CN106554958A - The primer and fast construction method of water/mud sample DNA amplicon sequencing library rapid build in a kind of sewage disposal system - Google Patents

The primer and fast construction method of water/mud sample DNA amplicon sequencing library rapid build in a kind of sewage disposal system Download PDF

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CN106554958A
CN106554958A CN201611071680.1A CN201611071680A CN106554958A CN 106554958 A CN106554958 A CN 106554958A CN 201611071680 A CN201611071680 A CN 201611071680A CN 106554958 A CN106554958 A CN 106554958A
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张徐祥
汤俊英
任洪强
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YIXING ENVIRONMENTAL PROTECTION RESEARCH INSTITUTE NANJING UNIVERSITY
Nanjing University
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YIXING ENVIRONMENTAL PROTECTION RESEARCH INSTITUTE NANJING UNIVERSITY
Nanjing University
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Abstract

The invention discloses in a kind of sewage disposal system water/mud sample DNA amplicon sequencing library rapid build primer and fast construction method, belong to high-flux sequence field.The present invention step be：First, the extraction of sample DNA；2nd, the amplification of purpose fragment；3rd, the purification of amplified production；4th, the Quality Identification of amplified production；5th, the mixing of amplified production；6th, mix the Quality Identification in library；7th, the preparation and sequencing in library are mixed.The invention discloses a kind of primer, including joint sequence, index sequence, heterogeneous intervening sequence, purpose fragment extension increasing sequence, so that the structure in library can be rapidly completed using the amplification of mono- steps of primer Jing, sequencing cost, a large amount of saving library construction times are reduced.Instant invention overcomes a highly diverse Bacterial community parsing difficult problem in complex wastewater/mud sample, meets the technical need that water in sewage disposal system/mud sample flora deep analysis are applied to based on high flux DNA sequencing technology.

Description

Water/mud sample DNA amplicon sequencing library rapid build in a kind of sewage disposal system Primer and fast construction method
Technical field
The invention belongs to high-flux sequence field, more particularly, it relates to water/mud sample in a kind of sewage disposal system The primer and fast construction method of the sub- sequencing library rapid build of DNA cloning.
Background technology
The secondary microarray dataset for comparing main flow in the market is public by Illumina companies and Life Technologies Department's production, it is long that these microarray datasets can produce longer reading.It is compared to the production of Life Technologies companies The individualized gene order-checking instrument of Ion Torrent, Hiseq 2500, the Hiseq 4000 produced based on Illumina platforms and Miseq sequenators run each circulation and can produce more data volumes, and wherein Miseq sequenators advantage is the test period It is short, can quickly analyze bacterial micro-organism structure of community and multiformity.
In bacterial genomes, the rDNA genes for encoding 16S rRNA have good evolutionary conservatism, fit analysis Length (about 1540bp) and the good variability matched with evolutionary distance, so becoming the standard logo of bacteria molecule identification Sequence.The sequence of 16S rDNA includes 9 hypervariable regions and 11 constant regions.Conserved sequence region is reflected between living species Sibship, and the difference between species can be embodied in hypervariable sequence region.16S rDNA hypervariable regions are entered using Miseq sequenators Row sequencing, can comprehensively be identified to the population structure of microorganism and multiformity.
To complete the sequencing of microorganism 16S rDNA, it is necessary first to carry out corresponding library structure based on Illumina sequenators Build, the banking process for being currently based on Illumina microarray datasets mainly there are two kinds, banking process and a step are expanded including two steps Amplification banking process.Storehouse is built in the amplification of two steps, i.e., carrying out two-wheeled PCR using two pairs of primers carries out the structure in library, and step is more numerous It is trivial and more through expanding the pollution of introducing twice so that follow-up accuracy of analysis is reduced.Second method expands for a step Increasing method, the method using than wide, i.e., complete to build storehouse process by step amplification, but the method needs are self-defined at present The sequencing primer of the sequencing primer of purpose fragment and each sample index sequence.
Water in sewage disposal system/mud sample contains the complicated organic and inorganic substances of a large amount of physicochemical properties, these things Matter can affect to carry out molecule manipulation (such as to DNA：Digestion with restriction enzyme, PCR amplifications etc.) material, such as heavy metal ion etc. Impurity and some extracellular free DNA.Therefore, it is still necessary to be optimized improvement to existing banking process, to provide one Plant the rapid build side of quick, the of reduced contamination water suitable for sewage disposal system/mud sample DNA amplicon sequencing library Method.
The content of the invention
1. problem to be solved
Build for water in current sewage disposal system/mud sample that storehouse quality is low, need the sequencing of self-defined purpose fragment to draw The problems such as thing, cumbersome, high cost, a kind of primer of amplicon sequencing library rapid build of present invention design, comprising joint Sequence, index sequence, heterogeneous intervening sequence, purpose fragment extension increasing sequence, it is therefore intended that overcome water/mud in sewage disposal system Sample builds the deficiencies such as low, cumbersome storehouse quality, easy pollution, cost height, using a step TRAP, without the need for self-defined purpose piece The sequencing primer of section, is directly sequenced using Illumina Miseq matched reagent boxes, and the sub- sequencing library of optimized expansion builds A kind of method, there is provided the fast construction method of water suitable for sewage disposal system/sub- sequencing library of mud sample amplification.
2. technical scheme
In order to solve the above problems, the technical solution adopted in the present invention is as follows：
The primer of water/mud sample DNA amplicon sequencing library rapid build in a kind of sewage disposal system, including upstream draws Thing and downstream primer, described forward primer and downstream primer according to from 5 ' hold to the 3 ' orders held include joint sequence, rope Draw sequence, heterogeneous intervening sequence, purpose fragment extension increasing sequence；Sequencing primer sequence is included in described joint sequence；It is described Index sequence by 12 base compositions；Base composition of the described heterogeneous interval sequence by 0-4bp；Described purpose fragment expands Increasing is classified as at 16S rDNA hypervariable regions：341F and 806R.
Further, described joint sequence is comprising can be with the flow in Illumina Miseq sequencing kits The sequence that cell is combined, and sequencing primer sequence, during amplification sublibrary and Illumina Miseq matched reagent boxes can be made Flow cell combine, and directly can be sequenced using Illumina Miseq matched reagent boxes, be wrapped in joint sequence Sequence containing sequencing primer, therefore without the need for self-defined sequencing primer.
Further, all include index sequence in forward primer and downstream primer, using the method for double indexes.
Further, described upstream primer sequence is any one in SEQ ID NO.1～SEQ ID NO.15； Described downstream primer sequence is any one in SEQ ID NO.16～SEQ ID NO.30.
Further, in sequence SEQ ID NO.1～SEQ ID NO.3 and SEQ ID NO.16～SEQ ID NO.18 Heterogeneous intervening sequence by 0 base composition；SEQ ID NO.4～SEQ ID NO.6 and SEQ ID NO.19～SEQ ID Heterogeneous intervening sequence in NO.21 is by 1 base composition；SEQ ID NO.7～SEQ ID NO.9 and SEQ ID NO.22～ Heterogeneous intervening sequence in SEQ ID NO.24 is by 2 base compositions；SEQ ID NO.10～SEQ ID NO.12 and SEQ Heterogeneous intervening sequence in ID NO.25～SEQ ID NO.27 is by 3 base compositions；SEQ ID NO.13～SEQ ID Heterogeneous intervening sequence in NO.15 and SEQ ID NO.28～SEQ ID NO.30 is by 4 base compositions.
A kind of fast construction method of the sub- sequencing library of DNA cloning, comprises the following steps：
(1) extraction of sample DNA；
(2) amplification of purpose fragment：The amplification of purpose fragment is carried out using PCR, primer is above-mentioned primer；
(3) purification of amplified production：Using silicones method adsorption of DNA, washing liquid washes away impurity；
(4) Quality Identification of amplified production：The concentration of amplified production, purity are determined using the method for absorbance, using solidifying The method identification fragment length of gel electrophoresis；
(5) mixing of amplified production：The quality such as different sample DNAs are mixed；
(6) mix the Quality Identification in library：Using real time fluorescent quantitative nucleic acid amplification detecting system (qPCR) to mixing text Storehouse carries out detection by quantitative；
(7) preparation and sequencing in library are mixed：First degeneration is carried out to mixing library, then matched somebody with somebody using Illumina Miseq Set test kit is diluted and the sequencing of upper machine to mixing library.
Further, described sample comes from the water/mud sample in sewage disposal system.
Further, extracted using silicon adsorption of DNA principle in step (1)；If sample is water sample, need to use The microporous filter membrane enriched microorganism of 0.22um, if sample is mud sample, needs centrifugation with enriched microorganism.
Further, the DNA purity requirements after extraction be absorbance=1.8 at absorbance/280nm at 260nm～ 2.0。
Further, enter the primer of performing PCR amplification in step (2) by SEQ ID NO.1～SEQ ID NO.15 Any one is formed with any one random combine in NO.16～SEQ ID NO.30.
Further, degeneration is carried out to mixing library using NaOH in step (4).
3. beneficial effect
Compared to prior art, beneficial effects of the present invention are：
(1) present invention is by joint sequence, index sequence, heterogeneous intervening sequence, purpose fragment extension increasing sequence are integrated On the pair of primers of upstream and downstream amplification so that library construction can be completed by a step TRAP using the primer, not only be carried High sequencing library quality and storehouse efficiency is built, and reduce and build Kucheng's sheet；
(2) present invention design primer in, joint sequence comprising can with Illumina Miseq sequencing kits in The sequence that flow cell are combined, and sequencing primer sequence, can make amplification sublibrary and Illumina Miseq matched reagents Flow cell in box are combined, and directly can be sequenced using Illumina Miseq matched reagent boxes, joint sequence In include sequencing primer sequence, therefore without the need for self-defined sequencing primer, operate easier；
(3) of the invention compared with conventional banking process, storehouse quality is low, behaviour to overcome water/mud sample in sewage disposal system to build Make the shortcoming of loaded down with trivial details, high cost, the method can accurately distinguish different samples and ensure that different sample sequencing bar numbers are average, There is in the sub- sequencing field of water/mud sample amplification in sewage disposal system wide application prospect.
Description of the drawings
Fig. 1 is the primer structural representation of the present invention；
Fig. 2 is to be embodied as middle water sample and mud sample by step amplification using library constructing method of the present invention and institute after purification The result of the amplicon sequencing library Jing electrophoresis detection for obtaining.
Specific embodiment
The present invention is further described below with reference to specific embodiment.
Embodiment 1
(1) in sewage disposal system water sample DNA extraction
1. certain sewage treatment plant inflow 500mL, water outlet and different processing units three are gathered and goes out water sample about 2L, used The filtering with microporous membrane water sample of 0.22um, the microorganism in water sample are enriched on microporous filter membrane, for the extraction of DNA, are compiled successively Number be water sample 1, water sample 2, water sample 3, water sample 4, water sample 5；
2. the FastDNA Soil Kit for being produced using MP Biomedicals companies of the U.S. carry out the extraction of water sample DNA, Instrument and reagent are got out to specifications；
3. filter membrane is shredded, is respectively put in different sample dissociation pipes, add 978 μ L sodium phosphate buffers and 122 μ L MT buffer, crushes in biological homogenizer, and broken condition is 5.5m/s, 45s；
4. the sample dissociation pipe after will be broken is put into centrifuge, is centrifuged 15 minutes, supernatant is turned under the conditions of 14000 × g Move on in 2mL centrifuge tubes, plus 250 μ L PPS buffer, hand 10 mixings liquid；
5. mixed liquor is centrifuged 5 minutes under the conditions of 14000 × g, takes 1mL supernatant and be transferred in 15mL centrifuge tubes, and To in 15mL centrifuge tubes, add 1mL to mix Binding Matrix, hand mixed liquor 5 minutes so as to mix homogeneously are subsequently quiet Put 3 minutes；
6. remaining liq in centrifuge tube, 700 μ L mixed liquors of transfer to SPIN are mixedTMIn Filter, under the conditions of 14000 × g Centrifugation 1 minute, abandons waste liquid, is repeated 2 times；
7. to SPINTMAdd the SEWS-M solution of 500 μ L in Filter, mixed with pipette tips pressure-vaccum, under the conditions of 14000 × g Centrifugation 1 minute, abandons waste liquid；
8. again by SPINTMFilter is centrifuged under the conditions of 14000 × g 2 minutes.DNA purification columns are placed in into cleaning In 1.5mL centrifuge tubes, drying at room temperature added 70 μ L distilled waters in purification column jecket inner cylinder central authorities after 3 minutes, after slowly mixing, Heat shock 5 minutes under the conditions of 55 DEG C；
9. it is centrifuged 1 minute under 14000 × g, the sample total DNA that gained is as extracted；
10. the concentration and purity of the DNA sample of microspectrophotometer Detection and Extraction are used.
(2) purpose fragment amplification
Primer construction schematic diagram in the present invention is as shown in figure 1, select five primer pairings in the present embodiment：Water sample 1 makes With SEQ ID NO.1, SEQ ID NO.16；Water sample 2 uses SEQ ID NO.1, SEQ ID NO.17；Water sample 3 uses SEQ ID NO.1、SEQ ID NO.18；Water sample 4 uses SEQ ID NO.2, SEQ ID NO.16；Water sample 5 uses SEQ ID NO.2, SEQ ID NO.17.Wherein SEQ ID NO.1, SEQ ID NO.2,1-58 bit bases are joint sequences, and 59-70 bit bases are ropes Draw sequence, 0 heterogeneous intervening sequence, 71-87 bit bases are purpose fragment extension increasing sequences；SEQ ID NO.16、SEQ ID NO.17, SEQ ID NO.18,1-58 bit bases are joint sequences, and 59-70 bit bases are index sequences, between 0 heterogeneous Every sequence, 71-90 bit bases are purpose fragment extension increasing sequences.
Amplification condition is：95℃2min；30 circulation (95 DEG C of 20s；51℃30s；72℃60s)；72℃5min.Amplification body It is for 30 μ L：2 × TransTaq High Fidelity (HiFi) PCR of Beijing Quan Shi King Companies production of 15 μ L SuperMix I, the forward primer (primer concentration is 1 μM) of 3 μ L, 3 μ L downstream primers (primer concentration is 1 μM), the DNA moulds of 5 μ L Plate (template concentrations are diluted to 25ng/ μ L)；Every group of sample do three it is parallel, and be all provided with blank, blank is i.e. with double Steam water and replace sample DNA templates, other conditions are constant.
(3) purification of amplified production；
1. using the QIAquick PCR purification kits of QIAGEN companies of Germany production；
2. prepare before operating：The dehydrated alcohol of designated volume is added according to body in Buffer PE；
3. the 25 μ L of pcr products in three pipe parallel samples are taken respectively, come to the centrifuge tube that 75 μ L add new 1.5mL；
4. the Buffer PB of 500 μ L are added in the centrifuge tube of 1.5mL, be vortexed mixing, low-speed centrifugal；
5. in the QIAquick spin column for adding test kit to provide aforesaid liquid, 17900 × g room temperature centrifugation 1 Minute, abandon waste liquid；
6. in QIAquick spin column add 750mL Buffer PE, 17900 × g to be centrifuged 1 minute, abandon useless Liquid；
7. then 17900 × g room temperatures are centrifuged 90s again, remove the Buffer PE of residual；
8. QIAquick column are placed in new clean 1.5mL centrifuge tubes, add 25 μ L Buffer EB, it is quiet Put 1 minute, 17900 × g is centrifuged 1 minute, and gained is exactly DNA after purification.
(4) Quality Identification of amplified production
(4a) using match Mo Feishier companiesDsDNA BR Assay Kit reagents are used2.0 fluorescence Meter is determined；
1. prepare working solution：By A reagents：B reagent=199：1 proportions working solution (note：A reagents are ds DNA BR Buffer, B reagent is ds DNA BR Reagent*200 ×)；
2. two models and the matching used qubit developmental tubes of exometer are taken, first respectively in two pipes before each addition The 190 μ L of working solution of preparation, be separately added into 10 μ L 2 reagent of 1 reagent of standard sample and 10 μ L standard sample (dsDNA BR Assay Kit self-contained reagents), under room temperature condition, after the concussion that is vortexed is mixed, low-speed centrifugal；
3. qubit developmental tubes are taken, the 198 μ L of working solution of preparation is added in pipe, add 2 μ L purpose fragment samples, room Under the conditions of temperature, after the concussion that is vortexed is mixed, low-speed centrifugal；
4. according to the volume for adding amplified production, determine the concentration of amplified production.
The concentration of amplified production is：Water sample 1 is 70ng/ul, water sample 2 is 98ng/ul, water sample 3 is 103ng/ul, water sample 4 It is 88ng/ul for 90ng/ul, water sample 5.
(4b) amplified production clip size is identified using gel electrophoresiss；
1. 1 gram of agarose powder is taken into 100mL 1 × TAE buffer, be heated to dissolving boiling completely, be cooled to 60 DEG C Left and right, adds ethidium bromide (commercially available) into solution, is 0.5ug/mL to concentration, pours glue into glue groove, and cooling half is little When, it is plugged comb and makes even level's glue surface；
2. gel is put into into electrophoresis tank so as to be loaded nose end and put addition 1 × TAE electrophoretic buffers in negative electrode section, groove, until Liquid level covered glue surface；
3. the 4 μ L of amplified production for taking after purification are mixed plus 1 μ L loading buffer (Quan Shi King Companies buy) and are added Loading hole, with DL2000DNA marker as reference material (Quan Shi King Companies buy), setting voltage 120V, electric current 400mA run glue Time is 20 minutes.
4. PCR experiment result is observed using ultraviolet gel imaging instrument.
In Fig. 2,6 glue holes represent respectively from left to right：First glue hole is DL 2000DNA marker, indicates DNA point The band of sub- size, is followed successively by from top to bottom：100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, second to the 6th Individual glue hole is water sample 1, water sample 2, water sample 3, water sample 4,5 amplified production of water sample result after purification respectively.Normal 16S rDNA The size in V3-V4 areas is 450bp or so, and upstream and downstream primer is always about 140bp, therefore, expand the overall length for obtaining and should be 590bp Left and right, the about 590bp or so of PCR primer size shown in Fig. 2, it is seen then that the 16S obtained using the above-mentioned primer amplification of the present invention The size of the amplicon sequencing library in rDNA V3-V4 areas is to meet expected.
Embodiment 2
(1) in sewage disposal system mud sample DNA extraction
4 mud sample about 50ml of certain sewage treatment plant's different processing units are gathered, 10000rpm is centrifuged and collects within 10 minutes big The sludge of about 500mg carries out the extraction of DNA；FastDNA of the extraction of DNA using the production of MP Biomedicals companies of the U.S. Soil Kit, numbering are mud sample 1, mud sample 2, mud sample 3, mud sample 4.
(2) amplification of purpose fragment
Select four primer pairings：Mud sample 1 uses SEQ ID NO.4, SEQ ID NO.19；Mud sample 2 uses SEQ ID NO.4、SEQ ID NO.20；Mud sample 3 uses SEQ ID NO.5, SEQ ID NO.19；Mud sample 4 uses SEQ ID NO.5, SEQ ID NO.20.Amplification condition is identical with embodiment 1 with amplification system.Wherein SEQ ID NO.4, SEQ ID NO.5,1-58 positions Base is joint sequence, and 59-70 bit bases are index sequences, and the 71st is heterogeneous intervening sequence, and 72-88 bit bases are Purpose fragment extension increasing sequence；SEQ ID NO.19, SEQ ID NO.20,1-58 bit bases are joint sequences, 59-70 positions alkali Base is index sequence, and the 71st is heterogeneous intervening sequence, and 72-91 bit bases are purpose fragment extension increasing sequences.
(3) purification of amplified production
The QIAquick PCR purification kits produced using German QIAGEN companies carry out purification to amplified production.
(4) Quality Identification of amplified production：
Using match Mo Feishier companiesDsDNA BR Assay Kit reagents are used2.0 fluorescence are measured Fixed, 1 concentration of mud sample is 150ng/ul, 2 concentration of mud sample is 145ng/ul, 3 concentration of mud sample is 161ng/ul, 4 concentration of mud sample is 157ng/ul；
In Fig. 2, the 7th glue hole starts to represent successively from left to right：First glue hole is DL 2000DNA marker, the Eight to the 11st glue holes are mud sample 1, mud sample 2, mud sample 3, the result after 4 amplification purification of mud sample respectively, it is seen then that using the present invention The size of the amplicon sequencing library in 16S rDNA V3-V4 areas that obtains of above-mentioned primer amplification be to meet expected.
Embodiment 3
(1) sample pre-treatments：
Collect the DNA (including embodiment 1, the sample in example 2) of 450 water samples and mud sample in sewage disposal system, with by Any one random group in any one in SEQ ID NO.1～SEQ ID NO.15 and NO.16～SEQ ID NO.30 The 225 pairs of primers for closing carry out the amplification of purpose fragment, purification, Quality Identification (see embodiment 1), each pair primer with twice, The size of all samples amplicon sequencing library meets expection.
(2) mixing of amplified production：
According to the concentration for determining, the sample obtained using different primers amplification is mixed according to identical quality, is obtained To mixing library A and mixing library B, each library includes 225 samples, and each library not using the primer for repeating Pairing.
(3) mix the Quality Identification in library：
(3a) using match Mo Feishier companiesDsDNA BR Assay Kit reagents are used2.0 fluorescence Meter carries out preliminary measure (embodiment 1) to mixing library；
(3b) using the KAPA Library Quantification Kit of Roche Holding Ag of Switzerland production to mixing library Concentration carries out accurate quantification：
1. preparation process：10X Primer Premix (1mL) in test kit are added 2X KAPAFAST In qPCR Master Mix (5mL), it is vortexed and mixes, be made into working solution Mix；
2. configure library diluent：The 10mM Tris-HCl of configuration pH=8.0, add20 (Suo Laibao companies Purchase) so that its ultimate density is 0.05%；
3. prepare sample：According to the concentration that (3a) is measured, with library diluent respectively library A, B dilute 100 times, 1000 Again, 10000 times respectively obtain library A1, library A2, library A3, library B1, library B2, library B3；
4. qPCR amplifications are carried out on ABI 7500qPCR instruments, amplification system is 20 μ L：The working solution Mix of 12 μ L, 3.6 μ L distilled waters, 0.4 μ L ROX Low (test kit is carried), (DNA is respectively 4 μ L DNA：Standard substance 1-6 is carried for test kit, text Storehouse A1, library A2, library A3, library B1, library B2, library B3)；Amplification condition is：95 DEG C of 5min, 35 circulate (95 DEG C 30s；60℃45s)；Every group of sample do three it is parallel, and be all provided with blank, blank replaces sample with distilled water DNA profiling, other conditions are constant.
5. the mass concentration according to standard substance, draws the molal weight concentration of final library：The molal weight concentration of library A Molal weight concentration for 92.9nM, library B is 126.7nM.
(4) preparation and sequencing in library are mixed
1. the molal weight concentration according to the mixing library measured in (3), library A, library B concentration dilution to 3nM；
2. 0.2M NaOH and 0.2M Tris-HCl (purchase of Suo Laibao companies) are prepared；
3. in two reaction tubes add the library DNA and 10 μ L 0.2M NaOH of 10 μ L 3nM to be vortexed respectively to shake, it is of short duration Centrifugation, stands 5min；
4. in above-mentioned reaction tube plus 10 μ L 0.2M Tris-HCl so that final mass concentration is 1nM；
5. the reagent HT1 for being carried with Illumina Miseq Reagent Kit v3 (600cycle), dilutes above-mentioned library DNA so that final mass concentration is 12pM；
6. go up machine sequencing：Respectively library A, library B are sequenced using the Miseq instruments of Illumina, sequencing strategy It is 300PE (two ends are sequenced, each base for surveying 300bp).Lower machine sequence is spliced with PANDAseq softwares, splicing yield difference For 92.99%, 93.74%, wherein sequencing data the results are shown in Table 1；And the sequence bar number of each sample is relatively average, all In 40000-50000 bars；After removing primer information, the average length of sequence is：429 ± 7bp, meets actual purpose fragment length.
1 library constructing method of the present invention of table is embodied as the sequence information result of middle amplicon sequencing library
Note：In high-flux sequence, often survey a base and can provide a corresponding mass value, this mass value is to weigh to survey Sequence accuracy, Q30 shows that the probability of wrong identification is 0.1%, i.e., accuracy is 99.9%.
SEQUENCE LISTING
<110>Nanjing University Yixing environmental protection academy；Nanjing University
<120>The primer and quick structure of water/mud sample DNA amplicon sequencing library rapid build in a kind of sewage disposal system Construction method
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<213>Synthetic
<400> 5
caagcagaag acggcatacg agatgtgact ggagttcaga cgtgtgctct tccgatctac 60
tttaagggtg tcctacgggn ggcwgcag 88
<210> 6
<211> 88
<212> DNA
<213>Synthetic
<400> 6
caagcagaag acggcatacg agatgtgact ggagttcaga cgtgtgctct tccgatctga 60
gcaacatcct tcctacgggn ggcwgcag 88
<210> NO. 7
<211> 89
<212> DNA
<213>Synthetic
<400> 7
caagcagaag acggcatacg agatgtgact ggagttcaga cgtgtgctct tccgatcttg 60
ttgcgtttct gtcctacggg nggcwgcag 89
<210> 8
<211> 89
<212> DNA
<213>Synthetic
<400> 8
caagcagaag acggcatacg agatgtgact ggagttcaga cgtgtgctct tccgatctat 60
gtccgaccaa gtcctacggg nggcwgcag 89
<210> 9
<211> 89
<212> DNA
<213>Synthetic
<400> 9
caagcagaag acggcatacg agatgtgact ggagttcaga cgtgtgctct tccgatctag 60
gtacgcaatt gtcctacggg nggcwgcag 89
<210> 10
<211> 90
<212> DNA
<213>Synthetic
<400> 10
caagcagaag acggcatacg agatgtgact ggagttcaga cgtgtgctct tccgatctac 60
agccacccat cgacctacgg gnggcwgcag 90
<210> 11
<211> 90
<212> DNA
<213>Synthetic
<400> 11
caagcagaag acggcatacg agatgtgact ggagttcaga cgtgtgctct tccgatcttg 60
tctcgcaagc cgacctacgg gnggcwgcag 90
<210> 12
<211> 90
<212> DNA
<213>Synthetic
<400> 12
caagcagaag acggcatacg agatgtgact ggagttcaga cgtgtgctct tccgatctga 60
ggagtaaagc cgacctacgg gnggcwgcag 90
<210> 13
<211> 91
<212> DNA
<213>Synthetic
<400> 13
caagcagaag acggcatacg agatgtgact ggagttcaga cgtgtgctct tccgatctgt 60
tacgtggttg atgacctacg ggnggcwgca g 91
<210> 14
<211> 91
<212> DNA
<213>Synthetic
<400> 14
caagcagaag acggcatacg agatgtgact ggagttcaga cgtgtgctct tccgatctta 60
ccgcctcgga atgacctacg ggnggcwgca g 91
<210> 15
<211> 91
<212> DNA
<213>Synthetic
<400> 15
caagcagaag acggcatacg agatgtgact ggagttcaga cgtgtgctct tccgatctcg 60
taagatgcct atgacctacg ggnggcwgca g 91
<210> 16
<211> 90
<212> DNA
<213>Synthetic
<400> 16
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctcc 60
taaactacgg ggactachvg ggtwtctaat 90
<210> 17
<211> 90
<212> DNA
<213>Synthetic
<400> 17
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttg 60
cagatccaac ggactachvg ggtwtctaat 90
<210> 18
<211> 90
<212> DNA
<213>Synthetic
<400> 18
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctcc 60
atcacatagg ggactachvg ggtwtctaat 90
<210> 19
<211> 91
<212> DNA
<213>Synthetic
<400> 19
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctgt 60
ggtatgggag aggactachv gggtwtctaa t 91
<210> 20
<211> 91
<212> DNA
<213>Synthetic
<400> 20
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctac 60
tttaagggtg aggactachv gggtwtctaa t 91
<210> 21
<211> 91
<212> DNA
<213>Synthetic
<400> 21
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctga 60
gcaacatcct aggactachv gggtwtctaa t 91
<210> 22
<211> 92
<212> DNA
<213>Synthetic
<400> 22
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttg 60
ttgcgtttct tcggactach vgggtwtcta at 92
<210> 23
<211> 92
<212> DNA
<213>Synthetic
<400> 23
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctat 60
gtccgaccaa tcggactach vgggtwtcta at 92
<210> 24
<211> 92
<212> DNA
<213>Synthetic
<400> 24
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctag 60
gtacgcaatt tcggactach vgggtwtcta at 92
<210> 25
<211> 93
<212> DNA
<213>Synthetic
<400> 25
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctac 60
agccacccat ctaggactac hvgggtwtct aat 93
<210> 26
<211> 93
<212> DNA
<213>Synthetic
<400> 26
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatcttg 60
tctcgcaagc ctaggactac hvgggtwtct aat 93
<210> 27
<211> 93
<212> DNA
<213>Synthetic
<400> 27
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctga 60
ggagtaaagc ctaggactac hvgggtwtct aat 93
<210> 28
<211> 94
<212> DNA
<213>Synthetic
<400> 28
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctgt 60
tacgtggttg gataggacta chvgggtwtc taat 94
<210> 29
<211> 94
<212> DNA
<213>Synthetic
<400> 29
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctta 60
ccgcctcgga gataggacta chvgggtwtc taat 94
<210> 30
<211> 94
<212> DNA
<213>Synthetic
<400> 30
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctcg 60
taagatgcct gataggacta chvgggtwtc taat 94

Claims (9)

1. in a kind of sewage disposal system water/mud sample DNA amplicon sequencing library rapid build primer, including forward primer And downstream primer, it is characterised in that：Described forward primer and downstream primer include connecing according to holding to the 3 ' orders held from 5 ' Header sequence, index sequence, heterogeneous intervening sequence, purpose fragment extension increasing sequence；Sequencing primer is included in described joint sequence Sequence；Described index sequence is by 12 base compositions；Base composition of the described heterogeneous interval sequence by 0-4bp；Described Purpose fragment extension increasing sequence is at 16S rDNA hypervariable regions：341F and 806R.
2. water/mud sample DNA amplicon sequencing library rapid build in a kind of sewage disposal system according to claim 1 Primer, it is characterised in that：Described joint sequence is included can be with the flow cell in Illumina Miseq sequencing kits With reference to sequence, and sequencing primer sequence.
3. water/mud sample DNA amplicon sequencing library rapid build in a kind of sewage disposal system according to claim 2 Primer, it is characterised in that：Described upstream primer sequence is any one in SEQ ID NO.1～SEQ ID NO.15；Institute The downstream primer sequence stated is any one in SEQ ID NO.16～SEQ ID NO.30.
4. water/mud sample DNA amplicon sequencing library rapid build in a kind of sewage disposal system according to claim 3 Primer, it is characterised in that：In sequence SEQ ID NO.1～SEQ ID NO.3 and SEQ ID NO.16～SEQ ID NO.18 Heterogeneous intervening sequence is by 0 base composition；SEQ ID NO.4～SEQ ID NO.6 and SEQ ID NO.19～SEQ ID Heterogeneous intervening sequence in NO.21 is by 1 base composition；SEQ ID NO.7～SEQ ID NO.9 and SEQ ID NO.22～ Heterogeneous intervening sequence in SEQ ID NO.24 is by 2 base compositions；SEQ ID NO.10～SEQ ID NO.12 and SEQ Heterogeneous intervening sequence in ID NO.25～SEQ ID NO.27 is by 3 base compositions；SEQ ID NO.13～SEQ ID Heterogeneous intervening sequence in NO.15 and SEQ ID NO.28～SEQ ID NO.30 is by 4 base compositions.
5. the fast construction method of the sub- sequencing library of a kind of DNA cloning, it is characterised in that：Comprise the following steps：
(1) extraction of sample DNA；
(2) amplification of purpose fragment：The amplification of purpose fragment is carried out using PCR, primer is any one in Claims 1 to 4 Described primer；
(3) purification of amplified production；
(4) Quality Identification of amplified production：Determine concentration, purity and the fragment length of amplified production；
(5) mixing of amplified production：The quality such as different sample DNAs are mixed；
(6) mix the Quality Identification in library：Detection by quantitative is carried out to mixing library；
(7) preparation and sequencing in library are mixed：First degeneration is carried out to mixing library, be then diluted and upper machine to mixing library Sequencing.
6. the fast construction method of the sub- sequencing library of a kind of DNA cloning according to claim 5, it is characterised in that：It is described Sample come from the water/mud sample in sewage disposal system.
7. the fast construction method of the sub- sequencing library of a kind of DNA cloning according to claim 5 or 6, it is characterised in that：Step Suddenly extracted using silicon adsorption of DNA principle in (1)；If sample is water sample, need the microporous filter membrane enrichment with 0.22um micro- Biology, if sample is mud sample, needs centrifugation with enriched microorganism.
8. the fast construction method of the sub- sequencing library of a kind of DNA cloning according to claim 7, it is characterised in that：Extract DNA purity requirements afterwards are absorbance=1.8～2.0 at absorbance/280nm at 260nm.
9. the fast construction method of the sub- sequencing library of a kind of DNA cloning according to claim 5, it is characterised in that：Step (2) enter the primer of performing PCR amplification in by any one in SEQ ID NO.1～SEQ ID NO.15 and NO.16～SEQ ID Any one random combine in NO.30 is formed.
CN201611071680.1A 2016-11-29 2016-11-29 The primer and fast construction method of water/mud sample DNA amplicon sequencing library rapid build in a kind of sewage disposal system Pending CN106554958A (en)

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CN108866051A (en) * 2018-06-19 2018-11-23 上海锐翌生物科技有限公司 Amplicon sequencing library and its construction method

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