CN108060218A - The screening technique of the nucleic acid fragment of preset range in nucleic acid sequencing library - Google Patents
The screening technique of the nucleic acid fragment of preset range in nucleic acid sequencing library Download PDFInfo
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Abstract
The present invention provides a kind of screening technique of the nucleic acid fragment of preset range in nucleic acid sequencing library, comprises the following steps：By the free nucleic acid structure nucleic acid sequencing library in predetermined source in biological sample, the screening of the nucleic acid fragment of preset range is carried out to the nucleic acid sequencing library, the nucleic acid sequencing library of preset range is obtained, the screening technique includes at least one of agarose gel electrophoresis screening method, polyacrylamide gel electrophoresis screening method, magnetic bead screening method.The present invention also provides a kind of methods of the free nucleic acid concentration in predetermined source in definite biological sample.Target nucleic acid fragment screening technique provided by the invention, the screening and enrichment of nucleic acid fragment can not only effectively be carried out, improve target nucleic acid fragment concentration, so that the result of detection is more accurate, the lowest detection limit of genetic test can also be significantly improved, increases subject group so that more crowds benefit from gene tester, also, this method can carry out target area domain dna screening to multiple samples simultaneously.
Description
Technical field
The present invention relates to biomedical sector, specifically, in nucleic acid sequencing library the nucleic acid fragment of preset range screening
The method of method and definite free nucleic acid concentration.
Background technology
In the past few years, there is the transformation of an essence in DNA sequencing technology, and hair is sequenced from traditional Sanger
So-called " next generation's sequencing " technology (Next Generation Sequencing, NGS) is opened up.Although with it is traditional
Sanger PCR sequencing PCRs are compared, and the relevant cost of NGS technologies is greatly reduced, and genome sequencing is still that cost is higher.In addition,
There are many application and genome sequencing is not required, but needs to be sequenced for the specific region of one or more samples.
Such as effective full extron (coded portions of all genes) sequencing is at present than major application, but for smaller
The enrichment sequencing of genome or genome area is also widely used.During enrichment is targeted, uninterested DNA fragmentation
It is removed to greatest extent, target area is enriched with from initial genome sequencing library to be come out so that follow-up NGS sequencings
Generated sequencing result is concentrated mainly on interested target area.
Therefore need carrying out target dna before sequencing targeting screening and enrichment, then pass through the target dna to enrichment
It is sequenced, obtains it and carry information, and desired information is understood according to the carrying information that sequencing obtains.
The content of the invention
It is an object of the present invention to provide a kind of simple and quick and can carry out target DNA fragments sieve to multiple samples simultaneously
The method of choosing.Meanwhile the method that the free nucleic acid concentration in predetermined source in a kind of definite biological sample is also provided.
Based on object above, one aspect of the present invention provides a kind of sieve of the nucleic acid fragment of preset range in nucleic acid sequencing library
Choosing method comprises the following steps：
By the free nucleic acid structure nucleic acid sequencing library in predetermined source in biological sample, the nucleic acid sequencing library is carried out
The screening of the nucleic acid fragment of preset range, obtains the nucleic acid sequencing library of preset range, and the screening technique is coagulated including agarose
At least one of gel electrophoresis screening method, polyacrylamide gel electrophoresis screening method, magnetic bead screening method.
The present invention also provides a kind of method of the free nucleic acid concentration in predetermined source in definite biological sample, including following step
Suddenly：
High-flux sequence is carried out to the nucleic acid sequencing library of the preset range of above-mentioned acquisition, to obtain by multiple sequencing numbers
According to the sequencing result of composition；
Based on the sequencing result, the window number divided in the sample on chromosome according to predetermined length is determined respectively
And fall into the sequence number of the window；
Number based on the window on chromosome in the sample and the sequence number for falling into the window, determine the biology
The free nucleic acid concentration in predetermined source described in sample.
The present invention provides a kind of methods of target nucleic acid fragment screening, can not only effectively carry out the sieve of nucleic acid fragment
Choosing and enrichment improve mark nucleic acid fragment concentration so that the result of detection is more accurate, moreover it is possible to significantly improve the minimum of genetic test
Detection limit increases subject group so that more crowds benefit from gene tester, also, this method can be right simultaneously
Multiple samples carry out target area domain dna screening.The method that the present invention carries out target nucleic acid fragment screening and enrichment has pervasive
Property, operation scheme is easily implemented, can be widely used.
Description of the drawings
Fig. 1 is the gel photograph of sample in one embodiment of the invention, and wherein sample 1 is the mixing library of 3 clinical samples,
Sample 2 is the mixing library of other 3 clinical samples.
Fig. 2 is the 2100 biological analysers detection peak figure of sample 1 in one embodiment of the invention.
Fig. 3 is the 2100 biological analysers detection peak figure of sample 2 in one embodiment of the invention.
Fig. 4 is the fetal concentrations raising figure for not screening group and screening group in one embodiment of the invention.
Main element symbol description
Nothing
Following specific embodiment will be further illustrated the present invention with reference to above-mentioned attached drawing.
Specific embodiment
The technical solution in the embodiment of the present invention is clearly and completely described below in conjunction with embodiment, it is clear that
Described embodiment is only part of the embodiment of the present invention, instead of all the embodiments.Based on the implementation in the present invention
Example, those of ordinary skill in the art's all other embodiments obtained without making creative work, belongs to
The scope of protection of the invention.
Unless otherwise defined, all of technologies and scientific terms used here by the article is with belonging to technical field of the invention
The normally understood meaning of technical staff is identical.Term used in the description of the invention herein is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein " and/or " include one or more phases
The arbitrary and all combination of the Listed Items of pass.
In the case where there is no conflict, the feature in following embodiment and embodiment can be mutually combined.
Following term and phrase have meanings shown below, unless in addition providing herein.Unless expressly stated otherwise,
Or be evident that from the context, following term and phrase are not excluded for what the term or phrase had had in its fields
Meaning.The scope of the present invention is only limited by the claims, defined below to be only used for helping to describe specific embodiment, it is no intended to limit
Make invention claimed.Unless otherwise defined, the meaning of all technical and scientific terms used herein with the present invention
As those of ordinary skill in the art are generally understood.
It should be noted that in the description of the present invention, unless otherwise indicated, " multiple " be meant that two or two with
On." unique aligned sequences " in the present invention, " uniquely comparing sequencing sequence " are also referred to as " sequence ", " sequencing sequence sometimes
Row ".
Term " maternal sample " in this article refers to such biological sample, pregnancy is obtained from, for example, women.
Term " X chromosome ", " Y chromosome " refer to sex chromosome respectively.Autosome refers to the dye unrelated with Sex Determination
Colour solid.
Term " window " refers to the chromosome overlapped or nonoverlapping segment, each segment definition is respectively cut into
For a window.
Term " GC amendments " refers to be modified the depth of sequencing according to G/C content.
Term " sequencing depth " refers to the item number of sequence in some window.
The present invention provides a kind of screening technique of the nucleic acid fragment of preset range in nucleic acid sequencing library, including following step
Suddenly：
By the free nucleic acid structure nucleic acid sequencing library in predetermined source in biological sample, the nucleic acid sequencing library is carried out
The screening of the nucleic acid fragment of preset range, obtains the nucleic acid sequencing library of preset range, and the screening technique is coagulated including agarose
At least one of gel electrophoresis screening method, polyacrylamide gel electrophoresis screening method, magnetic bead screening method.
According to a particular embodiment of the invention, the nucleic acid sequencing library is to carry sample mark for high-flux sequence
The nucleic acid library of label.The sample label refers to that the one or both ends to each sequencing template before PCR add in one section of spy
The different sequence for identification.The present invention sample label can be commission associated companies voluntarily synthesize, the technology for industry
Know the technology emphasis of technology also non-invention, therefore details are not described herein.
According to a particular embodiment of the invention, the biological sample is peripheral blood, and the free nucleic acid in the predetermined source is
The free nucleic acid of free fetal nucleic acid or mother source in maternal blood either tumor patient, suspected tumor patient or
The free nucleic acid of free tumour nucleic acid or non-tumour source in tumor screening person's peripheral blood.The preset range can be based on inspection
Survey needs to screen arbitrary segment, and the preferred preset range is 10bp-500bp.The nucleic acid is DNA.
Method using the present invention is capable of being screened to free nucleic acid described in the biological sample for precise and high efficiency, and
It is enriched with target nucleic acid fragment.Especially, multiple samples can be mixed and screened by the present invention simultaneously, efficient, cost
It is low.Present invention is particularly suitable for the free fetal nucleic acid in maternal blood and tumor patient, suspected tumor patients or swollen
The screening of free tumour nucleic acid in knurl examination person's peripheral blood or the target nucleic acid fragment in the free nucleic acid in non-tumour source with
Enrichment.
According to a particular embodiment of the invention, when the present invention is for detecting the free fetal nucleic acid in maternal blood,
Because the DNA molecular clip size of free pregnant woman is about 160-170bp, and free fetal dna molecule fragment specific ionization mother
DNA molecular segment is small, and to be enriched with free fetal dna molecule fragment, the fragment length of the screening is less than 160-170bp, such as
10bp-80bp, 20bp-100bp, 50-150bp ....
The present invention is especially suitable for sequencing sample in there are two types of or two or more different length scopes DNA fragmentation sieve
Choosing.Episomal DNA molecule segment of the length range in 10-500bp is especially sequenced, using target dna molecule fragment length than it
The principle that remaining non-targeted DNA molecular fragment length has differences, target dna piece is filtered out using screening techniques such as gel electrophoresises
Section, is then recycled, and achievees the purpose that be enriched with target dna molecule segment.
According to a particular embodiment of the invention, by the free nucleic acid structure nucleic acid sequencing library in predetermined source in biological sample
Comprise the following steps：
Obtain the free nucleic acid of biological sample to be measured；
End reparation is carried out to the free nucleic acid, obtains the free nucleic acid after end is repaired；
Free nucleic acid after being repaired to end carries out end and adds " A ", obtains the free nucleic acid that end adds " A "；
Add the free nucleic acid adjunction head of " A " to the end, obtain the free nucleic acid of adjunction head；
The connector contains the sequence label of specimen discerning；
The nucleic acid fragment of the multiple sequence label containing specimen discerning is mixed；
The free nucleic acid mixture of the adjunction head is expanded, the enriched product of the free nucleic acid of adjunction head is obtained, to described
Enriched product carries out magnetic bead recycling, obtains the nucleic acid sequencing library.
According to a particular embodiment of the invention, the end is repaired through T4 archaeal dna polymerases (T4 DNA
Polymerase) completed with T4 polynueleotide kinases (T4 Polynucleotide Kinase) reaction.
According to a particular embodiment of the invention, the end adds " A " to add in the end containing Klenow (3 ' -5 ' exo-)
It is carried out in " A " system.
According to a particular embodiment of the invention, the adjunction head passes through sequence measuring joints and T4DNA ligases (T4 DNA
Ligase) reaction is completed.
According to a particular embodiment of the invention, the amplification is containing Pfx archaeal dna polymerases (Platinum Pfx DNA
Polymerase carried out in amplification system).
According to a particular embodiment of the invention, the agarose gel electrophoresis screening method includes：With 2-3% Ago-Gels
The free nucleic acid of different fragments size is separated by electrophoresis, then carries out segment screening with the method for gel extraction, 2-3% agaroses coagulate
Gel electrophoresis condition is：Voltage is 100-130V, time 1-2h, and the segment cut between 250-270bp is recycled.
Preferably, the agarose gel electrophoresis screening method includes：Different fragments are separated with 2.5% agarose gel electrophoresis
The free nucleic acid of size, then carries out segment screening with the method for gel extraction, and 2.5% agarose gel electrophoresis condition is：Electricity
It presses as 100V, time 1.5h, the segment cut between 250-270bp is recycled.
According to a particular embodiment of the invention, the polyacrylamide gel electrophoresis screening method includes：With 8-10% poly- third
Acrylamide gel electrophoresis separates the free nucleic acid of different fragments size, then carries out segment screening, 8- with the method for gel extraction
10% polyacrylamide gel electrophoresis condition is：Voltage is 7-1V/cm, time 1.5-2.5h, is cut between 250-270bp
Segment is recycled.
According to a particular embodiment of the invention, the magnetic bead screening method includes：First use compared with 0.8-0.9 times of body of sample
Long-pending magnetic bead removal large fragment, then recycles target fragment using the magnetic bead of 1.1-1.2 times of volume.
The present invention also provides a kind of method of the free nucleic acid concentration in predetermined source in definite biological sample, including following step
Suddenly：
The nucleic acid sequencing library of preset range to being obtained using the above method carries out high-flux sequence, to obtain by more
The sequencing result that a sequencing data is formed；
Based on the sequencing result, the window number divided in the sample on chromosome according to predetermined length is determined respectively
And fall into the sequence number of the window；
Number based on the window on chromosome in the sample and the sequence number for falling into the window, determine the biology
The free nucleic acid concentration in predetermined source described in sample.
According to a particular embodiment of the invention, the sequencing includes but not limited to double end sequencings, single end sequencing or list
Molecule is sequenced, and the microarray dataset includes but not limited to the Hiseq2000/2500 of Illumina, Life Technologies
The microarray datasets such as Ion Torrent, single-molecule sequencing.
According to a particular embodiment of the invention, the window number divided in the sample on chromosome according to predetermined length is determined
Mesh and the sequence number for falling into the window, further include：
The sequencing result is compared with reference gene group, so as to build it is unique compare sequencing sequence collection, it is described only
One compares each sequencing sequence that sequencing sequence is concentrated only can be with a location matches of the reference gene group；
Reference gene group is divided into multiple windows according to predetermined length, the predetermined length is 1bp~5M；
It counts respectively and uniquely compares sequencing sequence in the sample on autosome, X chromosome and Y chromosome in the window
The number summation of row and the total number of the window.
According to a particular embodiment of the invention, count respectively in the sample on autosome, X chromosome and Y chromosome
After the number summation of sequencing sequence and the total number of the primary window are uniquely compared in the window, further include：
GC amendments are carried out to the unique comparison sequencing sequence number fallen into window and revised result is criticized
It is adjusted between secondary.The methods of GC, which is corrected, includes but not limited to local weighted recurrence, linear regression, logistic regression.
According to a particular embodiment of the invention, count respectively in the sample on autosome, X chromosome and Y chromosome
After the number summation of sequencing sequence and the total number of the window are uniquely compared in the window, further include：
According to the number of the window on the autosome, X chromosome and Y chromosome and fall into the sequence of the window
Number calculates the nucleic acid concentration of the X chromosome and Y chromosome；
According to the X chromosome and the nucleic acid concentration of Y chromosome, the free core in predetermined source in the biological sample is determined
Acid concentration.
According to a particular embodiment of the invention, the nucleic acid concentration of the X chromosome and Y chromosome is calculated, is further included：
According to the number of the window on the autosome and the sequence number of the window is fallen into, calculates the autosome
Mean depth da, da=described on autosome on total sequence number/autosome of window window number；
According to the number of the window on the X chromosome and the sequence number of the window is fallen into, calculates being averaged for X chromosome
On depth dx, dx=X chromosome on total sequence number/X chromosome of window window number；
According to the number of the window on the Y chromosome and the sequence number of the window is fallen into, calculates being averaged for Y chromosome
On depth dy, dy=Y chromosome on total sequence number/Y chromosome of window window number；
According to the mean depth dx of the X chromosome, nucleic acid concentration fx=2* (da-dx)/da of X chromosome is calculated；
According to the mean depth dy of the Y chromosome, the nucleic acid concentration fy==2*dy/da of Y chromosome is calculated.
According to a particular embodiment of the invention, according to the X chromosome and the nucleic acid concentration of Y chromosome, the life is determined
The free nucleic acid concentration in predetermined source described in object sample further includes：
Calculate free nucleic acid concentration f, the f=(nucleic acid concentration+X of Y chromosome in predetermined source described in the biological sample
The nucleic acid concentration of chromosome)/2.
According to a particular embodiment of the invention, determine respectively in the sample on autosome, X chromosome and Y chromosome
It the number of the window and falls into the sequence number of the window and further includes：
The sequence number that the window is fallen on removal autosome, X chromosome and Y chromosome is more than mean sequence number
5 times or more of window.
Following embodiment is not intended to is limited to the present invention by the scope of claims, and is intended as some embodiment party
The specific example of case.Any variation in the illustrative methods that those skilled in the art have found is within the scope of the present invention.
Embodiment 1
Obtain the free nucleic acid in predetermined source in biological sample to be measured；
End reparation is carried out to the free nucleic acid, obtains the free nucleic acid after end is repaired；
Free nucleic acid after being repaired to end carries out end and adds " A ", obtains the free nucleic acid that end adds " A "；
Add the free nucleic acid adjunction head of " A " to the end, obtain the free nucleic acid of adjunction head；
The nucleic acid fragment of the multiple sequence label containing sample is mixed；
The free nucleic acid mixture of the adjunction head is expanded, the enriched product of the free nucleic acid of adjunction head is obtained, to described
Enriched product carries out magnetic bead recycling, obtains nucleic acid sequencing library.
The screening of the nucleic acid fragment of preset range is carried out to the nucleic acid sequencing library, obtains the nucleic acid sequencing of preset range
Library, the screening technique include agarose gel electrophoresis screening method, polyacrylamide gel electrophoresis screening method, magnetic bead screening method
At least one of.
High-flux sequence is carried out to the nucleic acid sequencing library of the preset range after screening, to obtain by multiple sequencing datas
The sequencing result of composition；
The sequencing result is compared with reference gene group, so as to build it is unique compare sequencing sequence collection, it is described only
One compares each sequencing sequence that sequencing sequence is concentrated only can be with a location matches of the reference gene group；
Reference gene group is divided into multiple windows according to predetermined length, the predetermined length is 1bp~5M；
It counts respectively and uniquely compares sequencing sequence in the sample on autosome, X chromosome and Y chromosome in the window
The number summation of row and the total number of the window；
GC amendments are carried out to the unique comparison sequencing sequence number fallen into window and revised result is criticized
It is adjusted between secondary；
The sequence number that the window is fallen on removal autosome, X chromosome and Y chromosome is more than mean sequence number
5 times or more of window；
According to the number of the window on the autosome and the sequence number of the window is fallen into, calculates the autosome
Mean depth da, da=described on autosome on total sequence number/autosome of window window number；
According to the number of the window on the X chromosome and the sequence number of the window is fallen into, calculates being averaged for X chromosome
On depth dx, dx=X chromosome on total sequence number/X chromosome of window window number；
According to the number of the window on the Y chromosome and the sequence number of the window is fallen into, calculates being averaged for Y chromosome
On depth dy, dy=Y chromosome on total sequence number/Y chromosome of window window number；
According to the mean depth dx of the X chromosome, nucleic acid concentration fx=2* (da-dx)/da of X chromosome is calculated；
According to the mean depth dy of the Y chromosome, the nucleic acid concentration fy==2*dy/da of Y chromosome is calculated；
Calculate free nucleic acid the concentration f, f=(fy+fx)/2 in predetermined source in the biological sample.
Embodiment 2
6 samples are analyzed twice.I.e. same sample builds two libraries simultaneously, and one of library uses fine jade
Sepharose carries out target fragment screening, filters out target fragment of the scope in 130-150bp.Another library is without agar
Sugared gel screening, while upper machine is sequenced, judges whether the library by segment screening is contained than the library screened without segment
Higher fetal concentrations.
1st, sample collection and processing
Take maternal blood 5-10mL using Streck heparin tubes, after acquisition, according to two step centrifugal process of standard into
Promoting circulation of blood slurry separation.
2nd, the extraction of maternal plasma dissociative DNA
The extraction of maternal blood slurry dissociative DNA is carried out using TIANamp Micro DNA Kit, concrete operation step is such as
Under：
2.1 take 600 μ L maternal plasmas in 2mL centrifuge tubes, add in 20 μ L Proteinase K solution, fully
Shake mixing, of short duration centrifugation.
2.2 add in 600 μ L buffer solutions GB (the Carrier RNA storing liquids containing concentration for 1 μ g/ μ L, specific preparation side
Method is shown in specification), fully vibrate mixing, of short duration centrifugation.
2.3 56 DEG C of incubation 10min, and sample is shaken frequently.Brief centrifugation is to remove the drop of cap wall.
2.4 add in the freezing absolute ethyl alcohol of 300 μ L, overturn mixing, are placed at room temperature for 5min, brief centrifugation.
Previous step acquired solution is transferred to an adsorption column (adsorption column is put into collecting pipe), 12,000rpm centrifugations by 2.5
30sec abandons waste liquid, and adsorption column is put back in collecting pipe.
2.6 add in 500 μ L buffer solutions GD (please first checked whether before use and added in absolute ethyl alcohol) into adsorption column, and 12,
000rpm centrifuges 30sec, abandons waste liquid, adsorption column is put back in collecting pipe.
2.7 add in 600 μ L rinsing liquids PW (please first checked whether before use and added in absolute ethyl alcohol) into adsorption column, and 12,
000rpm centrifuges 30sec, abandons waste liquid, adsorption column is put back in collecting pipe.
2.8 repetitive operation step 2.7..
2.9 12,000rpm centrifuge 2min, outwell waste liquid.Adsorption column is placed in and is placed at room temperature for 2-5min, thoroughly to dry
Remaining rinsing liquid in sorbing material.
2.10 are transferred to adsorption column in one clean centrifuge tube, and 90 μ L elution buffers are added in adsorbed film centre position
Liquid TB, is placed at room temperature for 2-5min, and solution is collected into centrifuge tube, obtains maternal plasma by 12,000rpm centrifugation 2min
Dissociative DNA.
3rd, end is repaired
End is prepared according to table 1 and repairs reaction system, and end reparation is carried out to maternal plasma dissociative DNA, is obtained
The dissociative DNA that end is repaired.
Table 1
100 μ L ends dummies are tied up into 20 DEG C of incubation 30min in Thermomixer constant temperature dry bath devices, obtains to end and repaiies
Multiple dissociative DNA solution.The dissociative DNA that the end obtained using 150L AmpureXP beads purifying magnetic beads for purifying is repaired, and
With 36L Elution buffer (elution buffer) from AmpureXP beads elute the end repair dissociative DNA obtain it is pure
The dissociative DNA solution that end after change is repaired.
4th, end adds " A "
End is prepared according to table 2 and adds " A " reaction system, and carrying out end to the dissociative DNA for having carried out end reparation adds
" A " obtains the dissociative DNA that end adds " A ".
Table 2
| Reagent | Volume |
| The dissociative DNA solution that step 3 end is repaired | 34μL |
| 10X Blue Buffer | 5μL |
| dATP(1mM) | 10μL |
| Klenow(3’-5’exo-) | 1μL |
| Total volume | 50μL |
50L ends are added into " A " reaction system 37 DEG C of incubation 30min in Thermomixer, obtain the trip that end adds " A "
From DNA solution.The end obtained using 75L AmpureXP beads purifying adds the dissociative DNA of " A ", and with 24L Elution
Buffer elutes the dissociative DNA that the end that the end adds the dissociative DNA of " A " to obtain after purification adds " A " from AmpureXP beads
Solution.
5th, adjunction head reacts
Connector coupled reaction system is prepared according to table 3, specimen discerning sequence is carried to the dissociative DNA both ends connection of end plus " A "
The Nextseq CN500 sequence measuring joints of the 126bp of row, obtain the dissociative DNA solution of belt lacing.The connector of the present invention can pass through
Market business approach obtains.
Table 3
| Reagent | Volume |
| Step 4 end adds the dissociative DNA solution of " A " | 45μL |
| 2×Rapid ligation buffer | 50μL |
| T4DNA ligase | 4μL |
| Barcode-PE index Adapte(10pmol/ul) | 1μL |
| Total volume | 100μL |
100L connector coupled reaction systems are incubated into 15min for 20 DEG C in Thermomixer, obtain the free of belt lacing
DNA solution.The dissociative DNA for obtaining belt lacing is purified using 150L AmpureXP beads, and with 13L Elution buffer
The dissociative DNA that the belt lacing is eluted from AmpureXP beads obtains the dissociative DNA solution of belt lacing after purification.
6th, sample mixes
The sample for needing to carry out target fragment screening is mixed according to every 3 samples, i.e., 6 samples are mixed into two samples
Product, sample 1 and sample 2, i.e., sample to be screened have 2 parts.
7th, PCR reacts
PCR reaction systems are prepared according to table 4, PCR enrichments are carried out to the dissociative DNA of belt lacing, obtain the free of belt lacing
DNA enriched products form complete structure library.
Table 4
| Reagent | Volume |
| The dissociative DNA solution of step 5 belt lacing | 32.2μL |
| 10×Pfx ampilcation buffer | μL |
| dNTP Solution(10nM) | 2μL |
| MgSO4(50mM) | μL |
| Universal primer(10pM) | 4μL |
| Index primer(10pM) | 4μL |
| Pfx DNA Polymerse | 0.8μL |
| Total volume | 50μL |
The PCR reaction systems of 50L in PCR instrument are run into the program such as table 5, obtain the dissociative DNA enrichment production of belt lacing
Object.The dissociative DNA enriched product for obtaining belt lacing is purified using 50L AmpureXP beads, and with 22L Elution
Buffer elutes the dissociative DNA enriched product solution for the belt lacing that the enriched product obtains after purification from AmpureXP beads,
To this completion library construction.
Table 5
8th, target fragment is screened
A library progress segment screening to the same sample that step 7 obtains, carries out the library name of segment screening
For screening group, without screening, the library screened without segment, which is named as, does not screen group for another.Screening group is divided into as sample
1 and sample 2, that is, there are 2 parts of libraries to be screened, 6 parts of libraries are without screening.The library that need to be screened carries out Ago-Gel
Electrophoresis shows, gel extraction best using 2.5% Ago-Gel progress Piece Selection effect by multiple condition test
254-274bp target fragments, when operation, pay attention to more allowing blade replacement, avoid the pollution of other segments, obtain garbled peripheral blood blood
Starch dissociative DNA library.
8.1 prepare Ago-Gel：2.5g agaroses are weighed, are put into clear clean conical flask, are measuring 110mL's
1 × TAE buffer solutions of Fresh are poured into conical flask, are shaken up and are put into micro-wave oven high fire heating 4min dissolving agarose (fine jades
Until lipolysaccharide is completely dissolved), safety is should be noted in heating process, is suitably shaken up, is prevented bumping.Cold water cooling 2min after cooked,
Liquid is poured slowly into ready paving offset plate, pays attention to avoiding generating bubble, room temperature is cooled to curdled appearance.
8.2 electrophoresis：By the dissociative DNA enriched product of 20 μ L belt lacings obtained by step 6 and the yellow abundant mixing of 10 μ L bromines phenol
Centrifugation all clicks and enters glue hole, while carries out band instruction using 5 μ L D2000DNA ladder and 50bp ladder.Using new
The 1xTAE buffer solutions of fresh preparation, voltage 100V, time 2h, when bromine phenol yellow dye is moved at gel front 2cm
When, stop electrophoresis.
8.3 dye glue and gel imaging：The soak after electrophoresis is taken out in ethidium bromide dye liquor, dyes 15min.It takes out
It is put into gel automated imaging instrument, takes pictures as shown in Figure 1.As can be seen that the sample 1 and the DNA of sample 2 that filter out from photo
Segment is 254-274bp.
8.4 cut glue：The blob of viscose of 254-274bp segments is cut with knife blade under bale cutting instrument, is put into clean
In 1.5mL centrifuge tubes, continue glue recycling step.
8.5 glue recycle：Product recycling is carried out using GeneJET Gel Extraction Kit.Operating procedure is as follows：
8.5.1 respective amount Binding Buffer are separately added into and (add in 100 again by every 100mg glue into 1.5mL centrifuge tubes
μ L volumes calculate), abundant mixing centrifuges in short-term.
8.5.2 50 DEG C of 500rpm in Thermomixer are placed and are incubated to blob of viscose all dissolvings (about 15min).
8.5.3 take out be separately added into respective amount isopropanol (adding in 100 μ L volumes again by every 100mg glue to calculate) it is fully mixed
It is even, it centrifuges in short-term.
8.5.4 aforesaid liquid is all transferred in corresponding purification column, pays attention to corresponding pipe mark, 12,000rpm centrifugations
1min abandons filtrate, and purification column is put back to collecting pipe.
8.5.5 100 μ L Binding Buffer are added in, 12,000rpm centrifuge 1min, abandon filtrate, purification column is put back to receipts
Collector.
8.5.6 700 μ L Wash Buffer are added in, 12,000rpm centrifuge 1min, abandon filtrate, purification column is put back to collection
Pipe.
8.5.7 sky is got rid of.12,000rpm centrifuges 2min.
8.5.8 purification column is put into new 1.5mL centrifuge tubes, dries 2min, add in 20 μ L Elution Buffer,
3-5min is placed at room temperature for, 12,000rpm centrifugation 1min collect product.Obtain the plasma DNA library for machine for the treatment of.
9th, upper machine library quality inspection
Library Quality detection, 2100 inspections are carried out using 2100 biological analysers of Agilent and quantitative fluorescent PCR (QPCR)
Survey peak figure as Figure 2-3, as can be seen from the figure by the mean size (peak value) of sample product 1 and 2 be respectively 267bp and
264bp, it was demonstrated that the sample filtered out is in 254-274bp scopes, and mass concentration and molar concentration are satisfied by machine sequencing
It is required that.
10th, it is sequenced
Sequencing procedure is operated the computer in strict accordance with the Standard Operating Procedure that upper machine is sequenced.
11st, data analysis
The data that gained is sequenced with reference gene group are compared, build unique comparison sequencing sequence collection, it is described unique
Each sequencing sequence for comparing sequencing sequence concentration only can be with a location matches of the reference gene group；
Reference gene group is divided into multiple windows by 20kb；
It counts respectively and uniquely compares sequencing sequence in each sample on autosome, X chromosome and Y chromosome in the window
The number summation of row and the total number of the window.
GC amendments are carried out to the unique sequencing sequence number that compares fallen into window, carrying out batch to revised result
Adjustment.
According to the number of the window on the autosome and the sequence number of the window is fallen into, calculates the autosome
Mean depth da, da=described on autosome on total sequence number/autosome of window window number；
According to the number of the window on the X chromosome and the sequence number of the window is fallen into, calculates being averaged for X chromosome
On depth dx, dx=X chromosome on total sequence number/X chromosome of window window number；
According to the number of the window on the Y chromosome and the sequence number of the window is fallen into, calculates being averaged for Y chromosome
On depth dy, dy=Y chromosome on total sequence number/Y chromosome of window window number；
According to the mean depth dx of the X chromosome, calculate the fetal nucleic acid concentration fx=2* (da-dx) of X chromosome/
da；
According to the mean depth dy of the Y chromosome, the fetal nucleic acid concentration fy==2*dy/da of Y chromosome is calculated.
According to the X chromosome and the nucleic acid concentration of Y chromosome, the trip in predetermined source described in the biological sample is determined
Freestone acid concentration；
Calculate free nucleic acid concentration f, the f=(nucleic acid concentration+X of Y chromosome in predetermined source described in the biological sample
The nucleic acid concentration of chromosome)/2.
Need the sequence number for falling into the window on removal autosome, X chromosome and Y chromosome big during selected window
In the window of 5 times of mean sequence number or more.
Fetal concentrations result is calculated as shown in table 6 and Fig. 4：
Table 6
As shown in table 6 and Fig. 4, it is substantially unchanged not screen the fetal concentrations of No. 1 women in group and screening group, this is to calculate
Method causes, and can illustrate that X chromosome and Y chromosome segment will not have big fluctuation due to screening step, i.e., this method is to female's tire
Concentration has no too big influence.Wherein 2 to No. 6 are male fetus, it can be seen that the concentration of male fetus has apparent rise, explanation
The fetal concentrations of male are greatly improved after screening step, probably increase one times, can illustrate fetus segment
Selection has DNA concentration effects.
Embodiment 3
The present embodiment and the method for embodiment 2 are essentially identical, and difference lies in target fragment is screened using magnetic bead.
Magnetic bead screening technique step is as follows：
Sample to be selected is transferred in 1.5mL centrifuge tubes, with AMPure XP DNA Purification kit (SPRI
Beads) the sample after purifying amplification.
1st, the Ampure XP Beads of 4 DEG C of preservations are taken out, are placed at room temperature for 30min balances；
2nd, using preceding shaken well, magnetic bead and mixing is added according to 0.8~0.9 times of volume of sample volume, stands 2min, wink
When centrifuge 3 seconds.
3rd, the transfer of 1.5mL centrifuge tubes is placed on magnetic frame, stands 3-5min to clarification；
4th, supernatant is carefully sucked, is transferred in new 1.5ml centrifuge tubes, abandons former centrifuge tube；
5th, 0.3~0.4 times of magnetic bead is added in new centrifuge tube, is stored at room temperature 2~3min, brief centrifugation 3 seconds.
6th, centrifuge tube transfer is placed on magnetic frame, stands 3-5min to clarification；
7th, 500 μ L, 70% ethyl alcohol is added in, pipe lid is covered, magnetic frame is overturned 2-3 times, abandon supernatant (should delay when adding in ethyl alcohol
It is slow to add in, it tries not that liquid is allowed to add toward magnetic bead direction, magnetic bead otherwise can be made to depart from tube body and be lost.)
8th, step 7 is repeated, removes supernatant as far as possible.
9th, dry 5min of 37 DEG C of constant temperature blending instrument or so is put, (the dry and cracked situation of magnetic bead is examined until magnetic bead is dry and cracked, first removes
Supernatant part can it is dry must be than very fast, continuous heating has the potential risk for making magnetic bead calving well, causes damages and sample
Between pollute, individually non-dry hole can use from drying instrument stand air-dry).
10th, 24 μ L nuclease-free water are added in into 1.5mL centrifuge tubes, abundant mixing stands 5min, then
Magnetic frame about 5min is placed in clarification.
11st, 24 μ L clarified solutions are transferred in the new 1.5mL centrifuge tubes of preprepared (paid particular attention to during tube by
It is transferred in respective tube, avoids malfunctioning).
The above-described embodiments are merely illustrative of preferred embodiments of the present invention, not to the model of the present invention
It encloses and is defined, on the premise of design spirit of the present invention is not departed from, this field ordinary skill technical staff is to the technology of the present invention
The various modifications and improvement that scheme is made should all be fallen into the protection domain that claims of the present invention determines.
Claims (10)
1. the screening technique of the nucleic acid fragment of preset range in a kind of nucleic acid sequencing library, which is characterized in that comprise the following steps：
By the free nucleic acid structure nucleic acid sequencing library in predetermined source in biological sample, the nucleic acid sequencing library is made a reservation for
The screening of the nucleic acid fragment of scope, obtains the nucleic acid sequencing library of preset range, and the screening technique includes Ago-Gel electricity
At least one of swimming screening method, polyacrylamide gel electrophoresis screening method, magnetic bead screening method.
2. according to the method described in claim 1, it is characterized in that, the biological sample be peripheral blood, the predetermined source
Free nucleic acid is the free nucleic acid or tumor patient, doubtful swollen of the free fetal nucleic acid or mother source in maternal blood
Knurl patient or free tumour nucleic acid or the free nucleic acid in non-tumour source in tumor screening person's peripheral blood, the preset range
For 10bp-500bp.
3. according to the method described in claim 1, it is characterized in that, the free nucleic acid in the predetermined source is in maternal blood
Free fetal nucleic acid, the preset range be less than 160-170bp.
4. according to the method described in claim 1, it is characterized in that, the agarose gel electrophoresis screening method includes：Use 2-3%
Agarose gel electrophoresis separates the free nucleic acid of different fragments size, then carries out segment screening, 2- with the method for gel extraction
3% agarose gel electrophoresis condition is：Voltage is 100-130V, time 1-2h, and the segment cut between 250-270bp carries out
Recycling.
5. according to the method described in claim 1, it is characterized in that, the free nucleic acid in predetermined source in biological sample is built into core
Sour sequencing library comprises the following steps：
Obtain the free nucleic acid of biological sample to be measured；
End reparation is carried out to the free nucleic acid, obtains the free nucleic acid after end is repaired；
Free nucleic acid after being repaired to end carries out end and adds " A ", obtains the free nucleic acid that end adds " A "；
Add the free nucleic acid adjunction head of " A " to the end, obtain the free nucleic acid of adjunction head；
Expand the free nucleic acid of the adjunction head, obtain the enriched product of the free nucleic acid of adjunction head, to the enriched product into
Row magnetic bead recycles, and obtains the nucleic acid sequencing library.
6. a kind of method of the free nucleic acid concentration in predetermined source in definite biological sample, which is characterized in that comprise the following steps：
The nucleic acid sequencing library of preset range to being obtained in claim 1 carries out high-flux sequence, to obtain by multiple surveys
Ordinal number according to composition sequencing result；
Based on the sequencing result, the window number divided in the sample on chromosome according to predetermined length is determined respectively and is fallen
Enter the sequence number of the window；
Number based on the window on chromosome in the sample and the sequence number for falling into the window, determine the biological sample
Described in predetermined source free nucleic acid concentration.
7. it according to the method described in claim 6, it is characterized in that, determines to be drawn according to predetermined length on chromosome in the sample
Point window number and fall into the sequence number of the window, further include：
The sequencing result is compared with reference gene group, to build unique comparison sequencing sequence collection, unique ratio
Each sequencing sequence concentrated to sequencing sequence only can be with a location matches of the reference gene group；
Reference gene group is divided into multiple windows according to predetermined length, the predetermined length is 1bp~5M；
It counts respectively in the sample and uniquely compares sequencing sequence in the window on autosome, X chromosome and Y chromosome
The total number of number summation and the window.
8. the method according to the description of claim 7 is characterized in that count respectively autosome in the sample, X chromosome and
After uniquely comparing the number summation of sequencing sequence and the total number of the window on Y chromosome in the window, further include：
According to the number of the window on the autosome, X chromosome and Y chromosome and the sequence number of the window is fallen into, meter
Calculate the nucleic acid concentration of the X chromosome and Y chromosome；
According to the X chromosome and the nucleic acid concentration of Y chromosome, the free core in predetermined source described in the biological sample is determined
Acid concentration.
9. according to the method described in claim 8, it is characterized in that, calculate the nucleic acid concentration of the X chromosome and Y chromosome,
It further includes：
According to the number of the window on the autosome and the sequence number of the window is fallen into, is calculated described autosomal flat
Described in equal depth da, da=on autosome on total sequence number/autosome of window window number；
According to the number of the window on the X chromosome and the sequence number of the window is fallen into, calculates the mean depth of X chromosome
On dx, dx=X chromosome on total sequence number/X chromosome of window window number；
According to the number of the window on the Y chromosome and the sequence number of the window is fallen into, calculates the mean depth of Y chromosome
On dy, dy=Y chromosome on total sequence number/Y chromosome of window window number；
According to the mean depth dx of the X chromosome, nucleic acid concentration fx=2* (da-dx)/da of X chromosome is calculated；
According to the mean depth dy of the Y chromosome, the nucleic acid concentration fy=2*dy/da of Y chromosome is calculated.
10. according to the method described in claim 8, it is characterized in that, according to the X chromosome and the nucleic acid concentration of Y chromosome,
Determine that the free nucleic acid concentration in predetermined source described in the biological sample further includes：
Calculate free nucleic acid concentration f, the f=(nucleic acid concentration+X dyeing of Y chromosome in predetermined source described in the biological sample
The nucleic acid concentration of body)/2.
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