CN109680054A - A kind of detection method of low frequency DNA mutation - Google Patents
A kind of detection method of low frequency DNA mutation Download PDFInfo
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Abstract
The invention discloses a kind of connector mixtures for DNA library building, and the DNA mutation detection method using the connector mixture.The oxidative damage that the present invention passes through the DNA plerosis segment before building library, and the internalcontrol sequence of specificity is imported in every DNA fragmentation, it is aided with unique data analysing method, oxidative damage is excluded, the false positive mutation that PCR is introduced or sequencing generates, to improve the sensitivity and specificity of low frequency DNA mutation detection.
Description
Technical field
The invention belongs to technical field of gene detection, in particular to a kind of detection method for low frequency DNA mutation.
Background technique
With going deep into for molecular biology research, carry hereditary information DNA be proved it is closely related with human health, especially
It is that more and more diseases are proved related to the variation of gene or protein.In this context, pass through molecular biosciences
The potential applicability in clinical practice that method detects human DNA is boundless.Currently, widely applied DNA mutation detection method includes poly-
Polymerase chain reacts (PCR), fluorescence in situ hybridization technique (FISH), genetic chip (gene chip), Sanger sequencing etc..
Low frequency mutation refers to the extremely rare gene mutation being present in a large amount of wild gene sequence backgrounds.Low frequency mutation
Most commonly seen example is exactly the genetic mutation occurred in tumour cell, and many causes the somatic mutation of tumour to be all doping
Intracellular in wild type, a large amount of wild type DNA can be had in obtained DNA sample.
Tumour is a kind of disease for meeting darwinian evolution feature, and heterogeneity is one of feature of malignant tumour.Tumour exists
In growth course, by multiple division growth, the diversification in terms of daughter cell shows molecular biology and genome changes,
To make the speed of growth of tumour, invasive ability, produce sizable difference to various aspects such as sensibility, the prognosis of drug.
The heterogeneity of tumour is the primary restraining factors of current antineoplastic target medication effect, thus nothing in real time, highly sensitive
It is great to the diagnostic significance of tumour to create DNA detection method.
CtDNA (circulating tumor DNA) i.e. Circulating tumor DNA, is a kind of extracellular dna of cell-free state,
It is present in the body fluid such as blood, synovial fluid and cerebrospinal fluid.These ctDNA carry in tumour cell gene mutation believe
Breath can be equally used for early detection, targeting medication and the prognosis detection of tumour.CtDNA half-life period is shorter, can accurately reflect trouble
The current mutation status of person's tumour can be used as the real-time instruction information of oncotherapy.In addition, ctDNA is noninvasive sampling, from prominent
Become in information and says therefore the overall picture that can more reflect that oncogene is mutated has by the liquid Biopsy of test object of ctDNA
Its superiority, using being also increasingly taken seriously.And occur spreading for tumour or state is difficult to be resistant to the trouble of tissue biopsy
Person, ctDNA detection are even more unique selection instantly.
Although ctDNA detection technique has great potential, being widely used in clinic also needs to overcome many obstacles.
The DNA content that dissociates in blood is seldom, can only extract the total dissociative DNA of about 10ng or so in average 1ml blood plasma, and ctDNA Zhan is total
Dissociative DNA ratio is lower, and generally only 0.1%~10%.So the detection of low frequency mutational site is current ctDNA detection technique
The bottleneck encountered.
In low frequency mutation detection methods, Sanger PCR sequencing PCR is considered as " goldstandard ".But the sensitivity of Sanger sequencing
Limited, under the background of a large amount of wild type genes, Sanger sequencing is only capable of detecting the mutation containing 5%, so being unsuitable for pair
The detection of ctDNA.Technology currently used for ctDNA abrupt climatic change mainly has ARMS method (mainly Super-ARMS), digital pcr
(NGS) is sequenced in (dPCR, including BEAMing technology) and the second generation.Super-ARMS and digital pcr technology are easy quickly, specifically
Property and sensitivity are higher, the disadvantage is that the known mutations of finite number can only be detected, flux is low.NGS flux is high, detects gene number
It measures unrestricted, can detect known or unknown mutation, allow using liquid biopsy and analyze micro ctDNA and be possibly realized.
NGS is directed to the detection of polygenic mutation, and false negative result usually can be by simply increasing sequencing data amount, i.e.,
Increase the modes such as sequencing depth to be inhibited.But due to the particularity of ctDNA itself, sample size collected is usually lower, adds
Big sequencing data amount will cause a large amount of repetitive sequence (duplicate), and the increase of increase and data volume that depth is sequenced is not
With linear relationship.Therefore when practical application, the cost that generation is sequenced is very high, and not to the inhibitory effect of false negative
It is ideal.
And for false positive results, the defect as present in principal level still lacks simple control means at present.This
When having resulted in NGS applied to ctDNA detection, needed for detection sensitivity is not able to satisfy often, the appearance of false positive leads to mistake
Medical treatment guidance Strategy Risk increase the problems such as.
The problem of restricting NGS detection sensitivity at present and defect mainly have at following 3 points: (1) two generation microarray dataset error rate:
The single base sequencing error rate of the sequencing highest two generations microarray dataset degree Illumina NextSeq of precision is about 0.01% at present
Between~1%.In the case where 0.1% and detection below limit, it is difficult to mutually distinguish ctDNA mutation and sequencing mistake, restrict
The specificity of detection.(2) base mispairing that PCR is introduced: since the characteristic of ctDNA itself is limited, original samples amount is usual
It is less, thus be required to through PCR be means when carrying out library construction, the amplification of detection signal is carried out, it is anti-to meet sequencing
It answers needed for lower limit.And the high fidelity enzyme applied in round pcr at present, the duplication random error rate of DNA is about 10-6。PCR
The base mispairing for reacting introduced can mutually be obscured with the true mutation of low frequency ctDNA under normal tissue DNA background, greatly interfere
The sensitivity and specificity of detection.(3) oxidative damage of ctDNA: recent studies have shown that ctDNA in vitro the preservation of environment and
It will receive certain oxidative damage in enrichment treatment process.It is wherein most commonly seen with 8- hydroxyl-guanine (8-oxo-dG), it should
The PCR reaction through library preparation flow, easily mistake pairing adenine are damaged, artificial introducing G → T during building library is caused
Mutation, causes the appearance of final sequencing result false positive.Have been reported that the false positive mutation for even claiming the type, up to public gene
It is more than the G that group database is always included → T-type mutation one third.And it is examined caused by existing other kinds DNA oxidative damage
Measured data mistake cannot also despise (Fig. 1).
Summary of the invention
In order to solve the above problem present in the detection of low frequency DNA mutation, the present invention passes through the DNA plerosis segment before building library
Oxidative damage, and import in every DNA fragmentation the internalcontrol sequence of specificity, be aided with unique data analysing method, exclude
Oxidative damage, the false positive mutation that PCR is introduced or sequencing generates, to improve the sensitivity of low frequency DNA mutation detection and special
Property.
Connector used in the present invention is that the sequence of special designing synthesis anneal, it is that the present invention excludes false sun
The key of property mutation disturbance.
Therefore, the first aspect of the present invention provides a kind of connector mixture for DNA library building.The connector is mixed
Closing object includes a variety of Y type double-stranded adapters containing different internalcontrol sequences, and every kind of breeches joint is by with the first chain of flowering structure and the
Two chains form (Fig. 2):
First chain: the 5 ' area-A1 area the area-B1-C1-T-3 ',
Second chain: the 3 ' area-A2 area the area-B2-C2-p-5 '；
Wherein, the area A2 and the area A1 be not complementary, and the area B2 is complementary with the area B1, and the area C2 is complementary with the area C1, and T is thymidine, and p is indicated
5 ' terminal phosphates of the second chain；
The area A1 of a variety of double-stranded adapters in the connector mixture, the area A2, the area B1, the sequence difference in the area B2 are identical；C1
Area, the area C2 are internalcontrol sequence, are made of random nucleotides, and the internalcontrol sequence of a variety of double-stranded adapters is different.
In a specific embodiment, the area A1 includes P5 sequence, and the area A2 includes P7 complementary series.P5 sequence and P7 sequence
Column be it is widely used build library joint sequence, can for its design primer be used for amplified library.
When building library for multisample, the area A1 and/or the area A2 can also include sequence label, it is preferable that the label sequence
Column are made of 6 nucleotide, it is highly preferred that the sequence label is located at the 3 ' ends in the area A1 and/or the 5 ' ends in the area A2.It is optimal
Selection of land, the area A1 and the area A2 include different sequence labels, and two strip label sequences can carry out dual test, in this way can will be each
A stage is effectively identified due to the Reads of index misassignment caused by crosstalk, and is rejected, and guarantees to enter final
The Reads of analysis process can represent authentic sample.
In a specific embodiment, the sequence of the first chain and the second chain is respectively 5 '-AATGATACGGCGACCA
CCGAGATCTACACTCTTTCCCTACACGAGGCTCTTCCGATCNNNNNT-3 ' and 5 '-pNNNNNGATCGGAAGAGCACA
CGTTCTGAACTCCAGTCACCGATGTATCTCGTATGCCGTCTTCTGCTTG-3’。
When connector mixture of the invention is constructed for DNA library, every DNA fragmentation connection has unique internalcontrol sequence
Two connectors, the unique identification by the two internalcontrol sequences as the DNA fragmentation, and the DNA fragmentation is being expanded and surveyed
State before sequence secures.On this basis, after the sequencing is completed, by the analysis to Reads data, it can identify and arrange
Except false positive is mutated.
Further, the second aspect of the present invention provides a kind of detection method of low frequency DNA mutation, including following step
It is rapid:
(1) total DNA is extracted from sample；
(2) the oxidative damage reparation of total DNA and purifying；
(3) A base is added in the blunt end for the total DNA segment repaired and 3 ' ends；
(4) DNA fragmentation is attached with connector mixture of the invention；
(5) the target area hybrid capture based on probe；
(6) amplified library；
(7) machine is sequenced on；
(8) sequencing data is analyzed.
Detection sample can be any sample containing DNA, such as: blood, amniotic fluid, tissue etc..The low frequency DNA of detection is prominent
Change can be known mutations, be also possible to unknown mutation.
In a specific embodiment, detection sample is blood.Above-mentioned steps (1) are first from the blood sample of acquisition
In isolate blood plasma, then extraction purification goes out total dissociative DNA from blood plasma；Preferably, the separation of blood plasma uses two step centrifugal process,
Whole blood 1600g centrifugation 10min takes supernatant, and supernatant 16000g is centrifuged 10min again, and second step centrifugation gained supernatant is blood plasma.
If the total DNA molecule extracted in sample is longer, for the sequencing of upper machine, need to carry out fragmentation processing, piece to it
Duan Huake is carried out before or after oxidative damage reparation.The reading of the requirement view sample type and sequencing system of fragmentation processing is long
Depending on.In a specific embodiment, sample is the dissociative DNA in blood sample, and length is generally 170bp or so,
General two generations sequenator to the nucleic acid molecules of the length can direct Sequencing, so there is no need to fragmentation processing can carry out in next step
Operation.
Most of 8- hydroxyl-guanine that step (2) can be such that oxidation generates the oxidative damage reparation of total DNA is gone back
Original, this can greatly reduce the mutation of the false positive as caused by oxidative damage, to improve the accuracy of detection.Preferably, it walks
Suddenly (2) carry out oxidative damage reparation to total DNA using the FFPE DNA Repair Mix system of NEB company.
The total DNA fragment ends polishing of reparation is become into flush end in step (3), and adds A base in 3 ' ends, in order to
The connection of next step.In step (4), the A base of the T base of 3 ' end of the first chain of connector and the addition of 3 ' end of DNA fragmentation is utilized
It is connected by hydrogen bond, then connects DNA fragmentation and connector usually using T4DNA ligase.
In a specific embodiment, the connection product that step (5) obtains rna probe and above-mentioned steps (4) is miscellaneous
It hands over, then uses the affine magnetic capture Hybrid Library of biotin.Step (6) is designed using based on the first area chain A1 and the second area chain A2
Primer PCR expand library.
Method of the invention is suitable for various sequencing approaches, and the sequencing of especially two generations is best suited for Illumina company
NextSeq microarray dataset.
Since mutated-genotype is all mutating alkali yl on two chains of DNA fragmentation, the method according to the invention expands it
All segments obtained afterwards all include the mutation in the site, and the mutation that oxidative damage, PCR amplification or sequencing introduce then only exists
In on a chain of a DNA fragmentation, there was only Partial Fragment after amplification includes the mutation in the site.Therefore, in step
(8) in, the internalcontrol sequence that both ends are connected Reads Data Integration all the same or complementary, if this group of Reads data phase
Together, then a Group data are formed；If this group of Reads data are different, abandoned as abnormal data.Then, with
Group data are unit, are compared with Ref normal data, find mutated-genotype, and further with saltant type Group number
The ratio of the total Group number of Zhan calculates the frequency of mutation.
Detailed description of the invention
Fig. 1 is directed to ctDNA at present and detects common connector connection banking process, and the mistake that oxidative damage generates will cause vacation
It is positive.The principle is as follows:
(1) 8- hydroxyl-guanine (G that oxidation generates+) be randomly occurring in double-strand, T#To be really mutated；
(2) double-strand links " Y " font sequence measuring joints；
(3) it is reacted by PCR, 8- hydroxyl-guanine (G+) mistake is matched with adenine A, and then expanding is that thymus gland is phonetic
Pyridine T, TΔThe mispairing generated for PCR mistake；
(4) when sequencing result carries out data analysis, the pairing mistake of generation causes false positive.
Breeches joint Fig. 2 of the invention.The area A2 and the area A1 be not complementary, and the area B2 is complementary with the area B1, and the area C2 is complementary with the area C1, and T is
Thymidine, p indicate 5 ' terminal phosphates of the second chain.
Implementation process Fig. 3 of the invention.
Fig. 4 the principle of the present invention, greatly improves detection sensitivity.The principle is as follows:
(1) 8- hydroxyl-guanine (G that oxidation generates+) be randomly occurring in double-strand, T#To be really mutated；
(2) 8- hydroxyl-guanine (G that oxidation generates+) repaired major part is reduced normally, small part there are still not by
The oxidative damage of reparation；
(3) double-strand connects special designing, " Y " font sequence measuring joints with internalcontrol sequence and self annealing, herein internal reference sequence
Column are by taking " GTTCA " and " ATCGG " as an example；
(4) it is reacted by PCR, a small number of 8- hydroxyl-guanine (G+) mistake is matched with adenine A, and then expanding is chest
Gland pyrimidine T, and internalcontrol sequence introduces double-strand, TΔThe mispairing generated for PCR mistake；
(5) before being compared with Ref standard sequence, by containing same internalcontrol sequence " GTTCAATCGG " and complementation
The Reads data of " CAAGTTAGCC " are integrated, and are formed Group data or are abandoned abnormal data；
(6) it as unit of Group data, is compared with Ref normal data, it is special by the mutation analysis of target of ctDNA
The opposite sex greatlys improve.
The fragment length in Fig. 5 present invention processing library.
Fig. 6 present invention handles library quantitative data.
The site Fig. 7 digital pcr EGFR p.T790M verify data.
Specific embodiment
Below with reference to example, the present invention is further illustrated.
Embodiment 1DNA sample preparation
Negated Patients With Small Cell Carcinoma of The Lung whole blood 30ml is transported at room temperature, haulage time using 3 pipe of BCT pipe of streck company
About 46 hours.
The separation of blood plasma uses two step centrifugal process, i.e. whole blood 1600g centrifugation 10min takes supernatant；Preceding step supernatant 16000g from
Heart 10min, gained supernatant are blood plasma.
The blood plasma of separator well takes 2 parts of QIAamp Circulating Nucleic Acid for using Qiagen company immediately
Kit, carries out the extraction of total dissociative DNA respectively, and extracted amount is shown in Table 1.
Table 1
| Sample ID | Sample extraction amount |
| SLZ1 | 68.5ng |
| SLZ2 | 57.3ng |
Embodiment 2DNA oxidative damage reparation
A copy of it DNA sample prepared by Example 1, the proportion preparation DNA oxidative damage reparation reaction referring to table 2 are mixed
Close liquid.
Table 2
DNA repairs buffer and enzyme mixation is the FFPE DNA Repair Mix system of NEB company.
Above-mentioned system is moved into PCR instrument, 20 DEG C, 15 minutes, carries out the reparation of the oxidative damage of DNA.Then it is added 186
μ l AMPure XP magnetic bead (being purchased from Beckman) is purified, and is eluted with 50 μ l nuclease-free waters.
A base is added in the blunt end of embodiment 3DNA segment and 3 ' ends
Total dissociative DNA segment that Example 2 is repaired prepares reaction mixture referring to the proportion of table 3.End is repaired in this example
Again and adds A buffer and end to repair and A enzyme mixation is added to use KAPA Hyper Prep Kit.
Table 3
Above-mentioned system is moved into PCR instrument, program is set by table 4 and is reacted.
Table 4
The connection of 4 connector of embodiment
Connector used in the present invention is that the sequence of special designing synthesis anneal.In this example use comprising
The connector (referring to table 5) of Illumina Index 1 (ATCACG) sequence label, wherein " NNNNN " is internalcontrol sequence, N, that is, ATGC
The ambiguity base of four kinds of bases.
Table 5
Prepare reaction mixture according to the proportion of table 6.
Table 6
Connection buffer is 50mM Tris-HCl, 10mM MgSO4、1mM ATP。
The system prepared is divided into 2 pipes, every 55 μ l of pipe is put in PCR instrument, 25 DEG C of reaction 45min.After reaction, make
With 0.8 × AMPure XP magnetic beads for purifying DNA sample.
Target area hybrid capture of the embodiment 5 based on probe
The connector connection product that embodiment 4 obtains is mixed with 5 μ l closed reagents, is moved into PCR instrument, journey is set by table 7
Sequence is reacted.
Table 7
For target area hybrid capture rna probe can the official website Aglient company SureSelect (https: //
Earray.chem.agilent.com/suredesign/home.htm the target area designed, designed captured as needed on).
The probe sum that this example uses be 677, capture target area 28.583kb, covering EGFR, ERBB2, ALK, ROS1, RET,
The moiety site of 13 genes such as BRAF, KRAS, NRAS, PIK3CA, MET, DD2, PTEN, FGFR1.The target area of probe design
The specific range in domain is as shown in table 8.
Table 8
Prepare probe hybrid mixed liquid according to the proportion of table 9, is added in step in probe, after which is added closing
In connector connection product.Wherein, hybridization buffer is Aglient SureSelectXTReagent Kit, RNase inhibitor purchase
From Thermo Fisher Scientific.
Table 9
After reaction, using the affine magnetic capture Hybrid Library of biotin, Dynabeads MyOne is used in this example
Streptavidin T1(Thermo Fisher Scientific)。
6 amplified library of embodiment
Prepare amplified library reaction mixture according to the proportion of table 10.
Table 10
Archaeal dna polymerase and PCR buffer use KAPA Hyper Prep Kit in this example.
It is put into PCR instrument, program is set by table 11 and is reacted.
Table 11
After reaction, using 1.2 × AMPure XP magnetic bead amplified production is purified, finally library is dissolved in
In 50 μ l NF-water.Library is carried out using 3.0 fluorimeter of Thermo Fisher Scientific company Qubit accurate
It is quantitative.
Purified product is diluted to 1.5ng/ μ l, 1 μ l is taken to carry out segment using Aglient company 2100Bioanalyzer
Size detection (Fig. 5)；
The library quantification kit that 1 μ l uses KAPA company is taken out, with 480 II platform of Roche company, it is effective to carry out qPCR
Library quantitative detection (Fig. 6).
Embodiment 7 is sequenced
According to concentration measured by embodiment 6, by library be diluted to it is upper it is confidential ask after (2nM), in Illumina company
PE150 sequencing is carried out in NextSeq microarray dataset.
According to the used capture probe area size of this example, total amount of data, which is sequenced, should be greater than 5G, and target area data account for
Than be greater than 20%, average sequencing depth 30000 × more than.
Average same internalcontrol sequence supports 6 reads or more of number when merging identical internalcontrol sequence Group.
Averagely be sequenced after merging Group depth 3000 × more than, target area be sequenced depth be greater than 2000 × 90%
More than.
Sample Quality Control data in this example are shown in Table 12.
Table 12
| Data volume (G) | Target area | Average sequencing depth | Average sequencing depth after merging | Greater than 2000 × accounting |
| 5.7 | 26.7% | 53244 | 4126 | 92.7% |
Sample used in this example is analyzed through data, and detection abrupt information is shown in Table 13.
Table 13
| Annotation | With reference to genotype | Mutated-genotype | Total Group number | Saltant type Group number | The frequency of mutation |
| EGFR p.T790M | C | T | 4768 | 7 | 0.147% |
In this example, using the Raindrop digital pcr platform of Raindance company, to the detection knot of this example sample
Fruit is verified.Detection site EGFR p.T790M is chosen, the result of verifying is the EGFR with 0.1825% frequency
P.T790M Positive mutants (Fig. 7), this is very high with the testing result consistency of method provided by the invention.
The EGFR p.T790M frequency of mutation of digital pcr detection is slightly higher, is probably derived from the oxidative damage of ctDNA sample,
And through this flow processing, sequencing is damaged with sample and has been repaired.
Particularly, this banking process carries out common mutations test, detection knot using the ctDNA standard items of Horizon company
Fruit and expected consistent degree are very high (table 14).
Table 14
| Gene | pHGVS | Mutation Standard | Result | Wild Standard | Result |
| EGFR | L858R | 0.10% | 0.076% | 0% | 0.00% |
| EGFR | E746_A750del | 0.10% | 0.089% | 0% | 0.00% |
| EGFR | T790M | 0.10% | 0.085% | 0% | 0.00% |
| EGFR | V769_D770insASV | 0.10% | 0.077% | 0% | 0.00% |
| KRAS | G12D | 0.13% | 0.131% | 0% | 0.00% |
| NRAS | Q61K | 0.13% | 0.117% | 0% | 0.00% |
| NRAS | A59T | 0.13% | 0.136% | 0% | 0.00% |
| PIK3CA | E545K | 0.13% | 0.116% | 0% | 0.00% |
Sequence table
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<120>a kind of detection method of low frequency DNA mutation
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Claims (16)
1. a kind of connector mixture for DNA library building, it is characterised in that: the connector mixture includes a variety of by following
The Y type double-stranded adapters of first chain of structure and the second chain composition:
First chain: the 5 ' area-A1 area the area-B1-C1-T-3 ',
Second chain: the 3 ' area-A2 area the area-B2-C2-p-5 '；
Wherein, the area A2 and the area A1 be not complementary, and the area B2 is complementary with the area B1, and the area C2 is complementary with the area C1, and T is thymidine, and p indicates the 2nd
5 ' terminal phosphates of chain；
The area A1 of a variety of double-stranded adapters in the connector mixture, the area A2, the area B1, the sequence difference in the area B2 are identical；The area C1, C2
Area is internalcontrol sequence, is made of random nucleotides, and the internalcontrol sequence of a variety of double-stranded adapters is different.
2. the connector mixture of claim 1, wherein the area A1 includes P5 sequence, and the area A2 includes P7 complementary series, it is preferable that institute
P5 sequence and P7 complementary series are stated respectively as shown in SEQ ID NO:1 and 2.
3. the connector mixture of claims 1 or 2, wherein the area A1 and/or the area A2 include sequence label, it is preferable that the label
Sequence is made of 6 nucleotide, it is highly preferred that the sequence label is located at the 3 ' ends in the area A1 and/or the 5 ' ends in the area A2.
4. the connector mixture of claim 1, wherein the area B1 includes the sequence of SEQ ID NO:3, and the area B2 includes SEQ ID NO:
4 sequence.
5. the connector mixture of claim 1, wherein the random nucleotides that the area C1, the area C2 are made of 5 nucleotide.
6. a kind of detection method of low frequency DNA mutation, comprising the following steps:
(1) total DNA is extracted from sample；
(2) the oxidative damage reparation of total DNA and purifying；When necessary, it is carried out at fragmentation before reparation or to total DNA after repairing
Reason；
(3) A base is added in the blunt end for the total DNA segment repaired and 3 ' ends；
(4) the connector mixture of DNA fragmentation and any one of claim 1-5 is attached；
(5) the target area hybrid capture based on probe；
(6) amplified library；
(7) machine is sequenced on；
(8) sequencing data is analyzed.
7. the detection method of claim 6, wherein the sample is blood.
8. the detection method of claim 7, wherein step (1) isolates blood plasma from the blood sample of acquisition first, then from
Extraction purification goes out total dissociative DNA in blood plasma；Preferably, the separation of blood plasma uses two step centrifugal process, and whole blood 1600g is centrifuged 10min
Supernatant is taken, supernatant 16000g is centrifuged 10min again, and second step centrifugation gained supernatant is blood plasma.
9. the detection method of claim 6, wherein step (2) uses the FFPE DNA Repair Mix system pair of NEB company
Total DNA carries out oxidative damage reparation.
10. the detection method of claim 6, wherein step (4) uses T4 DNA ligase connection DNA fragmentation and connector.
11. the detection method of claim 6, wherein step (5) hybridizes rna probe with the connection product that step (4) obtains,
Then the affine magnetic capture Hybrid Library of biotin is used.
12. the detection method of claim 6, wherein step (6) is drawn using what is designed based on the first area chain A1 and the second area chain A2
Object PCR amplification library.
13. the detection method of claim 6, wherein step (7) is sequenced using two generation microarray datasets, it is preferable that
The NextSeq microarray dataset of Illumina company is sequenced.
14. the detection method of claim 6, wherein in step (8), the internalcontrol sequence that both ends are connected is all the same or complementary
Reads Data Integration forms a Group data if this group of Reads data are identical；If this group of Reads data
Difference is then abandoned as abnormal data.
15. the detection method of claim 14, wherein in step (8), further as unit of Group data, with Ref standard
Data are compared, and find mutated-genotype, then calculate the frequency of mutation with the ratio of the total Group number of saltant type Group number Zhan.
16. the detection method of any one of claim 6-15, wherein the low frequency DNA mutation is that the gene that ctDNA is carried is prominent
Become.
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