CN105861710B - Sequence measuring joints, its preparation method and its application in ultralow frequency variation detection - Google Patents
Sequence measuring joints, its preparation method and its application in ultralow frequency variation detection Download PDFInfo
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Abstract
The invention provides sequence measuring joints, its preparation method and its application in ultralow frequency variation detection.Wherein, sequence measuring joints include amplified library primer sequence, purpose fragment amplimer sequence and the miscue sequence being sequentially connected, miscue sequence is located at close to the side of purpose fragment, amplified library primer sequence is located remotely from the side of purpose fragment, and miscue sequence is the sequence of known base sequence.By being additionally arranged the miscue sequence of known base sequence close to the side of purpose fragment, miscue sequence can be that the DNA profiling of each double-strand adds distinctive exogenous marker.After the sequencing data for being easy to subsequently to obtain purpose fragment, whether identical miscue sequence screening is carried according to sequencing sequence or rejects the mutation that sequencing introduces in itself or in amplified library step, then will be defined as really being mutated in the site that making a variation all occur in two chain same positions, and amplification or sequencing error are regarded as into the site that only a chain has mutation, so as to improve variation accuracy of detection.
Description
Technical field
The present invention relates to high-throughput sequencing library to build field, in particular to a kind of sequence measuring joints, its preparation method
And its application in ultralow frequency variation detection.
Background technology
With the progressively maturation of high throughput sequencing technologies, its application field is also more and more extensive, can not only realize by big
Detection of the sample to trace sample is measured, and is also achieved by the detection of high frequency mutation to ultralow frequency variation.But due at present most
The error rate of the accurate single base of high-flux sequence in itself is just 10-3~10-2Left and right, along with well-known DNA gathers
The inherited error rate (10 of synthase-7~10-5), thus, conventional experiment banking process is impossible to accomplish 1% even more at present
High accuracy of detection, and this error make it that we are difficult to detect that some rare variations (are called and do ultralow frequency variation, dash forward
Variability is 0.5%~0.1%).Although at present had it is a variety of for ultralow frequency variation it is improved build storehouse sequence measurement, in reality
Many limitations still be present in the application of border.
The existing method for low frequency detection class is roughly divided into following three class, and its feature and corresponding limitation are as follows：
First, specific enrichment mutating molecule
Representational method includes the Multiplexed ICE COLD-PCR (MX- that Transgenomic companies use
ICP) and Boreal companies Synchronous coefficient of drag alteration (SCODA) technology, institute
Need sequencing data amount relatively low, but need extra instrument to be used for being enriched with mutating molecule, and be only used for detecting mononucleotide
Polymorphism (SNP), the extra buying of instrument and the single application for limiting the above method for detecting species.
2nd, overlap correction and information analysis optimization
Deep sequencing analysis method (the Cancer Personalized Profiling by deep 1. individuation is filed
Sequencing CAPP-seq) experiment flow do not carry out big change, simply to information on the premise of deep sequencing
Analysis process has carried out some optimizations, and this method can not eliminate the error that experimental method is brought；
2. by the original reads of lap that both-end is sequenced carry out overlap correction reduce sequencing error rate come
Accuracy of detection is improved, but this method can not eliminate the mistake that banking process is brought.
3rd, unimolecule rolling circle amplification (circle sequencing)
Template molecule is repeatedly expanded using the form of unimolecule rolling-circle replication, one kind is formed and derives from same template
The long-chain that links together of sequence, long-chain is entered to the structure in the laggard style of writing storehouse of Break Row processing, then to these from same
The amplicon of one template is sequenced, and the amplified production of the same template in these sources is carried out by the information analysis method in later stage
Analyze to reduce the mistake that sequencing and experiment are brought.But this method application has two limitation points：1) single chain molecule is cyclized
It is less efficient, template can be caused to lose for trace sample；2) long sequencing reading length is needed.
Thus, it is still necessary to the storehouse detection method of building of existing ultralow frequency variation is improved, ultralow frequency become with improving
Different accuracy of detection.
The content of the invention
It is a primary object of the present invention to provide a kind of sequence measuring joints, its preparation method and its in ultralow frequency variation detection
Application, to solve the problems, such as that accuracy of detection is not high enough in the prior art.
To achieve these goals, according to an aspect of the invention, there is provided a kind of sequence measuring joints, the sequence measuring joints bag
Include the amplified library primer sequence being sequentially connected, purpose fragment amplimer sequence and miscue sequence, miscue sequence
Row are located at is located remotely from the side of purpose fragment close to the side of purpose fragment, amplified library primer sequence, wherein, miscue
Sequence is the sequence of known base sequence.
Further, sequence measuring joints are Y-shaped joint.
Further, Y-shaped joint includes the first chain and second chain complementary with the first chain part, the first chain and the second chain
In the length of longer chain be 30~67bp, preferably 40~50bp, it is further preferred that the sequence of the first chain is SEQ ID NO:1：
5 '-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNT-3 ', the sequence of the second chain is SEQ ID NO:2：5’-
NNNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3 ', wherein, the sequence that 6 N are formed represents base sequence
Known miscue sequence.
Further, the length of miscue sequence is 4~12bp, preferably 6bp.
Further, the length of amplified library primer sequence is 15~30bp.
According to another aspect of the present invention, a kind of preparation method of sequence measuring joints is additionally provided, the preparation method includes：
Two single stranded sequences of sequence measuring joints are designed, include miscue sequence in every single stranded sequence, and miscue sequence is located at
For single stranded sequence close to the side for the purpose fragment to be connected, miscue sequence is the sequence of known base sequence；Close respectively
Into two single stranded sequences；Two single stranded sequences of synthesis are subjected to specific annealing, obtain the sequence measuring joints of double-strand.
Further, sequence measuring joints are multipair that preparation method also includes after the sequence measuring joints of double-strand are obtained：Will be multipair
Sequence measuring joints carry out equal proportion mixing, the step of the sequence measuring joints mixed.
According to an aspect of the present invention, a kind of sequence measuring joints are additionally provided, the sequence measuring joints pass through any of the above-described kind of system
Preparation Method is prepared.
According to a further aspect of the invention, a kind of kit for building ultralow frequency variation sequencing library is additionally provided, should
Kit includes sequence measuring joints, and the sequence measuring joints are any of the above-described kind of sequence measuring joints, or any of the above-described kind of sequence measuring joints
The sequence measuring joints that preparation method is prepared.
According to a further aspect of the invention, a kind of construction method of ultralow frequency variation sequencing library, the structure are additionally provided
Construction method includes：Step S1, build the sequencing library containing purpose fragment；Step S2, hybrid capture is carried out to sequencing library, obtained
Library after to capture；And step S3, library after capture is expanded, obtains purpose library；Wherein, using upper in step S1
State sequencing library of the kit structure containing purpose fragment of structure ultralow frequency variation sequencing library.
According to another aspect of the present invention, a kind of ultralow frequency variation sequencing library is additionally provided, ultralow frequency variation is surveyed
Preface storehouse is built-up using the construction method of above-mentioned ultralow frequency variation sequencing library.
Present invention also offers a kind of detection method of ultralow frequency variation, the detection method includes：Above-mentioned ultralow frequency is become
Different sequencing library carries out high-flux sequence, obtains sequencing data；And variant sites analysis is carried out to sequencing data, made a variation
Site data.
Apply the technical scheme of the present invention, by the inner side of purpose fragment sequencing primer (close to the side of purpose fragment)
The miscue sequence with known base sequence that purpose template can be marked is additionally arranged, miscue sequence is every
The DNA profiling of individual double-strand adds distinctive exogenous marker, because the base sequence of the exogenous marker is known, is easy to follow-up obtain
To after the sequencing data of purpose fragment, whether identical miscue sequence screening is carried according to sequencing sequence or rejects sequencing originally
The mutation for all occurring being mutated in the same position of two chains, is then defined as by the mutation introduced in body or amplified library step
Real mutation, and experimental error or sequencing error are regarded as in the mutation that only a chain has mutation, are improved very so as to realize
The accuracy of detection being mutated in fact.
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 show according to an embodiment of the invention 1 and reference examples method constructed by library electrophoresis detection result
Figure；And
Fig. 2A to Fig. 2 D shows the storehouse inspection knot in the library constructed by different samples in a kind of preferred embodiment of the present invention
Fruit；Wherein, the 2100 detection figures that Fig. 2A is library YF00CTDNACL00003 (0.1%)；Fig. 2 B are library
YF00CTDNACL00005 (5%) 2100 detection figures；2100 detections that Fig. 2 C are library YF00CTDNACL00006 (0.1%)
Figure；The 2100 detection figures that Fig. 2 D are library YF00CTDNACL00007 (5%).
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.The present invention is described in detail below in conjunction with embodiment.
It should be noted that joint sequence of the prior art generally includes amplified library primer sequence, purpose fragment expands
Increase primer sequence and sample label sequence (i.e. index sequences).Wherein, sample label sequence is expanded positioned at purpose fragment primer
Among increasing sequence and amplified library sequence, and purpose fragment amplimer sequence is the sequence being joined directly together with purpose fragment,
And amplified library sequence is the outermost sequence of purpose amplified fragments, such as P5 the and P7 sequences of Illumina microarray datasets.And this
Miscue sequence in application, also referred to as molecular template recognition sequence, its position are drawn in purpose fragment and purpose fragment amplification
Between thing sequence, i.e., miscue sequence is the sequence being directly connected to purpose fragment.
As background section is previously mentioned, the accuracy of detection that pin experiment flow makes a variation to ultralow frequency at present is not high enough, is
The above-mentioned present situation of improvement, in a kind of preferred embodiment of the application, there is provided a kind of sequence measuring joints, the sequence measuring joints include
Amplified library primer sequence, purpose fragment amplimer sequence and the miscue sequence being sequentially connected, miscue sequence
The side of purpose fragment is located remotely from positioned at close to the side of purpose fragment, amplified library primer sequence, wherein, miscue sequence
It is classified as the sequence of known base sequence.
The sequence measuring joints of the application in the inner side of purpose fragment sequencing primer (close to the side of purpose fragment) by setting up
What purpose template can be marked has the miscue sequence of known base sequence, and miscue sequence is each double
The DNA profiling of chain adds distinctive exogenous marker, because the base sequence of the exogenous marker is known, is easy to subsequently obtain mesh
Fragment sequencing data after, according to sequencing sequence whether with identical miscue sequence screening or reject sequencing in itself or
The mutation for all occurring being mutated in the same position of two chains, is then defined as truly by the mutation introduced in person's amplified library step
Mutation, and experimental error or sequencing error are regarded as in the mutation that only a chain has mutation, so as to realize improve it is true prominent
The accuracy of detection of change.
Above-mentioned sequence measuring joints are similar with existing amplified library sequence measuring joints, according to specifically used microarray dataset not
Together, it can be different existing double-stranded adapters or the double-stranded adapters being suitably modified according to existing double-stranded adapters, may be used also
To be the double-stranded adapters of experimenter's designed, designed.In this application, preferably upper sequencing chain joint is that widely used Y-shaped connects
Head.Similar with existing Y-shaped joint, Y-shaped joint includes the first chain and second chain complementary with the first chain part, and two
Generally there is the length of a chain relatively long in bar chain, in a kind of preferred embodiment of the application, preferred that longer chain
Length be 30~67bp, preferably 40~50bp, it is further preferred that the sequence of the first chain is：SEQ ID NO:1：5’-
ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNT-3 ', the sequence of the second chain are：SEQ ID NO:2：5’-
NNNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3 ', wherein, the sequence that N is formed has represented base sequence
The miscue sequence known.
For miscue sequence in above-mentioned sequence measuring joints length according to be actually needed carry out Reasonable adjustment, not
It is confined to a certain specific length.Miscue sequence length is longer, and sequence polymorphism is more, the template molecule that can be marked
It is more, but sequence is longer, takes the length for the target DNA molecule that the length of the sequencing data of output is also longer, actually obtains
Will accordingly it shorten.The length of miscue sequence is 4~8bp, preferably 6bp in a kind of preferred embodiment of the application.
By the control of miscue sequence length in the range of 4~8bp, the template DNA molecule used in all experiments is disclosure satisfy that substantially, and
And the influence of the length of the target DNA molecule to actually obtaining is relatively small.
In the application another kind preferred embodiment, it is used to expand purpose fragment sequencing primer sequence by above-mentioned
Sequence length control in the range of 5~20bp.So contribute to shorten the total length of constructed sequence measuring joints, so as to subtract
The mistake that few sequence measuring joints introduce in PCR amplifications, sequencing procedure, and cause the valid data amount for reducing sequencing output.
In another typical embodiment of the application, a kind of preparation method of sequence measuring joints, the preparation are additionally provided
Method includes：Two single stranded sequences of sequence measuring joints are designed, include miscue sequence in every single stranded sequence, and the mistake carries
Show that sequence is located at single stranded sequence close to the side for the purpose fragment to be connected, miscue sequence is the sequence of known base sequence
Row；It is respectively synthesized two single stranded sequences；Two single stranded sequences of synthesis are subjected to specific annealing, the sequencing for forming double-strand connects
Head.
Synthesis in above-mentioned preparation method refers to that chemical synthesis is typically that the company serviced by offer chemical synthesis synthesizes.And
Abiotic synthesis, i.e., do not refer to by the way that using a chain as template, another is synthesized by way of replicating and extending.The application
Above-mentioned sequence measuring joints preparation method, pass through every be respectively synthesized according to experiment demand in double-strand sequence measuring joints single-stranded sequence
Row, the sequence measuring joints of double-strand are then formed by way of specificity is annealed again.The joint preparation method is simple, and due to closing
Into single stranded sequence be to be synthesized according to pre-designed sequence, thus the miscue sequence carried on each joint
It is to determine.And then for build library to ultralow frequency variation detect when, can by by each template DNA molecule just
The base mutation that same position occurs on adopted chain and antisense strand is defined as really being mutated, and occurs independent on a chain
Mutation or the generation base mutation of diverse location on positive-sense strand and antisense strand are defined as pseudomutation.It just can so exclude text
Storehouse, which expands, or sequencing mistake is caused is mutated, and so as to improve the accuracy of detection, and helps to reduce false sun
Property rate.
Relative to conventional sequence measuring joints, the sequence measuring joints that the above-mentioned preparation method of the application is provided are used for abrupt climatic change
When, PCR mistakes, the sequencing mistake of target fragment can be not only identified, the miscue of known base sequence can also be passed through
Sequence, the self of miscue sequence is realized, so as to improve accuracy of detection.
In above-mentioned preparation method, each pair is respectively obtained by way of synthesizing single-stranded and single-stranded annealing forms double-stranded adapters
After sequence measuring joints, according to experimental implementation custom and the difference of experiment purpose, two steps are segmented into when using sequence measuring joints
The joint at both ends is connected, can also step completion connection.In the case where a step completes connection, surveyed to further improve both ends
The homogeneity of various different miscues entrained by sequence joint, in a kind of preferred embodiment of the application, sequence measuring joints have
Multipair, after the sequence measuring joints of double-strand are obtained, above-mentioned preparation method also includes：Multipair double-stranded adapters progress equal proportion is mixed
Close, the step of the sequence measuring joints mixed.
In above-mentioned preferred embodiment, when being connected using above-mentioned mixing sequence measuring joints to purpose fragment, to various different wrong
That misses prompting sequence utilizes probability equal, makes different mistakes on the DNA molecular fragment band identical from genome diverse location of source
The diversity increase of prompting sequence, and then the same template DNA molecular from diverse location can be excluded in first position
The base mutation of a certain position is dashed forward with the identical base of same position in the antisense strand of the second place and is erroneously interpreted as in positive-sense strand
Positive mutants, so as to improve accuracy of detection.
In another typical embodiment of the application, a kind of sequence measuring joints are additionally provided, the sequence measuring joints are using upper
Any preparation method is stated to be prepared.In the application in another typical embodiment, it is ultralow to additionally provide a kind of structure
The kit of frequency variation sequencing library, the kit include any of the above-described kind of sequence measuring joints or any of the above-described kind of preparation method
The sequence measuring joints being prepared.Above-mentioned sequence measuring joints or kit, have specific base suitable by being pre-designed, known
The sequence measuring joints that sequence is formed, are easy to the mutation introduced to experimental error subsequently during analyzing sequencing data to enter
Row screening and rejecting, improve the accuracy of true abrupt climatic change.
In another typical embodiment of the application, a kind of structure side of ultralow frequency variation sequencing library is additionally provided
Method, the construction method include：Step S1, build the sequencing library containing purpose fragment；Step S2, hybridizes to sequencing library
Capture, library after being captured；And step S3, library after capture is expanded, obtains purpose library；Wherein, step S1
It is middle that the sequencing library containing purpose fragment is built using mentioned reagent box.
The joint being prepared by using the preparation method of above-mentioned sequence measuring joints has sequence certainty, is easy to differentiation to connect
Header sequence builds base mutation introduced in storehouse or sequencing procedure in amplification, and then can exclude the mistake in laboratory operating procedures
Difference, meanwhile, the determination being mutated more conducively in subsequent analysis step to true positives and false positive is constructed so as to be advantageous to improve
The accuracy of detection that sequencing library makes a variation to ultralow frequency.
In the application in another typical embodiment, a kind of ultralow frequency variation sequencing library is additionally provided, this is ultralow
Frequency variation sequencing library is built-up using above-mentioned construction method, the detection that ultralow frequency variation sequencing library makes a variation to ultralow frequency
Rate is improved, and false positive rate reduces, and accuracy of detection is greatly enhanced.
In the application another preferred embodiment, a kind of detection method of ultralow frequency variation, the inspection are additionally provided
Survey method includes above-mentioned ultralow frequency variation sequencing library carrying out high-flux sequence, obtains sequencing data；Sequencing data is carried out
Variant sites are analyzed, and obtain variant sites data.The detection method is come by using the miscue sequence with known array
Each template DNA molecule is marked, during variant sites analysis is carried out to sequencing data, same DNA can not only be passed through
The miscue sequence at template molecule both ends excludes mistake that target DNA molecule is introduced into PCR amplifications, drawn in sequencing procedure
The mistake entered, but also whether the miscue sequence that can be obtained by detection sequencing is consistent with known base sequence come to mistake
Prompting sequence carries out self or rejecting by mistake, so as to improve the precision of variation testing result.
The above-mentioned sequence measuring joints of the application and its application in ultralow frequency abrupt climatic change, all species can be applied to
Various ultralow frequency mutation present in (such as plant, microorganism, animal or people) nucleotide, including single nucleotide mutation, insertion
Missing, Gene Fusion, copy number variation and chromosome translocation etc..During application, used sample DNA can be
The diversified forms such as tissue (FFPE) sample of FFPE, cell line, blood, urine, tissue fluid, saliva after formalin is fixed
DNA.
Further illustrate the beneficial effect of the application below in conjunction with specific embodiments.It is it should be noted that following
Agents useful for same in embodiment is commercially available prod unless otherwise indicated.Wherein, concentration unit μM is μM/L abbreviation.It is listed
Sequence unless otherwise specified, each means the sequence from 5 ' ends to 3 ' extreme directions.
The following example chooses the plasma sample (sample A, sample B) of two normal persons, and it is laggard that storehouse is built in the conventional capture of progress
Row sequencing and analysis of biological information, obtain the positive collection of two normal specimens, select two samples each distinctive 30 it is pure
Close site and be used for accuracy evaluation.
The present invention carries out the dilution of different proportion using A samples to B samples, finally gives the positive site frequency of 30, B samples
Rate is respectively 5% and 0.1% two samples, is respectively designated as sample 1 and sample 2.Using existing literature method as reference examples,
The present invention carries out detection ratio to the sample of two kinds of frequencies of mutation and detection frequency carries out statistical analysis, and idiographic flow is as follows：
First, joint synthesizes
1. the preparation method of reference examples joint (1)
(1) primer synthesizes
S51：SEQ ID NO:3
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT CT,
S55:SEQ ID NO:4
TCTTCTACAGTCAAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC。
Remarks：Thickened portion is the reverse complemental region of two chains.
(2) anneal
The powder of two primers is diluted using nuclease-free water, the mother liquor of 100uM concentration is obtained, takes equivalent respectively
2 primers in PCR pipe, be placed on ABI 2720PCR instrument and carry out Gradient annealing, 95 DEG C are down to 25 DEG C, per second to decline 0.1
℃.Reaction system such as table 1 below.
Table 1：
(3) extend
According to the form below 2 configures reaction solution in PCR pipe, and with rifle, gently pressure-vaccum mixes up and down.ABI 2720PCR instrument is put into,
37 DEG C of incubation 30min.
Table 2：
(4) absolute ethyl alcohol precipitates, purifying
Product after extension is transferred in 1.5ml centrifuge tube, adds 7 μ L 3M NaAc (PH 5.2) and 192.5 μ L
The absolute ethyl alcohol of precooling, after -20 DEG C are placed 30min, 12000g, 4 DEG C of centrifugation 30min, 75% ethanol washed once, dries.Add
Enter the water dissolving of 100 μ L nuclease frees.
(5) digestion
According to the form below 3 configures reaction solution in PCR pipe, and with rifle, gently pressure-vaccum mixes up and down.It is put into PCR instrument, 37 DEG C of incubations
16h。
Table 3：
(6) absolute ethyl alcohol precipitates, purifying
Product after digestion is transferred in new 1.5ml centrifuge tubes, adds 20 μ L 3M NaAc (PH 5.2) and 550 μ L
The absolute ethyl alcohol of precooling, after -20 DEG C are placed 30min, 12000g, 4 DEG C of centrifugation 30min, 75% ethanol washed once, dries.Add
Enter the water dissolving of 40 μ L nuclease frees.
The joint of embodiment 1 2. (2) preparation method
(1) joint sequence synthesizes
ERROR-top：ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNT(SEQ ID NO:5)；
ERROR-bot：P*NNNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC(SEQ ID NO:6：).It is standby
Note：P* is phosphorylation modification
The embodiment 1 carries out specifically being provided with 100 kinds specifically for the miscue sequence of N section, ERROR-bot labels
Sequence is with corresponding ERROR-top miscue sequence reverse complementals.Wherein, table 4 shows 100 kinds of ERROR-top sequences.Synthesis
When ERROR-top and corresponding ERROR-bot are respectively synthesized respectively.Wherein, in 7 bases that runic is shown, except last position
T be in order to purpose fragment 3 ' end protrusion A be connected outside, remaining 6 base is miscue sequence.
Table 4：
(2) powder of every primer is diluted using nuclease-free water, obtains the mother liquor of 100uM concentration, take respectively
The primer of the complementary pairing of equivalent is placed on ABI 2720PCR instrument in PCR pipe and carries out Gradient annealing, and 95 DEG C are down to 25 DEG C,
It is per second to decline 0.1 DEG C.Reaction system such as table 5 below.
Table 5：
(3) annealed 100 kinds of double-stranded adapters sequences are subjected to equal proportion mixing, joint completes.
2nd, plasma DNA (cfDNA) sample preparation
Each 24ml blood plasma of two Healthy Peoples of A, B is extracted using Qiagen plasma frees nucleic acid extraction kit.Then will carry
The cfDNA taken is mixed by 5% and 0.1%, obtains sample 1 and sample 2, and sample size is all 60ng, wherein 30ng conducts
Reference examples sample, 30ng is that the sample of embodiment 1 carries out follow-up capture and builds storehouse sequencing in addition.
3rd, library construction：
(1) end is repaired
According to the form below 6 configures reaction solution in PCR pipe, and with rifle, gently pressure-vaccum mixes up and down.
Table 6：
PCR instrument is put into, the program of according to the form below 7 is reacted.
Table 7：
(2) joint connects
According to the form below 8 configures reaction solution in PCR pipe, and with rifle, gently pressure-vaccum mixes up and down.
Table 8：
The reaction system configured is divided into 2 pipes, is put in PCR instrument, 20 DEG C of warm bath 15min.Finally, with 1 times of volume
AMPure XP magnetic beads for purifying connect the library of joint, the water elution of 23 μ L nuclease frees.
(4) library of joint has been gone up in amplification connection
According to the form below 9 configures reaction solution in PCR pipe, and with rifle, gently pressure-vaccum mixes up and down.
Table 9：
PCR instrument is put into, the program that according to the form below 10 is set is reacted.
Table 10：
By the PCR primer expanded 1.8 times of AMPure XP magnetic beads for purifying, the water elution of 30 μ L nuclease frees.Divide again
Library among 8 μ L is not taken, 2% agarose electrophoresis (115V, 35min) detection tabs residual condition, is as a result illustrated in fig. 1 shown below.
In Fig. 1, M is 100bp molecular labelings (Marker)；1 is control-sample 1；2 be control-sample 2；3 be embodiment 1- samples 1；4
For embodiment 1- samples 2.It was found that, illustrate that the present processes can significantly reduce joint pollution ratio by electrophoretogram.
(5) Library hybridization, capture and elution
Middle library is taken in a new centrifuge tube, draining machine with vacuum carries out concentration until the volume in library is 6.4 μ
L.Hybrid capture and elution are carried out using the QXT kits of Agilent company.It must ensure that lid covers tightly in crossover process, it is minimum
Change the evaporation for reducing hybrid mixed liquid product, otherwise will influence crossbreeding effect.Hybridization procedures such as table 11 below.
Table 11：
(6) PCR is expanded
On ice PCR reaction solutions are prepared according to table 12 below system.
Table 12：
Confirm after the reaction solution containing magnetic bead is mixed, pipe is put into PCR instrument and expanded, response procedures such as following table
Shown in 13.
Table 13：
With the product after the AMPure XP magnetic beads for purifying PCR amplifications of 1.8 times of volumes, the water elution of 20 μ L nuclease frees.Go out
Storehouse situation is as shown in table 14 below.
Table 14：
From the point of view of the outbound storage capacity of upper table Chinese library, the library storage capacity of embodiment 1 is significantly higher than reference examples.
4th, the upper machine of storehouse inspection
Upper machine after the inspection of library storehouse is qualified, the sequenators of nexseq 500 of upper machine platform selection illumina platforms, is sequenced plan
Slightly PE 75, each sample data volume are 5G.Result is examined as shown in Fig. 2A, Fig. 2 B, Fig. 2 C and Fig. 2 D in library storehouse.Wherein, scheme
The 2100 detection figures that 2A is library YF00CTDNACL00003 (0.1%)；Fig. 2 B are library YF00CTDNACL00005's (5%)
2100 detection figures；The 2100 detection figures that Fig. 2 C are library YF00CTDNACL00006 (0.1%)；Fig. 2 D are library
YF00CTDNACL00007 (5%) 2100 detection figures.What it is due to blood plasma cfDNA is broken in the form of nucleosome,
So cfDNA size is generally 170bp integral multiple, multiple peaks will be shown in 2100 detection figures, but because PCR was expanded
The amplification efficiency of small fragment is higher than long segment in journey, and small fragment can be in the majority in library, so the main peak of small fragment is highest.Peak
129bp is that joint has gone up the library main peak of joint for connection from the peak (having joint residual in library) connected, 294bp in Fig. 2A
(170bp+120bp), small peak below connect the peak for having gone up joint for larger purpose fragment.
5th, data results：
Storehouse sequence measurement is built using conventional to two biased samples, obtains the positive collection of two normal specimens.Share 30
SNP site.Performance comparision is carried out to the output data in the library constructed by existing method and the present processes respectively, compares knot
Fruit table 15 specific as follows is to table 18.
(1) SNP site analysis result
The SNP detections statistical result of reference examples is shown in Table 15：
Table 15：
The SNP detections statistical result of embodiment 1 is shown in Table 16：
Table 16：
The detection ratio of reference examples and embodiment 1 in table 15 and table 16 is compared respectively, comparative result such as table 17 below.
Table 17：
| Mixing ratio/method | Reference examples | Embodiment 1 |
| Positive site sum | 30 | 30 |
| 5% mixing library | 23 | 30 |
| 5% detection ratio | % | 100% |
| 0.1% mixing library | 11 | 28 |
| 0.1% detection ratio | 36.67% | 93.33% |
As can be seen from Table 17, the method for embodiment 1 is better than reference examples, and embodiment 1 in positive site detection ratio
The frequency in the mutational site of detection has good uniformity with blending ratio, thus in each site detection SNP site mutation
Frequency is also superior to comparative example 1.
In addition, also detection ratio of the two methods to false positive mutational site is counted and compared, comparative result
It is shown in Table 18.
Table 18：
| Mixing ratio/method | Reference examples | Embodiment 1 |
| Positive site sum | 30 | 30 |
| 5% mixing library | 108 | 0 |
| 5% false positive rate | 0.108% | 0.00% |
| 0.1% mixing library | 1321 | 2 |
| 0.1% false positive rate | 1.321% | 0.002% |
, can from above-mentioned comparative result because the length of the joint sequence in joint relative contrast's example in embodiment 1 is shorter
Reduce the pollution of output data center tap to find out, after truncation, thus, for dirt of the length to output data of detection tabs
The influence of dye, a series of joint that applicant devises different lengths (are specifically shown in table 19 below, sequence number is from top to bottom successively
For：SEQ ID NO:107~SEQ ID NO:114), using method same as Example 1, using 30 positive loci frequencies as
0.1% sample 2 carries out library construction for process object, and then the joint Contamination ratio in output data is compared.
Table 19：
Used in above-described embodiment 28 base N formed 100 miscue sequences and embodiment 5 used in 4
100 miscue sequences that base N is formed are respectively such as table 20 below, and 100 miscues used in embodiment 3 and 4
Sequence is identical with 100 miscue sequences used in embodiment 1, no longer repeats herein.
Table 20：
Then the data that the library built to the method for embodiment 2 to embodiment 5 is sequenced to obtain are in joint pollution rate, SNP
Recall rate and false positive rate etc. are counted, and statistical result is shown in Table 21.
Table 21：
| Embodiment | Total sequence number | SNP recall rates (%) | False positive rate (%) |
| Embodiment 2 | 45,979,351 | 90.00% | 0.005% |
| Embodiment 3 | 46,525,342 | 86.67% | 0.006% |
| Embodiment 4 | 46,241,752 | 93.33% | 0.004% |
| Embodiment 5 | 48,476,249 | 90.00% | 0.005% |
As can be seen from the above description, the above embodiments of the present invention realize following technique effect：Pass through basis
Experiment demand is respectively synthesized the single stranded sequence in double-stranded adapters, then forms double-stranded adapters by way of specificity is annealed again.
The joint preparation method is simple, and because the single stranded sequence of synthesis is synthesized according to pre-designed sequence, because
And the miscue sequence carried on each joint is to determine.And then ultralow frequency variation is detected for building library
When, it can be defined as very by the base mutation that same position on the positive-sense strand and antisense strand by each template DNA molecule occurs
Real mutation, and the base of mutation or the generation diverse location on positive-sense strand and antisense strand by independent generation on a chain
Mutation is defined as pseudomutation.It just can so exclude to be mutated caused by amplified library or sequencing mistake, so as to carry
The accuracy of high detection, and help to reduce false positive rate.
Compared with prior art, it is prepared by the joint that the preparation method of the above-mentioned joint of the application and profit are prepared in this way
Application in ultralow frequency variation detection library has following advantage：1) joint Making programme greatly simplifies, and improves joint quality；2)
Joint pollution is substantially reduced, storehouse quality is built in raising；3) SNP site recall rate is improved, accuracy of detection is improved, can accurately detect
The 0.1% even lower frequency of mutation.
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 (13)
1. a kind of sequence measuring joints, it is characterised in that the sequence measuring joints include amplified library primer sequence, the purpose being sequentially connected
Fragment amplification primer sequence and miscue sequence, the miscue sequence are located at close to the side of the purpose fragment,
The amplified library primer sequence is located remotely from the side of the purpose fragment, wherein, the miscue sequence is conventional base
The sequence of base sequence；The sequence measuring joints are Y-shaped joint, and the length of the miscue sequence is 4~12bp.
2. sequence measuring joints according to claim 1, it is characterised in that the Y-shaped joint include the first chain and with it is described
The second complementary chain of first chain part, the length of longer chain is 30~67bp in first chain and second chain.
3. sequence measuring joints according to claim 2, it is characterised in that longer chain in first chain and second chain
Length be 40~50bp.
4. sequence measuring joints according to claim 2, it is characterised in that the sequence of first chain is SEQ ID NO:1：
5 '-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNT-3 ', the sequence of second chain is SEQ ID NO:2：
5 '-NNNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3 ', wherein, the sequence that 6 N are formed represents base
The miscue sequence known to sequence.
5. sequence measuring joints according to claim 1, it is characterised in that the length of the miscue sequence is 6bp.
6. sequence measuring joints according to any one of claim 1 to 4, it is characterised in that the amplified library primer sequence
Length be 15~30bp.
A kind of 7. preparation method of the sequence measuring joints any one of claim 1 to 6, it is characterised in that the preparation side
Method includes：
Two single stranded sequences of sequence measuring joints are designed, miscue sequence, and the mistake are included in every single stranded sequence
Prompting sequence is located at the single stranded sequence close to the side for the purpose fragment to be connected, and the miscue sequence is conventional base
The sequence of base sequence；
It is respectively synthesized two single stranded sequences；
Two of the synthesis single stranded sequences are subjected to specific annealing, obtain the sequence measuring joints of double-strand.
8. preparation method according to claim 7, it is characterised in that the sequence measuring joints are multipair, the preparation method
Also include after the sequence measuring joints of double-strand are obtained：The multipair sequence measuring joints are subjected to equal proportion mixing, mixed
Sequence measuring joints the step of.
9. a kind of sequence measuring joints, it is characterised in that the sequence measuring joints are prepared by the preparation method described in claim 7 or 8
Form.
10. a kind of kit for building ultralow frequency variation sequencing library, the kit include sequence measuring joints, its feature exists
In, sequence measuring joints be sequence measuring joints any one of claim 1 to 6, or the preparation described in claim 7 or 8
The sequence measuring joints that method is prepared.
11. a kind of construction method of ultralow frequency variation sequencing library, it is characterised in that the construction method includes：
Step S1, build the sequencing library containing purpose fragment；
Step S2, hybrid capture, library after being captured are carried out to the sequencing library；And
Step S3, library after the capture is expanded, obtains purpose library；
Wherein, using the kit structure institute of the structure ultralow frequency variation sequencing library described in claim 10 in the step S1
State the sequencing library containing purpose fragment.
The sequencing library 12. a kind of ultralow frequency makes a variation, it is characterised in that the ultralow frequency variation sequencing library uses claim 11
Construction method it is built-up.
13. a kind of detection method of ultralow frequency variation, it is characterised in that the detection method includes：
High-flux sequence is carried out to the ultralow frequency variation sequencing library described in claim 12, obtains sequencing data；And
Variant sites analysis is carried out to the sequencing data, obtains variant sites data.
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| CN108070586A (en) * | 2016-11-18 | 2018-05-25 | 杭州拓宏生物科技有限公司 | Pcr amplification primer and its application |
| CN106755506A (en) * | 2016-12-27 | 2017-05-31 | 安诺优达基因科技(北京)有限公司 | Kit for detecting genetic mutation in tumour FFPE samples |
| CN107446996A (en) * | 2017-07-14 | 2017-12-08 | 艾吉泰康生物科技(北京)有限公司 | A kind of joint sequence and method of the ultralow frequency mutation for detecting target sequence |
| CN107354209B (en) * | 2017-07-14 | 2021-01-08 | 广州精科医学检验所有限公司 | Combinatorial tags, linkers and methods for determining nucleic acid sequences containing low frequency mutations |
| CN107858414B (en) * | 2017-10-18 | 2021-09-10 | 广州漫瑞生物信息技术有限公司 | High-throughput sequencing joint, preparation method thereof and application thereof in ultralow frequency mutation detection |
| CN107604046B (en) * | 2017-11-03 | 2021-08-24 | 上海交通大学 | Second-generation sequencing method for preparing bimolecular self-checking library for trace DNA ultralow frequency mutation detection and hybridization capture |
| CN108893466B (en) * | 2018-06-04 | 2021-04-13 | 上海奥根诊断技术有限公司 | Sequencing joint, sequencing joint group and detection method of ultralow frequency mutation |
| CN108728515A (en) * | 2018-06-08 | 2018-11-02 | 北京泛生子基因科技有限公司 | A kind of analysis method of library construction and sequencing data using the detection ctDNA low frequencies mutation of duplex methods |
| EP3859014A4 (en) * | 2018-09-27 | 2022-04-27 | BGI Shenzhen | Method for constructing sequencing library, obtained sequencing library and sequencing method |
| CN112852802A (en) * | 2018-12-06 | 2021-05-28 | 深圳海普洛斯医疗器械有限公司 | Linker for constructing sequencing library and construction method of sequencing library |
| CN109609598A (en) * | 2018-12-29 | 2019-04-12 | 北京优迅医学检验实验室有限公司 | A kind of rapidly and efficiently inexpensive library constructing method for two generation sequencing technologies |
| CN109680054A (en) * | 2019-01-15 | 2019-04-26 | 北京中源维康基因科技有限公司 | A kind of detection method of low frequency DNA mutation |
| CN110205683B (en) * | 2019-06-25 | 2022-12-20 | 广州燃石医学检验所有限公司 | Method for preparing DNA library and method for analyzing DNA library |
| CN111139533B (en) * | 2019-09-27 | 2021-11-09 | 上海英基生物科技有限公司 | Sequencing library adaptors with increased stability |
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