CN106754870A - A kind of method for building Multi-example total length transcript profile mixing library - Google Patents
A kind of method for building Multi-example total length transcript profile mixing library Download PDFInfo
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Abstract
The present invention relates to a kind of method for building Multi-example total length transcript profile mixing library, it carries out reverse transcription to the mRNA in different samples respectively in process of reverse-transcription using with the primer for differentiating label, synthesis cDNA, then the cDNA from different samples mixed and process obtain the library.The method of the present invention in reverse transcriptase primer by adding one section of special sequence label, so that distinctive label on reverse transcription product band, after to different samples using primer reverse transcription and subsequent PCR amplification with different sequence labels, all can differentiate which sample reverse transcription product and PCR primer initially come from by the label, so as to realize that biased sample builds storehouse.
Description
Technical field
The present invention relates to technical field of molecular biology, more specifically it relates to a kind of for building the transcription of Multi-example total length
The method in group mixing library.
Background technology
Transcript profile sequencing (Transcr iptomesequencing) mainly by high-flux sequence research particular organization or
Cell delayed early transcription mRNA out at certain.Transcript profile sequencing can rapidly obtain a certain species specific cells or tissue comprehensively
Almost all of transcript and gene order under a certain state, can be used for research gene structure and gene function, variable
Montage and the prediction of new transcript etc..
Traditional two generation transcript profiles sequencing when building storehouse due to the characteristics of it reads length, needing into Break Row, it is impossible to directly obtain
Obtain full sequence of the single rna molecule by the ˊ of 5 ˊ to 3.Total length transcript profile sequencing based on PacBio three generations's microarray datasets, without beating
It is disconnected, the full-length cDNA of reverse transcription can be directly read, the full sequence of high-quality single rna molecule can be effectively obtained,
In the generation of accurate discrimination two, is sequenced homology isomer (isoform), homologous gene, superfamily gene or the allele table of None- identified
The transcript for reaching.
Multi-example mixing Library development flow is not supported in existing total length transcription establishment storehouse at present, even if sample mixing builds storehouse, only yet
Only it is that sample is admixed together, sequencing data can not be distinguished effectively.In two generation transcript profile sequencing technologies, can be
Carry out Multi-example mixing and build storehouse, but because the total length transcription of three generations's sequencing is set up storehouse and need not be interrupted, according to two generations
The method addition sequence label for building storehouse can be very cumbersome, accordingly, it would be desirable to a kind of new structure Multi-example total length transcript profile mixing text
The method in storehouse.
The content of the invention
It is to solve problem above, the invention provides a kind of method for building Multi-example total length transcript profile mixing library,
Reverse transcription is carried out to the mRNA in different samples respectively using with the primer for differentiating label in process of reverse-transcription, synthesizes cDNA,
Then the cDNA from different samples is mixed and process and obtain the library.The method of the present invention is by inverse
One section of special sequence label is added in transcription primers so that distinctive label on reverse transcription product band, made to different samples
After with primer reverse transcription and subsequent PCR amplification with different sequence labels, can all differentiate that reverse transcription is produced by the label
Which sample thing and PCR primer initially come from, so as to realize that biased sample builds storehouse.
Preferably, it is described to include with lower curtate by 5 ' -3 ' order with the primer for differentiating label：Amplification region sequence, mark
Region sequence and Poly (A) grappling region sequence are signed, Poly (A) the grapplings region sequence includes multiple thymidines, the label region sequence
To distinguish the specific sequence of the cDNA from different samples, the amplification region sequence is the sequence for PCR amplifications.Wherein expand
Increasing region sequence is used for the grappling of the amplimer when the laggard performing PCR of reverse transcription is expanded, and Poly (A) grapplings region sequence is used for grappling
Poly (A) on mRNA, label region sequence is used to distinguish the source of sample.
Preferably, the 3 ' ends with the primer for differentiating label also include nucleotides V and N successively, wherein, V is represented
A, C or G, the N represent A, T, C or G, and the primer with discriminating label is all sequences of the condition for meeting V and N
Mixture.V and N is added by the end of primer 3 ' so that the initiation site of reverse transcription is located at the 5 ' of the Poly (A) of mRNA
End, rather than Poly (A) middle parts or 3 ' ends, reduces the inefficient length of reverse transcription product.
Preferably, the length of the label region sequence is 10-18nt, and label region sequence needs suitable length to keep
Its specificity each other, and the specificity between genome sequence.
Specifically, the described method comprises the following steps：
S1：Reverse transcription is carried out using the other mRNA in different samples of the primer with different labels, synthesizes cDNA；
S2：Obtain the cDNA mixed in equal amounts from each sample；
S3：Fragment is screened by clip size；
S4：PCR expands screened fragment；
S5：The fragment expanded to S4 adds joint；
S6：The fragment obtained with exonuclease digestion S5, purifying DNA fragment is to obtain the library after digestion.
Further, the size of the fragment of S3 screenings is 0.5-10KB.
Preferably, the total amount of mixing cDNA used is 0.1-10 μ g when S4PCR is expanded, and the setting of the cDNA total amounts is helped
Period during by PCR is limited in zone of reasonableness.
Preferably, it is further comprising the steps of before S5 addition sequence measuring joints after S4PCR amplifications：
S7：DNA damage is repaired；
S8：Repair end.
Preferably, S9 is also included after S6：Library quality inspection, determines library size.
The present invention sets up storehouse based on the transcription of three generations's total length need not enter the feature of Break Row, the first step started in experiment-complete
Sequence label is introduced in the amplification of cDNA long, sample mixing can be carried out after adding sequence label, effectively simplify operating process, saved
About reagent consumptive material consumption, reduces experimental cost, and realizes and multiple samples mixing are carried out in same library build storehouse
Flow.
Brief description of the drawings
Fig. 1 is the flow chart of the method for building Multi-example transcript profile mixing library；
Fig. 2 is the schematic diagram with the primer for differentiating sequence label；
Fig. 3 is the detection figure of Agilent 2100 after single sample PCR amplifications；
Fig. 4 is the detection figure of Agilent 2100 of PCR amplifications after the screening of biased sample fragment.
Specific embodiment
Principle of the invention and feature are described below in conjunction with example, example is served only for explaining the present invention, and
It is non-for limiting the scope of the present invention.
6 tissue-root, woody stems, bark, leaf, flower, the fruits different to plant mix after extracting total serum IgE respectively
Library construction, sets up three libraries, and 1-1.5kb, 1.5-2kb, 2-6kb distinguish 1 cell of each sequencing using PACBIO RSII,
Flow chart is as shown in Figure 1.Specific experiment process is as follows：
1. the cDNA of tape label is synthesized
The operation of this step is carried out in the super-clean bench without RNase, and different tissues sample separates individually operated, and each sample pair
The label answered is the structural representation of unique reverse transcriptase primer used without repetition, this experiment as shown in Fig. 2 particular sequence
As shown in table 1.
The primer of the tape label that the reverse transcription of table 1 is used
1) PCR pipe of 6 0.2mL is placed on ice, adds following reagent：The μ g of total serum IgE 1 of sample 1-6 (press concentration conversion
Volume), the μ L of primer (12 μM, BC1-BC6) 1 of tape label, nuclease free water be supplemented to 4.5 μ L.
2) soft vortex is mixed, and brief centrifugation is placed in PCR instrument, runs following program：72 DEG C of 3min, 42 DEG C of 2min, 72
DEG C it is set to 0.1 DEG C/s to 42 DEG C of rate of temperature fall；
3) after above-mentioned reaction terminates, product is added in the reverse transcription reaction system shown in table 2, soft vortex is mixed
Even, brief centrifugation runs following program in PCR instrument：42 DEG C of 90min, 70 DEG C of 10min；
The reverse transcription reaction system of table 2
4) the PCR amplifications of monocyte sample
New 1.5mL EP pipes are taken, PCR system is prepared by table 3, fully mixed, brief centrifugation, PCR programs are：98℃
2min；98 DEG C of 20s, 65 DEG C of 30s, 72 DEG C of 3.5min, 15 circulations；72℃5min.According to AMPure PB magnetic beads after the completion of reaction
Operating instruction is purified using the magnetic bead of 1 times of volume, is eventually adding the nuclease free water elution of 11 μ L；1 μ L purified products are taken,
Using nuclease free water dilute 10 times, take 2 μ L dilutions carry out Qubit quantify, determine the concentration and total amount of PCR primer；Take 1 μ L
Dilution carries out Agilent 2100 and detects, determines the size distribution of fragment.As shown in figure 3, the Agilent 2100 of all PCR primers
Detection fragment distribution is very consistent, and, by 0.7K-5K, its main peak is in 1.8K for fragment size distribution.
The monocyte sample PCR amplification system of table 3
2. equivalent sample mixing
According to Qubit quantitative results, the PCR primer that each sample respectively takes 500ng carries out sample mixing, the PCR primer after sample mixing
Total amount is 3 μ g.
3.BluePippin fragments are screened
Operating instruction according to Bluepippin carries out the screening of purpose fragment, and the segment ranges of this screening are 1-
1.5KB、1.5-2KB、2-6KB；Entered using the magnetic bead of 1 times of volume according to the operating instruction of AMPure PB magnetic beads after the completion of screening
Row purifying, is eventually adding the nuclease free water elution of 20 μ L.
4. the PCR amplifications of biased sample are amplified
PCR system, 2 are prepared by table 4) fully mix, brief centrifugation carries out following PCR programs：98℃2min；98℃
20s, 65 DEG C of 30s, 72 DEG C of Zmin, X circulations；72℃5min.Wherein, the extension of time of 1-2K is 2min, 6 circulations；2-3K's prolongs
The time is stretched for 3min, 8 circulations；The extension of time of 3-6K is 5min, 8 circulations.1 times of AMPure of volume is used after the completion of reaction
PB magnetic beads are purified, and are finally eluted using the nuclease free water of 37uL；Expanded after detecting screening using Agilent 2100
PCR primer distribution of lengths, as a result as shown in figure 4, its first peak be amplification 1-1.5K PCR primer, second peak is
The PCR primer of the 1.5-2K of amplification, the 3rd peak is the PCR primer of the 2-6K for expanding, and shows amplified fragments size with screening model
Enclose consistent.
The biased sample PCR system of table 4
5.DNA injury repairs
DNA damage being prepared by table 5 and repairing system, fully mixed, brief centrifugation, 37 DEG C of incubation 20min are placed on ice.
Table 5DNA injury repair systems
6. end is repaired
DNA damage is prepared by table 6 and repair system, fully mix, brief centrifugation, 25 DEG C of incubation 5min；Use 1 times of volume
AMPure PB magnetic beads are purified, and are finally eluted using the nuclease free water of 31uL.
Repair system in the end of table 6
7. jointing
Connector interfaces system is prepared by table 7, is fully mixed, brief centrifugation, 25 DEG C of incubation 15min, 65 DEG C are incubated 10min and make
Ligase is inactivated.
The connector interfaces system of table 7
8. exonuclease digestion
Exonuclease digestion system is prepared by table 8, is fully mixed, brief centrifugation, 37 DEG C of incubation 1h；Incubation makes after terminating
Purified with the AMPure PB magnetic beads of 1 times of volume, finally use the nuclease free water elution of 11 μ L.
The exonuclease digestion system of table 8
9. library quality inspection
Take 1 μ L libraries, dilute 5 times, take 2 μ L dilutions and carry out Qubit and quantify, calculate library concentration；1 μ L dilutions are taken to enter
Row 2100 detects, determines library size.
Machine sequencing on 10
According to the result of library quality inspection, according to the operating instruction of PacBio sequencings to 1-1.5KB, 1.5-2KB, 2-6KB
Sample mixing library carries out machine sequencing respectively, and three sample mixing libraries are respectively sequenced 1 chip；
Sequencing result is analyzed, the total length transcript identification proportional difference error of sample mixing library difference barcode exists
Within ± 30%.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all it is of the invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc. should be included within the scope of the present invention.
Claims (9)
1. a kind of method for building Multi-example total length transcript profile mixing library, it is characterised in that using band in process of reverse-transcription
Have the primer for differentiating label carries out reverse transcription to the mRNA in different samples respectively, synthesizes cDNA, then will be described from difference
CDNA in sample mixes and process and obtains the library.
2. method according to claim 1, it is characterised in that described with differentiating the order of the primer of label by 5 ' -3 '
Including with lower curtate：Amplification region sequence, label region sequence and Poly (A) grappling region sequence, Poly (A) the grappling region sequence bag
Multiple thymidines are included, the label region sequence is the specific sequence for distinguishing the cDNA from different samples, the amplification region sequence
It is the sequence for PCR amplifications.
3. method according to claim 2, it is characterised in that the 3 ' ends with the primer for differentiating label are also successively
Comprising nucleotides V and N, wherein, V represents A, C or G, and the N represents A, T, C or G, and described with the primer for differentiating label
To meet the mixture of all sequences of the condition of V and N.
4. method according to claim 2, it is characterised in that the length of the label region sequence is 10-18nt.
5. method according to claim 2, it is characterised in that comprise the following steps：
S1：Reverse transcription is carried out to the mRNA in different samples respectively with the primer for differentiating label using described, synthesizes cDNA；
S2：Fetch the cDNA mixed in equal amounts from each sample；
S3：Fragment is screened by clip size；
S4：PCR expands screened fragment；
S5：The fragment expanded to S4 adds joint；
S6：The fragment obtained with exonuclease digestion S5, purifying DNA fragment is to obtain the library after digestion.
6. method according to claim 5, it is characterised in that the size of the fragment of S3 screenings is 0.5-10KB.
7. method according to claim 5, it is characterised in that the total amount of mixing cDNA S4PCR used when expanding is
0.1-10μg。
8. method according to claim 5, it is characterised in that after S4PCR amplifications, before S5 addition sequence measuring joints, also
Comprise the following steps：
S7：DNA damage is repaired；
S8：Repair end.
9. the method according to any one of claim 5-8, it is characterised in that also include S9 after S6：Library quality inspection, really
Determine library size.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109056077A (en) * | 2018-09-13 | 2018-12-21 | 武汉菲沙基因信息有限公司 | A kind of amplicon sample mixing sequencing library construction method suitable for PacBio microarray dataset |
| CN109971849A (en) * | 2017-12-28 | 2019-07-05 | 安诺优达基因科技(北京)有限公司 | The unicellular multisample mixing banking process of liver cancer related gene |
| CN110396516A (en) * | 2018-04-25 | 2019-11-01 | 武汉康测科技有限公司 | A kind of absolute quantitation transcript profile library constructing method based on peculiar identification sequence |
| CN111440845A (en) * | 2020-04-09 | 2020-07-24 | 嘉兴菲沙基因信息有限公司 | Prokaryotic full-length initial transcript library building method suitable for PacBio sequencing platform and application |
| CN111455023A (en) * | 2020-04-09 | 2020-07-28 | 武汉菲沙基因信息有限公司 | Full-length amplicon rapid library construction method, primer and sequencing method suitable for PacBio platform |
| CN111549099A (en) * | 2020-04-23 | 2020-08-18 | 广州再生医学与健康广东省实验室 | Third-generation sequencing-based single-cell transcriptome sequencing method |
| WO2020164015A1 (en) * | 2019-02-13 | 2020-08-20 | 武汉华大医学检验所有限公司 | Fusion primer for third-generation sequencing library construction, and library construction method, sequencing method and library construction kit therefor |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109971849A (en) * | 2017-12-28 | 2019-07-05 | 安诺优达基因科技(北京)有限公司 | The unicellular multisample mixing banking process of liver cancer related gene |
| CN110396516A (en) * | 2018-04-25 | 2019-11-01 | 武汉康测科技有限公司 | A kind of absolute quantitation transcript profile library constructing method based on peculiar identification sequence |
| CN110396516B (en) * | 2018-04-25 | 2021-10-22 | 武汉康测科技有限公司 | Absolute quantitative transcriptome library construction method based on unique recognition sequence |
| CN109056077A (en) * | 2018-09-13 | 2018-12-21 | 武汉菲沙基因信息有限公司 | A kind of amplicon sample mixing sequencing library construction method suitable for PacBio microarray dataset |
| WO2020164015A1 (en) * | 2019-02-13 | 2020-08-20 | 武汉华大医学检验所有限公司 | Fusion primer for third-generation sequencing library construction, and library construction method, sequencing method and library construction kit therefor |
| CN113166756A (en) * | 2019-02-13 | 2021-07-23 | 武汉华大医学检验所有限公司 | Fusion primer for third-generation sequencing and library building, library building method, sequencing method and library building kit |
| CN113166756B (en) * | 2019-02-13 | 2023-10-13 | 武汉华大医学检验所有限公司 | Fusion primer for three-generation sequencing library construction, library construction method, sequencing method and library construction kit |
| CN111440845A (en) * | 2020-04-09 | 2020-07-24 | 嘉兴菲沙基因信息有限公司 | Prokaryotic full-length initial transcript library building method suitable for PacBio sequencing platform and application |
| CN111455023A (en) * | 2020-04-09 | 2020-07-28 | 武汉菲沙基因信息有限公司 | Full-length amplicon rapid library construction method, primer and sequencing method suitable for PacBio platform |
| CN111549099A (en) * | 2020-04-23 | 2020-08-18 | 广州再生医学与健康广东省实验室 | Third-generation sequencing-based single-cell transcriptome sequencing method |
| CN111549099B (en) * | 2020-04-23 | 2021-07-30 | 生物岛实验室 | Third-generation sequencing-based single-cell transcriptome sequencing method |
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