CN109207471A - A kind of method and its application constructing fragment section nucleic acid library - Google Patents
A kind of method and its application constructing fragment section nucleic acid library Download PDFInfo
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Abstract
The present invention relates to a kind of method and its application for constructing fragment section nucleic acid library, and in particular to a kind of method and application of the fragment section nucleic acid library building based on high-flux sequence platform, described method includes following steps: (1) being enriched with the nucleic acid of sample；(2) enriched product is interrupted；(3) adjunction head；(4) segment separates；(5) it expands；(6) amplified production is mixed.The present invention passes through during building library, different size of nucleic acid fragment is separated, isolated nucleic acid fragment is respectively completed library construction, it is inserted into the nucleic acid library of different fragments size respectively when preparing DNB link, proliferation time is changed using different rollings, signal strength is relatively uniform when realizing the homogenization of the unit recurring number on its DNB, that is, making its sequencing, the final homogeneity for improving data distribution.
Description
Technical field
The present invention relates to DNA sequencing technology fields, are related to a kind of method and its application for constructing fragment section nucleic acid library, tool
Body, it is related to the method and application of a kind of fragment section nucleic acid library building of microarray dataset based on single stranded circle amplified library.
Background technique
Based on the microarray dataset of single stranded circle amplified library, such as WES, RNA-seq of development and application at present,
SmallRNA-seq etc., conventional Library development flow mainly include the following steps: that (1) will originate DNA progress ultrasound or digestion is random
It interrupts；(2) it interrupts product and carries out Piece Selection, be clip size relatively uniformization；(3) Piece Selection product carries out end reparation
And add " A "；(4) specific linkers are connected to the both ends of DNA fragmentation with DNA ligase, are purified；(5) PCR and purifying；(6) literary
Library cyclisation.
CN 104946623A discloses a kind of small fragment DNA library based on Illumina Hiseq2500 microarray dataset
Construction method, including dissociative DNA in blood extract, clip size screening, end repair, 3 ' end addition connectors, PCR amplification, magnetic
Pearl purifying and etc..CN 105695448A discloses a kind of text of dissociative DNA in blood based on Ion ProtonTM microarray dataset
Base construction method, reagent and its application, building process include dissociative DNA in blood extraction, end reparation, Piece Selection, connector company
It connects, magnetic beads for purifying, amplified library, library detection, high-flux sequence.The above method directly uses the segment after adjunction head
Yu Jianku, Insert Fragment size Relatively centralized, but due to portioned product especially library and sequencing are built to PCR product, often due to
Different genes length is different so that Insert Fragment magnitude range is larger.
DNB technology be using rolling circle amplification (Rolling circle amplification, RCA) by single-stranded loop
2-3 order of magnitude of shape DNA cloning, generated amplified production are known as DNA nanosphere (DNAnanoball, DNB), final nanometer
Ball warp is crossed DNB loading technology and is fixed on the silicon chip of array.Compared with other two generations sequencing technologies, DNB sequencing technologies
With following advantage: (1) DNB enhances signal strength by increasing the copy number of DNA to be measured, so that it is quasi- to improve sequencing
Exactness；(2) it is different from PCR exponential amplification, the amplification mistake of rolling circle amplification will not accumulate；(3) DNB and activated sites on chip
The size of point is identical, and a DNB is only fixed in each site, does not generate and interferes with each other between guarantee signaling point；(4) array is sequenced
The combination of chip and DNB sequencing technologies, so that the area of imaging system pixel and sequence testing chip is maximally utilized.
Summary of the invention
The present invention is completed based on finding below inventor: inventor passes through to the sequencing based on DNB sequencing technologies
Platform is carefully studied, it is found that it is more sensitive to Insert Fragment size distribution range.When preparing DNB, due to be using
Constant-temperature amplification enzyme carries out rolling to single-stranded loop and changes amplification, under the identical reaction time, the rolling ring product length unanimous circumstances of formation
Under, the unit recurring number on the DNB in big Insert Fragment library is few, and the unit recurring number on the DNB in small insert library is more.
That is the position that sequencing primer combines is less, so that signal strength is lower when its sequencing, is easy to be judged as by software
Background signal filters out, and it is poor to ultimately cause data distribution homogeneity.
In order to solve the above technical problems, the present invention provides a kind of method and its application for constructing fragment section nucleic acid library,
The method and application of the fragment section nucleic acid library building of specially a kind of microarray dataset based on single stranded circle amplified library, this
Inventive method separates different size of nucleic acid fragment, and isolated nucleic acid fragment is expanded respectively again, by being arranged not
Same proliferation time realizes the homogenization of the unit recurring number between amplified production, makes each product signal strength phase in sequencing
To consistent, the homogeneity of data distribution is improved.
For this purpose, the invention adopts the following technical scheme:
In a first aspect, including the following steps: the present invention provides a kind of method of fragment section nucleic acid library building
(1) it is enriched with the nucleic acid of sample, obtains enriched product；
(2) enriched product is interrupted, the nucleic acid of fragmentation is obtained；
(3) connector is added at the nucleic acid both ends of the fragmentation, obtains " connector-sample fragmented nucleic acids-connector " shape
The adjunction head product of formula；
(4) segment separation is carried out to the adjunction head product, obtains the separation product with different nucleic acid fragment length；
(5) separation product is expanded respectively, obtains multiple amplified productions；And
(6) the multiple amplified production is mixed, obtains the fragment section nucleic acid library.
It according to the present invention, further include the nucleic acid for extracting sample before step (1), the nucleic acid is DNA and/or RNA,
The DNA and/or RNA be can extract sample of nucleic acid any source of people sample be all it is feasible, do not do particular determination herein, this
Field technical staff can need to extract according to experiment, and DNA of the invention and/or RNA come from peripheral blood, lymph, group
Knit in cell, hair or Oral Mucosal Cells any one or at least two combination, preferably peripheral blood.
According to the present invention, step (1) extracting method is the conventional method of this field, and those skilled in the art can root
According to being selected, particular determination is not done herein, and the present invention is extracted using paramagnetic particle method and/or boiling method.
Primer those skilled in the art that gene magnification of the present invention uses can set according to the segment of amplification
The primer of meter, different detection gene designs is also different, and does not do particular determination herein, of the inventionly poor genetic test library
The primer used when building is as follows:
HBA1-F:5'-AGCATAAACCCTGGCGCGC-3'(SEQ ID NO:1)；
HBA1-R:5'-ATGCCTGGCACGTTTGCTGAG-3'(SEQ ID NO:2)；
HBA2-F:5'-CAAGCATAAACCCTGGCGCGC-3'(SEQ ID NO:3)；
HBA2-R:5'-CCATTGTTGGCACATTCCGGGATA-3'(SEQ ID NO:4)；
HBB-F:5'-GCCAGTGCCAGAAGAGCC-3'(SEQ ID NO:5)；
HBB-R:5'-GCACTGACCTCCCACATTCC-3'(SEQ ID NO:6)；
According to the present invention, the enrichment is carried out using the method for method and/or the probe capture of PCR amplification.
According to the present invention, step (2) is described interrupts using ultrasound and/or digestion method, preferably ultrasonic method.
It according to the present invention, further include the nucleic acid progress end reparation to fragmentation, and at 3 ' ends before step (3)
End addition base A, specific steps are as follows: after the product after interrupting is mixed with reagent I, in 30-45 DEG C of 25-35min, 60-70 DEG C
10-20min, preferably in 37 DEG C of 30min, 65 DEG C of 15min.
According to the present invention, the reagent I is that the conventional reagent of " A " is repaired and added in end, is able to carry out end in this field
Repair and add " A " reagent be all it is feasible, those skilled in the art can select according to actual needs, the present invention use
T4 polynueleotide kinase, the final concentration of 5- of 10 × polynueleotide kinase buffer, dNTP, final concentration of 5-15U/ μ L
The T4DNA polymerase of the Klenow enzyme of 20U/ μ L, the rTaq polymerase of final concentration of 1-15U/ μ L and final concentration of 1-10U/ μ L,
T4 polynueleotide kinase, the final concentration of preferably 10 × polynueleotide kinase buffer, dNTP, final concentration of 8-12U/ μ L
It polymerize for the T4DNA of the Klenow enzyme of 3-10U/ μ L, the rTaq polymerase of final concentration of 3-10U/ μ L and final concentration of 2-6U/ μ L
Enzyme, further preferably 10 × polynueleotide kinase buffer, dNTP, the T4 polynucleotide of final concentration of 10U/ μ L are sharp
Enzyme, the Klenow enzyme of final concentration of 5U/ μ L, the T4DNA of the rTaq polymerase of final concentration of 5U/ μ L and final concentration of 3U/ μ L are poly-
Synthase.
Final concentration in the present invention, when the concentration of each reagent is use.
According to the present invention, the specific steps of step (3) the adjunction head are as follows: by the product and reagent after addition base " A "
II and connector mixing, in 15-30 DEG C of reaction 30-100min, preferably 20-26 DEG C reaction 50-80min, further preferably 23
DEG C reaction 60min.
The reaction temperature for example can be 15 DEG C, 16 DEG C, 17 DEG C, 18 DEG C, 19 DEG C, 20 DEG C, 21 DEG C, 22 DEG C, 23 DEG C, 24
DEG C, 25 DEG C, 26 DEG C, 27 DEG C, 28 DEG C, 29 DEG C or the specific point value between 30 DEG C and above-mentioned numerical value, as space is limited and for letter
Bright consideration, the specific point value that range described in the present invention no longer exclusive list includes.
The reaction time for example can be 30min, 35min, 40min, 45min, 50min, 55min, 60min,
Specific point value between 65min, 70min, 75min, 80min, 85min, 90min, 95min or 100min and above-mentioned numerical value,
As space is limited and for concise consideration, the specific point value that range described in the present invention no longer exclusive list includes.
According to the present invention, the reagent II is the ATP of 10 × polynueleotide kinase buffer, final concentration of 80-120mM
With the T4DNA ligase of final concentration of 550-650U/ μ L, preferably 10 × polynueleotide kinase buffer, final concentration of 90-
The T4DNA ligase of the ATP of 110mM and final concentration of 580-620U/ μ L, further preferably 10 × polynueleotide kinase are slow
The T4DNA ligase of fliud flushing, the ATP of final concentration of 100mM and final concentration of 600U/ μ L；
Preferably, the polyethylene glycol that the reagent II is also 40-60% containing mass fraction, preferably mass fraction are
The polyethylene glycol that the polyethylene glycol of 45-55%, further preferably mass fraction are 50%；
Preferably, the polyethylene glycol is any in Macrogol 4000, Macrogol 6000 or PEG 8000
It is a kind of or at least two combination, preferably PEG 8000.
Final concentration in the present invention, when the concentration of each reagent is use.
According to the present invention, the segment separation is carried out using paramagnetic particle method.
Preferably, realize that the segment separates with the volume ratio of magnetic bead by adjusting the adjunction head product；
It according to the present invention, further include by the separation product with different nucleic acid fragment length point before step (5)
It is not denaturalized, obtains the single nucleic acid strands with different length, and the single nucleic acid strands are cyclized respectively, obtaining has not
Circularized nucleic acid with length is single-stranded；
Preferably, using the cyclisation for mediating segment to realize the single nucleic acid strands, the mediation segment has respective complementary sequence
Arrange the both ends for connecting single nucleic acid strands；
Preferably, the cyclisation is the specific steps are mixing the separation product of step (4) with reagent III, 95 in PCR instrument
DEG C reaction 3~6 minutes, preferably 3 minutes, take out, be placed in cooled on ice 5~10 minutes, preferably 5 minutes, add reagent IV,
It mixes, 37 DEG C are reacted 50~80 minutes, preferably 60 minutes.
Preferably, the reagent III is to mediate segment；
Preferably, mediation segment nucleotide sequence as follows: 5 '-GCCATGTCGTTCTGTGAGCCAAGG-3 '
(SEQ ID NO.7)。
Preferably, final concentration of 2 μM~5 μM of the reagent III, preferably 3.5 μM.
Preferably, the reagent IV is cyclization reagent, including following components:
The T4DNA connection of 10 × TA buffer, the ATP of final concentration of 0.5mM~4mM, final concentration 3U/ μ L~10U/ μ L
Enzyme, the final concentration of 1.5mM of the ATP, the final concentration of of the T4DNA ligase is 6U/ μ L.
Preferably, when step (5) expands the separation product respectively, according to the nucleic acid fragment length of separation product
The different amplified reaction time is set.
Preferably, step (5) amplification is rolling circle amplification, single-stranded for mould with the circularized nucleic acid with different length
Plate carries out the rolling circle amplification, also, the different amplified reaction time is arranged according to the single-stranded length of the circularized nucleic acid.
According to the present invention, the separation of segment described in step (4) comprises the following specific steps that:
(1 ') is reacted after mixing well the product after adjunction head with magnetic bead, then is placed on magnetic frame, and the first supernatant is obtained
Liquid；The nucleic acid of magnetic bead absorption is eluted, the first nucleic acid solution is obtained；And
(2 ') magnetic bead is added into first supernatant, and mixing is placed on magnetic frame, obtains the second supernatant, elution
The nucleic acid of magnetic bead absorption, obtains the second nucleic acid solution；
Wherein, the nucleic acid fragment length in first nucleic acid solution and the nucleic acid fragment in second nucleic acid solution are long
Degree is different.
Preferably, the nucleic acid fragment length in first nucleic acid solution is greater than the nucleic acid piece in second nucleic acid solution
Segment length.
Preferably, the separation of segment described in step (4) further comprises: step (3 ') is by the second supernatant alternative steps
The first supernatant in (2 ') repeats step (2 ') at least once, obtains supernatant and nucleic acid solution；
Preferably, the volume ratio of adjunction head product and magnetic bead is 1:(0.5-0.8 in step (1 ')), such as can be 1:
0.5,1:0.6,1:0.7 or 1:0.8, preferably 1:(0.6-0.8), between further preferably 1:0.7 and above-mentioned numerical value
Specific point value, as space is limited and for concise consideration, the specific point value that range described in the present invention no longer exclusive list includes.
In the present invention, the magnetic bead is that this field all can be feasible, this field for isolating and purifying the magnetic bead of DNA
Technical staff, which can according to need, to be selected, and the magnetic bead includes, different magnetic bead brand such as Axygen, Beckman,
The result of Invitrogen has subtle gap, and the present invention is preferably the nucleic acid purification magnetic bead of Beckman.
In step (2 ') volume ratio of supernatant and magnetic bead be 1:(1-1.5), such as can be 1:1,1:1.1,1:1.2,
1:1.3,1:1.4 or 1:1.5, preferably 1:(1-1.3), it is specific between further preferably 1:1.1 and above-mentioned numerical value
Value, as space is limited and for concise consideration, the specific point value that range described in the present invention no longer exclusive list includes.
In the present invention, by adjusting the ratio of nucleic acid and magnetic bead, the nucleic acid with different fragments length can be divided
From, when the volume ratio of nucleic acid and magnetic bead be 1:(0.5-0.8) when, magnetic bead absorption be 400-1000bp nucleic acid, when nucleic acid with
The volume ratio of magnetic bead is 1:(1-1.5), magnetic bead absorption is 400bp nucleic acid below, nucleic acid separated by boundary of 400bp,
Rolling circle amplification is carried out respectively, using the different rolling circle amplification time, realizes that the unit of (i.e. DNA nanosphere) between amplified production follows
The homogenization of number of rings keeps each product signal strength in sequencing relatively uniform, the final homogeneity for improving data distribution.
According to the present invention, step (1 ') the first nucleic acid solution amplifying nucleic acid fragment length is 400bp or more, such as can be with
Be 400bp, 450bp, 500bp, 550bp, 600bp, 650bp, 700bp, 750bp, 800bp, 850bp, 900bp, 950bp or
1000bp, the specific point value between preferably 400-1000bp and above-mentioned numerical value, as space is limited and for concise consideration,
The specific point value that range described in the present invention no longer exclusive list includes.
According to the present invention, step (2 ') the second nucleic acid solution amplifying nucleic acid fragment length is 400bp hereinafter, for example can be with
Be 50bp, 55bp, 60bp, 65bp, 70bp, 75bp, 80bp, 85bp, 90bp, 95bp, 100bp, 150bp, 200bp, 250bp,
300bp, 350bp, 380bp or 390bp, preferably 50-400bp, between further preferably 75-200bp and above-mentioned numerical value
Specific point value, as space is limited and for concise consideration, the specific point value that range described in the present invention no longer exclusive list includes.
In the present invention, by the proportion for adjusting nucleic acid and magnetic bead, so that it may realize point using 400bp as the nucleic acid fragment of boundary
From.
It should be noted that inventors have found that being since the time sets when carrying out rolling circle amplification to single stranded circular nucleic acid
Consistent, the constant-temperature amplification enzyme used carries out rolling to single stranded circular nucleic acid and changes amplification, under the identical reaction time, the rolling of formation
Under circle amplification product length unanimous circumstances, the big library of Insert Fragment length, amplification cycles number is few, and Insert Fragment length
Small library, amplification cycles number are more.That is since the position of sequencing primer combination is less, so that Insert Fragment is long
It is lower to spend big library signal strength in sequencing, is easy to be judged as that background signal filters out by software, ultimately causes data point
Cloth homogeneity is poor.Therefore, for the library of different Insert Fragment sizes, the corresponding rolling circle amplification time is different.
When according to the present invention, using the nucleic acid in the first nucleic acid solution described in rolling circle amplification, the amplified reaction time is 35-
45min, for example, can be 35min, 36min, 37min, 38min, 39min, 40min, 41min, 42min, 43min, 44min or
45min, the specific point value between preferably 40min and above-mentioned numerical value, as space is limited and for concise consideration, the present invention
The no longer specific point value that includes of range described in exclusive list.
When according to the present invention, using the nucleic acid in the second nucleic acid solution described in rolling circle amplification, the amplified reaction time is 15-
25min, for example, can be 15min, 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min or
25min, the specific point value between preferably 20min and above-mentioned numerical value, as space is limited and for concise consideration, the present invention
The no longer specific point value that includes of range described in exclusive list.
In a specific embodiment, the method for the building fragment section nucleic acid library includes the following steps:
(1) using the nucleic acid of the method enrichment sample of PCR amplification, enriched product is obtained；
(2) ultrasound interrupts the enriched product, obtains the nucleic acid of fragmentation；
(3) end repairs 3 ' ends and adds base A: after the product after interrupting is mixed with reagent I, in 30-45 DEG C of 25-
35min, 60-70 DEG C of 10-20min；
Wherein, the reagent I is 10 × polynueleotide kinase buffer, dNTP, the T4 of final concentration of 5-15U/ μ L more
Polynucleotide kinase, the Klenow enzyme of final concentration of 5-20U/ μ L, final concentration of 1-15U/ μ L rTaq polymerase and final concentration
For the T4DNA polymerase of 1-10U/ μ L；
(4) connector is added at the nucleic acid both ends of the fragmentation, specifically included: product and examination after addition base " A "
Agent II and connector mixing obtain adding for " connector-sample fragmented nucleic acids-connector " form in 15-30 DEG C of reaction 30-100min
Connector product；
Wherein, the reagent II is that 10 × polynueleotide kinase buffer, the ATP of final concentration of 80-120mM and end are dense
Degree is the T4DNA ligase of 550-650U/ μ L；
(5) segment separation is carried out using paramagnetic particle method to the adjunction head product, obtaining has different nucleic acid fragment length
Separation product；
The segment separation includes the following steps:
It after (1 ') mixes well the adjunction head product and magnetic bead, then is placed on magnetic frame, obtains the first supernatant；It washes
The nucleic acid of de- magnetic bead absorption, obtains the first nucleic acid solution；And
(2 ') magnetic bead is added into first supernatant, and mixing is placed on magnetic frame, obtains the second supernatant, elution
The nucleic acid of magnetic bead absorption, obtains the second nucleic acid solution；
Wherein, the nucleic acid fragment length in first nucleic acid solution and the nucleic acid fragment in second nucleic acid solution are long
Degree is different；
(6) separation product is cyclized respectively, the cyclisation specific steps are as follows: by the separation product of step (5)
It is mixed with reagent III, is reacted 3~6 minutes for 95 DEG C in PCR instrument, take out, be placed in cooled on ice 5~10 minutes, add reagent
IV is mixed, and 37 DEG C are reacted 50~80 minutes；
Wherein, the mediation segment that the reagent III is final concentration of 2 μM~5 μM, the mediation segment have such as SEQ ID
Nucleotide sequence shown in NO.7, the reagent IV are cyclization reagent, including following components: 10 × TA buffer, and end is dense
Degree is the ATP of 0.5mM~4mM, the T4DNA ligase of final concentration 3U/ μ L~10U/ μ L；
(7) separation product is subjected to rolling circle amplification respectively, obtains multiple amplified productions；And
(8) the multiple amplified production is mixed, obtains the fragment section nucleic acid library.
Second aspect, the present invention provide a kind of sequencing library, and the method as described in first aspect is made.
The third aspect, the present invention provide a kind of method for nucleic acid sequencing, including carrying out sequencing library described in second aspect
The step of sequencing；
Preferably, it is sequenced using single stranded circle library microarray dataset；It is further preferred that using BGISEQ series
Microarray dataset is sequenced；
Preferably, further include the steps that sequencing result is assembled and/or spliced.
Fourth aspect, the present invention provide method as described in relation to the first aspect for construct poor genetic test library and/or
HLA Genotyping detects library.
Compared with prior art, the present invention at least has the advantages that
The present invention is by using the magnetic bead of different proportion, different size of nucleic acid fragment being divided during building library
From, isolated nucleic acid fragment is respectively completed library construction, the nucleic acid library of different fragments size is inserted into when carrying out rolling circle amplification,
Using the different amplified reaction time, the homogenization of the unit recurring number of (i.e. DNA nanosphere) between amplified production is realized, make each
Product signal strength in sequencing is relatively uniform, the final homogeneity for improving data distribution.
Figure of description
Fig. 1 is the flow chart of fragment section nucleic acid library of the present invention building；
Fig. 2 is the depth coverage diagram that the library of the method for the present invention obtains after sequencing, wherein Fig. 2 (a) is HBA1, Fig. 2
It (b) is HBA2, Fig. 2 (c) is HBB1, and Fig. 2 (d) is HBB2；
Fig. 3 is the details enlarged drawing of Fig. 2, wherein depth 0 to 500 × the case where, Fig. 3 (a) is HBA1, and Fig. 3 (b) is
HBA2, Fig. 3 (c) are HBB1, and Fig. 3 (d) is HBB2；
Fig. 4 is what the library of conventional method (i.e. conventional libraries construction method, no segment separating step) obtained after sequencing
Depth coverage diagram, wherein Fig. 4 (a) is HBA1, and Fig. 4 (b) is HBA2, and Fig. 4 (c) is HBB1, and Fig. 4 (d) is HBB2；
Fig. 5 is the details enlarged drawing of Fig. 4, wherein depth 0 to 500 × the case where, Fig. 5 (a) is HBA1, and Fig. 5 (b) is
HBA2, Fig. 5 (c) are HBB1, and Fig. 5 (d) is HBB2.
Specific embodiment
Of the invention for ease of understanding, it is as follows that the present invention enumerates embodiment.Those skilled in the art are it will be clearly understood that the implementation
Example is only to aid in the understanding present invention, should not be regarded as a specific limitation of the invention.
Embodiment 1: the fragment section nucleic acid library construction method based on BGISEQ-500 platform
1, sample: the present embodiment has used 95 people's peripheral blood samples altogether, joined 1 negative control N in test, negative
Control is the ultrapure water by sterilizing；
2, the fragment section nucleic acid library construction method, process is as shown in Figure 1, include the following steps:
(1) blood/dried blood spot/saliva DNA is extracted: people DNA is extracted from sample using paramagnetic particle method；
(2) PCR expansion hemoglobin-based gene-amplification: is carried out to DNA in step (1) using hemoglobin gene specific primer
Increase, amplified production magnitude range is 600bp-950bp；
Wherein specific primer is as follows:
HBA1-F:5'-AGCATAAACCCTGGCGCGC-3'(SEQ ID NO:1)；
HBA1-R:5'-ATGCCTGGCACGTTTGCTGAG-3'(SEQ ID NO:2)；
HBA2-F:5'-CAAGCATAAACCCTGGCGCGC-3'(SEQ ID NO:3)；
HBA2-R:5'-CCATTGTTGGCACATTCCGGGATA-3'(SEQ ID NO:4)；
HBB-F:5'-GCCAGTGCCAGAAGAGCC-3'(SEQ ID NO:5)；
HBB-R:5'-GCACTGACCTCCCACATTCC-3'(SEQ ID NO:6)；
(3) magnetic beads for purifying: PCR product obtained by step (2) is subjected to ultrasound and is interrupted, specific ultrasound parameter is as follows: duty
Than: 21, PIP:500, CPB:500, ultrasonic time: 20s, recurring number: 6, interrupt product electrophoresis detection, display product segment distribution
Within the scope of 100bp-950bp；
(4) end repairs and adds " A ": after reagent I mixing is added in product after step (4) is interrupted, 37 DEG C of 30min,
65 DEG C of 15min carry out end reparation and add " A "；
Wherein, reagent I are as follows: the T4 polymerized nucleoside of 10 × polynueleotide kinase buffer, dNTP, final concentration of 10U/ μ L
Acid kinase, the Klenow enzyme of final concentration of 5U/ μ L, the rTaq polymerase of final concentration of 5U/ μ L and final concentration of 3U/ μ L
T4DNA polymerase；
(5) adjunction head: reagent II and connector is added in system obtained by step (4), is reacted in 23 DEG C of 60min；
Wherein, reagent II are as follows: 10 × polynueleotide kinase buffer, the ATP of final concentration of 100mM, mass fraction are
The T4DNA ligase of 50% PEG 8000 and final concentration of 600U/ μ L；
(6) segment separates: 0.7 times of XP magnetic bead is added in the DNA fragmentation product for the adjunction head that (5) obtain, it is sufficiently mixed
Above magnetic frame is placed at room temperature for after 5min after even, and after 2min, Beads enrichment elutes magnetic bead；
(7) supernatant is transferred in a new EP pipe, 1.1 times of supernatant of magnetic bead is added, mixes well rear room temperature and puts
5min is set, upper magnetic frame, after 2min, Beads enrichment elutes magnetic bead；
Wherein, the DNA of the magnetic bead absorption in (6) is relatively large segment, and size is about 400bp-1000bp, is labeled as
400+, the DNA of (7) absorption are relatively small segment, and size is that 100bp-400bp is labeled as 400-；
(8) reagent III is added in the product eluted to (6) and (7) and carries out library cyclisation；
Wherein, the reagent III is the mating single-stranded cyclisation reagent preparation in library provided of BGISEQ-500 microarray dataset, examination
Agent III has nucleotide sequence as follows: 5 '-GCCATGTCGTTCTGTGAGCCAAGG-3 ' (SEQ ID NO.7)；Reagent
Final concentration of 3.5 μM of III；
(9) preparation that reagent IV carries out DNB is added into the product after the cyclisation of (8) library, wherein being labeled as the text of 400+
The library rolling circle amplification time is 40min, and the library rolling circle amplification labeled as 400- is 20min；
Wherein, the reagent IV is mating DNB (DNA nanosphere) reagent preparation provided of BGISEQ-500 microarray dataset,
Specifically include following component: the T4DNA ligase of 10 × TA buffer, the ATP of final concentration of 1.5mM, final concentration 6U/ μ L；
(10) the upper machine sequencing of DNB；
(11) Quality Control of machine data and analysis under carry out overburden depth analysis.As a result see Fig. 2 and Fig. 3.
Illustrated in Fig. 2 and Fig. 3 using the method for the present invention construct divide frag-ment libraries to be sequenced after, sequencing obtained
Depth covering.As it can be seen that sequencing depth position 300bp-550bp in the middle part of HBA1HBA2 gene, depth covering be greater than 100 × feelings
Condition is 95% or more, meets follow-up analysis and requires.
Embodiment 2: the conventional libraries nucleic acid library construction method based on BGISEQ-500 platform
1, sample: the present embodiment has used 95 people's peripheral blood samples altogether, joined 1 negative control N in test, negative
Control is the ultrapure water by sterilizing；
2, conventional libraries nucleic acid library construction method, step (1)-(5) are identical as step (1)-(5) in embodiment 1, step
Suddenly (6) do not need to carry out segment separation, but directly purify the product after adjunction head using magnetic bead, adjunction head product with
The volume ratio of magnetic bead is 1:1.Hereafter cyclisation, rolling circle amplification, sequencing etc. are identical as with step (8)-(11) in embodiment 1；
3, lower machine data Quality Control and analysis carry out overburden depth analysis.As a result see Fig. 4 and Fig. 5.
It illustrates in Fig. 4 and Fig. 5 and is existed using the library (not carrying out the conventional libraries of segment separation) that conventional method constructs
After sequencing, sequencing depth obtained covers result.As it can be seen that sequencing depth portion 300bp-550bp in the middle part of HBA1HBA2 gene
Position, depth covering are greater than for 100 × the case where for 5% and require hereinafter, being unable to satisfy follow-up analysis.
The Applicant declares that the present invention is explained by the above embodiments detailed process equipment and process flow of the invention,
But the present invention is not limited to the above detailed process equipment and process flow, that is, it is above-mentioned detailed not mean that the present invention must rely on
Process equipment and process flow could be implemented.It should be clear to those skilled in the art, any improvement in the present invention,
Addition, selection of concrete mode of equivalence replacement and auxiliary element to each raw material of product of the present invention etc., all fall within of the invention
Within protection scope and the open scope.
Claims (10)
1. a kind of method for constructing fragment section nucleic acid library, which comprises the steps of:
(1) it is enriched with the nucleic acid of sample, obtains enriched product；
(2) enriched product is interrupted, the nucleic acid of fragmentation is obtained；
(3) connector is added at the nucleic acid both ends of the fragmentation, obtains " connector-sample fragmented nucleic acids-connector " form
Adjunction head product；
(4) segment separation is carried out to the adjunction head product, obtains the separation product with different nucleic acid fragment length；
(5) separation product is expanded respectively, obtains multiple amplified productions；And
(6) the multiple amplified production is mixed, obtains the fragment section nucleic acid library.
2. it further include the nucleic acid for extracting sample the method according to claim 1, wherein before step (1),
The nucleic acid is DNA and/or RNA；
Preferably, the extraction of the nucleic acid is extracted using paramagnetic particle method and/or boiling method；
Preferably, the enrichment is carried out using the method for method or the probe capture of PCR amplification.
Preferably, step (2) is described interrupts using ultrasound and/or digestion method, preferably ultrasonic method；
It preferably, further include end reparation being carried out to the nucleic acid of fragmentation, and add alkali in 3 ' ends before step (3)
Base A；
Preferably, the end repairs and the step of base A is added in 3 ' ends are as follows: mixes the nucleic acid of fragmentation and reagent I
After conjunction, in 30-45 DEG C of 25-35min, 60-70 DEG C of 10-20min, preferably in 37 DEG C of 30min, 65 DEG C of 15min；
Preferably, the reagent I is the T4 poly of 10 × polynueleotide kinase buffer, dNTP, final concentration of 5-15U/ μ L
Nucleoside monophosphate kinase, the Klenow enzyme of final concentration of 5-20U/ μ L, the rTaq polymerase of final concentration of 1-15U/ μ L and final concentration of
The T4DNA polymerase of 1-10U/ μ L, preferably 10 × polynueleotide kinase buffer, dNTP, final concentration of 8-12U/ μ L
T4 polynueleotide kinase, the Klenow enzyme of final concentration of 3-10U/ μ L, the rTaq polymerase of final concentration of 3-10U/ μ L and end
Concentration is the T4DNA polymerase of 2-6U/ μ L, further preferably 10 × polynueleotide kinase buffer, dNTP, final concentration of
The T4 polynueleotide kinase of 10U/ μ L, the Klenow enzyme of final concentration of 5U/ μ L, final concentration of 5U/ μ L rTaq polymerase and
The T4DNA polymerase of final concentration of 3U/ μ L.
3. method according to claim 1 or 2, which is characterized in that the step of step (3) the adjunction head are as follows: will add
Product after base A is mixed with reagent II and connector, in 15-30 DEG C of reaction 30-100min, preferably 20-26 DEG C reaction 50-
80min, further preferably 23 DEG C reaction 60min；
Preferably, the reagent II is the ATP and final concentration of 10 × polynueleotide kinase buffer, final concentration of 80-120mM
For the T4DNA ligase of 550-650U/ μ L, preferably 10 × polynueleotide kinase buffer, final concentration of 90-110mM
The T4DNA ligase of ATP and final concentration of 580-620U/ μ L, further preferably 10 × polynueleotide kinase buffer, end
Concentration is the T4DNA ligase of the ATP and final concentration of 600U/ μ L of 100mM；
Preferably, the polyethylene glycol that the reagent II is also 40-60% containing mass fraction, preferably mass fraction are 45-
The polyethylene glycol that 55% polyethylene glycol, further preferably mass fraction are 50%；
Preferably, the polyethylene glycol is any one in Macrogol 4000, Macrogol 6000 or PEG 8000
Or at least two combination, preferably PEG 8000.
4. method according to any one of claim 1-3, which is characterized in that segment separation using paramagnetic particle method into
Row；
Preferably, realize that the segment separates with the volume ratio of magnetic bead by adjusting the adjunction head product；
It preferably, further include becoming the separation product with different nucleic acid fragment length respectively before step (5)
Property, the single nucleic acid strands with different length are obtained, and the single nucleic acid strands are cyclized respectively, obtaining has different length
Circularized nucleic acid is single-stranded；
Preferably, using mediating segment to realize, the mediation segment is used with respective complementary sequence for the cyclisation of the single nucleic acid strands
In the both ends of connection single nucleic acid strands；
Preferably, the cyclisation is the specific steps are mixing the separation product of step (4) with reagent III, and 95 DEG C instead in PCR instrument
3~6 minutes, preferably 3 minutes are answered, takes out, is placed in cooled on ice 5~10 minutes, preferably 5 minutes, reagent IV is added, mixes
Even, 37 DEG C are reacted 50~80 minutes, preferably 60 minutes；
Preferably, the reagent III is to mediate segment；
Preferably, the mediation segment has the nucleotide sequence as shown in SEQ ID NO.7；
Preferably, final concentration of 2 μM~5 μM of the reagent III, preferably 3.5 μM；
Preferably, the reagent IV is cyclization reagent, including following components:
The T4DNA ligase of 10 × TA buffer, the ATP of final concentration of 0.5mM~4mM, final concentration 3U/ μ L~10U/ μ L；
Preferably, the final concentration of 1.5mM of the ATP；
Preferably, the final concentration of of the T4DNA ligase is 6U/ μ L；
Preferably, when step (5) expands the separation product respectively, it is arranged according to the nucleic acid fragment length of separation product
The different amplified reaction time；
Preferably, step (5) amplification is rolling circle amplification；
Preferably, the rolling circle amplification is single-stranded for template with the circularized nucleic acid with different length, according to the cyclisation core
The different amplified reaction time is arranged in the single-stranded length of acid.
5. method according to any of claims 1-4, which is characterized in that segment described in step (4), which separates, includes
Following steps:
It after (1 ') mixes well the adjunction head product and magnetic bead, then is placed on magnetic frame, obtains the first supernatant；Elute magnetic
The nucleic acid of pearl absorption, obtains the first nucleic acid solution；And
(2 ') magnetic bead is added into first supernatant, and mixing is placed on magnetic frame, obtains the second supernatant, elutes magnetic bead
The nucleic acid of absorption obtains the second nucleic acid solution；
Wherein, the nucleic acid fragment length in the nucleic acid fragment length in first nucleic acid solution and second nucleic acid solution is not
Together；
Preferably, the nucleic acid fragment that the nucleic acid fragment length in first nucleic acid solution is greater than in second nucleic acid solution is long
Degree；
Preferably, the separation of segment described in step (4) further comprises: step (3 ') will be in the second supernatant alternative steps (2 ')
The first supernatant, repeat step (2 ') at least once, obtain supernatant and nucleic acid solution；
Preferably, the volume ratio of adjunction head product and magnetic bead is 1:(0.5-0.8 in step (1 ')), in step (2 ') supernatant with
The volume ratio of magnetic bead is 1:(1-1.5).
6. method according to claim 5, which is characterized in that the volume ratio of adjunction head product and magnetic bead in step (1 ')
For 1:(0.6-0.8), preferably 1:0.7；Preferably, step (1 ') the first nucleic acid solution amplifying nucleic acid fragment length is
400bp or more, preferably 400-1000bp；
Preferably, the volume ratio of supernatant and magnetic bead is 1:(1-1.3 in step (2 ')), preferably 1:1.1；
Preferably, step (2 ') the second nucleic acid solution amplifying nucleic acid fragment length is 400bp hereinafter, preferably 50-400bp,
Further preferably 75-200bp.
7. the method according to any one of claim 5-6, which is characterized in that molten using the first nucleic acid described in rolling circle amplification
When nucleic acid in liquid, the amplified reaction time is 35-45min, preferably 40min；
When preferably, using the nucleic acid in the second nucleic acid solution described in rolling circle amplification, the amplified reaction time is 15-25min, preferably
For 20min.
8. a kind of sequencing library, which is characterized in that be made by method of any of claims 1-7.
9. a kind of method for nucleic acid sequencing, which is characterized in that including the step that sequencing library according to any one of claims 8 is sequenced
Suddenly；
Preferably, it is sequenced using single stranded circle library microarray dataset；It is further preferred that using the survey of BGISEQ series
Sequence platform is sequenced；
Preferably, further include the steps that sequencing result is assembled and/or spliced.
10. method as claimed in any one of claims 1-9 wherein is for constructing poor genetic test library and/or HLA gene point
Type detects library.
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