CN104561294B - The construction method and sequence measurement of Genotyping sequencing library - Google Patents
The construction method and sequence measurement of Genotyping sequencing library Download PDFInfo
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Abstract
The invention discloses a kind of construction method and sequence measurement of Genotyping sequencing library.Wherein, construction method comprises the following steps：S1, while first time digestion the first endonuclease bamhi of generation is carried out to genomic DNA, sample label sequence is connected at the both ends of the first endonuclease bamhi, obtains the first endonuclease bamhi of tape label；S2, enter performing PCR amplification to the first endonuclease bamhi using the amplimer with library sequence label, obtain Genotyping sequencing library.The above-mentioned construction method of the present invention, by directly connecting sample label sequence at endonuclease bamhi both ends while first time digestion produces endonuclease bamhi, constructed library is enabled to be directly entered the reading of purpose endonuclease bamhi sequence after sample label information is read when upper machine is sequenced, and then cause the reading length increase of purpose endonuclease bamhi sequence, reduce invalid data amount, improve the effective dose of sequencing data.
Description
Technical field
The present invention relates to high-flux sequence field, in particular to a kind of construction method of Genotyping sequencing library
And sequence measurement.
Background technology
GBS (Genotyping-by-Sequencing) technology, which is mainly used in, to be carried out molecular markers development to sample, surpasses
Dense genetic map structure, population genetic analysis and colony GWAS analyses etc..Based on SLAF-seq (Specific Length
Amplified Fragments sequencing) it is a kind of method of extensive sample gene parting, it is that base is sequenced in two generations
Simplification gene order-checking method of one to grow up on plinth based on full-length genome restriction enzyme site.SLAF-seq general principle
It is：Digestion is carried out to genome DNA sample using restriction enzyme, genome complexity is reduced and carries out high-flux sequence,
And then extensive sample is marked exploitation and genetic map drafting, whole-genome association etc..
The major experimental flow of SLAF-seq library constructions is as follows：First, is carried out to genome with the first restriction endonuclease
Digestion, the genomic fragment both ends after digestion respectively connect the preceding paragraph catenation sequence, and the catenation sequence at both ends is identical, connection
After the completion of, 65 DEG C inactivate enzyme, terminate digestion coupled reaction；Then, above-mentioned connection sequence is connected with above-mentioned by PCR method
The both ends of the first time endonuclease bamhi of row connect sample label sequence；Then, with second and the third restriction enzyme pair
The genomic DNA endonuclease bamhi of the sequence to be formed and sample label sequence is combined with particular bases in the both ends that previous step obtains
Second of digestion is carried out, with to second and/or the third restriction enzyme digestion sites in such endonuclease bamhi be present
Fragment is removed；Then, the product after second of digestion is entered into performing PCR amplification, and it is identical in the connection of the both ends of PCR primer
Sample label sequence (barcode sequences) to distinguish samples sources；Next, using Omega purification kits (E.Z.N.ACycle Pure Kit) purified pcr product, mixed pond then is carried out to the above-mentioned PCR primer after purification of different samples；Most
Afterwards, solexa modular connection is connected；Connection product carries out 2% agarose gel electrophoresis after purification；Select 450-500bp's
Scope carries out cutting glue, and entering performing PCR again with the product after Qiagen glue reclaim kit recovery expands, linking library sequence label
(index)；Then Piece Selection is carried out again, and gel extraction after purification can outbound.
However, not only Library development flow is numerous for the construction method of above-mentioned SLAF-seq Genotypings sequencing library of the prior art
It is trivial, the time length, and in constructed library can with the limited amount of sample mixing, the sequencing data obtained after sequencing it is of low quality,
Often result in the waste of sequencing data.
Therefore, it is still necessary to which a kind of new library constructing method for carrying out parting to gene by being sequenced, on the one hand letter are provided
Change Library development flow, shortening is built place and taken time；On the other hand the quality of library sequencing data is improved, improves valid data amount.
The content of the invention
It is a primary object of the present invention to provide a kind of construction method and sequence measurement of Genotyping sequencing library, to carry
The effective dose of high library sequencing data.
To achieve these goals, according to an aspect of the invention, there is provided a kind of structure of Genotyping sequencing library
Construction method, the construction method comprise the following steps：S1, the first endonuclease bamhi is produced carrying out first time digestion to genomic DNA
While, sample label sequence is connected at the both ends of the first endonuclease bamhi, obtains the first endonuclease bamhi of tape label；And S2,
Enter performing PCR amplification to the first endonuclease bamhi using the amplimer with library sequence label, obtain Genotyping sequencing library.
Further, in step sl, the sequence that sample label sequence is formed for 6~12 base random combines, and phase
The type of adjacent base is different.
Further, sample label sequence also includes joint sequence on the direction away from the first endonuclease bamhi.
Further, when the restriction endonuclease used in first time digestion is two kinds of restriction endonucleases that can produce different cohesive ends
When, step S1 connects different sample label sequences at the both ends of the first endonuclease bamhi, obtains the first endonuclease bamhi of tape label.
Further, when the quantity of the first endonuclease bamhi caused by first time digestion is more than required quantity, structure side
Method also includes：The step of second of digestion purifies is carried out to the first endonuclease bamhi of tape label, obtains removing the band of unnecessary fragment
First endonuclease bamhi of label.
Further, the restriction endonuclease used in the step of second of digestion purifies is one or two kinds of, and second of enzyme
Restriction endonuclease used in the step of cutting purifying is different from the restriction endonuclease used in first time digestion step.
Further, step S2 includes：Performing PCR is entered to the second endonuclease bamhi using the amplimer with library sequence label
Amplification, obtain expanding library；And amplification library is purified using isometric magnetic bead, obtain Genotyping sequencing text
Storehouse.
Further, in step s 2, the amplimer with library sequence label includes sense primer and anti-sense primer, on
Trip primer is P5 sequences, and anti-sense primer is P7 sequences and library sequence label, and library sequence label is 6~12 base random groups
Close the sequence formed.
According to another aspect of the present invention, there is provided a kind of sequence measurement of Genotyping sequencing library, method includes will
Genotyping sequencing library carries out the step of machine sequencing, before the step of upper machine is sequenced, in addition to by multiple Genotypings
Sequencing library carries out the step of mixed pond, wherein, multiple Genotyping sequencing libraries using any of the above-described kind of construction method structure and
Into.
Further, the step of multiple Genotyping sequencing libraries being carried out into mixed pond includes：Multiple Genotypings are sequenced
Library is quantified；And equivalent is carried out to multiple Genotyping sequencing libraries and mixes pond.
Apply the technical scheme of the present invention, by first time digestion produce endonuclease bamhi while at endonuclease bamhi both ends
Directly connect sample label sequence so that constructed library can be straight after sample label information is read when upper machine is sequenced
Tap into the reading into purpose endonuclease bamhi sequence so that the length increase of purpose endonuclease bamhi sequence in sequencing data, reduce
Invalid data amount, improve the effective dose of sequencing data；Meanwhile banking process of the invention can be by first time digestion and sample
Sequence label is connected in same step and completed, and not only simplify Library development flow, and improves and build storehouse efficiency.
Brief description of the drawings
The Figure of description for forming the part of the application is used for providing a further understanding of the present invention, and of the invention shows
Meaning property embodiment and its illustrate be used for explain the present invention, do not form inappropriate limitation of the present invention.In the accompanying drawings：
Fig. 1 shows the stream of the construction method of Genotyping sequencing library in a kind of preferred embodiment according to the present invention
Journey schematic diagram；And
Fig. 2 is shown in experiment eight of the invention to the result figure of library size detection.
Embodiment
It should be noted that in the case where not conflicting, the feature in embodiment and embodiment in the application can phase
Mutually combination.Describe the present invention in detail below with reference to the accompanying drawings and in conjunction with the embodiments.
It is cumbersome for Library development flow existing for the construction method of Genotyping sequencing library in the prior art, time length, and
The sequencing data that constructed library is sequenced to obtain is of low quality, the shortcomings of causing data to waste, of the invention a kind of typical
In embodiment, there is provided a kind of construction method of Genotyping sequencing library, as shown in figure 1, this method comprises the following steps：
S1, while first time digestion the first endonuclease bamhi of generation is carried out to genomic DNA, connected at the both ends of the first endonuclease bamhi
Sample label sequence, obtain the first endonuclease bamhi of tape label；S2, using the amplimer with library sequence label to the first enzyme
Section section enters performing PCR amplification, obtains Genotyping sequencing library.
The construction method of the said gene parting sequencing library of the present invention, by producing endonuclease bamhi in first time digestion
Sample label sequence is directly connected at endonuclease bamhi both ends so that constructed library can read when upper machine is sequenced simultaneously
The reading of purpose endonuclease bamhi sequence is directly entered after sample label information so that purpose endonuclease bamhi sequence in sequencing data
Length increase, reduce invalid data amount, improve the effective dose of sequencing data；Meanwhile banking process of the invention can
First time digestion is connected in same step with sample label sequence and completed, not only simplify Library development flow, and improve
Build storehouse efficiency.
In the above-mentioned construction method of the present invention, above-mentioned sample label sequence is connected directly between the two of first time endonuclease bamhi
End, and the step of the prior art is that one section of fixed sequence program has been connected at the both ends of first time endonuclease bamhi, the section fixes sequence
The sequence that the cohesive end of row and endonuclease bamhi is formed no longer is the restriction enzyme site of the first restriction endonuclease, so as to prevent in first time enzyme
Cut and be again coupled to and (connect certainly) between endonuclease bamhi during connecting；Then again by PCR method by sample label sequence
Connect.The present invention is above-mentioned directly to connect sample label sequence at the both ends of endonuclease bamhi, although being inevitably present very small amount
Endonuclease bamhi between be again coupled to, but in the digestion system, digestion is with being dynamic process from connecting, in sample label sequence
In the case that the quantity of row is much larger than the quantity for the endonuclease bamhi being again coupled to, it is again coupled to ignore between endonuclease bamhi
Disregard.
The sample label sequence of above-mentioned first time endonuclease bamhi both ends connection, can be according to the more of pond sample size to be mixed
It is few to determine that sequence label used is identical or different.When identical, the sequence label at both ends can only one sample of mark, mixed in library
It can mix that the number of samples in pond is relatively fewer, and sequencing throughput is relatively low during pond.When different, the sequence label at both ends can mark
Two different samples, the number in library Suo Nenghun ponds greatly increase, and sequencing throughput also greatly improves, such as, if sample mark
The number for signing sequence is 10, when both ends sample label sequence is identical, is only capable of the library of 10 samples of mark, then mixed Chi Shiye
It is only capable of mixing the library of 10 samples；However, when the sample label sequence difference at both ends, 10 same sample label sequence energy
Enough combinations form the combination of 90 groups of difference sample label sequences, you can so that the flux of sequencing improves 9 times.Of the invention a kind of excellent
In the embodiment of choosing, in above-mentioned steps S1, sample label sequence is the sequence that 6~12 base random combines are formed, and adjacent
The type of base is different；It is further preferred that the sample label sequence at the both ends of above-mentioned endonuclease bamhi is different.Using adjacent base type not
Same sequence label can improve the sequencing quality of sample label sequence.
The present invention above-mentioned construction method in, above-mentioned sample label sequence can in the form of individual with the first digestion piece
Section connection, it can also close with joint sequence and be connected in a sequence with the first endonuclease bamhi.It is a kind of preferable real in the present invention
Apply in example, sample label sequence also includes joint sequence on the direction away from the first endonuclease bamhi, i.e., sample label sequence and
Joint sequence is closed in a sequence, so can complete digestion, sample label sequence by first step digestion Connection Step
Connection and the connection of joint sequence, simplify Library development flow, and storehouse efficiency is built in raising.
In the above-mentioned construction method of the present invention, the selection of restriction endonuclease is determined according to the quantity of endonuclease bamhi seeking to obtain
It is fixed.In a kind of preferred embodiment of the present invention, the restriction endonuclease used in above-mentioned first time digestion can produce different viscous for two kinds
The restriction endonuclease of property end, step S1 connect different sample label sequences at the both ends of the first endonuclease bamhi, obtain tape label
First endonuclease bamhi.When carrying out digestion using two kinds of different restriction endonucleases, caused endonuclease bamhi has three kinds, the first：
The both ends of endonuclease bamhi are all the cohesive ends after the first endonuclease digestion；Second：The both ends of endonuclease bamhi are all second
Cohesive end after kind endonuclease digestion；The third：Both ends one end of endonuclease bamhi is the viscosity after the first endonuclease digestion
End, the other end are the cohesive ends after second of endonuclease digestion.When being attached with different sample label sequences,
The third endonuclease bamhi can be screened in follow-up PCR amplification steps and carry out library construction sequencing.
In the above-mentioned construction method of the present invention, the quantity of the restriction endonuclease used in above-mentioned first time digestion step can be with
The value volume and range of product of how much carry out reasonable selection restriction endonucleases of the quantity of the fragment obtained according to be intended to digestion.When first time digestion
When the quantity of caused first endonuclease bamhi is more than required quantity, construction method also includes：To the first digestion piece of tape label
The step of second of digestion of Duan Jinhang purifies, obtain removing the first endonuclease bamhi of the tape label of unnecessary fragment.Pass through second
The sequence that digestion includes first time endonuclease bamhi the restriction enzyme site of the restriction endonuclease used in second of digestion removes, and it is suitable to obtain
The endonuclease bamhi of quantity.Therefore, the restriction endonuclease used in the step of second of digestion purifies and institute in first time digestion step
Restriction endonuclease used in the step of restriction endonuclease used is different, and second of digestion purifies is one or two.
In the above-mentioned construction method of the present invention, the step of second of digestion in restriction endonuclease used be for one or two
It is adjusted according to the number of the quantity for the first endonuclease bamhi to be removed.In a kind of preferred embodiment of the present invention, the
Restriction endonuclease in digestion step is Mse I and PstI, and restriction endonuclease used is Nla III and EcoR in the second digestion step
Ⅰ.This frequency of use of several enzymes in actual library construction is higher.
In the above-mentioned construction method of the present invention, in step s 2, the amplimer with library sequence label includes upstream
Primer and anti-sense primer, sense primer are P5 sequences, and anti-sense primer is P7 sequences and library sequence label, and library sequence label is
The sequence that 6~12 base random combines are formed.With the above-mentioned amplimer with library sequence label can with it is conventional
Other reagents combination of Illumina microarray datasets, simplifies sequencing flow, improves sequencing efficiency.
In the above-mentioned construction method of the present invention, the step of above-mentioned steps S1 can connect when prior art is in digestion
Suitably adjusted, it is applied to the change of the sample label sequence of the present invention.In a kind of preferable implementation of the invention
In example, above-mentioned steps S1 comprises the following steps：By the first restriction endonuclease, DNA ligase and sample label sequence and genomic DNA
Mixing, obtains mixture；Digestion coupled reaction is carried out to mixture, obtains the first endonuclease bamhi of both ends belt lacing.By digestion
Connection raw material used, which is mixed into same system, to react, and can not only realize the purpose of digestion connection, and can also improve effect
Rate.In a kind of preferred embodiment of the present invention, the outer end of above-mentioned sample label sequence further comprises joint sequence, thus,
The connection of joint can also be realized by above-mentioned step digestion coupled reaction, more saving builds place and uses the time.
In the above-mentioned construction method of the present invention, after first time digestion and second of digestion step, in addition to the
The step of restriction endonuclease in digestion and/or second of digestion step carries out denaturation treatment, it is preferred to use at 65~95 DEG C
Denaturation treatment is carried out under hot conditions.Because in above-mentioned digestion step, the enzyme in digestion system can play the work of digestion all the time
With, after digestion is complete, to above-mentioned restriction endonuclease carry out deactivation, be to prevent that reaction condition is not enzyme in subsequent steps
The suitable condition of reaction is cut, the presence of restriction endonuclease can have a negative impact to endonuclease bamhi.And in 65~95 DEG C of hot conditions
Lower progress denaturation treatment is convenient, simple, quick.
In the above-mentioned construction method of the present invention, in order to further improve effective profit of amplimer in following amplification step
It is more excellent preferably after step S1, and before step S2, in addition to the step of purified to the second endonuclease bamhi with rate
Choosing purifying is purified using magnetic bead to the second endonuclease bamhi.Second endonuclease bamhi is purified, can be by the second restriction endonuclease
And/or the 3rd the fragment of restriction endonuclease cut-out removed, the only remaining sequence that can not be cut off so that the second endonuclease bamhi it is pure
Du Genggao.Purified using magnetic bead, purification efficiency is high and time saving.
In the above-mentioned construction method of the present invention, step S2 includes：Using the amplimer with library sequence label to
Two endonuclease bamhis enter performing PCR amplification, obtain expanding library；Amplification library is purified using isometric magnetic bead, obtains base
Because of parting sequencing library.Amplification library is purified by using isometric magnetic bead, can be to being walked in amplification library in amplification
Suddenly enzyme or primer for introducing and other impurities are removed, while further optimize enrichment to the fragment of specific library size.
In another preferred embodiment of the present invention, a kind of sequencing side of Genotyping sequencing library is additionally provided
Method, this method includes Genotyping sequencing library carrying out the step of upper machine is sequenced, before the step of upper machine is sequenced, in addition to
The step of multiple Genotyping sequencing libraries are subjected to mixed pond, wherein, multiple Genotyping sequencing libraries utilize any of the above-described kind
Construction method is built-up.Surveyed using multiple Genotyping sequencing libraries constructed by the above-mentioned construction method of the present invention in upper machine
Before sequence, the flux of sequencing can be improved by mixing pond.Especially, when purpose endonuclease bamhi in multiple Genotyping sequencing libraries
Both ends connected sample label sequence difference when, the mixed pond quantity in library can be greatly improved.
In the above-mentioned sequence measurement of the present invention, the step of multiple Genotyping sequencing libraries are carried out into mixed pond, can basis
The conventional experience in this area carries out mixed pond, and mixed pond is carried out after can also quantifying.The step of preferably above-mentioned mixed pond of the invention, includes：
Multiple Genotyping sequencing libraries are quantified；And equivalent is carried out to multiple Genotyping sequencing libraries and mixes pond.By right
Library is quantified, and is then mixed pond according to each library equivalent, can be made the survey from different samples in sequencing the data obtained
Sequence data volume is relatively impartial, and the validity of data is higher.
Further illustrate beneficial effects of the present invention below in conjunction with specific embodiments, the following example is according to Fig. 1
Shown flow carries out building storehouse：
Test one first digestion with restriction enzyme genomic DNA, sample label sequence and jointing
1. being quantified by Qubit (fluorescent quantitation meter, life technologies) to genomic DNA, source is determined
In the concentration of 22 samples of corn be respectively 40.7ng/ μ l, 22.7ng/ μ l, 29.5ng/ μ l, 23.2ng/ μ l, 20.3ng/ μ
l、22.9ng/μl、26.3ng/μl、37.6ng/μl、34.6ng/μl、26.6ng/μl、35.5ng/μl、21.4ng/μl、
26.8ng/μl、24.6ng/μl、27.0ng/μl、20.0ng/μl、22.0ng/μl、27.4ng/μl、18.4ng/μl、20.3ng/
μl、21.4ng/μl、20.2ng/μl；
2. digestion, digestion system such as table 1 below are carried out to genomic DNA with restriction enzyme Mse I：
Table 1：
| Genomic DNA | 150ng |
| 10 × cutsmart buffer solutions | 5μl |
| MseⅠ(10000U/ml) | 0.5μl |
| PstI-HF(20000U/ml) | 1μl |
| ATP(10mM) | 5μl |
| The Y-shaped connector sequences 1 (10 μM) of Mse I | 2μl |
| PstI-HF joint sequences 2 (500nM) | 1ul |
| T4DNA ligases (40000U/ml) | 0.5μl |
| Deionized water is mended to cumulative volume | 50μl |
Wherein, the particular sequence of joint is：The sequence of the positive-sense strand of joint 1 such as SEQ ID NO：Shown in 1：5′-
ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNN-3’；Wherein, NNNNNN represents the sample label sequence at 5 ' ends,
Positive sequencing primer sequence is shown in 12 bases of black runic sign；And ACACTCTTTCCCTACACGAC is P5 joints
The partial sequence complementarity of amplimer in a part for sequence, with follow-up PCR amplification steps.
Equally, the sequence of the positive-sense strand of joint 2 such as SEQ ID NO：Shown in 2：5’-(PO4)
TANNNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTC-3′；Wherein, NNNNNN represents the sample label sequence at 3 ' ends
Reverse sequencing primer sequence is shown in row, 12 bases of black runic sign；And ACACGTCTGAACTCCAGTC is P7 connects
The partial sequence complementarity of amplimer in a part for header sequence, with follow-up PCR amplification steps.
N represents any one of A, T, C, G base in above-mentioned joint sequence, the both ends of each sample in 22 samples
Sequence label is different, and the sequence label of each sample is also different, contained each sample both ends in specific each joint sequence
Sequence label is referring to table 2.
Table 2：
| Sample number | 5 ' end sample sequence labels | 3 ' end sample sequence labels |
| Sample 1 | CAGATC | GATCTG |
| Sample 2 | GATCAG | CTGATC |
| Sample 3 | CTTGTA | TACAAG |
| Sample 4 | ATGTCA | TGACAT |
| Sample 5 | GTAGAG | CTCTAC |
| Sample 6 | CGTACG | CGTACG |
| Sample 7 | GGTAGC | GCTACC |
| Sample 8 | ATGAGC | GCTCAT |
| Sample 9 | CACTCA | TGAGTG |
| Sample 10 | CATGGC | GCCATG |
| Sample 11 | CTATAC | GTATAG |
| Sample 12 | CTCAGA | TCTGAG |
| Sample 13 | GACGAC | GTCGTC |
| Sample 14 | TCGGCA | TGCCGA |
| Sample 15 | GCTCCA | CTATCT |
| Sample 16 | AGATAG | TATATC |
| Sample 17 | GATATA | TTCTCG |
| Sample 18 | CGAGAA | TGCGCT |
| Sample 19 | AGCGCA | GACGAT |
| Sample 20 | ATCGTC | GAGCGC |
| Sample 21 | GCGCTC | GAGTCT |
| Sample 22 | TGCATA | TATGCA |
3. after above-mentioned system is fully mixed, it is placed in PCR instrument, 37 DEG C of reactions 6h, 65 degrees Celsius of denaturation treatment 20min；
Test 2 second of digestions
1. the endonuclease bamhi quantity obtained according to Maize genome size and be intended to digestion, choose the limitation shown in table 3 below
Property restriction endonuclease, carries out secondary digestion, polishing digestion linked system to 60 μ l.The system of second of endonuclease reaction such as table 3 below：
Table 3：
| 10 × cutsmart buffer solutions | 1μl |
| NlaⅢ(10000U/ml) | 0.5μl |
| EcoRⅠ(10000U/ml) | 0.5μl |
| Deionized water is mended to cumulative volume | 10μl |
2. after above-mentioned system is fully mixed, it is placed in PCR instrument, 37 DEG C are reacted 6h or stayed overnight, 65 DEG C of denaturation treatments
20min；
Test three digestions connection end-product purifying
With the XP magnetic beads for purifying of 0.8 times of volume once, dissolved using 50 μ l nuclease-free waters；Then again with 0.8 times
The XP magnetic beads for purifying of volume once, is dissolved using 20 μ l nuclease-free waters.
Test four PCR amplification linking library sequence labels
1. a pair above-mentioned purified product enters performing PCR amplification, reaction system, reaction system such as table 4 below are prepared on ice：
Table 4：
| The product of previous step after purification | 2~5ng |
| Phusion MM(2×) | 25μl |
| Universal Primer(25μM) | 1μl |
| Index Primer(25μM) | 1μl |
| Deionized water | 22μl |
| Altogether | 50μl |
Wherein, the upstream sequence of the amplimer sequence with library sequence label such as SEQ ID NO：Shown in 3：
5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’；
The downstream sequence of amplimer sequence with library sequence label such as SEQ ID NO：Shown in 4：5′-
CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’
2. after above-mentioned reaction system is fully mixed, it is placed in PCR instrument, the program such as table 5 below of PCR reactions：
Table 5：
Test five PCR primer magnetic beads for purifying
Above-mentioned PCR primer once, is dissolved using isometric XP magnetic beads for purifying using 30 μ l nuclease-free waters.
The quantitatively mixed pond of experiment six, Piece Selection
1. concentration mensuration is carried out to above-mentioned each sample after purification using Qubit；
2. carrying out equivalent according to the concentration measured mixes pond, sample mixing sample is concentrated using instrument is concentrated in vacuo, is concentrated into
Volume is 30ul.
3. the agarose that concentration is 2% carries out electrophoresis, 2 hours of 100 volts of electrophoresis, gel extraction 375~400bp models
Enclose, reclaimed using gel reclaims kit (Qiagen), eluted using 50 μ l nuclease-free waters.
4. using isometric above-mentioned recovery product of XP magnetic beads for purifying, dissolved using 20 μ l nuclease-free waters.
Test seven library outbound censorships
1 μ l are taken to carry out Qubit (fluorescent quantitation meter) quantitative, it is standby that with nuclease-free water library is diluted into 2ng/ μ l.It is suitable
Library is measured according to Qubit concentration dilutions to 2ng/ μ l, hands over supreme unit to carry out the upper machine of storehouse inspection.
Test eight library inserts and Concentration Testing
Library inserts size is detected with the biological analysers of Aglilent 2100, it is dense to library with q-PCR instrument
Degree is detected；Testing result is shown in Fig. 2, from figure 2 it can be seen that using the size in the library constructed by the present invention in 380bp
Left and right, no miscellaneous peak, main peak are obvious, non junction and primer dimer, meet the Insert Fragment size that both-end sequencing requires.
Test nine storehouses and examine qualified upper machine
Find out that the above embodiment of the present invention passes through to base in high-flux sequence field from the above-mentioned experimental result of the present invention
It is optimized and adjusts in the library constructing method of SLAF-seq technologies, devises the joint containing sample label sequence, profit
With the first endonuclease digestion genomic DNA, then realized by being connected in digestion in the connection of genomic fragment both ends containing not
With the joint of sample sequence label information；In compared with prior art, based on SLAF-seq (Specific Length
Amplified Fragments sequencing) extensive sample gene classifying method in, the process that is connected in digestion
Simply connect the sequence that the preceding paragraph is fixed, and sample label sequence is and the genome by follow-up PCR reaction formings
What DNA fragmentation both ends connected is the banking process of identical sample label sequence, both saves the time, adds sample mixing number again
Mesh, reduce and build Kucheng's sheet.
Moreover, the skill based on SLAF-seq (Specific Length Amplified Fragments sequencing)
What the library of art structure connected at the both ends of DNA fragmentation is one section of fixed sequence (about 20 bases).In sequencing procedure
In, preceding 6 bases survey of beginning is sample label sequence information, and what is surveyed since the 7th is this fixed sequence program, treats this
One fixed sequence program is only purpose fragment sequence information after having surveyed, thus, this section of fixed sequence program in sequencing data belongs to nothing
Data are imitated, waste data volume.And using the library constructed by the present invention start sequencing when before 6 bases be also survey sample label
Sequence information, but due to being not present that section of fixed sequence program between sample label sequence and purpose fragment sequence, thus from the 7th alkali
The data that base is surveyed are the sequencing datas of purpose fragment.Thus, using constructed by library constructing method provided by the present invention
Library can improve sequencing quality in sequencing, while the waste of data will not be also caused, so as to improve having for sequencing data
Effect amount.
The preferred embodiments of the present invention are these are only, are not intended to limit the invention, for those skilled in the art
For member, the present invention can have various modifications and variations.Any modification within the spirit and principles of the invention, being made,
Equivalent substitution, improvement etc., should be included in the scope of the protection.
Claims (10)
1. a kind of construction method of Genotyping sequencing library, it is characterised in that the construction method comprises the following steps：
S1, while first time digestion the first endonuclease bamhi of generation is carried out to genomic DNA, in first endonuclease bamhi
Both ends connect sample label sequence, obtain the first endonuclease bamhi of tape label；And
S2, enter performing PCR amplification to first endonuclease bamhi using the amplimer with library sequence label, obtain the gene
Parting sequencing library；
The sample label sequence is connected directly between the both ends of the first time endonuclease bamhi, and at least sample mark of one end
Sign between sequence and first endonuclease bamhi without intervening sequence.
2. construction method according to claim 1, it is characterised in that in the step S1, the sample label sequence
The sequence formed for 6~12 base random combines, and the type of adjacent base is different.
3. construction method according to claim 1 or 2, it is characterised in that the sample label sequence is away from described the
Also include joint sequence on the direction of one endonuclease bamhi.
4. construction method according to claim 1, it is characterised in that when the restriction endonuclease used in the first time digestion is two
When kind can produce the restriction endonuclease of different cohesive ends, the step S1 connects different at the both ends of first endonuclease bamhi
Sample label sequence, obtain the first endonuclease bamhi of the tape label.
5. construction method according to claim 1, it is characterised in that when the first digestion piece caused by the first time digestion
When the quantity of section is more than required quantity, the construction method also includes：
The step of second of digestion purifies is carried out to the first endonuclease bamhi of the tape label, obtains removing the described of unnecessary fragment
First endonuclease bamhi of tape label.
6. construction method according to claim 5, it is characterised in that used in the step of second of digestion purifies
Restriction endonuclease be one or two, and the step of second of digestion purifies used in restriction endonuclease and the first time enzyme
The restriction endonuclease cut used in step is different.
7. construction method according to claim 1, it is characterised in that the step S2 includes：
Enter performing PCR amplification to first endonuclease bamhi using the amplimer with library sequence label, obtain expanding library；With
And
The amplification library is purified using isometric magnetic bead, obtains the Genotyping sequencing library.
8. the construction method according to claim 1 or 7, it is characterised in that in the step S2, band library label
The amplimer of sequence includes sense primer and anti-sense primer, and the sense primer is P5 sequences, and the anti-sense primer is P7 sequences
Row and library sequence label, the library sequence label are the sequence that 6~12 base random combines are formed.
9. a kind of sequence measurement of Genotyping sequencing library, methods described includes carrying out the Genotyping sequencing library
The step of machine is sequenced, it is characterised in that be sequenced before the step of machine on described is sequenced, in addition to by multiple Genotypings
Library carries out the step of mixed pond, wherein, multiple Genotyping sequencing libraries are using any one of claim 1 to 8
Construction method it is built-up.
10. sequence measurement according to claim 9, it is characterised in that carry out multiple Genotyping sequencing libraries
The step of mixed pond, includes：
Multiple Genotyping sequencing libraries are quantified；And
Equivalent is carried out to the multiple Genotyping sequencing library and mixes pond.
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| CN105602936A (en) * | 2015-11-18 | 2016-05-25 | 中国人民解放军第四军医大学 | Construction method of dual-barcode next-generation sequencing library |
| CN105401222A (en) * | 2015-12-30 | 2016-03-16 | 安诺优达基因科技(北京)有限公司 | Method for constructing DNA library used for sequencing |
| CN106086189A (en) * | 2016-06-22 | 2016-11-09 | 杭州和壹基因科技有限公司 | A kind of RAD banking process improving data homogeneity |
| CN106367485B (en) | 2016-08-29 | 2019-04-26 | 厦门艾德生物医药科技股份有限公司 | Double label connector groups of a kind of more positioning for detecting gene mutation and its preparation method and application |
| CN106676099B (en) * | 2016-12-21 | 2019-07-02 | 中国水稻研究所 | Construct the method and kit for simplifying genomic library |
| CN107368706A (en) * | 2017-06-27 | 2017-11-21 | 中国水稻研究所 | Sequencing data interpretation of result method and apparatus, sequencing library structure and sequence measurement |
| CN108315240A (en) * | 2018-01-19 | 2018-07-24 | 武汉永瑞康华医学检验所有限公司 | A kind of flow quality control standard technology can be used for gene sequencing |
| CN109295050A (en) * | 2018-09-26 | 2019-02-01 | 刘强 | Both-end label specific linkers, kit and the banking process in the library Blood Trace cfDNA |
| CN109517882B (en) * | 2018-11-09 | 2021-08-17 | 广州燃石医学检验所有限公司 | Quality control method for detecting unique double-end library label combination and application |
| CN111524552B (en) * | 2020-04-24 | 2021-05-11 | 深圳市儒翰基因科技有限公司 | Simplified genome sequencing library construction and analysis method, detection equipment and storage medium |
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