CN105368929A - Compound label for high-throughput sequencing of macro transcription internal sequence and application of compound label - Google Patents
Compound label for high-throughput sequencing of macro transcription internal sequence and application of compound label Download PDFInfo
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- CN105368929A CN105368929A CN201510628325.9A CN201510628325A CN105368929A CN 105368929 A CN105368929 A CN 105368929A CN 201510628325 A CN201510628325 A CN 201510628325A CN 105368929 A CN105368929 A CN 105368929A
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Abstract
The invention discloses a compound label for high-throughput sequencing of a macro transcription internal sequence and application of the compound label. The sequences of the compound label are shown in the serial numbers from NO.1 to NO.48. An application method of the label includes the steps of extracting the total DNA of all samples, selecting any pair of sequences from 48 pairs of unselected sequences to serve as a primer for each sample, amplifying the total DNA of the corresponding samples, conducting electrophoresis detection on the amplification product of each sample, conducting purification and recovery on each amplification product, accurately quantifying the product obtained after each sample is purified and recovered through an ultraviolet spectrophotometer, establishing a sequencing library after all the samples are mixed equivalently, and conducting high-throughput sequencing on the established sequencing library. By means of the label, the sample throughput of ultrahigh-throughput sequencing is improved, ultrahigh-throughput sequencing cost is effectively reduced, fungal populations in the whole microorganism populations in the samples are effectively enriched, and the high-throughput analysis approach is provided for researching the fungal diversity in the environment.
Description
Technical field
The present invention relates to gene sequencing technology field, be specifically related to a kind of composite label of the high-flux sequence for grand internal transcribed spacer sequence, for ultra-high throughput gene sequencing.
Background technology
Microbial Community Diversity is one of emphasis of microbial ecology and environmentalism research.Utilize grand genome research microbial diversity more and more to receive the concern of scientists at present, but due to for same sample, in the result of grand genome research, often the quantity of bacterium is in the great majority and make the microbe species such as fungi far fewer than bacterium.This research for some field is very unfavorable, and as studied the microbial population in each period in wine brewing process, the effect of fungi is far above bacterium, larger on the impact of drinks quality.Therefore utilize the internal transcribed spacer sequence of fungi uniqueness for target sequence, become very effective method with method enrichment fungal DNA from complex samples of pcr amplification.
Handelman (HandelsmanJ, RondonMR, BradySF.molecularbiologialaccesstothechemistryofunkownso ilmicrobes:Anewfrontierfornaturalproducts [J] .1998,5 (10): R245-R249.) etc. (1998) propose grand genomic concept first, it is defined as " thegenomesofthetotalmicrobiotafoundinnature ", i.e. the summation of whole tiny organism genetic material in environment.It contains the genome summation of educable and not educable microorganism.The grand interior intervening sequence of indication of the present invention refers to the summation of intervening sequence in whole fungi in environment, for microbial diversity, population structure, evolutionary relationship, functionally active, mutually cooperation relation and and environment between pass be that research purpose provides a new thinking.
Ribosome-RNA(rRNA) (rRNA) is due to functionally high conservative, and in sequence, different positions has different variation rates, is most widely used molecule marker on Molecular Ecology of Microbiology at present.In eukaryotic gene group, the gene of encoding ribosomal RNA comprises 28SrDNA, 5SrDNA, 18SrDNA and 5.8SrDNA4 kind, they are from beginning to end, arranged in series on chromosome, (Kuang Zhizhou is separated each other by transcribed spacer, Xu Yang. the application of rrna rDNAITS sequence in mycology research [J]. the chemistry of life, 2004 (02): 120-122.).Wherein 18S, 5.8S and 28SrDNA genomic constitution transcriptional units, three's high conservative, systems analysis between the biotic population being suitable for higher level level, transcribed spacer is therebetween the Internal Transcribed Spacer (InternalTranscribedSpacer, ITS), comprise the Internal Transcribed Spacer 2 (ITS2) two portions between the Internal Transcribed Spacer 1 (ITS1) between 18S and 5.8S and 5.8S and 28S, because ITS does not add ripe rrna, so the selective pressure be subject to is less, evolutionary rate is very fast, sequence polymorphism very is widely shown in the eukaryote of the overwhelming majority.ITS sequence moderate length simultaneously, in various eukaryotes from the mankind to yeast, the sequence length of ITS is that 1000bp differs in size to being less than 300bp, people can obtain enough information in never oversize sequence, can be widely used in belonging between planting or the Phylogenetic Studies (IwenPC of kind in-group, HinrichsSH, RuppME.Utilizationoftheinternaltranscribedspacerregionsa smoleculartargetstodetectandidentifyhumanfungalpathogens [J] .MedMycol, 2002,40 (1): 87-109).
Summary of the invention
The invention provides a kind of composite label and application thereof of the high-flux sequence for grand internal transcribed spacer sequence, improve the sample flux of ultra-high throughput order-checking, effective reduction ultra-high throughput order-checking cost, fungal population in effective enrichment sample in whole microbial population, provides high throughput analysis approach for verifying fungal diversity research in environment.
For a composite label for the high-flux sequence of grand internal transcribed spacer sequence, its base sequence is as shown in following sequence numbering numbering NO.1 ~ NO.48:
The label that each sequence of composite label of the present invention is made up of left side 8-10 based composition tool uniqueness label and 21, the right side sequence coming from fungi the Internal Transcribed Spacer 1 is composited.
Table 1
The present invention also provides a kind of fungi high-throughput gene order surveying method, and its sequence is as shown in following sequence numbering numbering NO.1 ~ NO.48:
Utilize described multiple sample fungi internal transcribed spacer sequence composite label to carry out the method for ultra-high throughput gene sequencing, comprise the steps:
(1) STb gene of each sample is extracted;
(2) to each sample, Accessory Right to require shown in 1 48 to and do not selected arbitrary pair of sequences as primer by the sequence selected, the STb gene of corresponding sample is increased;
(3) electrophoresis detection is carried out to the amplified production of each sample, purifying recovery is carried out to amplified production;
(4) the product ultraviolet spectrophotometer accurate quantification after each Sample purification being reclaimed;
(5) sequencing library is set up by after all sample balanced mix;
(6) high-flux sequence is carried out to built sequencing library.
The grand internal transcribed spacer sequence of fungi of the present invention refers to all summations dropping on the Internal Transcribed Spacer 1 between fungi 18S and 5.8S, and composite label is as shown in table 1; Utilize the fungi of table 1 grand internal transcribed spacer sequence composite label to reach to detect the grand interior intervening sequence of the fungi in 1 ~ 48 sample in a ultra-high throughput order-checking simultaneously.
Utilizing current ultra-high throughput to check order self provides 96 kinds of labels (8-10 base) that check order, 96 samples can be made, but before ultra-high throughput order-checking, first 96 storehouses are built respectively to 96 samples, then balanced mix become 1 check order storehouse check order again simultaneously, and the present invention only need build 1 order-checking storehouse just can check order to 48 samples simultaneously.If 96 labels carried checking order and 48 composite labels of the present invention are combined, then can carry out ultra-high throughput order-checking to the internal transcribed spacer sequence of 96*48 sample simultaneously.
In step (1), common extraction procedure is adopted to each sample total DNA extraction.
In step (2) to each sample from shown in table 1 48 to and do not selected arbitrary pair of sequences as primer by the sequence selected, its selection principle is specially: the numbering determining each sample, to each numbering sample, from table 1, selecting the sequence 1 corresponding to a sequence numbering and sequence 2, (sequence 1 and the sequence 2 of NO.1 are a pair, sequence 1 and the sequence 2 of NO.2 are a pair, the sequence 1 of NO.3 and sequence 2 be a pair by that analogy), determine the pair of sequences of each sample; In the optional table 1 of sample of each numbering 48 centerings any a pair, but require not overlap when table 1 is selected between the sample of different numbering, namely same composite label do not selected by different numbering sample.
In step (2) to the amplification system that the STb gene of corresponding sample increases be:
Contain in 50 μ L systems of pcr amplification: 50ng sample STb gene, 4 μ LMg
2+, 5 μ L10 × Buffer, 4 μ L concentration are 5nmoldNTP, 1.5UTaq enzyme, and concentration is each 1.5 μ L, moisturizing to the 50 μ L of 10pmol primer.
Pcr amplification program: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 40 DEG C of renaturation 30s, 72 DEG C extend 1min, 30 circulations, and last 72 DEG C extend 5min, 4 DEG C of preservations.
In step (3), purifying is carried out to each sample amplified production and reclaim the general PCR primer recovery test kit of employing.
The order-checking platform that high-flux sequence employing of the present invention leads to sequenator based on IonPGM s-generation superelevation carries out.
Preferably, step (5) Zhong Jianku storehouse test kit of building used is that IonPGM s-generation superelevation leads to IonXpress corresponding to sequenator
tMplusgDNAFragmentLibraryPreparation test kit.
The amplification kit used that increases during order-checking in step (6) is that IonPGM s-generation superelevation leads to IonPGM corresponding to sequenator
tMtemplateOT2400Kit test kit.
The sequencing kit used that checks order during order-checking in step (6) is that IonPGM s-generation superelevation leads to IonPGMSequencing400Kit test kit corresponding to sequenator.
Described sample is wheat koji sample.Can certainly for other be containing the sample of fungi.
Compared with the conventional method, the present invention has following beneficial effect:
In environment, a large amount of microorganisms in (as soil) perch, and the distribution of these microorganisms and movable and Soil nutritional condition thereof, aboveground vegetation is closely related.Wetland and forest, ocean also claim the whole world the three large ecosystems, are called as " kidney of the earth ", have abundant species diversity.Present method with the Internal Transcribed Spacer of fungi in the microorganism in specific environment for research object, based on the Research Thinking of metagenomics, adopt the composite label described in present method, build a storehouse and can carry out ultra-high throughput order-checking to 48 samples at most simultaneously, carry out based on the fungal population Changing Pattern research in grand genomic environmental samples, for various environmental samples carries out similar research supplying method.
Accompanying drawing explanation
Fig. 1 is one of them sample electrophoresis of the present invention detected result figure.
Fig. 2 is the ultra-high throughput sequencing result overview of 8 samples.
Embodiment
Embodiment 1
The extraction of environmental samples STb gene can with reference to the method (DNArecoveryfromsoilsofdiversecomposition of Zhou, Appl.Environ.Microbiol., 1996,62,316-322), after extracting, the purifying of DNA can with reference to the method (Asimple of Jackson, efficientmethodfortheseparationofhumicsubstancesandDNAfr omenvironmentalsamples, Appl.Environ.Microbiol., 1997,63,4993-4995).Or the method extraction of test kit is extracted with reference to soil DNA.Key step is as follows:
1. take environmental sample sample 5g in 50mL centrifuge tube, add 15 milliliters of phosphoric acid buffers, violent vortex 5min on vortex instrument, then the centrifugal 10min of 12500rpm, abandons supernatant, the quartzite sand grind of precipitation sterilizing;
2. add the DNA extraction damping fluid of 13.5 milliliters, 100 microlitre 20mg/mL Proteinase Ks, 37 DEG C, 250 revs/min are shaken 30 minutes;
3. add 700 microlitre 20%SDS, 65 DEG C of water-bath 2h, interval 15min put upside down and shake up for several times;
Centrifugal 15 minutes of 4.6000 revs/min of room temperatures, liquid phase layer in the middle of collecting; In precipitation, add 5 milliliters of DNA extraction liquid, 300 microlitre 20%SDS, 65 DEG C of water-bath 30min, 6000 revs/min centrifugal, and liquid phase layer in the middle of collecting, merges; Repeat secondary;
5. in the liquid phase layer collected, add equal-volume chloroform: once, 8000 revs/min of centrifugal 10min, collect upper aqueous phase layer in primary isoamyl alcohol (24:1) extracting;
6. the 5th step collects the sodium acetate adding 0.1 times of volume in liquid phase layer, the Virahol of 0.6 times of volume, and room temperature places 1-2h.
7. overnight precipitation thing 12000 revs/min of centrifugal 15min, abandon supernatant; In precipitation, add the cold washing with alcohol of 10mL, 12000 revs/min of centrifugal 5min, abandon supernatant; 12000 revs/min of centrifugal 30sec,
8. natural drying at room temperature precipitation, adds 400 microlitre TE and dissolves in dry backward precipitation.
DNA extraction liquid forms: 100mMTris, 100mMEDTA, 200mMNaCl, 2%PVPP, 3%CTAB, pH9.0
Embodiment 2
Implement for 8 samples.
(1) 8 samples are numbered from 1 to 8, from table 1, in No.1 to No.48 sequence, choose a sequence numbering (as table 2) separately hand over correlated series Synesis Company to synthesize; The sequence of synthesis is diluted to 10pmol again with the water without DNA enzymatic; Sequence number in table 1 is associated to corresponding sample number, as table 2.
Table 2 sample is corresponding with composite label to be shown
| Sample number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| Sequence numbering | No.1 | No.2 | No.3 | No.4 | No.5 | No.6 | No.7 | No.8 |
(2) pcr amplification: get 8 200LPCR pipe, the often corresponding sample of pipe, and be the primer of pcr amplification by the sequence 1 in sequence numbering corresponding to above-mentioned sample number and sequence 2.Contain in 50 μ L systems of pcr amplification: respectively according to the DNA concentration of 8 samples, draw total amount be 50ng sample STb gene in respective tube, then add 4 μ LMg in every pipe
2+, 5 μ L10 × Buffer, 4 μ L concentration are 5nmoldNTP, 1.5UTaq enzyme, and concentration is each 1.5 μ L, moisturizing to the 50 μ L of 10pmol primer.
Pcr amplification program: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 40 DEG C of renaturation 30s, 72 DEG C extend 1min, 30 circulations, and last 72 DEG C extend 5min, 4 DEG C of preservations;
(3) take agarose 2 grams in Erlenmeyer flask, add water to 100mL, microwave heating, to thoroughly dissolving, adds appropriate fluorescence display agent mixing, pours in electrophoresis mould and cool.Draw above-mentioned pcr amplification terminate after liquid 5 μ L and electrophoretic buffer mix after add in the loading hole of sepharose, constant voltage electrophoresis is carried out by the voltage of every centimetre of 5V, put into by sepharose on ultraviolet visualizer after electrophoresis terminates and observe electrophoresis result, successful PCR reaction can obtain the amplified production of molecular weight distribution between 200bp to 500bp (as shown in Figure 1) through electrophoresis detection.
Embodiment 3
One, utilize general PCR primer to reclaim kits pcr amplification product, step is as follows:
(1) in the PCR reactant of 1 times of volume, add the BindingBuffer of 2 times of volumes, upset fully mixes.
(2) above-mentioned mixed solution is added in DNA purification column, if liquor capacity >700 μ is L, gradation transfer solution; Room temperature places 1-2min or longer time.
(3) centrifugal 1 minute of 13000g, proceeds in DNA purification column by the solution in collection tube after centrifugal end again, centrifugal, abandons waste liquid.
(4) in DNA purification column, add 650 μ LWashBuffer, centrifugal 30 seconds of 13,000g, abandons waste liquid, DNA purification column is put back to collection tube.Repeating step 4.
The centrifugal 3min of (5) 13,000g, to remove residual ethanol in post.
(6) purification column is put into new centrifuge tube, Xiang Zhuzhong is added in 30-50 μ LElutionBuffer or ddH of preheating at 60 DEG C
2o, room temperature places 1-2min or longer time.
The centrifugal 1min of (7) 13,000g, gained liquid is high purity DNA.
Two, by the ultraviolet spectrophotometer accurate quantification of the pcr amplification product after purifying, and to be adjusted to DNA total amount in solution that volume is 70 μ L with water be 100ng;
(8) IonXpress corresponding to sequenator is led to by IonPGM s-generation superelevation
tMplusgDNAFragmentLibraryPreparation test kit carries out building storehouse;
(9) IonPGM corresponding to sequenator is led to by IonPGM s-generation superelevation
tMtemplateOT2400Kit and IonPGMSequencing400Kit test kit carries out sequence amplification and order-checking, 18 chips check order, and 8 composite label sequence informations are inputted in high-flux sequence instrument sequence measurement used, high-flux sequence instrument according to the sequence information of input, will sort out corresponding sampled data.The sequencing result (Fig. 2) obtained according to above-mentioned sequence measurement shows, high-flux sequence instrument can according to composite label sequence corresponding to each sample, effectively can sort out the sequencing data mixed go out 8 samples sequencing result separately, reach the object of composite label application.
For the sequence measurement of other arbitrary sample quantity with embodiment 2 and embodiment 3.
Claims (4)
1. for a composite label for the high-flux sequence of grand internal transcribed spacer sequence, it is characterized in that, its base sequence is as shown in following sequence numbering NO.1 ~ NO.48:
2. utilize composite label described in claim 1 to carry out a method for ultra-high throughput gene sequencing, it is characterized in that, comprise the steps:
(1) STb gene of each sample is extracted;
(2) to each sample, Accessory Right to require shown in 1 48 to and do not selected arbitrary pair of sequences as primer by the sequence selected, the STb gene of corresponding sample is increased;
(3) electrophoresis detection is carried out to the amplified production of each sample, purifying recovery is carried out to amplified production;
(4) the product ultraviolet spectrophotometer accurate quantification after each Sample purification being reclaimed;
(5) sequencing library is set up by after all sample balanced mix;
(6) high-flux sequence is carried out to built sequencing library.
3. method according to claim 2, it is characterized in that, described sample is the sample containing fungi.
4. method according to claim 2, is characterized in that, to the PCR amplification system that the STb gene of corresponding sample increases be:
50ng sample STb gene, 4 μ LMg
2+, 5 μ L10 × Buffer, 4 μ L concentration are 5nmoldNTP, 1.5UTaq enzyme, and concentration is each 1.5 μ L, moisturizing to the 50 μ L of 10pmol primer.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104131008A (en) * | 2014-07-24 | 2014-11-05 | 深圳华大基因医学有限公司 | DNA labels, PCR primers and application thereof |
| CN104263726A (en) * | 2014-09-25 | 2015-01-07 | 天津诺禾致源生物信息科技有限公司 | Primer applied to amplicon sequencing library construction and method for constructing amplicon sequencing library |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104131008A (en) * | 2014-07-24 | 2014-11-05 | 深圳华大基因医学有限公司 | DNA labels, PCR primers and application thereof |
| CN104263726A (en) * | 2014-09-25 | 2015-01-07 | 天津诺禾致源生物信息科技有限公司 | Primer applied to amplicon sequencing library construction and method for constructing amplicon sequencing library |
Non-Patent Citations (6)
| Title |
|---|
| BJORN D.LINDAHL ET AL: "Fungal community analysis by high-throughput sequencing of amplified markers – a user’s guide", 《NEW PHYTOLOGIST》 * |
| CONRAD L.SCHOCH ET AL: "Nuclear ribosomal internal transcribed spacer(ITS) region as a universal DNA barcode marker for Fungi", 《PNAS》 * |
| D.LEE TAYLOR ET AL: "Increasing ecological inference from high throughput sequencing of fungi in the environment through a tagging approach", 《MOLECULAR ECOLOGY RESOURCES》 * |
| LEHO TEDERSOO ET AL: "Shotgun metagenomes and multiple primer pair-barcode combinations of amplicons reveal biases in metabarcoding analyses of fungi", 《MYCOKEYS》 * |
| X. IRIART ET AL: "Eumycetoma caused by Diaporthe phaseolorum (Phomopsis phaseoli): a case report and a mini-review of Diaporthe/Phomopsis spp invasive infections in humans", 《CLIN MICROBIOL INFECT》 * |
| 杜迎春: "传代对红色毛癣菌表型和核糖体基因序列的影响", 《中国优秀硕士学位论文全文数据库（电子期刊）》 * |
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Application publication date: 20160302 |