CN104946629B - A kind of method of minim DNA sample fragmentation and the method using minim DNA sample constructed dna library - Google Patents
A kind of method of minim DNA sample fragmentation and the method using minim DNA sample constructed dna library Download PDFInfo
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Abstract
Method the invention discloses a kind of method of minim DNA sample fragmentation and using minim DNA sample constructed dna library.Wherein, the method for minim DNA sample fragmentation comprises the following steps：S1, Carrier RNA are added in minim DNA sample, obtain treating the sample of fragmentation；S2, the sample for treating fragmentation carry out fragmentation processing.Inventor has found; pass through the Carrier RNA to adding external source in target DNA; target dna is mixed with Carrier RNA; carrying out fragmentation again can play a protective role to target dna; reduce to greatest extent and interrupt destruction of the instrument to minim DNA so that micro FFPE sample DNAs carry out full genome establishment storehouse sequencing or even hybrid capture sequencing becomes a reality.
Description
Technical field
The present invention relates to biological technical field, method and utilization in particular to a kind of minim DNA sample fragmentation
The method in minim DNA sample constructed dna library.
Background technology
It is to preserve tissue that formalin, which fixes FFPE (Formalin-fixed paraffin-embedded, FFPE),
A kind of important method of sample, this method is quite varied in clinical and scientific research field application, including morphology and pathology are ground
Study carefully the research with molecular level.But Molecular level study FFPE samples are very challenging, mainly there are three aspect reasons,
First, FFPE medical sample is most valuable, the content of each sample is limited, and many samples can not be obtained subsequently again；Second, should
Species sample begins to degrade before dipped into formalin is entered, formalin caused crosslinking order extraction when permeating
Work all becomes more difficult, in addition, the most FFPE Sample storages time is all very long, the environment of preservation and time are to sample
In DNA also have tremendous influence；Third, micro FFPE sample DNAs (20-50ng) are directly over the effective content after interrupting
Seldom, the difficulty of storehouse sequencing is continued after adding.
Conventionally, micro FFPE sample fragmentizations mainly have two kinds of thinkings：First, directly using ultrasound
The method of ripple is interrupted, and instrument is interrupted for example with Covaris ultrasounds, and parameter setting is with reference to Covaris S220 specifications, i.e.,：Peak
Incident Power (power peak)：175W；Duty factor (duty factor)：10%；Cycles per Burst (outbursts
Periodicity)：200；Treatment Times (processing time)：180s.Second, using the transposase structure generation of minim DNA two sequencing
The method in library, joint is added on DNA fragmentation while interrupting DNA using transposons.
But prior art has the following disadvantages：
First, the DNA fragmentation of instrument progress FFPE samples is interrupted using Covaris S220 ultrasounds, at present for FFPE samples
Ultrasonic wave interrupt the sample for being limited only to extracted amount in more than 200ng.
2nd, the method in transposase structure library is it was proved that there is the shortcomings that three aspects：1, the fragment length interrupted
Bad control；2, it is not high by interrupting and adding the DNA fragmentation of the joint amplification efficiency during performing PCR enrichment is entered, cause
The storage capacity for hybridizing preceding library is inadequate, it is impossible to carries out the probe hybridization and capture of next step；3, it is base that transposase, which carries out DNA fragmentation,
In complete genome, for the FFPE uncertain DNA of this palliating degradation degree, it is poor to interrupt effect.
Existing method has higher for the undegradable genomic DNA of the solution either higher FFPE samples of extracted amount
Success rate, also have been reported that FFPE samples can be used for the demonstration of target area capture sequencing at present, but for micro FFPE
Sample, because extracted amount is few, the complexity of FFPE samples, storehouse side is built currently without good for micro FFPE samples in addition
Method.
The content of the invention
The present invention is intended to provide method and the utilization minim DNA sample constructed dna library of a kind of minim DNA sample fragmentation
Method, build the difficult technical problem in storehouse with solve micro FFPE samples in the prior art.
To achieve these goals, according to an aspect of the invention, there is provided a kind of side of minim DNA sample fragmentation
Method.This method comprises the following steps：S1, Carrier RNA are added in minim DNA sample, obtain treating the sample of fragmentation；
S2, the sample for treating fragmentation carry out fragmentation processing.
Further, minim DNA sample is the DNA from FFPE samples.
Further, DNA amount is 20~50ng in minim DNA sample.
Further, Carrier RNA addition is 500~1000ng.
Further, the sample for treating fragmentation carries out fragmentation to handle being to interrupt instrument using ultrasonic wave to treat fragmentation
Sample carries out ultrasonic wave and interrupted.
According to another aspect of the present invention, there is provided a kind of method using minim DNA sample constructed dna library.The party
Method comprises the following steps：S1, fragmentation is carried out to minim DNA sample using the method for above-mentioned minim DNA sample fragmentation, obtained
DNA fragmentation；S2, is repaired to the end of DNA fragmentation and A bases are added at 3 ' ends, obtains the DNA fragmentation of end connection A bases；
S3, end is connected to the end jointing of the DNA fragmentation of A bases；S4, enter performing PCR according to the primers of joint and expand
Increase, obtain sample DNA library；S5, the probe hybrid capture target DNA fragments based on target area capture platform；S6, elution and
PCR expands target DNA fragments, using the primer of the sequences Design according to joint, target DNA fragments is entered with performing PCR amplification, is obtained
DNA library.
Further, target area capture platform is Agilent SureSelect solution hybridization capture platforms.
Further, this method includes：S8, DNA library is detected.
Further, carrying out detection to DNA library includes：Use Agilent 2100Bioanalyzer detection DNA libraries
Insert Fragment size；The yield of DNA library is quantitatively detected using qPCR.
Because the content of micro FFPE samples is extremely low, directly carrying out fragmentation can cause remaining target DNA content too low,
And clip size also is difficult to meet expection, follow-up storehouse of building can not be carried out and tested.Inventor has found, by target DNA
Add external source Carrier RNA, i.e., target dna is mixed with Carrier RNA, then carry out fragmentation can be to target
DNA plays a protective role, and reduces interrupt destruction of the instrument to minim DNA to greatest extent so that micro FFPE sample DNAs
Carry out full genome establishment storehouse sequencing or even target area capture sequencing becomes a reality.
Brief description of the drawings
The Figure of description for forming the part of the application is used for providing a further understanding of the present invention, and of the invention shows
Meaning property embodiment and its illustrate be used for explain the present invention, do not form inappropriate limitation of the present invention.In the accompanying drawings：
Fig. 1 shows the insertion for including joint using library constructed by the detection of the biological analyser of Agilent 2100 in example 1
Clip size；
Fig. 2A and Fig. 2 B show library GC or AT separating degree constructed in example 1 and G/C content；
Fig. 3 A and 3B show the sequencing depth profile in constructed library in example 1.
Embodiment
It should be noted that in the case where not conflicting, the feature in embodiment and embodiment in the application can phase
Mutually combination.Describe the present invention in detail below with reference to the accompanying drawings and in conjunction with the embodiments.
Due to the complexity of FFPE samples so that detect that the hereditary information that it is included is relatively difficult, and prior art is for carrying
Sample of the taken amount higher than 200ng has certain assurance, but can not carry out building storehouse survey well for the relatively low sample of extracted amount
Sequence and follow-up analysis.
Present invention is generally directed to the preparation of the library of micro FFPE samples (20-50ng), and skill can be captured with target area
Art and sequencing technologies are combined, and obtained sequencing data quality disclosure satisfy that subsequent bio bioinformatics analysis.The technological break-through can
To solve to lack the problem of experiment sample in molecular biology experiment, increase the reliability and accuracy of experiment, help to study
Personnel understand the genetic mechanism included in the FFPE samples after preserving for many years more fully hereinafter.
According to a kind of typical embodiment of the present invention, there is provided a kind of method of minim DNA sample fragmentation.This method bag
Include following steps：S1, Carrier RNA are added in minim DNA sample, obtain treating the sample of fragmentation；S2, treat fragmentation
Sample carry out fragmentation processing.
Because the content of micro FFPE samples is extremely low, directly carrying out fragmentation can cause remaining target DNA content too low,
And clip size does not meet expection yet, follow-up storehouse of building can not be carried out and tested.Inventor has found, by adding in target DNA
Add the Carrier RNA of external source, i.e., mixed target dna with Carrier RNA, then carry out fragmentation can be to target
DNA plays a protective role, and reduces interrupt destruction of the instrument to minim DNA to greatest extent so that micro FFPE sample DNAs
Carry out full genome establishment storehouse sequencing or even hybrid capture sequencing becomes a reality.
The method of minim DNA sample fragmentation of the present invention can be used for the fragmentation of any minim DNA sample, according to this hair
A kind of bright typical embodiment, minim DNA sample are the DNA from FFPE samples.
According to a kind of typical embodiment of the present invention, DNA amount is 20~50ng in minim DNA sample.
Carrier RNA addition is not particularly limited, according to a kind of typical embodiment of the present invention, it is preferred that
Carrier RNA addition is 500~1000ng.
According to a kind of typical embodiment of the present invention, the sample for treating fragmentation carries out fragmentation to handle being using ultrasound
Ripple interrupt instrument treat fragmentation sample carry out ultrasonic wave interrupt.Preferably, beaten using Covaris LE220R high fluxs ultrasound
Disconnected instrument carries out the fragmentation of sample DNA.
According to a kind of typical embodiment of the present invention, there is provided a kind of side using minim DNA sample constructed dna library
Method.This method comprises the following steps：S1, fragment is carried out to minim DNA sample using the method for above-mentioned minim DNA sample fragmentation
Change, obtain DNA fragmentation；S2, is repaired to the end of DNA fragmentation and A bases are added at 3 ' ends, obtains end connection A bases
DNA fragmentation；S3, end is connected to the end jointing of the DNA fragmentation of A bases；S4, entered according to the primers of joint
Performing PCR expands, and obtains sample DNA library；S5, sample DNA is included in using the probe hybrid capture of target area capture platform
Target DNA fragments in library；S6, elution and PCR amplification target DNA fragments, using the primer of the sequences Design according to joint,
Target DNA fragments are entered with performing PCR amplification, obtains DNA library.
Because the content of micro FFPE samples is extremely low, directly carrying out fragmentation can cause remaining target DNA content too low,
And clip size also is difficult to meet expection, follow-up storehouse of building can not be carried out and tested.Inventor has found, by target DNA
Add external source Carrier RNA, i.e., target dna is mixed with Carrier RNA, then carry out fragmentation can be to target
DNA plays a protective role, and reduces interrupt destruction of the instrument to minim DNA to greatest extent so that micro FFPE sample DNAs
Carry out full genome establishment storehouse sequencing or even target area capture sequencing becomes a reality.
Preferably, target area capture platform is Agilent (Agilent) SureSelect solution hybridization capture platforms.By
It is higher (750ng-1000ng) in the hybridization initial amount of Agilent SureSelect solution hybridizations capture platform requirement, as far as possible
Reduction PCR cycle number under conditions of, it is necessary to library carry out bulk crossing capture, so as to improve hybridization initial amount.
According to a kind of typical embodiment of the present invention, this method further comprises：S8, DNA library is detected.
Preferably, carrying out detection to DNA library includes：Using Agilent 2100Bioanalyzer, (Agilent 2100 is given birth to
Thing analyzer) detection DNA library Insert Fragment size；The yield of DNA library is quantitatively detected using qPCR.
Beneficial effects of the present invention are further illustrated below in conjunction with specific embodiment.
According to a typical embodiment of the invention, the method using minim DNA sample constructed dna library includes：
1) DNA sample is extracted, and in target DNA sample incorporation 500ng Carrier RNA；
2) DNA fragmentation
Interrupt instrument (Covaris LE220R) using ultrasonic wave and sample interrupted into parameter according to table 1 be broken into master tape and exist
150bp-300bp DNA fragmentation；
Table 1
3) DNA fragmentation end is repaired and A bases are added at 3 ' ends；
4) connection of joint
By joint (Illumina sequence measuring joints) known to the DNA random fragments end catenation sequence of end connection A bases；
5) PCR is expanded
Primer is designed according to known joint sequence, enters performing PCR amplification, obtains FFPE sample DNAs library；
6) the probe hybrid capture based on Agilent SureSelect target areas capture platform；
7) elution of product and PCR amplifications after capturing；
According to known joint sequence primer, enter performing PCR enrichment, obtain final library；
8) library detection
Insert Fragment size is detected using the biological analyser of Agilent 2100；Library yield is quantitatively detected using qPCR.
Embodiment 1
2, Patients with Non-small-cell Lung FFPE samples, sample ID and extracted amount are shown in Table 2.
Table 2
1) DNA fragmentation
Target dna and the Carrier RNA (500ng) of incorporation are pressed using sonicator (Covaris LE220R)
According to the fragmentation for specifically interrupting parameter progress DNA, the Covaris LE220R parameter setting that interrupts is shown in Table 1.
DNA after interrupting by 1.8 × AMPure XP magnetic beads for purifying after carry out next step operation.
2) end is repaired and 3 ' ends add A bases
Prepare reaction mixture with reference to table 3 below proportioning, gently pressure-vaccum mixes up and down with rifle.
Table 3
PCR instrument is put into, according to the form below setting program is reacted, and (Gai Wen is set to 70 DEG C, and preceding 30min does not cover hot lid, and temperature arrives
Up to 65 DEG C, hot lid is covered immediately), step is shown in Table 4.
Table 4
3) joint connects
Prepare reaction mixture according to the proportioning of table 5 below, gently pressure-vaccum mixes up and down with rifle.
Table 5
It is divided into 2 pipes, often pipe 55ul, is put in PCR instrument, 20 DEG C of reaction 15min.After reaction terminates, using 0.8 ×
AMPure XP magnetic beads for purifying DNA samples.
4) PCR is expanded
Prepare reaction mixture according to the proportioning of table 6 below, gently pressure-vaccum mixes up and down with rifle.
Table 6
PCR instrument is put into, according to the form below 7 sets program to react
Table 7
With 1.8 × AMPure XP magnetic beads amplified production is purified, library is finally dissolved in 50ul NF-water
In.Purified product progress Qubit is quantified.
5) Library hybridization
5.1 mix each library, are placed in concentrating instrument and are evaporated, and temperature setting is 45 DEG C, is generally required 1 hour or so.
5.2 add 1.4ul pure water into the sample being evaporated, then each add appropriate is closed for joint sequence
Blocking oligos (oligonucleotides sealer).
5.3 follow-up hybridization and catching method are with reference to Agilent company SureSeclect XT hybrid capture methods.I.e. 65 DEG C
Hybridize 16~24h, captured using DynabeadsMyone Streptavidin T1 Streptavidin MagneSpheres, used
SureSelect Wash1 room temperature elutions one time, SureSelect Wash2 are eluted three times at 65 DEG C.
6) Library PCR amplification after capturing
Reaction solution is configured according to table 8 below, gently pressure-vaccum mixes up and down with rifle.
Table 8
PCR instrument is put into, according to the form below 9 sets program to be reacted.
Table 9
With 1.8 × AMPure XP magnetic beads amplified production is purified, library is finally dissolved in 30ul NF-water
In.Purified product is subjected to Qubit accurate quantifications.
7) library detection
Purified product in step 6) is diluted to 2ng/ul, 1ul is taken out and uses the biological analyser (U.S. of Agilent 2100
Agilent company) detection Insert Fragment size, testing result as shown in figure 1, the library total length of structure in 300bp or so, symbol
Close PE150 sequencing strategy；Detected in addition, further taking out 1ul for qPCR, upper machine concentration is determined according to testing result.
According to the concentration obtained by upper step, by library be diluted to it is upper it is confidential ask after (2nM), existed according to the requirement of planning data amount
PE150 sequencings are carried out in Illumina microarray datasets.
8) machine result Quality Control on
Build library and carry out GC separating degrees and G/C content for 8.1 pairs and assess, as shown in Figure 2 A and 2B,
8.2 pairs of sequencing randomnesss for building library are assessed, as shown in Figure 3 A and Figure 3 B：
8.3 sequencing quality control files are shown in Table 10.
Table 10
As known from Table 10, two micro FFPE sample datas yield and target area coverage reach expected, target area
The sequencing depth in domain has also respectively reached 200X and more than 500X, and the repetitive rate of target area is also controlled 60% or so.Say
The bright present invention, which has been successfully established, to be captured for micro FFPE samples progress fragmentation and target area and carries out the skill of two generations sequencing
Art.
As can be seen from the above description, the above embodiments of the present invention realize following technique effect：
For the present invention by adding the Carrier RNA of external source in target dna, what control Cocaris interrupted instrument interrupts ginseng
Number, fragmentation is carried out to the FFPE samples of 20~50ng extracted amounts, and then using the micro banking process optimized, controlled as far as possible
Complete to build storehouse on the premise of PCR cycle number processed, be sequenced, obtained than better quality by follow-up bulk crossing capture and upper machine
Data.So as to fill up the technological gap that micro FFPE samples build storehouse hybrid capture.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.Within the spirit and principles of the invention, that is made any repaiies
Change, equivalent substitution, improvement etc., should be included in the scope of the protection.
Claims (9)
- A kind of 1. method of minim DNA sample fragmentation, it is characterised in that comprise the following steps：S1, Carrier RNA are added in the minim DNA sample, obtain treating the sample of fragmentation；S2, fragmentation processing is carried out to the sample for treating fragmentation.
- 2. according to the method for claim 1, it is characterised in that the minim DNA sample is the DNA from FFPE samples.
- 3. according to the method for claim 2, it is characterised in that DNA amount is 20~50ng in the minim DNA sample.
- 4. according to the method for claim 1, it is characterised in that the addition of the Carrier RNA be 500~ 1000ng。
- 5. according to the method for claim 1, it is characterised in that carrying out fragmentation processing to the sample for treating fragmentation is Instrument is interrupted using ultrasonic wave to interrupt the sample progress ultrasonic wave for treating fragmentation.
- A kind of 6. method using minim DNA sample constructed dna library, it is characterised in that comprise the following steps：S1, using the method for the minim DNA sample fragmentation as any one of claim 1 to 5 to the minim DNA sample This progress fragmentation, obtains DNA fragmentation；S2, is repaired to the end of the DNA fragmentation and A bases are added at 3 ' ends, obtains the DNA fragmentation of end connection A bases；S3, the end is connected to the end jointing of the DNA fragmentation of A bases；S4, enter performing PCR amplification according to the primers of the joint, obtain sample DNA library；S5, the probe hybrid capture target DNA fragments based on target area capture platform；S6, elution and PCR expand the target DNA fragments, using the primer of the sequences Design according to the joint, to the mesh Mark DNA fragmentation enters performing PCR amplification, obtains the DNA library.
- 7. according to the method for claim 6, it is characterised in that the target area capture platform is Agilent SureSelect solution hybridization capture platforms.
- 8. according to the method for claim 6, it is characterised in that methods described further comprises：S8, the DNA library is detected.
- 9. according to the method for claim 8, it is characterised in that carrying out detection to the DNA library includes：Use Agilent 2100Bioanalyzer detects the Insert Fragment size of the DNA library；The production of the DNA library is quantitatively detected using qPCR Amount.
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| CN106119345A (en) * | 2016-06-22 | 2016-11-16 | 杭州杰毅麦特医疗器械有限公司 | A kind of quick homogenization or the method for equal proportion DNA sample |
| CN106282163A (en) * | 2016-08-15 | 2017-01-04 | 天津诺禾医学检验所有限公司 | Construction method based on Ion Torrent order-checking fusion of platforms gene test library and the method for fusion gene detection |
| CN106498504A (en) * | 2016-12-13 | 2017-03-15 | 上海美迪维康生物科技有限公司 | Two generations sequencing database technology based on multiplex PCR |
| CN108251515A (en) * | 2016-12-27 | 2018-07-06 | 安诺优达基因科技(北京)有限公司 | A kind of method for building FFPE sample DNAs library |
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| CN109023537B (en) * | 2018-09-04 | 2021-10-08 | 上海交通大学 | Construction technology of trace DNA sample high-throughput sequencing library |
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| CN109810973A (en) * | 2019-03-19 | 2019-05-28 | 深圳因合生物科技有限公司 | A method of gDNA simulation cfDNA is interrupted using ultrasonic wave |
| CN110607563A (en) * | 2019-09-19 | 2019-12-24 | 北京市儿科研究所 | Sequencing library of filter paper sheet dry blood spots and construction method thereof |
| CN111041069B (en) * | 2019-12-26 | 2021-01-19 | 人和未来生物科技（长沙）有限公司 | High-throughput sequencing library construction method for low-initial-quantity DNA sample and application thereof |
| CN113106144A (en) * | 2020-12-30 | 2021-07-13 | 中南大学湘雅二医院 | DNA fragment targeted enrichment method and application thereof in genome targeted sequencing |
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