CN106282161A - Special capture and repeat replication low frequency DNA base variation method and application - Google Patents
Special capture and repeat replication low frequency DNA base variation method and application Download PDFInfo
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Abstract
The invention discloses a kind of special capture the method for repeat replication low frequency DNA base variation, belong to its step of molecular diagnosis field to include: (1) carries out thermal denaturation to DNA, then with the primer mixture with thermodynamics dynamic structure, target dna is hybridized, then repeat replication；(2) utilize and measure oligonucleotide that target 3 end mates the secondary DNA fragmentation of repeat replication is carried out specificity extension, tailing, and introducing one section of consensus at its 3 end；(3) sequencing library is built；(4) multiple order-checking readings is generated；(5) sequence difference between order-checking readings and reference sequences is identified；(6) sequence variants is determine whether；The present invention can reach 0.01% for the detection sensitivity of low frequency DNA base variation, to in normal sequence background may the qualification of low frequency variance in the sample containing a small amount of series of variation and illustrating, and the qualification to the low frequency variation under order-checking mistake background has help greatly.
Description
Technical field
The present invention relates to molecular biology and clinical diagnosis field, particularly relate to utilize structural primer from fragment DNA sample
It is directly used in the parallel sequenator of secondary high flux after capturing target dna, repeat replication, amplification in Ben to measure, with identification of dna fragment
The base of middle ULF variation replaces, lack, insert or the method for the DNA sequence such as mixed type mutation combination sudden change and answering
With.
Background technology
The oncogene variation of tumor tissues is the principal element driving tumor cell neoplasm.The cancer base of most of tumors
Because, in addition to there is a main driving variation, there is also other driving variation of multiple lower frequency, and these be relatively low
Other of frequency drives variation to have a great impact the therapeutic effect of tumor, and these variations are the masters of tumor cells pathology detection
Want object, be the basis of neoplasm targeted therapy.
Normal human peripheral blood's blood exists the dissociative DNA fragment (cell-free DNA is called for short cfDNA) of trace.Giving birth to
Reason or during pathological change, can determine related pathologies or the cfDNA of physiological tissue's cell-specific by plasma sample, therefore,
CfDNA can detect object as " the liquid biopsy " during pathophysiological change.Liquid biopsy is with the dissociative DNA in blood for inspection
Survey object, there is non-invasi, repeatable sampling, acceptance high, have become as noinvasive antenatal genetic diagnosis, tumor is examined
The focus of the research fields such as disconnected, tumor prognosis, and initially enter clinical practice.CfDNA identifies at the early diagnosis of tumor, medicine
The fields such as effect assessment, targeted therapy and prognosis evaluation also have broad application prospects.
The gene that in flesh tissue, frozen tissue, paraffin-embedded tissue, hydrothorax, blood plasma, exfoliated tumor cells, pulmonary carcinoma is relevant
Abrupt climatic change has been applied to instruct personalized targeting medication, and obtains such as cFDA, FDA and the accreditation of Yao Jian mechanism of European Union.Cut
Only in April, 2016, adjoint the diagnostic gene of the pulmonary carcinoma targeting medication that the administrative organization such as FDA is assert: EGFR, Kras, Braf,
The mutantional hotspot of Nras gene, PIK3CA gene, EML4-ALK fusion gene, ROS1, ALK/ROS1, BCR-ABL fusion gene,
PDGFRA, JAK2, C-KIT mutant gene etc..Oncology, the liquid biopsy to these gene mutation focuses is possibly used for prison
Survey the tumor burden in blood, as to the blood of patients with lung cancer sample epidermal growth factor receptor lacking biopsy diagnosis
(EGFR) detection driving sudden change has been used for the adjoint diagnosis of EGFR-casein kinase 2 enzyme inhibitor class medicine by the approval of Yao Jian mechanism
Method.But, before in blood, the liquid biopsy method of DNA analysis is as common cancers diagnostic application, it is necessary to solve
The problem of its effectiveness, operability and reliability, but liquid biopsy is at early diagnosis, pharmacodynamic assessment and the prognosis evaluation etc. of tumor
Field has highlighted special application prospect [Diehl F, Schmidt K, Choti MA, et al.Circ μ lating
mutant DNA to assess tumor dynamics.Nat Med 2008；14(9):985-90】.
Technically, owing to blood middle reaches freestone acid content is low, and free nucleic acid is easily disturbed by the dilution of wild-type nucleic acid,
And limited by traditional sensing techniques sensitivity, liquid biopsy receives certain restriction in clinical practice.With regard to catastrophe point
For the sensitivity of frequency measurement, qPCR technology can reach 0.1%, digital P CR is 0.01%, the parallel survey of secondary high flux
Sequence (being called for short, secondary order-checking) can reach the level of 0.001%.If cfDNA in blood plasma is detection sample, to sudden change detection skill
The sensitivity requirement of art is the highest, it is desirable to reach 0.01%, and according to this requirement, the most secondary sequencing technologies is the most permissible
High throughput assay for cfDNA.And due to the order-checking degree of depth and the obstacle of library background signal, liquid based on secondary sequencing technologies
State biopsy method is the most also only applicable to scientific research, also has a segment distance away from clinical diagnostic applications.
Except the clinical practice at diagnosing tumor, the detection of low frequency variant also has huge at other aspects biological
Application, such as somatic mutation, the detection etc. of sample contamination.The detection that mixing sample low and medium frequency is made a variation by secondary sequencing technologies
There is huge application prospect.But the error rate that the limitation of the method is method itself is high, reaches 0.1 1%, and low in sample
Frequency variation may as little as 0.01%.Applied sample amount is also had high requirements by the detection of low frequency variant, common library construction
Need the DNA sample of at least 50ng.But, DNA sample amount is few, variation frequency is low, DNA fragmentation is the common special of clinical sample
Property so that current secondary sequencing technologies, when the detection of low frequency variant, can produce unacceptable false positive rate.
As a example by the secondary order-checking platform of Illumina, the base of MiSeq order-checking platform substitutes error rate and reaches more than 0.1%
【Performance comparison of NGS platforms miSeq Ion Torrent-NBT 2012Loman】.Its
Producing cause has: the base reading mistake produced during 1.DNA sequent synthesis reading；During the amplification of 2.DNA chain cluster, polymerase causes
Resultant fault；3. the base pairing mistakes that in the preparation process of library, pcr amplification primer enters；4. library order-checking time sample between dirt
Dye.It is relevant with machine system that the base that the first two reason causes reads error, the polymerase itself that mainly earlier trials uses
Error rate produce, along with high-fidelity synzyme, such as application and the optimization of sequencing technologies platform of HiFi KAPA polymerase,
The error of this respect has been greatly lowered.Secondary sequencing library builds to be needed by PCR chain reaction DNA fragmentation tailing and amplification,
In PCR chain reaction, the DNA fragmentation that previous circulation produces can be used for the DNA profiling of later reaction cycle, and therefore PCR produces
Thing increases with index rank along with PCR cycle number.It is wrong that archaeal dna polymerase for DNA tailing and amplification can produce certain synthesis
By mistake, the Taq archaeal dna polymerase error rate as conventional is 2.84x10-4, under conditions of not optimizing, during 9000 bases of synthesis just
There is a mistake that [Tindall KR, Kunkel TA.Tindall KR, Kunkel TA.Fidelity of DNA occurs
synthesis by the Thermus aquaticus DNA polymerase.Biochemistry.1988 9；27(16):
6008-13.], mistake substitutes synthesis to have one section to occur in the fragment of about 100 90 bases of average synthesis.Ribonucleic acid is by four kinds
Base is constituted, and it is 2,1/7000th that the particular bases Mutation probability at an ad-hoc location occurs.And the KAPA of high-fidelity
The resultant error rate of HiFi polymerase is lower 100 times than Taq archaeal dna polymerase.On the other hand, in PCR chain reaction, previous follow
The DNA fragmentation that ring produces can be used for the DNA profiling of later reaction cycle, the DNA synthesis mistake therefore produced in a circulation
By mistake, can be inherited by circulation subsequently, in the way of index several levels, expand amplification.In PCR cycle mistake produce and produce time
Between, there is randomness, the background noise of NGS sequencing library is had significant effect, be that high-flux sequence produces background noise
Main cause.Except using high-fidelity DNA polymerase, there are two strategies can reduce nucleotide variation rate in the preparation process of library,
One is the global cycle number reducing PCR amplification, and one is the total amount improving sample medium and low frequency variant.The two must take into account ability simultaneously
The noise of reduction method background.But the blood plasma cfDNA of the rare and trace of clinical sample, has been doomed can not ad infinitum increase sample
Amount, the amplification step before order-checking is essential.A lot of to the amplification method of sequencing library at present, but all increase in amplification procedure
Variation frequency, causes higher false positive rate.The Lampyridea technology of An Keji (AccuraGen), utilizes the side of rolling circle amplification
Formula can optionally expand target fragment to introduce the cost of relatively low variation, but the follow-up library construction having remained a need for routine
And amplification step, this technology also can only to variation diagnostic sensitivity bring up to 0.02%[Lin, 2015].
Another factor having influence on NGS mensuration sensitivity is the coverage rate to surveyed target fragment, and i.e. check order the degree of depth.Survey
The sequence degree of depth directly determines the sensitivity of order-checking, and the order-checking degree of depth maximum of 100x can only provide the mensuration sensitivity of 1%.Theoretical
On, the order-checking degree of depth of secondary order-checking is unrestricted, can be easily more than 10,000x, as we utilize OmegaPlex to single
The order-checking degree of depth in site can be the highest more than 900,000, sees embodiments of the invention 4.Owing to genome is too big, about 3,200,000,000,
Being limited by sequencing throughput, mensuration price and data-handling capacity, said method is the most infeasible, must be right in practice
Library carries out selective enrichment.Existing enrichment method with lock onto target fragment, but can not reduce library noise signal, phase
Instead, it is also possible to additionally increase background, cause sequencing result that false positive occurs.
Summary of the invention
An object of the present invention, in that provide a kind of repeat replication special capture low frequency DNA base to make a variation
Method, to solve the problems referred to above.
To achieve these goals, the technical solution used in the present invention is such that a kind of repeat replication special capture
The method of low frequency DNA base variation, comprises the following steps:
(1) DNA is carried out thermal denaturation, then with the primer mixture with thermodynamics dynamic structure, target dna is carried out
Hybridization, carries out extending duplication for template with target dna with archaeal dna polymerase, repeats said process, completes to repeat template again
System；Thermal cycler is preferably employed when DNA is carried out degeneration；
(2) utilize and measure the target 3 ' oligonucleotide that mates of end and carry out spy to repeating duplicated secondary DNA fragmentation
The opposite sex extends, tailing, and the 3 ' ends producing thing secondarily introduce one section of consensus；
(3) carry out PCR amplification with the primer containing order-checking sequence of barcodes, complete the structure of sequencing library；
(4) sequencing library is carried out the parallel order-checking of high flux to generate multiple order-checking readings；
(5) sequence difference between order-checking readings and reference sequences is identified；
(6) poor with the sequence of 0.01% or higher frequency generation in the multiple readings that will obtain from described nucleic acid samples
Different it is judged to sequence variants.
As preferred technical scheme: the primer with thermodynamics dynamic structure described in step (1) is probe length
For 12nt-16nt omega primer, or the stem ring primer that probe length is 12nt-16nt, or probe length is
The omega primer of 12nt-16nt and probe length are the combination of the stem ring primer of 12nt-16nt.
As preferred technical scheme: the primer 5 ' end with thermodynamics dynamic structure described in step (1) is containing spy
Fixed combined sequence, in anchor series, sample sequence of barcodes or sequencing primer target sequence needed for the parallel order-checking of high flux
At least one.
As preferred technical scheme: step (1) described archaeal dna polymerase is high-fidelity DNA polymerase, or high-fidelity DNA
Polymerase and the combination of high efficiency polymerase.
As preferred technical scheme: step (1) described hybridization temperature is the scope of 4 DEG C to 35 DEG C, and with more than 50 DEG C
Temperature alternately.
As preferred technical scheme: step (1) described repeat replication be complete at low temperatures primer and DNA hybridization and
Extend, then carry out the process of thermal denaturation at high temperature；Or after two or more low temperatures repeatedly circulate, carry out height
Warm degeneration, described repeat replication is the repetition of once or more.
As preferred technical scheme: the primer with thermodynamics dynamic structure described in step (1) is to specific objective
The covering of fragment is two or more, completes in series.
As preferred technical scheme: the primer with thermodynamics dynamic structure described in step (1) is to specific double-strand
The covering of target fragment is for wherein chain, or for its complementary strand, or be to be simultaneous for the two.
The structure of secondary sequencing library is in the special anchor series of the two ends of DNA fragmentation to be measured assembling, sample bar code sequence
Row and sequencing primer site sequence.The primer sequence being fixed on stromal surface in anchor series sequenator secondary with Illumina is miscellaneous
Hand over and by DNA fragmentation grappling to be measured, expanded by bridge-type PCR, form DNA fragmentation bunch to be measured, read by archaeal dna polymerase synthesis
The base composition of each position, records the sequence of each fragment.Different secondary sequenators or mensuration platform are to fragment two ends
Anchor series requires different, but meets its order-checking require that the library constructing method of sequence targets does not the most limit to how to obtain
System, the present invention utilizes the primer (being also called " structural primer ") with thermodynamics dynamic structure, such as omega primer (patent
Application number: PCT/CN2013/070525), stem ring primer (Applied Biosystems, Inc, PCT/CN2013/070525)
Deng, introduce anchor series and sequencing primer target sequence, build principle and the method for sequencing library, it is adaptable to various secondary order-checkings
The structure in platform library.The method, also referred to as: OmegaPlex repeat replication detection method, for improving Sensitivity, enrichment spy
Set the goal, increase order-checking the degree of depth provide multiple facility, see Fig. 1.Multiplex PCR particular target concentration method is commonly used to particular target
The specific amplified of fragment, concentrates and analyzes fragment collection interested, can obtain the high order-checking degree of depth, but cannot change background and make an uproar
The frequency of sound, therefore helpless to low-frequency variant.The present invention uses structural omega or stem ring primer to carry out
The hybrid capture of target debris DNA and duplication, it is possible to use same fragment carries out the repeat replication of up to 200 times, is equal to
Add DNA applied sample amount Radix Achyranthis Bidentatae, see Fig. 2.Due to this duplication with same DNA fragmentation repeatedly as template, linear amplification, DNA
The resultant fault that polymerase produces is suppressed, and will not produce the amplification of mistake.In the synthesis of Taq archaeal dna polymerase, any one
It is 1/9000 that site occurs that base substitutes error rate, and the error rate of specific variation occurs in i.e. averagely the most newly synthesized DNA sequence
For 0.0037%=1/ (9000x 3).In the repeat replication method of the present invention, duplication each time all for same template, because of
This error rate every time replicated remains constant, not accumulation.
Table 1: archaeal dna polymerase base replacement mistake reasons for its use sudden change in repeat replication linear amplification (unit: ten thousand/
One).
With 10 in table 1, as a example by 000 molecular template contains the low frequency variation sample of a mutant, with mathematical calculation
Relation between the different polymerase reasons for its use signal of derivation and polymerase resultant fault rate own.It can be seen that polymerase
Unrelated with the DNA fragmentation length being replicated at the random substitution rate of a specific site, the most relevant with the resultant fault rate of enzyme itself.
As a example by Taq polymerase, producing a DNA fragmentation having random mutation, sudden change occurs the probability at specific site to be constant
0.0037%.To 10,000 DNA target repeat replications 100 times, produce 37 secondary fragments identical with target variant, but always
Body molecular number has expanded 1,000,000, and the error rate of newborn variant remains 0.0037%.If therefore with repeat replication
Mode expand, the frequency of the special variant newly increased is constant, only the most relevant with the error rate of polymerase.Therefore, use
The KAPA HiFi of fidelity or be applied in combination by different polymerases, optimizes the means such as enzyme reaction condition and reduces going out of polymerase
Error rate, can obtain a large amount of amplifications of sample size with extremely low background noise cost.Thus reduce subsequently needed for amplified library
PCR cycle number, plays the effect reducing background noise on the whole, and improves Sensitivity.On the contrary, Taq archaeal dna polymerase warp
When PCR cycle exponentially expands 10,000 DNA fragmentations 100 times, need 6-7 circulation, the probability of newborn fragment sudden change from
0.0037% to 0.50%, PCR cycle number residing when occurring to variation is relevant, because the template of PCR reaction is previous
The product of individual circulation, the sudden change occurred in early days can be inherited by recursive copying subsequently, suddenlys change and occurs the most early, to suddenly change in end-product
Percentage ratio the highest, see Fig. 2.The sudden change that PCR reaction introduces is the main cause causing secondary order-checking false positive rate high.And with same
One target carries out the mode of repeat replication can make template number be expanded in the case of producing less sudden change, improves letter
Number intensity, reduces the PCR amplification cycles number needed for the preparation of library simultaneously, reduces background signal, so that detection sensitivity is able to
Improve, see Fig. 2.
Multiplex PCR particular target concentration method carries out capture with linear primer to specific target and expands, it is difficult to avoid primer
Between dimeric formation, be not therefore suitable for the linear amplification mode of repeat replication.Omega primer is a kind of structural widow
Nucleotide primer, containing forming a length of 4-12 complementary base sequences thereof to the stem ring of base, and 3 ' 12 bases of end
Probe and probe separation district.Omega primer can be avoided dimer between startup and the primer of target spot in template DNA chain
Change, higher primer amplification efficiency and preferable specificity can be obtained with a small amount of primer, it is possible at a lower temperature
Carry out hybridizing and extending, improve synthesis specificity and sensitivity.The sequence length of omega probe is extended to 12nt 16nt
Time, omega primed probe with paratope dot blot in the chain of short segment DNA, can start the synthesis of primer and extend, therefore may be used
To be used for sheet segment DNA carries out capture duplication.Omega primer has thermodynamics dynamic structure, forms stem at a lower temperature
Ring, makes probe region independent with the sequences of 5 ' ends, as long as thus do not destroy loop-stem structure, can arbitrarily regulate 5 ' terminal sequences and
Do not affect the primer usefulness of probe.
In example of the present invention, we are at the 5 ' P5 anchor series introducing secondary order-checking held and Read1
Sequencing primer combines target sequence, improves and target fragment is built storehouse efficiency, see Fig. 1.Same, the stem ring formed under low temperature is tied
Structure, stops and forms dimer between primer, can be used to template is carried out repeat replication, and low temperature also contributes to the spirit of hybridization
The balance of the degree of depth that checks order between sensitivity and different target.
One of which implementation of the present invention illustrates as a example by omega primer.The first step that library is set up is by Europe rice
Composition needed for gal primer, sample to be tested DNA (fragment), Taq archaeal dna polymerase and synthesis is mixed together, degeneration Cheng Dan at 95 DEG C
Chain.Then when temperature is down to 60 DEG C, primer loop-stem structure is formed, and stops the hybridization extension between primer.Continue to be reduced to miscellaneous
Temperature needed for friendship, makes probe hybridize with target dna, then rises high-temperature to activate polymerase activity, completes the conjunction of DNA
Become to extend.Repeat the hybridization synthesis of above-mentioned 95 DEG C of degeneration and 4 DEG C, complete the linear amplification to target dna.Second step is special mesh
The synthesis of mark DNA, reaction utilizes and hybridizes containing the DNA primer with target dna downstream complementary series and sequencing primer sequence
And extension, produce and can carry out, with NGS anchor primer, the template that expands.It is finally to enter by the PCR primer with sample bar code
Row amplification, and mix sample bar code and anchor series, so far complete the structure to sequencing library.This invention takes sample
Linear repeat replication, greatly reduces follow-up to the PCR cycle number needed for amplified library, completes building from 1 genome (about 5pg)
Storehouse works, and only needs 12 PCR cycle.Utilizing low temperature to repeat synthesis, background signal drops to 2/100000ths, and the sensitivity in library can
Reach 0.01%.Preferably employ exo+ polymerase, such as KAPA HiFi polymerase, background signal can be lowered further, be shown in Table 1.
That compares current commercialization builds storehouse test kit or technology, and the present invention can reach the library of secondary order-checking with minimum PCR cycle
Quantity and optimal sensitivity, this is to the low frequency core in the sample that may contain a small amount of series of variation in normal sequence background
The qualification of acid variation and illustrating, and the qualification to the low frequency variation under order-checking mistake background has greatly help.
When the probe length of the stem ring primer of structural type is 5nt-8nt, has under low temperature and avoid primer dimer to generate, catch
Obtain the characteristic of short dna 3 ' end.The present invention finds, after stem ring primed probe extended length is 16nt, can be with short segment DNA
Paratope dot blot in chain, starts the synthesis of DNA.Stem ring primer through probe optimization have avoid primer dimer generate,
The characteristic of capture fragment DNA, may be used for the repeat replication of multiplex PCR.Linear primer does not the most possess this characteristic, therefore can not
Repeat replication for multiplex PCR.The present invention compares single probe omega primer, stem ring primer and linear primer to fragment
The acquisition sensitivity of DNA, it was demonstrated that the probe sequence of 16nt can reach to analyze the sensitivity of 1.6 copy numbers in sample.Further
The omega primer of different probe length is tested, finds that the acquisition sensitivity of 14 16nt probes reaches 1.6 copy numbers,
The probe sensitivity of 12nt is 33 copy numbers, and 3300 copy numbers are reduced in the probe sensitivity of 10nt.To through omega primer or stem
Check order through HiSeq and MiSeq in library prepared by ring primer, it was demonstrated that its feasibility, sees embodiment 3.
After secondary order-checking obtains order-checking readings, it is necessary to sequencing data is carried out bioinformatic analysis, from the number of magnanimity
Useful information is found out according to.In present invention application, from MiSeq or HiSeq sequencing result, first obtain FASTQ file, so
Afterwards with the software of quality testing, such as FastQC, Trimmomatic etc., sequencing result is carried out quality evaluation, and rejects matter
The amount order-checking readings less than QV30；Use BWA, bowtie or R software program by the sequence in FASTQ file and targeting reference sequence
Row (such as Kras, Braf and EGFR) are compared, and use comparison result to find the repeat region of each sequence or variation and ginseng thereof
Examine position.To find and merge with identical variation number in specific objective fragment all sequences reading, calculate variation frequency and
Frequency noise, by analyzing the QV30 of variation frequency and frequency noise and variation analysis, judges to make a variation as the positive with confirmation and dashes forward
Become or the probability of negative mutation.
Identify that the process of sequence variants includes comparing, to identify this one or more order-checking readings with reference sequences
Difference between the two, and identify contact.Generally, comparison is an order-checking readings sequence to be placed along reference sequences, sequentially
Whether ground gives a mark along two sequences match of each sequence pair, and carries out each position preferably along reference sequences
Repeat comparison.The coupling with optimal score is considered successful matching, and represents about order-checking readings sequence and reference sequence
The deduction of the degree of relationship between row.The reference sequences compared with order-checking readings is object reference genome, with reference to genome
Can be complete or incomplete.In some embodiments, with reference to genome only by the region comprising herbicide-tolerant polynucleotide
Composition, for example originating from reference to genome or the consensus sequence that is derived from the generation of analyzed order-checking readings.In some embodiments,
Reference sequences is only by the part with reference to genome, or the region composition corresponding with one or more analyzed target sequences.
In typical comparison, the base side in order-checking readings has the base of not mating in reference sequences to show at this point
There occurs replacement mutation.Similarly, when breach occurs in the base side that sequence is corresponding in its reference sequences, infer
There occurs deletion mutation.When extra base occurs in the base side that sequence is corresponding in its reference sequences, infer
There occurs insertion mutation.In some embodiments, the probable property setting number of displacement and insertion/deletion it is related to alignment score
Value.When single base is compared, coupling or do not mate cause according to replace probable property comparison score, itself it may be that
Such as, 1 for coupling 0 for not mate.Gap penalty and the probable property of displacement can be based on Heuristicses or based on about sequence such as
The a priori assumption of what sudden change.The comparison that their value impact produces.The example of the algorithm for comparing includes but not limited to
Bowtie algorithm, Smith-Waterman algorithm, Needleman-Wunsch algorithm, based on Burrows-Wheeler conversion
Algorithm and hash function comparative device such as Novoalign, ELAND, SOAP etc..
On the one hand, the invention provides a kind of method identifying low frequency series of variation, in view of the above, DNA target
Fragment can be single chain deoxynucleotide, it is possible to be double-strand Deoxydization nucleotide.Target fragment can also be RNA target fragment, for
Single-stranded nucleotide, it is possible to be double chain nucleotide.When target fragment is RNA, the synthesis of DNA complementary strand is by reverse transcription synthesis system
Retro DNA polymerase completes.And remaining step all follows the described standard method of the present invention.
On the other hand, the primer of same DNA target fragment can be carried out with the omega primer of multiple series connection simultaneously, when
Use with archaeal dna polymerase (such as: Vent (-the exo)) pairing with chain replacement activity (strain displacement), increase
Utilization rate to template, improves the sensitivity of detection further.
On the other hand, can be with multiple omega primers to same for target design primer with the complementary strand of DNA target fragment
One target expands simultaneously, increases the utilization rate to template, improves the sensitivity of detection further, and can be used for testing further
Card sudden change verity, increase low frequency variation measure credibility.
On the other hand, target present in sample can be expanded by the primer with DNA target fragment as target design,
And when To Template lacks, then will not produce non-specific amplification, in the presence of fusion gene fragment, can be expanded
Increase, take certain order-checking degree of depth, and when not this fusion gene fragment in sample, then will not produce any non-specific
Section, this characteristic of OmegaPlex sequencing can save the order-checking degree of depth.The most inevitably carry by the method for hybridization enrichment
Come Non-specific hybridization and the amplification of homologous sequence, the waste order-checking degree of depth.
The primed probe of loop-stem structure can shield PCR primer site, the initiation to DNA short-movie section.In the present invention one
Individual detailed description of the invention is to utilize the primed probe of loop-stem structure, substitutes omega primer and completes anchor series and sequencing primer
The introducing of site sequence.The experimental result of the present invention confirms, may be used under low temperature through suitably modifying the stem ring primer of design
Repeat replication to target fragment.Stem ring primer can also be used for the enforcement of the design planning of the present invention, can be with omega primer
Combination or exchange use.The design of stem ring primer needs increase extra 14-18nt base at 5 ' ends, formed containing enough
Thermodynamically stable matched sequence, in identical design of primers, stem ring primer would generally be longer 14-20nt than omega primer.
The thermodynamics dynamic structure primer of the present invention can be, but not limited to omega primer, stem ring primer；Nucleotide sample
Can be strand or double-strand；Arranging of primer can be many series connection, such as Fig. 3, it is also possible to is any in double-strand
Article one, strand, such as Fig. 4.In concrete enforcement, reference sequences is known reference sequences, by order-checking readings being carried out therewith
Comparison and the consensus sequence that formed
One of concrete application of the present invention is to can be used for the diagnosis of canceration focus in blood ctDNA.The enforcement of the present invention,
By to the capture of DNA fragmentation in blood plasma and amplification, with the flow process simplified, utilizing one or several react, easily and fast, clever
Quick and qualification pulmonary carcinoma canceration gene exactly the process that dynamically changes, helps clinician to find that extremely low concentration and extremely low sudden change are rich
The carcinogenesis XOR Resistance mutation of degree, instructs and carries out accurate medication in clinical therapy of tumor, see Fig. 5.
The two of the object of the invention, are to provide the application of a kind of said method, its technical scheme are: prepare any for examining
Survey mensuration reagent or the test kit suit of low frequency variance.The present invention can individually carry out commercialization utilization, it is also possible to makees
Ingredient for application-specific test kit.In addition, it is to be understood that read the above embodiment of the present invention teachings it
After, the present invention can be made various changes or modifications by those skilled in the art, and these equivalent form of values fall within appended by the application equally
Claims limited range.
Compared with prior art, it is an advantage of the current invention that: the present invention can reach secondary order-checking with minimum PCR cycle
Library quantity and optimal sensitivity, the detection sensitivity for the variation of low frequency DNA base can reach 0.01%, and
Demand for sample size is less, can detect corresponding target fragment in the sample of pg rank, and this is in normal sequence background
Middle may be containing the qualification of low frequency variance in the sample of a small amount of series of variation with illustrate, and at the order-checking mistake back of the body
The qualification of the low frequency variation under scape has help greatly.
Accompanying drawing explanation
Fig. 1: OmegaPlex NGS library construction flow chart；
Fig. 2: OmegaPlex repeat replication reduces the schematic diagram of library background signal；
Fig. 3: multiple series connection omega primers multiple copies schematic diagram to same target fragment；
Fig. 4: omega primer is to duplication schematic diagram with two complementary strands of DNA as target respectively；
Fig. 5: OmegaPlex low frequency variant overhaul flow chart；
The IMR-90 cell DNA that the H1299 cell of Fig. 6: ultrasonication, H549 cell DNA are broken with Fragmentase
Electrophoresis result figure；
The omega primer of Fig. 7: different probe length, stem ring primer and the efficiency of initiation comparison diagram of linear primer；
Fig. 8: the conventional BioAnalyzer 2100 chip assay method distribution of lengths analysis chart to OmegaPlex library；
Fig. 9: the fluorescent capillary electrophoresis tube assessment figure to Library Quality；
Figure 10: the repeat replication lifting result figure to method sensitivity and productivity；
Figure 11: amplification cycles number affects result figure to library yield；
Figure 12: amplification cycles number affects result figure to library yield；
Figure 13: in adding, circulation improves the order-checking degree of depth and sensitivity degree test result figure；
Figure 14: Her2_V777 region T2663 low frequency mixes the detection result figure of SNP.
Detailed description of the invention
Below in conjunction with accompanying drawing, the invention will be further described.
The mensuration analysis process that the variation of embodiment 1:OmegaPlex low frequency measures
Fragment DNA is captured, adds 3.5 μ l to the DNA sample (DNA sample weight range: 5pg-100ng) of 2 μ l purification and contain
Have the repeat replication reactant liquor of omega primer, centrifugal, be positioned over PCR instrument, run following PCR program: 95 DEG C 1 minute, 5 are followed
The capture of ring extends (8 DEG C 3 minutes, 55 DEG C 15 seconds), 95 DEG C of degeneration 15 seconds, repeats capture and extends and cycles of denaturation 40 100 times,
Enter 72 DEG C of final extensions of 10 minutes.Add 40 μ l object matching PCR reactant liquors, mix homogeneously, run at PCR thermal cycler
Following procedure: 95 DEG C 2 minutes, 5 circulation conversion extend (65 DEG C 3 minutes, 72 DEG C 1 minute, 95 DEG C 15 seconds), finally at 72 DEG C
Extend 10 minutes.With magnetic bead (Life Technologies, DynaBeads T1/C1) the purification PCR sample of streptomycin coupling,
After 2 times are washed, add 20 μ l sample bar code amplification PCR reactant liquors.Mix homogeneously, in PCR thermal cycler operation following procedure:
95 DEG C 2 minutes, 8-12 amplification cycles (65 DEG C 15 seconds, 72 DEG C 15 seconds, 95 DEG C 15 seconds), finally extend 10 minutes at 72 DEG C.Take
10 μ l PCR primer, add 2 μ l USB exoSap-IT, mixing, it then follows the explanation of manufacturer is reacted.Use Qubit
The product of purification is carried out quantitatively by 2.0DNA Quantification Assay (Life Technologies), and is aided with DNA
The fluorescent capillary electrophoresis tube such as agarose gel electrophoresis analysis, Agilent BioAnalyzer 2100 chip analysis, ABI 3730
Method carries out Analysis of quality control to clip size, the distribution of DNA product.So far, the DNA sequencing template structure of secondary order-checking sample is completed
Build, be used directly for the sequencing analysis of corresponding secondary sequenator by the DNA product of Quality Control.OmegaPlex sequencing library structure
Building principle parsing and see Fig. 1, flow process resolves sees Fig. 5.
After secondary order-checking completes order-checking reading, from MiSeq or HiSeq sequencing result, obtain FASTQ file, then use matter
The software of amount detection, such as FastQC, Trimmomatic etc., carries out quality evaluation to sequencing result, rejects quality less than QV30
Reading result.Use BWA, bowtie or R software program by the combination of the sequence in file and targeting reference sequences (as Kras,
Braf and EGFR) compare, find the repeat region of each sequence or variation and reference position thereof.To find and specific objective
In fragment all sequences reading, identical variation number merges, and calculates variation frequency and frequency noise, by variation frequency
Analyze and variation analysis with the QV30 of frequency noise, to confirm to make a variation as Positive mutants or the probability of negative mutation.This process
Automation mechanized operation can be carried out by computer language.
Described reactant liquor and archaeal dna polymerase are different because of experiment purpose in different experiments, in the present embodiment, represent
The solution compolision of property is:
Repeat replication reactant liquor containing omega primer: 10 μ l 2x JumpStart PCR buffer, 2 μ l of
100nM omega primer or mix primer, 1 μ l JumpStart Taq, 1 μ l RNase A (0.05ug), 2 μ l H20；Wherein,
Omega primer refers to number of patent application: the primer disclosed in PCT/CN2013/070525；
Object matching PCR reactant liquor: 15 μ l 2xPCR buffer, 0.2 μ l LS1980,0.2 μ l LS1976,0.6 μ l
JumpStart Taq, 15 μ l H2O, containing the primer sets of specific objective coupling；
Sample bar code amplification PCR reactant liquor: 15 μ l 2xPCR buffer, 0.3 μ l LS1985,0.3 μ l LS1959,0.6
μl JumpStart Taq,15μl H2O。
Prepared by embodiment 2:DNA, ultrasonication and enzyme fragmentation process
Blood plasma used in the embodiment of the present invention, organize or cultivate the extraction of cell DNA all with DNeasy Blood Tissue
Kit (Qiagen) processes and obtains.The method recommended in strict accordance with handbook of extracting of plasma dna performs.Cultivate what cell DNA extracted
Flow process is slightly changed on the basis of supplier's handbook.
Specific as follows: 1, the cell cultivated in 100mm plate is resuspended in the PBS of 200 μ l, join the tube pipe of 2ml,
Labelling respectively, often pipe adds 20 μ l PK；2, adding 200 μ l buffer A L, concussion 15 seconds, get off solution centrifugal the most up and down,
56 DEG C of hatching 10min, period every 3min concussions are centrifugal once；3, crushing with Ultrasonic Cell Disruptor, broken condition is intensity 40, opens 15
Second/stop 45 seconds, 2 minutes altogether；4, often pipe adds 200 μ l ethanol (96%-100%), up and down concussion 15 seconds, centrifugal；5, will be mixed
Liquid is transferred in the 2ml collecting pipe with Filter column, and 6000g is centrifuged 1 minute；6, Filter column is put in a new 2ml collect
In pipe, adding 500 μ l buffer A W1,6000g is centrifuged 1min, loses waste liquid and collecting pipe；7, Filter column is put in one new
In 2ml collecting pipe, adding 500 μ l buffer A W2,14000rpm is centrifuged 3min, loses waste liquid and collecting pipe；8, Filter column is put
In the centrifuge tube of the low absorption of DNA, add 50 μ l H2O, incubation at room temperature 1min, 6000g are centrifuged 1min；9, take out 2 μ l to use
Qubit measures concentration；10, with 2% agarose gel electrophoresis, run 100V 40 minutes, applied sample amount 1-2 μ l；
Then taking 5 μ g Fragmentase restriction endonucleases to process, system for handling is: 1 μ l restriction endonuclease buffer, 1 μ
LFragmentase, 8 μ l DNA samples, it is incubated 30 minutes at 37 DEG C.With EDTA (10 μ l systems add 2.5 μ l 0.5M EDTA)
Terminate reaction.Sample PBS after being processed by enzyme action is made into 200 μ l, adds 20 μ l E.C. 3.4.21.64s, mixing, centrifugal.Through DNeasy
Blood Tissue Kit purification again, the DNA fragmentation mean size of gained is 700-1000bp, sees Fig. 6.In Fig. 6,1:2ng
H1299 cell DNA fragment；2:2ng A549 cell DNA fragment；3:2ng IMR-90 cell DNA fragment, fragment length scope:
500–1,000bp。
Embodiment 3: the efficiency of initiation contrast of the omega primer of different probe length, stem ring primer and linear primer
For measuring different primers, and the omega primer of different probe length causes sensitivity and efficiency of initiation.We
Utilizing copy number is 1) 3,300；2)330；3)33；4)6.6；5) the H1299 cell debris DNA of 1.6 makees template, draws by difference
Thing, and the omega primer of different probe length, the sequence of closing on carrying out EGFR G719N site carries out amplification and builds storehouse.Used
Primer sequence be shown in Table 5.
Table 5: the primer sequence used by embodiment 3
In omega primer sequence, AATGATACGGCGACCACCGAGATCT is Illumina secondary order-checking platform P5 anchor
Fixed sequence；ACACTCTTTCCCTACACGACGCTCTTCCGATCT is Read1 sequencing primer site；
CAAGCAGAAGACGGCATACGAGAT is P7 anchor series；GTGACTGGAGTTCAGACGTGTGCTCT is that Read2 order-checking is drawn
Thing site.
In the present embodiment, repeat replication reactant liquor: 10 μ l 2x JumpStart PCR buffer, 2 μ l of 100nM
The omega primer of different length probe or stem ring primer or the monolithic entity of linear primer, 1 μ l JumpStart Taq, 1 μ l
RNase A(0.05ug)、2μl H20；
Object matching PCR reactant liquor: 15 μ l 2xPCR buffer, 0.2 μ l LS1984,0.2 μ l LS2060,0.6 μ l
JumpStart Taq, 15 μ l H2O, containing the primer sets of specific objective coupling.
Add, containing different copy number DNA samples, the repeat replication reactant liquor that 3.5 μ l contain various primer to 2 μ l, centrifugal, put
Be placed in PCR instrument, run following PCR program: 95 DEG C 1 minute, the capture of 40 circulations extends (8 DEG C 3 minutes, 55 DEG C 15 seconds), 72
DEG C extend 10 minutes.Add 40 μ l object matching PCR reactant liquors, PCR thermal cycler run following procedure: 95 DEG C 2 minutes, 40
Individual amplification cycles (65 DEG C 3 minutes, 72 DEG C 1 minute, 95 DEG C 15 seconds), finally extend 10 minutes at 72 DEG C.Agarose with 2% coagulates
Gel electrophoresis is analyzed, and deposition condition: 100v 30 minutes, result is shown in Fig. 7.In Fig. 7, sample is the H1299 cell of different copy number
Fragment DNA, applied sample amount is respectively as follows: 1) 3,300 copies；2) 330 copy；3) 33 copy；4) 6.6 copy；5) 1.6 copy.
In order to compare the sensitivity of various primers objectively, the present embodiment does not use the mode of repeat replication, but adopts
The sensitivity of primer is measured with the circulation under 40 low temperature.From the result of Fig. 7 it can be seen that the probe length pair of omega primer
The capture of target dna has a great impact.The omega primer of the probe of 14-16nt can reach the sensitive of odd number mesh copy
Degree.The omega primer of the probe of 12nt has had significantly reduction.And the sensitivity of the probe of 10nt is 330 copy numbers, no
Have use value.Have a stem ring primer of 16nt probe and linear primer all can reach the sensitivity of single copy number.Therefore exist
In the well-designed single or multi-PRC reaction of a small amount of target, both can be used alternatingly with omega primer.But along with
Number of targets increases, the difficulty of design can increase in logarithmic series, make linear primer no longer have actual experiment meaning, especially
The repeat replication to template cannot be used at low temperatures.In the present invention, stem ring primer can be interchangeable with omega primer,
Or be used in mixed way, it is that the sequence before probes held by 5 ' end needs of stem ring primer and 3 ' that stem ring primer is applied one of disadvantageous factor
Row complementary pairing, it is therefore desirable to the base of additional number forms double-stranded stem structure, same design, needs more than omega primer
Go out 10-20 base.Cost can be increased when this is to the detection of multiple target, and the oeverall quality of primer is had an impact.Because drawing
Thing synthesis is the process of complicated chemical polymerization, and primer is the longest, and the mistake of introducing is the most.
Embodiment 4: stem ring primer builds and sequencing analysis for the secondary sequencing library of Kras G12N fragment
In the present invention, as the test trial to OmegaPlex Method And Principle, we utilize stem ring primer thin to H1299
Single goal gene (Kras G12NG13N) site of born of the same parents and A549 cell DNA carries out the assembling synthesis of sequencing library formula.Used
Primer sequence is shown in Table 6.Primer LS1953 is (Applied Biosystems, Inc, the PCT/ of the principle design by stem ring primer
CN2013/070525), the sequence of underscore can match at low temperatures mutually, forms stably double-strand and folds, so that whole
Primer has the structure of stem, ring and probe.
Table 6: the primer sequence used by embodiment 4
In the present embodiment, repeat replication reactant liquor: 10 μ l 2x JumpStart PCR buffer, 2 μ l of 100nM
LS1953 stem ring primer, 1 μ l JumpStart Taq, 1 μ l RNase A (0.05ug), 2 μ l H20；
Object matching PCR reactant liquor: 15 μ l 2xPCR buffer, 0.2 μ l LS1980,0.2 μ l LS1976,0.6 μ l
JumpStart Taq,15μl H2O；
Sample bar code amplification PCR reactant liquor: 15 μ l 2xPCR buffer, 0.3 μ l LS1985,0.3 μ l sample bar code is drawn
Thing LS1959-LS1963,0.6 μ l JumpStart Taq, 15 μ l H2O；
Sample 1:50ng H1299 cell debris DNA；
Sample 2:50ng A549 cell debris DNA；
Sample 3:100ng H1299 cell debris DNA+1ng A549 cell debris DNA；
Sample 4:100ng H1299 cell debris DNA+0.1ng A549 cell debris DNA；
Sample 5:100ng H1299 cell debris DNA+0.01ng A549 cell debris DNA；
In the DNA sample/sample mixtures of 2 μ l purification, add 3.5 μ l contain the repeat replication reactant liquor of stem ring primer,
Centrifugal, place and PCR instrument, run following PCR program: 95 DEG C 1 minute, capture extension that 40 circulate (8 DEG C 3 minutes, 55 DEG C 15
Second), 95 DEG C of degeneration 15 seconds, enters 72 DEG C of 10 minutes finally extend.Add 40 μ l object matching PCR reactant liquors, mix homogeneously,
PCR thermal cycler run following procedure: 95 DEG C 2 minutes, 5 circulation conversion extend (65 DEG C 3 minutes, 72 DEG C 1 minute, 95
DEG C 15 seconds), finally extend 10 minutes at 72 DEG C.With magnetic bead (Life Technologies, the DynaBeads of streptomycin coupling
T1/C1) purification PCR sample, after 2 times are washed, adds 20 μ l sample bar code amplification PCR reactant liquors.Mix homogeneously, in PCR warm
Circulating instrument runs following procedure: 95 DEG C 2 minutes, 15 amplification cycles (65 DEG C 15 seconds, 72 DEG C 15 seconds, 95 DEG C 15 seconds), finally exist
72 DEG C extend 10 minutes.Take 10 μ l PCR primer, add 2 μ l USB exoSap-IT, mixing, it then follows the explanation of manufacturer is carried out
Reaction.It is fixed to carry out the product of purification with Qubit 2.0DNA Quantification Assay (Life Technologies)
Amount.
Measure chip by BioAnalyzer 2100DNA fragment and H1299Kras G12N library fragments is carried out distribution of lengths
Analyzing, result demonstrates extraordinary specificity.The library built up with conventional connection methods, the most all contains a small amount of single stranded primer
Thing and the miscellaneous band of macromole, be shown in the B in Fig. 8；And library background signal that OmegaPlex method is built up is relatively low, sees the A in Fig. 8.
Analyze through HiSeq fast chip, obtain the order-checking readings of 120 140 ten thousand respectively, all exist with reference sequences pairing rate
More than 83%, the order-checking degree of depth is more than 900,000, and it the results are shown in Table 2.Table 3 lists in A549 cell and H1299 cell DNA
The frequency that Kras G12N measures.The G12 site of H1299 cell DNA based on GGT, but also have low-frequency CGT, TGT and
AGT.The G12 site of A549 cell DNA is also based on GGT, but has the CGT of 4.75%, the TGT's and 38.08% of 4.62%
AGT.When H1299 cell DNA is mixed into the A549 cell DNA of 1%, in mixture, the mensuration frequency of CGT is 0.36%, and ratio is with pure
Have more 0.05% during H1299DNA, and this part conform exactly to that the A549 cell DNA of 1% is contributed 0.0475%.Mixture
The mensuration frequency of middle TCGT is 1.95%, and ratio has more 0.08% during with pure H1299 cell DNA, and this part conforms exactly to 1%
A549 cell DNA contributed 0.0462%.The mensuration frequency of mixture AGT is 3.86%, and ratio is during with pure H1299 cell DNA
Have more 0.53%, and this part meet that the A549 cell DNA of 1% is contributed 0.38%.Thus prove OmegaPlex order-checking side
Method can measure the resolution of 0.05%.In the present embodiment, use Taq archaeal dna polymerase, there is higher resultant fault
Rate, when replacing with exo+ polymerase, such as KAPA HiFi, can reduce background signal further.
The OmegaPlex sequencing analysis of table 3:Kras G12N fragment
Embodiment 5: agarose gel electrophoresis, Bioanalyzer 2100, fluorescent capillary electrophoresis tube method are to OmegaPlex literary composition
The com-parison and analysis of storehouse DNA mass
Agarose gel electrophoresis, Bioanalyzer 2100 are generally used for the Quality Control of secondary sequencing library, but operate bothersome
Time-consuming, flux is low.The present invention has carried out fluorescent labeling to PCR primer, utilizes fluorescent capillary electrophoresis tube method to OmegaPlex product
Carry out finer assessment.Compared with the former two, higher analysis precision is not only provided, and there is operation automatization, price
Cheaply, flux is high, the advantage that finding speed is fast.Fig. 9 is the analysis result of OmegaPlex, can show the quantitative of different band
And distribution.
Embodiment 6: repeat replication improves library yield
Utilizing copy number is 1) 330；2)33；3)6.6；4) the H1299 cell debris DNA of 3.3 makees template, uses 16nt probe
Omega primer, EGFR G719N site is closed on sequence and carries out amplification and build storehouse.Primer sequence used is shown in Table 7.At this
In test, compare repeat replication and the conventional copy mode impact on library productivity.Except condition described below, tool
The operation of body is identical with described in test experiments 1.
Table 7: the primer sequence used by embodiment 6
The conventional capture replicated extends condition and is: 95 DEG C 1 minute, the capture of 40 circulations extends (8 DEG C 3 minutes, 55 DEG C 30
Second), 72 DEG C extend 10 minutes；
The capture of repeat replication extends condition: 95 DEG C 1 minute, the capture of 20 circulations extends (8 DEG C 3 minutes, 55 DEG C 30
Second, 95 DEG C 15 seconds), 72 DEG C extend 10 minutes.
Result shows, the conventional mode replicated can be measured to the sample of 33 copies, and the mode of repeat replication is permissible
Reach the sensitivity of 3.3 copies.Two ways all shows the positive correlation of library yield and template number.The weight of 20 times
Multiple duplication can promote nearly 10 times of productivity, sees Figure 10.
Embodiment 7: interior circulation increases the amplification concordance in library
The present embodiment checking object matching PCR cycle number, and sample bar code amplification PCR reaction cycle number is to library productivity
With the relation between the relation of quality, and repeat replication temperature and productivity, with optimization optimal proportion.Primer sequence used
It is shown in Table 8a, 8b.
Table 8a:OmegaPlex-1 primer sets
Table 8b:LPM-314 primer sets
Step is: captures fragment DNA, adds 3.5 μ l and contain Europe in 2 μ l (5ng) IMR-90 cell debris DNA samples
The repeat replication reactant liquor of rice gal primer, centrifugal, place and PCR instrument, run following PCR program: 95 DEG C 1 minute, 5 circulate
Capture extends (35 DEG C 3 minutes, 55 DEG C 15 seconds), 95C degeneration 15 seconds, repeats capture and extends and cycles of denaturation 100 times, enters 72 DEG C
The final extension of 10 minutes.Add 40 μ l object matching PCR reactant liquors, mix homogeneously, run following journey at PCR thermal cycler
Sequence: 95 DEG C 2 minutes, n time circulation conversion extend (65 DEG C 3 minutes, 72 DEG C 1 minute, 95 DEG C 15 seconds), finally 72 DEG C extend 10
Minute.With magnetic bead (DynaBeads T1/C1, Life Technologies) the purification PCR sample of streptomycin coupling, wash through 2 times
After washing, add 20 μ l sample bar code amplification PCR reactant liquors.Mix homogeneously, in PCR thermal cycler operation following procedure: 95 DEG C 2 points
Clock, m amplification cycles (65 DEG C 15 seconds, 72 DEG C 15 seconds, 95 DEG C 15 seconds), finally extend 10 minutes at 72 DEG C.Take 10 μ l PCR to produce
Thing, adds 2 μ l USB exoSap-IT, mixing, it then follows the explanation of manufacturer is reacted.Use Qubit 2.0DNA
The product of purification is carried out quantitatively by Quantification Assay (Life Technologies), and it is solidifying to be aided with DNA agarose
The fluorescent capillary electrophoresis tube methods such as gel electrophoresis analysis, ABI 3730 carry out Analysis of quality control to clip size, the distribution of DNA product.n
For period, it is equal to: 5,10, or 15, m is period, equal to 5, and 10, or 15.
In the present embodiment, repeat replication reactant liquor: 10 μ l 2x JumpStart PCR buffer, 2 μ l of 100nM
OmegaPlex-1,1 μ l JumpStart Taq, 1 μ l RNase A (0.05ug), 2 μ l H20；
Object matching PCR reactant liquor: 6 μ l 5xHiFi buffer, 0.3 μ l LS1980 [biotin], 0.3uM LPM-
314,0.6μl KAPA HiFi,1μl dNTP(10mM),24μl H2O.Primer sets containing specific objective coupling；
Sample bar code amplification PCR reactant liquor: 6 μ l 5xHiFi buffer, 0.3 μ l LS1985,0.3 μ l sample bar code is drawn
Thing, 0.6 μ l KAPA HiFi, 1 μ l dNTP (10mM), 24 μ l H2O；
As a example by Illumina platform, required sample is 2ng/ μ l.Experimental result shows, circulates (5 through minimum 10
Join circulation+5 amplification cycles) OmegaPlex yield be 1.75ng/ μ l, cumulative volume 20 μ l, can meet the requirement of loading.
Increasing PCR cycle sum, the yield in library is consequently increased, and sees Figure 11.Sample carried out point with fluorescent capillary electrophoresis tube simultaneously
Analysis, in the range of being tested, period is the least on the impact of each fragment yield, and difference can be ignored, and result is shown in Figure 12.Often
The increase of individual PCR cycle, always causes the rising of background level, and therefore controlling PCR cycle sum is to reduce having of background level
One of efficacious prescriptions formula.In this test, DNA sample amount is 5ng, the DNA fragmentation that the amplification through 10 15 circulations is obtained, yield
Between 1.75ng/ μ l 8.11ng/ μ l, fully meet follow-up Quality Control, sequencing demands.
It addition, during repeat replication, add the shadow that the interior circulation of low temperature, the degree of depth that also checks order library and sensitivity are useful
Ring, see Figure 13.Sample 1 and sample 13 are same sample, and 5ng H1299 cell debris DNA, sample 1 is through 100 repeat replications
Circulation (16 DEG C of x 1 second 55 DEG C 10 seconds 95 DEG C 15 seconds), and sample 13 circulate through 100 repeat replications, addition in each circulation
5 interior circulations [(16 DEG C x 1 second 55 DEG C 10 seconds) 5 circulations of x 95 DEG C 15 seconds] of 16 DEG C to 55 DEG C.The fluorescence hair of sample
Cons electrophoresis analysis result shows, in adding, the homogeneity of the sample 13 of circulation exceedes the sample 1 not having interior circulation.
The order-checking precision of embodiment 8:OmegaPlex and the order-checking degree of depth
Utilizing reaction condition in the same manner as in Example 7, (primer sequence is listed in table 8a and table to same primer mixture
8b), through 5 pairing circulations and 15 bar code amplification cycles, we are prepared for IMR-90 cell and IMR-90 cell and A549
The DNA mixing sample of cell and the library of plasma dna sample.And the library MiSeq of preparation is analyzed, result arranges
In table 4.
FASTQ file is obtained, through fastQC quality analysis, QV30 from MiSeq > 85%.4 libraries obtain 50 230 respectively
The readings that effectively checks order of ten thousand, in addition to plasma dna sample, with reference sequences pairing rate all more than 31%, averagely check order the degree of depth
More than 10,000, it is shown in Table 4.The order-checking degree of depth of Her2_V777L fragment reaches more than 80,000, and 4 sample frequency noise statistical results are respectively
For: the BACKGROUNDFrequency meansigma methods that N > G changes is 0.0050% (0.0045%, 0.0066%, 0.0040%, 0.0048%)；N>C
The BACKGROUNDFrequency meansigma methods of conversion: 0.0050% (0.0041%, 0.0072%, 0.0045%, 0.0044%).N > T conversion
BACKGROUNDFrequency meansigma methods: 0.0064% (0.0047%, 0.0116%, 0.0047%, 0.0048%), the background frequency of N > A conversion
Rate meansigma methods: 0.0054% (0.0039%, 0.0100%, 0.0040%, 0.0038%).First of next-door neighbour's omega probe
The mutation frequency of base is more abnormal, hence it is evident that higher than meansigma methods, eliminates the frequency in this site to general background in statistics
Impact.Check analysis IMR-90 sample, can measure the gene SNP T2663 of the A549 that low frequency mixes.SNP T2663 detection
Frequency is 0.032% and 0.03%, sees Figure 14.Although adding the Europe rice of the fusion gene fragment of 9 couples of EML4-ALK in Shi Yan
Gal primer, but in all samples, all find no any fragment matched therewith.
Table 4: the OmegaPlex sequencing result of cell and plasma dna
Should be appreciated that, after the teachings having read the above embodiment of the present invention, those skilled in the art are permissible
Making various changes or modifications the present invention, these equivalent form of values fall within the model that the application appended claims is limited equally
Enclose.
Claims (9)
1. a special capture the method for repeat replication low frequency DNA base variation, it is characterised in that comprise the following steps:
(1) DNA is carried out thermal denaturation, then with the primer mixture with thermodynamics dynamic structure, target dna is hybridized,
Carry out extending duplication for template with target dna with archaeal dna polymerase, repeat above-mentioned degeneration and crossover process, complete the weight to template
Replicate；
(2) utilize and measure the oligonucleotide that target 3 end mates and carry out specificity to repeating duplicated secondary DNA fragmentation
Extension, tailing, and produce 3 ends one section of consensus of introducing of thing secondarily；
(3) carry out PCR amplification with the primer containing order-checking bar code, complete the structure of sequencing library；
(4) sequencing library is carried out the parallel order-checking of high flux to generate multiple order-checking readings；
(5) sequence difference between order-checking readings and reference sequences is identified；
(6) sequence difference occurred with 0.01% or higher frequency in the multiple readings that will obtain from described nucleic acid samples is sentenced
It is set to sequence variants.
Method the most according to claim 1, it is characterised in that: there is thermodynamics dynamic structure described in step (1)
Primer be probe length be 12nt-16nt omega primer, or the stem ring primer that probe length is 12nt-16nt, or
Be probe length be omega primer and the probe length of 12nt-16nt be the combination of the stem ring primer of 12nt-16nt.
Method the most according to claim 1, it is characterised in that: there is thermodynamics dynamic structure described in step (1)
Primer 5 end contains specific combined sequence, for anchor series, sample sequence of barcodes or sequencing primer needed for the parallel order-checking of high flux
At least one in target sequence.
Method the most according to claim 1, it is characterised in that: step (1) described archaeal dna polymerase is high-fidelity DNA polymerization
Enzyme, or the combination of high-fidelity DNA polymerase and high efficiency polymerase.
Method the most according to claim 1, it is characterised in that: step (1) described hybridization temperature is the model of 4 DEG C to 35 DEG C
Enclose, and the temperature with more than 50 DEG C is alternately.
Method the most according to claim 1, it is characterised in that: step (1) described repeat replication is to complete at low temperatures to draw
The hybridization of thing and DNA and extension, then carry out the process of thermal denaturation at high temperature；Or carry out many at two or more low temperatures
Carrying out high-temperature hot degeneration after secondary circulation, described repeat replication is the repetition of once or more.
Method the most according to claim 1, it is characterised in that: there is thermodynamics dynamic structure described in step (1)
Primer is two or more to the covering of specific objective fragment, completes in series.
Method the most according to claim 1, it is characterised in that: there is thermodynamics dynamic structure described in step (1)
Primer is for wherein chain to the covering of specific double stranded target fragment, or for its complementary strand, or be simultaneously
For the two.
9. the application of the method described in claim 1-8 any one, it is characterised in that: preparation is used for detecting low frequency DNA alkali
The mensuration reagent of base variation or test kit suit.
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| CN111534569A (en) * | 2020-05-29 | 2020-08-14 | 安徽安龙基因科技有限公司 | Oligonucleotide primer, kit and application |
| WO2021203461A1 (en) * | 2020-04-09 | 2021-10-14 | 江苏先声医学诊断有限公司 | Position anchoring bar code system for nanopore sequencing library construction |
| CN115125314A (en) * | 2022-06-01 | 2022-09-30 | 四川大学华西医院 | Detection method for base heterozygosis in heterogeneous drug-resistant bacteria |
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| CN110699426B (en) * | 2019-01-02 | 2022-01-28 | 上海臻迪基因科技有限公司 | Gene target region enrichment method and kit |
| CN114250269A (en) * | 2021-12-28 | 2022-03-29 | 上海市肺科医院 | Probe composition, second-generation sequencing library based on probe composition and application of second-generation sequencing library |
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