CN109943628A - The method that PGS and methylation analysis are carried out to biopsy cells using DedscRRBS analytic approach - Google Patents
The method that PGS and methylation analysis are carried out to biopsy cells using DedscRRBS analytic approach Download PDFInfo
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Abstract
Use " test-tube baby " blastaea ectoderm cell for raw material the present invention relates to a kind of, representative bisulfite sequencing technologies are simplified using double digestion, blastaea ectoderm cell methylation state of DNA is parsed using epigenetics analytical technology, while to science of heredity screening before chromatin state progress embryo implantation.Multi-angle fully assesses the potentiality of development of embryo, to select the embryo of " correct " to provide new reference in supplementary reproduction, provides strong backing to the success rate in raising " test-tube baby " period.
Description
Technical field
The present invention relates to biomedical and molecular cytobiology fields, and in particular to a kind of couple of test-tube baby embryo is grown
It supports layer biopsy sample and carries out the method that unicellular double digestion simplifies representative bisulfite sequencing, utilize epigenetic credit
Analysis technology parses blastaea ectoderm cell methylation state of DNA, while sieving to science of heredity before chromatin state progress embryo implantation
It looks into.
Background technique
There is the medical treatment across significance of times to invent human fertility's history as one, tube-test baby techniques are considered " existing
For the milestone of medical development ".This technology has been the countless growing barriers in the whole world in nearly 40 years since 1978 are born
Family brings dawn, also has found effective solution for increasingly prominent human fertility's problem.With technology
Rapid development, test-tube baby also experienced two generation an of generation, nowadays entered three generations.
One test tube baby cycle checks comprehensively from parent both sides, ovulation induction secures good health ovum obtains sperm, is external
Antenatal Serial monitoring inspection after the processes such as fertilization, fertilized eggs and Embryo Culture, the suitable embryo transfer of selection and transplanting etc. is straight
1 year or so is at least needed to healthy babies birth, so during complexity, the development that factors influence embryo is latent
Energy.The main standard of assessment embryonic development potential is morphological criteria at present, but with the development of technology and clinical analysis
Deeply, researcher has found that the normal embryo chromosome ortholoidy of morphology is not necessarily normal, and the chromosome aneuploidy of embryo
Property frequently can lead to graft failure.Therefore, science of heredity screening (PGS) is come into being before embryo implantation, which can comprehensive screening
Possible abnormal chromosome in embryo, selects good embryo and carries out palace intraluminal grafting, reduce embryo diapause, risk of miscarriage.But
It is that while that the technology substantially increases the success rate of third generation test-tube baby, but the overall expectation that people are still not achieved, mesh
Preceding success rate generally only has 40% or so.
After PGS technology, still there is the normal embryo quality graft failure of chromosome.Its main cause may be auxiliary
The outer after fertilization of gonophore is helped, embryo will undergo non-maternal environment culture in 3-6 days in vitro, and epigenetic situation is interfered,
To influence the potentiality of development of embryo.Wherein, DNA methylation is as important " epigenetics label ", in human body early embryo
(the Guo et al.Nature.2014Jul 31 of dynamic change as exactly experiencing roller-coaster in growth course；511(7511):
606-610；Zhu P et al.Nature Genetics.2018 Jan；50 (1): 12-19.), it is most likely that by extraneous ring
The influence in border and mutation occurs, and then lead to that some morphology are high-quality, chromatin state normal embryo transfer failure.And mesh
Before preceding implantation in screening, all the epigenetic situation of embryo is not analyzed.
As " goldstandard " of DNA methylation detection, bisulfite is modified using high-flux sequence platform of new generation as base
Plinth draws genomic DNA methylation level map in conjunction with the processing of genome bisulfite and biological data analysis；Meanwhile
The main information of DNA methylation sequencing is DNA sequence dna information, and the C not methylated only is substituted for T to distinguish methylation
With non-methylation.DNA methylation sequencing data can be used to assess large-scale chromosome copies number variation (CNV), even
The single nucleotide mutation (SNV) of single base resolution ratio.
DNA methylation high throughput sequencing technologies on individual cell level specifically include that unicellular full-length genome weight sulfurous acid
(Single-cell whole genome bisulfite sequencing, scWGBS) and unicellular simplified representative is sequenced in salt
Property bisulfite sequencing (Single-cell reduced-representation bisulfite sequencing,
scRRBS).Unicellular full-length genome methylation sequencing (scWGBS) is the complete genome DNA based on individual cell level
(Smallwood SA,Lee HJ et al.,Nature Methods.2014 Aug；11 (8): 817-820.), it is available
The full-length genome methylation profiles of single base resolution ratio, but this technology genome coverage rate is low, sequencing cost is high；It is slender
The simplified representative bisulfite sequencing (scRRBS) of born of the same parents, which refers to, utilizes specific DNA restriction enzyme (Msp I) to carry out digestion
(Guo et al., Nature Protocols, 2015,10 (5): 645-59.) are enriched with out in full-length genome greatly
The site CpG with important information, is a kind of relative ease, economic DNA methylation research method, but this skill at present
Art obtains sequence and genome matching rate and also there was only 70% or so, and redundancy is higher, and the valid data amount of acquisition is relatively
It is few；And when carrying out PGS analysis, SD value is larger, especially can not accurately detect lesser CNV.
It is had been reported about research of the DNA methylation sequencing in supplementary reproduction field.North doctor three Yuan Qiaojie study groups with
The cooperation discovery of Liu Jiang study group, Joint Genome Institute, the methylation state of blastaea trophocyte can react entire embryo's
Methylation state, while related to quality of blastocysts, DNA abnormal methylation may be cause one of embryonic development failure it is important
Factor (Li et al., Journal ofGenetics and Genomics, 2017 Oct 20；44 (10): 475-481.),
The research points out that embryo quality overall dna methylation level concentrates within particular range, and most low quality embryos
Overall dna methylation level shows as excessively high or too low；Meanwhile DNA abnormal methylation region and embryonic development approach phase
It closes.Tang Fuchou seminar, Beijing University has delivered unicellular DNA methylation research (the Zhu P et al.Nature of human body early embryo
Genetics.2018 Jan；50 (1): 12-19.), result of study is shown, chromosomal copy number once increases in body early embryo,
The DNA methylation level of full-length genome entirety can reduce, on the contrary, chromosomal copy number reduces then whole DNA methylation level
Rise.Therefore, on the basis of traditional PGS, in addition DNA methylation assay can not only improve the standard of chromosomal aneuploidy screening
True property, while epigenetics information can be obtained, further increase Success Rate of Embryo Transfer.
In conclusion technology still has shortcoming at present:
(1) tradition PGS is only assessed from chromatin state, is had ignored in embryo's early stage highly important apparent something lost
The progress of disease cannot provide more references for more comprehensively assessment embryonic development potential；
(2) either unicellular full-length genome bisulfite sequencing (scWGBS) or single endonuclease digestion (Msp I) handle and are
Genome matching rate obtained by the simplified expression sulphite sequencing (scRRBS) on basis is all relatively low, the valid data amount of acquisition
It is relatively fewer；And when carrying out PGS analysis, SD value is larger, cannot obtain more specific chromosomal aneuploidy result.
Therefore, it is badly in need of a kind of easy, efficiently, economic unicellular DNA methylation high-flux sequence method, to obtain more
High genome matching rate, wider array of genome coverage rate, more comprehensive CpG site information；Simultaneously to the chromosome of embryo
Aneuploidy situation and DNA methylation situation carry out double analysis, provide multi-angle with reference to detection embryonic development potential.
Summary of the invention
The present invention uses " test-tube baby " blastaea ectoderm cell for raw material, simplifies representative weight sulfurous using double digestion
(Double-enzyme digestion single-cell reduced-representation bifulfite is sequenced in hydrochlorate
Sequencing, DedscRRBS) technology, blastaea ectoderm cell DNA methylation is parsed using epigenetics analytical technology
State, while to science of heredity screening (Preimplantation genetic before chromatin state progress embryo implantation
Screening, PGS).Multi-angle fully assesses the potentiality of development of embryo, to select the embryo of " correct " to mention in supplementary reproduction
New reference has been supplied, strong backing is provided to the success rate in raising " test-tube baby " period.
To solve problems of the prior art, the completely new improvement that the present invention carries out the prior art, with double enzymes
It cuts and (I/ApeKI of Msp, Msp I/Xma I, Msp I/Taq α I, Xma I/Taq α I, Xma I/ApeKI, Taq α I/ can be selected
A variety of enzymes combinations such as ApeKI) the simplified representative bisulfite sequencing (DedscRRBS) of unicellular double digestion is carried out, not only
It is easier, efficient, economical, and genome coverage is improved comprehensively, more comprehensive CpG site information is obtained, while again
Chromosomal aneuploidy situation can be analyzed.
Simplify representative bisulfite sequencing the present invention provides a kind of completely new unicellular double digestion and chromosome is non-
Ortholoidy analyzes (Double-enzyme digestion single-cell reduced-representation
Bifulfite sequencing&Preimplantation genetic screening, DedscRRBS-PGS) method,
Include the following steps:
The acquisition of blastaea biopsy cells: fertilized eggs are obtained by Intracytoplasmic sperm injection method (ICSI), fertilized eggs are in ovum
It is cultivated in blastomere culture medium (G1 culture medium) 3 days after arriving the blastomere phase, then embryo is transferred in blastocyst culture base (G2 culture medium)
Culture is to the 5th day until the blastaea stage of ripeness is arrived in development.Take Trophectoderm cells 3-5 of the separate inner cell mass of blastaea
It is transferred to spare in PCR pipe；
The building of DedscRRBS sequencing library: according to sample volume, being proportionally added into lysis buffer and lyases, mixes
Lyases is inactivated after incubation, to pyrolysis product carries out double digestion, end is repaired plus A, connector connection, CT conversion processing and purifying
Recycling, PCR amplification, purifying and second of amplification, recycling purpose product；
(3) library is sequenced: being carried out using technologies such as the sequencing of two generations or nucleic acid chip, immune-blotting methods to sequencing result
Parsing, analyzes chromosomal condition and DNA methylation composes situation.
Preferably, in step (2), the main component of lysis buffer (Buffer A) includes final concentration 2-200mM
Tris-EDTA, 1-50mM KCl, surfactant such as Triton X-100 of 0.1wt%-5wt%, SDS, Tween-20,
One of NP40 or a variety of.
Lyases is selected from Proteinase K, Qiagen Protease, pepsin, papain, trypsase and bacteriolyze
One of enzyme is a variety of, and enzyme concentration used is 1-30 μ g/ml.Incubation temperature is 37-65 DEG C, and the time is 30min to 12h, is lost
Temperature living is the time 10-45min no more than 80 DEG C.
In any of the above-described scheme preferably, the buffer in step (2) uses general Buffer B, mainly
Ingredient includes: 10-40mM Tris-acetate, 1-10mM Magnesium Acetate, 1-100mM Potassium
Acetate, 0.1-1mg/ml BSA.
In double digestion system, the preferred BufferB of buffer is sample as the non-methylation λ DNA additional amount referring to DNA
About the 1% of middle DNA, 37-75 DEG C of digestion temperature, time 1-6 hour, 60-80 DEG C of deactivation temperature, time 10-30min.
In any of the above-described scheme preferably, end repairs and A is added to use archaeal dna polymerase Klenow segment, buffer
It is preferred that BufferB, reaction temperature is 37 DEG C, repair time 20-60min, 60-80 DEG C inactivation 5-30min.
In any of the above-described scheme preferably, connector connection is connected using methylation connector with T4DNA ligase, excellent
Process flow: 16 DEG C of pre-connection 30min is selected, then 4 DEG C of connections are overnight or not less than 8 hours, after the completion of connection at 50-75 DEG C
Reason 20min inactivates enzyme.
In any of the above-described scheme preferably, CT conversion is carried out, it is preferable to use Invitrogen using bisulfite
MethylCode Kit or EZ DNA Methlyation-Direct Kit, and illustrate to carry out referring to kit.
In any of the above-described scheme preferably, PCR amplification uses KAPA HiFi U+master Mix, Phusion
high-fidelity(HF)PCR master Mix with HF buffer、KAPA HiFi fridelity
The one or two of Buffermsupplied with polymerase.First time amplification program is as follows: 98 DEG C, 2min；98
DEG C, 20s, 60 DEG C, 30s, 72 DEG C, 60s, 6-12 circulations；72 DEG C, 5min；4 DEG C of preservations.
Complete after expanding for the first time, amplified production preferably through 0.8-1.5 times of volume XP magnetic beads for purifying, purified product into
Second of amplification of row.Second of amplification program is as follows: 98 DEG C, 2min；98 DEG C, 20s, 60 DEG C, 30s, 72 DEG C, 60s, 15-21
Circulation；72 DEG C, 5min；4 DEG C of preservations.
In any of the above-described scheme preferably, step returns the target DNA fragment 200-450bp in (2)
It receives, it is preferable that using magnetic bead recovery purifying, first step 0.3-0.6X XPbeads in two steps, remove excessive DNA, second
Step adds 0.4-0.7x again in the supernatant of the first step, and finally recycling DNA length is 200-450bp.
In any of the above-described scheme preferably, it is sequenced in step (3) using NGS, key instrument XTEN, NEXT-
The Illumina such as seq, Highseq-2500, Miseq series and corresponding reagent consumptive material, referring to instrument and reagent consumptive material SOP into
Row operation.
The present invention provides dual points that a kind of couple of embryo carries out chromosomal aneuploidy situation and DNA methylation situation
The method of analysis simplifies representative weight Asia including using method of the invention to carry out unicellular double digestion to embryonic feeder confluent monolayer cells
Sulfate sequencing.
It in certain embodiments, further include according to chromosomal aneuploidy situation and DNA methylation situation
The step of analysis result assesses the potentiality of development of embryo.
The present invention uses the blastaea ectoderm biopsy cells in " test-tube baby " operation for raw material, can be simultaneously to embryo's
Chromosomal aneuploidy situation and DNA methylation situation carry out double analysis, which can be used for a variety of purposes, both
It can be used for screening before embryo implantation, it can also be used to correlative study of embryonic development etc. in laboratory.With the PGS of current mainstream
It compares, the present invention can also be from the angle of DNA methylation point while providing same chromosomal aneuploidy and testing and analyzing
Analysis, thus from epigenetic and the potentiality of development of embryo and the completely new angle estimator embryo of culture environment reacted, to assist
It selects the embryo of " correct " to provide new reference in reproduction, strength is provided to the success rate in raising " test-tube baby " period
It supports.
Detailed description of the invention
Figure 1A and B is the same 1PN but PGS analysis is trophoderm biopsy cells (sample 1) difference of the useless embryo of normal medicine
The sequencing obtained using mainstream PGS analysis product SurePlex-Veriseq (Figure 1A) and DedscRRBS (Figure 1B) experimental arrangement
The chromosomal aneuploidy analysis that data carry out；
Fig. 2A and B is the same 1PN but PGS analysis divides for the trophoderm biopsy cells (sample 2) of the useless embryo of abnormal normal medicine
It Cai Yong not the chromosome aneuploidy that carries out of the sequencing data that obtains of routine PGS (Fig. 2A) and DedscRRBS (Fig. 2 B) experimental arrangement
Property analysis；
Fig. 3 is the number for having chosen the trophoderm biopsy cells (TE) of 9 embryos and DedscRRBS experimental arrangement being used to obtain
According to the methylation sequencing analysis of progress；
Fig. 4 is that the 9 TE samples chosen carry out methylation data analysis, and it is each in the genome to analyze the site CpG emphatically
Distribution and the overall methylated level of sample on a function original part；
Fig. 5 is the further part of Fig. 4；
Fig. 6 is main agents involved in the present invention.
Specific embodiment
In order to be further understood that summary of the invention of the invention, the present invention is elaborated below in conjunction with specific embodiment.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, or according to manufacturer
Proposed condition.
It takes 2 blastaea biopsy cells (sample 1 and sample 2), carries out DedscRRBS-PGS sequencing analysis, concrete operations are such as
Under:
1. the acquisition of blastaea biopsy cells: obtaining fertilized eggs by Intracytoplasmic sperm injection method (ICSI), fertilized eggs exist
It is cultivated in blastomere culture medium (G1 culture medium) 3 days after arriving the blastomere phase, then embryo is transferred to blastocyst culture base (G2 culture medium)
Middle culture is to the 5th day until the blastaea stage of ripeness is arrived in development.Take the Trophectoderm cells 3-5 of the separate inner cell mass of blastaea
It is a to be transferred to (using the useless embryo of 1PN medicine in this research) spare in PCR pipe；
2.DedscRRBS-PGS library construction: according to the volume of sample, be added 10 × lysis buffer (BufferA) and
The protease of 1/20 volume, make final concentration of 1 ×, protease 2mg/ml.It is collected by centrifugation after mixing in tube bottom.
Sample is placed in PCR instrument, 50 DEG C of 1.5h lytic cell released dnas, 75 DEG C of 30min inactivate protease.
Msp I, ApeKI, 10 × BufferB, non-methylation λ DNA to final concentration of digestion Msp are added into pyrolysis product
Each 9U of I/ApeKI, 1 × BufferB, non-methylation λ DNA are 1% or so of sample DNA estimator.Sample is placed in PCR instrument,
37 DEG C of 2h, 75 DEG C of 2h.
Archaeal dna polymerase Klenow exo- is added into digestion products, 10 × BufferB to final concentration is respectively 5U, 1
×, 40 μM of dATP of dNTP final concentration, 4 μM of dGTP, 4 μM of dCTP are repaired in end.It mixes well, is slightly collected by centrifugation in tube bottom,
37 DEG C 40 minutes in PCR instrument, 75 DEG C 15 minutes.
T4DNA ligase, 10 × BufferB, ATP, methylation connector are added in product to repairing, after mixing, is placed in
In PCR instrument, 16 DEG C 30 minutes, 4 DEG C overnight or be not less than 8 hours, after the completion, 65 DEG C, 20min inactivates enzyme.Reference
The operation of Invitrogen MethylCode Kit (or EZ DNAMethlyation-Direct Kit) specification carries out CT and turns
Change, in brief, bisulfite transformation mixture is added into connection product to 150 μ l of total volume, slightly centrifugation is received after mixing
Combine in tube bottom, 98 DEG C in PCR instrument, be denaturalized 10 minutes, 64 DEG C, bisulfite converts 2.5 hours, 4 DEG C place to 20 hours after
Continuous conversion.Converted product combines through centrifugal column, washing, purifies elution recycling, and 24 μ l of eluent is for expanding.
It is used for first time PCR amplification after converted product is purified, using 50 μ l systems, 2 × Kapa HiFi U+ is added
Master Mix 25 μ l, 15 μM of each 0.5 μ l of Universal primer and Index primer, 24 μ l purified products.Reference
Following reaction amplification: 98 DEG C, 2min；98 DEG C, 20s, 60 DEG C, 30s, 72 DEG C, 60s, 6 circulations；72 DEG C, 5min；4 DEG C of preservations.
Product 0.8 × XP magnetic beads for purifying PCR product, with 33 μ l water washings.
5X KAPA HiFi Fidelity buffer is added to final concentration 1 using 50 μ l systems in second of PCR amplification
×, 10mM dNTP Mix1.5 μ l, 1U/ul KAPA HiFi HotStart DNA polymerase to 1U, 10 μM
Each 2.5 μ l of Universal primer and Index primer, 32.5 μ l purified products.Referring to following reaction amplification: 98 DEG C,
2min；98 DEG C, 20s, 60 DEG C, 30s, 72 DEG C, 60s, 18 circulations；72 DEG C, 5min；4 DEG C of preservations.
Target DNA fragments are recycled after the completion of amplification, it is preferable that use the magnetic bead recovery purifying first step in two steps
With 0.5X XPbeads, excessive DNA is removed, second step adds 0.5X XPbeads again in the supernatant of the first step, finally returns
Receipts DNA length is 200-450bp.
It is sequenced after obtaining library using NEXT-seq.
Data parse data using methylation analysis process, obtain the methylation state of DNA of sample；Data use simultaneously
General PGS analysis process analyzes the chromosomal aneuploidy state of sample.From the PGS scatter plot of Fig. 1 and Fig. 2 it can be found that
No matter for sample 1 or sample 2, the result multiplicity for carrying out PGS analysis to trophoderm is quite high, therefore uses methylation
Analysis process can make accurate judgement to the chromatin state of embryo.
It chooses multiple embryo biopsy samples and carries out double digestion methylation sequencing analysis, 9 sample genome matching rates exist
80% or more, it is significantly higher than unicellular full-length genome methylation sequencing reported at present and unicellular single endonuclease digestion simplifies representative
Property methylation sequencing；The average methyl level in the site CpG in 20% or so (17-27%), this with the human body early of report
Close (the Guo et al.Nature.2014Jul 31 of tire methylation level data；511 (7511):606-610)；In addition it converts
Rate is 98% or more, and the SD value for PGS analysis is respectively less than 2, and as a result stable, multiplicity is high.
It will be apparent to those skilled in the art that of the invention carries out PGS to biopsy cells using DedscRRBS analytic approach
And the method for methylation analysis includes summary of the invention and specific embodiment part and the attached drawing institute of aforementioned present invention specification
Any combination of each section shown, as space is limited and to keep specification concise without these combining each scheme constituted
It describes one by one.All within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should all include
Within protection scope of the present invention.
Claims (10)
1. a kind of method for carrying out PGS and methylation analysis to biopsy cells using DedscRRBS analytic approach, according to sequencing
The following steps are included:
Step (1): fertilized eggs are obtained by Intracytoplasmic sperm injection method, fertilized eggs are cultivated in blastomere culture medium to the spilting of an egg
After the ball phase, then embryo is transferred to culture in blastocyst culture base and is taken in blastaea to development to the blastaea stage of ripeness far from inner cell mass
Trophectoderm cells be transferred to it is spare in PCR pipe；
Step (2): the cell sample that step (1) obtains successively is passed through into the product that enzymatic lysis obtains and carries out digestion, end reparation
And add A, connector connection, CT conversion processing and purification and recovery, PCR amplification, purifying and second of amplification, recycling purpose product；
Step (3): the DedscRRBS-PGS sequencing library to step (2) building is that obtained purpose product is sequenced.
2. the side according to claim 1 that using DedscRRBS analytic approach biopsy cells are carried out with PGS and methylation analysis
Method, which is characterized in that in step (2), the lyases that cracking process is selected is selected from Proteinase K, Qiagen Protease, stomach egg
One of white enzyme, papain, trypsase and lysozyme or a variety of combinations, enzyme concentration used are 1-30 μ g/ml；It splits
The lysis buffer that solution preocess is selected is Buffer A, and the Buffer A includes: 2-200mM Tris-EDTA, 1-50mM
The surfactant of KCl, 0.1wt%-5wt%, the surfactant include Triton X-100, SDS, Tween-20,
One of NP40 or a variety of；
The lyases incubation temperature is 37-65 DEG C, and the time is 30min to 12h, and for no more than 80 DEG C, the time is deactivation temperature
10-45min。
3. the side according to claim 1 that using DedscRRBS analytic approach biopsy cells are carried out with PGS and methylation analysis
Method, which is characterized in that in step (2), the restriction enzyme of digestion process selects MspI/ApeKI, MspI/Taq α I, MspI/
The combination of any one or more combination in XmaI, preferably MspI/ApeKI；The buffer that digestion process is selected is Buffer
B, the Buffer B include: 10-40mM Tris-acetate, 1-10mM Magnesium Acetate, 1-100mM
Potassium Acetate, 0.1-1mg/ml BSA, 37-75 DEG C of the digestion temperature of digestion process, time 1-6 hour, inactivation temperature
60-80 DEG C of degree, time 10-30min.
4. the side according to claim 1 that using DedscRRBS analytic approach biopsy cells are carried out with PGS and methylation analysis
Method, which is characterized in that in step (2), the archaeal dna polymerase that end repairs and A process is added to use is Klenow exo-, reaction temperature
Degree is 37 DEG C, repair time 20-60min, 60-80 DEG C inactivation 5-30min.
5. the side according to claim 1 that using DedscRRBS analytic approach biopsy cells are carried out with PGS and methylation analysis
Method, which is characterized in that in step (2), connector connection procedure selects methylation connector, is connected with T4 DNA ligase, and 16 DEG C pre-
30min is connected, then 4 DEG C of connections are overnight or not less than 8 hours, and 50-75 DEG C of processing 20min inactivates enzyme after the completion of connection.
6. the side according to claim 1 that using DedscRRBS analytic approach biopsy cells are carried out with PGS and methylation analysis
Method, which is characterized in that CT conversion process selects bisulfite, it is preferable to use Invitrogen MethylCode Kit or EZ
DNA Methlyation-Direct Kit。
7. the side according to claim 1 that using DedscRRBS analytic approach biopsy cells are carried out with PGS and methylation analysis
Method, which is characterized in that in step (2), PCR amplification process uses KAPA HiFi U+master Mix, Phusion high-
fidelity(HF)PCR master Mix with HF buffer、KAPA HiFi fridelity Buffermsupplied
The one or two of with polymerase.
8. the side according to claim 7 that using DedscRRBS analytic approach biopsy cells are carried out with PGS and methylation analysis
Method, which is characterized in that PCR amplification program is as follows: 98 DEG C, 2min；98 DEG C, 20s, 60 DEG C, 30s, 72 DEG C, 60s, 6-12 are followed
Ring；72 DEG C, 5min；4 DEG C of preservations；Second of amplification program is as follows: 98 DEG C, 2min；98 DEG C, 20s, 60 DEG C, 30s, 72 DEG C, 60s,
15-21 circulation；72 DEG C, 5min；4 DEG C of preservations.
9. the side according to claim 1 that using DedscRRBS analytic approach biopsy cells are carried out with PGS and methylation analysis
Method, which is characterized in that in step (2), recycling purpose product process is recycled between the target DNA fragments 200-450bp,
Preferably, using magnetic bead, recovery purifying, first step 0.3-0.6X XPbeads remove excessive DNA in two steps, and second step exists
Add 0.4-0.7x in the supernatant of the first step again, finally recycling DNA length is 200-450bp.
10. according to claim 1 carry out PGS and methylation analysis to biopsy cells using DedscRRBS analytic approach
Method, which is characterized in that in step (3), NGS sequencing is selected in sequencing.
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