CN106191045B - Index and primer for multiple nucleic acid sequencing - Google Patents
Index and primer for multiple nucleic acid sequencing Download PDFInfo
- Publication number
- CN106191045B CN106191045B CN201610645107.0A CN201610645107A CN106191045B CN 106191045 B CN106191045 B CN 106191045B CN 201610645107 A CN201610645107 A CN 201610645107A CN 106191045 B CN106191045 B CN 106191045B
- Authority
- CN
- China
- Prior art keywords
- primer
- seq
- classified
- sequence
- primers
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to technical field of molecular biology, and in particular to for the Index of multiple nucleic acid sequencing and the PCR primer of building 16SrRNA multiple nucleic acid sequencing library.Index provided by the invention fully takes into account sequence difference degree, base discrimination and sequencing reaction between index tab to the data output Preference of different Index sequences, it will be it includes after being sequenced or building library primer, the sequencing data balance that mixing sequencing reaction obtains is relatively preferable.PCR primer provided by the invention is highly suitable for pedotheque, high specificity, and purpose amplified band is clear, without diffusing phenomenon, so that Phylogenetic diversity of bacteria research is more efficiently convenient accurate in pedotheque.Contain Index provided by the invention in PCR primer provided by the invention, contain in the amplified production of acquisition there are two Index label, which is used to mix sample sequencing, can be realized the differentiation to different sequencing libraries.
Description
Technical field
The present invention relates to technical field of molecular biology, and in particular to Index and building for multiple nucleic acid sequencing
The PCR primer of 16SrRNA multiple nucleic acid sequencing library.
Background technique
The numerous areas such as microbial diversity, especially Phylogenetic diversity of bacteria be micro- and agriculture, medicine, food and industrial production
There are close ties.With the continuous development of Protocols in Molecular Biology, microbial diversity research method is also gradually improved and maturation.
During microbial evolution, rRNA (the especially 16SrRNA of bacterium or the 18S rRNA of fungi) has centainly
Evolutionary conservatism, conserved region sequence are to exist as caused by evolving between conserved sequence common to all same organisms
The Variable Area of sequence difference between species, therefore be considered as one of the gene for being most suitable for genealogical classification.By to these sequences
The measurement and comparison of column Variable Area, can probe into and disclose the diversity of Soil Microorganism species and structure of community.With
The development of gene sequencing technology, high throughput sequencing technologies are also introduced into the investigative technique of microbial diversity.
In high throughput sequencing technologies, in order to save sequencing cost or research needs, it will usually mix different samples and survey
Sequence, referred to as multiple sequencing (Multiplexedsequencing) technology, by PCR primer, are used that is, when constructing sequencing library
After different index tabs (Index) marks different samples, different samples are mixed into upper machine and are sequenced, are passed through after sequencing
Index is identified to distinguish the different sample datas in same swimming lane (Lane).Multiple sequencing technologies high degree reduces
Sequencing cost saves sequencing resource, and to the convenient means that biological study provides.For example, carrying out the microorganisms such as bacterium
When Study on Diversity, it usually needs the diversity of hundreds of samples is analyzed, if without multiple sequencing technologies, scientific research cost
It will be greatly increased with time cost.
On the one hand, practical sequencing reaction has certain data output Preference to different Index sequences, causes to detect
The fluctuation of light intensity parameter when Insert Fragment influences the quality of data and balance of output, i.e. Index sequence influences mixed to a certain degree
The accuracy of the sequencing data of sample is closed, not any nucleotide sequence is suitable for being used as Index.Therefore it provides more may be used
It is extremely important to multiple sequencing technologies field with Index sequence.
On the other hand, inventor has found when carrying out Microbial diversity Journal of Sex Research: conventional at present is more for bacteria samples
The 16S rRNA amplimer (the 16S rRNA amplimer that such as Illunima company provides) of sample Journal of Sex Research is although can be to intestines
Road sample obtains relatively good expanding effect；But when the pedotheque more compared with strong, impurity for complexity, amplified production
Specificity is not high, and electrophoresis showed amplified band disperse is serious, this will seriously affect subsequent sequencing and precision of analysis.
In conclusion providing the relatively better Index sequence of one group of sequencing data balance and one group is suitable for building
The PCR primer of the multiple sequencing library of bacterial 16 S rRNA in pedotheque enables to the work of soil bacteria Study on Diversity more
It is 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 " Index " in the present invention refers to index tab, refers specifically to one section for distinguishing the nucleosides of different sequencing samples
Acid sequence；Term " PCR " refers to polymerase chain reaction；Term " connexon " refer to any one section and nucleotide sequence to be sequenced it
Between there is no be more than continuous 5 bases more than specific pairs nucleotide sequence.
The present invention considers influence of the index tab to the amplification efficiency of PCR primer and the Preference of data output simultaneously, mentions
One group is supplied for marking the index tab of nucleic acid samples to be sequenced, a group index label includes following 34 Index
In 2 or multiple；The nucleotide sequence of 34 Index is as shown in the table:
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 bacterial 16 S rRNA sequence.
Due in addition to bacteria, also containing the inhereditary material of a large amount of fungies, animal, plant etc. in soil, utilizing
When 16SrRNA sequencing carries out Phylogenetic diversity of bacteria research, pedotheque often has more compared to the other kinds of sample such as enteron aisle
High complexity.When the 16SrRNA amplimer of existing building sequencing library is used for intestinal samples, it can guarantee certain spy
It is anisotropic；However when being used for pedotheque, due to its complexity, ideal amplified production can not be often obtained, the amplification of acquisition produces
Object poor specificity, electrophoretic band disperse are serious.
Another object of the present invention is to provide one group for constructing the PCR primer of the multiple sequencing library of 16SrRNA.Institute
One group stated includes forward primer group and reverse primer group for constructing the PCR primer of the multiple sequencing library of 16SrRNA；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 16S rRNA in sample；The upstream and downstream of the amplified production of acquisition respectively contains an Index, this
Specific marker of two Index as the sample, for being distinguished with other samples to be sequenced.The amplified production of acquisition connects
After connecing sequence measuring joints, machine sequencing can be mixed；It cannot be containing comprising identical in the forward primer group and reverse primer group
The primer of Index.
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；
Primers F 30: including Index30 of the present invention, nucleotide sequence is as shown in SEQ ID NO:30；
Primers F 31: including Index31 of the present invention, nucleotide sequence is as shown in SEQ ID NO:31；
Primers F 32: including Index32 of the present invention, nucleotide sequence is as shown in SEQ ID NO:32；
Primers F 33: including Index33 of the present invention, nucleotide sequence is as shown in SEQ ID NO:33；
Primers F 34: including Index34 of the present invention, nucleotide sequence is as shown in SEQ ID NO:34.
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:35；
Primer R2: including Index2 of the present invention, nucleotide sequence is as shown in SEQ ID NO:36；
Primer R3: including Index3 of the present invention, nucleotide sequence is as shown in SEQ ID NO:37；
Primer R4: including Index4 of the present invention, nucleotide sequence is as shown in SEQ ID NO:38；
Primer R5: including Index5 of the present invention, nucleotide sequence is as shown in SEQ ID NO:39；
Primer R6: including Index6 of the present invention, nucleotide sequence is as shown in SEQ ID NO:40；
Primer R7: including Index7 of the present invention, nucleotide sequence is as shown in SEQ ID NO:41；
Primer R8: including Index8 of the present invention, nucleotide sequence is as shown in SEQ ID NO:42；
Primer R9: including Index9 of the present invention, nucleotide sequence is as shown in SEQ ID NO:43；
Primer R10: including Index10 of the present invention, nucleotide sequence is as shown in SEQ ID NO:44；
Primer R11: including Index11 of the present invention, nucleotide sequence is as shown in SEQ ID NO:45；
Primer R12: including Index12 of the present invention, nucleotide sequence is as shown in SEQ ID NO:46；
Primer R13: including Index13 of the present invention, nucleotide sequence is as shown in SEQ ID NO:47；
Primer R14: including Index14 of the present invention, nucleotide sequence is as shown in SEQ ID NO:48；
Primer R15: including Index15 of the present invention, nucleotide sequence is as shown in SEQ ID NO:49；
Primer R16: including Index16 of the present invention, nucleotide sequence is as shown in SEQ ID NO:50；
Primer R17: including Index17 of the present invention, nucleotide sequence is as shown in SEQ ID NO:51；
Primer R18: including Index18 of the present invention, nucleotide sequence is as shown in SEQ ID NO:52；
Primer R19: including Index19 of the present invention, nucleotide sequence is as shown in SEQ ID NO:53；
Primer R20: including Index20 of the present invention, nucleotide sequence is as shown in SEQ ID NO:54；
Primer R21: including Index21 of the present invention, nucleotide sequence is as shown in SEQ ID NO:55；
Primer R22: including Index22 of the present invention, nucleotide sequence is as shown in SEQ ID NO:56；
Primer R23: including Index23 of the present invention, nucleotide sequence is as shown in SEQ ID NO:57；
Primer R24: including Index24 of the present invention, nucleotide sequence is as shown in SEQ ID NO:58；
Primer R25: including Index25 of the present invention, nucleotide sequence is as shown in SEQ ID NO:59；
Primer R26: including Index26 of the present invention, nucleotide sequence is as shown in SEQ ID NO:60；
Primer R27: including Index27 of the present invention, nucleotide sequence is as shown in SEQ ID NO:61；
Primer R28: including Index28 of the present invention, nucleotide sequence is as shown in SEQ ID NO:62；
Primer R29: including Index29 of the present invention, nucleotide sequence is as shown in SEQ ID NO:63；
Primer R30: including Index30 of the present invention, nucleotide sequence is as shown in SEQ ID NO:64；
Primer R31: including Index31 of the present invention, nucleotide sequence is as shown in SEQ ID NO:65；
Primer R32: including Index32 of the present invention, nucleotide sequence is as shown in SEQ ID NO:66；
Primer R33: including Index33 of the present invention, nucleotide sequence is as shown in SEQ ID NO:67；
Primer R34: including Index34 of the present invention, nucleotide sequence is as shown in SEQ ID NO:68.
The present invention also provides a kind of multiple sequencing library of bacterial 16 S rRNA construct kit, it is characterised in that: it is described
Kit include one group of the present invention for constructing the PCR primer of the multiple sequencing library of 16SrRNA.
A kind of multiple sequencing library building kit of above-mentioned bacterial 16 S rRNA can also comprising extracting genome DNA reagent,
Archaeal dna polymerase, 16SrRNA sequence measuring joints add reagent.
The present invention also provides a kind of multiple sequencing library building kits of above-mentioned bacterial 16 S rRNA to examine in Phylogenetic diversity of bacteria
Application in survey.
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；It will be it includes after being sequenced or building library primer, mixing sequencing reaction obtains
The sequencing data balance arrived is relatively preferable.
(2), it is expanded provided by the present invention for constructing the PCR primer of the multiple sequencing library of 16SrRNA in conventional 16SrRNA
It is added to Index provided by the invention on the basis of primer and length is the joint sequence of 28-36 base, and surprisingly sends out
The PCR primer now thus constituted is other than it can obtain the higher 16SrRNA amplified production of specificity to samples such as enteron aisles, also
It is highly suitable for pedotheque, when expanding the 16SrRNA in pedotheque, high specificity, purpose amplified band is clear, without more
Phenomenon is dissipated, dramatically ensure that the accuracy of subsequent sequencing reaction and result of study, so that bacterium multiplicity in pedotheque
Journal of Sex Research is more efficiently convenient accurate.
(3), it provided by the present invention for the PCR primer of building 16S rRNA sequencing library, is combined by forward primer anti-
Primer into primer sets introduces Index provided by the invention, containing there are two Index labels in the amplified production of acquisition, by this
Index realizes the differentiation to different sequencing libraries for mixing sample sequencing.
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 " ddH2O " in the present invention 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.
DNA Kit is purchased from Omega company；KOD-Plus-Neo is purchased from TOYOBO company；Primer synthesis
Student on commission work bioengineering limited liability company carries out；The pedotheque of 100 separate sources is ground by Chinese Academy of Sciences's Beijing genome
Study carefully and is provided.
1 one groups of embodiment for constructing the PCR primer of the multiple sequencing library of 16SrRNA
It is made of 20 primers,
Wherein forward primer group is made of following 10 primers:
Primers F 1 of the present invention, nucleotide sequence is as shown in SEQ ID NO:1；
Primers F 2 of the present invention, nucleotide sequence is as shown in SEQ ID NO:2；
Primers F 4 of the present invention, nucleotide sequence is as shown in SEQ ID NO:4；
Primers F 5 of the present invention, nucleotide sequence is as shown in SEQ ID NO:5；
Primers F 7 of the present invention, nucleotide sequence is as shown in SEQ ID NO:7；
Primers F 8 of the present invention, nucleotide sequence is as shown in SEQ ID NO:8；
Primers F 9 of the present invention, nucleotide sequence is as shown in SEQ ID NO:9；
Primers F 10 of the present invention, nucleotide sequence is as shown in SEQ ID NO:10；
Primers F 12 of the present invention, nucleotide sequence is as shown in SEQ ID NO:12；
Primers F 13 of the present invention, nucleotide sequence is as shown in SEQ ID NO:13；
Wherein reverse primer group is made of following 10 primers:
Primer R14 of the present invention, nucleotide sequence is as shown in SEQ ID NO:48；
Primer R15 of the present invention, nucleotide sequence is as shown in SEQ ID NO:49；
Primer R16 of the present invention, nucleotide sequence is as shown in SEQ ID NO:50；
Primer R18 of the present invention, nucleotide sequence is as shown in SEQ ID NO:52；
Primer R19 of the present invention, nucleotide sequence is as shown in SEQ ID NO:53；
Primer R20 of the present invention, nucleotide sequence is as shown in SEQ ID NO:54；
Primer R22 of the present invention, nucleotide sequence is as shown in SEQ ID NO:56；
Primer R23 of the present invention, nucleotide sequence is as shown in SEQ ID NO:57；
Primer R25 of the present invention, nucleotide sequence is as shown in SEQ ID NO:59；
Primer R26 of the present invention, nucleotide sequence is as shown in SEQ ID NO:60.
The detection application of 1 Phylogenetic diversity of bacteria of experimental example
Construct the multiple sequencing library of bacterial 16 S rRNA of the pedotheque of 100 separate sources
(1), the extraction of sample gene group DNA:
The genomic DNA in pedotheque is extracted with Soil DNA Kit, extraction step is referring to the description of product
Book.
(2), PCR amplification 16S rRNA:
The genomic DNA extracted using step (1) expands sample as template, using the PCR primer provided in the embodiment of the present invention 1
16S rRNA in product, wherein the primer in forward primer group and reverse primer group matches two-by-two, obtains 100 different primers
Right, for 100 different samples PCR amplification.
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, gum concentration 0.8% are carried out to 100 amplified productions of acquisition, electrophoresis result display uses
The 16SrRNA amplified product band that primer provided by the invention obtains is clear, no diffusing phenomenon.Since sample size is more,
This shows the electrophoresis result of 1-24 sample, as shown in Figure 1, purpose band is clear, no diffusing phenomenon；25-100 sample
Electrophoresis result and No. 1-24 it is same or similar.
(3), sequence measuring joints are added:
Respectively using 100 amplified productions that step (2) obtain as template, PCR amplification is carried out using following primer pair, with
Sequence measuring joints containing Illumina MiSeq microarray dataset in lower primer.
5 '-CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTG-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
It is the multiple sequencing library of bacterial 16 S rRNA of the pedotheque of 100 separate sources after a amplified production mixing.
Comparative example 1
Following primer pair 1 and primer pair 2 are respectively that routine 16SrRNA amplimer and Illunima company provide
16SrRNA sequencing library constructs primer.
Primer pair 1:
5’-CCTACGGGNGGCWGCAG-3’
5’-GGACTACHVGGGTWTCTAAT-3’
Primer pair 2:
5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3'；
5’-GTCTCGTGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3’。
Using in the pedotheque of 100 separate sources used in above-mentioned two primer pair experimental example 1 of the present invention with
16SrRNA in 50 pedotheques of machine is expanded.
The extracting method of sample gene group DNA is with 1 step of experimental example (1) of the present invention, and PCR system and PCR program are the same as this hair
Bright 1 step of experimental example (2).
2 parts 50 of acquisition different amplified productions are subjected to agarose gel electrophoresis, gum concentration and deposition condition with experiment
1 step of example (2).Electrophoresis result is shown: the 16SrRNA amplified production purpose band obtained using primer pair 1 and primer pair 2 is unclear
Clear, disperse is serious.Since sample size is more, show that primer pair 1 is to the amplified production electricity of 1-24 sample in comparative example 1 herein
Swimming is as a result, disperse is serious as shown in Fig. 2, amplified production purpose band is unintelligible；The electrophoresis result of remaining sample in this comparative example
It is same or similar with Fig. 2.
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 bacterial 16 S rRNA 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 one or more in following primer:
Primers F l: its nucleotides sequence is classified as sequence shown in SEQ ID NO:1, wherein containing Index1:TGTACGCT；
Primers F 2: its nucleotides sequence is classified as sequence shown in SEQ ID NO:2, wherein containing Index2:GACTAGTC；
Primers F 4: its nucleotides sequence is classified as sequence shown in SEQ ID NO:4, wherein containing Index4:ACTAGATG；
Primers F 5: its nucleotides sequence is classified as sequence shown in SEQ ID NO:5, wherein containing Index5:GATGTACA；
Primers F 6: its nucleotides sequence is classified as sequence shown in SEQ ID NO:6, wherein containing Index6:GATCATGC；
Primers F 7: its nucleotides sequence is classified as sequence shown in SEQ ID NO:7, wherein containing Index7:CATGTCTA；
Primers F 8: its nucleotides sequence is classified as sequence shown in SEQ ID NO:8, wherein containing Index8:TATGTCAC；
Primers F 9: its nucleotides sequence is classified as sequence shown in SEQ ID NO:9, wherein containing Index9:TCTAGCAT；
Primers F 10: its nucleotides sequence is classified as sequence shown in SEQ ID NO:10, wherein containing Index10:GATCACTA；
Primers F 12: its nucleotides sequence is classified as sequence shown in SEQ ID NO:12, wherein containing Index12:TCTACAGA；
Primers F 13: its nucleotides sequence is classified as sequence shown in SEQ ID NO:13, wherein containing Index13:TACAGCGT；
The reverse primer group is made of one or more in following primer:
Primer R14: its nucleotides sequence is classified as sequence shown in SEQ ID NO:48, wherein containing Index14:GTGACTAT；
Primer R15: its nucleotides sequence is classified as sequence shown in SEQ ID NO:49, wherein containing Index15:TGCTACAT；
Primer R16: its nucleotides sequence is classified as sequence shown in SEQ ID NO:50, wherein containing Index16:TGATCTAC；
Primer R18: its nucleotides sequence is classified as sequence shown in SEQ ID NO:52, wherein containing Index18:TCGTGATA；
Primer R19: its nucleotides sequence is classified as sequence shown in SEQ ID NO:53, wherein containing Index19:ACTGCATA；
Primer R20: its nucleotides sequence is classified as sequence shown in SEQ ID NO:54, wherein containing Index20:ATAGTGCT；
Primer R22: its nucleotides sequence is classified as sequence shown in SEQ ID NO:56, wherein containing Index22:ACTCATGA；
Primer R23: its nucleotides sequence is classified as sequence shown in SEQ ID NO:57, wherein containing Index23:GTACATGA；
Primer R25: its nucleotides sequence is classified as sequence shown in SEQ ID NO:59, wherein containing Index25:CACGTGAT；
Primer R26: its nucleotides sequence is classified as sequence shown in SEQ ID NO:60, wherein containing Index26:CATCGAGT.
2. one group of PCR primer as described in claim 1, it is characterised in that: the PCR primer when in use: it is described just
Any one primer into primer sets and any one primer in reverse primer group are composed of primer pair two-by-two, pass through
PCR is reacted for expanding the 16S rRNA in sample；The upstream and downstream of the amplified production of acquisition respectively contains an Index, this
Specific marker of two Index as the sample, for being distinguished with other samples to be sequenced.
3. being used to construct the multiple sequencing library of bacterial 16 S rRNA in pedotheque for one group described in any one of claim 1 and 2
PCR primer Phylogenetic diversity of bacteria detection in application.
4. being used to construct the multiple sequencing library of bacterial 16 S rRNA in pedotheque for one group described in any one of claim 1 and 2
PCR primer preparing the application in Phylogenetic diversity of bacteria detection kit.
5. a kind of multiple sequencing library of bacterial 16 S rRNA constructs kit, it is characterised in that: the kit includes claim
Described in 1 and 2 any one one group for constructing the PCR primer of the multiple sequencing library of bacterial 16 S rRNA in pedotheque.
6. a kind of multiple sequencing library building kit of bacterial 16 S rRNA described in claim 5 is various in pedotheque bacterium
Property detection in application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610645107.0A CN106191045B (en) | 2016-08-08 | 2016-08-08 | Index and primer for multiple nucleic acid sequencing |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610645107.0A CN106191045B (en) | 2016-08-08 | 2016-08-08 | Index and primer for multiple nucleic acid sequencing |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106191045A CN106191045A (en) | 2016-12-07 |
| CN106191045B true CN106191045B (en) | 2019-10-11 |
Family
ID=57514659
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610645107.0A Expired - Fee Related CN106191045B (en) | 2016-08-08 | 2016-08-08 | Index and primer for multiple nucleic acid sequencing |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106191045B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102008451B1 (en) * | 2017-05-26 | 2019-08-07 | 주식회사 엠디헬스케어 | Method for diagnosis of Autism using analysis of bacteria metagenome |
| CN112735530A (en) * | 2021-01-22 | 2021-04-30 | 中国科学院北京基因组研究所(国家生物信息中心) | Method for tracing sample based on flora structure |
| CN113293238B (en) * | 2021-07-16 | 2022-04-12 | 烟台海关技术中心 | Soil virus detection technology based on macroviromics |
| CN117004700A (en) * | 2023-10-07 | 2023-11-07 | 北京爱博生生物技术有限公司 | Method for high-throughput sequencing of monoclonal antibody variable region genes, composition and kit used by method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105316320A (en) * | 2014-07-30 | 2016-02-10 | 天津华大基因科技有限公司 | DNA tags, PCR primer and application thereof |
| CN105671644A (en) * | 2016-02-26 | 2016-06-15 | 武汉冰港生物科技有限公司 | Preparation method of genome mixing sequencing library |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107075566B (en) * | 2014-01-27 | 2021-06-15 | 阿谢尔德克斯有限责任公司 | Isothermal methods for preparing nucleic acids and related compositions |
-
2016
- 2016-08-08 CN CN201610645107.0A patent/CN106191045B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105316320A (en) * | 2014-07-30 | 2016-02-10 | 天津华大基因科技有限公司 | DNA tags, PCR primer and application thereof |
| CN105671644A (en) * | 2016-02-26 | 2016-06-15 | 武汉冰港生物科技有限公司 | Preparation method of genome mixing sequencing library |
Non-Patent Citations (3)
| Title |
|---|
| Bacterial 16S rRNA Amplicon Sequencing;Herlemann等;《Standard Protocol》;20150316;第1-8页 * |
| Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex;Hamady M等;《Nature methods》;20080210;第5卷(第3期);第235-237页 * |
| Hamady M等.Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex.《Nature methods》.2008,第5卷(第3期),第235-237页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106191045A (en) | 2016-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cai et al. | Biased diversity metrics revealed by bacterial 16S pyrotags derived from different primer sets | |
| CN106191045B (en) | Index and primer for multiple nucleic acid sequencing | |
| CN106192022B (en) | The construction method of the multiple sequencing libraries of 16SrRNA | |
| CN104293783A (en) | Primer applicable to amplicon sequencing library construction, construction method, amplicon library and kit comprising amplicon library | |
| CN104263726A (en) | Primer applied to amplicon sequencing library construction and method for constructing amplicon sequencing library | |
| KR20200047686A (en) | Normalization for sequencing library | |
| CN101921860B (en) | Multicolor fluorescence composite detection method and kit of 44 SNPs (Single Nucleotide Polymorphism) loci | |
| Carrera et al. | Differentiation of smoked Salmo salar, Oncorhynchus mykiss and Brama raii using the nuclear marker 5S rDNA | |
| CN107304443A (en) | Storehouse PCR primer and banking process are built in the sequencing of the generation of chromaffin cell Disease-causing gene two | |
| CN108315399A (en) | Chondriogen detection kit and application method | |
| CN106282332B (en) | Label and primer for multiple nucleic acid sequencing | |
| Di Lenola et al. | Comparison of two molecular methods to assess soil microbial diversity | |
| Joseph et al. | Multilocus Sequence Typing (MLST) for Cronobacter spp. | |
| CN109136390A (en) | It is a kind of at the same identify chicken, duck, sheep Species origin PCR-STR method | |
| CN106192023A (en) | A kind of multiple sequencing library construction method based on multidimensional Index | |
| CN106191037B (en) | A kind of construction method of multiple sequencing library | |
| US20180291443A1 (en) | Library Quantitation And Qualification | |
| CN109576388B (en) | China fir EST-SSR molecular marker combination | |
| CN108060212B (en) | DNA typing identification kit | |
| Diaz et al. | Detecting genetic variability among different Mycobacterium tuberculosis strains using DNA microarrays technology | |
| Kumar et al. | Molecular Tools for Microbial Diversity Analysis | |
| CN110257489A (en) | A kind of detection technique system of 30 Multiple-allele SNP sites based on the sequencing of two generations | |
| Ngom et al. | Techniques for tracking microbial community structure and function in natural environment and engineered systems | |
| CN116083602B (en) | Species-specific target sequence for identifying deer based on time-base method, kit and application | |
| Inuwa et al. | Medical Biotechnology, Biopharmaceutics, Forensic Science and Bioinformatics |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Wang Xumin Inventor after: Liu Guiming Inventor after: Zhao Beilun Inventor after: Wang Guoliang Inventor after: Yu Jun Inventor before: Wang Xumin |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20191011 Termination date: 20200808 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |