CN105861486A - Method for obtaining target nucleic acid form mixed nucleic acid - Google Patents
Method for obtaining target nucleic acid form mixed nucleic acid Download PDFInfo
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- CN105861486A CN105861486A CN201510026998.7A CN201510026998A CN105861486A CN 105861486 A CN105861486 A CN 105861486A CN 201510026998 A CN201510026998 A CN 201510026998A CN 105861486 A CN105861486 A CN 105861486A
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Abstract
The invention discloses a method for obtaining a lot of target nucleic acids form mixed nucleic acid, the mixed nucleic acid comprises the target nucleic acid and non-target nucleic acid. The method comprises the following steps: gathering the target nucleic acid from the mixed nucleic acid, wherein the gathering means usage of methyl binding domain protein for binding the non-target nucleic acid, removing a formed methyl binding domain protein- non-target nucleic acid bonder; amplifying the enriched target nucleic acid, wherein the amplifying means usage of a N6 random primer; wherein, a lot of target nucleic acids means that the target nucleic acid amount reaches a minimum nucleic acid amount which exceeds sequencing requirement, and the N6 random primer is a sequence with the length being 6 bp combined by A,T,C and G with random arrangement. The invention also discloses a method for identifying low-abundance strain in an anthropogenic genome sample.
Description
Technical field
The present invention relates to biological sample process field, concrete, the present invention relates to one and obtain a large amount of target from mixing nucleic acid
The method of low abundance bacterial classification in the method for nucleic acid, and a kind of surveyor source grand genome sample.
Background technology
Grand genome (Metagenome is called for short meta), also referred to as microbial environment genome or unit's genome, it is defined as spy
Determining the summation of whole tiny organism inhereditary materials in environment, it comprises the genome of all species of microbiologic population.
Derive from the grand genome sample (people source grand genome sample) of people, such as the core from the skin of people, ight soil, saliva etc.
Acid sample, the microbial nucleic acids comprised is trace, and the main bacteria seed in the people source meta sample of separate sources is different, has
The abundance of bacterial classification extremely low, it is difficult to obtain effective amplification, it is difficult to be detected.
In the meta sample of trace people source, host DNA accounts for the overwhelming majority, and the sequencing analysis to people source meta sample utilizes grand base
Being analyzed because setting up the low abundance bacterial classification in the meta sample of storehouse sequencing technologies people low-abundance to trace source, strategy before is
Taking out host DNA in mixing nucleic acid sequencing data, for sample trace, low-abundance, there is wave in this strategy
Taking data, cost is high, the problem of low abundance microbiological analysis difficulty.And, when sequencing library builds, because sample rises
Beginning nucleic acid amount is too low, and the success rate of library construction is the lowest, even and if build sequencing library meet confidential asking, typically exist
Rule of thumb can only increase the order-checking degree of depth on the basis of conventional correlated samples research background during order-checking and obtain a large amount of sequencing data,
Followed by analysis of biological information technology, the sequence from low abundance bacterial classification comprised in sequencing data is analyzed, checks order into
This height, and owing to data volume big analysis workload is big.
Summary of the invention
Present invention seek to address that at least one the problems referred to above or propose a kind of business selection.
According to an aspect of of the present present invention, the present invention provides a kind of method obtaining a large amount of target nucleic acid from mixing nucleic acid, described
Mixing nucleic acid includes target nucleic acid and non-targeted nucleic acid, said method comprising the steps of: be enriched with institute from described mixing nucleic acid
Stating target nucleic acid, described enrichment includes utilizing methyl binding domain protein to combine described non-targeted nucleic acid, removes the methyl knot formed
Close territory albumen-non-targeted nucleic conjugate；Expanding the target nucleic acid through described enrichment, described amplification includes utilizing N6 random
Primer；Wherein, described a large amount of target nucleic acid refers to that the amount of described target nucleic acid is up to or over the minimum nucleic acid required for order-checking
Amount, described N6 random primer is the sequence of a length of 6bp being combined into by A, T, C and G random alignment.
In one embodiment of the invention, described mixing nucleic acid from people source meta sample, such as application on human skin meta sample,
Nucleic acid in application on human skin meta sample is mixed with in a large number from the nucleic acid of people, and target nucleic acid therein is meta nucleic acid.People source meta
In sample, the amount of people's nucleic acid contained before enrichment meta nucleic acid be probably 5 times of amount of target meta nucleic acid, 10 times,
20 times, 30 times, 40 times, 50 times, 100 times, 150 times, 200 times or 250 times.General, if non-targeted core
The amount of acid reaches 5 times of the amount of target nucleic acid, and what order-checking realized is just substantially the sequence obtaining non-targeted nucleic acid.
Described methyl binding domain protein includes can be specific binding with the cystine that methylates of the CpG-in DNA
MeCP2, Methyl-CpG binding domain protein 1 (MBD1), MBD2, MBD3 or MBD4 (US7,670,773),
In one embodiment of the invention, described methyl binding domain protein is elevation of methyl-CpG binding domain 2 (MBD2),
MBD2 can be combined in methylated CpG region on people DNA.In one embodiment of the invention, first by MBD2
Be attached on magnetic bead (Magnetic Beads), the combination of MBD2 and magnetic bead be by magnetic bead is coated upper albumin A or
Protein G, is combined MBD2 with antibody Fc portion, and then pass through Fc and albumin A or Protein G is implemented in combination with MBD2
With the combination of magnetic bead, a coated magnetic bead can be combined with multiple MBD2, is subsequently added into people source meta DNA sample,
Host DNA (people DNA) in sample is combined with MBD2-magnetic bead, under magnetic frame effect, and host DNA-MBD2-
The adsorbed sedimentation of bead complexes, enrichment obtains the meta DNA being present in supernatant, it is achieved described target nucleic acid enrichment.
Albumin A is a kind of cell wall protein, has the character not being combined with immunoglobulin (Ig) at antigen-combining site.Protein G can be with
The Fc district of the IgG of many animals combines.Magnetic bead refers to the super paramagnetic microsphere with fine particle size, and magnetic bead typically has super
Strong paramagnetism, can assemble rapidly in magnetic field, can aid in again the dispersed of Magneto separate after leaving magnetic field, secondly,
There is suitable and that difference is less particle diameter, if particle diameter is 4.0-4.5 μm or 3.0-3.9 μm, it is ensured that sufficiently strong magnetic response
Property will not settle again.The existence of salt is proved to be able to the combination strengthening MBD2-magnetic bead with people DNA, at one of the present invention
In embodiment, the buffer solution that used of enrichment comprises 150-400mM NaCl and nonionic surfactant less than 1%, wherein,
Nonionic surfactant can be Tween 20 (polysorbas20), Tween 80 or Tween 100.Utilize above-mentioned comprising
The buffer solution of salt and surfactant carries out described enrichment, it is possible to significantly reduce non-methylated CpG nucleic acid and MBD2
Non-specific binding.
In some embodiments of the invention, described amplification is that multiple displacement amplification (MDA), MDA can be to limited nucleic acid
Sample carry out quick, uniform, unbiased to isothermal whole genome amplification.Described MDA amplification comprises a plurality of N6 random primer
With the cohesive process of sex change target dna after enrichment, and the chain using Phi 29 polymerase to carry out at a constant temperature puts
Changing building-up process, displacement chain combines primer as template, thus generates substantial amounts of amplification target dna.Phi 29 polymerase
Come from bacteriophage, be the one archaeal dna polymerase with 3' end → 5' end 5 prime excision enzyme activity (proofreading activity), using the teaching of the invention it is possible to provide
The fidelity of high 100 times than Taq archaeal dna polymerase.A plurality of N6 random primer (random hexamer) can be attached to (mutually
Benefit is paired to) multiple positions of template, it is beneficial to the overall unbiased of target dna to amplification, a plurality of N6 random primer is permissible
Be 2,3,4,5,10,15,20,30,40,50,60,70,80
Bar, 100,150,200,250 or 300 random primers, substantially, the bar number of N6 random primer is permissible
Size according to target dna adjusts, and utilizes the N6 random primer of the different bar number of many groups to test, makes adjacent two N6
Binding site between sequence can effectively be expanded.In one embodiment of the invention, one group share N6 with
Machine sequence is ATCACG, CGATGT, TTAGGC, TGACCA, ACAGTG, GCCAAT, CAGATC,
ACTTGA, GATCAG, TAGCTT, GGCTAC and CTTGTA.In one embodiment of the invention, amplification
The pH value of buffer solution system be 5～10, comprise Mg2+0.1-25mM, NaCl or KCl 1-150mM and ammonium salt, such as,
Amplification buffer system is Tris, pH 8.5,10mM MgCl2, 50mM KCl, 20mM (NH4)2SO4.Phi 29 polymerase
In conjunction with this buffer system, it is possible to overcome the secondary structure difficulty such as hair fastener ring one class easily, avoid sliding in amplification procedure
The generation of the phenomenons such as de-, polymerase dissociates.The most agonic DNA fragmentation can be blended into the length of 100kb.
In one embodiment of the invention, described minimum nucleic acid amount is 20ng.The method utilizing this one side of the present invention can
From the meta sample of trace people source, simply accurately obtain the meta nucleic acid more than 20ng, allow to meet general library construction pair
The requirement of initial nucleic acid amount, it is easy to meta library construction, order-checking, it is thus achieved that target nucleic acid sequence, compared with conventional method, carries
Height builds Kucheng's power, reduces order-checking cost, and, because the major part in sequencing data is target nucleic acid data, do not have
The interference of a large amount of host data so that follow-up analysis based on sequencing data becomes relatively easy.
Can be by the agent prescription related in this one side of the invention described above or arbitrary detailed description of the invention, enzyme, reactant
System and/or sequence etc. are combined into kit, and kit also can farther include amplification of nucleotide substrate dNTP, amplified production inspection
Survey primer, other buffer solution etc..This kit may be used for obtaining a large amount of target nucleic acid, particularly from micro-from mixing nucleic acid
Amount people source meta sample obtains the meta DNA of trace.
According to another aspect of the present invention, the present invention provides a kind of method building grand gene order-checking library, and the method includes:
Obtaining the nucleic acid in grand genome sample, described nucleic acid comprises the nucleic acid from host and the nucleic acid from microorganism；Enrichment institute
Stating the nucleic acid from microorganism in nucleic acid, described enrichment includes, utilizes methyl binding domain protein to combine the nucleic acid from host,
Remove the methyl binding domain protein-host nucleic acids bond formed；Expanding the nucleic acid through described enrichment, described amplification includes profit
Use N6 random primer；The amplified production obtained is carried out sequencing library structure, to obtain described grand gene order-checking library.
According to another aspect of the invention, the present invention provides a kind of sequence measurement, and it includes, utilizes above-mentioned grand gene order-checking
Library constructing method builds grand gene order-checking library, and checks order the sequencing library obtained.Can be according to selected
Order-checking platform carries out corresponding sequencing library structure, for example, it is possible to carry out single-ended sequencing library structure, double end library construction
Deng, order-checking can select existing platform to carry out, and includes but not limited to Illumina Hiseq2000/2500, Life Technologies
Ion Torrent, Complete Genomics CGA and Roche 454 check order platform.Aforementioned to one aspect of the present invention from mixed
Synkaryon acid obtains technical characteristic and the description of advantage of the method for a large amount of target nucleic acid, this one side of the equally applicable present invention
Sequencing library construction method and sequence measurement, do not repeat them here.
According to another aspect of the present invention, the present invention provides the method for low abundance bacterial classification in the grand genome sample of a kind of surveyor source,
Including: obtaining the nucleic acid in the grand genome sample of described people source, described nucleic acid comprises from the nucleic acid of people with from microorganism
Nucleic acid；The nucleic acid from microorganism being enriched with in described nucleic acid, described enrichment includes, utilizes methyl binding domain protein to combine
From the nucleic acid of people, remove the methyl binding domain protein-people's nucleic conjugate formed；Expand the nucleic acid through described enrichment, described
Amplification includes utilizing N6 random primer；Nucleic acid after amplification is carried out sequencing, it is thus achieved that sequencing data；By described order-checking
Data and microorganism reference sequences comparison, it is thus achieved that comparison result；Described low abundance bacterial classification is identified according to described comparison result.Ratio
To utilizing SOAP (Short OligonucleotideAnalysis Package), the software such as bwa, GATK is carried out, this
This is not restricted by invention.Microorganism reference sequences refers to known microorganism sequence, can be the microbial gene being obtained ahead of time
Group or fragment, for example, it is possible to for oneself measuring multiple-microorganism that is that assemble or that can obtain from public data storehouse
Genome or the combination of fragment.Further, according to the position of people source meta samples sources, the main bacteria seed that may comprise
Or bacterial classification, can be pre-configured with the sequence library comprising more reference sequences, contributes to obtaining identification and analysis result more accurately.
Aforementioned to the method obtaining a large amount of target nucleic acid from mixing nucleic acid in one aspect of the present invention or arbitrary detailed description of the invention
Technical characteristic and the description of advantage, this method on the one hand of the equally applicable present invention.In one embodiment of the invention, profit
The bacterial classification of abundance as little as 0.4% in the meta sample of people source can be identified by this method of the present invention.
The present invention this method provides low abundance bacterial classification sequencing analysis strategy in the meta sample of a kind of trace people source, the most first goes
Major part (more than 90% can be reached) host DNA in the meta sample of eliminating minute people source, then expand trace meta DNA,
The total amount making trace meta DNA reaches the demand that sequencing library builds.This inventive method improves well to be passed through before
Increase sequencing data amount, to improve the strategy of the identification rate of low abundance bacterial classification in the grand genome sample of trace people source, reached to subtract
Few sequencing data waste, trace people source grand genome sample sequencing analysis cost, and improve trace people source grand genome sample
In the effect of identification rate of low abundance bacterial classification.
Accompanying drawing explanation
Above-mentioned and/or the additional aspect of the present invention and advantage will become bright from combining the accompanying drawings below description to embodiment
Aobvious and easy to understand, wherein:
Fig. 1 is the electrophoresis result figure of the amplified production after the MDA in one embodiment of the present of invention, and wherein M1 swimming lane is
λ Hind III digestion DNA, 1 swimming lane is meta DNA after enrichment, and 2 swimming lanes are enrichment descendant DNA, and 3 swimming lanes are negative right
According to, M2 swimming lane is standard molecular weight Maker (D2000), unit bp；
Fig. 2 is the house-keeping gene detection electrophoretogram of the amplified production in one embodiment of the present of invention, and wherein M swimming lane is standard
Molecular weight (Maker, unit bp), 1 swimming lane is YH standard items, 2 swimming lanes be enrichment after meta DNA full-length genome expand
Increasing (WGA) product, 3 swimming lanes are the WGA product of enrichment descendant's source DNA, and 4 swimming lanes are NTC (negative control)
WGA product.
Detailed description of the invention
Carry out in detail below in conjunction with concrete sample is carried out target nucleic acid acquisition, low abundance strain idenfication etc. according to the method for the present invention
Thin description and result are shown.Example below, is only used for explaining the present invention, and is not considered as limiting the invention.This
In the description of invention, except as otherwise noted, " multiple " are meant that two or more.
Except as otherwise explaining, the reagent explained the most especially that relates in following example, sequence (joint, label and primer), soft
Part and instrument, be all conventional commercial product or disclosed, such as builds purchased from the hiseq2000 order-checking platform of Illumina company
Storehouse related kit carries out sequencing library structure etc..
Embodiment
(1) material prepares
Obtaining trace people source meta sample, acquisition is that human body skin meta sample packages is total to 32ng containing nucleic acid, and concentration is
1.1ng/μl。
(2) method can divide following step
1, the meta nucleic acid of gathering trace
Utilize this step to eliminate people's source DNA of more than 90%, make the content of meta DNA be greatly improved, it is achieved described richness
Collection trace meta DNA.
Concrete, MBD2-Fc is combined with the magnetic bead being coated with albumin A, combines 10min with under vertical suspendible instrument room temperature,
With combining buffer solution 2 times, in conjunction with buffer solution by 10mM Tris, pH7.5,1mM EDTA, 0.2%Tween20 and
300mM NaCl forms, and then to combine buffer solution resuspended MBD2-Fc-magnetic bead, is combined 50min with DNA sample, will
Reaction tube is placed on magnetic frame to stand and clarifies to liquid, draws supernatant and i.e. can get meta DNA.Magnetic bead protease in pipe
K (20mg/ml, 2 μ l) and TE buffer (10mMTris, 1mM EDTA, pH8.0) hatches 20min at 75 DEG C,
It is placed in reaction tube on magnetic frame to stand and clarifies to liquid, draw supernatant and i.e. can get host DNA.To supernatant and back dissolving liquid
Carry out nucleic acid extraction purifying, such as, purify with AMPure XP Bead and can get the DNA sample that can carry out subsequent reactions.
The people DNA reclaimed in supernatant and back dissolving liquid is carried out quantitatively, Qubit detectable concentration: obtain 18ul 0.76ng/ul's
Meta DNA, altogether 14.4ng；Obtaining people's source DNA of 9.5ul 0.749ng/ul, altogether 7.12ng, other nucleic acid are
Loss in experiment.
2, MDA amplification
Carry out respectively with meta DNA to obtaining of one group of N6 stochastic ordering primer and Phi29DNA polymerase and host DNA
Whole genome amplification, one group of N6 random primer include ATCACG, CGATGT, TTAGGC, TGACCA, ACAGTG,
GCCAAT, CAGATC, ACTTGA, GATCAG, TAGCTT, GGCTAC and CTTGTA.First, will
Sample is dissolved in TE buffer, adds denaturing soln and carries out the alkaline denaturation of gentleness, it is to avoid fragmentation and infringement occur during sex change
Template DNA, denaturing soln comprises 0.25mM KOH and 0.625 μM of EDTA.Then, by neutralization buffer by sample
With, then hatch at 30 DEG C with MDA amplification reaction solution, neutralization buffer is 3mol/LNaAc, pH=5.2, MDA
Amplification reaction solution is by Tris, pH 8.5,10mM MgCl2, 50mM KCl, 20mM (NH4)2SO4Composition.Use multiple
Displacement amplification (MDA) technology, random primer is combined with denatured DNA, utilizes Phi29 polymerase to carry out under isothermal environment
Replacement synthesis.The strand of each displacement, as template, is combined with primer, and amplification generates the DNA of high yield.Amplification is anti-
Should: 30 DEG C of 16h, 65 DEG C of 3min, maintain 4 DEG C.
Amplified production is detected by the agarose electrophoresis utilizing 0.6%, and testing result such as Fig. 1, in figure, M1 is λ Hind III digestion
DNA, 1 for enrichment after meta DNA, 2 for enrichment descendant DNA, 3 be NTC (negative control, microbial DNA),
M2 is Marker (D2000), and the amplified production that full-length genome is expanded or obtains as can be seen from Figure 1 is large fragment.
Utilizing host's house-keeping gene to detect amplified production, 2% agarose electrophoresis detection amplification, result such as Fig. 2, in figure
Each swimming lane, M be Maker, 1 for YH (Yan Di and Huang Di, two legendary rulers of remote antiquity, from Yan Di and Huang Di, two legendary rulers of remote antiquity's project) standard items, 2 for enrichment after meta DNA
Whole genome amplification (WGA) product, 3 be the enrichment WGA product of descendant's source DNA, 4 be the WGA of NTC
Product.
The primer of amplification house-keeping gene can use the multipair house-keeping gene primer in CN201010619689, or commercially available people pipe
Primer in family's gene detecting kit.
As can be seen from Figure 2, the house-keeping gene testing result of the host DNA after MDA amplification shows 8 house-keeping gene bands,
And the house-keeping gene testing result of the meta DNA after MDA amplification enrichment shows without house-keeping gene band, after enrichment is described
Meta DNA is substantially free of host DNA, at least without host DNA under agarose electrophoresis detection sensitivity.Pass through
Meta DNA after whole genome amplification uses HiSeq sequencer, analysis of biological information result after building small fragment library
Display enrichment can remove the host DNA of 91%, reduces the requirement to sequencing data amount of the people source meta sample, and can
Analysis obtains the most low-abundance microorganism sequence.From this human body skin meta sample, have found the most low-abundance acinetobacter calcoaceticus
Belong to.In the dermatological specimens of people source, acinetobacter content is the lowest, content be less than 0.5%, the most same sample identical nucleic acid content,
Repeatedly utilize current research method, including repeatedly building storehouse, increasing sequencing data amount, and/or from a large amount of mixing sequencing datas
Removing people's DNA data, result can not find acinetobacter calcoaceticus or the acinetobacter calcoaceticus found less than 3 kinds.But, utilizing should
Exemplary method, total amount of data is less than 1/10 before but can have found 7 kinds of acinetobacter calcoaceticus.In this example, identify meta
Whether comprise the standard of a certain Pseudomonas and usual standard of perfection in data is consistent, for example, it is possible to based in sequencing data
There is reading section or have a certain proportion of reading section comparison to the special reference sequences of this Pseudomonas, illustrating in this people source meta sample
Comprise this Pseudomonas.It is explained above the storehouse of building utilizing the inventive method to be capable of trace people source meta sample to check order, it is possible to fall
The low demand to sequencing data amount, and the detection sensitivity of low abundance microorganism can be improved.
Claims (10)
1. the method obtaining a large amount of target nucleic acid from mixing nucleic acid, described mixing nucleic acid includes target nucleic acid and non-targeted
Nucleic acid, it is characterised in that comprise the following steps:
Being enriched with described target nucleic acid from described mixing nucleic acid, it is described non-that described enrichment includes utilizing methyl binding domain protein to combine
Target nucleic acid, removes the methyl binding domain protein-non-targeted nucleic conjugate formed；
Expanding the target nucleic acid through described enrichment, described amplification includes utilizing N6 random primer；Wherein,
Described a large amount of target nucleic acid refers to the minimum nucleic acid amount that the amount of described target nucleic acid requires up to or over order-checking,
Described N6 random primer is the sequence of a length of 6bp being combined into by A, T, C and G random alignment.
2. the method for claim 1, it is characterised in that described mixing nucleic acid is from people source meta sample, described target core
Acid is meta nucleic acid.
3. the method for claim 2, it is characterised in that described methyl binding domain protein is combined on magnetic bead；
Optional, described in be removed by magnetic force and realize described methyl binding domain protein-non-targeted nucleic conjugate and described target
The separation of nucleic acid.
4. the method for claim 2, it is characterised in that described amplification is multiple displacement amplification.
5. the method for claim 2, it is characterised in that carry out described amplification and include utilizing a plurality of N6 random primer.
6. the method for claim 2, it is characterised in that the polymerase that described amplification utilizes is Phi29 polymerase.
7. claim 1-6 either method, it is characterised in that described minimum nucleic acid amount is 20ng.
8. the method building grand gene order-checking library, it is characterised in that include,
Obtaining the nucleic acid in grand genome sample, described nucleic acid comprises the nucleic acid from host and the nucleic acid from microorganism；
The nucleic acid from microorganism being enriched with in described nucleic acid, described enrichment includes, utilize methyl binding domain protein combine from
The nucleic acid of host, removes the methyl binding domain protein-host nucleic acids bond formed；
Expanding the nucleic acid through described enrichment, described amplification includes utilizing N6 random primer；
The amplified production obtained is carried out sequencing library structure, to obtain described grand gene order-checking library.
9. a sequence measurement, it is characterised in that include,
The method utilizing claim 8 builds grand gene order-checking library,
The sequencing library obtained is checked order.
10. the method for low abundance bacterial classification in a surveyor source grand genome sample, it is characterised in that include,
Obtaining the nucleic acid in the grand genome sample of described people source, described nucleic acid comprises the nucleic acid from people and the core from microorganism
Acid；
The nucleic acid from microorganism being enriched with in described nucleic acid, described enrichment includes, utilize methyl binding domain protein combine from
The nucleic acid of people, removes the methyl binding domain protein-people's nucleic conjugate formed；
Expanding the nucleic acid through described enrichment, described amplification includes utilizing N6 random primer；
Nucleic acid after amplification is carried out sequencing, it is thus achieved that sequencing data；
By described sequencing data and microorganism reference sequences comparison, it is thus achieved that comparison result；
Described low abundance bacterial classification is identified according to described comparison result；
Optional, abundance that described low abundance bacterial classification is included in the grand genome sample of the described people source bacterial classification more than 0.4%.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019000254A1 (en) * | 2017-06-28 | 2019-01-03 | 中国医学科学院药用植物研究所 | Method for quality control of chinese patent medicine based on metagenome |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533960A (en) * | 2010-12-31 | 2012-07-04 | 深圳华大基因科技有限公司 | Single-cell genome analysis method and kit |
| CN102732506A (en) * | 2011-04-02 | 2012-10-17 | 新英格兰生物实验室公司 | Methods and compositions for enriching either target polynucleotides or non-target polynucleotides from a mixture of target and non-target polynucleotides |
| CN104152437A (en) * | 2014-08-12 | 2014-11-19 | 上海派森诺生物科技有限公司 | Methylated DNA enrichment method used in methyl binding protein sequencing |
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2015
- 2015-01-19 CN CN201510026998.7A patent/CN105861486A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533960A (en) * | 2010-12-31 | 2012-07-04 | 深圳华大基因科技有限公司 | Single-cell genome analysis method and kit |
| CN102732506A (en) * | 2011-04-02 | 2012-10-17 | 新英格兰生物实验室公司 | Methods and compositions for enriching either target polynucleotides or non-target polynucleotides from a mixture of target and non-target polynucleotides |
| CN104152437A (en) * | 2014-08-12 | 2014-11-19 | 上海派森诺生物科技有限公司 | Methylated DNA enrichment method used in methyl binding protein sequencing |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019000254A1 (en) * | 2017-06-28 | 2019-01-03 | 中国医学科学院药用植物研究所 | Method for quality control of chinese patent medicine based on metagenome |
| US11398294B2 (en) | 2017-06-28 | 2022-07-26 | Institute Of Medicinal Plant Development, Chinese Academy Of Medical Science | Method for controlling the quality of traditional Chinese patent medicines based on metagenomics |
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