Full-length genome methylates sequencing library and its construction methodTechnical field
The present invention relates to high-throughput sequencing library builds field, methylate sequencing in particular to a kind of full-length genomeLibrary and its construction method.
Background technology
DNA methylation is a kind of main epigenetic modification form of genomic DNA.In recent years, numerous studies show DNAMethylate modification for maintaining normal cell function, transfer gene group genetic imprinting, fetal development and human tumor to occur, risesVital effect.Methylate the focus studied into epigenetics, and corresponding technology is also to emerge in an endless stream, according to realityTest the difference of purpose, these technology substantially can be divided into two classes：Full-length genome methylates investigative technique and specific methylationSite investigative technique.
WGBS (Whole genome bisulfite sequencing), i.e. full-length genome bisulfite sequencing.ExperimentPrinciple is：Early stage sulfiting again, will not occur methylated C base to change into U in genome, after entering performing PCR amplificationBecome T, the C base having the modification that methylates with script makes a distinction；Based on second filial generation high-flux sequence platform, in conjunction with completeGenome sulfiting and biological data analytical technology again, carries out the full-length genome of low cost, high efficiency, high accuracyThe horizontal collection of illustrative plates of DNA methylation is drawn.
With developing rapidly of secondary sequencing, the cost that is sequenced significantly declines, and WGBS technology can be drawn single base and be differentiatedThe complete genome DNA methylation profiles of rate, have absolute technical advantage for research.From chip to second filial generation high fluxSequencing, methylate from specific methylation site to full-length genome research, and corresponding technology is also to emerge in an endless stream.Sequencing cost is bigAmplitude declines the research making to methylate group (methylome) and is possibly realized.At present, the genome based on second filial generation microarray datasetMethylate research, mainly includes tri- kinds of technology of WGBS, RRBS and MeDIP, as shown in table 1 below, for the different regions that methylates,Different technology can be selected to realize：
Mention the sequencing that methylates, necessarily " goldstandard " the full-length genome bisulfite sequencing (whole expecting firstGenome bisulfite sequencing), abbreviation BS-seq.The experimental principle of the method is at early stage BisulfiteReason, will not occur methylated C base transition to become U in genome, become T after entering performing PCR amplification, has to methylate with script and repaiiesThe C base of decorations makes a distinction, and in conjunction with high throughput sequencing technologies, is particularly well-suited to draw the full-length genome of single base resolutionDNA methylation collection of illustrative plates.WGBS technology can draw the complete genome DNA methylation profiles of single base resolution, comes for researchSay that there is absolute technical advantage.
Up to the present, the sequencing of full-length genome bisulfite is that DNA methylation studies reasonable method, but the partyThere is design defect in itself in method, lead to reduce the accuracy of methylation assessment during bioinformatic analysis, hinder itApplication, thus it is still necessary to sequence measurement that existing full-length genome is methylated improves, commented with improving follow-up methylationThe accuracy estimated.
Table 1：
Content of the invention
Sequencing library and its construction method present invention is primarily targeted at a kind of full-length genome of offer methylates, to reduceThe accuracy of constructed library impact follow-up full-length genome methylation assessment in prior art.
To achieve these goals, the sequencing literary composition according to an aspect of the invention, it is provided a kind of full-length genome methylatesThe construction method in storehouse, this construction method comprises the following steps：S1, carries out fragmentation to complete genome DNA, obtains DNA fragmentation；S2, carries out end reparation using the dNTP being mixed to form by dATP, dTTP and dGTP and carries out end reparation to DNA fragmentation, obtainRepair fragment；S3, carries out 3 ' ends to reparation fragment and adds " A ", obtain 3 ' end band " A " fragments；S4, arrives " T " using through " C "The joint sequence of conversion processing carries out joint connection to 3 ' end band " A " fragments, obtains the fragment of belt lacing；S5, to belt lacingFragment carries out " C " and arrives " T " conversion processing, obtains processing fragment；S6, expands to processing fragment, obtains full-length genome methylChange sequencing library.
Further, after step S1, and before step S2, construction method also includes carrying out purification to DNA fragmentationStep.
Further, the step of purification carries out purification using DNA affinity column.
Further, the step of purification carries out purification using QIAquick PCR purification kit to DNA fragmentation.
Further, in step s 2, the dNTP being mixed to form using dATP, dTTP and the dGTP in End-It test kitEnd reparation is carried out to DNA, obtains repairing fragment.
Further, upon step s 2, and before step S3, construction method also includes carrying out purification to reparation fragmentStep.
Further, after step s 3, and before step S4, also include using Ampure XP magnetic bead to 3 ' endsThe step that band " A " fragment carries out purification.
Further, after step s4, and before step S5, construction method also includes belt lacing fragment being carried out determineThe step of amount.
Further, in step s 5, using EZ DNA Methylation-LightningTMTest kit is to belt lacing pieceDuan Jinhang " C " arrives " T " conversion processing, obtains processing fragment.
According to a further aspect in the invention, there is provided a kind of full-length genome methylates sequencing library, using any of the above-described kindConstruction method is built-up.
Application technical scheme, by end repair step in by the dNTP raw material of end repair enzyme byThe mixture of dATP, dCTP, dTTP and dGTP is improved to the mixture of dATP, dTTP and dGTP, and then during repairing, keeps awayExempt from non-methylated C and introduce DNA fragmentation end, thus improve the accuracy of follow-up methylation analysis and assessment.
Brief description
The Figure of description constituting the part of the application is used for providing a further understanding of the present invention, and the present invention showsMeaning property embodiment and its illustrate for explaining the present invention, does not constitute inappropriate limitation of the present invention.In the accompanying drawings：
Fig. 1 shows the schematic flow sheet of the library construction that methylates according to the full-length genome of the present invention；
Fig. 2 shows the electrophoresis result figure after the genomic fragmentization process in present invention experiment one；
Fig. 3 shows the electrophoresis result figure after the PCR amplification in present invention experiment three；
Fig. 4 shows in present invention experiment four that constructed full-length genome methylates GC degree of distribution and G/C content in libraryFigure；And
Fig. 5 shows the schematic diagram of the comparison that methylates in present invention experiment four.
Specific embodiment
It should be noted that in the case of not conflicting, the embodiment in the application and the feature in embodiment can phasesMutually combine.To describe the present invention below with reference to the accompanying drawings and in conjunction with the embodiments in detail.
In prior art, during the library construction that carries out methylating, repair in step using conventional bag in endDNTP containing dATP, dCTP, dTTP and dGTP repairs to the DNA fragmentation of fragmentation as the raw material of end repair enzyme, suddenlyOmit and non-methylated C can be artificially introduced on the DNA fragmentation of fragmentation using this dNTP, and then to follow-up methylationThe accuracy of assessment has a negative impact.
For the problems referred to above of the prior art, in a kind of typical embodiment of the present invention, there is provided a kind of full baseBecause group methylates the construction method of sequencing library, as shown in figure 1, this construction method comprises the following steps：S1, to full-length genomeDNA carries out fragmentation, obtains DNA fragmentation；S2, is carried out to DNA fragmentation using the dNTP being mixed to form by dATP, dTTP and dGTPEnd is repaired, and obtains repairing fragment；S3, carries out 3 ' ends to reparation fragment and adds " A ", obtain 3 ' end band " A " fragments；S4, adoptsWith the joint sequence through " C " to " T " conversion processing, joint connection is carried out to 3 ' end band " A " fragments, obtain belt lacing fragment；S5, carries out " C " and arrives " T " conversion processing to belt lacing fragment, obtains processing fragment；S6, expands to processing fragment, obtains completeGenomic methylation sequencing library.
The above-mentioned construction method of the present invention, by end repair step in by end repair enzyme comprise dATP, dCTP,The dNTP raw material of dTTP and dGTP is improved to the dNTP mixing with dATP, dTTP and dGTP, and then during repairing, keeps awayExempt from non-methylated C and introduce DNA fragmentation end, thus ensure that the accuracy of follow-up methylation analysis and assessment.
In the above-mentioned construction method of the present invention, the step that complete genome DNA is carried out with fragmentation can be beaten using physicsThe mode that disconnected or chemistry interrupts carries out fragmentation.In a kind of preferred embodiment of the present invention, it is preferred to use the mode that physics interruptsCarry out fragmentation, by the way of physics crushes, carry out fragmentation so that clip size is concentrated mainly between 150～300bp,Simple to operation, and repeatability is good with controllability.
In the construction method of the present invention, after step S1, and before step S2, preferably above-mentioned construction method also wrapsInclude the step that purification is carried out to DNA fragmentation.Carry out this purification step the DNA fragmentation being unsatisfactory for clip size can be goneRemove, thus obtaining the DNA fragmentation that fragment is more concentrated and purity is higher.
In above-mentioned purification step, the method for concrete purification adopts the conventional purification process in this area.Preferably above-mentioned pureChange and purification is carried out using DNA affinity column, carrying out purification using DNA affinity column makes the purity of DNA piece degree higher.A kind of in the present inventionIn preferred embodiment, the step of above-mentioned purification carries out purification using QIAquick PCR purification kit to DNA fragmentation.QIAquick PCR purification kit is more preferable to the DNA fragmentation purification effect of fragmentation.
In the construction method of the present invention, in step s 2, when DNA fragmentation is carried out with end reparation, as long as adoptDo not contain dCTP in dNTP, so can avoid being artificially introduced non-methylated C, and then affect non-methyl in sequencing dataThe content of the C changing, thus lead to the accuracy of methylation analysis to decline.In a kind of preferred embodiment of the present invention, adoptThe dNTP being mixed to form with dATP, dTTP and dGTP in End-It test kit carries out end reparation to DNA, obtains repairing pieceSection.When using dNTP in this test kit, it is added without dCTP therein, make with the end repair enzyme cooperation in this test kitWith repair rate is higher, and fragment, especially few to difficulty of drawing materials, quality sample are repaired in the end that can obtain high-load, it is possible to increaseBuild the success rate in storehouse.
In the construction method of the present invention, the step that 3 ' ends add " A " after being repaired, can be entered.In order that plusMore preferably, after above-mentioned steps S2, and before step S3, preferably this construction method is also included to reparation the connection effect of " A "The step that fragment carries out purification.Purification is carried out by the reparation fragment after end is repaired, can directly get rid of and not be repairedFragment, be enriched with the fragment that repairs, to improve follow-up plus " A " efficiency.In a kind of preferred embodiment of the present invention, onThe step stating purification carries out purification using Ampure XP magnetic bead to repairing fragment.Adopt magnetic beads for purifying in this step, by reasonableRatio between the consumption of adjustment AmpureXP magnetic bead and the amount of reparation fragment to be purified, can utilize Ampure XP magnetic beadScreening obtains the fragment of purpose fragment size, removes excessive or too small reparation fragment further, improves purpose size fragmentEnrichment journey.
In the construction method of the present invention, after end is repaired, the step that 3 ' ends add " A " is carried out using conventional methodSuddenly, obtain the fragment of 3 ' end bands " A ", then carry out joint Connection Step, obtain belt lacing fragment.Now, build to saveThe storehouse time, " C " can be directly entered and arrive " T " conversion processing step." C " to " T " conversion processing is the warp of detection gene methylationAllusion quotation processing mode, its principle is：With bisulfites or weight bisulf iotate-treated genomic DNA, all methylateCytosine (C) be converted into uracil (U), and methylated cytosine is then constant.Thus, sub- through bisulfites or weightAfter disulfate is processed, methylated site produces the polynucleotides polymorphism (SNP) similar to a C/T.Genomic DNA warpAfter sulfiting, expand purpose fragment, now uracil (U) is completely converted into thymus pyrimidine (T), finally to PCR primerIt is sequenced it may determine that whether methylating.
In joint Connection Step, the joint of connection is to pass through the joint that " C " arrives " T " conversion processing.ConventionalFull-length genome methylate sequencing library build when, take into account the non-methylated C in the joint being connected in joint Connection StepNeed to process through bisulfites or bisulfite, and have ignored the non-of introducing in end reparation step and methylateC also can affect the calculating of finally methylated C content.Thus, in the DNA fragmentation after fragmentation, there is single stranded DNA, have flatThe double-stranded DNA of end, also both-end all carry the non-flat terminal double link DNA of prominent base, have bases G in these prominent bases, non-methylated C will be artificially introduced during reparation.
In another kind of preferred embodiment of invention, before carrying out above-mentioned joint Connection Step, also include adoptingThe step that Ampure XP magnetic bead carries out purification to above-mentioned 3 ' end band " A " fragments, by Ampure XP magnetic beads for purifying step energyEnough some fragments that 3 ' ends are not connected " A " remove.
In another preferred embodiment of the present invention, after above-mentioned steps S4, and before step S5, this structure sideMethod also includes carrying out quantitative step to belt lacing fragment.Carrying out before " C " arrive " T " conversion processing, pending band being connectHead fragment carries out quantitation, can accurately calculate required bisulfites or the amount of weight bisulfites, can make belt lacing pieceC in section changes into U, will not be excessively used bisulfites or bisulfite again, thus reducing waste, cost-effective.
Above-mentioned " C " arrives in the step of " T " conversion processing, can be using the bisulfites of oneself configuration or bisulfiteSolution is processed, it would however also be possible to employ the conventional treatment kits that methylate are processed, as long as handled band can be made to connectThe non-methylated C and methylated C in purpose fragment in head fragment makes a distinction.In the present invention, another is preferredIn embodiment, adopt EZ DNA Methylation-Lightning in step s 5TMTest kit carries out " C " to belt lacing fragmentArrive " T " conversion processing, obtain processing fragment.EZ DNA Methylation-LightningTMTest kit treatment effeciency is high.
In another kind of typical embodiment of the present invention, there is provided a kind of full-length genome methylates sequencing library, and this is completeGenomic methylation sequencing library adopts any of the above-described kind of construction method built-up.The full-length genome of the present invention methylates sequencingLibrary is because repairing in step using the dNTP not containing dCTP in end, and then avoids and introduce non-methylating during Jian KuC so that the methylated C content that constructed full-length genome methylates in sequencing library, closer to its real content, carriesThe high accuracy of the analysis of full-length genome methylation or assessment.
Further illustrate the effect of the present invention below in conjunction with specific embodiment.It should be appreciated that these embodiments are onlyFor the present invention being described rather than limiting the scope of the present invention.
Experiment one
1. with arabidopsiss seed DNA as sample, each sample takes 5.2 μ g genomic DNAs, respectively plus 26ng (0.5%)Negative control (λ DNA), then uses water polishing to 130 μ l, is added in Covaris pipe.
2. carry out physics to crush with Covaris S220 equipment, the experience summarized according to many experiments, by CovarisThe parameter of S220 is set to period 8%～12%, 170～180 watts of peak value incident power；Period/outburst 200～220；PlaceReason time 250～330s；When in the range of 4～8 DEG C of temperature, all can crush the clip size obtaining mainly 150～300bp itBetween, it is configured according to the data in table 2 below in this experiment.
Table 2：
3. take 20-30ng (1 μ l) sample to carry out 2% agarose gel electrophoresiies (120V, 25min), detect to interrupt whether collectIn, concrete testing result is shown in Fig. 2.
In fig. 2, Far Left is the big tick marks of DNA molecular, and 5 stripe size from bottom to up are respectively：100bp、200bp, 300bp, 400bp and 500bp, from figure 2 it can be seen that the size of the DNA fragmentation after interrupting through Covaris S220All concentrate between 150～300bp.
4. use the description purifying DNA fragment of QIAquick PCR purification kit.
5. carry out end reparation with End-It test kit (Epicenter), on ice according to following components ratio in PCR pipeForm reaction system, and mixed with pipettor, room temperature is placed 45 minutes.
The consisting of of above-mentioned reaction system：
6. use Ampure XP magnetic beads for purifying end to repair product.
Comprise the following steps that：
(1) add 60ul Ampure XP magnetic bead in reaction system, repeat to mix with pipettor, room temperature stands 5 minutes.
(2) PCR pipe is placed on magnetic stand, stands 5 minutes, become clarification to solution.Will be upper with pipettorClear liquid suctions out and abandons.
(3) (keep PCR pipe on magnetic frame) and add 200ul 80% ethanol, stand 30 seconds, ethanol is suctioned out and abandons.
(4) repeat step 3) once.Inhaled again once with 10ul pipette tips it is ensured that not having ethanol solution to remain.
(5) (continue to keep PCR pipe on magnetic frame) room temperature to stand and be dried to magnetic bead.
(6) add 39ul water on the magnetic bead being dried, PCR pipe taken off from magnetic frame, with pipettor by the water in pipe andMagnetic bead mixes, static 2 minutes of room temperature, is placed on absorption magnetic bead 5 minutes on magnetic frame, 37ul supernatant is transferred in new PCR pipe.
7.3 ' ends add A.
Add reagent on ice by following system in PCR pipe.If (sample more it should other reagent are blended togetherMix, then be dispensed among each reaction system)
Mixed with pipettor, PCR pipe is placed in PCR instrument 37 DEG C and reacts 30 minutes.
8. add A product with Ampure XP magnetic beads for purifying 3 ' end.
(1) add 50ul Ampure XP magnetic bead in reaction system, repeat to mix with pipettor, room temperature stands 5 minutes.
(2) PCR pipe is placed on magnetic stand, standing adsorption magnetic bead 5 minutes, becomes clarification to solution.With movingSupernatant is suctioned out and abandons by liquid device.
(3) (keep PCR pipe on magnetic frame) and add 200ul 80% ethanol, stand 30 seconds, ethanol is suctioned out and abandons.
(4) repeat step 3) once.Inhaled again once with 10ul pipette tips it is ensured that not having ethanol solution to remain.
(5) (continue to keep PCR pipe on magnetic frame) room temperature to stand and be dried to magnetic bead.
(6) add 33ul water on the magnetic bead being dried, PCR pipe taken off from magnetic frame, with pipettor by the water in pipe andMagnetic bead mixes, static 2 minutes of room temperature, is placed on absorption magnetic bead 5 minutes on magnetic frame, 31ul supernatant is transferred in new PCR pipe.
9. joint connects.According to following reaction system, each constituent is added in PCR pipe.
Mixed with pipettor, be placed in PCR instrument, 16 DEG C connect overnight.
Wherein, the sequence of joint 24 (NEXTflexTM Bisulfite-Seq Barcodes 12, Bioo) of methylating is：SEQ ID NO.1：
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT；SEQ IDNO.2：
GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGTAGCATCTCGTATGCCGTCTTCTGCTTG.
10. use Ampure XP magnetic beads for purifying adjunction head product quantitation
(1) add 40ul Ampure XP magnetic bead in reaction system, repeat to mix with pipettor, room temperature stands 5 minutes.
(2) PCR pipe is placed on magnetic stand, standing adsorption magnetic bead 5 minutes, becomes clarification to solution.With movingSupernatant is suctioned out and abandons by liquid device.
(3) (keep PCR pipe on magnetic frame) and add 200ul 80% ethanol, stand 30 seconds, ethanol is suctioned out and abandons.
(4) repeat step 3) once.Inhaled again once with 10ul pipette tips it is ensured that not having ethanol solution to remain.
(5) (continue to keep PCR pipe on magnetic frame) room temperature to stand and be dried to magnetic bead.
(6) add 51ul water on the magnetic bead being dried, PCR pipe taken off from magnetic frame, with pipettor by the water in pipe andMagnetic bead mixes, static 2 minutes of room temperature, is placed on absorption magnetic bead 5 minutes on magnetic frame, 50ul supernatant is transferred in new PCR pipe.
(7) add 50ul Ampure XP magnetic bead in PCR pipe, fully mixed with pipettor, room temperature stands 5 minutes.
(8) repeat step 2)～5)
(9) add 22.5ul water on the magnetic bead being dried, PCR pipe is taken off from magnetic frame, with pipettor by the water in pipeMix with magnetic bead, static 2 minutes of room temperature, be placed on absorption magnetic bead 5 minutes on magnetic frame, 20ul supernatant is transferred to new PCR pipeIn.
(10) 2.5ul in pipe on magnetic frame is suctioned out 1ul Qubit HsDNA quantitative.
Experiment two
CT conversion processing is (with EZ DNA Methylation-LightningTMKit, Zymo Research)
1. preparation：
(1) subpackage Lightning Conversion Reagent
Often pipe Lightning Conversion Reagent is the amount of 10 reactions, if the sample of conversion is few, permissibleShift to an earlier date subpackage and keep in Dark Place.
(2) prepare M-Wash Buffer
Dehydrated alcohol：M-Wash Buffer=4：1
96ml dehydrated alcohol is added in 24ml M-Wash Buffer.
2. experimental procedure：
(1) 400ngDNA sample is taken to carry out CT conversion according to quantitative result, less than 400ng with all 20ul adjunction headsProduct, more than the product taking 400ng of 400ng, the benefit that adds water puts 20ul.Plus 130 μ l Lightning ConversionIn Reagent to 20 μ l DNA sample, fully mix sample with pipettor.The volume of reaction not can exceed that PCR instrument effectivelyBig reaction volume, otherwise to be dispensed into reaction in multiple pipes (for example, maximum reaction volume be 50ul PCR instrument in, Ying Jiang150ul reaction system is averagely divided in 3 PCR pipe).
(2) by sample cell be put into temperature cycling device and as shown in table 3 below step operation：
Table 3：
Reaction terminates back balance and carries out operations described below to room temperature or store (the longest 20 hours) at 4 DEG C
(3) by Zymo-SpinTMIC Column is placed in the Collection Tube of test kit offer, and addsThe M-Binding Buffer of 600 μ l is in pillar.
(4) reactant liquor in step (2) is added the Zymo-Spin containing M-Binding BufferTMIC ColumnIn, close the lid and pillar is overturned biased sample for several times.(Optional：If initial amount of DNA is less, should be first by reactant liquorMiddle addition 8ug Carrier RNA, then by reactant liquor be added in Column with M-Binding Buffer mix)
Note：Step (3) and step (4) can not overturn.
(5) centrifugation 30 seconds (>10,000g), outwell liquid.
(6) add 100 μ l M-Wash Buffer in post, be centrifuged 30 seconds.
(7) add 200 μ l M-Desulphonation Buffer to place in post and under room temperature (20 DEG C -30 DEG C)15-20 minute.After incubation terminates, it is centrifuged 30 seconds.
(8) add the M-Wash Buffer of 200 μ l in post, be centrifuged 30 seconds.It is repeated once.
(9) sky gets rid of 1min.
(10) pillar is put in new 1.5ml Eppendorf pipe, open lid and stand 2 minutes
(11) add the M-Elution Buffer of 13 μ l in pillar, stand 2min, be centrifuged 1 minute eluted dna.
(12) repeat step 11, are obtained the recovery product of about 23ul, if less than 23ul, the benefit that adds water to 23ul.
Experiment three
PCR expands
1. add following reagent on ice and reagent (if sample is more, is first blended together mix, then is dispensed into each by fully mixingAmong individual reaction system).
The DNA 23ul of weight bisulf iotate-treated
KAPA HiFi HotStart Uracil+ReadyMix(2X) 25ul
NEXTflexTMPrimer mixture 2ul
PCR response procedures such as table 4 below：
Table 4：
Fragment after PCR is expanded goes 1ul to carry out the size of electrophoresis detection final library, and testing result is shown in Fig. 3.
In figure 3, Far Left is the big tick marks of DNA molecular, and 5 stripe size from bottom to up are respectively：100bp、200bp, 300bp, 400bp and 500bp.From figure 3, it can be seen that the size in the library obtaining after the amplification of the present invention is all concentratedBetween 250～450bp.
2. use Ampure XP magnetic beads for purifying PCR primer.
(1) add 50ul Ampure XP magnetic bead in reaction system, repeat to mix with pipettor, room temperature stands 5 minutes.
(2) PCR pipe is placed on magnetic stand, standing adsorption magnetic bead 5 minutes, becomes clarification to solution.With movingSupernatant is suctioned out and abandons by liquid device.
(3) (keep PCR pipe on magnetic frame) and add 200ul 80% ethanol, stand 30 seconds, ethanol is suctioned out and abandons.
(4) repeat step 3) once.Inhaled again once with 10ul pipette tips it is ensured that not having ethanol solution to remain.
(5) (continue to keep PCR pipe on magnetic frame) room temperature to stand and be dried to magnetic bead.
(6) add 22ul water on the magnetic bead being dried, PCR pipe taken off from magnetic frame, with pipettor by the water in pipe andMagnetic bead mixes, static 2 minutes of room temperature, is placed on absorption magnetic bead 5 minutes on magnetic frame, 20ul supernatant is transferred in new PCR pipe.
(7) liquid remaining in the PCR pipe on magnetic frame is taken 1ul Qubit hsDNA quantitative.
3. outbound
To bank number, original text storehouse is managed with 1.5ml, the literal arts of a 2ng/ μ l of dilution, with 500ul pipe dress.
Experiment four
The obtained full-length genome sequencing library that methylates is carried out upper machine sequencing, sequencing data must be beaten, and by sequencingData carries out GC separating degree to built library and G/C content is estimated, as shown in figure 4, constructed in the above embodiment of the present inventionThe full-length genome sequencing library display GC that methylates separate, and G/C content is low.Because the library after bisulfite is processedIn, C, G content can be low, and because the directivity that the sequence (reads) obtaining in the strategy of storehouse remains chain is built in test, so GC dividesOn Butut, the C content of the first terminal sequence (read1) is very low, and T content is very high, the second terminal sequence (read2) be G content veryLow, A content is very high.
Bioinformatic analysis：Using Bimark (Krueger, 2011, bottom calls Bowtie2), carry out methylating dataReference gene group comparison analysis.Analysis principle is as shown in figure 5, the result of sequencing and reference gene group are all entered by BismarkGo the conversion of C to T and G to A (reverse complemental), the sequencing result after conversion and genome have been compared respectively two-by-two, obtainsThe comparison result arriving such as table 5 below.
Table 5：
The various types of methylation level of each chromosome from upper table 5 can be seen that and can be detected by compared to existing technologyTo each chromosome on CG, GHG and CHH three types methylation, using the construction method of the present invention, due to avoidingThe content of the non-methylated C that anthropic factor increases, constructed full-length genome methylates each dyeing that sequencing library obtainsThe methylation of CG, the GHG and CHH three types on body is higher, and closer to its true horizon, thus detection accuracy is moreHigh.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for the skill of this areaFor art personnel, the present invention can have various modifications and variations.All within the spirit and principles in the present invention, made any repairChange, equivalent, improvement etc., should be included within the scope of the present invention.