CN104673785A - Method for researching APA within strand-specific whole-genome range - Google Patents

Abstract

The invention discloses a method SS-3T-seq for researching APA within a strand-specific whole-genome range. The method is used for researching APA changes within a whole-genome range and comprises the following steps: firstly building a special magnetic bead containing oligo dT; screening RNA containing a polymer A by using the magnetic bead; synthesizing a first-stranded cDNA by reverse transcription on the magnetic bead, and replacing normal dCTP with methylated dCTP in a reverse transcription system; synthesizing double-stranded cDNA by using special dNTP (dTTP is replaced by dUTP); after breaking, taking the remaining part on the magnetic bead as a fragment containing an APA locus; removing the polymer A structure by Gsu I enzyme excision; releasing a target fragment from the magnetic bead; modifying the two ends and adding an Illumina sequencing joint; screening fragments according to size and performing USER enzyme treatment to remove the dUTP in the second strand; leaving the first strand for PCR amplification to obtain a library which contains strand information and can be used for Illumina next-generation sequencing. The method is simple in experiment operation, has the advantages of reducing loss caused by complex steps and also reducing the experiment difficulty and the manufacturing cost, and is quite suitable for popularization.

Description

The method of APA is studied within the scope of a kind of chain specificity full-length genome

Technical field

SS-3T-seq of the present invention (Strand-specific 3'Terminal Sequencing) relates to the construction process of a kind of chain specific mrna 3' end non-coding region high-throughput sequencing library, for studying selectivity Polyadenylation (Alternative Polyadenylation, APA) within the scope of full-length genome.

Background technology

Within the scope of full-length genome, study APA change existing technology and have multiple, but Method And Principle is all close, now lists two kinds of most representational.

Fig. 1 and Fig. 2 illustrates the experimental principle figure of these two kinds of methods:

Fig. 1 is SAPAS (Sequencing APA Sites), after being ruptured by RNA direct heating, goes out the first chain cDNA, the method synthetic double chain cDNA of strand displacement with the primer reverse transcription containing oligo dT.Then to be connected with sequence measuring joints with the oligo dT (TTTTCTTTTTTCTTTTTT) of sudden change and to increase as PCR primer, then not containing poly A (poly A) structure in the product amplified, avoid poly structure on the impact of sequencing quality with this.The shortcoming of SAPAS method thoroughly can not eliminate poly A not excise impact on high-flux sequence quality.

Fig. 2 is 3P-Seq (Poly (A) Position Profiling by sequencing), after the RNA containing poly A sequence being filtered out with oligo dT, 3' end adds the RNA joint that band vitamin H (Biotin) is modified, then after RNase T1 ruptures RNA, the fragment of poly A is contained with magnetic bead (Beads) screening containing Streptavidin, by the reverse transcription system only containing dTTP, poly A structure reverse transcription is become heterozygosis duplex again, utilize the characteristic hydrolysis poly A structure of RNase H, fragment containing APA site is released from magnetic bead, two ends add synthetic double chain cDNA after RNA joint, the library that can be used for checking order is after pcr amplification.The shortcoming of 3P-Seq method is rna level trivial operations, need to connect and enzyme be cut through the RNA of multistep, to the damage of RNA and loss also comparatively large, be not suitable for the structure in a small amount of sample library, and this method difficulty comparatively greatly, cost is higher.

In addition, the fracture mode of above two kinds of methods all has limitation, and RNase T1 enzyme cuts off the method splitting (3P-Seq) and RNA direct heating fracture (SAPAS) can not accomplish that can not damage RNA can rupture again simultaneously immediately.And a lot of genome the same area has the transcript of positive minus strand simultaneously, and the directivity information of all unknowable transcript chain of art methods, the sequence of the gained that namely checks order is from the normal chain of DNA double chain or minus strand.

Therefore those skilled in the art are devoted to exploitation one and reduce rna level operation as far as possible, and remove poly A sequence at DNA level, eliminate the poly structure method on the impact of order-checking, the method can retain again the directivity information of chain simultaneously.

Summary of the invention

The object of this invention is to provide a kind of method studying APA within the scope of chain specificity full-length genome, the method removes poly A sequence at DNA level, can retain again the directional information of chain simultaneously.

Method provided by the invention is called chain specificity 3' and holds sequencing (SS-3T-seq, Strand-specific 3'Terminal Sequencing), and the method is used for the change studying APA within the scope of full-length genome.

Said method comprising the steps of:

(1) build a kind of special magnetic bead containing oligo dT, with the RNA of this magnetic bead screening containing poly A, described magnetic bead can screen the RNA containing poly A, removes again the poly A structure in library containing restriction enzyme site simultaneously;

(2) on magnetic bead, reverse transcription synthesizes the first chain cDNA, substitutes normal dCTP in reverse transcription system with methylated dCTP;

(3) with special dNTP synthetic double chain cDNA, described special dNTP refers to and wherein replaces dTTP with dUTP, the fragment be containing APA site after fracture, magnetic bead stayed;

(4) go poly A structure through the excision of Gsu I enzyme again, object fragment is released from magnetic bead;

(5) Illumina sequence measuring joints is added in object fragment two ends after modifying;

(6) after clip size screening, with USER ferment treatment to remove the dUTP in the second chain, only residue the first chain does pcr amplification, namely obtains the library for the order-checking of Illumina bis-generation.

Preferably, before the inventive method is carried out, first carry out quality examination to the total serum IgE of extracting, the method for described quality examination can be agarose-formaldehyde denaturing electrophoretic or detect with Agilent 2100Bioanalyzer, and it is very important to Accurate Analysis APA change that high-quality RNA does parent material.

Preferably, the inventive method is applicable to the total serum IgE of 10 μ g-100 μ g as parent material.

Preferably, in step (3) synthetic double chain cDNA process in adopt RNase H, Ecoli.DNA Polymerase, Ecoli.DNA ligase tri-kinds of enzymes.

Preferably, described in step (5), two of object fragment terminal modified referring to form flat end, and add phosphate group at 5' end, hold interpolation base A at 3'; The concrete grammar of described interpolation Illumina sequence measuring joints is the object fragment after modifying be connected with Illumina P5/P7 joint, and ligase enzyme is preferably T4DNA ligase.

Preferably, the agarose gel electrophoresis isolated fragment of the screening of clip size described in step (6) employing 2.5%.

The schematic diagram of the inventive method, see Fig. 3, below further describes principle of the present invention.

(1) about the structure of special oligo dT magnetic bead

Common business oligo dT magnetic bead is for screening the RNA containing poly A, and the oligo dT magnetic bead after improving can screen the RNA containing poly A, removes the poly A structure in library containing restriction enzyme site simultaneously.

The special magnetic bead of structure of the present invention has following characteristics:

(1) the 3' end of oligo dT is modified with thiophosphoric acid between latter two T, surprisingly can not excised by exonuclease in protection APA site when later stage fragment ends is modified, retain 4 T screening for data during later data process on side, APA site simultaneously;

(2) the 5' end of oligo dT sequence adds that the enzyme of a Gsu I cuts the protection sequence of recognition site and 9 bases, in order to removing of poly A structure;

(3) the 5' end of primer adds biotin modification, in order to connect the magnetic bead with Streptavidin.

Further, the primer sequence be connected with magnetic bead is as follows:

5'-Biotin-GAGCTAGTTCTGGAGTTTTTTTTTTTTTTTTTTTTVN-3, wherein modifies with thiophosphoric acid between the 19th T and the 20th T.

(2) about the selection of fracture mode

The DNA break mode that can select comprises the method such as ultrasonic fracture, DNase I process, if but the method for ultrasonic fracture controls bad being easy to rupture whole DNA molecular from magnetic bead, DNase I reaction is acutely not easy controlled fracturing blasting product clip size.

In order to reduce the damage to RNA, the present invention preferably adopts ruptured in the DNA stage.After double-strand cDNA synthesis, double-strand cDNA fracture enzyme is used double-strand cDNA to be fragmented into the fragment of 200-400bp, after magnetic bead screening, the fragment hung on magnetic bead is near RNA 3' end and contains the fragment in poly A site, and wherein said double-strand cDNA fracture enzyme is preferably the product of NEB company.

(3) about the strategy removing poly A structure

Conveniently operate and reduce experiment difficulty, the present invention selects to remove poly A structure at DNA level.When reverse transcription, the enzyme introducing Gsu I at the 5' end of oligo dT cuts recognition site, Gsu I is a kind of 3rd type restriction enzyme, at the sticky end of the position of 16 bases in downstream of recognition site cutting formation two bases, and has one and to methylate closure property.When the base C in recognition site (CTGGAG) is methylated, then this recognition site is closed, None-identified.Also library fragments is released from magnetic bead due to while the removing of poly A structure, in order to library fragments when preventing from removing poly structure also causing the false positive in APA site containing this restriction enzyme site, the present invention uses methylated dCTP to substitute normal dCTP when reverse transcription, and gain normal dCTP when the second chain cDNA synthesizes, so just can ensure that the Gsu I restriction enzyme site of design makes other recognition sites methylated closed while intact, the generation of the false positive results avoided.

(4) TTTT label accurately locates APA site for garbled data

The present invention is designed in the primer of reversion containing 20 T, Gsu I cuts 16 when removing poly A, the sticky end leaving 4 T is cut when end modified, thus really stay on library fragments be containing thiophosphoric acid modify 4 T in order to mark the APA site of library fragments.

The present invention has following Advantageous Effects:

1, experimental implementation is simple, and rna level operation is less, simplifies experiment flow, reduces the loss that complex steps causes, and reduces experiment difficulty and cost simultaneously, makes its range of application wider;

2, DNA level removes poly A structure, improves sequencing quality, makes the qualification of APA more accurate;

3, DNA level ruptures the damage decreased compared with existing fracture mode RNA, improves productive rate;

4, add special dNTP (dUTP replaces dTTP) during two chain synthesis, last USER ferment treatment, removes the dUTP in two chains, finally only has the first chain to do PCR reaction, remains the directivity information of chain.

Accompanying drawing explanation

Fig. 1 is the schematic diagram of SAPAS method;

Fig. 2 is the schematic diagram of 3P-Seq method;

Fig. 3 is the schematic diagram of SS-3T-seq method of the present invention.

Embodiment

Below embodiments of the invention are elaborated: the present embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.

The detailed step of the present embodiment is as follows:

1. detect RNA quality from cell or after organizing extracted total RNA through agarose-formaldehyde denaturing electrophoretic or Agilent 2100Bioanalyzer.

2. the oligo dT primer cutting recognition site containing Gsu I enzyme directly synthesizes in commercial company, 5' end needs biotin modification, synthetic primer is dissolved in the solution that other 10mM Tris-HCl (pH=7) of RNA fraction is made into 100 μMs, then get 5 μ L and the magnetic bead (invitrogen) of the M280 Streptavidin of 50 μ L mix after room temperature be combined and spend the night, namely make the special magnetic bead that can be used for this experiment after being washed away by the Excess primer be not combined with magnetic bead.

3. the special magnetic bead using the 2nd step to make screens the RNA containing poly A from total serum IgE.

4. the mixed solution normal dATP, dGTP, dTTP and methylated dCTP mixing being made into 2.5mM concentration is used for reverse transcription, cuts recognition site with the Gsu I enzyme in block gene sequence.Reverse transcription system, with reference to the specification sheets of reversed transcriptive enzyme, without particular requirement, is reacted and is directly carried out on magnetic bead, without the need to by DNA fragmentation from wash-out magnetic bead.

5. wash magnetic bead with elution buffer, remove methylated dCTP.Again with special dNTP (dUTP replaces dTTP) synthetic double chain cDNA, adopt RNase H, Ecoli.DNA Polymerase, Ecoli.DNA ligase (being all purchased from NEB) three kinds of enzymes for two chains synthesis.

6. with double-strand DNA cleavage enzyme (NEB), double-strand cDNA is fragmented into the fragment of 200-400bp size, the specification sheets of method reference enzyme, stay after washing fragment on magnetic bead namely for the purpose of fragment.

7. cut with Gsu I enzyme (Fermentas) enzyme, excision poly A sequence, discharges object fragment from magnetic bead simultaneously.Enzyme cuts the specification sheets of system reference enzyme.

8. adopt end modified test kit (NEB) make object fragment two ends form flat end and 5' end add phosphate group, for the connection of Illumina sequence measuring joints.

9. hold interpolation base A, for the connection of Illumina sequence measuring joints with the object fragment 3' of Taq archaeal dna polymerase (Fermentas) after end modified.This step can be combined into a step with the 8th step and accomplish, to reduce the numerous and diverse loss caused of step, had test kit (NEB) to complete at present.

10. the object fragment after end modified be connected with Illumina P5/P7 joint, ligase enzyme selects T4DNA ligase (Fermentas), and system is with reference to specification sheets.

11. with 2.5% agarose gel electrophoresis excessive separation joint, carry out the screening of library fragments size, the library within the scope of 200-500bp is reclaimed in rubber tapping simultaneously.

Behind the library of reclaiming in 12.USER ferment treatment 11 step, adopt Q5 to surpass proofreading polymerase amplification, namely the polyacrylamide gel electrophoresis purifying by 8% can be used for the two-way order-checking of Illumina 1x50 after reclaiming.Polyacrylamide gel electrophoresis can screen clip size (200-500bp) according to Marker instruction when reclaiming purifying again, is conducive to improving sequencing quality to the accurate control of library fragments size.

More than describe preferred embodiment of the present invention in detail.Should be appreciated that those of ordinary skill in the art just design according to the present invention can make many modifications and variations without the need to creative work.Therefore, all technician in the art, all should by the determined protection domain of claims under this invention's idea on the basis of existing technology by the available technical scheme of logical analysis, reasoning, or a limited experiment.

Claims (10)

1. study a method of APA within the scope of chain specificity full-length genome, it is characterized in that, comprise the following steps:

(1) build a kind of special magnetic bead containing oligo dT, with the RNA of this magnetic bead screening containing poly A, described magnetic bead can screen the RNA containing poly A, removes again the poly A structure in library containing restriction enzyme site simultaneously;

(2) on magnetic bead, reverse transcription synthesizes the first chain cDNA, substitutes normal dCTP in reverse transcription system with methylated dCTP;

(3) with special dNTP synthetic double chain cDNA, described special dNTP refers to and wherein replaces dTTP with dUTP, the fragment be containing APA site after fracture, magnetic bead stayed;

(4) go poly A structure through the excision of Gsu I enzyme again, object fragment is released from magnetic bead;

(5) Illumina sequence measuring joints is added in object fragment two ends after modifying;

(6) after clip size screening, with USER ferment treatment to remove the dUTP in the second chain, only residue the first chain does pcr amplification, namely obtains the library for the order-checking of Illumina bis-generation.

2. study the method for APA within the scope of chain specificity full-length genome as claimed in claim 1, it is characterized in that, the special magnetic bead built described in step (1) has following characteristics:

(1) the 3' end of oligo dT is modified with thiophosphoric acid between latter two T, surprisingly can not excised by exonuclease in protection APA site when later stage fragment ends is modified, retain 4 T screening for data during later data process on side, APA site simultaneously;

(2) the 5' end of oligo dT sequence adds that the enzyme of a Gsu I cuts the protection sequence of recognition site and 9 bases, in order to removing of poly A structure;

(3) the 5' end of primer adds biotin modification, in order to connect the magnetic bead with Streptavidin.

3. study the method for APA within the scope of chain specificity full-length genome as claimed in claim 2, it is characterized in that, the primer sequence be connected with the special magnetic bead of described structure is:

5'-Biotin-GAGCTAGTTCTGGAGTTTTTTTTTTTTTTTTTT tTvN-3, wherein modifies with thiophosphoric acid between the 19th T and the 20th T.

4. the method for research APA in chain specificity full-length genome as claimed in claim 1, is characterized in that, the method for fracture described in step (3) be ultrasonic, with DNase I process or with double-strand cDNA fracture ferment treatment.

5. within the scope of chain specificity full-length genome as claimed in claim 1, study the method for APA, it is characterized in that, described in step (5), two of object fragment terminal modified referring to form flat end, and add phosphate group at 5' end, hold interpolation base A at 3'; The concrete grammar of described interpolation Illumina sequence measuring joints is the object fragment after modifying be connected with Illumina P5/P7 joint.

6. study the method for APA within the scope of chain specificity full-length genome as claimed in claim 1, it is characterized in that, the agarose gel electrophoresis isolated fragment of the screening of clip size described in step (6) employing 2.5%.

7. study the method for APA within the scope of chain specificity full-length genome as claimed in claim 1, it is characterized in that, before described method is carried out, first quality examination is carried out to the total serum IgE of extracting.

8. study the method for APA within the scope of chain specificity full-length genome as claimed in claim 7, it is characterized in that, the method for described quality examination is agarose-formaldehyde denaturing electrophoretic or detects with Agilent 2100Bioanalyzer.

9. study the method for APA within the scope of chain specificity full-length genome as claimed in claim 1, it is characterized in that, described method is applicable to the total serum IgE of 10 μ g-100 μ g as parent material.

10., for the magnetic bead containing oligo dT that APA within the scope of full-length genome studies, it is characterized in that there is following characteristics:

(1) the 3' end of oligo dT is modified with thiophosphoric acid between latter two T, surprisingly can not excised by exonuclease in protection APA site when later stage fragment ends is modified, retain 4 T screening for data during later data process on side, APA site simultaneously;

(2) the 5' end of oligo dT sequence adds that the enzyme of a Gsu I cuts the protection sequence of recognition site and 9 bases, in order to removing of poly A structure;

(3) the 5' end of primer adds biotin modification, in order to connect the magnetic bead with Streptavidin.