CN108315320A - A kind of preparation method of RNA high-throughput sequencing libraries - Google Patents
A kind of preparation method of RNA high-throughput sequencing libraries Download PDFInfo
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Abstract
The present invention provides the method that one kind can carry out high-throughput sequencing library preparation to all RNA without poly (A) tail, and the method for the present invention covers all RNA without poly (A) tail, including linear rna and circular rna.The linear RNA without poly (A) tail can directly carry out high-throughput sequencing library preparation using the method for the present invention or after interrupting；Circular rna can carry out high-throughput sequencing library preparation after interrupting using the method for the present invention.First, end extension is carried out, above-mentioned RNA is made to extend one section of homopolymer sequence in 3 ' ends.Then, after being attached reaction, make its 5 ' end connection top connection R5A.After connection is reacted, above-mentioned RNA connection products are purified, recycles special reverse transcriptase primer RTP to carry out reverse transcription reaction, obtains mono- chains of corresponding cDNA.Finally, PCR amplification is carried out using with the matched primer of high-flux sequence platform, you can obtain high-throughput sequencing library.It is noted that the method for the present invention has obtained larger promotion in sensitivity, it is particularly suitable for the preparation of the high-throughput sequencing library of low initial amount RNA in micro-example.
Description
Technical field
The present invention relates to molecular biology field, the RNA high passes in particular to one kind without poly (A) tail measure
The high-flux sequence of low initial amount RNA in preface library preparation method, more particularly to micro-example such as excretion body and extracellular vesica
The preparation in library.
Technical background
MicroRNA (small RNA) is a major class regulatory molecule, is present in almost all of organism, length
Generally 18-40 nucleotide (nucleotide, nt).From 1993 for the first time in caenorhabditis elegan (Caenorhadits
Elegans after finding small RNA in), people recognize the important function of small RNA more and more.They are with difference
In the mode of action of " classical RNA ", participates in gene's expression and control, the processing of RNA and shearing, protein translation, heredity and " enter
Invade " inhibit, the biological processes such as gamete occurs, in the physiology mistake such as generation of gene expression, bion development, metabolism and disease
Journey plays an important role.Can be 4 classes according to the characterization of molecules of tiny RNA：Microrna (microRNA, miRNA),
PiRNA (piwi-associated RNA), siRNA (small interfering RNA, siRNA) and small nucleolar RNA
(snoRNA, small nucleolar RNA), the wherein research of miRNA is more, and progress is most fast.
As typically linearly without the RNA of poly (A) tail, miRNA be a kind of length be 19-25nt microRNA,
It is found in nematode earliest.This 2 kinds of miRNA of lin-4 and let-7 are attached to 3 ' non-codings of said target mrna by partial sequence complementarity
Area regulates and controls elegans development process.Before high throughput sequencing technologies birth, the discovery of miRNA and research speed are relatively slow always, this
It is to be spent greatly, sequencing depth is limited, less efficient since traditional Sanger PCR sequencing PCRs are complicated for operation.Since two thousand five, high
Flux sequencing technologies are widely used for excavating miRNA.High throughput sequencing technologies are fast, at low cost with speed, coverage is deep, output
The advantages that huge, therefore it is very suitable for miRNA sequencings, miRNA especially low to those copy numbers.End in December, 2016,
Sanger miRBase databases (http://www.mirbase.org/) included more than 2800 kinds of miRNA, wherein the mankind at
Ripe body miRNA has more than 2500 kinds.Studies have shown that the unconventionality expression of miRNA and specific cancer, the occurrence and development phase of disease
It closes.The express spectra of miRNA is analyzed using high-flux sequence means in tumor research field, finding can in certain tumours
The miRNA of unconventionality expression can occur, it is possible to find the new biomarkers as diagnosing tumor and Index for diagnosis.Currently,
A very important means of research miRNA are had become with high throughput sequencing technologies combination bioinformatic analysis.
Compared with linear rna, the concern that circular rna (circular RNA, circRNA) is subject to is fewer, also more difficult
In research.Circular rna is a kind of RNA with special unknown function, and is objective a large amount of existing.Circular rna is by precursor
Then RNA connects tail by shearing by the head of linear rna and is formed.Circular rna is difficult to be distinguished with other RNA, amplification or piece
Duan Huahui destroys RNA rings, and the parser of early RNA sequencing can filter out the significant sequence of circular rna.Due to technology
With methodology problem, circular rna is always treated as being rare shearing mistake, to which the RNA of this part objective reality is ignored
.As depth RNA sequencings and the development of scale biology information technology, researcher just really have found largely to deposit in vivo
In cricoid RNA molecule, circular rna is very stablized in vivo due to forming the ring-type being closed.
With the help of bioinformatics, biochemical analysis and deep sequencing, researchers have sky to these mysterious molecules
Preceding understanding.For the function of circular rna, there is the saying of several hypothesis at present：(1) circular rna can be used as the sea " sponge "
Silk floss inhales miRNA, inhibits its function；(2) circular rna passes through other rna levels of base pair complementarity direct regulation and control；(3) circular rna
It can be combined with protein, inhibit protein active, raise the component of protein complex or the activity of regulation protein；(4) ring
Shape RNA also can be used as the synthesis of the template guided protein of translation.On the basis of to circRNA structure and function researchs, have
Researcher has found that they all send out in the diseases generating process such as atherosclerosis, nerve problems, diabetes and tumour
Wave very important effect.Have great importance to circular rna research：(1) the unique competitiveness of circular rna is endogenous
(ceRNA) feature can provide new thinking for drug development；(2) tissue specificity of circular rna and stability there is a possibility that
CircRNA becomes a kind of good biomarker；(3) research of circular rna provides new direction for the Study on Evolution of life.
At present for there are mainly two types of the RNA high-throughput sequencing library preparation methods without poly (A) tail, i.e. random primer
Method and connector connection method.The first random priming is to utilize random primer reverse transcription at cDNA RNA, then synthesizes double-strand
DNA, in addition connector, to complete the preparation in library.Although this method is simple, during prepared by library, it is easy
The RNA of small fragment is caused to lose.Second method is connector connection method, and this method is also that current mainstream is built library kit and adopted
Method.This method is to add RNA connectors at the 3 ' ends of dephosphorylation RNA, then carry out phosphorylation to 5 ' ends of connection product
Processing makes it add the RNA connectors at 5 ' ends, reverse transcription reaction and PCR amplification is then carried out, to complete the preparation in library.
The main problem of this method is easy to happen between connector from connecting, to influence the quality and output of subsequent sequencing data.
In addition, the banking process initial amount of RNA is usually required that it is higher, it is general to require in 10ng or more.However, being led in liquid biopsy
The rna content in domain, body fluid source is usually all relatively low, usually needs considerable humoral sample using conventional RNA banking process.
For example, using traditional RNA banking process based on connector connection, whole blood sample at least needs 10ml that could meet to build library and want
It asks.Therefore, the sensitivity of traditional banking process cannot be satisfied the requirement for construction data base of such low abundance RNA.Liquid biopsy field
Urgently a kind of highly sensitive be especially can be suitably used for all high-throughput sequencing library preparation methods without poly (A) tails RNA.
Invention content
For existing in the prior art to carrying out building library sequencing sensitivity and effective percentage without the RNA of poly (A) tail
Relatively low problem, especially can not be efficiently to the RNA without poly (A) tail in the micro-examples such as excretion body and extracellular vesica
The problem of carrying out building library, the present invention provides a kind of can be directed to obtain all RNA without poly (A) tail, and to its efficient structure
The method for building sequencing library.
The preparation method of RNA high-throughput sequencing libraries of the present invention includes the following steps：(1) end is carried out to extend instead
It answers, poly (A)-RNA is made to extend one section of sequence in its 3 ' end；(2) it is attached reaction, above-mentioned RNA is made to connect at its 5 ' end
Connector R5A is connected, RNA connection products are obtained；(3) above-mentioned RNA connection products are purified；(4) special reverse transcription is utilized
Primer RTP carries out reverse transcription reaction to above-mentioned RNA connection products after purification, obtains mono- chains of corresponding cDNA；(5) PCR is carried out
Amplification enrichment is to get to high-throughput sequencing library.Fig. 1 illustrates to carry out RNA high-throughput sequencing library systems using the inventive method
Standby flow chart.
In the method, for RNA after the extension of end, 3 ' ends are one section of homopolymer.The homopolymer by A, T,
C, any one composition in this five kinds of bases of G or U, it is therefore preferable to base A.End extension method in the present invention theoretically can be with
Unbiased poorly makes all RNA all extend one section of homopolymer at 3 ' ends, thus can completely, accurately RNA in response sample
The time of day of transcript.
In the method, 5 ' end connector R5A are by two sections of Sequence compositions：Only positioned at the distinguished sequence at 5 ' ends and positioned at 3 ' ends
One identification sequence.Wherein, 5 ' terminal specific sequences are the 3 ' terminal sequences or intact full-length sequence of high-flux sequence plain adapter；3 ' ends are only
One identification sequence is the random sequence by these four base compositions of A, T, C and G, length 5nt.Particularly, in the method, by
In R5A 5 ' end be there is no 3 ' end connectors without phosphorylation modification, and in coupled reaction system, therefore will not cause connector it
Between connect certainly, so as to avoid the influence to sequencing data quality.In addition, one section of continuous stochastic ordering is contained at the ends R5A 3 '
On the one hand row can be used as unique recognition sequence, another convenient for rejecting the influence that PCR deviations are brought in subsequent data analysis
Aspect can also improve its joint efficiency between RNA to a certain extent.Further, R5A sequences include Seq ID
No:1。
In the method, distinguished sequence two parts sequence structure that reverse transcriptase primer RTP is held by the distinguished sequence at 5 ' ends and 3 '
At.Wherein, 5 ' terminal specific sequences are the 3 ' terminal sequences or intact full-length sequence of high-flux sequence plain adapter；The distinguished sequence at 3 ' ends
Sequence is captured by RNA connection products and anchor series two parts form.RNA connection products capture sequence in RTP and above-mentioned RNA
3 ' end extension sequences it is mutually complementary, the length of the capture sequence is 2-50nt, it is therefore preferable to 10-30nt.The 1st of anchor series
Base is the degeneracy base that homopolymer complementation is not held with RNA 3 ' (from 5 ' ends to 3 ' extreme directions)；2nd bit base to 3 ' least significant ends
For by the random sequence of this 4 kinds of base compositions of A, T, C and G, sequence length 1-5nt, it is therefore preferable to 1-3nt, more preferably
1nt.Further, RTP sequences include Seq ID No:2、Seq ID No:3、Seq ID No:4 and Seq ID No:5 etc.
Sequence.
In the method, the PCR primer for being used for amplified library is the 5 ' terminal sequences or complete of high-flux sequence platform connector
Full length sequence.After PCR reactions, you can sequencing library be enriched with, complete.By the segment screening in library, after purification
With upper machine sequencing can be carried out after quality inspection.
One of feature of the method for the present invention is can be to carrying out building library sequencing without the RNA of poly (A) tail, and covering is all
Without the RNA of poly (A) tail, including linear rna and circular rna.The linear RNA without poly (A) tail can be utilized directly
The method of the present invention or as needed interrupt after carry out high-throughput sequencing library preparation；Nonlinear RNA, can into after Break Row
To carry out high-throughput sequencing library preparation using this method.The two of the feature of the method for the present invention are that sensitivity prepared by library obtains
To larger promotion, less (initial amount can be down to 1-10ng or even 0.5-2ng) is required to RNA initial amounts, can be realized to excretion
RNA in the micro-examples such as body and extracellular vesica carries out the preparation of high-throughput sequencing library.
Description of the drawings
The specific implementation mode of the present invention is described in further detail below in conjunction with the accompanying drawings.
The accompanying drawings which form a part of this application are used only to provide a further understanding of the present invention, not structure
At inappropriate limitation of the present invention.In the accompanying drawings：
Fig. 1 shows the flow chart that RNA high-throughput sequencing library preparations are carried out using the inventive method.
Fig. 2 shows the nano particle trace analysis instrument testing result figures of blood plasma excretion body.
Fig. 3 shows the analyzing biochips system detectio figure of blood plasma excretion body small RNA.
Fig. 4 shows the PAGE electrophoretograms (Illumina platforms) in library prepared by the method for the present invention and NEB kits.M
For 20bp DNA ladder；1-3 swimming lanes are library prepared by the method for the present invention, wherein No. 1 swimming lane is negative control, No. 2 swimming
Road and No. 3 swimming lane miRNA initial amounts are respectively 10ng and 2ng；4-6 swimming lanes are library prepared by NEB RNA isolation kits, wherein 4
Number swimming lane and No. 5 swimming lane miRNA are respectively 10ng and 2ng, and No. 6 swimming lanes are negative control.
Fig. 5 shows the analyzing biochips system detectio figure in the library prepared by the method for the present invention and NEB kits
(Illumina platforms).(A)-(B) utilizes library prepared by the method for the present invention, and miRNA initial amounts are respectively 10ng and 2ng；
(C)-(D) utilizes library prepared by NEB kits, and miRNA initial amounts are respectively 10ng and 2ng.
Fig. 6 shows the Venn diagram (Illumina platforms) in library prepared by the method for the present invention and NEB kits.(A)-
(B) library for utilizing the method for the present invention to prepare, miRNA initial amounts are respectively 10ng and 2ng；(C)-(D) utilizes NEB kit systems
Standby library, miRNA initial amounts are respectively 10ng and 2ng.
Fig. 7 shows the PAGE electrophoretograms (Illumina platforms) in library prepared by the method for the present invention.M is 20bp DNA
ladder；1-3 swimming lanes are library prepared by the method for the present invention, wherein No. 1 swimming lane is negative control, No. 2 swimming lanes and No. 3 swimming lanes
MiRNA initial amounts are respectively 10ng and 2ng.
Fig. 8 shows the analyzing biochips system detection results (Illumina platforms) of the circular rna after fragmentation；
Fig. 9 shows circular rna library electrophoresis pattern (Illumina platforms) prepared by the method for the present invention.M is 20bp
DNA ladder；1-2 swimming lanes are library prepared by the method for the present invention, and circular rna initial amount is respectively 50ng and 10ng；3-4
Number swimming lane is library prepared by NEB RNA isolation kits, and circular rna initial amount is respectively 50ng and 10ng.
Figure 10 shows the PAGE electrophoresis results (Ion Torrent platforms) in library prepared by the method for the present invention.M is 20bp
DNAladder；No. 1 swimming lane is negative control；No. 2 swimming lanes are that initial amount is 10ng miRNA；It is 2ng that No. 3 swimming lanes, which are initial amount,
miRNA。
Figure 11 shows analyzing biochips system detection results (the Ion Torrent in library prepared by the method for the present invention
Platform).(A) initial amount is the library of 10ng miRNA, and (B) initial amount is the library of 2ng miRNA.
Figure 12 shows the Venn diagram (Ion Torrent platforms) in the library prepared by the method for the present invention.(A) initial amount is
The library of 10ng miRNA, (B) initial amount are the library of 2ng miRNA.
Specific implementation mode
The embodiment and gain effect further illustrated the present invention below in conjunction with specific embodiment, example is only
Validity for explaining the present invention is not intended to limit use and the protection domain of the present invention.
Lifted microarray dataset is used only to the versatility of demonstration the method for the present invention in embodiment, is not intended to limit the present invention
Use and protection domain, the method for the present invention can be compatible with all high-flux sequence platforms.
Experimental method used in embodiment and experiment condition are unless otherwise specified conventional method and condition, or
Abide by corresponding instructions selection.
Room temperature described in this hair embodiment means normal temperature laboratory, i.e., 20-30 DEG C.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1
MiRNA high-throughput sequencing library preparation process in the blood plasma excretion body that this example passes through a kind of exemplary embodiment
For, to describe the principle and feature that the method for the present invention prepares the linear libraries RNA without poly (A) tail.In this example,
Connector matches with Illumina microarray datasets, and 3 ' end end homopolymers are made of base A；The capture at 5 ' ends in reverse transcriptase primer
Sequence is by 20 T base compositions, anchor series VN(1)。
Correlated series involved in this example：
5 ' end connector R5A-1,5 '-CCUUGGCACCCGAGAAUUCCANNNNN；
Reverse transcriptase primer RTP-1,5 '-CAGAGTTCTACAGTCCGACGATCT(20)VN(1)。
1. the collection of plasma sample and its separation of excretion body
Inventor has collected in the first affiliated hospital of University Of Suzhou the complete of the normal physical examination Healthy People of 10ml in May, 2016
Blood.Whole blood sample is loaded on EDTA- anticoagulant tubes, has detached corresponding plasma sample respectively by the method for centrifugation, plasma sample is set
Next experiment is preserved or is directly entered under the conditions of -80 DEG C.
From plasma sample detach excretion body concrete operations be：
1) blood plasma of freeze thawing state 2000 × g at 4 DEG C completely is centrifuged 20 minutes, removes cell；
2) supernatant is taken to move to new centrifuge tube, 16500 × g is centrifuged 30 minutes under the conditions of 4 DEG C, and it is broken further to remove cell
Piece；
3) it takes supernatant liquid to be transferred to new centrifuge tube, the phosphate buffer of 0.5 times of volume, vortex mixing is added；
4) sample is passed through into 0.22um disposable filters, removes big vesica, collect clear liquid；
5) clear liquid of collection is centrifuged 70 minutes at 110000 × g, abandons supernatant, the PBS for adding 5 times of volumes of whole blood is molten
Liquid cleaning precipitation, 110000 × g are centrifuged 70 minutes again, reject supernatant；
6) use 100ul phosphate buffers that all precipitations are resuspended, -20 DEG C save backup.
7) take above-mentioned 1ul excretions weight suspension, after 1000 times of dilution after, take 1ml dilutions, utilize nano particle tracking point
Analyzer (Marlven companies, model NS300) is detected excretion body, analyzes separated excretion body grain size and quantity, specifically
Testing result is as shown in Figure 2.
2. the extraction of total serum IgE in excretion body
1) 2000 μ l TRIzol solution are added in above-mentioned remaining excretion weight hangs solution, firmly concussion is equal to presenting
One liquid phase is stored at room temperature 5 minutes；
2) 400 μ l of chloroform are added to be mixed well with forced oscillation centrifuge tube, are stored at room temperature 5 minutes；
3) under the conditions of 4 DEG C, 12000rpm high speed centrifugations are drawn after 15 minutes in upper strata aqueous phase to new centrifuge tube, are paid attention to not
The protein layer being drawn onto between two layers of water phase.
4) isometric isopropanol is added, fully reverse mixing stands two hours under the conditions of -20 DEG C；
5) under the conditions of 4 DEG C, 12000rpm high speed centrifugations carefully discard supernatant after 15 minutes, and the 75% of 1ml precoolings is added
Ethyl alcohol washing precipitates, and 7500rpm is centrifuged 5 minutes at 4 DEG C；
6) liquid is discarded, 5 minutes is placed at room temperature fully to dry precipitation, the sterile water without RNA enzyme of 20 μ l is added
It is dissolved；
7) analyzing biochips system Bioanalyzer 2100 is utilized to coordinate Small RNA analysis kits (Agilent
Company, article No. 5067-1548) detect Small RNA purity, concentration and segment distribution situation in total serum IgE, testing result such as Fig. 3
It is shown.RNA sample is placed in freeze preserves or is directly entered next experiment in -80 DEG C.
3. carrying out miRNA high-throughput sequencing library preparations using present patent application method
The preparation of high-throughput sequencing library is carried out with the above-mentioned RNA of the miRNA equivalents of 2ng and 10ng respectively.
1) same polyadenylic acid sequence is added at the ends RNA 3 '
It is formulated as follows reaction system：
| Ingredient | Volume (ul) | Final concentration |
| 10x poly(A)polymerase buffer(NEB) | 0.5 | 1x |
| ATP(10mM,NEB) | 0.5 | 100uM |
| SuperaseIn(Ambion,20U/μL) | 0.1 | 2U |
| miRNA | 3.8ul | 2ng/10ng |
| poly(A)polymerase(20U/μL,NEB) | 0.1 | 2U |
| Total volume | 5.0 |
Above-mentioned reaction solution is uniformly mixed, is reacted as follows on thermostat：37 DEG C 5 minutes then 65 DEG C 20 minutes.
2) 5 ' end connectors are connected
It is formulated as follows reaction solution：
| Ingredient | Volume (ul) | Final concentration |
| 10x RNA ligase buffer(NEB) | 0.5 | 1x |
| R5A-1(10μM) | 0.5 | 100nM |
| PEG (50%, NEB) | 2 | 2% |
| ATP(10Mm,NEB) | 1 | 200uM |
| RNA ligase1(20U/μL,NEB) | 1 | 2U |
| DTT(100Mm,NEB,) | 0.2 | 0.4mM |
| SuperaseIn(20U/μL,Ambion) | 0.2 | 4U |
| Total volume | 5ul |
Above-mentioned reaction solution is uniformly mixed, following procedure is executed on thermostat：22 DEG C 60 minutes.
3) purifying of connection product
It utilizesOligo(dT)25Magnetic bead (Thermo Fisher companies, article No. 61002) to connection product into
Row purifying, is used in combination the sterile water without RNA enzyme of 5.5ul to be eluted.
4) reverse transcription reaction
According to the form below prepares connection reaction solution.
Above-mentioned reaction solution is uniformly mixed, following procedure is executed on thermostat：42 DEG C 60 minutes then 95 DEG C 3 minutes.
5) the amplification enrichment in library
According to the form below prepares connection reaction solution.
| Ingredient | Volume (ul) | Final concentration |
| 5x Phusion HF Buffer(NEB) | 10 | 1x |
| SR Primer for Illumina(10μM,NEB) | 2.5 | 200nM |
| Index(X)Primer(10μM,NEB) | 2.5 | 200nM |
| dNTP Mix(10mM,NEB) | 1.25 | 250uM |
| Phusion DNA Polymerase(2U/μL,NEB) | 0.5 | 1U |
| ddH2O | 23.25 | - |
| Total volume | 40 |
Above-mentioned reaction solution is uniformly mixed, following procedure is executed in PCR instrument：98 DEG C of 30 seconds pre-degenerations；98 DEG C 10 seconds, 62
DEG C 30 seconds, 72 DEG C 15 seconds, totally 15 cycles；72 DEG C 60 seconds.
4. carrying out miRNA high-throughput sequencing library preparations using NEB kits
As a comparison, the libraries NEB small RNA reagent preparation is selected in this example：Multiplex
Small RNA Library Prep Kit for Illumina (NEB companies, article No. E7560S) are carried out with the method for the present invention
Comparison, to illustrate that the method for the present invention has the advantage that in sensitivity.Respectively using the miRNA of 2ng and 10ng as initial amount into
The preparation of row high-throughput sequencing library, method and operating method step are carried out with reference to kit specification.
5. library PAGE is purified
PAGE glue (6%) electricity is carried out by invention method and using the libraries miRNA prepared by NEB kits respectively
Swimming, 300V 1 hour, after waiting for electrophoresis, the corresponding target stripe of recovery purifying.
The results are shown in Figure 4, and library band prepared by the method for the present invention is located near 170bp, prepared by NEB kits
Library band is located near the positions 140bp, with expected consistent.For the library that miRNA initial amounts are 2ng, the method for the present invention
The library band of preparation is clearly apparent, and the library band prepared by NEB kits is faint.
6. library quality inspection and the sequencing of upper machine
Using analyzing biochips system Bioanalyzer2100 cooperation high-sensitivity DNA assay kit (Agilent companies,
Article No. 5067-4626) distribution of lengths in library is determined.The results are shown in Figure 5, and library distribution of lengths is the same as expected consistent.
Utilize high-throughput sequencing library quantification kit:(Kapa is public by KAPA Library Quantification Kits
Department, article No. KK4824) copy number in library is determined, upper machine amount is sequenced to determine.It is carried out using illumina sequenators
Sequencing, sequencing strategy HiSeq2500SE50 are sequenced according to the applied sample amount of each library 8M Raw Reads.
7. bioinformatic analysis
1) Raw data quality assessment and filtration treatment
Quality evaluation and filtering are carried out to sequencing initial data using FastQC, cutadapt, FASTX-Toolkit software
Processing.Statistical result is as shown in table 1, the method for the present invention with NEB kits data in Clean Reads quantity and Q30 ratios
It is more consistent on the two major parameters.
1 sequencing data statistic of attribute result of table
(A)-(B) library that prepared by the method for the present invention, miRNA initial amounts are respectively 10ng and 2ng；(C)-(D) NEB reagents
Library prepared by box, miRNA initial amounts are respectively 10ng and 2ng.
2) reference sequences are compared
Compared with newest miRBase databases and the full reference gene group GRCh37 (hg19) of the mankind using Bowtie softwares
It is right.Statistical result is as shown in table 2, for the initial amount of 10ng miRNA, the text prepared by the method for the present invention and NEB kits
Library, comparison result is in desirable level；When miRNA initial amounts are 2ng, the comparison result in library prepared by the method for the present invention is still
It is more satisfactory, it is suitable with above-mentioned two groups of library results, and library comparison result prepared by NEB kits is poor.
2 clean reads comparison results of table count
(A)-(B) library that prepared by the method for the present invention, miRNA initial amounts are respectively 10ng and 2ng；(C)-(D) NEB reagents
Library prepared by box, miRNA initial amounts are respectively 10ng and 2ng.
3) different banking process and different being compared to each other between the library of library initial amount is built
Unique and overlapping miRNA relationships between four groups of libraries are as shown in fig. 6, utilize the method for the present invention system
Standby miRNA initial amounts are the library in the library and 2ng of 10ng, the text for being 10ng with miRNA initial amounts prepared by NEB kits
Library has preferable plyability between three；And for the initial amount of 2ng miRNA, library prepared by NEB kits,
MiRNA types are considerably less than other three kinds of libraries.Pearson correlation analysis is as shown in table 3, above-mentioned three kinds of libraries miRNA tables
Higher up to spectrum similarity, the initial amount for being substantially distinguished from the preparation of NEB kits is the library of 2ng miRNA.
Table 3 utilizes the Spearson correlation analysis in library prepared by the method for the present invention and NEB kits
(A)-(B) utilizes library prepared by the method for the present invention, and miRNA initial amounts are respectively 10ng and 2ng；(C)-(D) sharp
The library prepared with NEB kits, miRNA initial amounts are respectively 10ng and 2ng.
Embodiment 2
It is illustration the method for the present invention to high-flux sequence platform compatibility and compatibility, the present embodiment is still with blood plasma excretion
In body for miRNA high-throughput sequencing libraries preparation process, except that connector is substituted for and Ion Torrent microarray datasets
Sequence, other conditions are the same as shown in embodiment 1.
Correlated series involved in this example：
5 ' end connector R5A-2,5 '-CCUUGGCACCCGAGAAUUCCANNNNN；
Reverse transcriptase primer RTP-2,5 '-CAGAGTTCTACAGTCCGACGATCT(30)VN(1)。
1. the collection of plasma sample and its separation of middle excretion body are the same as example 1.
2. the extraction of excretion body total serum IgE is the same as example 1.
3. carrying out the preparation of miRNA high-throughput sequencing libraries with example 1 using present patent application method.
4. library PAGE is purified with embodiment 1.
Under the primer sequence combination of the present embodiment, library length is in 180bp or so.Electrophoresis result after PAGE is as schemed
Shown in 7, band is consistent with being expected, and illustrates successfully prepare miRNA high-throughput sequencing libraries in the case where the primer sequence combines.
Embodiment 3
It is prepared by the high-throughput sequencing library of the circular rna in the blood plasma excretion body that this example passes through a kind of exemplary embodiment
For process, to describe to prepare the principle and feature in the nonlinear libraries RNA without poly (A) tail using the method for the present invention.
In this example, connector matches with Illumina microarray datasets, and 3 ' end end homopolymers are made of base A；Reverse transcriptase primer
In 5 ' end capture sequences by 20 T base compositions, anchor series VN(1)。
Correlated series involved in this example：
5 ' end connector R5A-1,5 '-CCUUGGCACCCGAGAAUUCCANNNNN；
Reverse transcriptase primer RTP-1,5 '-CAGAGTTCTACAGTCCGACGATCT(20)VN(1)。
1. the collection of plasma sample and its separation of middle excretion body
Inventor has collected the complete of the normal physical examination Healthy Peoples of 50ml in May, 2016 in the first affiliated hospital of University Of Suzhou
Blood, whole blood sample are loaded on EDTA- anticoagulant tubes.The method and operating procedure of excretion body are the same as embodiment 1 in whole blood.
2. the extraction of total serum IgE in excretion body
Method and operating procedure are the same as embodiment 1.
3. the rRNA in the above-mentioned RNA of removal
Kit is removed using rRNA：Ribo-Zero rRNA Removal Kit (Human, Mouse, Rat) are tried
Agent box (Illumina companies, article No. K1550-01) removes the rRNA in above-mentioned total serum IgE, and method and steps is with reference to explanation
Book carries out, and is finally dissolved without RNA enzyme water with 20ul.The RNA sample of above-mentioned removal rRNA is placed in -80 DEG C
Save backup or be directly entered next experiment.
4. removing poly (A) RNA in total serum IgE
1) it mixes wellOligo(dT)25Magnetic bead (Thermo Fisher companies, article No. 61002), then
It takes in 30 μ l to 1.5ml centrifuge tubes, is placed on magnetic frame, stand 3 minutes, carefully drawn with pipette tips and abandon supernatant.
2) 100 μ l combination buffers are added and fully blow and beat mixing, brief centrifugation stands 3 minutes, use rifle on magnetic frame
Careful draw of head abandons supernatant.
3) it repeats the above steps primary.
4) 50 μ l combination buffers are added, mixing is fully blown and beaten.
5) the total serum IgE sample of above-mentioned removal rRNA is mended without RNA enzyme water to 50 μ l.
6) sample is added to above-mentionedOligo(dT)25In magnetic bead, mixing is fully blown and beaten, 65 DEG C are incubated 5 points
Clock is placed in 2 minutes on ice at once after incubation.
7) it is incubated at room temperature 10 minutes on blending instrument, brief centrifugation, 3 minutes is stood on magnetic frame.
8) clear liquid is transferred to another 1.5ml centrifuge tube, 180 μ l is adjusted to using no RNA enzyme water.The 3M of 18 μ l is added
Sodium acetate, the glycogen (10mg/ml) of 2 μ l, three times volume ice precooling absolute ethyl alcohol, slight vortex mixed, -20 DEG C are quiet
Set 1 hour.16000 × g is centrifuged 30 minutes under the conditions of 4 DEG C, and careful draw abandons supernatant.With 20 μ l without RNA after drying
Enzyme water dissolution RNA precipitate is to get having arrived Poly (A)-RNA sample.
5. then carrying out RNase R digestion
Linear rna is digested using RNase R (Epicentre companies, article No. RNR07250).According to the form below prepares reaction system：
Pressure-vaccum mixing is carried out to above-mentioned 20 μ l reaction systems, brief centrifugation is placed on 37 DEG C and is incubated 1 hour.After digestion
20 μ L phenol-chloroforms-isoamyl alcohol (Thermo Fisher companies, article No. 15593-031), concussion mixing is added to be digested to terminate,
16000 × g, 4 DEG C centrifuge 5 minutes.Supernatant is abandoned, 6 μ L 4M lithium chlorides, 1 μ L glycogens (10mg/ml), the nothing of 90 μ L precoolings are precipitated
Water-ethanol is reverse to be resuspended, and -80 DEG C of precipitations centrifuge 30 minutes for 1 hour, 16000 × g, 4 DEG C, and two are washed with 75% ethyl alcohol of precooling
It is secondary.It is dissolved to get to treated circular rna sample without RNase enzyme water with 20 μ L after drying.By above-mentioned ring-type
RNA sample preserves or is directly entered next experiment under the conditions of being placed in -80 DEG C.
6.RNA fragmentations
Utilize the RNA fragmentation reagents boxes of NEB：RNase III RNAFragmentation Module
(NEB companies, article No. E6146) carries out fragmentation processing to above-mentioned RNA.RNA fragmentation systems are established according to following table：
| Component | Volume (μ l) |
| Above-mentioned RNA after purification | 17 |
| RNase III(1unit/μl) | 1 |
| RNase III Reaction Buffer(10X) | 2 |
| Total volume | 20 |
Above-mentioned reaction solution is uniformly mixed, is incubated 5 minutes for 37 DEG C on thermostat, the EDTA of 10mM is added after reaction
Terminate reaction.It is purified using RNA purification kits (Qiagen companies, article No. 74204), with the water for going RNA enzyme of 10 μ L
It is dissolved to get to the RNA of fragmentation.Coordinate pico RNA cores using analyzing biochips system Bioanalyzer2100
Total serum IgE sample after piece (Agilent companies, article No. 5067-1548) removal rRNA is detected.As a result such as Fig. 8 institutes
Show, the circular rna clip size after fragmentation is in 200-300bp.RNA sample after above-mentioned fragmentation is placed in -80 DEG C of preservations
Or it is directly entered next experiment.
7. the preparation in library
High-throughput sequencing library is carried out using the circular rna after the above-mentioned fragmentation of 10ng and 50ng as initial amount respectively
It prepares.As a comparison, library kit is built using the RNA of NEB simultaneously in this example：NEBNext Ultra Directional
RNA Library Prep Kit (NEB companies, article No. E7420S) carry out the preparation in library, are carried out pair in the method for the same present invention
Than.
8. the PAGE electrophoresis in library and purifying are the same as embodiment 1.
The results are shown in Figure 9, and prepared by the library that the present invention can be used successfully to the high-flux sequence of circular rna, the present invention
Library band prepared by method and NEB kits is respectively positioned at 300bp to the positions 400bp, it is expected the band obtained.
In addition, the circular rna sample for building the fragmentation that library initial amount is 10ng, the library band prepared using the method for the present invention are clear
It is clear, and utilize the library band prepared by NEB kits faint.Therefore, the method for the present invention compares conventional method, and sensitivity obtains
Larger promotion.
Embodiment 4
It is illustration the method for the present invention to high-flux sequence platform compatibility and compatibility, this example is with Ion Torrent
Microarray dataset illustrates for representative.The present embodiment is still with miRNA high-throughput sequencing library preparation process in blood plasma excretion body
For, except that connector matches with Ion Torrent microarray datasets, remaining condition is the same as embodiment 1.
Correlated series involved in this example：
5 ' end connector R5A-3, CCTCTCTATGGGCAGTCGGTGATNNNNN；
Reverse transcriptase primer RTP-3, CCAUCUCAUCCCUGCGUGUCUCCGACT(20)VN(1)。
1. the collection of plasma sample and its separation of excretion body are the same as embodiment 1.
2. the extraction of excretion body total serum IgE is the same as embodiment 1.
3. carrying out the preparation of miRNA high-throughput sequencing libraries with embodiment 1 using present patent application method.
4. library PAGE is purified with embodiment 1.
The results are shown in Figure 10 after PAGE electrophoresis, and library band is located near 140bp, clear apparent, with expected one
It causes.
5. library quality inspection and high-flux sequence
Using analyzing biochips system Bioanalyzer2100 cooperation high-sensitivity DNAs assay kit, (Agilent is public
Department, article No. 5067-4626) distribution of lengths in library is determined.As a result as shown in figure 11, library distribution of lengths is the same as expected one
It causes, concentration meets confidential ask.
Utilize high-throughput sequencing library quantification kit:(Kapa is public by KAPA Library Quantification Kits
Department, article No. KK4827) copy number in library is determined, upper machine amount is sequenced to determine.Utilize Ion ProtonTMSequenator into
Row sequencing, is sequenced according to the applied sample amount of each library 10M raw reads.
6. bioinformatic analysis
1) Raw data quality assessment and filtration treatment
Quality evaluation and filtering are carried out to sequencing initial data using FastQC, cutadapt, FASTX-Toolkit software
Processing.Treated that statistical result is as shown in table 4 through filtering for the lower machine data of sequencing, is 10ng and 2ng miRNA for initial amount
Library, the two in Clean Reads numbers and Q30 ratios the two mainly to assess result in parameter suitable.
4 Raw data quality statistical result of table
(A) initial amount is the library of 10ng miRNA, and (B) initial amount is the library of 2ng miRNA.
2) reference sequences are compared
Using Bowtie softwares by sequence with miRNA databases miRBase and the full reference gene group GRCh37 of the mankind
(hg19) it is compared.The results are shown in Table 5, and for the library that miRNA initial amounts are 10ng and 2ng, comparison result is equal
Reach desirable level.
5 clean reads comparison results of table count
(A) initial amount is the library of 10ng miRNA, and (B) initial amount is the library of 2ng miRNA.
3) correlation analysis between library
Unique and overlapping miRNA correlations between two groups of libraries are as shown in figure 12, risen for building library
Beginning amount is the library of 10ng miRNA and 2ng miRNA, and the miRNA that two kinds of libraries are identified has preferably consistent in type
Property.
Pearson correlation analysis is as shown in table 6, and the miRNA express spectra similarities in above two library are higher.
Spearson correlation analysis between library prepared by 6 the method for the present invention of table
(A) initial amount is the library of 10ng miRNA, and (B) initial amount is the library of 2ng miRNA.
<110>Li Hui brightness Liu Yue stars
<120>A kind of preparation method of RNA high-throughput sequencing libraries
<141> 2016-12-10
<160> 10
<210> 1
<211> 26
<212> RNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (22)..(26)
<223>N=a or g or c or t
<400> 1
ccuuggcacc cgagaauucc annnnn 26
<210> 2
<211> 30-77
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (29)..(77)
<223>N=a or g or c or t, v=a or g or c
<400> 2
cagagttcta cagtccgacg atct(5-50)vn(1-3) 30-77
<210> 3
<211> 30-77
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (29)..(77)
<223>N=a or g or c or t, d=a or g or t
<400> 3
cagagttcta cagtccgacg atcc(5-50)dn(1-3) 30-77
<210> 4
<211> 30-77
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (29)..(77)
<223>N=a or g or c or t, h=a or c or t
<400> 4
cagagttcta cagtccgacg atcg(5-50)hn(1-3) 30-77
<210> 5
<211> 30-77
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (29)..(77)
<223>N=a or g or c or t, b=g or c or t
<400> 5
cagagttcta cagtccgacg atca(5-50)bn(1-3) 30-77
Claims (19)
1. a kind of preparation method of RNA high-throughput sequencing libraries, which is characterized in that include the following steps：(1) end extension is carried out
Reaction, makes the RNA without poly (A) tail extend one section of sequence in 3 ' ends；(2) it is attached reaction, makes above-mentioned RNA at it
5 ' end connection top connection R5A, obtain RNA connection products；(3) above-mentioned RNA connection products are purified；(4) it utilizes specifically
Reverse transcriptase primer RTP carries out reverse transcription reaction to above-mentioned RNA connection products after purification, obtains mono- chains of corresponding cDNA；(5) into
The enrichment of row PCR amplification is to get to high-throughput sequencing library.
2. the preparation method of RNA high-throughput sequencing libraries according to claim 1, which is characterized in that 3 ' end extension sequences
For one section of homopolymer sequence.
3. homopolymer sequence according to claim 2, it is characterised in that homopolymer by adenine (A), thymidine (T),
Any one composition in this five kinds of bases of cytimidine (C), guanine (G) or uracil (U), it is therefore preferable to base A.
4. the preparation method of NRA high-throughput sequencing libraries according to claim 1, which is characterized in that 5 ' end connector R5A by
Two sections of Sequence compositions：One section of unique recognition sequence of one section of distinguished sequence at 5 ' ends and 3 ' ends.
5. 5 ' end connector R5A according to claim 4, which is characterized in that 5 ' terminal specific sequences flush for high-flux sequence
3 ' the terminal sequences or intact full-length sequence of head.
6. 5 ' end connector R5A according to claim 4, which is characterized in that 3 ' end unique recognition sequences are by A, T, C and G
The random sequence of these four base compositions, length 5nt.
7. 5 ' the end connector R5A according to claim 5 and claim 6, which is characterized in that its sequence includes Seq ID
No:1。
8. the preparation method of RNA high-throughput sequencing libraries according to claim 1, which is characterized in that reverse transcriptase primer RTP
By distinguished sequence two parts Sequence composition of the distinguished sequence and 3 ' ends at 5 ' ends.
9. reverse transcriptase primer RTP according to claim 7, which is characterized in that the distinguished sequence at 5 ' ends is high-flux sequence
3 ' the terminal sequences or intact full-length sequence of platform connector.
10. reverse transcriptase primer RTP according to claim 7, which is characterized in that the distinguished sequence at 3 ' ends is produced by RNA connections
Object captures sequence and anchor series two parts composition.
11. RNA connection products according to claim 10 capture sequence, which is characterized in that the sequence with the ends RNA3 ' by prolonging
Stretch the complementary base composition of sequence phase.
12. RNA connection products according to claim 10 capture sequence, which is characterized in that the length of the sequence is 2-
50nt, it is therefore preferable to 10-30nt.
13. anchor series according to claim 10, which is characterized in that its 1st bit base (from 5 ' ends to 3 ' extreme directions)
For the degeneracy base not with RNA homopolymer complementations.
14. anchor series according to claim 10, which is characterized in that its 2nd bit base (from 5 ' ends to 3 ' extreme directions)
To the sequences of 3 ' least significant ends, by A, T, C and G, these four bases form at random.
15. anchor series according to claim 10, which is characterized in that the sequence length is 1-5nt, it is therefore preferable to 1-
3nt, more preferably 1nt.
16. according to claim 9 and reverse transcriptase primer RTP according to any one of claims 10, which is characterized in that its sequence includes Seq
ID No:2、Seq ID No:3、Seq ID No:4 and Seq ID No:5.
17. the preparation method of RNA high-throughput sequencing libraries according to claim 1, which is characterized in that be used for amplified library
PCR primer be high-flux sequence platform connector 5 ' terminal sequences or intact full-length sequence.
18. the preparation method of RNA high-throughput sequencing libraries according to claim 1, which is characterized in that this method can be right
All RNA without poly (A) tail carry out high-throughput sequencing library preparation, including the linear RNA without poly (A) tail and
Circular rna.
19. the preparation method of RNA high-throughput sequencing libraries according to claim 19, which is characterized in that linear
It, can be directly using the method for the present invention or into carrying out high-throughput sequencing library system after Break Row without the RNA of poly (A) tail
It is standby；The nonlinear RNA without poly (A) tail can carry out high-throughput sequencing library preparation after interrupting using this method.
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