CN104532360A - Whole-genome methylation sequencing library and construction method thereof - Google Patents
Whole-genome methylation sequencing library and construction method thereof Download PDFInfo
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Abstract
The invention provides a whole-genome methylation sequencing library and a construction method thereof. The construction method disclosed by the invention comprises the following steps of: S1, fragmenting whole-genome DNA so as to obtain a DNA fragment; S2, repairing the tail end of the DNA fragment by adopting dNTP formed by mixing dATP, dTTP with dGTP so as to obtain a repaired fragment; S3, adding A at the 3' tail end of the repaired fragment so as to obtain a fragment with A at the 3' tail end; S4, connecting the joint of the fragment with A at the 3' tail end by adopting a joint sequence transferred from C to T so as to obtain a fragment with a joint; S5, transferring the fragment with the joint from C to T to obtain a processed fragment; and S6, amplifying the processed fragment to obtain the whole-genome methylation sequencing library. Because the tail end is repaired by adopting the dNTP without dCTP, introduce of non-methylated C is avoided; and the analysis accuracy of methylation degree is increased.
Description
Technical field
The present invention relates to high-throughput sequencing library and build field, to methylate sequencing library and construction process thereof in particular to a kind of full-length genome.
Background technology
DNA methylation is the main epigenetic modification form of one of genomic dna.In recent years, large quantity research shows that DNA methylation is modified and occurs for maintenance normal cell function, transfer gene group genetic imprinting, fetal development and human tumor, plays vital effect.Methylate the focus studied into epigenetics, and corresponding technology also emerges in an endless stream, experimentally the difference of object, and these technology can be divided into two classes substantially: full-length genome methylates investigative technique and specific methylation site investigative technique.
WGBS (Whole genome bisulfite sequencing), i.e. full-length genome bisulfite order-checking.Experimental principle is: early stage, with heavily treating with sulfurous acid, changing into U by there is not methylated C base in genome, becoming T after carrying out pcr amplification, has the C base of modifying that methylates make a distinction with script; Based on s-generation high-flux sequence platform, in conjunction with full-length genome heavily treating with sulfurous acid and biological data analytical technology, carry out the complete genome DNA methylation level collection of illustrative plates drafting of low cost, high-level efficiency, split hair caccuracy.
Along with the develop rapidly of two generations order-checking, order-checking cost significantly declines, and WGBS technology can draw the complete genome DNA methylation profiles of single base discrimination rate, has absolute technical superiority for research.From chip to s-generation high-flux sequence, methylate from specific methylation site to full-length genome research, and corresponding technology also emerges in an endless stream.Significantly decline make the to methylate research of group (methylome) of order-checking cost becomes possibility.At present, based on the genomic methylation research of s-generation order-checking platform, mainly comprise WGBS, RRBS and MeDIP tri-kinds of technology, as shown in table 1 below, for the different regions that methylates, different technology can be selected realize:
Mention the order-checking that methylates, necessarily " gold standard " full-length genome bisulfite order-checking (whole genomebisulfite sequencing) first expected, is called for short BS-seq.The experimental principle of the method is Bisulfite process in early stage, U is become by there is not methylated C base transition in genome, T is become after carrying out pcr amplification, with script, there is the C base of modifying that methylates to make a distinction, again in conjunction with high throughput sequencing technologies, be specially adapted to the complete genome DNA methylation profiles drawing single base discrimination rate.WGBS technology can draw the complete genome DNA methylation profiles of single base discrimination rate, has absolute technical superiority for research.
Up to the present, the order-checking of full-length genome bisulfite is that DNA methylation studies reasonable method, but itself there is design defect in the method, cause the accuracy that can reduce methylation assessment during bioinformatic analysis, hinder its application, thus, still need to improve the existing full-length genome sequence measurement that methylates, to improve the accuracy of follow-up methylation assessment.
Table 1:
Summary of the invention
Main purpose of the present invention is to provide a kind of full-length genome to methylate sequencing library and construction process thereof, to reduce the accuracy that library constructed in prior art affects the assessment of follow-up full-length genome methylation.
To achieve these goals, according to an aspect of the present invention, provide a kind of full-length genome and to methylate the construction process of sequencing library, this construction process comprises the following steps: S1, carries out fragmentation, obtain DNA fragmentation to complete genome DNA; S2, adopts the dNTP be mixed to form by dATP, dTTP and dGTP to carry out end reparation and carries out end reparation to DNA fragmentation, obtains repairing fragment; S3, carries out 3 ' end to reparation fragment and adds " A ", obtain 3 ' end strips " A " fragment; S4, adopts the joint sequence through " C " to " T " conversion processing to carry out joint connection to 3 ' end strips " A " fragment, obtains the fragment of belt lacing; S5, carries out " C " to belt lacing fragment and arrives " T " conversion processing, obtains processing fragment; S6, increases to process fragment, obtains full-length genome and to methylate sequencing library.
Further, after step S1, and before step S2, construction process also comprises the step of DNA fragmentation being carried out to purifying.
Further, the step of purifying adopts DNA affinity column to carry out purifying.
Further, the step of purifying adopts QIAquick PCR purification kit to carry out purifying to DNA fragmentation.
Further, in step s 2, the dNTP adopting dATP, dTTP and dGTP in End-It test kit to be mixed to form carries out end reparation to DNA, obtains repairing fragment.
Further, upon step s 2, and before step S3, construction process also comprises the step of reparation fragment being carried out to purifying.
Further, after step s 3, and before step S4, the step adopting Ampure XP magnetic bead 3 ' end strips " A " fragment to be carried out to purifying is also comprised.
Further, after step s4, and before step S5, construction process also comprises and carries out quantitative step to belt lacing fragment.
Further, in step s 5, EZ DNA Methylation-Lightning is adopted
tMtest kit carries out " C " to belt lacing fragment and arrives " T " conversion processing, obtains processing fragment.
According to a further aspect in the invention, provide a kind of full-length genome and to methylate sequencing library, adopt any one construction process above-mentioned to build and form.
Apply technical scheme of the present invention, by repairing the mixture in step, the dNTP raw material of end repair enzyme being improved to dATP, dTTP and dGTP by the mixture of dATP, dCTP, dTTP and dGTP at end, and then in repair process, avoid non-methylated C and introduce DNA fragmentation end, thus improve the accuracy of follow-up methylation analysis and assessment.
Accompanying drawing explanation
The Figure of description forming a application's part is used to provide a further understanding of the present invention, and schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 shows full-length genome according to the present invention and to methylate the schematic flow sheet of library construction;
Fig. 2 shows the electrophoresis result figure after the genomic fragment process that the present invention tests in;
Fig. 3 shows the electrophoresis result figure after the pcr amplification that the present invention tests in three;
Fig. 4 shows the present invention and to test in four constructed full-length genome and to methylate in library GC degree of distribution and GC containing spirogram; And
Fig. 5 shows the schematic diagram that the present invention tests in four comparison that methylates.
Embodiment
It should be noted that, when not conflicting, the embodiment in the application and the feature in embodiment can combine mutually.Below with reference to the accompanying drawings and describe the present invention in detail in conjunction with the embodiments.
In prior art, carrying out methylating in the process of library construction, repairing in step at end adopts the conventional dNTP comprising dATP, dCTP, dTTP and dGTP to repair as the DNA fragmentation of raw material to fragmentation of end repair enzyme, have ignored and adopt this dNTP on the DNA fragmentation of fragmentation, artificially can introduce non-methylated C, and then the accuracy that follow-up methylation is assessed is had a negative impact.
For the problems referred to above of the prior art, in a kind of typical embodiment of the present invention, provide a kind of full-length genome and to methylate the construction process of sequencing library, as shown in Figure 1, this construction process comprises the following steps: S1, carries out fragmentation, obtain DNA fragmentation to complete genome DNA; S2, adopts the dNTP be mixed to form by dATP, dTTP and dGTP to carry out end reparation to DNA fragmentation, obtains repairing fragment; S3, carries out 3 ' end to reparation fragment and adds " A ", obtain 3 ' end strips " A " fragment; S4, adopts the joint sequence through " C " to " T " conversion processing to carry out joint connection to 3 ' end strips " A " fragment, obtains belt lacing fragment; S5, carries out " C " to belt lacing fragment and arrives " T " conversion processing, obtains processing fragment; S6, increases to process fragment, obtains full-length genome and to methylate sequencing library.
Above-mentioned construction process of the present invention, by repairing in step at end, the dNTP raw material comprising dATP, dCTP, dTTP and dGTP of end repair enzyme is improved to the dNTP mixed with dATP, dTTP and dGTP, and then in repair process, avoid non-methylated C and introduce DNA fragmentation end, thus ensure that the accuracy of follow-up methylation analysis and assessment.
In above-mentioned construction process of the present invention, the step of complete genome DNA being carried out to fragmentation can adopt the mode that physics interrupts or chemistry interrupts to carry out fragmentation.In a kind of preferred embodiment of the present invention, the mode preferably adopting physics to interrupt carries out fragmentation, adopts the mode of physics fragmentation to carry out fragmentation, clip size is mainly concentrated between 150 ~ 300bp, simple to operation, and repeatability and controllability good.
In construction process of the present invention, after step S1, and before step S2, preferred above-mentioned construction process also comprises the step of DNA fragmentation being carried out to purifying.Carry out this purification step the DNA fragmentation not meeting clip size to be removed, thus obtain fragment and more concentrate and the higher DNA fragmentation of purity.
In above-mentioned purification step, the method for concrete purifying adopts the purification process of this area routine.Preferred above-mentioned purifying adopts DNA affinity column to carry out purifying, adopts DNA affinity column to carry out purifying and makes the purity of DNA sheet degree higher.In a kind of preferred embodiment of the present invention, the step of above-mentioned purifying adopts QIAquick PCR purification kit to carry out purifying to DNA fragmentation.The DNA fragmentation purification effect of QIAquick PCR purification kit to fragmentation is better.
In construction process of the present invention, in step s 2, when end reparation is carried out to DNA fragmentation, as long as not containing dCTP in the dNTP adopted, can avoid so artificially introducing non-methylated C, and then affect the content of non-methylated C in sequencing data, thus the accuracy causing methylation to be analyzed declines.In a kind of preferred embodiment of the present invention, the dNTP adopting dATP, dTTP and dGTP in End-It test kit to be mixed to form carries out end reparation to DNA, obtains repairing fragment.When using the dNTP in this test kit, do not add dCTP wherein, with the end repair enzyme in this test kit with the use of, repair rate is higher, the end that can obtain high-content repairs fragment, especially to difficulty of drawing materials, sample that quality is few, can improve the success ratio of building storehouse.
In construction process of the present invention, the step that 3 ' end adds " A " after repairing, can be entered.In order to make the connection better effects if adding " A ", after above-mentioned steps S2, and before step S3, preferably this construction process also comprises the step of reparation fragment being carried out to purifying.By carrying out purifying to the reparation fragment after end reparation, can directly get rid of the fragment be not repaired, the fragment that enrichment repairs, follow-up adding " A " efficiency to improve.In a kind of preferred embodiment of the present invention, the step of above-mentioned purifying adopts Ampure XP magnetic bead to carry out purifying to reparation fragment.Magnetic beads for purifying is adopted in this step, by the ratio between the consumption of Reasonable adjustment AmpureXP magnetic bead and the amount of reparation fragment to be purified, Ampure XP magnetic bead can be utilized to screen the fragment obtaining object clip size, the reparation fragment that further removal is excessive or too small, improves the enrichment journey of object size fragment.
In construction process of the present invention, after end reparation, the step adopting conventional method to carry out 3 ' end to add " A ", obtains the fragment of 3 ' end strips " A ", then carries out joint Connection Step, obtain belt lacing fragment.Now, building the storehouse time to save, directly can enter " C " and arriving " T " conversion processing step." C " to " T " conversion processing detects the classical processing mode of gene methylation, its principle is: with hydrosulphite or heavy bisulf iotate-treated genomic dna, all methylated cytosine(Cyt)s (C) that do not occur are converted into uridylic (U), and methylated cytosine(Cyt) is then constant.Thus, after hydrosulphite or heavy bisulf iotate-treated, methylated site produces the polynucleotide polymorphism (SNP) being similar to a C/T.Genomic dna is after sulfiting, and amplification object fragment, now uridylic (U) is all converted into thymus pyrimidine (T), finally carries out order-checking to PCR primer and just can judge whether to methylate.
In joint Connection Step, the joint of connection is pass through the joint that " C " arrives " T " conversion processing.When the full-length genome of routine methylates sequencing library structure, the non-methylated C that take into account at joint Connection Step in the joint be connected into needs through hydrosulphite or bisulfite process, and have ignored the calculating that the non-methylated C introduced in end reparation step also can affect final methylated C content.Thus, in the DNA fragmentation after fragmentation, there is single stranded DNA, have the double-stranded DNA of flat end, also have both-end all with the non-flat terminal double link DNA of outstanding base, in these outstanding bases, have bases G, during reparation, artificially will introduce non-methylated C.
In the another kind of preferred embodiment of invention, before carrying out above-mentioned joint Connection Step, also comprise the step adopting Ampure XP magnetic bead above-mentioned 3 ' end strips " A " fragment to be carried out to purifying, some fragments 3 ' end can not being connected " A " by Ampure XP magnetic beads for purifying step are removed.
In another preferred embodiment of the present invention, after above-mentioned steps S4, and before step S5, this construction process also comprises and carries out quantitative step to belt lacing fragment.Carrying out before " C " arrive " T " conversion processing, carry out quantitatively to pending belt lacing fragment, accurately can calculate the amount of required hydrosulphite or heavy hydrosulphite, the C in belt lacing fragment can be made all to change into U, unlikely excessive use hydrosulphite or bisulfite again, thus cut the waste, cost-saving.
Above-mentioned " C " arrives in the step of " T " conversion processing, the hydrosulphite that oneself configures or bisulfite solution can be adopted to process, also the conventional treatment kits that methylates can be adopted to process, as long as the non-methylated C in the object fragment in handled belt lacing fragment and methylated C can be made to make a distinction.In another preferred embodiment of the present invention, adopt EZ DNA Methylation-Lightning in step s 5
tMtest kit carries out " C " to belt lacing fragment and arrives " T " conversion processing, obtains processing fragment.EZ DNA Methylation-Lightning
tMtest kit processing efficiency is high.
In the another kind of typical embodiment of the present invention, provide a kind of full-length genome and to methylate sequencing library, this full-length genome sequencing library that methylates adopts any one construction process above-mentioned to build to form.Full-length genome of the present invention methylates sequencing library because repairing in step the dNTP used not containing dCTP at end, and then avoid and introduce non-methylated C building in the process of storehouse, thus make methylated C content that constructed full-length genome methylates in sequencing library closer to its real content, improve full-length genome methylation and analyze or the accuracy of assessment.
Effect of the present invention is further illustrated below in conjunction with specific embodiment.Should be understood that, these embodiments are only not used in for illustration of the present invention and limit the scope of the invention.
Experiment one
1. with Arabidopis thaliana seed DNA for sample, 5.2 μ g genomic dnas got by each sample, add the negative control (λ DNA) of 26ng (0.5%) respectively, then use water polishing to 130 μ l, be added in Covaris pipe.
2. carrying out physics fragmentation with Covaris S220 equipment, according to the experience that great many of experiments is summed up, is cycle number 8% ~ 12% by the optimum configurations of Covaris S220, peak value incident power 170 ~ 180 watts; Cycle number/outburst 200 ~ 220; Treatment time 250 ~ 330s; Time within the scope of temperature 4 ~ 8 DEG C, the clip size that all can broken obtain, mainly between 150 ~ 300bp, is arranged according to the data in following table 2 in this experiment.
Table 2:
3. whether get 20-30ng (1 μ l) sample and carry out 2% agarose gel electrophoresis (120V, 25min), detect to interrupt and concentrate, concrete detected result is shown in Fig. 2.
In fig. 2, Far Left is the large tick marks of DNA molecular, 5 stripe size are from bottom to up respectively: 100bp, 200bp, 300bp, 400bp and 500bp, and as can be seen from Figure 2, the size of the DNA fragmentation after Covaris S220 interrupts all concentrates between 150 ~ 300bp.
4. with the specification sheets purifying DNA fragment of QIAquick PCR purification kit.
5. carry out end reparation with End-It test kit (Epicenter), on ice in PCR pipe according to following component ratio forming reactions system, and with pipettor mixing, room temperature places 45 minutes.
Consisting of of above-mentioned reaction system:
6. repair product with Ampure XP magnetic beads for purifying end.
Concrete steps are as follows:
(1) in reaction system, add 60ul Ampure XP magnetic bead, repeat mixing with pipettor, room temperature leaves standstill 5 minutes.
(2) PCR pipe is placed on magnetic stand, leaves standstill 5 minutes, become clarification to solution.With pipettor, supernatant liquor sucking-off is abandoned.
(3) (keep PCR pipe on magnetic frame) and add 200ul 80% ethanol, leave standstill 30 seconds, ethanol sucking-off is abandoned.
(4) repeating step 3) once.Inhale again once with 10ul rifle head, ensure do not have ethanolic soln to remain.
(5) (continue to keep PCR pipe on magnetic frame) room temperature to leave standstill to magnetic bead dry.
(6) on the magnetic bead of drying, add 39ul water, PCR pipe taken off from magnetic frame, with pipettor by the water in pipe and magnetic bead mixing, static 2 minutes of room temperature, is placed on magnetic frame and adsorbs magnetic bead 5 minutes, transferred in new PCR pipe by 37ul supernatant.
7.3 ' end adds A.
In PCR pipe, reagent is added by system below on ice.(if sample is more, other reagent should be blended together mix, then be dispensed among each reaction system)
With pipettor mixing, PCR pipe to be placed in PCR instrument 37 DEG C of reactions 30 minutes.
8. add A product with Ampure XP magnetic beads for purifying 3 ' end.
(1) in reaction system, add 50ul Ampure XP magnetic bead, repeat mixing with pipettor, room temperature leaves standstill 5 minutes.
(2) PCR pipe is placed on magnetic stand, standing adsorption magnetic bead 5 minutes, becomes clarification to solution.With pipettor, supernatant liquor sucking-off is abandoned.
(3) (keep PCR pipe on magnetic frame) and add 200ul 80% ethanol, leave standstill 30 seconds, ethanol sucking-off is abandoned.
(4) repeating step 3) once.Inhale again once with 10ul rifle head, ensure do not have ethanolic soln to remain.
(5) (continue to keep PCR pipe on magnetic frame) room temperature to leave standstill to magnetic bead dry.
(6) on the magnetic bead of drying, add 33ul water, PCR pipe taken off from magnetic frame, with pipettor by the water in pipe and magnetic bead mixing, static 2 minutes of room temperature, is placed on magnetic frame and adsorbs magnetic bead 5 minutes, transferred in new PCR pipe by 31ul supernatant.
9. joint connects.According to reaction system below, each moiety is added in PCR pipe.
With pipettor mixing, be placed in PCR instrument, 16 DEG C of connections are spent the night.
Wherein, the sequence of joint 24 (NEXTflexTM Bisulfite-Seq Barcodes – 12, Bioo) of methylating is: SEQ IDNO.1:
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT；SEQ ID NO.2：
GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGTAGCATCTCGTATGCCGTCTTCTGCTTG。
10. add joint product also quantitatively with Ampure XP magnetic beads for purifying
(1) in reaction system, add 40ul Ampure XP magnetic bead, repeat mixing with pipettor, room temperature leaves standstill 5 minutes.
(2) PCR pipe is placed on magnetic stand, standing adsorption magnetic bead 5 minutes, becomes clarification to solution.With pipettor, supernatant liquor sucking-off is abandoned.
(3) (keep PCR pipe on magnetic frame) and add 200ul 80% ethanol, leave standstill 30 seconds, ethanol sucking-off is abandoned.
(4) repeating step 3) once.Inhale again once with 10ul rifle head, ensure do not have ethanolic soln to remain.
(5) (continue to keep PCR pipe on magnetic frame) room temperature to leave standstill to magnetic bead dry.
(6) on the magnetic bead of drying, add 51ul water, PCR pipe taken off from magnetic frame, with pipettor by the water in pipe and magnetic bead mixing, static 2 minutes of room temperature, is placed on magnetic frame and adsorbs magnetic bead 5 minutes, transferred in new PCR pipe by 50ul supernatant.
(7) in PCR pipe, add 50ul Ampure XP magnetic bead, fully mix with pipettor, room temperature leaves standstill 5 minutes.
(8) repeating step 2) ~ 5)
(9) on the magnetic bead of drying, add 22.5ul water, PCR pipe taken off from magnetic frame, with pipettor by the water in pipe and magnetic bead mixing, static 2 minutes of room temperature, is placed on magnetic frame and adsorbs magnetic bead 5 minutes, transferred in new PCR pipe by 20ul supernatant.
(10) by quantitative for the 2.5ul sucking-off 1ul Qubit HsDNA in pipe on magnetic frame.
Experiment two
CT conversion processing is (with EZ DNA Methylation-Lightning
tMkit, Zymo Research)
1. preparation work:
(1) packing Lightning Conversion Reagent
Often pipe Lightning Conversion Reagent is the amount of 10 reactions, if the sample transformed is few, can shifts to an earlier date packing and keep in Dark Place.
(2) M-Wash Buffer is prepared
Dehydrated alcohol: M-Wash Buffer=4:1
96ml dehydrated alcohol is added in 24ml M-Wash Buffer.
2. experimental procedure:
(1) get 400ngDNA sample according to quantitative result and carry out CT conversion, add joint product less than 400ng with all 20ul, more than the product getting 400ng of 400ng, the benefit that adds water puts 20ul.Add 130 μ l Lightning Conversion Reagent in 20 μ l DNA sample, fully mix sample with pipettor.The volume of reaction can not exceed the effective maximum reaction volume of PCR instrument, otherwise will be dispensed into reaction (such as, be in the PCR instrument of 50ul at maximum reaction volume, 150ul reaction system average mark should be contained in 3 PCR pipe) in multiple pipe.
(2) sample hose be put into temperature cycling device and operate by with step shown in following table 3:
Table 3:
| 98℃ | 8min |
| 54℃ | 60min |
Reaction terminates back balance and carries out following operation to room temperature or store (the longest 20 hours) at 4 DEG C
(3) by Zymo-Spin
tMiC Column is placed in the Collection Tube that test kit provides, and the M-Binding Buffer adding 600 μ l is in pillar.
(4) reaction solution in step (2) is added the Zymo-Spin containing M-Binding Buffer
tMin IC Column, pillar is put upside down and is carried out biased sample for several times by cover lid.(Optional: if initiate dna amount is less, first should will add 8ug Carrier RNA in reaction solution, then reaction solution is joined in Column mix with M-Binding Buffer)
Attention: step (3) and step (4) can not be put upside down.
(5) centrifugal 30 seconds (>10,000g), outwells liquid.
(6) 100 μ l M-Wash Buffer are added in post, centrifugal 30 seconds.
(7) to add in 200 μ l M-Desulphonation Buffer to post and place 15-20 minute under room temperature (20 DEG C-30 DEG C).After incubation terminates, centrifugal 30 seconds.
(8) the M-Wash Buffer of 200 μ l is added in post, centrifugal 30 seconds.Repeat once.
(9) sky gets rid of 1min.
(10) pillar is put into new 1.5ml Eppendorf pipe, uncap leaves standstill 2 minutes
(11) add the M-Elution Buffer of 13 μ l in pillar, leave standstill 2min, centrifugal 1 minute eluted dna.
(12) repeating step 11, obtains the recovery product of about 23ul altogether, if less than 23ul, adds water and mends to 23ul.
Experiment three
Pcr amplification
1. add following reagent on ice and fully mixing (if sample is more, first reagent is blended together mix, then be dispensed among each reaction system).
The DNA 23ul of heavy bisulf iotate-treated
KAPA HiFi HotStart Uracil+ReadyMix(2X) 25ul
NEXTflex
tMprimer mixture 2ul
PCR response procedures is as following table 4:
Table 4:
Go 1ul to carry out the size in electrophoresis detection final library the fragment after pcr amplification, detected result is shown in Fig. 3.
In figure 3, Far Left is the large tick marks of DNA molecular, and 5 stripe size are from bottom to up respectively: 100bp, 200bp, 300bp, 400bp and 500bp.As can be seen from Figure 3, the size in the library obtained after amplification of the present invention all concentrates between 250 ~ 450bp.
2. by Ampure XP magnetic beads for purifying PCR primer.
(1) in reaction system, add 50ul Ampure XP magnetic bead, repeat mixing with pipettor, room temperature leaves standstill 5 minutes.
(2) PCR pipe is placed on magnetic stand, standing adsorption magnetic bead 5 minutes, becomes clarification to solution.With pipettor, supernatant liquor sucking-off is abandoned.
(3) (keep PCR pipe on magnetic frame) and add 200ul 80% ethanol, leave standstill 30 seconds, ethanol sucking-off is abandoned.
(4) repeating step 3) once.Inhale again once with 10ul rifle head, ensure do not have ethanolic soln to remain.
(5) (continue to keep PCR pipe on magnetic frame) room temperature to leave standstill to magnetic bead dry.
(6) on the magnetic bead of drying, add 22ul water, PCR pipe taken off from magnetic frame, with pipettor by the water in pipe and magnetic bead mixing, static 2 minutes of room temperature, is placed on magnetic frame and adsorbs magnetic bead 5 minutes, transferred in new PCR pipe by 20ul supernatant.
(7) liquid remaining in the PCR pipe on magnetic frame is got 1ul Qubit hsDNA quantitative.
3. outbound
To bank number, original text storehouse 1.5ml manages, and dilutes the literal arts of a 2ng/ μ l, fills with 500ul pipe.
Experiment four
The sequencing library that methylated by obtained full-length genome carries out upper machine order-checking, sequencing data must be beaten, and by sequencing data GC resolution is carried out to built library and GC content is assessed, as shown in Figure 4, full-length genome constructed in the above embodiment of the present invention sequencing library display GC that methylates is separated, and GC content is low.Because in the library after bisulfite process, C, G content can be low, and build owing to testing the directivity that the sequence (reads) obtained in the strategy of storehouse remains chain, so the C content of first end sequence (read1) is very low on GC distribution plan, T content is very high, second terminal sequence (read2) is that G content is very low, and A content is very high.
Bioinformatic analysis: adopt Bimark (Krueger, 2011, bottom calls Bowtie2) to carry out methylating the genomic compare of analysis of reference of data.As shown in Figure 5, Bismark is by the result of order-checking and the conversion having carried out C to T and G to A (reverse complemental) with reference to genome, and the sequencing result after conversion and genome are carried out comparison between two respectively, and the comparison result obtained is as following table 5 for analysis principle.
Table 5:
As can be seen from the various types of methylation level of each karyomit(e) in upper table 5, the methylation of CG, GHG and CHH three types on each karyomit(e) that can detect compared to existing technology, adopt construction process of the present invention, owing to avoiding the content of the non-methylated C that human factor increases, the methylate methylation of CG, GHG and CHH three types on each karyomit(e) that sequencing library obtains of constructed full-length genome is higher, closer to its true horizon, thus detection accuracy is higher.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. full-length genome methylates a construction process for sequencing library, and it is characterized in that, described construction process comprises the following steps:
S1, carries out fragmentation to described complete genome DNA, obtains DNA fragmentation;
S2, the dNTP adopting dATP, dTTP and dGTP to be mixed to form carries out end reparation to described DNA fragmentation, obtains repairing fragment;
S3, carries out 3 ' end to described reparation fragment and adds " A ", obtains 3 ' end strips " A " fragment;
S4, adopts the joint sequence through " C " to " T " conversion processing to carry out joint connection to described 3 ' end strips " A " fragment, obtains the fragment of belt lacing;
S5, carries out " C " to described belt lacing fragment and arrives " T " conversion processing, obtains processing fragment;
S6, increases to described process fragment, obtains described full-length genome and to methylate sequencing library.
2. construction process according to claim 1, is characterized in that, after described step S1, and before described step S2, described construction process also comprises the step of described DNA fragmentation being carried out to purifying.
3. construction process according to claim 2, is characterized in that, the step of described purifying adopts DNA affinity column to carry out purifying.
4. construction process according to claim 2, is characterized in that, the step of described purifying adopts the described DNA affinity column in QIAquick PCR purification kit to carry out purifying to described DNA fragmentation.
5. construction process according to claim 1, is characterized in that, in described step S2, the dNTP adopting dATP, dTTP and dGTP in End-It test kit to be mixed to form carries out end reparation to described DNA, obtains described reparation fragment.
6. construction process according to claim 1 or 5, it is characterized in that, after described step S2, and before described step S3, described construction process also comprises the step of described reparation fragment being carried out to purifying.
7. construction process according to claim 1, is characterized in that, after described step S3, and before described step S4, also comprises the step adopting Ampure XP magnetic bead described 3 ' end strips " A " fragment to be carried out to purifying.
8. construction process according to claim 1, is characterized in that, after described step S4, and before described step S5, described construction process also comprises and carries out quantitative step to described belt lacing fragment.
9. construction process according to claim 1, is characterized in that, in described step S5, adopts EZ DNAMethylation-Lightning
tMtest kit carries out " C " to described belt lacing fragment and arrives " T " conversion processing, obtains described process fragment.
10. full-length genome methylates a sequencing library, it is characterized in that, adopts the construction process according to any one of claim 1 to 9 to build and forms.
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