CN105238859A - Method for acquiring chicken whole genome high-density SNP marker sites - Google Patents
Method for acquiring chicken whole genome high-density SNP marker sites Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, and provides a method for acquiring chicken whole genome high-density SNP marker sites. The method comprises the following steps: (1) predicting distribution of restriction fragments acquired from double-restriction chicken genomes of EcoRI and MseI; (2) designing a universal joint, a barcode joint and a PCR amplification primer according to the distribution characteristics of the restriction fragments of EcoRI and MseI; (3) constructing a simplified genome sequencing library; (4) sequencing by virtue of the library constructed in the step (3) through a computer; and (5) according to a sequencing result, acquiring SNP marker sites. The method disclosed by the invention provides a universal strategy for the construction of a whole genome high-density SNP map by virtue of double-restriction GBS for different varieties of chickens, so that the cost on acquiring every SNP marker site is reduced by an order of magnitude compared with a conventional chip technology; and the method is stable in technology and high in repeatability.
Description
Technical field
The present invention relates to biological technical field, concrete, relate to a kind of method of the acquisition chicken full-length genome high-density SNP marker site based on sequenced genes typing method.
Background technology
As the model animals of bird, chicken became the agricultural economical animal that first completes full genome order-checking in 2004, because different chicken breeds has huge species diversity, it is as the Genetics Model of high-quality, is applied to the fields such as quantitative genetics and molecular breeding, functional gene location, the regulation and control of gene and growth more and more.Molecule marker is the important tool studying biological heritable variation, single nucleotide polymorphism (Singlenucleotidepolymorphisms, SNP) as third generation molecule marker, there is the features such as quantity is many, distribution is wide, inheritance stability, be widely used in the genetic breeding fields such as linkage analysis, whole-genome association and genome selection.Therefore understanding the SNP information in chicken genome, is unusual the key link in the genetic breeding process of chicken.
The chicken full-length genome SNP typing method of current main flow mainly contains gene typing chips and two generations and to check order two kinds of methods.The feature of gene typing chips is consistent, result repetition rate is high, but the cost of a chip technology somatotype experiment sample is very high, for population genetic study field, the cost price of colony's somatotype is too large, and chip technology limit due to technology, also there is SNP polymorphic site poor universality in different groups, mark density low (the SNP chip density of current chicken main flow is 60kSNP chip), can not meet the problems such as meticulous functional gene location and whole-genome association.The development of sequencing technologies of future generation makes the research of genomics and transcription group more to go deep into, and order-checking can obtain the high density marker collection of illustrative plates of full-length genome level, but also there is the shortcoming of unit sample high cost simultaneously.
The qualification and the somatotype that simplify the high-throughput molecule marker of the covering full-length genome that genomic sequencing technique (reduced-representationsequencing) makes population analysis institute need become possibility.But different simplification gene order-checking methods all has bigger difference in the selection etc. of building storehouse strategy, the combination selection of single endonuclease digestion/double digestion, order-checking platform, these all can the efficiency of the follow-up somatotype of remarkably influenced and cost.For example, the method for RAD order-checking to build storehouse strategy complicated, too much step can interfere with subsequent experimental result; Different restriction enzymes enzyme in different species gene groups cuts frequency and distribution all has relatively big difference, for specific species, selects which kind of enzyme to carry out testing the determinative just becoming and determine experiment acquisition SNP quantity and cost; 2b-RAD technology uses II Type B restriction enzyme, but the clip size that this enzyme is cut only has 25-35bp, although 2b-RAD technology can obtain the endonuclease bamhi of full-length genome level, but according to the frequency of full-length genome variation, too short endonuclease bamhi is difficult to be rich in SNP site, causes mass data to lose, simultaneously because endonuclease bamhi is too short, also can bring many mistakes in the comparison of genome repeat region, SNP somatotype reliability be declined to a great extent, severe jamming downstream application.
Therefore be necessary the analytical procedure developing a kind of new chicken genome SNP marker site, the enzymes combinations of applicable chicken genome SNP marker Locus Analysis in Shoots is provided, to reduce the cost of gene type, for the downstream application after gene type provides convenient.
Summary of the invention
For the deficiencies in the prior art, the object of the present invention is to provide a kind of method obtaining chicken full-length genome high-density SNP marker site based on sequenced genes typing method.
Sequenced genes somatotype (GenotypingBySequencing, GBS) technology is developed by people such as the Elshire of Cornell University, its banking process is the simplest, DNA is after enzyme is cut, given joint in connection, by the time extended in control PCR process, select the part of 100-500bp in digestion products, thus realize the object simplifying gene order-checking; But the defect of this method is, small segment digestion products not only to build amplification efficiency in the process of storehouse higher, also comparatively fast grows in the template amplification of sequenator, easily cause sequencing data to utilize part is less, the quality of data is poor problem.
The invention provides a kind of method of the acquisition chicken full-length genome high-density SNP marker site based on sequenced genes typing method, comprise the following steps:
(1) the endonuclease bamhi distribution situation that obtains of the double digestion chicken genome of prediction EcoRI and MseI;
(2) according to the design of endonuclease bamhi characteristic distributions universal joint, barcode joint and the pcr amplification primer of EcoRI and MseI;
(3) simplification gene order-checking library is built;
(4) upper machine order-checking is carried out in the library utilizing step (3) to build;
(5) SNP marker site is obtained according to sequencing result.
Optionally, the universal joint described in step (2) is with the cohesive end sequence identical with restriction enzyme MseI, and described barcode joint is with the cohesive end sequence identical with restriction enzyme EcoRI.
Optionally, described universal joint is the double-stranded DNA formed by sequence anneals shown in SEQIDNO:1 and SEQIDNO:2, and wherein SEQIDNO:1 is through 5 ' phosphorylation modification.
Optionally, described barcode joint is the double-stranded DNA formed by sequence anneals shown in SEQIDNO:3 and SEQIDNO:4; Wherein SEQIDNO:4 is through 5 ' phosphorylation modification, n and m in SEQIDNO:3 and SEQIDNO:4 represents that length is any short Nucleotide bar code sequence of 6-9bp.
Optionally, the pcr amplification primer described in step (2) is as shown in SEQIDNO:5 and SEQIDNO:6.
Optionally, comprise the following steps in step (3):
A () utilizes restriction enzyme combination EcoRI-MseI to carry out enzyme to chicken genome and cuts;
B () prepares universal joint and barcode joint;
C universal joint and barcode joint and digestion products are carried out ligation by () respectively, obtain and connect product;
D connection product equal proportion is carried out mixed pond by (), obtain the connection product behind mixed pond;
E the magnetic bead adding 1.2-1.4 times of volume in () connection product behind mixed pond carries out the first purifying and obtains the first purified product;
F magnetic bead that () adds 0.8-0.9 times of volume in described first purified product carries out the second purifying and obtains the second purified product;
G () is carried out pcr amplification to the second purified product and is obtained PCR primer;
H magnetic bead that () adds 1.2-1.4 times of volume in PCR primer carries out the 3rd purifying acquisition the 3rd purified product;
I magnetic bead that () adds 0.8-0.9 times of volume in the 3rd purified product carries out the 4th purifying and is simplified gene order-checking library.
Optionally, described first purifying is identical with the step of the 3rd purifying, specifically comprises: after adding magnetic bead, on gyroscope incubated at room 18-22min obtain hatch after system; Be placed on supernatant discarded on magnetic frame after hatching end, add 70% ethanol of 480-520 μ L, leave standstill slow circumvolve after 30-40s, magnetic bead is moved on tube wall, after solution clarification, remove supernatant liquor, then repeat this step and once obtain precipitation; LowTE is added again in obtained precipitation, after inhaling up and down beat with pipettor, vibration 10s, centrifugal rear standing clarification acquisition supernatant liquor; Wherein, precipitate described in 100 μ L, the addition of LowTE is 140-160 μ L.
Optionally, the second purifying is identical with the step of the 4th purifying, specifically comprises: after adding magnetic bead, incubated at room 13-16min on gyroscope; Be placed on supernatant discarded on magnetic frame after hatching end, add 70% ethanol of 480-520 μ L, slow circumvolve after standing 30-40s, makes magnetic bead move on tube wall, after solution clarification, removes supernatant liquor, repeats this step and once obtain precipitation; LowTE is added again in obtained precipitation, after inhaling up and down beat with pipettor, vibration 10s, centrifugal rear standing clarification acquisition supernatant liquor; Wherein, precipitate described in 100 μ L, the addition of LowTE is 30-50 μ L.
Optionally, the annealing system of the universal joint described in step (c) is: 100 μMs of SEQIDNO:15 μ L; 100 μMs of SEQIDNO:25 μ L, 5 × AnnealingBuffer10 μ L, nuclease free water 30 μ L; Cycle of annealing is: be heated to 95 DEG C, and is cooled to 25 DEG C with the speed of 1 DEG C/min, in 4 DEG C of preservations after 25 DEG C of insulation 30min.
The annealing system of barcode joint is: 100 μMs of SEQIDNO:35 μ L; 100 μMs of SEQIDNO:45 μ L, 5 × AnnealingBuffer10 μ L, nuclease free water 30 μ L; Response procedures is: 95 DEG C of 3min, with the cooling of the speed of 1 DEG C/min, until drop to 25 DEG C, in 4 DEG C of preservations after 25 DEG C of insulation 30min.
System AdaptersMix: universal joint 0.8 μ L of joint mixing, barcode joint 15 μ L, nuclease free water 84.2 μ L, total system 100 μ L.
Optionally, the system of the ligation described in step (c) is: digestion products 20 μ L, 5 × DNALigaseReactionBuffer8 μ L, DNA ligase 2 μ L, nuclease free water 5 μ L, joint mixture 5 μ L; Mixing is placed on PCR, and response procedures is: 22 DEG C of insulation 1h, and 65 DEG C of insulation 30min, are cooled to 4 DEG C of preservations.
This invention exploits a kind of method of carrying out gene type based on the order-checking of EcoRI – MseI double digestion, chicken for different varieties utilizes double digestion GBS to build full-length genome high-density SNP collection of illustrative plates and provides a kind of general strategy, the cost obtaining each SNP marker site is made to reduce an order of magnitude than traditional die technology, the method is consistent, and repeatability is high.
Accompanying drawing explanation
Fig. 1 is sequencing library Agilent2100 detected result provided by the invention.
Fig. 2 is sequencing quality provided by the invention report.
Embodiment
Below in conjunction with embodiment, the preferred embodiment of the present invention is described in detail.It will be appreciated that providing of following examples is only object in order to play explanation, being not used to limit scope of the present invention.Those skilled in the art, when not deviating from aim of the present invention and spirit, can carry out various amendment and replacement to the present invention.
The sequencing kit used in following examples is NextSeq500HighOutputKit (75cycles).
The genomic software of sequencing data comparison used in following examples is bowtie2 (version number bowtie2-2.2.3) (based on (SuSE) Linux OS), and SNP identifies that software is Tassel (version number tassel-4.3.13).
Embodiment 1
Embodiment 1 is for illustration of method of the present invention
1, experiment material:
Gather Red Jungle-fowl, the white Leghorn of business layer breed, commercial meat chickens kind Chinese mugwort pulls out broiler chicken, Lingnan Yellow chicken increasingly, Chinese Native Chicken Breeds Huiyang beard chicken, Wenchang Chicken, Henan cockfighting, Qingyuan Chicken, black langshan chicken, camellia chicken, Beijing Fatty Chicken, Tibetan chicken, Silky fowl, shouguang chicken, bamboo silk chicken, Shi Qi mix chicken, Xianju Chicken, stealthy Cold boiled chicken, each 4-6 of a yellow dwarf chickens individual blood sample, amount to 96 individualities, extract genome, and it is for subsequent use genome concentration to be diluted to 50ng/ μ L.
2, joint and primer sequence:
Synthesize a pair universal linker sequence, 96 pairs of barcode joint sequences, one couple of PCR primers sequence.
3, sequencing library builds:
96 chicken sample extraction postgenomes are carried out genome enzyme cut.Reaction system is 20 μ L, comprises 15 μ LNuclease-freewater, 2 μ L10 × CutSmartBuffer, 0.5 μ L enzyme 1,0.5 μ L enzyme 2 (0.5 μ LEcoRI, 0.5 μ LMseI), 200ng sample DNA, mixing, centrifugal, be placed in PCR instrument, reaction conditions is: 37 DEG C of 90min, 65 DEG C of 30min, 4 DEG C of preservations.
4, joint is annealed and is mixed:
Universal joint reaction system is: totally 50 μ L, comprise 30 μ L nuclease free water, 10 μ L5 × Annealingbuffer, SEQIDNO:1 (100 μMs) 5 μ L, SEQIDNO:2 (100 μMs) 5 μ L, mix centrifugal, reaction conditions is 95 DEG C of 3min, and decline 1 DEG C/min, until drop to 25 DEG C, 25 DEG C of 30min, 4 DEG C of preservations.
Barcode joint reaction system is: totally 50 μ L, comprise 30 μ L nuclease free water, 10 μ L5 × Annealingbuffer, SEQIDNO:3 (100 μMs) 5 μ L, SEQIDNO:4 (100 μMs) 5 μ L, mix centrifugal, reaction conditions is 95 DEG C of 3min, and decline 1 DEG C/min, until drop to 25 DEG C, 25 DEG C of 30min, 4 DEG C of preservations.
System AdaptersMix: universal joint 0.8 μ L of joint mixing, barcode joint 15 μ L, nuclease free water 84.2 μ L, total system 100 μ L.
5, joint connects:
Reaction system is 40 μ L, comprise 20 μ L digestion products, 5 μ LNuclease-freewater, 8 μ L5 × DNALigaseReactionBuffer, 2 μ LExpressLinkT4DNALigase, 5 μ LAdaptersMix, abundant mixing, centrifugal, reaction conditions is 22 DEG C of insulation 1h, 65 DEG C of 30min, 4 DEG C of preservations.
6, mixed pond:
By admixed together for 5 μ L each in the connection product of 96 samples, take out 240 μ L for lower step purifying.
7, magnetic beads for purifying connects product:
Connect in product at 240 μ L and add 312 μ LAMPureXPBeads, centrifuge tube is placed on gyroscope, hatches 20min for 15-25 DEG C, be then positioned over 3min on magnetic frame, abandon supernatant; Add 500 μ L70% ethanol, be placed in by centrifuge tube on magnetic frame, slowly rotary tube after 30s, revolves and takes two turns, magnetic bead is moved on tube wall, after solution clarification, removes supernatant liquor, then this step is repeated once again; Take off centrifuge tube, of short duration centrifugal, centrifuge tube is positioned on magnetic frame, removes residual ethanol with lancet head, dry 3min; Add 150 μ LLowTE, inhale up and down beat several times with rifle head, concussion 10s, be of short durationly centrifugally placed on magnetic frame, 3min, after solution clarification, transfers to supernatant liquor in new centrifuge tube; In 150 μ LLowTE elutriants, add 120 μ LAMPureXPBeads, centrifuge tube is placed on gyroscope, hatch 15min for 15-25 DEG C, be then positioned over 3min on magnetic frame, abandon supernatant; Add 500 μ L70% ethanol, be placed in by centrifuge tube on magnetic frame, slowly rotary tube after 30s, revolves and takes two turns, magnetic bead is moved on tube wall, after solution clarification, removes supernatant liquor, then this step is repeated once again; Take off centrifuge tube, of short duration centrifugal, centrifuge tube is positioned on magnetic frame, removes residual ethanol with lancet head, dry 3min; Add 50 μ LLowTE, inhale up and down with rifle head and beat several times, concussion 10s, of short duration centrifugal, be placed on magnetic frame, 3min, after solution clarification, supernatant liquor is transferred in new centrifuge tube, then centrifuge tube is placed in 2min on magnetic frame, supernatant is transferred to new centrifuge tube, obtains the connection product after purifying.
Concentration determination and pcr amplification.Qubit2.0 measures the connection production concentration after purifying, in order to connect the amount of product after determining PCR process purifying.Amplification system is 60 μ L, comprise 50 μ LPlatinumPCRSuperMixHighFidelity, the connection product after 10ng purifying, 1.2 μ L10 μM PrimerA, 1.2 μ L10 μM PrimerB, mend nuclease free water to 60 μ L, reaction conditions is 95 DEG C of 5min, 17 × (95 DEG C of 30s, 62 DEG C of 30s, 68 DEG C of 30s), 72 DEG C of 5min, 4 DEG C of preservations.
Repeating step 5) purifying, finally use 30 μ LLowTE wash-outs.Qubit2.0 measures library concentration, and Agilent2100 detects library fragments size distribution.Examining report is shown in accompanying drawing 1.8, the selection of order-checking platform:
Utilize the NextSeq500 sequencing system of Illumina bis-generation order-checking platform, use single-ended 75bp sequencing kit.Because NextSeq500 sequenator single can produce the order-checking reads of 400M, therefore this test platform and method maximizing reduce order-checking cost, also faster relative to Hiseq sequencing system speed.Accompanying drawing 2 is shown in sequencing quality report.
9, the mining analysis of SNP marker:
Utilize TASSEL software to carry out the excavation of SNP to sequencing data, genome mapping software adopts bowtie2 to carry out.Detect 291 altogether, 772 SNP marker, after detecting SNP, it is carried out to analysis and the functional annotation of position distribution, known SNP is evenly distributed on genome, the results detailed in Table 1, illustrate and utilize analytical procedure provided by the present invention can obtain the high SNP marker Locus Analysis in Shoots result of the quality of data.
Show 1SNP site in the distribution of coloured differently body and annotation result.
| Karyomit(e) | Chromosome length | SNP number | SNP spacing (bp) |
| 1 | 195276750 | 58839 | 3318 |
| 2 | 148809762 | 45818 | 3247 |
| 3 | 110447801 | 34272 | 3222 |
| 4 | 90216835 | 38865 | 3125 |
| 5 | 59580361 | 19024 | 3131 |
| 6 | 34951654 | 11675 | 2993 |
| 7 | 36245040 | 11281 | 3212 |
| 8 | 28767244 | 8776 | 3277 |
| 9 | 23441680 | 7518 | 3118 |
| 10 | 19911089 | 6244 | 3188 |
| 11 | 19401079 | 5881 | 3298 |
| 12 | 19897011 | 5958 | 3339 |
| 13 | 17760035 | 5256 | 3379 |
| 14 | 15161805 | 4337 | 3495 |
| 15 | 12656803 | 3616 | 3500 |
| 16 | 535270 | 134 | 3994 |
| 17 | 10454150 | 2749 | 3802 |
| 18 | 11219875 | 3913 | 3851 |
| 19 | 9983394 | 2658 | 3755 |
| 20 | 14302601 | 3914 | 3654 |
| 21 | 6802778 | 1983 | 3430 |
| 22 | 4081097 | 825 | 4946 |
| 23 | 5723239 | 1568 | 3650 |
| 24 | 6323281 | 1533 | 4124 |
| 25 | 2191139 | 401 | 5464 |
| 26 | 5329985 | 1123 | 4746 |
| 27 | 5209285 | 1176 | 4429 |
| 28 | 4742627 | 1073 | 4419 |
| Z | 82363669 | 12313 | 6689 |
| W | 1248174 | 49 | 25472 |
| Add up to | 1003035513 | 291772 | 3437 |
Comparative example 1 traditional die method obtains and chicken genome SNP marker
Comparative example 1 is the reference examples of embodiment 1.In comparative example 1, sample is identical with embodiment 1, and all samples all use the 60KSNP chip of Illumina company chicken to carry out gene type.Hybridization, the Scanning Detction work of chip are completed by Canadian DNALandmarks company (DNALandmarksInc., Quebec, Canada).Through Quality Control, finally remain 47,965 SNP can be used for next step data analysis.The fund cost that each SNP obtains is about 10 times in embodiment 1, and experimental period cost is about 5 times in embodiment 1.
The selection of the suitableeest restriction endonuclease combination of embodiment 2 chicken genome
Embodiment 2 is for illustration of enzymes combinations used in the present invention.
Contriver considers different restriction enzyme site recognition point (as identified base number, GC content, methylation status) etc., design 8 groups of double digestion combinations altogether, the order-checking carrying out different enzymes combinations by 3 Lingnan Yellow chickens and Huiyang beard chicken individuals is tested, experiment flow is with embodiment 1, and experimental result is as shown in table 2.Known, the SNP number of EcoRI – MseI enzymes combinations is 134,291 (SNP number can change along with the change of experimental subjects number), endonuclease bamhi number is 414,294, the highest with genomic comparison rate, can carry out the typing assay of 96 samples in a Nextseq500 order-checking experiment, test input-output ratio reaches optimum.
The different enzymes combinations of table 2 genotyping result in chicken genome
| Enzymes combinations | SNP number | Endonuclease bamhi | Comparison rate |
| Pst I–Mse I | 402,083 | 1,247,742 | 97.26％ |
| Pst I–ApeK I | 195,960 | 761,797 | 96.90％ |
| EcoR I–Mse I | 134,291 | 414,294 | 98.37％ |
| Bgl II–ApeK I | 133,770 | 436,503 | 97.82％ |
| Pst I–Mse I | 117,571 | 498,114 | 94.28％ |
| HinP1 I-Mse I | 94,724 | 491,451 | 95.03％ |
| HinP1 I-ApeK I | 71,751 | 389,479 | 91.77％ |
| EcoR I–Mse I | 26,112 | 96,527 | 96.25％ |
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. obtain the method in chicken full-length genome high-density SNP marker site, it is characterized in that, comprise the following steps:
(1) the endonuclease bamhi distribution situation that obtains of the double digestion chicken genome of prediction EcoRI and MseI;
(2) according to the design of endonuclease bamhi characteristic distributions universal joint, barcode joint and the pcr amplification primer of EcoRI and MseI;
(3) simplification gene order-checking library is built;
(4) upper machine order-checking is carried out in the library utilizing step (3) to build;
(5) SNP marker site is obtained according to sequencing result.
2. analytical procedure according to claim 1, it is characterized in that, universal joint described in step (2) is with the cohesive end sequence identical with restriction enzyme MseI, and described barcode joint is with the cohesive end sequence identical with restriction enzyme EcoRI.
3. analytical procedure according to claim 1 and 2, is characterized in that, described universal joint is the double-stranded DNA formed by sequence anneals shown in SEQIDNO:1 and SEQIDNO:2, and wherein SEQIDNO:1 is through 5 ' phosphorylation modification.
4. analytical procedure according to claim 1 and 2, is characterized in that, described barcode joint is the double-stranded DNA formed by sequence anneals shown in SEQIDNO:3 and SEQIDNO:4; Wherein SEQIDNO:4 is through 5 ' phosphorylation modification, n and m in SEQIDNO:3 and SEQIDNO:4 represents that length is any short Nucleotide bar code sequence of 6-9bp.
5. analytical procedure according to claim 1, is characterized in that, the pcr amplification primer described in step (2) is as shown in SEQIDNO:5 and SEQIDNO:6.
6. analytical procedure according to claim 1, is characterized in that, step comprises the following steps in (3):
A () utilizes restriction enzyme combination EcoRI-MseI to carry out enzyme to chicken genome and cuts;
B () prepares universal joint and barcode joint;
C universal joint and barcode joint and digestion products are carried out ligation by () respectively, obtain and connect product;
D connection product equal proportion is carried out mixed pond by (), obtain the connection product behind mixed pond;
E the magnetic bead adding 1.2-1.4 times of volume in () connection product behind mixed pond carries out the first purifying and obtains the first purified product;
F magnetic bead that () adds 0.8-0.9 times of volume in described first purified product carries out the second purifying and obtains the second purified product;
G () is carried out pcr amplification to the second purified product and is obtained PCR primer;
H magnetic bead that () adds 1.2-1.4 times of volume in PCR primer carries out the 3rd purifying acquisition the 3rd purified product;
I magnetic bead that () adds 0.8-0.9 times of volume in the 3rd purified product carries out the 4th purifying and is simplified gene order-checking library.
7. analytical procedure according to claim 6, is characterized in that, described first purifying is identical with the step of the 3rd purifying, specifically comprises: after adding magnetic bead, on gyroscope incubated at room 18-22min obtain hatch after system; Be placed on supernatant discarded on magnetic frame after hatching end, add 70% ethanol of 480-520 μ L, leave standstill slow circumvolve after 30-40s, magnetic bead is moved on tube wall, after solution clarification, remove supernatant liquor, then repeat this step and once obtain precipitation; In obtained precipitation, add LowTE again, inhale up and down play rear vibration with pipettor, centrifugal rear standing clarification obtains supernatant liquor; Wherein, precipitate described in 100 μ L, the addition of LowTE is 140-160 μ L.
8. method according to claim 6, is characterized in that, the second purifying is identical with the step of the 4th purifying, specifically comprises: after adding magnetic bead, incubated at room 13-16min on gyroscope; Be placed on supernatant discarded on magnetic frame after hatching end, add 70% ethanol of 480-520 μ L, slow circumvolve after standing 30-40s, makes magnetic bead move on tube wall, after solution clarification, removes supernatant liquor, repeats this step and once obtain precipitation; In obtained precipitation, add LowTE again, inhale up and down play rear vibration with pipettor, centrifugal rear standing clarification obtains supernatant liquor; Wherein, precipitate described in 100 μ L, the addition of LowTE is 30-50 μ L.
9. method according to claim 6, is characterized in that, the annealing system of the universal joint described in step (c) is: 100 μMs of SEQIDNO:15 μ L; 100 μMs of SEQIDNO:25 μ L, 5 × AnnealingBuffer10 μ L, nuclease free water 30 μ L; Cycle of annealing is: be heated to 95 DEG C, and is cooled to 25 DEG C with the speed of 1 DEG C/min, in 4 DEG C of preservations after 25 DEG C of insulation 30min;
The annealing system of barcode joint is: 100 μMs of SEQIDNO:35 μ L; 100 μMs of SEQIDNO:45 μ L, 5 × AnnealingBuffer10 μ L, nuclease free water 30 μ L; Response procedures is: 95 DEG C of 3min, with the cooling of the speed of 1 DEG C/min, until drop to 25 DEG C, in 4 DEG C of preservations after 25 DEG C of insulation 30min;
System AdaptersMix: universal joint 0.8 μ L of joint mixing, barcode joint 15 μ L, nuclease free water 84.2 μ L, total system 100 μ L.
10. method according to claim 9, is characterized in that, the system of the ligation described in step (c) is: digestion products 20 μ L, 5 × DNALigaseReactionBuffer8 μ L, DNA ligase 2 μ L, nuclease free water 5 μ L, joint mixture 5 μ L; Mixing is placed on PCR, and response procedures is: 22 DEG C of insulation 1h, and 65 DEG C of insulation 30min, are cooled to 4 DEG C of preservations.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105671174A (en) * | 2016-03-14 | 2016-06-15 | 中国农业大学 | Chicken creeping character gene and DNA molecular marker relevant to chicken creeping character |
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| CN106191253B (en) * | 2016-07-14 | 2019-04-16 | 中国农业大学 | Beijing duck based on GBS technology simplifies gene order surveying method |
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| CN111225986A (en) * | 2017-10-10 | 2020-06-02 | 中国农业科学院北京畜牧兽医研究所 | Chicken whole genome SNP chip and application thereof |
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| CN110452990A (en) * | 2018-05-07 | 2019-11-15 | 华中农业大学 | For selecting hen to lay eggs the SNP marker and its application of later period laying rate |
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| CN108913797A (en) * | 2018-06-22 | 2018-11-30 | 中国农业科学院蔬菜花卉研究所 | The method that GBS obtains Chinese cabbage group genome SNP building finger-print |
| WO2020062160A1 (en) * | 2018-09-29 | 2020-04-02 | 中国农业大学 | Laying hen whole genome snp chip and use thereof |
| CN109680041A (en) * | 2018-12-25 | 2019-04-26 | 上海派森诺生物科技股份有限公司 | A kind of processing method based on the sequencing sample for simplifying gene order-checking |
| CN113322333A (en) * | 2021-07-06 | 2021-08-31 | 广西大学 | CNV molecular marker combination related to Guangxi hemp chicken body size and slaughter traits based on whole genome sequencing screening and application |
| CN113322333B (en) * | 2021-07-06 | 2022-07-05 | 广西大学 | CNV molecular marker combination related to Guangxi hemp chicken body size and slaughter traits based on whole genome sequencing screening and application |
| WO2023165128A1 (en) * | 2022-03-04 | 2023-09-07 | 江苏省家禽科学研究所 | Chicken low-density snp liquid-phase chip based on target capture sequencing and use thereof |
| CN115679012A (en) * | 2022-10-18 | 2023-02-03 | 武汉市农业科学院 | Hot pepper whole genome SNP-Panel and application thereof |
| CN115679012B (en) * | 2022-10-18 | 2023-07-04 | 武汉市农业科学院 | Chilli whole genome SNP-Panel and application thereof |
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