CN104946629A - Method for fragmenting trace DNA sample and method for establishing DNA library by utilizing trace DNA sample - Google Patents
Method for fragmenting trace DNA sample and method for establishing DNA library by utilizing trace DNA sample Download PDFInfo
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- CN104946629A CN104946629A CN201510413466.9A CN201510413466A CN104946629A CN 104946629 A CN104946629 A CN 104946629A CN 201510413466 A CN201510413466 A CN 201510413466A CN 104946629 A CN104946629 A CN 104946629A
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Abstract
The invention discloses a method for fragmenting a trace DNA sample and a method for establishing a DNA library by utilizing the trace DNA sample. The method for fragmenting the trace DNA sample comprises the steps that 1, Carrier RNA is added into the trace DNA sample to obtain a sample to be fragmented; 2, fragmenting processing is conducted on the sample to be fragmented. An inventor discovers that after the exogenetic Carrier RNA is added into the target DNA, that is to say, the target DNA is mixed with the Carrier RNA and then fragmenting is conducted on the mixture, the protective action to the target DNA can be achieved, the destructive action of a fragmenting instrument to the trace DNA is reduced to the maximum extent, and full gene establishing library sequencing and even hybrid capture sequencing can be conducted on DNA of a trace FFPE sample.
Description
Technical field
The present invention relates to biological technical field, in particular to a kind of minim DNA sample fragmentation method and utilize the method in minim DNA sample constructed dna library.
Background technology
Paraffin embedding (Formalin-fixed paraffin-embedded fixed by formalin, FFPE) be a kind of important method of preserving tissue sample, the method is very extensive in application that is clinical and scientific research field, comprises the research of morphology and pathological research and molecular level.But Molecular level study FFPE sample is very challenging, mainly contains three reasons, one is that the medical sample of FFPE is most valuable, and the content of each sample is limited, and a lot of sample cannot follow-uply obtain again; Two is that this kind sample just started to degrade before entering dipped into formalin, when formalin infiltration, the crosslinked of generation makes extraction work all become more difficult, in addition, the most FFPE sample retention time is all very long, and also there are tremendous influence the environment of preservation and time to the DNA in sample; Three is that the effective content of FFPE sample DNA (20-50ng) directly after interrupting of trace is few, continues the difficulty of storehouse order-checking after adding.
Conventionally, the FFPE sample fragment of trace mainly contains two kinds of thinkings: one is directly adopt hyperacoustic method to interrupt, such as adopt that Covaris is ultrasonic interrupts instrument, optimum configurations with reference to Covaris S220 specification sheets, that is: Peak Incident Power (power peak): 175W; Duty factor (duty ratio): 10%; Cycles per Burst (outburst cycle life): 200; Treatment Times (treatment time): 180s.Two is the methods adopting transposase to build minim DNA two generation sequencing library, is added on DNA fragmentation by joint while utilizing transposon to interrupt DNA.
But there is following shortcoming in prior art:
One, adopt that Covaris S220 is ultrasonic interrupts the DNA fragmentation that instrument carries out FFPE sample, at present the ultrasonic wave of FFPE sample is interrupted to being only confined to the sample of extracted amount at more than 200ng.
Two, transposase builds the method in library through testing the shortcoming proving to have three aspects: 1, the bad control of the fragment length interrupted; 2, not high through the DNA fragmentation amplification efficiency in the process of carrying out PCR enrichment interrupting and add joint, cause the storage capacity of hybridizing front library inadequate, next step probe hybridization can not be carried out and catch; 3, it is based on complete genome that transposase carries out DNA fragmentation, for the uncertain DNA of this palliating degradation degree of FFPE, interrupts effect poor.
Existing method is for solving undegradable genomic dna or the higher FFPE sample of extracted amount has higher success ratio, also report FFPE sample is had to can be used for the demonstration that order-checking is caught in target area at present, but for the FFPE sample of trace, because extracted amount is few, in addition the complicacy of FFPE sample, the banking process for micro-FFPE sample not good at present.
Summary of the invention
The present invention aims to provide a kind of method of minim DNA sample fragmentation and utilizes the method in minim DNA sample constructed dna library, to solve the technical problem of building storehouse difficulty of micro-FFPE sample in prior art.
To achieve these goals, according to an aspect of the present invention, a kind of method of minim DNA sample fragmentation is provided.The method comprises the following steps: S1, adds Carrier RNA in minim DNA sample, obtains the sample treating fragmentation; S2, the sample treating fragmentation carries out fragmentation process.
Further, minim DNA sample is the DNA from FFPE sample.
Further, in minim DNA sample, the amount of DNA is 20 ~ 50ng.
Further, the add-on of Carrier RNA is 500 ~ 1000ng.
Further, it is that adopt ultrasonic wave to interrupt sample that instrument treats fragmentation carries out ultrasonic wave and interrupts that the sample treating fragmentation carries out fragmentation process.
According to a further aspect in the invention, a kind of method utilizing minim DNA sample constructed dna library is provided.The method comprises the following steps: S1, adopts the method for above-mentioned minim DNA sample fragmentation to carry out fragmentation to minim DNA sample, obtains DNA fragmentation; S2, repairs the end of DNA fragmentation and 3 ' end adds A base, obtains the DNA fragmentation that end connects A base; S3, connects the end jointing of the DNA fragmentation of A base by end; S4, the primers according to joint carries out pcr amplification, obtains sample DNA library; S5, the probe hybridization target acquisition DNA fragmentation of based target areas captured platform; S6, wash-out and pcr amplification target DNA fragments, utilize the primer of the sequences Design according to joint, carries out pcr amplification, obtain DNA library to target DNA fragments.
Further, target area capture platform is Agilent SureSelect solution hybridization capture platform.
Further, the method comprises: S8, detects DNA library.
Further, carry out detection to DNA library to comprise: use Agilent 2100Bioanalyzer to detect the Insert Fragment size of DNA library; Use the output of qPCR detection by quantitative DNA library.
Because the content of micro-FFPE sample is extremely low, directly carry out fragmentation and remaining target DNA content can be caused too low, and clip size is also difficult to meet expection, follow-up storehouse of building cannot be carried out and test.Contriver finds; by to the Carrier RNA adding external source in target DNA; mix with Carrier RNA by target dna; carry out fragmentation again to play a protective role to target dna; reduce to greatest extent and interrupt the destruction of instrument to minim DNA, make the FFPE sample DNA of trace carry out full genome and set up storehouse order-checking and even target area is caught order-checking and become a reality.
Accompanying drawing explanation
The Figure of description forming a application's part is used to provide a further understanding of the present invention, and schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 shows in example 1 the Insert Fragment size using the constructed library of Agilent 2100 biological analyser detection to comprise joint;
Fig. 2 A and Fig. 2 B to show in example 1 constructed library GC or AT resolution and GC content;
Fig. 3 A and 3B shows the order-checking depth profile in constructed library in example 1.
Embodiment
It should be noted that, when not conflicting, the embodiment in the application and the feature in embodiment can combine mutually.Below with reference to the accompanying drawings and describe the present invention in detail in conjunction with the embodiments.
Due to the complicacy of FFPE sample, make to detect its genetic information comprised more difficult, prior art has certain assurance for extracted amount higher than the sample of 200ng, but can not well carry out building storehouse order-checking and follow-up analysis for the sample that extracted amount is lower.
The present invention is prepared mainly for the library of micro-FFPE sample (20-50ng), and can combine with target area capture technique and sequencing technologies, and the sequencing data quality obtained can meet subsequent bio bioinformatics analysis.This technological breakthrough can solve in molecular biology experiment the difficult problem lacking experiment sample, increases reliability and the accuracy of experiment, contributes to researchist and understands the genetic mechanism comprised in the FFPE sample after preserving for many years more all sidedly.
According to a kind of typical embodiment of the present invention, provide a kind of method of minim DNA sample fragmentation.The method comprises the following steps: S1, adds Carrier RNA in minim DNA sample, obtains the sample treating fragmentation; S2, the sample treating fragmentation carries out fragmentation process.
Because the content of micro-FFPE sample is extremely low, directly carry out fragmentation and remaining target DNA content can be caused too low, and clip size does not meet expection yet, follow-up storehouse of building cannot be carried out and test.Contriver finds; by to the Carrier RNA adding external source in target DNA; mix with Carrier RNA by target dna; carry out fragmentation again to play a protective role to target dna; to greatest extent reduce interrupt the destruction of instrument to minim DNA, make trace FFPE sample DNA carry out full genome set up storehouse order-checking so that hybrid capture order-checking become a reality.
The method of minim DNA sample fragmentation of the present invention may be used for the fragmentation of any minim DNA sample, and according to a kind of typical embodiment of the present invention, minim DNA sample is the DNA from FFPE sample.
According to a kind of typical embodiment of the present invention, in minim DNA sample, the amount of DNA is 20 ~ 50ng.
The addition of Carrier RNA is not particularly limited, and according to a kind of typical embodiment of the present invention, preferably, the add-on of Carrier RNA is 500 ~ 1000ng.
According to a kind of typical embodiment of the present invention, it is that adopt ultrasonic wave to interrupt sample that instrument treats fragmentation carries out ultrasonic wave and interrupts that the sample treating fragmentation carries out fragmentation process.Preferably, employing Covaris LE220R high-throughput is ultrasonic interrupts the fragmentation that instrument carries out sample DNA.
According to a kind of typical embodiment of the present invention, provide a kind of method utilizing minim DNA sample constructed dna library.The method comprises the following steps: S1, adopts the method for above-mentioned minim DNA sample fragmentation to carry out fragmentation to minim DNA sample, obtains DNA fragmentation; S2, repairs the end of DNA fragmentation and 3 ' end adds A base, obtains the DNA fragmentation that end connects A base; S3, connects the end jointing of the DNA fragmentation of A base by end; S4, the primers according to joint carries out pcr amplification, obtains sample DNA library; S5, utilizes the probe hybridization of target area capture platform to catch the target DNA fragments be included in sample DNA library; S6, wash-out and pcr amplification target DNA fragments, utilize the primer of the sequences Design according to joint, carries out pcr amplification, obtain DNA library to target DNA fragments.
Because the content of micro-FFPE sample is extremely low, directly carry out fragmentation and remaining target DNA content can be caused too low, and clip size is also difficult to meet expection, follow-up storehouse of building cannot be carried out and test.Contriver finds; by to the Carrier RNA adding external source in target DNA; mix with Carrier RNA by target dna; carry out fragmentation again to play a protective role to target dna; reduce to greatest extent and interrupt the destruction of instrument to minim DNA, make the FFPE sample DNA of trace carry out full genome and set up storehouse order-checking and even target area is caught order-checking and become a reality.
Preferably, target area capture platform is Agilent (Agilent) SureSelect solution hybridization capture platform.Due to the hybridization initial amount higher (750ng-1000ng) that Agilent SureSelect solution hybridization capture platform requires, under the condition reducing PCR cycle number as much as possible, need library to carry out bulk crossing and catch, thus improve hybridization initial amount.
According to a kind of typical embodiment of the present invention, the method comprises further: S8, detects DNA library.
Preferably, carry out detection to DNA library to comprise: use Agilent 2100Bioanalyzer (Agilent 2100 biological analyser) to detect the Insert Fragment size of DNA library; Use the output of qPCR detection by quantitative DNA library.
Beneficial effect of the present invention is further illustrated below in conjunction with specific embodiment.
According to the typical embodiment of the present invention one, the method in minim DNA sample constructed dna library is utilized to comprise:
1) extract DNA sample, and mix 500ng Carrier RNA at target DNA sample;
2) DNA fragmentation
Use ultrasonic wave to interrupt instrument (Covaris LE220R) sample is interrupted parameter according to table 1 to be broken into the DNA fragmentation of master tape at 150bp-300bp;
Table 1
3) DNA fragmentation end is repaired and 3 ' end interpolation A base;
4) connection of joint
End is connected the joint (Illumina sequence measuring joints) that the DNA random fragment end catenation sequence of A base is known;
5) pcr amplification
According to known joint sequence design primer, carry out pcr amplification, obtain FFPE sample DNA library;
6) probe hybridization based on Agilent SureSelect target area capture platform is caught;
7) wash-out and the pcr amplification of after product is caught;
According to known joint sequence primer, carry out PCR enrichment, obtain final library;
8) library detection
Agilent 2100 biological analyser is used to detect Insert Fragment size; Use qPCR detection by quantitative library output.
Embodiment 1
Patients with Non-small-cell Lung FFPE sample 2 example, sample ID and extracted amount are in table 2.
Table 2
1) fragmentation of DNA
Use sonicator (Covaris LE220R) by target dna and the Carrier RNA (500ng) that mixes according to the fragmentation specifically interrupting parameter and carry out DNA, Covaris LE220R interrupts optimum configurations in table 1.
DNA after interrupting through 1.8 × AMPure XP magnetic beads for purifying after carry out next step operation.
2) end reparation and 3 ' end add A base
With reference to following table 3 proportioning ready reaction mixed solution, with rifle gently pressure-vaccum mixing up and down.
Table 3
Put into PCR instrument, according to the form below setting program carries out reacting (Gai Wen is set to 70 DEG C, and front 30min does not cover heat lid, and temperature arrives 65 DEG C, covers heat lid immediately), and step is in table 4.
Table 4
3) joint connects
According to the proportioning ready reaction mixed solution of following table 5, with rifle gently pressure-vaccum mixing up and down.
Table 5
Be divided into 2 pipes, often pipe 55ul, be put in PCR instrument, 20 DEG C of reaction 15min.Reaction terminate after, use 0.8 × AMPure XP magnetic beads for purifying DNA sample.
4) pcr amplification
According to the proportioning ready reaction mixed solution of following table 6, with rifle gently pressure-vaccum mixing up and down.
Table 6
Put into PCR instrument, according to the form below 7 setting program reacts
Table 7
With 1.8 × AMPure XP magnetic bead purifying is carried out to amplified production, finally library is dissolved in 50ul NF-water.Purified product is carried out Qubit quantitative.
5) Library hybridization
Each library mixes by 5.1, is placed in concentrating instrument evaporate to dryness, and temperature is set to 45 DEG C, general needs 1 hours.
5.2 add 1.4ul pure water in the sample of evaporate to dryness, respectively add the appropriate Blocking oligos (oligonucleotide encapsulant) carrying out closing for joint sequence.
5.3 follow-up hybridization and catching method are with reference to Agilent company SureSeclect XT hybrid capture method.I.e. 65 DEG C of hybridization 16 ~ 24h, use DynabeadsMyone Streptavidin T1 Streptavidin MagneSphere to catch, adopt SureSelect Wash1 room temperature elution one time, and SureSelect Wash2 is 65 DEG C of wash-outs three times.
6) rear Library PCR amplification is caught
Reaction solution is configured, with rifle gently pressure-vaccum mixing up and down according to following table 8.
Table 8
Put into PCR instrument, according to the form below 9 setting program reacts.
Table 9
With 1.8 × AMPure XP magnetic bead purifying is carried out to amplified production, finally library is dissolved in 30ul NF-water.Purified product is carried out Qubit accurate quantification.
7) library detection
By step 6) in purified product be diluted to 2ng/ul, taking out 1ul adopts Agilent 2100 biological analyser (Agilent company of the U.S.) to detect Insert Fragment size, as shown in Figure 1, the library total length of structure, at about 300bp, meets the sequencing strategy of PE150 to detected result; In addition, then take out 1ul and detect for qPCR, determine upper machine concentration according to detected result.
According to the concentration of upper step gained, library is diluted to upper confidential ask after (2nM), require to check order on platform at Illumina to carry out PE150 order-checking according to planning data amount.
8) upper machine result Quality Control
GC resolution is carried out in the 8.1 pairs of libraries of building and GC content is assessed, as shown in Figure 2 A and 2B,
8.2 pairs build library order-checking randomness assess, as shown in Figure 3 A and Figure 3 B:
8.3 sequencing quality control files are in table 10.
Table 10
As known from Table 10, two micro-FFPE sampled data output and target area coverage all reach expection, and the order-checking degree of depth of target area also reaches 200X and more than 500X respectively, and the repetition rate of target area also controls about 60%.Illustrate that the present invention successfully establishes and carry out fragmentation for micro-FFPE sample and target area is caught and carries out the technology of two generations order-checking.
As can be seen from the above description, the above embodiments of the present invention achieve following technique effect:
The present invention by adding the Carrier RNA of external source in target dna, what control Cocaris interrupted instrument interrupts parameter, fragmentation is carried out to the FFPE sample of 20 ~ 50ng extracted amount, and then adopt optimised micro-banking process, complete under the prerequisite of control PCR cycle number of trying one's best and build storehouse, caught by follow-up bulk crossing and the order-checking of upper machine, obtain more high-quality data.Thus fill up the technological gap that storehouse hybrid capture built by micro-FFPE sample.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. a method for minim DNA sample fragmentation, is characterized in that, comprises the following steps:
S1, adds Carrier RNA, obtains the sample treating fragmentation in described minim DNA sample;
To described, S2, treats that the sample of fragmentation carries out fragmentation process.
2. method according to claim 1, is characterized in that, described minim DNA sample is the DNA from FFPE sample.
3. method according to claim 2, is characterized in that, in described minim DNA sample, the amount of DNA is 20 ~ 50ng.
4. method according to claim 1, is characterized in that, the add-on of described Carrier RNA is 500 ~ 1000ng.
5. method according to claim 1, is characterized in that, to treat that the sample of fragmentation carries out fragmentation process be adopt ultrasonic wave to interrupt instrument and treat that the sample of fragmentation carries out ultrasonic wave and interrupts to described to described.
6. utilize the method in minim DNA sample constructed dna library, it is characterized in that, comprise the following steps:
S1, adopt the method for the minim DNA sample fragmentation according to any one of claim 1 to 5 to as described in minim DNA sample carry out fragmentation, obtain DNA fragmentation;
S2, repairs the end of described DNA fragmentation and 3 ' end adds A base, obtains the DNA fragmentation that end connects A base;
S3, connects the end jointing of the DNA fragmentation of A base by described end;
S4, the primers according to described joint carries out pcr amplification, obtains sample DNA library;
S5, the probe hybridization target acquisition DNA fragmentation of based target areas captured platform;
S6, target DNA fragments described in wash-out and pcr amplification, utilizes the primer of the sequences Design according to described joint, carries out pcr amplification to described target DNA fragments, obtain described DNA library.
7. method according to claim 6, is characterized in that, described target area capture platform is Agilent SureSelect solution hybridization capture platform.
8. method according to claim 6, is characterized in that, described method comprises further:
S8, detects described DNA library.
9. method according to claim 8, is characterized in that, carries out detection comprise described DNA library: use Agilent 2100Bioanalyzer to detect the Insert Fragment size of described DNA library; Use the output of DNA library described in qPCR detection by quantitative.
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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 |
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| CN106636442A (en) * | 2017-02-23 | 2017-05-10 | 上海鼎晶生物医药科技股份有限公司 | Combined detection kit for human tumor gene mutation |
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| CN109023537A (en) * | 2018-09-04 | 2018-12-18 | 上海交通大学 | A kind of constructing technology of minim DNA sample high-throughput sequencing library |
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