CN106282332B - Label and primer for multiple nucleic acid sequencing - Google Patents
Label and primer for multiple nucleic acid sequencing Download PDFInfo
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- CN106282332B CN106282332B CN201610645098.5A CN201610645098A CN106282332B CN 106282332 B CN106282332 B CN 106282332B CN 201610645098 A CN201610645098 A CN 201610645098A CN 106282332 B CN106282332 B CN 106282332B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
Abstract
The present invention relates to technical field of molecular biology, and in particular to the PCR primer for the label of multiple nucleic acid sequencing and for constructing the multiple sequencing library of fungi ITS sequence.Index provided by the invention is examined by the practical sequencing of vast number, will be it includes after being sequenced or building library primer, and the sequencing data balance that mixing sequencing reaction obtains is relatively preferable, and then being capable of fungi composition situation in more true and reliable ground response sample.Higher than the specificity of existing routine ITS primer provided by the present invention for the PCR primer of the building multiple sequencing library of fungi ITS sequence, sample type that can not only be relatively low to complexity such as animal intestinal tract, water body and yeast obtains high special amplified production；It to the sample type of the high complexities such as soil and mud also specificity with higher, therefore has ensured the accuracy of subsequent sequencing result, and then relatively more accurate analysis result and more true fungi composition situation can be obtained.
Description
Technical field
The present invention relates to technical field of molecular biology, and in particular to for the label of multiple nucleic acid sequencing and for constructing
The PCR primer of the multiple sequencing library of fungi ITS sequence.
Background technique
The numerous areas such as microbial diversity, especially fungal diversity and agricultural, medicine, food and industrial production have
Close ties.With the continuous development of Protocols in Molecular Biology, microbial diversity research method is also gradually improved and maturation.In
During microbial evolution, rRNA has certain evolutionary conservatism, and conserved region sequence is all same organism institutes
It is shared, there is the Variable Area of sequence difference between the species as caused by evolving between conserved sequence, is considered as
It is most suitable for one of the gene of genealogical classification.By the measurement and comparison to these sequence Variable Areas, it can probe into and disclose soil
The diversity of microbial species and structure of community in earth.With the development of gene sequencing technology, high throughput sequencing technologies rely on it
Unique advantage is also introduced into the investigative technique of microbial diversity.
High throughput sequencing technologies (High-throughput sequencing), also known as " next generation " sequencing technologies ("
Next-generation " sequencing technology), with can be once parallel to hundreds of thousands to millions of DNA moleculars
Progress sequencing and generally reading length are shorter to be waited to indicate.With the continuous development of high throughput sequencing technologies, in order to save sequencing
Cost or research need to have developed multiple sequencing (Multiplexed sequencing) technology, usually in building sequencing library
When, by PCR primer, after marking different samples with different labels (Index), different samples is mixed into upper machine and are sequenced,
The different sample datas in same swimming lane (Lane) are distinguished by identification Index after sequencing.Multiple sequencing technologies pole
Big degree reduces sequencing cost, saves sequencing resource, and to the convenient means that biological study provides.For example, into
When the actual fungal diversity research of row, it usually needs analyze the diversity of hundreds of samples, if without multiple sequencing
Technology, scientific research cost and time cost will greatly increase.
Label (Index) for distinguishing different samples is actually one section of nucleotide sequence, on the one hand in practical application
Middle discovery, sequencing reaction have certain data output Preference to different Index sequences, cause the detection Insert Fragment time
The fluctuation of strong parameter influences the quality of data and balance of output, i.e. Index sequence influences to mix the sequencing of sample to a certain degree
The accuracy of data, therefore not any nucleotide sequence is suitable for being used as Index.In view of existing at present by practical sequencing
Verifying Index number preferable to data balance simultaneously it is few, therefore provide more on data output balance influence compared with
Small Index sequence is extremely important to multiple nucleic acid sequencing technologies.
On the other hand, it is often used genetic marker of the 18S rRNA as taxonomic identification in fungal diversity research early stage, with
The continuous development of Protocols in Molecular Biology, gradually using ITS sequence replace 18S rRNA as genetic marker.Although at present
There are many ITS sequence amplification universal primer, but its be used for different types of sample sequencing library building when, can not all obtain
Preferable expanding effect.Such as when being used for the sequencing library building of pedotheque, due to the complexity of pedotheque itself, at present
The pcr amplification product disperse that conventional ITS universal primer obtains is serious, and specific amplification is relatively poor.And specific amplification is straight
The accuracy for influencing taxonomic identification is connect, and then influences the accuracy detected to fungal diversity in sample.Therefore it provides one group high
The PCR primer of the multiple sequencing library building of ITS sequence specific, for fungal diversity detection, can effectively improve
It is provided to the accuracy that fungal diversity in sample detects, and then to the related application of agricultural, medicine, food and industrial circle
More scientific and effective guidance.
In another aspect, the kit of Index is only added to bacterial 16 S rRNA sequencing library sample in the prior art,
The Nextera XT Index Kit and Nextera XT v2Index Kit of Illunima company；However, not useful at present
In the kit to the multiple sequencing library sample addition Index of fungi ITS sequence.
In conclusion providing the relatively better Index of one group of sequencing data balance and the higher structure of a group-specific
The PCR primer of the multiple sequencing library of ITS sequence and the kit to fungi ITS sequence sequencing library addition Index are built, it can
So that multiple nucleic acid sequencing technologies and soil fungal diversity research work are more efficiently convenient.
Summary of the invention
Unless otherwise defined, all technical and scientific terms used herein have and the technical field of the invention
The normally understood identical meaning of those of ordinary skill.
Term " label " in the present invention is also referred to as Index, refers specifically to one section for distinguishing the nucleosides of different sequencing samples
Acid sequence；Term " PCR " refers to polymerase chain reaction；Term " connexon " refers between any one section and aim sequence to be sequenced
There is no the nucleotide sequences for the specific pairs for being more than continuous 5 bases or more.
For distinguishing the label of different samples, also referred to as Index, it is actually one section of nucleotide sequence, on the one hand exists
It is found in practical application, sequencing reaction has certain data output Preference to different Index sequences, and detection is caused to be inserted into
The fluctuation of light intensity parameter when segment, influences the quality of data and balance of output, i.e. Index sequence influences aggregate sample to a certain degree
The accuracy of the sequencing data of product, therefore not any nucleotide sequence is suitable for being used as Index, however existing process at present
The Index number preferable to data balance of practical sequence verification is simultaneously few.
The present invention considers influence of the Index to the amplification efficiency of PCR primer and the Preference of data output simultaneously, provides
One group for marking the labels of nucleic acid samples to be sequenced, a group of labels include 2 or more in following 29 Index
It is a:
The present invention also provides above-mentioned Index for the building of sequencing library and/or for the purposes of sequencing: will be described
Index is included in for expanding in the PCR primer to sequencing sequence, to constitute corresponding Index-PCR primer, is passed through
Above-mentioned Index is introduced into in sequencing sequence by PCR method；The Index-PCR primer 3 ' the end primers that may be used as PCR
Or 5 ' end primer, or simultaneously be used as 3 ' end primers and 5 ' end primers.
Wherein, in the Index-PCR primer, the Index passes through or does not pass through connexon and is used to expand
5 ' the ends or 3 ' ends for increasing the PCR primer to sequencing sequence are connected.
Wherein, described to sequencing sequence is preferably fungi ITS sequence.
Fungi ITS sequence is used as the important genetic marker of classification of fungi identification, and currently used ITS section is ITS1-
ITS4, however the existing primer for expanding ITS1-ITS4 this section can not carry out Gao Te to various types of samples
Specific amplification, such as the pedotheque that complexity is relatively high.
In order to solve this technical problem, it does invention and additionally provides one group for constructing the PCR of the multiple sequencing library of ITS sequence
Primer.Described one group includes forward primer group and reverse primer for constructing the PCR primer of the multiple sequencing library of ITS sequence
Group cannot contain the primer comprising identical Index in the forward primer group and reverse primer group.When in use, described
Any one primer in forward primer group and any one primer in reverse primer group are composed of primer pair two-by-two, pass through
PCR is reacted for expanding the ITS sequence in sample；The upstream and downstream of the amplified production of acquisition respectively contains an Index, this two
Specific marker of a Index as the sample, for being distinguished with other samples to be sequenced.
The forward primer group is made of one or more in following primer:
Primers F 1: including Index1 of the present invention, nucleotide sequence is as shown in SEQ ID NO:1；
Primers F 2: including Index2 of the present invention, nucleotide sequence is as shown in SEQ ID NO:2；
Primers F 3: including Index3 of the present invention, nucleotide sequence is as shown in SEQ ID NO:3；
Primers F 4: including Index4 of the present invention, nucleotide sequence is as shown in SEQ ID NO:4；
Primers F 5: including Index5 of the present invention, nucleotide sequence is as shown in SEQ ID NO:5；
Primers F 6: including Index6 of the present invention, nucleotide sequence is as shown in SEQ ID NO:6；
Primers F 7: including Index7 of the present invention, nucleotide sequence is as shown in SEQ ID NO:7；
Primers F 8: including Index8 of the present invention, nucleotide sequence is as shown in SEQ ID NO:8；
Primers F 9: including Index9 of the present invention, nucleotide sequence is as shown in SEQ ID NO:9；
Primers F 10: including Index10 of the present invention, nucleotide sequence is as shown in SEQ ID NO:10；
Primers F 11: including Index11 of the present invention, nucleotide sequence is as shown in SEQ ID NO:11；
Primers F 12: including Index12 of the present invention, nucleotide sequence is as shown in SEQ ID NO:12；
Primers F 13: including Index13 of the present invention, nucleotide sequence is as shown in SEQ ID NO:13；
Primers F 14: including Index14 of the present invention, nucleotide sequence is as shown in SEQ ID NO:14；
Primers F 15: including Index15 of the present invention, nucleotide sequence is as shown in SEQ ID NO:15；
Primers F 16: including Index16 of the present invention, nucleotide sequence is as shown in SEQ ID NO:16；
Primers F 17: including Index17 of the present invention, nucleotide sequence is as shown in SEQ ID NO:17；
Primers F 18: including Index18 of the present invention, nucleotide sequence is as shown in SEQ ID NO:18；
Primers F 19: including Index19 of the present invention, nucleotide sequence is as shown in SEQ ID NO:19；
Primers F 20: including Index20 of the present invention, nucleotide sequence is as shown in SEQ ID NO:20；
Primers F 21: including Index21 of the present invention, nucleotide sequence is as shown in SEQ ID NO:21；
Primers F 22: including Index22 of the present invention, nucleotide sequence is as shown in SEQ ID NO:22；
Primers F 23: including Index23 of the present invention, nucleotide sequence is as shown in SEQ ID NO:23；
Primers F 24: including Index24 of the present invention, nucleotide sequence is as shown in SEQ ID NO:24；
Primers F 25: including Index25 of the present invention, nucleotide sequence is as shown in SEQ ID NO:25；
Primers F 26: including Index26 of the present invention, nucleotide sequence is as shown in SEQ ID NO:26；
Primers F 27: including Index27 of the present invention, nucleotide sequence is as shown in SEQ ID NO:27；
Primers F 28: including Index28 of the present invention, nucleotide sequence is as shown in SEQ ID NO:28；
Primers F 29: including Index29 of the present invention, nucleotide sequence is as shown in SEQ ID NO:29.
The reverse primer group is made of one or more in following primer:
Primer R1: including Index1 of the present invention, nucleotide sequence is as shown in SEQ ID NO:30；
Primer R2: including Index2 of the present invention, nucleotide sequence is as shown in SEQ ID NO:31；
Primer R3: including Index3 of the present invention, nucleotide sequence is as shown in SEQ ID NO:32；
Primer R4: including Index4 of the present invention, nucleotide sequence is as shown in SEQ ID NO:33；
Primer R5: including Index5 of the present invention, nucleotide sequence is as shown in SEQ ID NO:34；
Primer R6: including Index6 of the present invention, nucleotide sequence is as shown in SEQ ID NO:35；
Primer R7: including Index7 of the present invention, nucleotide sequence is as shown in SEQ ID NO:36；
Primer R8: including Index8 of the present invention, nucleotide sequence is as shown in SEQ ID NO:37；
Primer R9: including Index9 of the present invention, nucleotide sequence is as shown in SEQ ID NO:38；
Primer R10: including Index10 of the present invention, nucleotide sequence is as shown in SEQ ID NO:39；
Primer R11: including Index11 of the present invention, nucleotide sequence is as shown in SEQ ID NO:40；
Primer R12: including Index12 of the present invention, nucleotide sequence is as shown in SEQ ID NO:41；
Primer R13: including Index13 of the present invention, nucleotide sequence is as shown in SEQ ID NO:42；
Primer R14: including Index14 of the present invention, nucleotide sequence is as shown in SEQ ID NO:43；
Primer R15: including Index15 of the present invention, nucleotide sequence is as shown in SEQ ID NO:44；
Primer R16: including Index16 of the present invention, nucleotide sequence is as shown in SEQ ID NO:45；
Primer R17: including Index17 of the present invention, nucleotide sequence is as shown in SEQ ID NO:46；
Primer R18: including Index18 of the present invention, nucleotide sequence is as shown in SEQ ID NO:47；
Primer R19: including Index19 of the present invention, nucleotide sequence is as shown in SEQ ID NO:48；
Primer R20: including Index20 of the present invention, nucleotide sequence is as shown in SEQ ID NO:49；
Primer R21: including Index21 of the present invention, nucleotide sequence is as shown in SEQ ID NO:50；
Primer R22: including Index22 of the present invention, nucleotide sequence is as shown in SEQ ID NO:51；
Primer R23: including Index23 of the present invention, nucleotide sequence is as shown in SEQ ID NO:52；
Primer R24: including Index24 of the present invention, nucleotide sequence is as shown in SEQ ID NO:53；
Primer R25: including Index25 of the present invention, nucleotide sequence is as shown in SEQ ID NO:54；
Primer R26: including Index26 of the present invention, nucleotide sequence is as shown in SEQ ID NO:55；
Primer R27: including Index27 of the present invention, nucleotide sequence is as shown in SEQ ID NO:56；
Primer R28: including Index28 of the present invention, nucleotide sequence is as shown in SEQ ID NO:57；
Primer R29: including Index29 of the present invention, nucleotide sequence is as shown in SEQ ID NO:58.
The ITS sequence refers to the nucleic acid sequence in this region ITS1-ITS4.
The sample is including but not limited to Types Below: pedotheque, intestinal samples, water body example, mud sample, greatly
Bent sample.
The present invention also provides a kind of multiple sequencing libraries of ITS sequence to construct kit, which is characterized in that the reagent
Box includes one group of the present invention for constructing the PCR primer of the multiple sequencing library of fungi ITS sequence.
A kind of above-mentioned multiple sequencing library building kit of ITS sequence can also include extracting genome DNA reagent, DNA
Polymerase, ITS sequence measuring joints add reagent.
The present invention also provides a kind of multiple sequencing library building kits of above-mentioned ITS sequence in fungal diversity detection
Application.
Compared with prior art, the invention has the benefit that
(1), Index provided by the invention fully takes into account sequence difference degree between index tab, base discrimination
And sequencing reaction is to the data output Preference of different Index sequences.The sequence of Index provided by the invention avoids sequence
Between there is the appearance of 3 or 3 or more continuous identical bases, high degree reduces in its sequent synthesis or sequencing procedure
The error rate being likely to occur；Label include after building in library or sequencing primer be avoided as much as occurring hairpin structure or with
Sequencing primer and its identical phenomenon of reverse complementary sequence.Index provided by the invention is examined by the practical sequencing of vast number
It tests, it will be it includes after being sequenced or building library primer, the sequencing data balance that mixing sequencing reaction obtains is relatively preferable.
(2), provided by the present invention for constructing the PCR primer of the multiple sequencing library of fungi ITS sequence for expanding ITS sequence
ITS1-ITS4 this section in column, this section will be significantly better than 18SrRNA or ITS1- to the effect that classification of fungi is identified
ITS2 section；It wherein also include Index provided by the invention, the primer pair constituted using upper forward primer and reverse primer is logical
It crosses 1 wheel PCR reaction and introduces two Index to it while expanding ITS sequence, the two Index are as the special of the sample
Property label, for other mix sequencing samples distinguish；Since it contains Index provided by the invention, mixed in library
The balance of data output is relatively preferable when sequencing, so can the fungi in more true and reliable ground response sample form feelings
Condition.
(3), provided by the present invention for constructing the PCR primer of the multiple sequencing library of fungi ITS sequence than existing routine
The specificity of ITS primer is higher.Conventionally used for expanding the PCR primer of ITS1-ITS4 stronger for complexity such as soil, mud
Sample when, specificity is bad, and purpose band electrophoresis disperse is serious, is often possible to cause sequencing result inaccurate, can not obtain
Reliable analysis forms situation as a result, also can not just obtain fungi in more true sample, to a certain degree to food, health,
The application in the fields such as agricultural causes adverse effect.Provided by the present invention for constructing the PCR of the multiple sequencing library of fungi ITS sequence
Primer can not only be relatively low to complexity such as animal intestinal tract, water body and yeast sample type obtain high special amplification
Product；To the sample type of the high complexities such as soil and mud also specificity with higher, amplified production specificity is high, amplification
Band is clear, no diffusing phenomenon, therefore has ensured the accuracy of subsequent sequencing result, and then can obtain relatively more accurately
It analyzes result and more true fungi forms situation.
Detailed description of the invention
Fig. 1 is the agarose gel electrophoresis figure of the amplified production in 1 step of experimental example (2).
Fig. 2 is the agarose gel electrophoresis figure of the amplified production in comparative example 1.
Specific embodiment
The explanation of following embodiment is merely used to help understand method and its core concept of the invention.It should be pointed out that pair
For those skilled in the art, without departing from the principle of the present invention, the present invention can also be carried out
Some improvements and modifications, these improvements and modifications also fall within the scope of protection of the claims of the present invention.To disclosed implementation
The following the description of example, enables those skilled in the art to implement or use the present invention.Various modifications to these embodiments
It will be readily apparent to those skilled in the art, the general principles defined herein can not depart from this
In the case where 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 be applied to meet broader model consistent with the principles and novel features disclosed in this article
It encloses.Although can be used in implementation or test of the invention and heretofore described similar or of equal value any method and material
Material, place enumerates preferred method and material herein.
Unless otherwise defined, all technical and scientific terms used herein have and the technical field of the invention
The normally understood identical meaning of those of ordinary skill.
Term " ddH in the present invention2O " refers to distilled water.
In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art
(such as with reference to J. Pehanorm Brooker etc. write, " Molecular Cloning:A Laboratory guide " that Huang Peitang etc. is translated, the third edition, Science Press) or
Person carries out according to product description.
Soil DNA Kit is purchased from Omega company；KOD-Plus-Neo is purchased from TOYOBO company；Primer
Student on commission's work bioengineering limited liability company is synthesized to carry out；The pedotheque of 100 separate sources is by Chinese Academy of Sciences's Beijing gene
Group research institute provides.
1 one groups of embodiment for constructing the PCR primer of the multiple sequencing library of ITS sequence
It is made of 20 primers,
Wherein forward primer group is made of following 10 primers:
It is of the present invention:
Primers F 1, nucleotide sequence is as shown in SEQ ID NO:1；
Primers F 2, nucleotide sequence is as shown in SEQ ID NO:2；
Primers F 3, nucleotide sequence is as shown in SEQ ID NO:3；
Primers F 7, nucleotide sequence is as shown in SEQ ID NO:7；
Primers F 8, nucleotide sequence is as shown in SEQ ID NO:8；
Primers F 9, nucleotide sequence is as shown in SEQ ID NO:9；
Primers F 12, nucleotide sequence is as shown in SEQ ID NO:12；
Primers F 13, nucleotide sequence is as shown in SEQ ID NO:13；
Primers F 14, nucleotide sequence is as shown in SEQ ID NO:14；
Primers F 23, nucleotide sequence is as shown in SEQ ID NO:23；
Wherein reverse primer group is made of following 10 primers:
It is of the present invention:
Primer R15, nucleotide sequence is as shown in SEQ ID NO:44；
Primer R17, nucleotide sequence is as shown in SEQ ID NO:46；
Primer R18, nucleotide sequence is as shown in SEQ ID NO:47；
Primer R19, nucleotide sequence is as shown in SEQ ID NO:48；
Primer R21, nucleotide sequence is as shown in SEQ ID NO:50；
Primer R25, nucleotide sequence is as shown in SEQ ID NO:54；
Primer R26, nucleotide sequence is as shown in SEQ ID NO:55；
Primer R27, nucleotide sequence is as shown in SEQ ID NO:56；
Primer R28, nucleotide sequence is as shown in SEQ ID NO:57；
Primer R29, nucleotide sequence is as shown in SEQ ID NO:58.
The detection application of 1 fungal diversity of experimental example
The multiple sequencing library of ITS sequence is constructed to 100 different samples
Laboratory sample: the pedotheque of 100 separate sources
Experimental procedure:
(1), sample gene group DNA is extracted:
WithSoil DNA Kit extracts the genomic DNA in pedotheque, obtains 100 different genes
Group DNA sample, extraction step is referring to its product description；
(2), PCR amplification ITS1-ITS4 sequence, while 2 Index are added to each library sample using PCR primer:
The 100 different genes group DNA samples obtained respectively using step (1) will be provided as template in the embodiment of the present invention 1
20 PCR primers match two-by-two, 100 different primers pair are constituted, for expanding the ITS1-ITS4 sequence in template；
PCR reaction system is as shown in the table:
| Ingredient | Content |
| Genomic DNA (10ng/ μ L) | 2μL |
| Primer (10 μM) | Each 0.75 μ L |
| MgSO4(25mM) | 3μL |
| dNTPs(2mM) | 5μL |
| 10×KOD-Plus-Neo Buffer | 5μL |
| KOD-Plus-Neo | 1μL |
| ddH2O | 32.5μL |
| It is total | 50μL |
PCR program is as shown in the table:
Agarose gel electrophoresis is carried out to 100 different amplified productions of acquisition, gum concentration 0.8%, electrophoresis result is shown
The ITS1-ITS4 amplified product band obtained using primer provided by the invention is clear, no diffusing phenomenon.Due to sample size compared with
It is more, the electrophoresis result of 1-10 sample is shown herein, as shown in Figure 1, purpose band is clear, no diffusing phenomenon；No. 11-100
The electrophoresis result of sample and No. 1-10 it is same or similar.
(3), sequence measuring joints are added:
Using the amplified production that step (2) obtain as template, PCR amplification, following primer pair are carried out using following primer pair
In the sequence measuring joints containing Illumina MiSeq microarray dataset.
5’-CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAG ACGTG-3’
5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC-3’
PCR reaction system is as shown in the table:
| Ingredient | Content |
| The amplified production (1ng/ μ L) of step (2) | 2μL |
| Primer (10 μM) | Each 0.75 μ L |
| MgSO4(25mM) | 3μL |
| dNTPs(2mM) | 5μL |
| 10×KOD-Plus-Neo Buffer | 5μL |
| KOD-Plus-Neo | 1μL |
| ddH2O | 32.5μL |
| It is total | 50μL |
PCR program is as shown in the table:
100 amplified productions obtained using Agencourt AMPure XP magnetic beads for purifying step (3), after purification 100
After a amplified production mixing, the multiple sequencing library of the ITS sequence of the pedotheque of as 100 separate sources.
Comparative example 1
Following primer pair is the amplimer of conventional use of ITS1-ITS4 sequence in the prior art:
5’-TCCGTAGGTGAACCTGCGG-3’
5’-TCCTCCGCTTATTGATATGC-3’
Using above-mentioned primer pair to random in the pedotheque of 100 separate sources used in experimental example 1 of the present invention
The ITS1-ITS4 sequence of 50 samples is expanded: the extracting method of sample gene group DNA is the same as 1 step of experimental example of the present invention
(1), PCR system and PCR program are the same as 1 step of experimental example (2) of the present invention.50 of acquisition different amplified productions are subjected to agarose
Gel electrophoresis, gum concentration and deposition condition are the same as 1 step of experimental example (2).Since sample size is more, wherein 10 samples are shown herein
The amplified production electrophoresis result of product, as shown in Fig. 2, amplified production purpose band is unintelligible, disperse is serious；Remaining in this comparative example
The electrophoresis result of sample and Fig. 2 are same or similar, and amplified production purpose band is unintelligible, and disperse is serious.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (6)
1. one group for constructing the PCR primer of the multiple sequencing library of fungi ITS sequence in pedotheque, it is characterised in that: described
One group of PCR primer include forward primer group and reverse primer group；
The forward primer group is made of following primer:
Primers F 1: its sequence is nucleotide sequence shown in SEQ ID NO:1, wherein containing Index1:ACGATGCT；
Primers F 2: its sequence is nucleotide sequence shown in SEQ ID NO:2, wherein containing Index2:AGACTGTC；
Primers F 3: its sequence is nucleotide sequence shown in SEQ ID NO:3, wherein containing Index3:AGCATCGT；
Primers F 7: its sequence is nucleotide sequence shown in SEQ ID NO:7, wherein containing Index7:ATCGTAGC；
Primers F 8: its sequence is nucleotide sequence shown in SEQ ID NO:8, wherein containing Index8:CAGACTGT；
Primers F 9: its sequence is nucleotide sequence shown in SEQ ID NO:9, wherein containing Index9:CGATGCTA；
Primers F 12: its sequence is nucleotide sequence shown in SEQ ID NO:12, wherein containing Index12:CGTAGCAT；
Primers F 13: its sequence is nucleotide sequence shown in SEQ ID NO:13, wherein containing Index13:CTACGATG；
Primers F 14: its sequence is nucleotide sequence shown in SEQ ID NO:14, wherein containing Index14:GACAGTCT；
Primers F 23: its sequence is nucleotide sequence shown in SEQ ID NO:23, wherein containing Index23:GCATCGTA；
The reverse primer group is made of following primer:
Primer R15: its sequence is nucleotide sequence shown in SEQ ID NO:44, wherein containing Index15:CTCTGCCT；
Primer R17: its sequence is nucleotide sequence shown in SEQ ID NO:46, wherein containing Index17:CCTACTAT；
Primer R18: its sequence is nucleotide sequence shown in SEQ ID NO:47, wherein containing Index18:CGATGCTG；
Primer R19: its sequence is nucleotide sequence shown in SEQ ID NO:48, wherein containing Index19:ATGCTAAT；
Primer R21: its sequence is nucleotide sequence shown in SEQ ID NO:50, wherein containing Index21:GCGTCTAT；
Primer R25: its sequence is nucleotide sequence shown in SEQ ID NO:54, wherein containing Index25:GGAGTCCT；
Primer R26: its sequence is nucleotide sequence shown in SEQ ID NO:55, wherein containing Index26:GCTCGAGC；
Primer R27: its sequence is nucleotide sequence shown in SEQ ID NO:56, wherein containing Index27:AAGGCATA；
Primer R28: its sequence is nucleotide sequence shown in SEQ ID NO:57, wherein containing Index28:ACTGCGGA；
Primer R29: its sequence is nucleotide sequence shown in SEQ ID NO:58, wherein containing Index29:AGGCGCTA.
2. PCR primer as described in claim 1, it is characterised in that: the PCR primer when in use: the forward direction is drawn
Any one primer in object group and any one primer in reverse primer group are composed of primer pair two-by-two, anti-by PCR
Applied to the ITS sequence in amplification sample；The upstream and downstream of the amplified production of acquisition respectively contains an Index, the two
Specific marker of the Index as the sample, for being distinguished with other samples to be sequenced；The ITS sequence refers to ITS1-
The nucleic acid sequence in this region ITS4.
3. for constructing the multiple sequencing library of fungi ITS sequence in pedotheque described in any one of claim 1 and 2
Application of the PCR primer in fungal diversity detection.
4. for constructing the multiple sequencing library of fungi ITS sequence in pedotheque described in any one of claim 1 and 2
Application of the PCR primer in preparation fungal diversity detection kit.
5. a kind of multiple sequencing library of ITS sequence constructs kit, which is characterized in that the kit includes claim 1
With the PCR primer for being used to construct the multiple sequencing library of fungi ITS sequence in pedotheque described in 2 any one.
6. a kind of multiple sequencing library building kit of ITS sequence described in claim 5 is examined in pedotheque fungal diversity
Application in survey.
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| WO2014086037A1 (en) * | 2012-12-07 | 2014-06-12 | 深圳华大基因科技服务有限公司 | Method for constructing nucleic acid sequencing library and applications thereof |
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| WO2014086037A1 (en) * | 2012-12-07 | 2014-06-12 | 深圳华大基因科技服务有限公司 | Method for constructing nucleic acid sequencing library and applications thereof |
Non-Patent Citations (3)
| Title |
|---|
| Brant C. Faircloth, Travis C. Glenn.Not All Sequence Tags Are Created Equal: Designing and Validating Sequence Identification Tags Robust to Indels.《PLOS ONE》.2012,第7卷(第8期),补充数据File S1. * |
| Emergence Shapes the Structure of the Seed Microbiota;Matthieu Barret et al.;《Applied and Environmental Microbiology》;20141212;第81卷(第4期);第1258页右栏第3段 * |
| Not All Sequence Tags Are Created Equal: Designing and Validating Sequence Identification Tags Robust to Indels;Brant C. Faircloth, Travis C. Glenn;《PLOS ONE》;20120810;第7卷(第8期);补充数据File S1 * |
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