CN106192023A - A kind of multiple sequencing library construction method based on multidimensional Index - Google Patents
A kind of multiple sequencing library construction method based on multidimensional Index Download PDFInfo
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- CN106192023A CN106192023A CN201610645867.1A CN201610645867A CN106192023A CN 106192023 A CN106192023 A CN 106192023A CN 201610645867 A CN201610645867 A CN 201610645867A CN 106192023 A CN106192023 A CN 106192023A
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Abstract
The invention belongs to biology field, be specifically related to a kind of multiple sequencing library construction method based on multidimensional Index.It is an object of the invention to for the deficiencies in the prior art, a kind of multiple sequencing library construction method that sample carries out Index labelling is provided, described multiple sequencing library construction method designs based on multidimensional Index, expanded by two-wheeled PCR, when often taking turns PCR amplification, same sample is introduced 12 Index, so that use relatively small number of Index number, it is possible to obtain relatively more Index combination, being greatly improved multiple order-checking can the sample size of hybrid analysis.The method that the present invention provides is capable of using 40 Index to complete up to 10000 different samples and is marked.
Description
Technical field
The invention belongs to biology field, be specifically related to a kind of multiple sequencing library based on multidimensional Index and build
Method.
Background technology
High throughput sequencing technologies (High-throughput sequencing) is also known as " of future generation " sequencing technologies (" Next-
Generation " sequencing technology), once parallel hundreds of thousands to millions of DNA molecular can be carried out sequence
Row measure and the long shorter grade of general reading is mark, study for genomics and post-genomic science and bring new research methods reconciliation
Certainly scheme.In animals and plants research field, high-flux sequence has led the scientific research model innovation once with milestone significance,
Scientific research personnel may utilize this technology and launches in fields such as genome, transcript profile and apparent gene groups multi-level many-sided multilevel
Research.At present, described high throughput sequencing technologies is primarily referred to as 454 Life Sciences companies, ABI company and Illumina
The second filial generation sequencing technologies of company's release and Helicos HeliscopeTMThe list released with Pacific Biosciences divides
Sub-sequencing technologies.
In order to save order-checking cost, order-checking platform is made to run more efficiently, or demand based on scientific research, it will usually
Use multiple order-checking (Multiplexedsequencing) technology, i.e. when building sequencing library, by PCR primer, by difference
Index tab (Index) labelling difference sample after, upper machine that difference sample mix is got up check order, order-checking terminate after by identify
Index distinguishes the different sample datas in same swimming lane (Lane).
Multiple sequencing technologies high degree reduce order-checking cost, save order-checking resource, and carry to biological study
The convenient means of confession.Such as, when carrying out Microbial diversity Journal of Sex Research, it usually needs the multiformity of hundreds of samples is carried out point
Analysis, if not having multiple sequencing technologies, fund of scientific research cost and time cost will be greatly increased.
Although current multiple sequencing library constructing technology improve order-checking platform biased sample order-checking ability, but
Actual application still can not meet the demand that larger number biased sample is checked order by scientific research or business well.Such as Illunima
The Nextera XT Index Kit of company, wherein comprises 20 (12+8) different Index, using the teaching of the invention it is possible to provide 96 kinds (12 × 8)
Index combines, and is i.e. capable of 96 different samples and checks order in the mixing of same swimming lane.In the face of practical study (such as soil
The diversity analysis of earth microorganism or enteric microorganism etc.) time, need the most hundreds of of the sample size measured, the most thousands of
Individual, the most current multiple sequencing library constructing technology, cannot meet demand.Meanwhile, actual sequencing reaction finds,
Index sequence to a certain degree affects the sequencing data balance of biased sample, and the most any nucleotide sequence is suitable for being used as
Index.The Index sequence provided in the test kit adding Index of some maturations is the most all to select through a large amount of order-checking tests
The more stable sequence gone out.
In sum, utilize Index sequence the most efficiently, a kind of novel multiple sequencing library structure side of exploitation
Method so that obtain Index labelling as much as possible combination by the fewest Index quantity, improves mixing order-checking further and can divide
The biased sample quantity of analysis, reduces analysis cost, shortens the scientific research cycle, is that urgently to be resolved hurrily important of biology field is asked
Topic.
Summary of the invention
Unless otherwise defined, all technology used herein and scientific terminology have and the technical field of the invention
The same meaning that those of ordinary skill is generally understood that.
Term " Index " in the present invention refers to index tab, refers specifically to one section for the nucleoside distinguishing different order-checking sample
Acid sequence；Term " PCR " refers to polymerase chain reaction；Term " genes of interest " refers to the gene order needing to carry out checking order；Term
" the forward amplimer of genes of interest " and " the reverse amplimer of genes of interest " refers to amplify genes of interest by PCR
Pair of primers.
It is an object of the invention to for the deficiencies in the prior art, it is provided that a kind of sample is carried out resurveying of Index labelling more
Preface base construction method, described multiple sequencing library construction method is designed based on multidimensional Index, is expanded by two-wheeled PCR,
When often wheel PCR expands, the gene samples to the same preface and table of contents to be measured introduces 1-2 Index, so that using relatively small number of Index
Number, it is possible to obtain relatively more Index combination, being greatly improved multiple order-checking can the sample size of hybrid analysis.
Described multiple sequencing library construction method comprises the following steps:
(1), amplification PCR: expand genes of interest to be checked order by PCR method, and utilize primer 1 and primer 2 to introduce two
Individual Index；The genes of interest in primer 1 and 2 pairs of testing samples is used to carry out the nucleotide sequence of the amplified production that amplification obtains
Comprise genes of interest and two Index, and genes of interest is between two Index；
(2), storehouse PCR is built: the amplified production obtained using step (1), as template, uses primer 3 and primer 4 to step (1)
The amplified production obtained carries out PCR amplification, and utilizes primer 3 and primer 4 to introduce two Index；Use primer 3 and 4 pairs of steps
(1) after the amplified production obtained expands, it is thus achieved that the nucleotide sequence of amplified production comprise genes of interest, step (1) is drawn
Two Index that two Index entered and this step introduce.
The described forward amplimer that primer 1 is the genes of interest comprising an Index；Wherein said Index leads to
Cross or do not connected by 5 ' ends of connexon and the forward amplimer of gene.
Described primer 2 is the reverse amplimer of the genes of interest comprising an Index；Wherein said Index leads to
Cross or do not connected by 5 ' ends of connexon and the reverse amplimer of gene.
In the art, when building sequencing library, often can be by introducing various terminal sequence.Described primer 1 and drawing
Thing 2 can also comprise joint sequence.
The described Index in primer 1 is different with the nucleotide sequence of the Index in primer 2.
The a length of 1-15bp of the described Index in primer 1；It is preferably 2-14bp；More preferably 3-13bp；Further
It is preferably 4-12bp；Much further preferably from 5-10bp；Further it is preferably 6-9bp.
The a length of 1-15bp of the described Index in primer 2；It is preferably 2-14bp；More preferably 3-13bp；Further
It is preferably 4-12bp；Much further preferably from 5-10bp；Further it is preferably 6-9bp.
The nucleotide sequence of described primer 3 comprises 5 ' continuous 8-25 the cores held of an Index and described primer 1
Thuja acid.
The nucleotide sequence of described primer 4 comprises 5 ' continuous 8-25 the cores held of an Index and described primer 2
Thuja acid.
Described primer 3 and primer 4 can also comprise joint sequence, such as sequence measuring joints.
The described Index in the primer 3 and a length of 0-15bp of the Index in primer 4；It is preferably 2-14bp；More excellent
Elect 3-13bp as；More preferably 4-12bp；Much further preferably from 5-10bp；Further it is preferably 6-9bp；Described
The length of the Index in the primer 3 and Index in primer 4 can not be 0bp simultaneously.
The described Index in primer 3 is different with the nucleotide sequence of the Index in primer 4.
The nucleotide sequence of the described Index in primer 1-4 is different.
Compared with prior art, the present invention is expanded by two-wheeled PCR, introduces Index twice, enabling the sample of labelling
Quantity is multiplied.The multiple sequencing library construction method that the present invention provides can utilize relatively small number of Index number indicia
Sample to be checked order.Such as use 12 Index, it is possible to obtain the Index combination that 3*3*3*3 kind is different, the most just
It is to use 12 different Index (alternatively 12 primers) just can 81 different samples be marked.When Index's
When number sharp increase adds as 25, the method provided by the present invention is then obtained in that the Index combination that 5*5*5*5 kind is different, even if
It is capable of 625 different samples are marked with 20 different Index (alternatively 20 primers).And Illunima
The Nextera XT Index Kit that company provides, 20 Index of same use, but its method provided can only realize 96
Different samples are marked.When adding as 10 if being increased severely by the number of Index1, Index2, Index3 and Index4 further,
The method provided by the present invention can be obtained in that the Index combination that 10*10*10*10 kind is different, i.e. use 40 different
Index (alternatively 40 primers) is capable of being marked up to 10000 different samples.And Illunima company carries
For Nextera 42 equally primers of XT v2 Index Kit, be merely able to 384 samples of labelling.
As can be seen here, using in the case of equal number of Index, the method that the present invention provides can the sample of labelling
Number is far longer than prior art, and then multiple order-checking blendable sample number is greatly improved.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of multiple sequencing library construction method of the present invention.Wherein A: build storehouse joint 1；B:
Index1；The forward amplimer of C: genes of interest；The reverse amplimer of D: genes of interest；E:Index2；F: build storehouse joint
2；G: sequence measuring joints；H:Index3；I: build continuous 8-25 the nucleotide that the 5 ' of storehouse joint 1 is held；J: build what the 5 ' of storehouse joint 2 was held
8-25 nucleotide continuously；K:Index4；L: sequence measuring joints.
Detailed description of the invention
The explanation of following example is only intended to help to understand method and the core concept thereof of the present invention.It is right to it should be pointed out that,
For those skilled in the art, under the premise without departing from the principles of the invention, it is also possible to the present invention is carried out
Some improvement and modification, these improve and modify in the protection domain also falling into the claims in the present invention.To disclosed enforcement
The description below of example, makes professional and technical personnel in the field be capable of or uses the present invention.Multiple amendment to these embodiments
Will be apparent from for those skilled in the art, generic principles defined herein can be without departing from this
In the case of the spirit or scope of invention, realize in other embodiments.Therefore, the present invention is not intended to be limited to illustrated herein
These embodiments in, but can apply to meet the broader model consistent with principles disclosed herein and features of novelty
Enclose.Although implementing or can use and heretofore described any method similar or of equal value and material in test in the present invention
Material, place enumerates preferred method and material herein.
Unless otherwise defined, all technology used herein and scientific terminology have and the technical field of the invention
The same meaning that those of ordinary skill is generally understood that.
Term " ddH in the present invention2O " refer to distilled water.
Unreceipted concrete technology or condition person in embodiment, according to the technology described by the document in this area or condition
(such as with reference to works such as J. Pehanorm Brookers, " the Molecular Cloning: A Laboratory guide " that yellow training hall etc. is translated, the third edition, Science Press) or
Person is carried out according to product description.
KOD-Plus-Neo is purchased from TOYOBO company；Agencourt AMPure XP magnetic bead is purchased from Beckman company；Scorpion
Sub-intestinal samples is provided by Beijing Joint Genome Institute of the Chinese Academy of Sciences；Primer synthesis student on commission work biological engineering limited company enters
OK.
Embodiment 1 this method is studied for intestine microbial diversity
Use 10 primers, wherein have in 9 primers and to comprise an Index, the Scorpio to source, 27 different regions
Intestinal samples adds Index label, builds multiple sequencing library.
One, design of primers:
Primer 1-1, its nucleotide sequence is as shown in SEQ ID NO:1；Primer 1-2, its nucleotide sequence such as SEQ ID
Shown in NO:2；Primer 1-3, its nucleotide sequence is as shown in SEQ ID NO:3；Primer 2-1, its nucleotide sequence such as SEQ ID
Shown in NO:4；Primer 2-2, its nucleotide sequence is as shown in SEQ ID NO:5；Primer 2-3, its nucleotide sequence such as SEQ ID
Shown in NO:6；Primer 3-1, its nucleotide sequence is as shown in SEQ ID NO:7；Primer 4-1, its nucleotide sequence such as SEQ ID
Shown in NO:8；Primer 4-2, its nucleotide sequence is as shown in SEQ ID NO:9.Primer 4-3, its nucleotide sequence such as SEQ ID
Shown in NO:10.
Primer 1-1,1-2,1-3,2-1,2-2,2-3,4-1,4-2 and 4-3 wherein respectively contains 1 Index sequence, the most just
It is to utilize 9 Index, uses 10 primers to realize 27 different samples are marked, build sequencing library.
1-19bp in primer 1-1,1-2 and 1-3 is for building storehouse joint 1 sequence；20-27bp is Index sequence；28-44 is
The degenerate primer sequence of amplification bacterial 16 S rRNA.
1-22bp in primer 2-1,2-2 and 2-3 is for building storehouse joint 2 sequence；23-30bp is Index sequence；31-50 is
The degenerate primer sequence of amplification bacterial 16 S rRNA.
1-30bp in primer 3-1 is sequence measuring joints sequence；31-46bp is 16 continuous kernels that 5 ' in primer 2-1 are held
Thuja acid, does not has Index sequence in this primer.
1-24bp in primer 4-1,4-2 and 4-3 is sequence measuring joints sequence；25-30bp is Index sequence；31-47bp is
17 continuous nucleotides that in primer 1-1 5 ' are held.
27 samples are the Scorpio intestinal samples in source, 27 different regions.
Two, the structure of sequencing library:
1, the extraction of sample gene group DNA:
Scorpio intestinal samples uses QIAamp DNA Stool Mini Kit (50) test kit (purchased from Qiagen company) to carry
Take, obtain 27 different genes groups DNA altogether；
2, amplification PCR:
9 primers pair below primer 1-1,1-2,1-3,2-1,2-2 and 2-3 combination of two is constituted:
Primer is to 1: primer 1-1 and primer 2-1；
Primer is to 2: primer 1-1 and primer 2-2；
Primer is to 3: primer 1-1 and primer 2-3；
Primer is to 4: primer 1-2 and primer 2-1；
Primer is to 5: primer 1-2 and primer 2-2；
Primer is to 6: primer 1-2 and primer 2-3；
Primer is to 7: primer 1-3 and primer 2-1；
Primer is to 8: primer 1-3 and primer 2-2；
Primer is to 9: primer 1-3 and primer 2-3；
27 samples are randomly divided into 9 groups, and often 3 samples of group, with above-mentioned 9 primers as primer, obtain with step 1 respectively
27 genomic DNAs obtained, as template, carry out PCR amplification, the 16S rRNA in amplification sample, it is thus achieved that 9 groups, totally 27 amplifications
Product；
Amplification PCR reaction system is as shown in the table:
| Composition | Content |
| Genomic DNA (10ng/ μ L) | 2μL |
| Primer (10 μMs) | Each 0.75 μ L |
| MgSO4(25mM) | 3μL |
| dNTPs(2mM) | 5μL |
| 10×KOD-Plus-Neo Buffer | 5μL |
| KOD-Plus-Neo | 1μL |
| ddH2O | 32.5μL |
| Amount to | 50μL |
Amplification PCR program is as shown in the table:
3, storehouse PCR is built:
Primer 3-1 and primer 4-1,4-2 and 4-3 are combined and constitute following 3 primers pair:
Primer is to 10: primer 3-1 and primer 4-1；
Primer is to 11: primer 3-1 and primer 4-2；
Primer is to 12: primer 3-1 and primer 4-3；
Respectively with 27 amplified productions obtaining with step 2 as template, use that step 2 is obtained by primer by 1-3 respectively 9
3 amplified productions of often group in group carry out PCR amplification；
Each sample is at amplification PCR and to build the primer used in the PCR of storehouse as shown in the table:
Build storehouse PCR reaction system as shown in the table:
Build storehouse PCR program as shown in the table:
Use 27 amplified productions that Agencourt AMPure XP magnetic beads for purifying step 3 obtains, 27 expansions after purification
After volume increase thing mixing, it is multiple sequencing library, Illumina MiSeq order-checking platform can be used to check order.
Fig. 1 is the schematic flow sheet of the present embodiment, the Index, Index2 during wherein Index1 guides thing 1-1,1-2,1-3
The Index, Index3 in thing 2-1,2-2,2-3 is guided to guide the Index in thing 3-1 (without Index in this primer, therefore
The a length of 0bp of Index3), Index4 guides the Index in thing 4-1,4-2,4-3, and genes of interest refers to 16SrRNA to be checked order
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all essences in the present invention
Within god and principle, any modification, equivalent substitution and improvement etc. made, should be included within the scope of the present invention.
Claims (9)
1. the construction method of a multiple nucleic acid sequencing library, it is characterised in that: described construction method is reacted by two-wheeled PCR
The gene samples of one preface and table of contents to be measured is introduced 3 or 4 Index.
2. construction method as claimed in claim 1, it is characterised in that: described construction method comprises the following steps:
(1), amplification PCR: expand sequence genes of interest to be measured by PCR method, and utilize primer 1 and primer 2 to introduce two
Index；The genes of interest in primer 1 and 2 pairs of testing samples is used to carry out the nucleotide sequence bag of the amplified production that amplification obtains
Containing genes of interest and two Index, and genes of interest is between two Index；
(2) storehouse PCR, is built: step (1) is obtained using the amplified production that step (1) obtains as template, use primer 3 and primer 4
Amplified production carry out PCR amplification, and utilize primer 3 and primer 4 to introduce two Index；Primer 3 and 4 pairs of steps (1) are used to obtain
Amplified production expand after, it is thus achieved that the nucleotide sequence of amplified production comprise genes of interest, step (1) introduces two
Two Index that individual Index and this step introduce.
3. construction method as claimed in claim 2, it is characterised in that: described primer 1 is the purpose containing an Index
The forward amplimer of gene；Wherein said Index passes through or not by connexon and the 5 ' of the forward amplimer of gene
End connects.
4. construction method as claimed in claim 2, it is characterised in that: described primer 2 is the purpose containing an Index
The reverse amplimer of gene；Wherein said Index passes through or not by connexon and the 5 ' of the reverse amplimer of gene
End connects.
5. construction method as claimed in claim 2, it is characterised in that:
The nucleotide sequence of described primer 3 comprises 5 ' continuous 8-25 the nucleoside held of an Index and described primer 1
Acid.
6. construction method as claimed in claim 2, it is characterised in that:
The nucleotide sequence of described primer 4 comprises 5 ' continuous 8-25 the nucleoside held of an Index and described primer 2
Acid.
7. the banking process as described in claim 2-6 any one, it is characterised in that: in described primer 1 and primer 2
The a length of 1-15bp of Index；The a length of 0-15bp of the Index in described primer 3 and primer 4；In described primer 3
The length of the Index in Index and primer 4 can not be 0bp simultaneously.
8. the banking process as described in claim 2-6 any one, it is characterised in that: the described Index's in primer 1-4
Nucleotide sequence is different.
9. the banking process as described in claim 2-6 any one, it is characterised in that: described primer 1, primer 2, primer 3
With primer 4 also comprises joint sequence.
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Cited By (3)
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| CN107904298A (en) * | 2017-12-29 | 2018-04-13 | 苏州普瑞森基因科技有限公司 | A kind of kit and its application for being used to analyze enteric microorganism |
| CN107937582A (en) * | 2017-12-29 | 2018-04-20 | 苏州普瑞森基因科技有限公司 | A kind of primer sets and its application for being used to analyze enteric microorganism |
| EP4211239A4 (en) * | 2020-09-08 | 2024-09-11 | Seqwell Inc | Sequencing oligonucleotides and methods of use thereof |
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| CN107904298A (en) * | 2017-12-29 | 2018-04-13 | 苏州普瑞森基因科技有限公司 | A kind of kit and its application for being used to analyze enteric microorganism |
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