CN108251515A - A kind of method for building FFPE sample DNAs library - Google Patents
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Abstract
The present invention relates to a kind of methods for building FFPE sample DNAs library, and in general, the present invention improves the construction method in FFPE sample DNAs library, and end reparation, purifying are carried out to low quality FFPE sample DNAs；3' ends add A；Adjunction head, purifying and PCR amplification, purifying.Wherein, adjunction head after reaction, is purified using 0.9 × magnetic bead.The present invention reduces the loss of DNA fragmentation by the optimization to building library step, even if remaining able to obtain the library yield that enough high-flux sequences use using low quality FFPE sample DNAs structure library, makes it possible research to low quality FFPE samples.
Description
Technical field
The invention belongs to biology fields, and in particular to a kind of method for building FFPE sample DNAs library.
Background technology
Formalin fixes paraffin embedding (Formalin-fixed and Paraffin-embedded, FFPE) processing side
Method can preserve tissue or prepare the tissue specimen needed for examining for a long time, so in clinical pathology inspection, oncogene
It is often used during detection and medical scientific, is the material source of a reliable molecular biology research.In the world
In the range of, about billions of parts of tissue samples are stored in hospital or tissue sample library.Wherein the overwhelming majority is to use good fortune
Your Malin fixes the sample of the method processing of paraffin embedding.FFPE samples have typically represented biomedicine that is precious and deriving from a wealth of sources
Research material, the filing FFPE samples of enormous amount are retrospective study, illustrate disease mechanisms, find therapeutic targets and indicate pre-
Valuable resource is provided afterwards etc..
However, the special production method of FFPE samples and preserving process and all causing various shadows to the nucleic acid in sample
It rings.Degradation takes place in tissue medicine sample after in vitro, and the fixation of formalin makes nucleic acid and nucleic acid or core in tissue
It is crosslinked between acid and albumen, the infiltration process of paraffin further speeds up the degradation of nucleic acid, and holding time and environment are in sample
Nucleic acid also has significant effect, these above-mentioned reasons eventually lead to the Nucleic acid quality obtained by sample and decline to a great extent.
With the development of two generation sequencing technologies, sample sequencing speed is greatly increased, reduces sequencing cost so that should
Sequencing is carried out to FFPE sample DNAs with two generation sequencing approaches to be possibly realized.In recent years, using two generation sequencing approaches to FFPE samples
What DNA was sequenced has a extensive future, and the banking process for being increasingly being applied to the FFPE sample DNAs of two generations sequencing is met the tendency of
And it gives birth to.The FFPE sample banking process of common FFPE samples banking process such as Illumina companies, mainly comprises the following steps：(1)
The sample having no progeny of fighting each other carries out end reparation, using 1.8 × magnetic beads for purifying, obtains flat terminal DNA fragments；(2) to flat end DNA
Segment carries out plus A, using 1.8 × magnetic beads for purifying, obtains adding A products；(3) pair plus A products carry out adjunction head, use 1.8 × magnetic
Pearl purifies, and obtains adjunction head product；(4) PCR is carried out to adjunction head product, using 0.9 × magnetic beads for purifying, obtains library.
The usual integralities of DNA are very poor in serious FFPE samples of degrading, and there are many small fragments, and library is built using existing
The usual homogeneity in library that method obtains is poor (DNA fragmentation difference in size causes), library low output, it is impossible to reach follow-up study
Demand.Existing banking process can be used for common FFPE samples are carried out building library, for serious low quality FFPE samples of degrading
This DNA, which builds library, can not then obtain the library yield of enough follow-up studies.The usual total amount of medical sample of paraffin embedding is all very limited,
Each sample is very precious due to its irreplaceability, so how to be established by the DNA that low-quality FFPE samples obtain
Qualified library has become sequencing research field urgent problem to be solved.
Invention content
In view of above-mentioned problems of the prior art, the purpose of the present invention is to provide a kind of structure FFPE sample DNAs
The method in library carries out FFPE sample DNAs end reparation, purifying；Add A, adjunction head, purifying and PCR amplification, purifying.Its
In, adjunction head reaction after after, use 0.9 × magnetic beads for purifying.By the method for above-mentioned optimization, low quality FFPE samples can be made
The library yield of sequencing research follow-up enough that this DNA is obtained ensures the output in library.
That is, the present invention includes：
1. a kind of method for building FFPE sample DNAs library, including：
Step A：The DNA of FFPE samples is extracted, obtains sample genomic dna segment；
Step B：Sample genomic dna segment is subjected to end reparation, purifying obtains flat terminal DNA fragments；
Step C：Flat terminal DNA fragments are subjected to 3' ends and add A, obtain the DNA fragmentation that 3' ends add A；
Step D：It will add 3' ends that the DNA fragmentation of A is added to carry out adjunction head, purifying obtains adjunction head DNA fragmentation；
Step E：The DNA fragmentation of adjunction head is subjected to PCR amplification, purifying obtains DNA fragmentation library,
Wherein, the purifying in step D uses 0.9 × magnetic beads for purifying.
2. according to the method described in item 1, wherein, purifying in step B is using 1.8 × magnetic beads for purifying, the purifying in step E
Using 0.9 × magnetic beads for purifying.
3. according to the method described in item 1, the FFPE samples are low quality FFPE samples.
4. according to the method described in item 1, wherein, the amount of sample DNA segment is 1 μ g in the step A.
5. according to the method described in item 4, wherein, in the step C reactions plus A uses Klenow segments (3'-5'exo-),
The usage amount of the Klenow segments is 1~2 μ L, it is preferred to use is measured as 1.5~2 μ L.
6. according to the method described in item 4, wherein, adjunction head uses T4DNA ligases in the step D, and the T4DNA connects
The usage amount for connecing enzyme is 6~7.5 μ L, the amount of being preferably used is 6.5~7 μ L, and the connector usage amount is 40~100pmol, excellent
Selection of land connector usage amount is 40~80pmol.
7. one kind builds library kit, wherein, the kit includes：Reagent for end reparation, the examination for adding A
Agent, the reagent for adjunction head, the reagent for PCR amplification and the reagent for purifying.
8. a kind of sequencing DNA library is built by the method described in any one of item 1~6.
9. a kind of sequencing approach of DNA library, uses library according to any one of claims 8 to be sequenced for object.
Beneficial effects of the present invention：The present invention provides a kind of be applicable in by optimizing FFPE sample DNA library constructing methods
In the method that low quality FFPE sample DNAs build library, it ensure that the output for building library text library, provide safeguard for follow-up study, make low-quality
The research of amount FFPE sample DNAs is possibly realized.
Specific embodiment
With reference to embodiments, the present invention will be described in further detail.It should be appreciated that specific reality described herein
Example is applied only to explain the present invention, is not intended to limit the present invention.
On the one hand, the invention discloses a kind of method (method of the invention) for building FFPE sample DNAs libraries, including：
Step A：The DNA of FFPE samples is extracted, obtains sample genomic dna segment；
Step B：Sample genomic dna segment is subjected to end reparation, purifying obtains flat terminal DNA fragments；
Step C：Flat terminal DNA fragments are subjected to 3' ends and add A, obtain the DNA fragmentation that 3' ends add A；
Step D：It will add 3' ends that the DNA fragmentation of A is added to carry out adjunction head, purifying obtains adjunction head DNA fragmentation；
Step E：The DNA fragmentation of adjunction head is subjected to PCR amplification, purifying obtains DNA fragmentation library,
Wherein, the purifying in step D uses 0.9 × magnetic beads for purifying.
The FFPE samples include conventional FFPE samples and low-quality FFPE samples.In the present invention, low quality is
Refer to the DNA in FFPE samples and serious degradation occurs, integrality is destroyed and the situation of serious fragmentation.Inventor has found
In low-quality FFPE samples, DNA fragmentation size distribution is inhomogenous, can there are many small fragment (being less than 100bp), inventors
It was found that these small fragments are the key that influence library output.By the present invention to building the optimization of library step, text can be significantly improved
The yield in library.
The specific method of DNA fragmentation to extracting FFPE samples is not particularly limited, and those skilled in the art may be used
The method of generally use, for example, full recycling total nucleic acid separating kit (the Recover All Total of Ambion companies
Nucleic Acid Isolation Kit), QIAGEN companiesDNA FFPE Tissue kits or
GeneRead DNA FFPE Kit kits etc..
The amount of sample DNA segment is the μ g of 500ng~1 in step B, it is preferable that is desired with building the sample DNA segment in library
It measures as 1 μ g.
3' ends add the reaction reagent of A to include in step C：DATP (1mM), Klenow segments (3'-5'exo-), buffer solution,
And flat terminal DNA fragments.Wherein, the usage amount of Klenow segments (3'-5'exo-) is 1~2 μ L.Preferred Klenow segments
The usage amount of (3'-5'exo-) is 1.5~2 μ L.
The reaction reagent of adjunction head includes in step D：Buffer solution, T4DNA ligases, connector and 3' ends add the DNA of A
Segment, wherein, the usage amount of T4DNA ligases is 6~7.5 μ L, 40~100pmol of usage amount of connector, it is preferable that T4DNA
The usage amount of ligase is 6.5~7 μ L, and the usage amount of connector is 40~80pmol.Herein, butt joint (Adapter) does not have
It is specifically limited, it may be used to realize this hair as long as the connector of PCR amplification step that the sequencing of two generations is built during library can be used for
It is bright.
Step E may be used any using PCR method well known by persons skilled in the art and purification process progress.
In another aspect, the present invention, which provides one kind, builds library kit, including：The reagent repaired for end, for adding A's
Reagent, the reagent for adjunction head, the reagent for PCR amplification and the reagent for purifying, wherein,
The reagent repaired for end mainly includes：Klenow segments, T4 polynueleotide kinases, T4DNA polymerases.
Reagent for adding A mainly includes：DATP, Klenow segment (3'-5'exo-).
It is mainly included for the reagent packet of adjunction head：T4DNA ligases, connector.
Mainly include for the reagent of PCR amplification：Amplimer, archaeal dna polymerase.
Mainly include for the reagent of purifying：1.8 × magnetic bead, 0.9 × magnetic bead, EB solution.
The sequencing of the present invention with DNA library may be used the method in structure FFPE sample DNAs library of the invention come
Structure.
Finally, the present invention provides a kind of sequencing approach (sequencing approach of the invention), wherein, it is used with the sequencing of the present invention
DNA library is sequenced as object.
Other than using the sequencing of the present invention by the use of DNA library as object, this skill may be used in sequencing approach of the invention
The conventional method in art field carries out.Preferably, sequencing approach of the invention may be used both-end and be sequenced, such as using
The sequencing that Illumina platforms (such as HiSeq2500 or NextSeq500) carry out.
Embodiment 1
1.FFPE sample DNAs extract
Three parts of different cancerous tissue FFPE samples for preserving 3 years is taken (to be named as：Sample 1, sample 2, sample 3), it uses
Method and reagent the extraction DNA that GeneRead DNA FFPE Kit kits (Qiagen companies) provide, obtain FFPE samples
Genomic DNA.
2.FFPE sample genomic dnas interrupt
Instrument is interrupted into Break Row using Biorupter, and setting interrupts 30 cycles of condition, and 30s ON/30s OFF will
The segment that FFPE sample DNAs are broken into, the DNA fragmentation after being interrupted.
3. end is repaired
It prepares end and repairs mixture：Reagent needed for being taken out from the kit of -20 DEG C of preservations in advance, places it on ice
It thaws and abundant mixing, single sample amount of preparation is referring to table 1.
Repair reaction system in 1 end of table
Repair reaction in end：Reaction system is placed in Thermomixer 20 DEG C of warm bath 30 minutes.It uses after reaction
DNA in 1.8 × magnetic bead recovery purifying reaction system, 20 μ L EB dissolve.
4.3' ends add " A " (A-Tailing)
It prepares 3' ends and adds " A " mixed liquor：Reagent needed for being taken out from the kit of -20 DEG C of preservations in advance, places it in ice
On thaw and abundant mixing, single sample amount of preparation is referring to table 2.
2 end of table adds " A " reaction system
3' ends add " A " to react：Sample is placed in Thermomixer 37 DEG C of warm bath 30 minutes.
The connection of 5.Adapter
Prepare the connection mixture of Adapter (connector)：Reagent needed for being taken out from the kit of -20 DEG C of preservations in advance,
It places it in and thaws on ice and abundant mixing.Single sample amount of preparation is referring to table 3.
The coupled reaction system of 3 Adapter of table
The coupled reaction of Adapter：Sample is placed in Thermomixer 20 DEG C of warm bath 15 minutes.Use 0.9 × magnetic bead
DNA in recovery purifying reaction system is dissolved in 30 μ L EB solution.
6.PCR reacts
PCR amplification is carried out using HiFi DNA Polymerase Mix kits, single sample amount of preparation is referring to table 4.
4 PCR reaction systems of table
Ann index-41 sequences (SEQ ID NO:1)：
5'-CAAGCAGAAGACGGCATACGAGATGTTGCAACGTGACTGGAGTTCAGA
CGTGTGCTCTTCCGATCT-3'；
Ann index-42 sequences (SEQ ID NO:2)：
5'-CAAGCAGAAGACGGCATACGAGATCTCAATTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
-3'；
Ann index-43 sequences (SEQ ID NO:3)：
5'-CAAGCAGAAGACGGCATACGAGATCAAGTCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
-3'；
Ann consensus primers (SEQ ID NO:4)：
5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'
PCR reaction conditions are as follows：
PCR product in 0.9 × magnetic bead recovery purifying reaction system is dissolved in 30 μ L EB solution.
7. library quantifies
2100Bioanalyzer (Agilent)/LabChip GX (Caliper) detections, the production of detection library are carried out to library
Amount, the results are shown in Table 5, and the library yield that the method for sample 1, sample 2 and sample 3 through this embodiment obtains is respectively
0.345 μ g, 0.374 μ g and 0.397 μ g.
Comparative example 1
It (is respectively designated as using three samples in embodiment 1：3) control 1, control 2, control are built using existing FFPE
Library method carries out library construction, and obtained library yield is as shown in table 5.
Table 5 the results show that the library yield of sample 1 uses the side of comparative example 1 for 0.345 μ g in the method for embodiment 1
The library yield for the control 1 (sample 1) that method obtains is 0.197 μ g；The library yield of the sample 2 of embodiment 1 is 0.374 μ g, is made
The library yield that 2 (samples 2) of control are obtained with the method for comparative example 1 is 0.234 μ g；The 3 library yield of sample of embodiment 1 is
0.379 μ g, the library yield of control 3 (sample 3) obtained using the method for comparative example 1 are 0.245 μ g.In summary, pass through
The library that the method for the present invention obtains is significantly increased in the yield of library.The library structure of FFPE sample DNAs provided by the invention
Construction method even if being low-quality in FFPE samples, can obtain the library (example of the yield of sequencing research follow-up enough
Such as, the minimum library amount that capture sequencing needs is 250 μ g).
5 library output situation of table
The preferred embodiment of the present invention has shown and described in above description, as previously described, it should be understood that the present invention is not office
Be limited to form disclosed herein, be not to be taken as the exclusion to other embodiment, and available for various other combinations, modification and
Environment, and can be changed in the scope of the invention is set forth herein by the above teachings or related fields of technology or knowledge
It is dynamic.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of the present invention, then it all should be appended by the present invention
In scope of the claims.
Sequence table
<110>It is excellent up to Gene science to pacify promise（Beijing）Co., Ltd
<120>A kind of method for building FFPE sample DNAs library
<130> 1605-3TGCN
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 66
<212> DNA
<213>Artificial sequence
<400>Ann index-41 sequences
CAAGCAGAAGACGGCATACGAGATGTTGCAACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT 66
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400>Ann index-42 sequences
CAAGCAGAAGACGGCATACGAGATCTCAATTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT 66
<210> 3
<211> 66
<212> DNA
<213>Artificial sequence
<400>Ann index-43 sequences
CAAGCAGAAGACGGCATACGAGATCAAGTCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT 66
<210> 4
<211> 58
<212> DNA
<213>Artificial sequence
<400>Ann consensus primers
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT 58
Claims (9)
1. a kind of method for building FFPE sample DNAs library, including：
Step A：The DNA of FFPE samples is extracted, obtains sample genomic dna segment；
Step B：Sample genomic dna segment is subjected to end reparation, purifying obtains flat terminal DNA fragments；
Step C：Flat terminal DNA fragments are subjected to 3' ends and add A, obtain the DNA fragmentation that 3' ends add A；
Step D：It will add 3' ends that the DNA fragmentation of A is added to carry out adjunction head, purifying obtains adjunction head DNA fragmentation；
Step E：Adjunction head DNA fragmentation is subjected to PCR amplification, purifying obtains DNA fragmentation library,
Wherein, the purifying in step D uses 0.9 × magnetic beads for purifying.
2. according to the method described in claim 1, it is characterized in that, the purifying in step B is using 1.8 × magnetic beads for purifying, step E
In purifying use 0.9 × magnetic beads for purifying.
3. according to the method described in claim 1, it is characterized in that, the FFPE samples are low quality FFPE samples.
4. according to the method described in claim 1, it is characterized in that, the amount of sample DNA segment is 1 μ g in the step A.
5. according to the method described in claim 4, it is characterized in that, A is added to use Klenow segments (3'- in step C reactions
5'exo-), the usage amount of the Klenow segments is 1~2 μ L, it is preferred to use is measured as 1.5~2 μ L.
6. according to the method described in claim 4, it is characterized in that, adjunction head uses T4DNA ligases, institute in the step D
The usage amount for stating T4DNA ligases is 6~7.5 μ L, the amount of being preferably used is 6.5~7 μ L, the connector usage amount for 40~
100pmol, preferably connector usage amount are 40~80pmol.
7. one kind builds library kit, which is characterized in that the kit includes：The reagent repaired for end, for adding A's
Reagent, the reagent for adjunction head, the reagent for PCR amplification and the reagent for purifying.
8. a kind of sequencing DNA library is built by claim 1~6 any one of them method.
9. a kind of sequencing approach of DNA library, which is characterized in that library according to any one of claims 8 is used to be sequenced for object.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109610011A (en) * | 2018-12-28 | 2019-04-12 | 厦门胜芨科技有限公司 | A kind of NanoDNA overlength is adjoint to build library kit and its application method |
| CN110669830A (en) * | 2019-10-24 | 2020-01-10 | 裕策医疗器械江苏有限公司 | Low-quality FFPE DNA processing method and device and storage medium |
| CN112680794A (en) * | 2020-12-28 | 2021-04-20 | 深圳海普洛斯医学检验实验室 | Ultramicro nucleic acid sample library building method applied to NGS platform |
| WO2023098492A1 (en) | 2021-12-01 | 2023-06-08 | 江苏为真生物医药技术股份有限公司 | Sequencing library construction method and application |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109610011A (en) * | 2018-12-28 | 2019-04-12 | 厦门胜芨科技有限公司 | A kind of NanoDNA overlength is adjoint to build library kit and its application method |
| CN110669830A (en) * | 2019-10-24 | 2020-01-10 | 裕策医疗器械江苏有限公司 | Low-quality FFPE DNA processing method and device and storage medium |
| CN110669830B (en) * | 2019-10-24 | 2023-05-23 | 裕策医疗器械江苏有限公司 | Processing method, device and storage medium of low-quality FFPE DNA |
| CN112680794A (en) * | 2020-12-28 | 2021-04-20 | 深圳海普洛斯医学检验实验室 | Ultramicro nucleic acid sample library building method applied to NGS platform |
| WO2023098492A1 (en) | 2021-12-01 | 2023-06-08 | 江苏为真生物医药技术股份有限公司 | Sequencing library construction method and application |
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Application publication date: 20180706 |