CN106350590A - DNA library construction method for high-throughput sequencing - Google Patents
DNA library construction method for high-throughput sequencing Download PDFInfo
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Abstract
The invention discloses a DNA library construction method for high-throughput sequencing. The DNA library construction method includes the steps of carrying out multiple PCR (polymerase chain reaction) to a DNA template obtained from a to-be-tested sample through multiple pairs of fusion primers, then recycling PCR products to obtain a sequencing library. The multiple pairs of fusion primers respectively aim at different target fragments of the DNA template, and each fusion primer comprises specificity primer sequence aiming at the target fragment and linker sequence for sequencing. The multiple PCR is carried out on reaction conditions: reacting at 95 DEG C for 2 minutes; reacting within 38 cycles, wherein each cycle includes processes of preserving at the temperature of 95 DEG C for 30 seconds, cooling to the temperature from 76 DEG C to 55-58 DEG C slowly at the speed of 0.1 DEG C each second, holding the temperature for 20 seconds once cooling to the temperature of 58 DEG C, and then preserving at 72 DEG C for 30 seconds; after that, preserving at 72 DEG C for 2 minutes, and finally preserving at 4 DEG C. The invention further discloses application of the library construction method in testing STR locus and paternity identification on the basis of high-throughput sequencing.
Description
Technical field
The present invention relates to field of biological detection is and in particular to a kind of dna library constructing method for high-flux sequence.
Background technology
The research and analysis object of legal medical expert's dna inspection technology is mainly the polymorphism of biological internal dna.In general, base
The dna labelling of cause or noncoding region position on chromosome is referred to as locus, and is located on same locus different sequences
Gene be referred to as allele.Dna polymorphism just refers to there are multiple allele as the locus of genetic marker, analysis
On locus, the difference of allele is exactly to realize the same Basic of Biology [1].
STR (short tandem repeats, str) [2] in microsatellite dna is that a kind of dna is polymorphic
Property, be widely present on 23 pairs of chromosomes of human genome, generally by 2-6bp recurring units (or claim core sequence, core
Sequence) form, the repetition number that the difference between Different Individual (or allele) typically only shows as recurring units is poor
Different.Str locus are a kind of important genetic markers, and on the high locus of polymorphism degree, its individual identification ability is strong, and
And simply can use pcr technology for detection out, thus be widely used.Str locus have the following characteristics that str polymorphism
Locus quantity is many, and fragment length is generally less than 400bp it is easy to amplification, is suitable to the inspection of micro sample；Allele size ratio
It is closer to, less allele preferential amplification is inconspicuous；The str locus fragment difference in length of different loci is less, is easy to multiple
Close amplification, improve detection efficiency.
The typing inspection technology of str locus important role in legal medical expert dna inspection, paternity identification.China is most
Number paternity identification laboratory is all used limited loci str typing method as Main Means, to different str locus polymorphic
It is analyzed, compares and calculate paternity index (pi value, the i.e. ratio of true father's probability and false father's probability).According to many in the world
The boundary line whether number laboratory [3] accepted standard (pi value >=10 000) " has one's own relation " as differentiation.
More than traditional str typing method based on pcr technology and capillary electrophoresis technique, using fluorescently-labeled primer
Sample is carried out with multiple pcr amplification, produces different size of amplified fragments and separate in capillary electrophoresis, thus realizing dividing
Type, but this method flux is relatively low, and due to analysis to as if fragment length it is impossible to detect nucleic acid primary structure further
Fine difference, limit the resolution of detection, additionally, be likely to occur stutter peak (fragment analysis when there is biased sample
When come across the small peak before main peak) interference.And sanger sequencing due to due to flux, cost it is difficult to be used for str typing
Analysis.
Secondary high throughput sequencing technologies (next generation sequencing) technology can be once parallel to tens
Ten thousand to millions of dna molecules carry out sequencing, promote the high speed development of sequencing technologies.Using secondary high-flux sequence skill
Art can be sequenced to full-length genome.When researcher is only interested in specific genome area, it is possible to use expand
Increase son sequencing (amplicon sequencing) method and only respective regions are carried out with sequencing research.By designing base interested
Because organizing the primer in region, carry out pcr amplification, target area is enriched with, be then directed to the pcr product of length-specific or catch
The fragment obtaining carries out building storehouse, and high-flux sequence simultaneously carries out sequence analysis.
Current amplicon sequencing is all by conventional banking process, after target area is enriched to by pcr amplification,
Carry out sample mixing, then carry out end reparation and add a, then adjunction head, purification of samples, reclaim purpose band, then pcr amplification, then
Run glue reclaim master tape, this conventional banking process complex steps, lose time, workload big, be unfavorable for the survey of a large amount of samples
Sequence builds storehouse.
Content of the invention
It is an object of the invention to provide a kind of construction method of amplicon sequencing library and this library constructing method exist
High-flux sequence detects the application in str locus, and the method can realize the structure of amplicon sequencing library by once amplification
Build, simplify the operating process building storehouse, reduce time and the cost building storehouse, do not affect sequencing quality simultaneously.
According to an aspect of the present invention, provide a kind of construction method of amplicon sequencing library, including using multipair fusion
The dna template that primer pair obtains from testing sample carries out multiple pcr, reclaims pcr product, obtains sequencing library；Wherein said many
To merging the different purpose fragments that primer is respectively directed in dna template, each pair merges primer and comprises successively to survey from 5' end to 3' end
Sequence joint sequence and the specific primer sequence for purpose fragment.
In preferred embodiments, multiple pcr is carried out by following reaction conditions: 95 DEG C of 2min；38 circulations, each follows
Ring is 95 DEG C of 30s, then from 76 DEG C to the arbitrary temp slow cooling 55 DEG C and 58 DEG C, 0.1 DEG C of fall per second, and mesh to be down to
Temperature after keep 20s, then 72 DEG C of 30s；72℃2min.
In the present invention, described " multipair fusion primer " refer to two to, three to or more to merge primer.
In a more preferred embodiment, multiple pcr is carried out by following reaction conditions: 95 DEG C of 2min；38 circulations, each
Circulate as 95 DEG C of 30s, the then slow cooling from 76 DEG C to 58 DEG C, 0.1 DEG C of fall per second, keep 20s after being down to 58 DEG C, then 72
℃30s；72℃2min.
In some embodiments, testing sample include but is not limited to blood, body fluid, saliva, seminal fluid, hair, muscle or
Histoorgan.In preferred embodiments, dna template is the genome dna extracting from testing sample.
In the present invention, each pair merges primer and comprises respectively for the specific forward primer sequence of purpose fragment and reversely draw
Thing sequence.
In some embodiments, comprise index sequence in sequence measuring joints sequence, to identify different dna templates, be easy to
Different sample mix are sequenced.
Merge the sequence measuring joints sequence included in primer in the present invention and may is that 5'- in forward primer
Cctctctatgggcagtcggtgat-3', 5'-ccatctcatccctgcgtgtctccgactcag-index in downstream primer
Sequence-gat-3'.
Merge sequence measuring joints sequence included in primer it may also is that 5'- in forward primer in the present invention
Aatgatacggcgaccaccgagatctacac- the first index sequence-
Acactctttccctacacgacgctcttccgatct-3', 5'-caagcagaagacggcatacgagat+ in downstream primer
Two index sequence+gtgactggagttcagacgtgtgctcttccgatct-3'.
Wherein the first index sequence is different index sequences from the 2nd index sequence.
In some embodiments, multiple pcr reaction divides one or more reaction systems to complete.
In preferred embodiments, comprise two, three, four or five in a reaction system and be directed to different mesh
Fragment fusion primer pair.
In preferred embodiments, described purpose fragment is str locus.
In preferred embodiments, purpose fragment includes two or more the str genes selected from following str locus
Seat:
csf1po(genbank x14720)、fga(genbank m64982)、th01(genbank d00269)、tpox
(genbank m68651)、d3s1358(nt_005997positions 754983-755121)、d5s818(genbank
ac008512)、d7s820(genbank ac004848)、d8s1179(genbank af216671)、 d13s317(genbank
al353628)、d16s539(genbank ac024591)、d18s51(genbank ap001534)、d21s11(genbank
ap000433)、d2s1338(genbank ac010136)、cd4(genbank m86525)、d12s391(genbank
m08921)、pla2a1(genbank m22970)、fabp(genbank m18079)、d18s865(genbank
) and vwa (genbank m25858) ac012289.12.
In a more preferred embodiment, purpose fragment includes following all str locus:
csf1po(genbank x14720)、fga(genbank m64982)、th01(genbank d00269)、tpox
(genbank m68651)、d3s1358(nt_005997positions 754983-755121)、d5s818(genbank
ac008512)、d7s820(genbank ac004848)、d8s1179(genbank af216671)、d13s317(genbank
al353628)、d16s539(genbank ac024591)、d18s51(genbank ap001534)、d21s11(genbank
ap000433)、d2s1338(genbank ac010136)、cd4(genbank m86525)、d12s391(genbank
M08921), pla2a1 (genbank m22970) and fabp (genbank m18079).
In a more preferred embodiment, purpose fragment includes following all str locus:
csf1po(genbank x14720)、fga(genbank m64982)、th01(genbank d00269)、tpox
(genbank m68651)、d3s1358(nt_005997positions 754983-755121)、d5s818(genbank
ac008512)、d7s820(genbank ac004848)、d8s1179(genbank af216671)、d13s317(genbank
al353628)、d16s539(genbank ac024591)、d18s51(genbank ap001534)、d21s11(genbank
ap000433)、d2s1338(genbank ac010136)、cd4(genbank m86525)、d12s391(genbank
M08921), d18s865 (genbank ac012289.12) and vwa (genbank m25858).
The specific forward primer sequence and the reverse primer sequences that are wherein directed to each str locus are as follows:
Csf1po: positive 5'-3':tagcaggttgctaaccaccc, reverse 5'-3':
tcagaccctgttctaagtacttc；
Fga: positive 5'-3':cccataggttttgaactcacag, reverse 5'-3':
gtgatttgtctgtaattgccagc；
Th01: positive 5'-3':gggcaaaattcaaagggtatctg, reverse 5'-3':tgcaggtcacagggaacac；
Tpox: positive 5'-3':aggcacttagggaaccctc, reverse 5'-3':tccttgtcagcgtttatttgcc；
D3s1358: positive 5'-3':caagaccctgtctcatagatag, reverse 5'-3':
tcaacagaggcttgcatgtatc；
D5s818: positive 5'-3':gtgacaagggtgattttcctctt, reverse 5'-3':
gtgattccaatcatagccacag；
D7s820: positive 5'-3':ggtcaggctgactatggag, reverse 5'-3':
tcctcattgacagaattgcacc；
D8s1179: positive 5'-3':tctttttgcccacacggcc, reverse 5'-3':
ctgtagattattttcactgtgggg；
D13s317: positive 5'-3':atttctttagtgggcatccgtg, reverse 5'-3':
ccttcaacttgggttgagcc；
D16s539: positive 5'-3':cagatcccaagctcttcctc, reverse 5'-3':
gcatgtatctatcatccatctctg；
D18s51: positive 5'-3':cacttcactctgagtgacaaattg, reverse 5'-3':
gtgtggagatgtcttacaataacag；
D21s11: positive 5'-3':tcaattccccaagtgaattgcc, reverse 5'-3':
tgttctccagagacagactaatag；
D2s1338: positive 5'-3':gtggatttggaaacagaaatggc, reverse 5'-3':
gtggcccataatcatgagttattc；
Cd4: positive 5'-3':aggggtacttgtgttaattgttgg, reverse 5'-3':
gcgttttccagtctgaaaaaagtg；
D12s391: positive 5'-3':gaatcaacaggatcaatggatgc, reverse 5'-3':
cctccatatcacttgagctaattc；
Fabp: positive 5'-3':ttgtaagctccatgaggttagag, reverse 5'-3':
agcctccctaggtcagatag；
Pla2a1: positive 5'-3':tagtatcagtttcatagggtcacc, reverse 5'-3':
agttcgtttccattgtctgtcc；
D18s865: positive 5'-3':caaatgtagatcttgggacttgtc, reverse 5'-3':
attctcaaacatccccattaccttc；
Vwa: positive 5'-3':tcagtatgtgacttggattgatc, reverse 5'-3':
caggttagatagattagacagacag.
In preferred embodiments, multiple pcr reaction divides one or more reaction systems to complete.The plurality of reaction
System includes the one or more reaction systems being selected from the group:
Comprise the reaction system merging primer and the fusion primer for d18s51 locus for tpox locus；
Comprise the reactant merging primer and the fusion primer for d21s11 locus for d8s1179 locus
System；
Comprise merging primer, the fusion primer for d18s51 locus, being directed to d8s1179 base for tpox locus
The reaction system merging primer and the fusion primer for d21s11 locus because of seat；
Comprise merging primer, the fusion primer for d3s1358 locus, being directed to for csf1po locus
The reaction system merging primer and the fusion primer for d12s391 locus of d13s317 locus；
Comprise merging primer, the fusion primer for th01 locus, being directed to d5s818 locus for fga locus
Merge primer and for pla2a1 locus fusion primer reaction system；
Comprise merging primer, the fusion primer for th01 locus, being directed to d5s818 locus for fga locus
Merge primer and for vma locus fusion primer reaction system；
Comprise merging primer, the fusion primer for d16s539 locus, being directed to for d7s820 locus
The fusion primer of d2s1338 locus, the reaction system merging primer and fabp primer for cd4 locus；With
Comprise merging primer, the fusion primer for d16s539 locus, being directed to for d7s820 locus
The fusion primer of d2s1338 locus, the fusion primer merging primer and being directed to d18s865 locus for cd4 locus
Reaction system.
In a more preferred embodiment, the plurality of reaction system includes the one or more reactants being selected from the group
System:
Comprise 1.5 μm be directed to tpox locus merge the fusion primer that primers and 3.5 μm are directed to d18s51 locus
Reaction system；
Comprise 1.5 μm of fusion primers being directed to d8s1179 locus and 3.5 μm of fusion primers for d21s11 locus
Reaction system；
Comprise 1 μm be directed to tpox locus merge primer, 1.5 μm be directed to the fusion primer of d18s51 locus, 1 μm of pin
Fusion primer to d8s1179 locus and 1.5 μm of reaction systems for the fusion primer of d21s11 locus；
Comprise 1 μm be directed to csf1po locus merge primer, 2 μm be directed to d3s1358 locus fusion primer, 1 μm
Fusion primer for d13s317 locus and 1 μm of reaction system for the fusion primer of d12s391 locus；
Comprise 1 μm be directed to csf1po locus merge primer, 1 μm be directed to d3s1358 locus fusion primer, 1 μm
Fusion primer for d13s317 locus and 2 μm of reaction systems for the fusion primer of d12s391 locus；
Comprise 1 μm be directed to fga locus merge primer, 1.5 μm be directed to the fusion primer of th01 locus, 1.5 μm of pins
Fusion primer to d5s818 locus and 1 μm of reaction system for the fusion primer of pla2a1 locus；
Comprise 1.5 μm be directed to fga locus merge primer, 1 μm be directed to the fusion primer of th01 locus, 1.5 μm of pins
Fusion primer to d5s818 locus and 1 μm of reaction system for the fusion primer of vma locus；
Comprise 1 μm be directed to d7s820 locus merge primer, 1 μm be directed to d16s539 locus fusion primer, 1 μm
For the fusion primer of d2s1338 locus, 1 μm of reactant merging primer and 1 μm of fabp primer for cd4 locus
System；With
Comprise 1 μm be directed to d7s820 locus merge primer, 1 μm be directed to d16s539 locus fusion primer, 1 μm
Merge primer, 1 μm of fusion primer being directed to cd4 locus and 1 μm for d2s1338 locus are directed to d18s865 locus
Fusion primer reaction system.
Other str locus can also according to circumstances be selected.Alternative str locus select to see network address: http: //
Www.cstl.nist.gov/biotech/strbase/seq_info.htm, this network address is the medicolegal authoritative net of the U.S.
Stand, which provide the str locus being related in legal medical expert's Relationship iden- tification.The reference sequences information of these str locus can
To obtain from network address http://www.cstl.nist.gov/biotech/strbase/seq_ref.htm.
The construction method of described amplicon sequencing library is applied to the high-flux sequence to any amplicon, for example, can be used for
Str locus, snp, snv, the mutation of a range of insertion and deletion etc. are detected by high-flux sequence, but not limited to this.
The method that another aspect of the present invention provides amplicon high-flux sequence, comprising: using the sequencing of above-mentioned amplicon
Library constructing method prepares sequencing library, then carries out high-flux sequence and carries out data analysiss to sequencing data.
The target fragment of described amplicon high-flux sequence including but not limited to comprises str locus or comprises snp site
Fragment.In preferred embodiments, the target fragment of described amplicon high-flux sequence is multiple str locus.
Another aspect of the present invention provides the detection method of str locus, comprising: using above-mentioned amplicon sequencing library
Construction method preparation, for the sequencing library of multiple str locus, then carries out high-flux sequence and enters line number to sequencing data
According to analysis.
In preferred embodiments, described data analysiss include obtaining the plurality of str locus core repeat sequence
Number of repetition.
In some embodiments, the shadow band that described data analysiss also include chain slipped mispairing is led to is analyzed.
In some embodiments, described data analysiss further include to obtain the mutation in the plurality of str locus
Information.
In some embodiments, the detection method of the method for amplicon high-flux sequence of the present invention and str locus
It is non-diagnostic purpose.
The method of the detection str locus of the present invention can be used for legal medical expert and other is related to the correlation of str locus detection
Field, the such as field such as gene diagnosises, genetic map construction and population genetic study.In preferred embodiments, the present invention
Detection str locus method be used for paternity identification.
The present invention also provides a kind of method for paternity test, comprising: detect gene by the detection method of above-mentioned str locus
Number of repetition in group, then calculates paternity index (pi value).
The invention has the beneficial effects as follows:
(1) present invention entirely build storehouse process only pass through once multiple pcr can complete, greatly reduce amplicon sequencing
Build time and the cost in storehouse.When being applied to str genotype analysis and the identification of parental right relation, can simple flow reduces cost.
(2) the amplicon banking process of the present invention is optimized design it is adaptable to various expansion to multiple pcr reaction condition
Increase the high-flux sequence of son, there is no selectivity to primer, all can pass through the banking process one of the present invention using different primers
Storehouse is built in secondary amplification.
Brief description
Fig. 1 is the agarose gel electrophoresis figure of multiple pcr product in embodiment 1.
Fig. 2 is the library quality inspection figure in embodiment 1.
Fig. 3 is the scattergram of different numbers of repetition in the sequencing data of each str in embodiment 1.
Fig. 4 is the polyacrylamide page gel nucleic acid electrophoretogram of multiple pcr product in embodiment 2.
Fig. 5 is the library quality inspection figure in embodiment 2.
Specific embodiment
In order to be more clearly understood that the technology contents of the present invention, it is described with reference to the accompanying drawings especially exemplified by following examples.
It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.Unreceipted in the following example
The experimental technique of actual conditions, generally according to normal condition, or according to the condition proposed by manufacturer.Used in embodiment
Various chemical reagent and biological preparation, be commercially available prod.
Embodiment 1: legal medical expert's Relationship iden- tification
The present embodiment purpose is the number of repetition of repetitive sequence in detection str, wherein using ngs sequencing scheme.
First, mouth epithelial cells collection and process
(1) buccal swab collection tube is used to gather the mouth epithelial cells of people.
(2) buccal swab genome extracts kit (dp322) is used to extract mouth epithelial cells dna.
(3) use nanodrop 2000 type spectrophotometric determination od260nm and od280nm, confirm that its purity is high, and
Measure its concentration.
2nd, multiple pcr reacts design of primers
(1) design of primers becomes to merge primer, the specific primer sequence including purpose fragment and sequence measuring joints sequence, wherein
Containing index sequence.
(2) merging primer construction is:
Forward primer: 5 '-cctctctatgggcagtcggtgat-3'+ purpose fragment specific forward primer；
Downstream primer: 5'-ccatctcatccctgcgtgtctccgactcagctaaggtaacGat-3'+ purpose fragment is special
Different in nature reverse primer；
Wherein underlined sequences are index sequence.
(3) the str locus with sequencing to be amplified and corresponding specific primer are shown in Table 1.
The corresponding specific primer of table 1str locus
| Str locus | Forward primer 5'-3' | Reverse primer 5'-3' |
| csf1po | tagcaggttgctaaccaccc | tcagaccctgttctaagtacttc |
| fga | cccataggttttgaactcacag | gtgatttgtctgtaattgccagc |
| th01 | gggcaaaattcaaagggtatctg | tgcaggtcacagggaacac |
| tpox | aggcacttagggaaccctc | tccttgtcagcgtttatttgcc |
| d3s1358 | caagaccctgtctcatagatag | tcaacagaggcttgcatgtatc |
| d5s818 | gtgacaagggtgattttcctctt | gtgattccaatcatagccacag |
| d7s820 | ggtcaggctgactatggag | tcctcattgacagaattgcacc |
| d8s1179 | tctttttgcccacacggcc | ctgtagattattttcactgtgggg |
| d13s317 | atttctttagtgggcatccgtg | ccttcaacttgggttgagcc |
| d16s539 | cagatcccaagctcttcctc | gcatgtatctatcatccatctctg |
| d18s51 | cacttcactctgagtgacaaattg | gtgtggagatgtcttacaataacag |
| d21s11 | tcaattccccaagtgaattgcc | tgttctccagagacagactaatag |
| d2s1338 | gtggatttggaaacagaaatggc | gtggcccataatcatgagttattc |
| cd4 | aggggtacttgtgttaattgttgg | gcgttttccagtctgaaaaaagtg |
| d12s391 | gaatcaacaggatcaatggatgc | cctccatatcacttgagctaattc |
| fabp | ttgtaagctccatgaggttagag | agcctccctaggtcagatag |
| pla2a1 | tagtatcagtttcatagggtcacc | agttcgtttccattgtctgtcc |
3rd, storehouse and nucleic acid agarose gel electrophoresiies are built in multiple pcr reaction
Five groups are divided to carry out multiple pcr reaction, packet situation and various primer concentration are as shown in table 2.
The multiple pcr primer of table 2 is grouped situation
Multiple pcr agents useful for same is dreamtaq green pcr master mix 2x (thermo fisher, article No.
K1081), reaction system is shown in Table 3:
The multiple pcr reaction system of table 3
Multiple pcr reaction is carried out using slow cooling method, reaction condition is as shown in table 4.
The multiple pcr reaction condition of table 4
Pcr product is entered with row agarose gel electrophoresis, the condition of electrophoresis is set as: voltage 120v, electric current 400ma,
Time is 30min, and gum concentration is that 1.5%, marker band standard is from bottom to up successively: 100bp, 200bp, 300bp,
400bp, 500bp, 700bp, 1kb, 1600bp, 2kb, 5kb, 8kb, 10kb, result is as shown in figure 1,5 groups of multiple pcr reactions are equal
Purpose band can be amplified, and the position that purpose band occurs is all in expected scope.
It was pre-mixed with sanprep pillar pcr Product Purification Kit (work, article No. b518141 are given birth in Shanghai) purification
Five groups of multiple pcr products, are illustrated to carry out purification by manufacturer, obtain the dna library for sequencing.Nanodrop spectrophotometer
The dna library concentration recording after purification is 128.73ng/ μ l, and 260/280 is worth for 1.944.
4th, library quality inspection
Library quality inspection to be completed by agilent 2100, and quality inspection result is as shown in Fig. 2 by figure it can be seen that multiple pcr
Expanding effect is more homogeneous, does not expand the Preference of very different, can directly carry out high-flux sequence.
5th, large scale sequencing
(1) carry out high-flux sequence using the dna sequencing library of above-mentioned preparation with ion torrent platform se400, sequencing
After the completion of, result is output as fastq formatted file, sequencing data amount 67m, records and matches the reads quantity of str locus and be
490536.
6th, sequencing data is processed
(1) intercept the forward and backward 20-30 base of each read thus positioning which str locus each read belongs to, if
This read cannot navigate to and then discard on target str locus.The reads number that every kind of str matches is shown in Table 5.
The reads number that each str locus of table 5 are positioned
(2) because multiple pcr reacts during primer extension, primer strand or template strand slip, and lead to one to repeat list
The base non-matching ring that position is formed, i.e. chain slipped mispairing mechanism, finally produce shadow band (stutter).For convenience of analysis shadow
The impact of band, finally to analyze the number of repetition class of the core recurring units obtaining on the corresponding reads of each str in sequencing data
Type is abscissa, and the percentage ratio of the reads number corresponding to each type, as vertical coordinate, does rectangular histogram, such as shown in Fig. 3 (a-q).
(4) number of times of each str locus recurring unit such as table 6 in test sample:
The number of times of each str locus recurring unit of table 6
| Str locus | Number of repetition |
| csf1po | 10,11 |
| fga | 21,23 |
| th01 | 7,9 |
| tpox | 8 |
| d3s1358 | 16 |
| d5s818 | 11 |
| d7s820 | 10 |
| d8s1179 | 13,14 |
| d13s317 | 8,10 |
| d16s539 | 9,10 |
| d18s51 | 18 |
| d21s11 | 31,32 |
| d2s1338 | 19,23 |
| cd4 | 5 |
| d12s391 | 18 |
| fabp | 10 |
| pla2a1 | 11,14 |
Embodiment 2: detect which kind of cell line is unknown cell line belong to
The present embodiment uses 2 unknown cell lines of people, by detecting the number of repetition of repetitive sequence in str thus confirming
Which kind of cell line is unknown cell line belong to.Wherein using ngs sequencing scheme.
First, the extraction of human cell line dna
(1) human cell line dna extracts and adopts peripheral blood dna test kit (qiagen, article No. 937236).
(2) use nanodrop 2000 type spectrophotometric determination od260nm and od280nm, confirm that its purity is high, and
Measure its concentration, ultimate density is 51ng/ μ l.
2nd, storehouse and polyacrylamide page gel nucleic acid electrophoresis are built in multiple pcr reaction
(1) design of primers becomes to merge primer, the specific primer sequence including purpose fragment and sequence measuring joints sequence, wherein
Containing index sequence.
(2) merging primer construction is:
Sample one:
Forward primer: 5 '-aatgatacggcgaccaccgagatctacac-3'+index sequences 1 (ctctctat)+
Acactctttccctacacgacgctcttccgatct+ purpose fragment specific forward primer；
Downstream primer: 5'-caagcagaagacggcatacgagat-3'+index sequence 2 (tcgcctta)+
Gtgactggagttcagacgtgtgctcttccgatct+ purpose fragment specific reverse primers；
Sample two:
Forward primer: 5 '-aatgatacggcgaccaccgagatctacac-3'+index sequences 1 (tatcctct)+
Acactctttccctacacgacgctcttccgatct+ purpose fragment specific forward primer；
Downstream primer: 5'-caagcagaagacggcatacgagat-3'+index sequence 2 (ctagtacg)+
Gtgactggagttcagacgtgtgctcttccgatct+ purpose fragment specific reverse primers；
(3) the str locus with sequencing to be amplified and corresponding specific primer are shown in Table 7.
The corresponding specific primer of table 7str locus
3rd, storehouse and polyacrylamide page gel nucleic acid electrophoresis are built in multiple pcr reaction
(1) multiple pcr reacts using dreamtaq green pcr master mix (2x) reagent (the silent winged generation that of match, goods
Number k1081) carry out pcr reaction, points of 4 groups multiple pcr react and to complete the dna library construction of target str, packet situation and each
Plant primer concentration as shown in table 8, multiple pcr reaction condition is such as the reaction condition in example 1.
The multiple pcr primer of table 8 is grouped situation
Multiple pcr reaction system and reaction condition are with example 1.
(2) product of multiple for two samples pcr reactions is all mixed, carry out polyacrylamide page gel
Nucleic acid electrophoresis, gum concentration is 8%, runs 4 hours glue time, wherein, and electric current 16ma, 20 minutes；30ma, 3 hours 40 minutes.Result
As shown in figure 4,4 groups of multiple pcr reactions all can amplify purpose band, and the position that purpose band occurs is all in expected model
In enclosing.
4th, pcr product (str correlation dna library) purification
The pcr product mixtures that the product of pcr reactions multiple to two samples all mixes carry out purification, according to
Sanprep pillar pcr Product Purification Kit (Shanghai give birth to work, article No. b518141) description operating guidance completing,
The dna library concentration that nanodrop spectrophotometer records after purification is 96.44ng/ μ l, and 260/280 is worth for 1.826.
5th, library quality inspection
(1) library quality inspection to be completed by agilent 2100, and quality inspection result is as shown in figure 5, by figure it can be seen that multiple
Pcr expanding effect is more homogeneous, does not expand the Preference of very different, can directly carry out high-flux sequence.
6th, large scale sequencing
(1) purpose dna library good for purification is carried out high-flux sequence with illumina miseq platform pe250, sequencing
After the completion of, result is output as fastq formatted file, sequencing data amount 63.6m.
(2) the reads quantity matching str locus measured by is: 686347.
7th, sequencing data analysis
(1) read that in both-end sequencing, one end can cover str core repeat sequence is selected to be analysis target, the other end
Read discards without intercepting the forward and backward 20-30 base of each target read thus positioning which str base each read belongs to
Because of seat, if this read cannot navigate on target str locus, discard.The reads number that every kind of str matches is shown in Table 9:
The reads number that each str locus of table 9 are positioned
| Str locus | The reads number being positioned |
| csf1po | 9646 |
| fga | 58728 |
| th01 | 55099 |
| tpox | 39217 |
| d3s1358 | 15413 |
| d5s818 | 62128 |
| d7s820 | 108465 |
| d8s1179 | 57787 |
| d13s317 | 76639 |
| d16s539 | 45218 |
| d18s51 | 13047 |
| d21s11 | 28294 |
| d2s1338 | 15417 |
| cd4 | 22561 |
| d12s391 | 36707 |
| d18s865 | 12472 |
| vwa | 29509 |
(2) impact of the stutter band with regard to producing in pcr course of reaction, with reference to the method analysis in example 1, and
Eventually analysis obtain cell line locus str allele the number of repetition of core recurring units and with cell in atcc cell bank
Be corresponding str allelic gene typing result (analyzed target str is: csf1po, d13s317, d16s539, d5s818,
D7s820, th01, tpox, vwa) mutually compare, the results are shown in Table 10,11.
The number of times of each str locus recurring unit of 10 1 unknown cell lines of table
| Str locus | Number of repetition |
| csf1po | 10 |
| d13s317 | 11,12 |
| d16s539 | 9,13 |
| d5s818 | 13 |
| d7s820 | 10 |
| th01 | 8 |
| tpox | 8,11 |
| vwa | 16,17 |
The number of times of each str locus recurring unit of another unknown cell line of table 11
So, two unknown cell lines can be judged respectively by the corresponding allelic gene typing result of above-mentioned str locus
Belong to nci-h292 cell line and a549 cell line.
Claims (9)
1. a kind of construction method of amplicon sequencing library, merges, including with multipair, the dna mould that primer pair obtains from testing sample
Plate carries out multiple pcr, reclaims pcr product, obtains sequencing library；
Wherein said multipair merge the different purpose fragments that primer is respectively directed in dna template, each pair fusion primer from 5' end to
3' end comprises sequence measuring joints sequence and the specific primer sequence for purpose fragment successively；
Wherein multiple pcr is carried out by following reaction conditions: 95 DEG C of 2min；38 circulations, each circulates as 95 DEG C of 30s, Ran Houcong
76 DEG C to the arbitrary temp slow cooling between 55 DEG C and 58 DEG C, 0.1 DEG C of fall per second, keep 20s after being down to purpose temperature, so
72 DEG C of 30s afterwards；72℃2min.
2. the construction method of claim 1, wherein dna template are the genome dna extracting from testing sample.
3. the construction method of claim 1, wherein multiple one or more reaction systems of pcr reaction point complete.
4. the amplicon high-flux sequence method of non-diagnostic purpose, comprising: using the method preparation sequencing literary composition of claim 1 or 2
Storehouse, then carries out high-flux sequence.
5. the method for claim 4, the purpose fragment of wherein said amplicon high-flux sequence is multiple str locus.
6. the detection method of the str locus of non-diagnostic purpose, comprising: using the preparation of above-mentioned amplicon sequencing library construction method
For the sequencing library of multiple str locus, then carry out high-flux sequence and data analysiss are carried out to sequencing data.
7. the detection method of claim 6, wherein said data analysiss include obtaining the plurality of str locus core repetition sequence
The number of repetition of row.
8. the method for claim 7, the shadow band that described data analysiss also include chain slipped mispairing is led to is analyzed.
9. a kind of method for paternity test, comprising: measure the repetition of str locus in genome using the method for claim 7 or 8
Number of times, then calculates paternity index (pi value).
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