CN109680041A - A kind of processing method based on the sequencing sample for simplifying gene order-checking - Google Patents
A kind of processing method based on the sequencing sample for simplifying gene order-checking Download PDFInfo
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- CN109680041A CN109680041A CN201811594696.XA CN201811594696A CN109680041A CN 109680041 A CN109680041 A CN 109680041A CN 201811594696 A CN201811594696 A CN 201811594696A CN 109680041 A CN109680041 A CN 109680041A
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Abstract
The invention discloses a kind of processing methods based on the sequencing sample for simplifying gene order-checking, include the following steps: step 1: extracting target gene group DNA；Step 2: genomic DNA obtained by step (1) is quantified；Step 3: according to step (2) DNA concentration measurement result, 500ng DNA is chosen as digestion initial amount, is combined digestion, at least three hour digestion time using EcoR1 and Mse1；Step 4: it is cut with DNA enzymatic obtained by step (3) and continues library after product carries out, and upper machine is sequenced.The beneficial effects of the present invention are: the present invention passes through the sequencing initial amount in optimization sequencing, digestion time, the base balance of connector, it will be able to the output of the data of the effective valid data ratio for increasing library and high quality.
Description
Technical field
The invention belongs to gene sequencing fields, and in particular to a kind of processing based on the sequencing sample for simplifying gene order-checking
Method.
Background technique
Simplify gene order-checking (Reduced-Representation Genome Sequencing, RRGS) as its name suggests
It is that sequencing is carried out to portion gene group.It refers to using bioinformatics method, designs marker development scheme, and enrichment is special
Property long fragment, obtain magnanimity sequence label using high throughput sequencing technologies to represent the sequencing of target species full-length genome information
Method.
Simplify gene order-checking (Reduced-Representation Genome Sequencing, RRGS) and refers to utilization
Restriction enzyme interrupts genomic DNA, carries out high-flux sequence to specific fragment and obtains magnanimity genetic polymorphism sequence label
Sufficiently represent the sequencing strategy of target species full-length genome information.
The method experimental procedure is simple, at low cost, and can not depend on reference to genome, can obtain full-length genome model
Interior genetic polymorphism label is enclosed, thus is widely used in ecology, the fields such as theory of evolution and genomics, but the number of its sequencing
It is always a problem according to accuracy precision.
Technology path such as Fig. 1 of traditional simplification gene order-checking.
Applicant passes through the discovery of concentrating on studies of many years: simplified gene order-checking initial amount, the digestion time, suitable in connector
Base balance can effectively increase the output of the valid data ratio in library and the data of high quality.
Summary of the invention
In order to overcome drawbacks described above of the existing technology, the purpose of the present invention is to provide one kind based on simplified genome
The processing method of the sequencing sample of sequencing.
In order to achieve the object of the present invention, used technical solution is: a kind of based on the sequencing for simplifying gene order-checking
The processing method of sample, includes the following steps:
Step 1: target gene group DNA is extracted；
Step 2: genomic DNA obtained by step (1) is quantified；
Step 3: according to step (2) DNA concentration measurement result, 500ng DNA is chosen as digestion initial amount, is used
EcoR1 and Mse1 is combined digestion, at least three hour digestion time；
Step 4: it is cut with DNA enzymatic obtained by step (3) and continues library after product carries out, and upper machine is sequenced.
In a preferred embodiment of the invention, in the step 1, the method that the target gene group DNA is extracted is
General extraction methods.
In a preferred embodiment of the invention, the general extraction methods be using various commercial reagent boxes or other
Routine techniques extracts target gene group DNA.
In a preferred embodiment of the invention, the target gene group DNA includes Animal genome DNA, plant base
Because of any one or more in a group DNA, microbe genome DNA.
In a preferred embodiment of the invention, the target gene group DNA includes having with reference to genome sequence
Genomic DNA and or the genomic DNA without reference genome sequence.
In a preferred embodiment of the invention, the restriction enzyme in the step 3 is purchased from NEB (New
England Biolabs) company.
The beneficial effects of the present invention are:
For the present invention by the sequencing initial amount in optimization sequencing, digestion time, enzymes combinations can effectively increase height
The output of quality sequencing data.
Detailed description of the invention
Fig. 1 is flow diagram of the invention.
Specific embodiment
The present invention is further illustrated below by embodiment:
Embodiment 1: rice (OryzasativaLcv.Nipponbare, Oryza.Sativa L.spp.japonica)
In order to solve the above technical problems, the present invention builds library DNA initial amount by increasing, extend the digestion time, selection is most closed
Suitable enzymes combinations, the method includes the following steps:
(1) target gene group DNA is extracted；
(2) genomic DNA obtained by step (1) is quantified；
(3) according to step (2) DNA concentration measurement result, choose 500ng DNA and be used as digestion initial amount, using EcoR1 with
Mse1 is combined digestion, 3 hours digestion time or 3 hours or more；
(4) it is cut with DNA enzymatic obtained by step (3) and continues library after product carries out, and upper machine is sequenced.
Wherein step (1) is to extract target gene group DNA.The method that the target gene group DNA is extracted is traditional extraction
Method.Preferably target gene group DNA is extracted using various commercial reagent boxes or other routine techniques.The target gene group
DNA is preferably Animal genome DNA, plant genome DNA or microbe genome DNA；The target gene group DNA includes
Genomic DNA with reference genome sequence, or the genomic DNA without reference genome sequence.
Wherein the reference genome is this field conventional conception, refers to that the genome sequence of species is surveyed by full-length genome
Sequence.Species gene group DNA with reference genome sequence refers to that the species have been sequenced by full genome；Without referring to gene
The genomic DNA of group sequence refers to that the species are not sequenced by full genome.
Restriction enzyme used in step (3) is purchased from NEB (New England Biolabs) company.
Simplifying genomic library initial amount at least 500ng, the digestion time, it is more than hour to be preferably increased to 3 hours or 3,
For rice (OryzasativaLcv.Nipponbare, Oryza.Sativa L.spp.japonica), the enzymes combinations effect of EcoR1 and Mse1 are most
It is good.
1,200ng builds library initial amount and 500ng builds influence of the library initial amount to upper machine valid data
This experiment enzymes combinations is identical, and the digestion time is constant, and unique variable is digestion initial amount, respectively 200ng and
500ng the results are shown in Table 1.
Table 1: different initial amounts build library
Table 1
| Initial amount | Enzymes combinations | The digestion time |
| 500ng | Pst and Bfa1 | 3 hours |
| 200ng |
Table 2 is that difference builds influence of the library initial amount to high quality valid data；
Table 2
Finally pass through library construction, upper machine testing, data are analyzed, as can be seen from the results the high quality in the library 500ng
Valid data ratio is 76.31%, hence it is evident that higher than the 70.69% of the library 200ng.
2, the relationship between digestion time and valid data
This experiment digestion initial amount, enzymes combinations are identical, and unique variable is the digestion time, respectively processing 1 hour, and 3
Hour, 5 hours and 12 hours.
The different digestion time-triggered protocols of table 3；
Table 3
Finally pass through library construction, upper machine testing, data analysis, from result it can be found that after 1 hour of digestion it is high-quality
It is minimum to measure valid data ratio, followed by digestion 3 hours, digestion 5 hours, and digestion 12 hours ratio highests.Therefore with
The trend gradually increased can be presented in the extension of digestion time, high quality valid data ratio.But simplify carrying out high-volume
During gene order-checking, 5 hours, 12 small the reaction times were too long, influenced efficiency, therefore, comprehensively considered 3 hours digestion time
It is most suitable for.
Influence of the different digestion times of table 4 to high quality valid data
Table 4
| The digestion time | High quality valid data ratio |
| 1 hour | 74.52% |
| 3 hours | 76.31% |
| 5 hours | 76.56% |
| 12 hours | 78.77% |
3. different restriction enzymes combine the influence to high quality sequencing data.
This experiment digestion initial amount, digestion time consistency, unique variable is enzymes combinations, be respectively adopted EcoR I and
Mse I, EcoR I and Msp I, Hind III and Bfa I, Pst I and Bfa I, Pst I and Msp I, Sbf I and Bfa I six
Kind combination, is shown in Table 5.
5 enzymes combinations detail of table
Table 5
Finally pass through library construction, upper machine testing, data analysis, it can be found that different enzymes combinations constructed from result
Library, high quality valid data ratio are different.The high quality valid data ratio of EcoR1 and Mse1 enzymes combinations is
82.92%, highest；The library high quality valid data ratio minimum 43.76% of Sbf1 and Bfa1 combination, it is minimum, it is shown in Table 6.
Influence of the different enzymes combinations of table 6 to sequencing result；
Table 6
Therefore, final determine simplifies genome digestion optimum reaction condition are as follows: 500ng DNA is as initial amount, when digestion
Between control in 3h, be most suitable for simplifying genomic library sequencing for the enzymes combinations of rice plants EcoR I and Mse I.
Claims (6)
1. a kind of processing method based on the sequencing sample for simplifying gene order-checking, which comprises the steps of:
Step 1: target gene group DNA is extracted；
Step 2: genomic DNA obtained by step (1) is quantified；
Step 3: according to step (2) DNA concentration measurement result, choosing 500ng DNA and be used as digestion initial amount, using EcoR1 with
Mse1 is combined digestion, at least three hour digestion time；
Step 4: it is cut with DNA enzymatic obtained by step (3) and continues library after product carries out, and upper machine is sequenced.
2. a kind of processing method based on the sequencing sample for simplifying gene order-checking as described in claim 1, which is characterized in that
In the step 1, the method that the target gene group DNA is extracted is general extraction methods.
3. a kind of processing method based on the sequencing sample for simplifying gene order-checking as claimed in claim 2, which is characterized in that
The general extraction methods are to extract target gene group DNA using various commercial reagent boxes or other routine techniques.
4. a kind of processing method based on the sequencing sample for simplifying gene order-checking as described in claim 1, which is characterized in that
The target gene group DNA includes Animal genome DNA, plant genome DNA, any one in microbe genome DNA
Or it is a variety of.
5. a kind of processing method based on the sequencing sample for simplifying gene order-checking as described in claim 1, which is characterized in that
The target gene group DNA includes with the genomic DNA with reference to genome sequence and or without reference genome sequence
Genomic DNA.
6. a kind of processing method based on the sequencing sample for simplifying gene order-checking as described in claim 1, which is characterized in that
Restriction enzyme in the step 3 is purchased from NEB (New England Biolabs) company.
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Cited By (1)
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