, the inventors discovered that mixing one kind
The apparent modification of activated form and a kind of suppressive is more preferred.It is lost for single modification fineness, but for
Method of the overwhelming majority based on Linkage mapping gene is also applicable.The present inventor is by widely comparing different modifying in gene
It neighbouring positioning and is associated with gene expression, discovery H3K4me3 covering gene is widest in area, cost performance highest.
Specific antibody be can use to capture apparent modified regions in the genome, for example, coprecipitated by being immunized
The method in shallow lake.It is as shown in Figure 1 using a kind of embodiment of co-immunoprecipitation method.
Product for carrying out co-immunoprecipitation can be commercialization, for example, when being used to capture histone modification region
When, the product includes but may be not limited to: activated form apparently modify antibody (one or more) H3K4me1/2/3, H3K27ac,
The antibody H3K27me1/2/3, H3K9me1/2/3 that H3K9ac, H3K36me1/2/3 and suppressive are apparently modified.
In a part of preference of the invention, the DNA sequence dna of target area is effectively obtained using the antibody；It is right
In highly expressed gene, obtained by the following activated form modification of co-immunoprecipitation: H3K4me3, H3K4me1, H3K4me2,
H3K27ac and H3K36me3；The gene that do not express for low expression or can be repaired by co-immunoprecipitation suppressive H3K27me3
Decorations obtain.In the embodiment of transformation, the type that can be captured is more than that.
To the method that the nucleic acid sequence building library of acquisition can use some building libraries known in the art, such as with
That closes Illumina sequencing system utilizes the methods of double-stranded specific nuclease (duplex-specificnuclease (DSN))
It is enriched with the building that low abundance nucleic acid sequence (removal high abundance segment) carries out library, to improve low abundance nucleic acid content of fragment.
In a preferred embodiment of the invention, after acquisition sequencing result, before progress sequence assembly, further includes: go
Except low quality sequence and short sequence；Preferably, removal sequencing quality < 25 and connector, will be shorter than the sequencing fragment of 20bp later
All removals.
In a preferred embodiment of the invention, the assembling and it is subsequent fill up extension include: for long segment sequence,
If the sequencing fragment of wheat is 150 bases longs, first with Abyss (http://www.bcgsc.ca/platform/
Bioinfo/software/abyss) spliced；Second, sequencing fragment is compared into the sequence spliced again, is passed through
Both-end sequencing fragment is connected, longer splicing result is obtained.For the segment that co-immunoprecipitation obtains, need further to remove special
Other low cover degree (the 1/3 of sequencing fragment coverage<mean coverage) and especially high coverage (sequencing fragment coverage>it is flat
5 times of equal coverage) false positive result.Third is filled up: primitive sequencer segment being compared onto the long segment of splicing, is led to
The information for crossing the original series compared fills neutral gear present in above-mentioned splice segment.4th, extend: by primitive sequencer segment
It compares again and arrives previous step splice segment obtained, using the primitive sequencer segment of comparison to splice segment end to splicing piece
Duan Jinhang extends.The threshold value of the extension is that primitive sequencer segment base ratio identical as splice segment is greater than original sheet segment length
The 95% of degree, the sequencing fragment number of least support is 5.
It is used to obtain the core gene group sequence information of rice for short-movie section sequence, such as the method, sequencing fragment is
Single-ended 50 bases longs are spliced using Velvet (https: //www.ebi.ac.uk/~zerbino/velvet/)；Compared with
Goodly, splicing parameter is the coverage for being at least 4 times；Spliced using the Kmer of gradient, selecting 27 and 31, the two are best
Result；By merging two as a result, taking longer sequence.
Method of the invention is suitable for all DNA sequence dnas for capturing apparent modification combination using Immunoprecipitation and carries out
Later stage work, including combine other technologies carry out map based cloning, mix pond sequencing, design chips probe etc..
Method of the invention is also applied for the group high for polymorphism and the genetic analyses of species is not sequenced comparing.
Method of the invention is also applied for big genome wheat as used in example of the present invention, high polymorphic species and not
The analysis of genetic polymorphisms of species is sequenced.
Method of the invention is also applied for the evolution of gene and control region during systematic comparison spore or domestication.
Method of the invention utilizes co-immunoprecipitation mixing or the DNA of single apparent modification enrichment gene and near zone
Sequence.Acquisition is not sequenced the promoter region and remote expression regulation of kind gene and control region, especially short distance
Sequence is of great significance.
Main apparent modification complex is generally guarded in eucaryote, is suitable for extensive object with the inventive method
Kind, if be stabilized in its genome or conservative existing for apparent modification of the present invention.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part such as J. Pehanorm Brooker etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002, or
According to the normal condition proposed by manufacturer.
Antibody
H3K4me3 (Abcam, Cat.ab8580)；
H3K4me1 (Millipore, 07-436)；
H3K27me3 (Upstate, USA, Cat.07-449)；
H3K27ac (Upstate, USA, Cat.07-360)；
H3K36me3 (Abcam, Cat.ab9050)；
Above-mentioned antibody is the commercial antibodies apparently modified.
Vegetable material and condition of culture
The kind of wheat (Triticum aestivum) is China spring (Chinese Spring, abbreviation CS).Seed is existed
30%H2O2Middle disinfection 10 minutes, then wash with distilled water 5 times.3 are germinateed under constant temperature in 22 DEG C of water after disinfection
It, is transferred in soil later.After the condition of 9 days long-day, take the aerial part of wheat as experimental material.
The kind of rice is Huang Huazhan (Huanghuazhan, one of abbreviation HHZ, indica cultivar) He Jizi respectively
1560 (one of jizi1560, JZ-1560, japonica cultivars).It germinates in the water of the seed of rice after sterilization, later
It is transferred in experimental field, is grown under natural conditions.The fringe of water intaking rice is as experimental material.
Embodiment 1, genetic polymorphism experimental method of the invention and calculation process
One, chromatin immune co-precipitation (ChIP)
1. the material of period respective organization needed for choosing.
2. material to be put in the Fix Buffer (each material 100ml Fix Buffer) being pre-chilled on ice.As material is inclined
Greatly, material can be shredded.
3. vacuum suction (10min), covers bottleneck with gauze, prevent material when pumping from spraying.
4. 5ml 2.5M glycine is added, continue to be evacuated 5min.
5. being cleaned 5 times with deionized water, it is eliminated as much as extra water for the last time.
6. the abundant milling material of liquid nitrogen is milled at least 4 times.
7. powder is poured into 50ml Falcon pipe, it is quantitative.Process operates in liquid nitrogen.It can -80 DEG C of preservations.
8. the CLB1 that 15ml is pre-chilled on ice is added, it is immediately placed in vortex instrument and is stirred, be sufficiently stirred 5 times or more, 4 DEG C
Turn 30min.
9. being managed with 100 μm of BD Falcon Cell Strainer filterings in another 50ml Falcon, then with 40 μm
It filters and (is operated on ice) in another 50ml Falcon pipe.
10.4000rpm (3000g or so) centrifugation, 4 DEG C, 20min.
11. supernatant is sufficiently removed, 700 μ l CLB2 suspension is added and is deposited in 1.5ml EP pipe, the patient soft (ice of process
Upper operation).300 μ l CLB2,4000rpm centrifugation of supplement addition, 4 DEG C, 20min.
12. supernatant is sufficiently removed, the patient soft precipitating that suspends (operating on ice) of 500 μ l CLB2,700 μ l are added in supplement
CLB2,4000rpm centrifugation, 4 DEG C, 20min.This step repeats once.
13. supernatant is sufficiently removed, 200 μ l CLB2 suspension precipitating is added, 20 μ l 10%SDS are added, and (SDS is final concentration of
1%), soft to mix.Into ultrasonic procedure.
14. 700 μ l CLB2 are added in supplement, mix well, 13200rpm, 10min, 4 DEG C.Taking-up is placed on ice, at this time
Precipitating bottom has a little partially black.
15. supernatant moves into another 1.5ml EP pipe, 13200rpm is centrifuged, 10min, 4 DEG C.Taking-up is placed on ice, this
When have very little precipitating.
16. supernatant is moved into another 2ml EP pipe, 1ml CLB2 is added in supplement, and (total volume 2ml, SDS is dense eventually at this time
Degree is 0.1%), to be placed on ice.
17. taking out 40 μ l chromatin (10%Input), 400 μ l Elution Buffer, 20 μ l5M are added
NaCl, is put into 65 DEG C of blending instruments after mixing, 300rpm is greater than 5h.
18.400 μ l phenol-chloroforms take out it is primary after use the anhydrous EtOH of 1ml, 40 μ l NaAc, be added 1 μ l 20ug/ μ l tRNA or
1 μ l 15mg/ml glycogen, after -20 DEG C of placement 20min, 13200rpm is centrifuged 10min, and 70% ethyl alcohol of 1ml is washed once,
13200rpm is centrifuged 5min, after discarding supernatant, firmly gets rid of, blots remaining liq with pipette tips.It places room temperature and blows 2-5min, sink
It forms sediment and 35 μ l water is added after doing.
19. take out 5 μ l, it is added 1 μ l50 μ g/ml RNase, 37 DEG C, 5min, electrophoresis (1%gel).Remaining 30 μ l conducts
1%Input.
20. electrophoresis result DNA is distributed in about 1~2 nucleosome between 100~300bp.Take the DNA in the region, sample
Between amount of DNA want equal.
21. 40 μ l Protein A Beads are added in remaining about 2ml Chromatin, 4 DEG C turn 1h.
22.500g is centrifuged 4 DEG C of 2min, and supernatant chromatin is moved to new 2ml EP and is managed.
23. running cementing fruit for chromatin packing (it is recommended that using 400 μ l-1000 μ l chromatin/ according to ultrasound
Pipe).First pipe: negative control is added without antibody, or IgG control antibodies is added；Second pipe: antibody is added (for apparent modification
The antibody in site) 5 μ l.4 DEG C, low speed rotation is overnight (> 14h).Remaining chromatin can be put in -80 DEG C of preservations.
24. 40 μ l Protein A Beads/ pipe is added, 4 DEG C, turn 1h.
25.500g be centrifuged 2min, 4 DEG C.It softly sucks supernatant, is added 820 μ l Low Salt Wash Buffer, 4 DEG C,
Low speed rotation 5min (is operated) on ice.
26.500g be centrifuged 2min, 4 DEG C.800 μ l supernatants are softly sucked, 800 μ l High Salt Wash are added
Buffer, 4 DEG C, low speed rotation 5min (is operated) on ice.
27.500g be centrifuged 2min, 4 DEG C.It softly sucks 800 μ l supernatants, is added 800 μ l LiCl Wash Buffer, 4 DEG C,
Low speed rotation 5min (is operated) on ice.
28.500g be centrifuged 2min, 4 DEG C.It softly sucks 800 μ l supernatants, is added 800 μ l 1X TE, 4 DEG C, low speed rotation 5min
(operating on ice).
29.500g be centrifuged 2min, 4 DEG C.It softly sucks 800 μ l supernatants, is added 800 μ l 1X TE, 4 DEG C, low speed rotation 5min
(operating on ice).
30.500g be centrifuged 2min, 4 DEG C.800 μ l supernatants are softly sucked, 250 μ l Elution Buffer are added.It is put into 65
DEG C blending instrument, 15min, 1400rpm are kept stirred, and prevent beads from sinking to tube bottom.
31.500g room temperature is centrifuged 2min, and 200 μ l supernatants are moved into another new 1.5ml EP and are managed.
32. adding 200 μ l Elution Buffer.65 DEG C of blending instruments are put into, 15min, 1400rpm are kept stirred, and are prevented
Only beads sinks to tube bottom.
33.500g room temperature is centrifuged 2min, the EP that 200 μ l supernatants are merged into step 31 is managed, every pipe has 400 μ l at this time
chromatin。
34. 20 μ l 5M NaCl are added, mix.It is put in 65 DEG C of blending instruments, 300rpm, 5h or more.
35.400 μ l phenol-chloroforms take out it is primary after use 1ml dehydrated alcohol, 40 μ l NaAc, be added 1 μ l 20ug/ μ l tRNA or
1 μ l 15mg/ml glycogen, after -20 DEG C of placement 20min, 13200rpm is centrifuged 10min, and 70% ethyl alcohol of 1ml is washed once,
13200rpm is centrifuged 5min, after discarding supernatant, firmly gets rid of, blots remaining liq with pipette tips.It places room temperature and blows 2-5min, sink
It forms sediment and 30 μ l water is added after doing.
36. carrying out PCR, conventional 30 circulations.Together with Input, negative control and plus antibody group.
37. as needed, carrying out real-time PCR.
Two, ChIP-seq library construction and sequencing
At least 10ng ChIP DNA is used for the building of sequencing library, and library construction process is referring to official, Illumina company
The sequencing library building process of website (http://www.illumina.com).It is double using the utilization of Illumina sequencing system
Chain specific nucleic acid enzyme (duplex-specific nuclease (DSN)) (http://nextgen.mgh.harvard.edu/
Attachments/DSN_Normalization_Sampl ePrep_Guide_15014673_B.pdf) method enrichment it is low rich
Spend the building that nucleic acid sequence carries out library.
Three, from the beginning CGT-Seq splices
Wheat
1. mass controls: using Trim Galore v0.4.4 and Sickle remove low-quality base (mass fraction <
25) especially short sequencing fragment (length < 20bp), is screened out.
2. the data of wheat are spliced using Abyss v2.0.2, are spliced into contig.
3. comparing original sequencing fragment into the sequence spliced, obtained result is further built with BESST
Scaffold, while removing the result of the potential false positive of special low cover degree (Coverage) and especially high coverage.
4.GapCloser fills Gap with the result of BESST.
5. pair or more as a result, the Python version for having used the main flow of SSPACE to reconstruct to above
Scaffolds is further extended, and the threshold value of extension is identical base ratio > 0.95, and the reads of least support is a
Number is 5.
Rice
1. mass controls: being removed low-quality base (< 25), sieved using Trim Galore v0.4.4 and Sickle
Fall especially short Reads (< 20bp).
2. the data of rice, are spliced with Velvet, splicing parameter is the coverage for being at least 4 times, and length is at least
200bp.Spliced using the Kmer of gradient, select 27 and 31 the two it is best as a result, using Amos merge two as a result,
Take longer sequence.
Four, weight sequencing technologies
Full-length genome weight sequencing technologies simplify sequencing technologies relative to existing mainstream, including RAD-seq and exon are caught
Technology is obtained, it being capable of more comprehensively covering gene group.Therefore, method of the invention is compared by the present inventor with weight sequencing technologies.
Using rice as research object, weight sequencing steps are specifically included that through Mechanical Crushing complete genome DNA segment, screening
The segment of 350-500 bases longs builds library sequencing approach with conventional Illumina and obtains sequencing fragment.
The measurement analysis of embodiment 2, wheat
In the present embodiment, by taking Chinese spring as an example, the important assessment ginseng after simplifying genomic sequencing protocol is successively analyzed
Number.
Chinese spring is obtained, is exempted from H3K4me3 and H3K27me3 antibody using method described in previous embodiment 1
Epidemic disease co-precipitation.Later, ChIP-seq library construction and sequencing are carried out.CGT-Seq is carried out again from the beginning to splice.Later, group is carried out
The step of filling, fill up, extending.
Recruitment evaluation is as follows:
1, accuracy
With Circos (http://circos.ca/) mapping software, the sequence that will be obtained using the method for above-described embodiment 1
Compared with the Chinese spring data (the RefSeq version v1.0 of the international wheat sequencing newest announcement of alliance) being sequenced, as a result such as
Shown in Fig. 2, the segment (the 4th and the 5th circle outside in) obtained using H3K4me3 and H3K27me3 antibody capture, by from the beginning
Splice the splicing sequence (the 3rd circle outside in) obtained, has with the position (the 2nd circle outside in) with the gene annotation information having
There is the high goodness of fit.
By taking gene NAM-A1 as an example, obtained by the segment for obtaining H3K4me3 and H3K27me3 antibody capture, and splicing
Splicing sequence, compare on the Chinese spring genome being sequenced, as a result such as Fig. 3, it is seen that trapping region, splicing result and base
Because position and sequence are identical.
2, susceptibility
Susceptibility is to measure the ratio that can obtain target area (promoter region before gene and gene), will be of the invention real
The sequence that the method for applying example obtains is compared with Chinese spring data, calculates the ratio for obtaining target area.As a result as schemed
4.The results show that the gene sequence of Wheat volatiles 95% can be covered using the sequencing fragment that the method for above-described embodiment 1 obtains
Arrange the promoter region sequence with 89%.
Genomic information is not depended on completely, therewith the immediate sequencing based on restriction enzyme site of purpose, be in full-length genome
Relatively random distribution, it can not specific enrichment gene and promoter region.
3, specific
Further, the present inventor carries out specific analysis and the sequencing fragment of much ratios is located at target area (gene
And the promoter region before gene).
Analyze result such as Fig. 5, it is seen that the sequencing fragment that the technology of the present inventor obtains have 50% or more in wheat cdna and
Promoter region before gene, and in whole gene group, only 5% sequence is located at target area.If the mode being sequenced with digestion,
It is relatively random because being distributed, so result is similar with full-length genome background 5%.
4. cost accounting, comparison
Full-length genome weight is compared for big genome species, such as hexaploid wheat full-length genome 17Gb, the present inventor
The cost of CGT-seq that sequencing and the present invention use, in the case where 10 times of coverage rates are reached near gene, CGT-seq at
(15800 yuan) reduction an order of magnitude (table 1) are sequenced in this (4762 yuan) specific gravity.
Table 1 resurveys sequence and CGT-seq sequencing cost (for Chinese spring)
The measurement analysis of embodiment 3, rice
Chinese spring is obtained, it is coprecipitated to be immunized with H3K4me3, H3K4me1, H3K27me3, H3K27ac, H3K36me3
Shallow lake antibody carries out co-immunoprecipitation using method described in previous embodiment 1.Later, carry out ChIP-seq library construction and
Sequencing, then carry out CGT-Seq and from the beginning splice.
Recruitment evaluation is as follows:
Firstly, by being compared (Fig. 6) with gene location with expression conditions, it can be seen that H3K4me3,
H3K4me1, H3K27ac and H3K36me3 modification are positively correlated with gene expression, are distributed mainly near cance high-expression gene, wherein
The gene range of H3K4me3 modification is the most extensive；H3K27me3 and gene expression are negatively correlated, are distributed mainly on and do not express and low table
Up near gene.Thus in whole subsequent experimentals, analyzed using H3K4me3 and H3K27me3 combination.
Secondly, determining the ability of the capture new gene of method of the invention with rice Huang Hua Zhanwei measure object.Huang Huazhan
For long-grained nonglutinous rice, and the best rice varieties of existing sequencing quality are japonica rice OryzasativaLcv.Nipponbare, CGT-seq will be used capture and spliced yellow magnificent
Account for kind core gene group sequence and existing japonica rice OryzasativaLcv.Nipponbare sequence (http://rice.plantbiology.msu.edu
/, MSU7.0) compare, it is found that the access of the new gene obtained enrichment is mainly the resistant gene (R gene) for participating in resistance mechanism,
Its representative configurations domain includes NB-ARC and LRR (Fig. 7).This is consistent with being contemplated to be, because disease-resistant gene is generally by key player on a team
It selects, evolutionary rate is fast, and it is high in object interspecies variation, also say that CGT-seq has the good function of excavating new gene.
Moreover, CGT-seq can be used in excavating new gene control region.Key GW5 wide as decision rice grain weight grain
Gene, expression has notable difference between japonica rice and long-grained nonglutinous rice, and the splicing result of the present inventor is shown, in japonica rice OryzasativaLcv.Nipponbare kind
The promoter region of GW5 gene has the missing (Fig. 8) of 1212 bases longs, and (the GW5acts in that fits like a glove has been reported
the brassinosteroid signaling pathway to regulate grain width and weight in
rice.Nature plants,3,17043).The regulation difference of this long segment utilizes and resurveys sequence, exon trapping and RNA-
Seq is difficult to obtain, because first two mode is relied on reference to genome, and RNA-seq can only obtain coding region sequence.
To sum up, illustrate the ability that method of the invention has very strong capture rice new gene and regulating and controlling sequence.
In addition to identifying new sequence, finding molecular labeling with a high credibility is that important in genetics research of sequencing technologies answers
With the present invention can find more polynucleotides polymorphism sites compared with weight sequencing technologies near gene and control region
(Fig. 9 A), these sites are predominantly located at the high position of polymorphism (Fig. 9 B).
The application prospect of embodiment 4, the present invention in other species
In addition to rice and wheat in above-described embodiment, by obtaining arabidopsis, the table of tree cotton and corn from public data
See modification ChIP-seq data.Data source is respectively as follows:
Arabidopsis: https: //www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE79259
Https: //www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE68370
Https: //www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE75071
Https: //www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE80056
Corn: https: //www.ncbi.nlm.nih.gov/sra/? term=SRP103594
Tree cotton: https: //www.ncbi.nlm.nih.gov/sra/? term=SRP114409
The apparent modification ChIP-seq data of the species are compared with gene location and expressing information, discovery is quasi-
In southern mustard, corn and tree cotton, the distribution pattern of main apparent modification H3K4me3 and H3K27me3 and pass corresponding with gene activity
It is (Figure 10 A~C) similar with rice and wheat.
(Epigenetic is guarded since main apparent modification complex is relatively common in high animals and plants
Reprogramming in plant and animal development.Science, 330,622-627), so of the invention
The applicable extensive species range of method.
It should be understood that after reading the above teachings of the present invention, those skilled in the art can make the present invention
Various changes or modification, these equivalent forms also fall within the scope of the appended claims of the present application.
Claims (17)
1. a kind of method for obtaining core gene group sequence information, which is characterized in that the method includes:
(1) nucleic acid sequence of apparent modified regions in genome is captured；
(2) library, sequencing are constructed using the nucleic acid sequence that step (1) obtains；
(3) sequencing result is obtained according to step (2), carries out sequence assembly, assembles, fills up and extend, obtain core gene group sequence
Column information.
2. the method as described in claim 1, which is characterized in that in step (1), capture base using the method for co-immunoprecipitation
Because of the nucleic acid sequence of modified regions apparent in group.
3. the method as described in claim 1, which is characterized in that in step (1), described is apparently modified to single apparent modification
Or a variety of apparent modifications.
4. the method as described in claim 1, which is characterized in that described being apparently modified in genome is stabilized or guards
It is apparently modified existing for property；Preferably, including the apparent modification of chromatin and DNA, transcription factor is combined, and enrichment is incorporated in
Gene nearby shears closely related protein complexes with transcription initiation, extension, RNA using RNA polymerase as representative.
5. the method as described in claim 1, which is characterized in that the apparent modification includes histone modification；Preferably, institute
The histone modification stated includes: the apparent modification or the apparent modification of suppressive of activated form.
6. method as claimed in claim 5, which is characterized in that the apparent modification of the activated form includes below one or more
Kind: the one/bis- of the 3rd No. 4 lysine of subunit of histone/tri-methylated；One/bis-/front three of the 3rd No. 36 lysine of subunit of histone
Base；The acylation of the 3rd No. 27 lysine of subunit of histone；The acylation of the 3rd subunit 9 lysine of histone or other in base
Because organizing there is the activated form of stable bond apparently to modify.
7. method as claimed in claim 5, which is characterized in that the apparent modification of the suppressive includes: that histone the 3rd is sub-
The one/bis- of No. 27 lysine of base/tri-methylated；The one/bis- of the 3rd subunit 9 lysine of histone/tri-methylated or other
There is genome the suppressive of stable bond apparently to modify.
8. the method as described in claim 1, which is characterized in that the apparent modification includes the apparent of at least one activated form
Modification and/or the apparent modification of at least one suppressive.
9. the method as described in claim 1, which is characterized in that in step (1), utilize spy of the specific enrichment near gene
Heterogenetic antibody captures the apparent modified regions.
10. the method as described in claim 1, which is characterized in that in step (2), using utilization double-stranded specific nuclease etc.
Method is enriched with the building that low abundance nucleic acid sequence carries out library.
11. the method as described in claim 1, which is characterized in that in step (3), before carrying out the splicing, further includes:
Remove low quality sequence and short sequence；Preferably, removal base of the sequencing quality score less than 25, removes sequence measuring joints, on
The segment for stating step acquisition removes if shorter than 20.
12. the method as described in claim 1, which is characterized in that the assembling includes, and after completing splicing, both-end is surveyed
The segment of sequence again compares sequencing fragment into the sequence spliced, and by connecting both-end sequencing fragment, obtains longer spelling
Binding fruit.
13. method as claimed in claim 12, which is characterized in that for the sequence fragment of acquisition, further removal is especially low
The result of the false positive of coverage and especially high coverage.
14. the method as described in claim 1, which is characterized in that described filling up includes: to compare primitive sequencer segment to spelling
In the long segment connect, neutral gear present in above-mentioned splice segment is filled by the information of the original series compared.
15. method as claimed in claim 14, which is characterized in that the extension includes: to compare primitive sequencer segment again
To splice segment obtained after filling up step, the primitive sequencer segment of splice segment end is arrived to splicing piece using comparison
Duan Jinhang extends.
16. the method as described in claim 1, which is characterized in that after step (3), further includes:
(4) the core gene group sequence information of acquisition is analyzed, comprising: determine the genetic polymorphism position of gene and control region
Species population genetics research is not sequenced for point, compares the heredity variation of gene and control region during spore or domestication.
17. the application of any method of claim 1~16, is used for: determining the genetic polymorphism position of gene and control region
Species population genetics research is not sequenced for point, compares the heredity variation of gene and control region during spore or domestication.
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