CN107904668A - A kind of microbial diversity library constructing method and its application - Google Patents

A kind of microbial diversity library constructing method and its application Download PDF

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CN107904668A
CN107904668A CN201810000905.7A CN201810000905A CN107904668A CN 107904668 A CN107904668 A CN 107904668A CN 201810000905 A CN201810000905 A CN 201810000905A CN 107904668 A CN107904668 A CN 107904668A
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李静
陈昌岳
王芳
张璐璐
胡秋萍
张祥林
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SHANGHAI MAJORBIO PHARM TECHNOLOGY Co Ltd
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Abstract

The present invention relates to technical field of molecular biology, more particularly to a kind of microbial diversity library constructing method and its application.The new microbial diversity library constructing method of the present invention, microbial diversity library construction is carried out to grand genome using single-stranded connection and special tag primer connector, the program is suitable for conventional sample, micro-example, FFPE samples and a small amount of cell mass of less than 150, expands the application range of Microbial diversity Journal of Sex Research；Different population purpose fragment can be carried out the less enrichment of skewed popularity by single-stranded connection and the introducing of special tag, ensure that the authenticity of Species richness and sample different plant species abundance.In addition, also have the advantages that initial amount is low and can carry out several species discriminating to same sample.

Description

A kind of microbial diversity library constructing method and its application
Technical field
The present invention relates to microbial diversity research field, more particularly to a kind of microbial diversity library constructing method And its application.
Background technology
The structure of community and diversity of microorganism and the function and metabolic mechanism of microorganism are the research of microbial ecology Hot spot.For a long time, it is also not comprehensive to biological community structure and multifarious understanding due to being subject to technology restriction, to micro- life What is understood in terms of thing function and metabolic mechanism is also seldom.Sequencing technologies development in recent years is swift and violent, it has also become the weight of biological study Want means.The rise of high throughput sequencing technologies, make the grand genome sequence of extensive, inexpensive research and microbial diversity into For possibility.Analysis of Microbial Diversity is for the purpose of the type and quantity of microorganism in research environment, is disclosed with environmental change The process for causing biological community structure to change.Its research object is extensive, including soil, water body, zymotic fluid, plant surface, dynamic Thing intestines and stomach, skin etc..Conventional identity process is the extraction of grand genome, utilizes 16S rRNA/18S rRNA/ITS etc. at present Amplimer is expanded and is sequenced to gene order.Environmental microorganism structure of community is detected to send out with the change of external environment Raw extremely faint change, relation, environmental improvement and the utilization of microbial resources for our microorganisms and environment And human medical's health has important theory and realistic meaning.
At home, it is many to be related to agricultural, soil, forestry, ocean, mine, human medical etc. for the research of microbial diversity Field.By taking the application in medical field as an example, by it is relatively normal with morbid state or the micro- life of human body in the different processes of disease The 26S Proteasome Structure and Function change of thing group, can carry out the micropopulation diversity in normal population and some Disease bodies Comparative analysis, research obtain human microflora's change with the relation between disease；Can also be rapidly by deep sequencing It was found that and detection encountered pathogenic and emerging infectious disease pathogenic microorganism.
Research for microbial diversity at present is more demanding to sample DNA amount, need to usually reach ng and more than.Sample Total amount is relatively low or when cell number is less, and amplification can seem particularly difficult.Currently used solution is as follows：1. adjustment is moved back Fiery temperature；2. increase period；3. first carrying out full genome amplification, microbial diversity PCR amplification is then carried out again.But this 3 kinds There are drawback for scheme：1. adjusting annealing temperature, more amplified productions can be obtained to a certain extent by reducing annealing temperature, But what the reduction of annealing temperature was brought is increasing for non-specific amplification and increasing for primer dimer, to follow-up library structure Build and the utilization of sequencing data bring greatly it is unfavorable；2. the adjustment of period to a certain extent can also improve product amount Storehouse level is continued after, but the increase of period also can increase non-specific amplification product, primer dimer, trace sample Copy number may only have each digit, and the excessive increase of period is likely to bring the skewed popularity and negative control product of amplification Occur, cause the missing of different microorganisms abundance and distortion in sequencing result.
Conventional microbiological diverse libraries can only be directed to a certain specific regions such as 16S rRNA/18S rRNA/ITS and be expanded Increase, can only just carry out single analysis to populations such as the bacterium of sample, fungi, ancient bacterium under such circumstances, if it is desired to while to more Kind population analyze at the same time, and the method that can use multiplex PCR, is expanded in same system using corresponding primer on multiple populations Increase.But this scheme can cause the difference of annealing temperature due to the difference of different amplimer Tm values, easily cause non-specificity Product and primer dimer increase.And the introducing of multiplex PCR easily causes amplification skewed popularity, and it is true to influence subsequent data analysis Reality.
Mainly include currently used for the multifarious Protocols in Molecular Biology of microorganisms：Denaturing gradient gel electrophoresis system (DGGE), temperature gradient gel electrophoresis (TGGE), time-temperature gradient electrophoresis (TTGE), restricted length polymorphism (Terminal Restriction Fragment Length Polymorphism, T-RFLP), SSCP, FISH, the marking are miscellaneous Friendship, quantitative PCR, genetic chip etc..One in microbial ecology of TTGE time-temperature gradient gel electrophoresis systems technologies Analysis biological community structure composition is mainly used for, which is widely used in each microbial ecosystem, including soil, Activated sludge, human body and animal intestinal tract, hot spring, root system of plant, ocean, fresh water lake, oil reservoir etc..Overwhelming majority research all passes through The 16S rRNA genes of amplification bacterium or archeobacteria study bacterium or the archeobacteria community diversity in each ecosystem.Also have 18S rRNA genes are expanded with the universal primer of fungi, so as to study the community diversity of fungi.
DGGE equimolecular fingerprint pattern technologies, often comprise only tens of band in its experimental result, can only reflect The information of minority advantage bacterium in sample；On the other hand, due to the error of resolution ratio, not only one may be included in the electrophoretic band of part Kind 16S rDNA sequences, therefore to learn specific strain information in electrophoresis pattern, also need to build clone library to each band, And screening and cloning is sequenced, this experimental implementation is relatively cumbersome；In addition, the microorganism in sample can not be cooked in this way To absolute quantitation.Biochip is the information that microbial diversity is obtained by the probe being fixed on chip, " is able to validate only It is known that can not but explore unknown ", the method judges the abundance of microorganism nor very accurate by signal strength.
With the fast development of high throughput sequencing technologies, microbial diversity is become using two generation sequencing technologies conventional Research approach.Sequencing is carried out to microbiologic population includes two classes, and one kind is carried out by 16s rDNA, 18s rDNA, ITS regions Expand the Canopy structure and diversity of sequencing analysis microorganism；Also one kind is grand gene order-checking, is without being separately cultured Microorganism, and all microbial DNAs are sequenced, so as to analyze microbiologic population's composition, gene is formed, and excavation has using valency The genetic resources of value.Old process is special using 16s rDNA, 18s rDNA, ITS regions respectively after being stripped to sample Property primer expanded after carry out again two generation sequencing libraries structure, gained library sequencing after be used for Microbial Community Diversity and structure Into analysis.
But old process has the following disadvantages：
(1) initial amount is high：Conventional diverse libraries structure needs initial amount DNA total amounts to be more than 100ng, and concentration is more than 10ng/ul.DNA total amounts are less after some preciousness sample and trace sample extractings, or even do not reach kit and carry out DNA extractings It is required that this part sample cannot carry out the structure of diverse libraries, the application of diversity analysis is limited.
(2) several species discriminating cannot be carried out to same sample：Conventional diverse libraries structure is that whole group DNA is carried out PCR amplification, can only carry out amplification respectively and build storehouse for a certain region such as 16s rDNA, 18s rDNA, ITS regions.Can not be to sample Several species such as whole samples such as bacterium, fungi, ancient bacterium in this are analyzed comprehensively.
(3) sample skewed popularity is stronger：Conventional diverse libraries structure expands conserved sequence region using PCR method Increase, the less information of abundance is often since the skewed popularity of PCR causes information on the low side or even lacks in sample group, it is impossible to accurate Reflected sample truth.
(4) there are error for sample abundance：Conventional diverse libraries structure carries out library construction using PCR method, involved PCR primer does not add coherent reference correction sequence since the differences such as Tm values be easy to cause deviation during design of primers, make analysis As a result there is deviation.
The content of the invention
In view of the foregoing deficiencies of prior art, it is an object of the invention to provide microbial diversity library construction side Method and its application.
In order to achieve the above objects and other related objects, the first aspect of the present invention provides a kind of microbial diversity text Base construction method, includes the following steps：
(1) genomic DNA of micro-biological samples is handled, becomes single stranded DNA；
(2) single stranded DNA obtained in step (1) is attached with special tag primer connector, obtains one end and be connected with The single stranded DNA of special tag primer connector；
(3) single stranded DNA that special tag primer connector is connected with to the one end obtained in step (2) extends, and is formed One end is connected with the double-stranded DNA of special tag primer；
(4) double-stranded DNA obtained to step (3) is attached with double-stranded adapters, is obtained both ends and is respectively connected with the double of connector Chain DNA；
(5) both ends obtained to step (4) are respectively connected with the double-stranded DNA of connector and expand, and obtain microbial diversity Library.
Further, the micro-biological samples can be the trace sample containing microorganism, FFPE samples and less than 150 A small amount of cell mass.
The genomic DNA of the micro-biological samples is cracked or extracted by micro-biological samples and obtained.
Furthermore, it is possible to be before step (1) is carried out, first micro-biological samples are cracked or extracted, so as to obtain institute State the genomic DNA of micro-biological samples.It can also use by other people using technology known in the art via microorganism sample The sample genomic dna that this cracking is obtained or obtained by micro-biological samples extracting.
Further, can Direct Pyrolysis acquisition microorganism sample when the cell number of micro-biological samples is below 100,000 This genomic DNA, without being stripped.
The cell can be prokaryotic or eukaryotic.
Further, the quality of the genomic DNA of the micro-biological samples can be more than or equal to 6pg.
Further, in the step (1), the length of the single stranded DNA can be 500nt or so.
Further, in the step (1), those skilled in the art can use known technology, for example, it is high-temperature denatured after The schemes such as sudden cold, NaOH solution alkaline denaturation make the genomic DNA of micro-biological samples be denatured into single stranded DNA.The high temperature is usually Refer to 60 DEG C -98 DEG C.Preferably 80 DEG C -98 DEG C.More preferably 90 DEG C -98 DEG C.Processing time is generally 30s-10min.Institute It is 0.2~0.6M NaOH, 1~1.5MNaCl to state NaOH solution.Preferably 0.3~0.5M NaOH, 1~1.5MNaCl.More Preferably 0.4M NaOH, 1.5MNaCl.
Further, in the step (2), the special tag primer connector, the special tag primer connector, is band There are the double-stranded adapters of cohesive terminus,cohesive termini, one end of the special tag primer connector is the first cohesive terminus,cohesive termini, the first stickiness end Hold and include at least special end sequence, target area of the special end sequence for identifying microbe genome DNA, and with The single stranded DNA of the target area is attached by complementary pairing.
Further, the target area is selected from 16S rRNA regions, 18S rRNA regions, ITS regions, functional gene etc. Region.
Further, the structure of the special end sequence is " optional sequence-identification target sequence ", the optional sequence The Tm between Tm, each connector of guarantee for connector where adjusting is close, and the identification target sequence is used to identify microorganism base It is attached because of the target area of group DNA, and with the single stranded DNA of the target area by complementary pairing.
Further, the length of the optional sequence is 1-2nt.It is preferred that can be 2nt.
Further, the length of the special end sequence is 2-30nt, preferably 4-20nt, more preferably 5- 10nt。
Further, the special end sequence such as SEQ ID NO.8~57 is any shown.
Further, the other end of the special tag primer connector can be flat end or the second stickiness end End.When second cohesive terminus,cohesive termini is arbitrary sequence or the repetitive sequence of A/T/C/G oligonucleotides, preferably repetitive sequence； Length is preferably 2-10nt, more preferably 2-5bt.
Further, in the special tag primer connector, the single-stranded length with the first cohesive terminus,cohesive termini is 10- 70nt.Can be further 20-40nt.Can be further 20-35nt.
Further, in the special tag primer connector, the single-stranded annealing temperature with the first cohesive terminus,cohesive termini is 10- 100℃.It can be further 20-80 DEG C.It can be further 30-60 DEG C.
Further, in the special tag primer connector, single-stranded 5 ' with the first cohesive terminus,cohesive termini are held into arm in the ranks (spacer) modify.Preferably, using C3Spacer or C6Spacer or Spacer C12 or Spacer 12 or Spacer C18 Modification.More preferably, in the special tag primer connector, 5 ' the single-stranded ends with the first cohesive terminus,cohesive termini are using 2-5 The modification of Spacer C12 or 2 C18Spacer or 10 C3Spacer or 5 C6Spacer.More preferably, the special mark Sign in primer connector, the single-stranded 5 ' end with the first cohesive terminus,cohesive termini using the modification that methylates repetition A/T/C/G sequences with The combination of the modification of C3Spacer or C6Spacer or Spacer 12 or Spacer 12 or Spacer C18.Preferably, use The repetition A/T/C/G sequences number of modification of methylating is preferred for 2-8；It is highly preferred that it can also be used after random primer The modification of AmC3 or AmC6 or AmC7 or thio-modification.
Further, it is single-stranded referred to as another without that of the first cohesive terminus,cohesive termini in the special tag primer connector Bar is single-stranded.
Further, in the special tag primer connector, another single-stranded length is 10-70nt.Preferably 10-30nt.It can be 10-25nt to be further preferably.
Further, in the special tag primer connector, another single-stranded annealing temperature is 10-100 DEG C.It is excellent Elect 20-80 DEG C as.It is 30-60 DEG C to be further preferably.
Further, in the special tag primer connector, described another 5 ' single-stranded ends carry out phosphorylation modification.
Further, in the special tag primer connector, described another single-stranded 3 ' is held into arm in the ranks (spacer) Modification.Preferably, in the special tag primer connector, described another single-stranded 3 ' end can use C3Spacer or The modification of C6Spacer or Spacer C12 or Spacer 12 or Spacer C18.More preferably, the special tag primer connects In head, described another 3 ' single-stranded ends can use 2 Spacer C12 or 2 C18Spacer or 10 C3Spacer or 5 The modification of C6Spacer.
Further, in the special tag primer connector, described another 3 ' single-stranded ends carry out biotinylation (bioten) modify.
Further, in the special tag primer connector, described another 3 ' the single-stranded existing arms (spacer) in end Modification has biotinylation (bioten) modification again.Preferably, arm (spacer) modification is positioned at the 3 ' of another chain between described Between end terminal sequence and biotinylation (bioten) modification.More preferably, between described arm (spacer) modification and biotin it Between be attached with TEG.
Further, in step (2), single stranded DNA and special tag primer connector are attached using ligase.
Further, the ligase be selected from HiFi Taq DNA ligases, T4 RNA ligases 2, T4 RNA ligases,Ligase, 9 ° of NTMDNA ligase, Taq DNA ligases, T7 DNA ligases, T3 DNA ligases, electricity turn to connect Meet enzyme, flat end/TA ligases premixed liquid, instantaneous stickiness ligase premixed liquid, T4 DNA ligases, Circligase ssDNA Any of ligase, 5 ' AppDNA/RNA thermostabilization ligases are a variety of.
Further, in step (3), when extension DNA extension reagents used be selected from archaeal dna polymerase, dNTPs, DNA polymerize Enzyme buffer liquid, extension primer.
Further, the archaeal dna polymerase be selected from vent archaeal dna polymerases, T7 archaeal dna polymerases, Bsu archaeal dna polymerases, T4 archaeal dna polymerases, DNA polymerase i, Klenow large fragments, DNA polymerase i (E.coli), TherminatorTMDNA polymerize Enzyme, Sulfolobus DNA polymerase i V, phi29DNA polymerase, Bst2.0Archaeal dna polymerase, Bst 2.0 Archaeal dna polymerase, Bst archaeal dna polymerases, Bst archaeal dna polymerases, Deep VentR (exo -) archaeal dna polymerase, Deep VentRTM Archaeal dna polymerase, VentR (exo -) archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Taq DNA polymerize Enzyme,Thermal starting Taq archaeal dna polymerases,Taq archaeal dna polymerases, thermal starting Taq archaeal dna polymerases, Taq archaeal dna polymerases large fragment, Taq archaeal dna polymerases, Taq archaeal dna polymerases,Thermal starting archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Flex archaeal dna polymerases,Super fidelity dna polymerase, Thermal starting surpass fidelity dna polymerase,The combination of the one or more enzyme such as super fidelity dna polymerase.
Further, the dNTPs can be 2.5mM~10mM.The DNA polymerase buffer liquid is selected most to be corresponding Good matching buffer solution.
Further, the sequence of the extension primer and the sequence of the non-stickiness part of any one chain of cohesive terminus,cohesive termini connector Row are all or part of complementary.The non-stickiness part refers to the double stranded section of cohesive terminus,cohesive termini connector.
Further, in step (4), when amplification DNA cloning reagent used be selected from archaeal dna polymerase, dNTPs, DNA polymerize Enzyme buffer liquid, amplimer.
Further, the archaeal dna polymerase be selected from vent archaeal dna polymerases, T7 archaeal dna polymerases, Bsu archaeal dna polymerases, T4 archaeal dna polymerases, Klenow fragments, DNA polymerase i (E.coli), TherminatorTMArchaeal dna polymerase, Sulfolobus DNA polymerase i V, phi29DNA polymerase, Bst2.0Archaeal dna polymerase, Bst 2.0DNA polymerases, Bst Archaeal dna polymerase, Bst archaeal dna polymerases, Deep VentR (exo -) archaeal dna polymerase, Deep VentRTMArchaeal dna polymerase, VentR (exo -) archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Taq archaeal dna polymerases,Heat opens Dynamic Taq archaeal dna polymerases,Taq archaeal dna polymerases, thermal starting Taq archaeal dna polymerases, Taq archaeal dna polymerases are large stretch of Section, Taq archaeal dna polymerases, Taq archaeal dna polymerases,Thermal starting archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Flex archaeal dna polymerases,Super fidelity dna polymerase,Thermal starting surpasses fidelity dna polymerization Enzyme,The combination of one or more of enzymes in super fidelity dna polymerase etc..
Further, the sequence of the amplimer and the sequence of the non-stickiness part of any one chain of cohesive terminus,cohesive termini connector Row are all or part of complementary.The non-stickiness part refers to the double stranded section of cohesive terminus,cohesive termini connector.
Further, before the double-stranded DNA obtained to step (3) expands, double-stranded adapters connection is first carried out, is obtained Obtain the double-stranded DNA that both-end is connected with connector.
Further, the end of the double-stranded adapters is flat end or cohesive terminus,cohesive termini.
The sequence of the double-stranded adapters can apply to the joint sequence of illumina microarray datasets or other The DNA of duplex structure.
Further, the length of the cohesive terminus,cohesive termini is 2-10nt, is preferably 2-5nt.
Further, the cohesive terminus,cohesive termini can be the cohesive terminus,cohesive termini with a T, Y types cohesive terminus,cohesive termini or be A or T or C Or the repetitive sequence of G.
For example, one end of the double-stranded adapters can be flat end or the cohesive terminus,cohesive termini with a T.The double-stranded adapters The other end be flat end or Y types cohesive terminus,cohesive termini or cohesive terminus,cohesive termini, the cohesive terminus,cohesive termini is the repetitive sequence of A or T or C or G.
Further, the double-stranded adapters include first chain and Article 2 chain.
Further, in the double-stranded adapters, first single-stranded length is 10-70nt.Preferably 10-30nt.It is excellent again Elect 10-25nt as.
Further, in the double-stranded adapters, first single-stranded annealing temperature is 10-100 DEG C.Preferably 20-80 DEG C. It is further preferably 30-60 DEG C.
Further, in the double-stranded adapters, first 5 ' single-stranded end carries out phosphorylation modification.
Further, in the double-stranded adapters, hold into arm in the ranks (spacer) for first single-stranded 5 ' and modify.
Further, in the double-stranded adapters, first 5 ' single-stranded end carries out biotinylation (bioten) modification.
Further, in the double-stranded adapters, first single-stranded existing of 5 ' end arm (spacer) modification has life again Thing elementization (bioten) is modified.Preferably, 3 ' end terminal sequences of arm (spacer) modification positioned at first chain between described Between being modified with biotinylation (bioten).More preferably, carried out between described between arm (spacer) modification and biotin with TEG Connection.
Further, in the double-stranded adapters, the single-stranded length of Article 2 is 10-70nt.Preferably 10-30nt.It is excellent again Elect 10-25nt as.
Further, in the double-stranded adapters, the single-stranded annealing temperature of Article 2 is 10-100 DEG C.Preferably 20-80 ℃.It is further preferably 30-60 DEG C.
Further, in the double-stranded adapters, Article 2 it is single-stranded 3 ' hold into arm in the ranks (spacer) modify.It is preferred that Ground, using the modification of C3Spacer or C6Spacer or Spacer C12 or Spacer 12 or Spacer C18.More preferably, 5 ' single-stranded ends of the Article 2 use 2-5 Spacer C12 or 2 C18Spacer or 10 C3Spacer or 5 The modification of C6Spacer.More preferably, the Article 2 it is single-stranded 5 ' end using methylate modification repetition A/T/C/G sequences with The combination of the modification of C3Spacer or C6Spacer or Spacer 12 or Spacer 12 or Spacer C18.Preferably, use The repetition A/T/C/G sequences number of modification of methylating is preferred for 2-8.
Further, 3 ' single-stranded ends of the Article 2 carry out biotinylation (bioten) modification.
Further, described two single-stranded existing of 3 ' end arm (spacer) modifications have biotinylation (bioten) to repair again Decorations.
Preferably, arm (spacer) modification can be located at the Article 2 single-stranded 5 ' end terminal sequences and biotin between described Between change (bioten) modification.More preferably, it is attached between described between arm (spacer) modification and biotin with TEG.
The second aspect of the present invention, there is provided aforementioned micro organism diverse libraries are used for microbial diversity sequencing or microorganism Purposes in diversity analysis.
The third aspect of the present invention, there is provided a kind of method of microbial diversity sequencing, includes the following steps：Using foregoing After method establishes library, the library obtained is sequenced.
The fourth aspect of the present invention, there is provided a kind of method of Analysis of Microbial Diversity, includes the following steps：Using foregoing After method establishes library, the library obtained is sequenced, diversity analysis is then carried out according to sequencing result.
The fifth aspect of the present invention, there is provided a kind of microbial diversity library construction Kit, including：Special tag primer Connector, ligase, DNA extensions reagent, DNA cloning reagent.
Further, the kit further includes cell pyrolysis liquid or genome DNA extraction reagent.The cell pyrolysis liquid For being cracked to micro-biological samples, micro-biological samples genomic DNA is obtained.The genome DNA extraction reagent be used for pair Micro-biological samples extract, and obtain micro-biological samples genomic DNA.
Further, the special tag primer connector is used for the genomic DNA denaturation for being connected to micro-biological samples On single stranded DNA, the single stranded DNA that one end carries special tag primer connector is obtained.
Further, the special tag primer connector is as described above.
Further, the ligase is used to connect the special tag primer connector and the single stranded DNA.
Further, the ligase be selected from HiFi Taq DNA ligases, T4 RNA ligases 2, T4 RNA ligases,Ligase, 9 ° of NTMDNA ligase, Taq DNA ligases, T7 DNA ligases, T3 DNA ligases, electricity turn to connect Meet enzyme, flat end/TA ligases premixed liquid, instantaneous stickiness ligase premixed liquid, T4 DNA ligases, Circligase ssDNA Any of ligase, 5 ' AppDNA/RNA thermostabilization ligases are a variety of.
Further, the single stranded DNA that the DNA extensions reagent is used to be connected with described one end connector extends, shape The double-stranded DNA of connector is connected with into one end.
Further, the DNA extends reagent as described above.
Further, the DNA cloning reagent is used to expand the double-stranded DNA, obtains microbial diversity and surveys Preface storehouse.
Further, the DNA cloning reagent is as described above.
Further, the kit may also include double-stranded adapters.The double-stranded adapters are connected with for being connected to one end The other end of the double-stranded DNA of special primer label, the double-stranded DNA of connector is connected with for obtaining both-end.
Further, the double-stranded adapters are as described above.
The fifth aspect of the present invention, there is provided a kind of microbial diversity sequencing products, including the structure examination of foregoing sequencing library Agent box.
Further, the sequencing can be generation sequencing, the sequencing of two generations or three generations's sequencing.
Further, the microarray dataset can be illumina microarray datasets.
Compared with prior art, the present invention has the advantages that：
(1) initial amount is low：
The new microbial diversity library constructing method of the present invention, pg grades can be reduced to by sample DNA initial amount, fitted For some precious samples and trace sample, multifarious application is greatly enlarged.
(2) several species discriminating can be carried out to same sample：
The new microbial diversity library constructing method of the present invention, can be to group using special tag primer connector In all sample conservative region such as 16s rDNA, 18s rDNA, ITS regions be carried out at the same time amplification and build storehouse.Can be in sample Several species such as whole samples such as bacterium, fungi, ancient bacterium are analyzed comprehensively.
(3) sample skewed popularity is uniformed
The new microbial diversity library constructing method of the present invention, utilizes single-stranded connection and special tag primer connector pair Grand genome carries out microbial diversity library construction, and the program is suitable for conventional sample, micro-example, FFPE samples and 150 A following a small amount of cell mass, expands the application range of Microbial diversity Journal of Sex Research；Single-stranded connection and the introducing of special tag Different population purpose fragment the less enrichment of skewed popularity be can be subjected to, Species richness and sample different plant species abundance ensure that Authenticity.
Brief description of the drawings
Fig. 1：The structure diagram of the special tag primer connector of the present invention, NNNN are special end sequence, 4 on figure N is display structure, and the restriction not to special end sequence partial-length.
Fig. 2：The single stranded DNA connection special tag primer connector of the displaying present invention simultaneously builds the substantially process in storehouse, wherein special Tag primer connector was connected in the step of single stranded DNA, and the special end sequence NNNN in special tag primer connector, is only used as tying Structure is illustrated, not as restriction；The modification such as biotin can also be connected on special tag primer connector, subsequently can be affine with strepto- The magnetic beads such as element are connected, and are purified；If after special tag primer connector is connected or extend into after double-stranded DNA or double-stranded adapters connect It can meet to apply after connecing, then can stop subsequent experimental accordingly, and not necessarily proceed to exponential amplification.
Fig. 3：Major microorganisms percentage composition block diagram in sample.
Embodiment
Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe Embodiment, the protection domain being not intended to be limiting of the invention；In description of the invention and claims, unless in text In addition explicitly point out, singulative "one", " one " and " this " include plural form.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using and Scientific terminology is identical with the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment, Outside material, according to grasp of the those skilled in the art to the prior art and the record of the present invention, it can also use and this Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method using this technology lead Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc. MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The present invention provides a kind of microbial diversity library constructing method, include the following steps：
(1) genomic DNA of micro-biological samples is handled, becomes single stranded DNA；
(2) single stranded DNA obtained in step (1) is attached with special tag primer connector, obtains one end and be connected with The single stranded DNA of special tag primer connector；
(4) double-stranded DNA obtained to step (3) is attached with double-stranded adapters, is obtained both ends and is respectively connected with the double of connector Chain DNA；
(5) both ends obtained to step (4) are respectively connected with the double-stranded DNA of connector and expand, and obtain microbial diversity Library.
In one example, the microorganism can be bacterium, fungi, ancient bacterium, conveyor screw, mycoplasma, Richettsia, clothing Substance, actinomyces etc..The microbial diversity refers to analyze microorganism contained therein for a certain micro-biological samples Species and accounting.
In one example, the micro-biological samples can be the trace sample containing microorganism, FFPE samples and 150 Following a small amount of cell mass.
In one example, the genomic DNA of the micro-biological samples is cracked by micro-biological samples and obtained or by microorganism Sample extracting obtains.
It can be before step (1) is carried out, first micro-biological samples are cracked or micro-biological samples are extracted, so as to obtain The genomic DNA of the micro-biological samples.It can also use by other people using technology known in the art via microorganism Sample cracking obtains or is extracted by micro-biological samples the genomic DNA of the micro-biological samples obtained.
In one example, in the step (1), the length of the single stranded DNA can be 500nt or so.
In one example, in step (2), the special tag primer connector, the special tag primer connector, is band There are the double-stranded adapters of cohesive terminus,cohesive termini, one end of the special tag primer connector is the first cohesive terminus,cohesive termini, the first stickiness end Hold and include at least special end sequence, target area of the special end sequence for identifying microbe genome DNA, and with The single stranded DNA of the target area is attached by complementary pairing.
In one example, the target area can be 16S rRNA regions, 18S rRNA regions or ITS regions, work( The regions such as energy gene.
In one example, the structure of the special end sequence is " optional sequence-identification target sequence ", described optional Sequence is used for the Tm of connector where adjusting, ensures that the Tm between each connector is close, and the identification target sequence is used to identify micro- life The target area of thing genomic DNA, and be attached with the single stranded DNA of the target area by complementary pairing.
In one example, the length of the optional sequence is 1-2nt.It is preferred that can be 2nt.
The length of the special end sequence can be adjusted according to the diversity of target sample, if target sample is various Property height then special end sequence length can relative increase, the length of special end sequence can if the diversity of target sample is low It is opposite to reduce.The length of the usual special end sequence is 2-30nt, preferably 4-20nt, more preferably 5-10nt.
In one example, the special end sequence such as SEQ ID NO.8~57 is any shown.
Further, the other end of the special tag primer connector can be flat end or the second stickiness end End.When second cohesive terminus,cohesive termini is arbitrary sequence or the repetitive sequence of A/T/C/G oligonucleotides, preferably repetitive sequence； Length is preferably 2-10nt, more preferably 2-5bt.
The length of first chain can make choice as needed.
In one example, in the special tag primer connector, the single-stranded length with the first cohesive terminus,cohesive termini is 10- 70nt.Can be further 20-40nt.Can be further 20-35nt.
In one example, in the special tag primer connector, the single-stranded annealing temperature with the first cohesive terminus,cohesive termini For 10-100 DEG C.It can be further 20-80 DEG C.It can be further 30-60 DEG C.
In one example, in the special tag primer connector, single-stranded with the first cohesive terminus,cohesive termini carries out appropriate Modification, to reduce the non-specific amplification of primer.For example, 5 ' the single-stranded ends for carrying the first cohesive terminus,cohesive termini can be into the ranks Arm (spacer) is modified, as using C3Spacer or C6Spacer or Spacer C12 or Spacer 12 or Spacer C18 Modification.Further, 5 ' the single-stranded ends with the first cohesive terminus,cohesive termini can use 2-5 Spacer C12 or 2 C18Spacer Or the modification of 10 C3Spacer or 5 C6Spacer.Yet further, using the repetition A/T/C/G sequences for the modification that methylates With the combination of the modification of C3Spacer or C6Spacer or Spacer 12 or Spacer 12 or Spacer C18；Wherein use first The repetition A/T/C/G sequences number of baseization modification is preferred for 2-8；It is highly preferred that it can also be used after special end sequence The modification of AmC3 or AmC6 or AmC7 or thio-modification prevent primer from degrading and form certain space structure, advantageously form double Chain structure.
In one example, it is single-stranded referred to as without that of the first cohesive terminus,cohesive termini in the special tag primer connector Another single-stranded.
In one example, in the special tag primer connector, another single-stranded length is 10-70nt.Into One step can be 10-30nt.Can be further 10-25nt.
In one example, in the special tag primer connector, another single-stranded annealing temperature is 10-100 ℃.It can be further 20-80 DEG C.It can be further 30-60 DEG C.
In one example, in the special tag primer connector, described another single-stranded to carry out appropriate modification, To reduce the non-specific amplification of primer.For example, in the special tag primer connector, described another 5 ' single-stranded end can be into Row phosphorylation modification, in the special tag primer connector, described another 3 ' single-stranded ends can be repaiied into arm in the ranks (spacer) Decorations, such as use the modification of C3Spacer or C6Spacer or Spacer C12 or Spacer 12 or Spacer C18, it is preferred to use The modification of 2 Spacer C12 or 2 C18Spacer or 10 C3Spacer or 5 C6Spacer.
In one example, in the special tag primer connector, described another 3 ' single-stranded ends can also carry out biology Elementization (bioten) is modified.When in the special tag primer connector, described another single-stranded 3 ' is held existing arms (spacer) modification has biotinylation (bioten) modification again when, an arm (spacer) modification can be located at the 3 ' of another chain Between end sequence and biotinylation (bioten) modification.Further, between described between arm (spacer) modification and biotin It is attached with TEG.
The cited joint sequence for applying to illumina microarray datasets, is only used for showing in some embodiments of the invention Meaning, it is not limited to these specific sequences.Cited modification mode is also only used for illustrating in embodiment.Can be according to actual need Carry out other modifications.
In one example, in step (2), single stranded DNA and special tag primer connector are attached using ligase.
In one example, the ligase is selected from HiFi Taq DNA ligases, T4 RNA ligases 2, T4 RNA and connects Connect enzyme,Ligase, 9 ° of NTMDNA ligase, Taq DNA ligases, T7 DNA ligases, T3 DNA ligases, electricity Turn ligase, flat end/TA ligases premixed liquid, instantaneous stickiness ligase premixed liquid, T4 DNA ligases, Circligase Any of ssDNA ligase, 5 ' AppDNA/RNA thermostabilization ligases are a variety of.
In one example, in step (3), when extension, DNA extension reagents used were selected from archaeal dna polymerase, dNTPs, DNA Polymerase buffer, extension primer.
In one example, the archaeal dna polymerase is selected from vent archaeal dna polymerases, T7 archaeal dna polymerases, Bsu DNA polymerizations Enzyme, T4 archaeal dna polymerases, Klenow fragments, DNA polymerase i (E.coli), TherminatorTMArchaeal dna polymerase, Sulfolobus DNA polymerase i V, phi29DNA polymerase, Bst2.0Archaeal dna polymerase, Bst2.0 DNA gather Synthase, Bst archaeal dna polymerases, Deep VentR (exo -) archaeal dna polymerase, Deep VentRTMArchaeal dna polymerase, VentR (exo -) archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Taq archaeal dna polymerases,Heat opens Dynamic Taq archaeal dna polymerases,Taq archaeal dna polymerases, thermal starting Taq archaeal dna polymerases, Taq archaeal dna polymerases are large stretch of Section, Taq archaeal dna polymerases, Taq archaeal dna polymerases,Thermal starting archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Flex archaeal dna polymerases,Super fidelity dna polymerase,Thermal starting surpasses fidelity dna polymerization Enzyme,The combination of the one or more enzyme such as super fidelity dna polymerase.
In one example, the dNTPs can be 2.5mM~10mM.The DNA polymerase buffer liquid is selected to be corresponding Best match buffer solution.Those skilled in the art can select the concentration of dNTPs according to being actually needed, as long as not limiting this hair Bright purpose.
In one example, the sequence of the extension primer and the non-stickiness part of any one chain of cohesive terminus,cohesive termini connector Sequence it is all or part of complementary.The non-stickiness part refers to the double stranded section of cohesive terminus,cohesive termini connector.
In one example, in step (4), when amplification, DNA cloning reagent used was selected from archaeal dna polymerase, dNTPs, DNA Polymerase buffer, amplimer.
In one example, the archaeal dna polymerase is selected from vent archaeal dna polymerases, T7 archaeal dna polymerases, Bsu DNA polymerizations Enzyme, T4 archaeal dna polymerases, DNA polymerase i, Klenow large fragments, DNA polymerase i (E.coli), TherminatorTMDNA gathers Synthase, Sulfolobus DNA polymerase i V, phi29DNA polymerase, Bst2.0Archaeal dna polymerase, Bst 2.0 Archaeal dna polymerase, Bst archaeal dna polymerases, Deep VentR (exo -) archaeal dna polymerase, Deep VentRTMArchaeal dna polymerase, VentR (exo -) archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Taq archaeal dna polymerases,Heat opens Dynamic Taq archaeal dna polymerases,Taq archaeal dna polymerases, thermal starting Taq archaeal dna polymerases, Taq archaeal dna polymerases are large stretch of Section, Taq archaeal dna polymerases, Taq archaeal dna polymerases,Thermal starting archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Flex archaeal dna polymerases,Super fidelity dna polymerase,Thermal starting surpasses fidelity dna polymerization Enzyme,The combination of one or more of enzymes in super fidelity dna polymerase etc..
In one example, the sequence of the amplimer and the non-stickiness part of any one chain of cohesive terminus,cohesive termini connector Sequence it is all or part of complementary.The non-stickiness part refers to the double stranded section of cohesive terminus,cohesive termini connector.
In one example, before the double-stranded DNA obtained to step (3) expands, double-stranded adapters connection is first carried out, Obtain the double-stranded DNA that both-end is connected with connector.
In one example, the end of the double-stranded adapters is flat end or cohesive terminus,cohesive termini.
The sequence of the double-stranded adapters can apply to the joint sequence of illumina microarray datasets or other The DNA of duplex structure.
In one example, the length of the cohesive terminus,cohesive termini is 2-10nt, is preferably 2-5nt.
In one example, the cohesive terminus,cohesive termini can be the cohesive terminus,cohesive termini with T, Y types cohesive terminus,cohesive termini or for A or The repetitive sequence of T or C or G.
For example, one end of the double-stranded adapters can be flat end or the cohesive terminus,cohesive termini with a T.The double-stranded adapters The other end be flat end or Y types cohesive terminus,cohesive termini or cohesive terminus,cohesive termini, the cohesive terminus,cohesive termini is the repetitive sequence of A or T or C or G.
In one example, the double-stranded adapters include first chain and Article 2 chain.
In one example, in the double-stranded adapters, first single-stranded length is 10-70nt.Preferably 10-30nt. It is further preferably 10-25nt.
In one example, in the double-stranded adapters, first single-stranded annealing temperature is 10-100 DEG C.Preferably 20- 80℃.It is further preferably 30-60 DEG C.
In one example, in the double-stranded adapters, first 5 ' single-stranded end carries out phosphorylation modification.
In one example, in the double-stranded adapters, hold into arm (spacer) between arm in the ranks for first single-stranded 5 ' and modify.
In one example, in the double-stranded adapters, first 5 ' single-stranded end carries out biotinylation (bioten) and repaiies Decorations.
In one example, in the double-stranded adapters, first single-stranded existing of 5 ' end arm (spacer) modification is again There is biotinylation (bioten) modification.Preferably, arm (spacer) modification is positioned at 3 ' end ends of first chain between described Between sequence and biotinylation (bioten) modification.More preferably, between described TEG is used between arm (spacer) modification and biotin It is attached.
In one example, in the double-stranded adapters, the single-stranded length of Article 2 is 10-70nt.Preferably 10-30nt. It is further preferably 10-25nt.
In one example, in the double-stranded adapters, the single-stranded annealing temperature of Article 2 is 10-100 DEG C.Preferably 20-80℃.It is further preferably 30-60 DEG C.
In one example, in the double-stranded adapters, Article 2 it is single-stranded 3 ' hold into arm in the ranks (spacer) modify.It is excellent Selection of land, using the modification of C3Spacer or C6Spacer or Spacer C12 or Spacer 12 or Spacer C18.Further preferably Ground, 5 ' single-stranded ends of the Article 2 use 2-5 Spacer C12 or 2 C18Spacer or 10 C3Spacer or 5 The modification of C6Spacer.More preferably, the Article 2 it is single-stranded 5 ' end using methylate modification repetition A/T/C/G sequences with The combination of the modification of C3Spacer or C6Spacer or Spacer 12 or Spacer 12 or Spacer C18.Preferably, use The repetition A/T/C/G sequences number of modification of methylating is preferred for 2-8.
In one example, 3 ' single-stranded ends of the Article 2 carry out biotinylation (bioten) modification.
In one example, described two single-stranded existing of 3 ' end arm (spacer) modifications have biotinylation again (bioten) modify.
In one example, between described arm (spacer) modification can be located at 5 ' single-stranded end terminal sequences of the Article 2 with Between biotinylation (bioten) modification.More preferably, it is attached between described between arm (spacer) modification and biotin with TEG.
The present invention still further provides aforementioned micro organism diverse libraries and be used for microbial diversity sequencing or microorganism Purposes in diversity analysis.
Present invention also offers a kind of microbial diversity sequencing approach, include the following steps：Established using preceding method Behind library, the library obtained is sequenced.
Present invention also offers a kind of Analysis of Microbial Diversity method, include the following steps：Established using preceding method Behind library, the library obtained is sequenced, then learning result according to survey carries out Analysis of Microbial Diversity.
Present invention also offers a kind of microbial diversity library construction Kit, including：Special tag primer connector, connect Connect enzyme, DNA extensions reagent, DNA cloning reagent.
In one example, the kit further includes cell pyrolysis liquid or genome DNA extraction reagent.The cell is split Solve liquid to be used to crack microbial single-cell sample, obtain microbial single-cell sample genomic dna.The genomic DNA is taken out Carry reagent to be used to extract microorganism many cells sample, obtain microorganism many cells sample genomic dna.
In one example,
Further, those skilled in the art can use known technology, such as high-temperature denatured rear sudden cold, NaOH solution alkali The schemes such as denaturation make the genomic DNA of micro-biological samples be denatured into single stranded DNA.The high temperature generally refers to 60 DEG C -98 DEG C.It is preferred that Ground is 80 DEG C -98 DEG C.More preferably 90 DEG C -98 DEG C.Processing time is generally 30s-10min.The NaOH solution for 0.2~ 0.6M NaOH, 1~1.5MNaCl.Preferably 0.3~0.5M NaOH, 1~1.5MNaCl.More preferably 0.4M NaOH, 1.5MNaCl.Those skilled in the art can select genomic DNA of the suitable mode to micro-biological samples according to being actually needed Handled, become single stranded DNA, as long as not limiting the purpose of the present invention.
In one example, the special tag primer connector is used for the genomic DNA denaturation for being connected to micro-biological samples Into single stranded DNA on, obtain one end carry special tag primer connector single stranded DNA.
In one example, the special tag primer connector is as described above.
In one example, the ligase is used to connect the special tag primer connector and the single stranded DNA.
In one example, the ligase is selected from HiFi Taq DNA ligases, T4 RNA ligases 2, T4 RNA and connects Connect enzyme,Ligase, 9 ° of NTMDNA ligase, Taq DNA ligases, T7 DNA ligases, T3 DNA ligases, electricity Turn ligase, flat end/TA ligases premixed liquid, instantaneous stickiness ligase premixed liquid, T4 DNA ligases, Circligase Any of ssDNA ligase, 5 ' AppDNA/RNA thermostabilization ligases are a variety of.
In one example, the single stranded DNA that the DNA extensions reagent is used to be connected with described one end connector prolongs Stretch, form the double-stranded DNA that one end is connected with connector.
In one example, the DNA extends reagent as described above.
In one example, the DNA cloning reagent is used to expand the double-stranded DNA, obtains Microbial diversity Property sequencing library.
In one example, the DNA cloning reagent is as described above.
In one example, the kit may also include double-stranded adapters.The double-stranded adapters are used to be connected to one end company The other end of the double-stranded DNA of special primer label is connected to, the double-stranded DNA of connector is connected with for obtaining both-end.
In one example, the double-stranded adapters are as described above.
The present invention furthermore provides a kind of microbial diversity sequencing products, including foregoing sequencing library structure kit.
In one example, the sequencing can be generation sequencing, the sequencing of two generations or three generations's sequencing.
Further, the microarray dataset can be illumina microarray datasets.
Compared with prior art, microbial diversity library constructing method of the invention, has following advantage：
(1) compared to traditional diverse libraries constructing plan, it can apply to the sample (as low as pg ranks) of low initial amount.
(2) compared to traditional diverse libraries constructing plan, library construction can be carried out to multiple groups in sample at the same time, more Comprehensively analyze sample composition.
(3) added in design optional compared to traditional diverse libraries constructing plan, special tag primer splice combinations Sequence so that the Tm values of each special tag primer connector ensure that the relatively uniform property of amplification in equivalency range, can be to a greater extent Reservation sample diversity, reduce due to library skewed popularity caused by primer reason.
Hereafter the technical solution of the embodiment of the present invention is further illustrated with specific embodiment 1.
Embodiment 1
First, special tag primer connector synthesizes：
The special tag primer connector, for the double-stranded adapters with cohesive terminus,cohesive termini, the special tag primer connector One end is the first cohesive terminus,cohesive termini, and first cohesive terminus,cohesive termini includes at least special end sequence, and the special end sequence is used for Identify the target area of microbe genome DNA, and be attached with the single stranded DNA of the target area by complementary pairing. The target area can be 16S rRNA regions, 18S rRNA regions or ITS regions etc..The structure of the special end sequence For " optional sequence-identification target sequence ", the optional sequence is used for the Tm between the Tm of connector where adjusting, each connector of guarantee Close, the identification target sequence is used for the nucleotide sequence complementary pairing with target area.
1st, as an example, synthesizing special tag primer connector using Primer1 and Primer2,
Primer1：5’-AGCTCAGAGT-3’-bioten(SEQ ID NO.1)；
Primer2：The special end sequences -3 ' of 5 '-ACTCTGAGC- (SEQ ID NO.2)；Wherein, the special end sequence Row, length be 2-30nt, preferably 4-20nt, be more preferably 5-10nt.
Under normal circumstances, those skilled in the art can be according to the target area for the microbe genome DNA to be identified, to select Corresponding special end sequence is selected, so as to form special tag primer connector group, in this group, contains several special marks Primer connector is signed, special end sequence can be different in each special tag primer connector.For example, when the target area to be identified is thin During bacterium 16S rRNA regions, two kinds of special tag primer connectors can be prepared, the special end in a kind of special tag primer connector Sequence is " CCTGGCTCAG ", optional sequence therein is " CC ", target knowledge sequence is " TGGCTCAG ", it is special to be formed at this time In Tag primer connector, the single-stranded global order with the first cohesive terminus,cohesive termini is classified as 5 '-ACTCTGAGC-CCTGGCTCAG-3 '. Special end sequence in another connector is " CCGTAGGAGT ", optional sequence therein is " CC ", target identification sequence is In " GTAGGAGT ", the special tag primer connector formed at this time, the single-stranded global order with the first cohesive terminus,cohesive termini is classified as 5 '- ACTCTGAGC-CCGTAGGAGT-3’.And so on.Different special end sequences in table 1 below, can form one and contain The special tag primer connector group of 52 special tag primer connectors, using this special tag primer connector group, you can at the same time Identify 26 different target areas.
Table 1
2nd, according to the form below 2 adds configuration reaction solution：
Table 2
Reagent Volume (ul)
Primer1(10μM) 1
Primer2(10μM) 1
TE buffer 7
5M NaCl 1
Cumulative volume 10
The TE buffer used in wherein above-mentioned reaction can also be by water, tris-hcl (0.05mM, PH7.0-8.0) etc. Buffer solution with identity function is substituted.
3rd, according to the form below 3 is reacted：
Table 3
Temperature Time Period
95℃ 60s 1
95℃-16℃ 0.5℃/s 1
16℃ forever 1
Obtain the special tag primer connector in identification different microorganisms genomic DNA different target region, its common structure For：
The special end sequences -3 ' of 5 '-ACTCTGAGC-
bioten-3’-GAAGGCTAGA-5’。
Wherein, the target area of identification is that two kinds of special tag primer connectors in bacterial 16 S rRNA regions are respectively：
5’-ACTCTGAGC-CCTGGCTCAG-3’
bioten-3’-GAAGGCTAGA-5’。
5’-ACTCTGAGC-CCGTAGGAGT-3’
bioten-3’-GAAGGCTAGA-5’。
And so on, form a special tag primer connector group containing 52 special tag primer connectors.
Can unloading in -20 DEG C preservation.
Reaction condition in table 3 is intended only as example, similar scheme can also be used to carry out, and the overall principle is first high temperature Slowly annealing forms complementary DNA double chain special tag primer connector after denaturation.
2nd, the preparation of double-stranded adapters
The end of the double-stranded adapters is flat end or cohesive terminus,cohesive termini.
1st, as an example, double-stranded adapters are prepared by Primer3 and Primer4, one end is flat end, and one end is stickiness End.
Primer3：5’-CTACTCTGAGC-3’(SEQ ID NO.3)；
Primer4：5’-GGAAGAGCGTCGTGTAGGGAAAGAGTG-3’(SEQ ID NO.4)；
Cs of the Primer3 at 3 ' ends preferably by phosphorylation modification, can more preferably use bi-deoxyribose nucleic acid C carries out the synthesis of primer；Primer4 can select to carry out thio-modification or without thio-modification, preferably carry out thio Modification, 3 bases that more preferably thio-modification 3 ' is held；5 ' the ends of primer4 are preferably by phosphorylation modification.
2nd, according to the form below 4 adds configuration reaction solution：
Table 4
Reagent Volume (ul)
Primer3(10μM) 5
Primer4(10μM) 5
TE buffer 9
5M NaCl 1
Cumulative volume 20
2nd, according to the form below 5 is reacted：
Table 5
Temperature Time Period
95℃ 60s 1
95℃-16℃ 0.5℃/s 1
16℃ forever 1
Obtain double-stranded adapters can unloading in -20 DEG C preservation.
Reaction condition in upper table 5 is intended only as example, similar scheme can also be used to carry out, and the overall principle is first high Slowly annealing forms complementary DNA double chain structure after temperature denaturation.
3rd, DNA is extracted
1st, the scheme that conventional sample DNA extracting can use those skilled in that art general is stripped, you can is walked Rapid four processing；
2nd, trace sample, FFPE samples and a small amount of cell mass of less than 150：14 μ L cells are added in each sample to split Solution liquid (50mM KAc, 20mM Tris-Ac, 10mM MgAc, 0.2%Trition X-100), 10mg glass beads are placed in Each 200 μ L PCR pipes, most high speed mix 30s and carry out broken wall treatment to sample.Add 1 μ L cell cracking enzymes (QIAGEN protease,10mg/ml；Lysozyme, 20mg/ml) centrifuge after mixing, be placed in PCR instrument, 25 DEG C be incubated 30 minutes, 50 DEG C be incubated 50 minutes cell lysis, 80 DEG C be incubated 10min make albumen enzyme denaturation.After obtaining sample genomic dna, step 4 is carried out Processing.
3rd, can certainly be can also use by other people using technology known in the art via trace sample, The sample genomic dna that cracking obtains in FFPE samples and a small amount of cell mass sample of less than 150 or extracting obtains.
The modes such as the 4th, gained genomic DNA can be interrupted by digestion, machinery, ultrasound interrupts, by genomic DNA interrupt for The double-strand of 500bp or so.
4th, single stranded DNA is attached using special tag primer connector group
1st, according to the form below 6 adds configuration reaction solution：
Table 6
Reagent Volume (ul)
10x T4 DNA ligation buffer 4
FastAP(1U/μl) 1
Sample genomic dna 15
Cumulative volume 20
DNA sample dephosphorylation can be prevented connecting certainly between sample by this operating procedure.
2nd, after sample being flicked mixing, according to the form below 7 is reacted：
Table 7
Temperature Time
37℃ 30min
95℃ 5min
Sample is immediately transferred on ice.
Sample genomic dna can be changed into single stranded DNA by this operating procedure.
3rd, according to the form below 8 adds configuration reaction solution：
Table 8
Reagent Volume (ul)
50%PEG-4000 4
100mM ATP 0.1
The special tag primer connector group obtained in embodiment 1 1
T4 DNA ligases 1
Seedless sour water 13.9
Cumulative volume 20
Flick mixing, cumulative volume 40ul.
4th, according to the form below 9 is reacted
Table 9
Temperature Time Period
37℃ 30min 1
95℃ 5min 1
4℃ forever 1
Can be by sample in -20 DEG C of preservations, or directly carry out next step reaction.
Sample genomic dna can not have to FastAP processing, preferably when being connected with special tag primer connector group To utilize FastAP processing.The buffering such as the 1%Tween 20 that is used in reaction, 1%NP40,1%triton x-100 Liquid is with homogeneous system；T4 DNA ligases can be substituted by other enzymes with double-strand or single-stranded connection enzymatic activity, preferably Have HiFi Taq DNA ligases, T4 RNA ligases 2, T4 RNA ligases,Ligase, 9 ° of NTMDNA connections Enzyme, Taq DNA ligases, T7 DNA ligases, T3 DNA ligases, electricity turn ligase, flat end/TA ligases premixed liquid, Instantaneous stickiness ligase premixed liquid, T4 DNA ligases, Circligase ssDNA ligase, 5 ' AppDNA/RNA thermostabilizations Any of ligase is a variety of.
5th, magnetic capture
1st, the magnetic bead for modifying Streptavidin, at least balances 30 minutes in room temperature, is vortexed and mixes.
2nd, take in the EP pipes of beads to the 1.5ml of 20ul (20ul/ samples).
3rd, 1xBWT+SDS (1M NaCl, 10mM Tris-HCl (pH 8.0), 1mM the EDTA (pH of 500 μ l of Beads 8.0), 0.05%Tween-20and 0.5%SDS) wash 2 times.
4th, beads is resuspended in the 1xBWT+SDS of 250ul (250ul/20ul magnetic beads).
5th, sample (40ul) is heated into 1min at 95 DEG C, is quickly transferred on ice, place at least 2-5min.
6th, the beads that sample is completed with washing is mixed.
7th, in incubation at room temperature 20min, and constantly mix.
8th, wink from, EP pipes are placed on magnetic frame, in removal please.
9th, 0.1xBWT+SDS (0.1M NaCl, 10mM Tris-HCl (pH 8.0), 1mM the EDTA (pH of 200 μ l are added 8.0), 0.05%Tween-20and 0.5%SDS), it is transferred to ThermoMixer (25 DEG C of set to；MKR 13,HLC/ Ditabis on), vortex 8s is that beads is resuspended.
10th, wink from, EP pipes are transferred on magnetic frame, remove supernatant.
The magnetic bead of wherein Streptavidin modification can be DynabeadsTM M-280Streptavidin、 DynabeadsTM MyOneTM Streptavidin C1、DynabeadsTMThe commercial products such as M-270Streptavidin, It can be the similar Magnetic bead sample for the Streptavidin modification being voluntarily coupled.
6th, extend
According to the form below 10 adds configuration reaction solution：
Table 10
Reagent Volume (ul)
10x Klenow reaction buffer 5
10mM dNTPs 0.4
1%Tween-20 2.5
Primer5(10μM) 1
Water 39.1
Cumulative volume 48
Primer5：5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-3’(SEQ ID NO.5).
Optionally, primer5 can carry out thio-modification, also can preferably carry out thio-modification, more without thio-modification Preferably whole primer carries out thio-modification, and more preferably 10 bases at 3 ends carry out thio-modification；More preferably 3 5 bases at end carry out thio-modification；A base at more preferably 3 ends carries out thio-modification.
1. beads is transferred on magnetic frame, please in removal.
2. adding the mixture in upper table 10, vortex makes beads fully mix.
3. 95 DEG C are transferred the sample into, 2min.
4. it is quickly transferred on ice, at least place 2-5min.
5. sample is transferred in EP pipes in room temperature.
6. adding the Klenow fragment (10U/ μ l) of 2ul, mix, beads is resuspended.
7.25 DEG C of incubation 5min, 2s, 1500rpm are mixed per 60s.
8.35 DEG C of incubation 25min, 2s, 1500rpm are mixed per 60s.
Wherein Klenow fragment can also be substituted by other polymerases [polymerase species], such as vent DNA Polymerase, T7 archaeal dna polymerases, Bsu archaeal dna polymerases, large fragment, T4 archaeal dna polymerases, DNA polymerase i, (Klenow) are large stretch of Section, DNA polymerase i (E.coli), TherminatorTMArchaeal dna polymerase, Sulfolobus DNA polymerase is V, phi29 DNA Polymerase, Bst2.0Archaeal dna polymerase, 2.0 archaeal dna polymerases of Bst, Bst archaeal dna polymerases, Deep VentR (exo -) archaeal dna polymerase, Deep VentRTMArchaeal dna polymerase, VentR (exo -) archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Taq archaeal dna polymerases,Thermal starting Taq archaeal dna polymerases,Taq DNA Polymerase, thermal starting Taq archaeal dna polymerases, Taq archaeal dna polymerases large fragment, Taq archaeal dna polymerases (are provided without magnesium ion ThermoPol II buffer solutions), Taq archaeal dna polymerases (provideBuffer solution),Thermal starting DNA gathers Synthase,Archaeal dna polymerase,Thermal starting Flex archaeal dna polymerases,Super fidelity dna polymerase,Thermal starting surpass fidelity dna polymerase,The combination of the one or more enzyme such as super fidelity dna polymerase.
Above-mentioned reaction system is served only for example displaying, rather than limits.
7th, cleaned after expanding
1. sample wink from, be transferred on magnetic frame, remove supernatant.
2. add 0.1xBWT2. (0.1M NaCl, 1mM Tris-HCl (pH 8.0), 0.1mM the EDTA (pH of 200 μ l 8.0), 0.005%Tween-20).
3. by sample blending, wink is from being transferred on magnetic frame, remove supernatant.
4. add the Stringency wash buffer (0.1xSSC and 0.1%SDS) of 100 μ l.
5. sample is incubated at room temperature 3min.
6. wink from, be transferred on magnetic frame, in removal please.
7. add 0.1xBWT2. (0.1M NaCl, 1mM Tris-HCl (pH 8.0), 0.1mM the EDTA (pH of 200 μ l 8.0), 0.005%Tween-20), it is vortexed and mixes.
Above-mentioned cleaning program only with example show rather than limit.
8th, double-stranded adapters connect
According to the form below 11 adds configuration reaction solution：
Table 11
Reagent Volume (ul)
10x T4 DNA ligase buffer 5
50%PEG-4000 5
Double-stranded adapters (10uM) 2
1%Tween-20 2
T4 DNA ligase(5U/μl) 2
Water 34
Cumulative volume 50
1. beads is gone supernatant, mixed with the mix in upper table 11.
2. sample is paid attention to mixing in 25 DEG C of incubation 1h, incubation.
T4 DNA ligases can be substituted by other enzymes with double-strand or single-stranded connection enzymatic activity, preferably there is HiFi Taq DNA ligases, T4 RNA ligases 2, T4 RNA ligases,Ligase, 9 ° of NTMDNA ligase, Taq DNA ligase, T7 DNA ligases, T3 DNA ligases, electricity turn ligase, flat end/TA ligases premixed liquid, instantaneous stickiness In ligase premixed liquid, T4 DNA ligases, Circligase ssDNA ligase, 5 ' AppDNA/RNA thermostabilization ligases It is any one or more.
9th, washing and sample elution after connecting
1. by sample wink from, be transferred on magnetic frame, in removal please.
2. add 0.1xBWT2. (0.1M NaCl, 1mM Tris-HCl (pH 8.0), 0.1mM the EDTA (pH of 200 μ l 8.0), 0.005%Tween-20).
3. by sample blending.
4. wink from, be transferred on magnetic frame, remove supernatant.
5. add the Stringency wash buffer (0.1xSSC and 0.1%SDS) of 100 μ l.
6. by sample at 45 DEG C, 3min is incubated.
7. wink from, be transferred on magnetic frame, in removal please.
8. add 0.1xBWT2. (0.1M NaCl, 1mM Tris-HCl (pH 8.0), 0.1mM the EDTA (pH of 200 μ l 8.0), 0.005%Tween-20).
9. transferring the sample into mixing, it is transferred on magnetic frame, please in removal.
10. add the seedless sour water of 50 μ l.
11. by sample after 10min is incubated at room temperature, 95 DEG C of heating 2min.
12. transferring the sample on magnetic frame, supernatant is transferred in the EP pipes of clean low suction.
Above-mentioned double-stranded adapters connection scheme is served only for example explanation, rather than limits.
Tenth, sample amplification (optional step)
According to the form below 12 mixes pcr amplification raw materials：
Table 12
Sample Volume (ul)
KAPA HiFi HotStart ReadyMix(2x) 25
Primer6(10μM) 1
Primer7(10μM) 1
NF water 23
Cumulative volume 50
By the sample of elution or directly with the following reaction of magnetic bead progress, (each sample can do 2 or multiple parallel PCR Amplified reaction, can increase the yield of sample).
Sample is mixed, according to the form below 13 is expanded：
Table 13
Primer6：
5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTACTCTGAGCT-3’(SEQ ID NO.6)；
Primer7：
5’-CAAGCAGAAGACGGCATACGAGATtgccgaGTGACTGGAGTTCAGACGTGTGCTACTCTGAGC-3’ (SEQ ID NO.7)；
Wherein underscore part is index sequences, could alternatively be other index sequences, specifically refers to The index sequences that illumina sequencing instruments are applicable in.
Wherein KAPA HiFi HotStart ReadyMix (2x) can also be substituted by other polymerases, such as vent Archaeal dna polymerase, T7 archaeal dna polymerases, Bsu archaeal dna polymerases, large fragment, T4 archaeal dna polymerases, DNA polymerase i, (Klenow) Large fragment, DNA polymerase i (E.coli), TherminatorTMArchaeal dna polymerase, Sulfolobus DNA polymerase is V, phi29 Archaeal dna polymerase, Bst2.0Archaeal dna polymerase, 2.0 archaeal dna polymerases of Bst, Bst archaeal dna polymerases, total length, Bst Archaeal dna polymerase, Deep VentR (exo -) archaeal dna polymerase, Deep VentRTMArchaeal dna polymerase, VentR (exo -) DNA polymerizations Enzyme,Archaeal dna polymerase,Thermal starting Taq archaeal dna polymerases,Thermal starting Taq DNA polymerize Enzyme,Taq archaeal dna polymerases, thermal starting Taq archaeal dna polymerases, Taq archaeal dna polymerases large fragment, Taq DNA gather Synthase (providing the ThermoPol II buffer solutions without magnesium ion), Taq archaeal dna polymerases (provideBuffering Liquid),Thermal starting archaeal dna polymerase,Archaeal dna polymerase,Thermal starting Flex archaeal dna polymerases,Super fidelity dna polymerase,Thermal starting surpass fidelity dna polymerase,Super fidelity dna polymerase, KAPA The combination of the one or more enzyme such as HiFi Uracil+ReadyMix (2x), PfuTurbo Cx thermal starting archaeal dna polymerases.
Above-mentioned reaction is served only for example displaying, rather than limits.
11, Ampure XP beads purification of samples
1. AMPure XP BeadsDNA are purified into magnetic bead in equilibrium at room temperature at least 30min.
2. in the sample for taking 40 μ lDNA purifying magnetic bead Agencourt AMPure XP Beads to 50 μ l, it is vortexed and mixes. If same sample has carried out parallel PCR reactions, multiple samples can be mixed into a pipe.Wherein DNA purifying magnetic beads Agencourt The dosage of AMPure XP Beads can have more needs and be adjusted, preferably 40-50ul.
3. room temperature places 5min, sample is set fully to be combined with DNA.
4. transfer the sample on magnetic frame, the supernatant discarding after sample clarification.
5. with the ethanol (now with the current) of fresh 200ul 80% (vol/vol), beads is cleaned, can be turned upside down, turned Dynamic EP pipes, make washing abundant.
6. supernatant discarding.
7. with the ethanol (now with the current) of fresh 200ul 80% (vol/vol), beads is cleaned, can be turned upside down, turned Dynamic EP pipes, make washing abundant.Supernatant discarding.
8. wink, from once, residual ethanol was discarded after sample clarification.
The wait 9. room temperature is uncapped, makes ethanol fully volatilize, and small slight crack occurs to surface.(overdrying or there can not be the ethanol residual Stay).
10. adding suitable seedless sour water, fully mixed with sample.
11. in incubation at room temperature at least 2min.
12. supernatant is transferred in the EP pipes of clean NF, remaining 2ul carries out quantitative and quality inspection.
Wherein DNA purifying magnetic beads can be that AMPure XP Beads can be by waiting other to have effects that identical magnetic bead replaces In generation, except that can use magnetic beads for purifying sample, can also use has effects that identical kit or scheme substitute.
Embodiment 2
(1) take fecal sample 10ng to be equally divided into 6 parts, be grouped according to table 14 below.Every group of setting negative control, point N1＆N2 is not named as it：
Table 14
Wherein, quantified after the extracting of A-C samples using picogreen double-strand quantitative stains, quantitative result is below examining Survey limit.
It is as follows that routine builds storehouse scheme：
1. the following PCR reaction mixtures of configuration：
Table 15
Sample Volume (ul)
KAPA HiFi HotStart ReadyMix(2x) 25
Primer storehouse (10 μM) 2
NF water 23
Cumulative volume 50
Wherein, primer storehouse represents well known to those skilled in the art to bacterium, fungi, ancient bacterium, denitrifying bacteria, fixed nitrogen The primer pair that the microorganisms such as bacterium, nitrate reductase bacterium, ammonia oxidizing bacteria, clump branch fungi are expanded.Each primer pair difference One reaction mix of configuration.
2. carry out PCR amplification according to following condition：
Table 16
Period is 35 or so when routine builds storehouse, since sample initial amount is relatively low in the present embodiment, therefore increases by 8 circulations Number is horizontal to reach magnetic beads for purifying.
3. a library will be mixed into after above-mentioned product purification, quantitative sequencing, uses miseq 2x250 platforms.
4.D, E, F sample use to be built storehouse mode and carries out in patent formula, and quantitative sequencing, uses miseq 2x250 platforms.
5. library outbound information such as table 17 below：
Table 17
Sample number Concentration Total amount
A 12.1 363
B 11.4 342
C 8.3 249
N1 9.7 291
D 9.1 273
E 7.9 237
F 8.8 264
N2 0.8 2.4
As shown above, trace sample is built storehouse scheme using difference and is expanded, and it is higher routinely to build storehouse scheme concentration of specimens, But it is also very high with batch negative control sample concentration, show more serious non-specific amplification occurred；The present invention builds storehouse side Concentration of specimens obtained by case is homogeneous, have with negative control compared with it is preferably comparative, it was initially believed that building Kucheng's work(.
Above-mentioned sample carries out miseq 2*250 sequencings, and data volume 300M data are with preliminary analysis library components, as a result such as Shown in table 18 below：
Table 18
If certain base mass value is 30 (Q30), then it represents that the probability of base sequencing error is 0.001.Comparison efficiency： The data of energy money order receipt to be signed and returned to the sender to genome account for the ratio of lower machine data.Redundancy：Energy money order receipt to be signed and returned to the sender repetitive sequence into the data of genome accounts for The ratio of total amount of data.
Micro fecal sample is poor using conventional storehouse mode comparison efficiency of building it can be seen from upper table 16, and redundancy is sequenced Degree is higher；The library sequencing result carried out using the present invention program shows that comparison efficiency is preferable, and redundancy is in acceptable water It is flat.
(2) 3 parts of row environmental samples of making even, every part of 1ng or so, are named as T1-T3, negative control is named as NC.According to this The experimental program that inventive embodiments 1 provide carries out library construction：
Library outbound information is as follows：
Table 19
Sequencing data such as table 20 below：
Table 20
Sample diversity information such as table 21 below after analysis：
Table 21
Sample main component percentage block diagram is as shown in Figure 3.
It can be seen from the above results using this programme preferably trace sample can be carried out diverse libraries structures, Sequencing and analysis.
In conclusion the present invention effectively overcomes various shortcoming of the prior art and has high industrial utilization.
The above-described embodiments merely illustrate the principles and effects of the present invention, not for the limitation present invention.It is any ripe Know the personage of this technology all can carry out modifications and changes under the spirit and scope without prejudice to the present invention to above-described embodiment.Cause This, those of ordinary skill in the art is complete without departing from disclosed spirit and institute under technological thought such as Into all equivalent modifications or change, should by the present invention claim be covered.
Sequence table
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Claims (23)

1. a kind of microbial diversity library constructing method, includes the following steps：
(1) genomic DNA of micro-biological samples is handled, becomes single stranded DNA；
(2) single stranded DNA obtained in step (1) is attached with special tag primer connector, it is special that acquisition one end is connected with The single stranded DNA of Tag primer connector；
(3) single stranded DNA that special tag primer connector is connected with to one end in step (2) extends, and forms one end and is connected with The double-stranded DNA of special tag primer；
(4) double-stranded DNA obtained to step (3) is attached with double-stranded adapters, obtains the double-strand that both ends are respectively connected with connector DNA；
(5) both ends obtained to step (4) are respectively connected with the double-stranded DNA of connector and expand, and obtain microbial diversity library.
2. microbial diversity library constructing method according to claim 1, it is characterised in that in the step (2), institute Special tag primer connector is stated, for the double-stranded adapters with cohesive terminus,cohesive termini, one end of the special tag primer connector is first Cohesive terminus,cohesive termini, first cohesive terminus,cohesive termini include at least special end sequence, and the special end sequence is used to identify microorganism The target area of genomic DNA, and be attached with the single stranded DNA of the target area by complementary pairing.
3. microbial diversity library constructing method according to claim 2, it is characterised in that the special tag primer The other end of connector is flat end or the second cohesive terminus,cohesive termini.
4. microbial diversity library constructing method according to claim 3, it is characterised in that when the special tag is drawn When the other end of thing connector is the second cohesive terminus,cohesive termini, any one of following characteristics or multinomial are further included：(1) second stickiness End is arbitrary sequence or the repetitive sequence of A/T/C/G oligonucleotides, preferably repetitive sequence；(2) the second stickiness end The length at end is 2-10nt, is preferably 2-5bt.
5. microbial diversity library constructing method according to claim 4, it is characterised in that the special end sequence Structure be " optional sequence-identification target sequence ", the optional sequence be used for the Tm of connector where adjusting, ensure each connector it Between Tm it is close, the identification target sequence is used to identifying the target area of microbe genome DNA, and with the target area Single stranded DNA be attached by complementary pairing.
6. microbial diversity library constructing method according to claim 5, it is characterised in that the special end sequence Length be 2-30nt, preferably 4-20nt, more preferably 5-10nt.
7. microbial diversity library constructing method according to claim 6, it is characterised in that the length of the optional sequence Spend for 1-2nt, preferably can be 2nt.
8. microbial diversity library constructing method according to claim 2, it is characterised in that the special tag primer In connector, the single-stranded length with the first cohesive terminus,cohesive termini is 10-70nt, is preferably 10-30nt, more preferably 10-25nt.
9. microbial diversity library constructing method according to claim 2, it is characterised in that the special tag primer In connector, the single-stranded annealing temperature with the first cohesive terminus,cohesive termini is 10-100 DEG C, is preferably 20-80 DEG C, more preferably 30-60 ℃。
10. microbial diversity library constructing method according to claim 2, it is characterised in that further include following characteristics Any one of or it is multinomial：(1) in the special tag primer connector, single-stranded 5 ' with the first cohesive terminus,cohesive termini are held into the ranks Arm (spacer) is modified；(2) in the special tag primer connector, 5 ' the single-stranded ends with the first cohesive terminus,cohesive termini carry out biology Elementization is modified.
11. microbial diversity library constructing method according to claim 2, it is characterised in that the special tag is drawn It is single-stranded without that single-stranded referred to as another of the first cohesive terminus,cohesive termini in thing connector.
12. microbial diversity library constructing method according to claim 11, it is characterised in that the special tag is drawn In thing connector, another single-stranded length is 10-70nt, is preferably 10-30nt, more preferably 10-25nt.
13. microbial diversity library constructing method according to claim 11, it is characterised in that the special tag is drawn In thing connector, another single-stranded annealing temperature is 10-100 DEG C, is preferably 20-80 DEG C, more preferably 30-60 DEG C.
14. microbial diversity library constructing method according to claim 11, it is characterised in that further include following characteristics Any one of or it is multinomial：(1) in the special tag primer connector, 5 ' ends of another chain carry out phosphorylation modification； (2) in the special tag primer connector, the 3 ' of another chain holds into arm in the ranks (spacer) modification；(3) it is described special In Tag primer connector, 3 ' ends of another chain carry out biotinylation modification.
15. microbial diversity library constructing method according to claim 1, it is characterised in that in step (2), use Single stranded DNA and special tag primer connector are attached by ligase.
16. microbial diversity library constructing method according to claim 15, it is characterised in that the ligase is selected from HiFi Taq DNA ligases, T4 RNA ligases 2, T4 RNA ligases,Ligase, 9 ° of NTMDNA ligase, Taq DNA ligases, T7 DNA ligases, T3 DNA ligases, electricity turn ligase, flat end/TA ligases premixed liquid, instantaneous Stickiness ligase premixed liquid, T4 DNA ligases, Circligase ssDNA ligase, 5 ' AppDNA/RNA thermostabilizations connection Any of enzyme is a variety of.
17. microbial diversity library constructing method according to claim 1, it is characterised in that obtained to step (3) Double-stranded DNA expanded before, first carry out double-stranded adapters connection, obtain both-end and be connected with the double-stranded DNA of connector.
18. as described in claim any one of 1-17 microbial diversity library constructing method be used for microbial diversity be sequenced or Purposes in Analysis of Microbial Diversity.
19. a kind of microbial diversity sequencing approach, includes the following steps：Using micro- life as described in claim any one of 1-17 After thing diverse libraries construction method establishes library, the library obtained is sequenced.
A kind of 20. Analysis of Microbial Diversity method, it is characterised in that using the microorganism as described in claim any one of 1-17 After diverse libraries construction method establishes library, the library obtained is sequenced, is then carried out according to sequencing result various Property analysis.
21. a kind of microbial diversity library construction Kit, including：Special tag primer connector, ligase, DNA extension examinations Agent and DNA cloning reagent.
22. sequencing library according to claim 21 builds kit, it is characterised in that the kit further includes cell Lysate or genome DNA extraction reagent.
23. sequencing library according to claim 21 builds kit, it is characterised in that the kit further includes double-strand Connector.
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Application publication date: 20180413