CN102719528A - emPCR method of 454 high-throughput sequencing based on pyrophosphoric acid principle - Google Patents
emPCR method of 454 high-throughput sequencing based on pyrophosphoric acid principle Download PDFInfo
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- CN102719528A CN102719528A CN2012101302669A CN201210130266A CN102719528A CN 102719528 A CN102719528 A CN 102719528A CN 2012101302669 A CN2012101302669 A CN 2012101302669A CN 201210130266 A CN201210130266 A CN 201210130266A CN 102719528 A CN102719528 A CN 102719528A
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Abstract
The invention discloses an emPCR method of 454 high-throughput sequencing based on a pyrophosphoric acid principle, the method is characterized in that during the process of preparing emPCR amplification mixed liquor, the volumes of ultrapure water, 5*amplification liquid, an amplimer, an emPCR amplification mixed liquor and PPiase are adjusted; and the amplification condition is changed from one-step amplification into two-steps amplification during the amplification reaction. The employed emPCR method is capable of effectively reducing the non-specific amplification signal, reducing the redundancy data, raising the output of the effective data by 30%, the experiment efficiency can be increased, and the experiment cost is saved.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of round pcr.In particular, the present invention relates to a kind of emPCR method that is used for based on 454 high-flux sequences of tetra-sodium principle.
Background technology
The tetra-sodium sequencing technologies is a dna sequence analysis technology of new generation, and this technology need not be carried out electrophoresis, and dna fragmentation also need not carry out fluorescent mark, operates very easy.The tetra-sodium sequencing technologies is by the enzyme cascade chemiluminescence reaction in 4 kinds of enzymatic same reaction systems; Take turns in the sequencing reaction at each; Only add a kind of dNTP; If this dNTP and template are matched, polysaccharase just can be incorporated into it in primer strand and the tetra-sodium group (PPi) of mole number such as discharges.PPi can finally be converted into visible light signal, and generates a peak value through optical system.The Nucleotide number that mixes is directly proportional in the height of each peak value and the reaction, adds a kind of dNTP down then, continues the synthetic of DNA chain.
Employed template need prepare through the method for a kind of emPCR of being called in the tetra-sodium order-checking.Belong to routine techniques based on the emPCR method in 454 high-flux sequences of tetra-sodium order-checking principle at present, and also have the sophisticated relatively commodity of exploitation directly to use on the market.Test kit---RocheGS Titanium LV emPCR Kit (Lib-L) v2 that for example adopts Roche Holding Ag to release by the procedure of its specification sheets record, can make the required emPCR product of order-checking.Utilize the step of the used emPCR product of RocheGSTitanium LV emPCR Kit (Lib-L) v2 test kit preparation order-checking following:
1) oil in water emulsion, emPCR amplification mixed solution and reagent are prepared, and wherein emPCR amplification mixed solution comprises following each component:
2) catch in the DNA library;
3) emulsification;
4) amplification, the program of amplified reaction is as follows:
①94℃，4min；
②94℃，30s；
③60℃，10min；
Step is 2. to 3. totally 50 circulations;
4. 10 ℃ of preservations;
5) magnetic bead reclaims;
6) DNA library enrichment with magnetic bead;
7) sequencing primer annealing promptly makes the used emPCR product of order-checking.
Existing this method based on emPCR in 454 high-flux sequences of tetra-sodium principle is relatively harsher for the requirement of clip size in the constructed library; If the clip size difference is excessive, will cause on the magnetic bead that emPCR amplifies the sequence signal strength difference very big, and then the reading efficiency and the accuracy of signal when influencing follow-up order-checking; Produce more redundant data; Valid data are reduced more than 30%, cause the waste of reagent and experimental period simultaneously, increase experimental cost.
Summary of the invention
Technical problem to be solved by this invention is above-mentioned based on the technological deficiency in 454 high-flux sequences of tetra-sodium principle in order to overcome, and a kind of new emPCR method based on 454 high-flux sequences of tetra-sodium principle that is used for is provided.
The present invention solves the problems of the technologies described above one of technical scheme of being taked: a kind of method that is used for based on 454 high-flux sequence emPCR of tetra-sodium principle, it comprises the steps:
1) oil in water emulsion, emPCR amplification mixed solution and reagent are prepared;
2) catch in the DNA library;
3) emulsification;
4) amplification;
5) magnetic bead reclaims;
6) DNA library enrichment with magnetic bead;
7) sequencing primer annealing promptly makes the used emPCR product of order-checking;
Wherein, the described emPCR amplification of step 1) mixed solution comprises following each component: emPCRAdditive, 30.6%～43.0% (v/v); 5 * amplification liquid, 18.4%～21.5% (v/v); Amplimer, 2.0%～7.5% (v/v); EmPCR enzyme mixed solution, 2.6%～5.9% (v/v); PPiase, 0.1%～0.3% (v/v).Above-mentioned each component is RocheGS Titanium LV emPCR Kit (Lib-L) v2 test kit from carries product.
Preferable, emPCR amplification mixed solution comprises following each component: emPCR Additive, 38.4% (v/v); 5 * amplification liquid, 20.3% (v/v); Amplimer, 4.8% (v/v); EmPCR enzyme mixed solution, 5.3% (v/v); PPiase, 0.2% (v/v); Surplus is a water.
Among the present invention, described water is the conventional water that is used to check order in this area, preferred ultrapure water.
Among the present invention, the TV of described emPCR amplification mixed solution is the conventional TV that is adopted in 454 high-flux sequences of this area based on the tetra-sodium principle, is generally 930 μ l～980 μ l, and that the present invention is best is 937 μ L.
A preferred embodiments of the present invention is that the TV of the described emPCR amplification of step 1) mixed solution is 937 μ L, comprises following each component: 360 μ LemPCRAdditive; 180～200 μ L5 * amplification liquid; 35～55 μ L amplimers; 50 μ LemPCR enzyme mixed solutions; 2 μ LPPiase; All the other are water.Each component concentration is as shown in the table:
A preferred example of the present invention is that the TV of the described emPCR amplification of step 1) mixed solution is 937 μ L, comprises following each component: 290 μ L ultrapure waters; 360 μ LemPCRAdditive; 190 μ L5 * amplification liquid; 45 μ L amplimers; 50 μ LemPCR enzyme mixed solutions; 2 μ LPPiase.Each component concentration is as shown in the table:
Preferably, in the present invention, the response procedures of the said amplification of step 4) is as follows:
①90-95℃，3-6min；
②93-95℃，28-32s；
③57-59℃，4-5min；
④66-70℃，28-32s；
Step is 2. to 4. 45-55 circulation altogether;
5. 4-10 ℃ of preservation.
More preferably, the amplified reaction programstep 2. in 4. cycle number be 48-52, most preferably 50.
More preferably, the amplified reaction programstep is 2. to 4. being 2. 94-95 ℃, 28-32s; 3. 57-58 ℃, 4-5min; 4. 67-69 ℃, 28-32s; Most preferably 2. 94 ℃, 30s; 3. 58 ℃, 4.5min; 4. 68 ℃, 30s.
In the preferred example of the present invention, the response procedures of the said amplification of step 4) is:
①94℃，4min；
②94℃，30s；
③58℃，4.5min；
④68℃，30s；
Step is 2. to 4. totally 50 circulations;
5. 10 ℃ of preservations.
The emPCR method that is used for based on 454 high-flux sequences of tetra-sodium principle of the present invention; Other step all adopts this area ordinary method and condition; Can consult the workbook among the test kit RocheGSTitanium LV emPCR Kit of Roche Holding Ag (Lib-L) v2---the method for being put down in writing on " Roche, emPCRAmp-Lib L SV Method Manual_XL+_May2011 ".
EmPCR method of the present invention is used for 454 high-flux sequences based on the tetra-sodium principle; Described 454 high-flux sequences can be that 454 high-throughputs are transcribed the group order-checking; Also can be 454 high throughput genome sequencings or other 454 high-flux sequences, preferred 454 high-throughputs be transcribed the group order-checking.
Positive progressive effect of the present invention is: the present invention optimizes and revises the consumption of emPCR Additive, 5 * amplification liquid, amplimer, emPCR enzyme mixed solution and ultrapure water in the process of preparation emPCR amplification mixed solution; When carrying out amplified reaction, amplification program has been carried out optimizing improvement, changed the amplification of two steps into by step amplification.Adopt emPCR method of the present invention can effectively reduce the non-specific amplification signal, reduce redundant data, make the output of valid data improve 30%, improved conventional efficient simultaneously, practiced thrift experimental cost.
Embodiment
Below in conjunction with embodiment the present invention is described further.Should be understood that following examples only are used to the present invention is described but not are used to limit scope of the present invention.The experimental technique of unreceipted actual conditions among the following embodiment carries out according to normal condition or according to the condition that manufacturer advises usually.
Embodiment 1
Present embodiment adopts the method for the invention preparation based on the required emPCR product of 454 high-flux sequences of tetra-sodium principle; Agents useful for same if no special instructions; All adopt the reagent that carries in Roche GS Titanium LVemPCR Kit (Lib-L) the v2 test kit, may further comprise the steps.
1, oil in water emulsion, emPCR amplification mixed solution and reagent are prepared;
1) oil in water emulsion is put on the TissueLyser tube frame (production of Qiagen company).
2) the frequency oscillation 2min of 25Hz.
3) remove the oil in water emulsion that concussion is accomplished.
4) with 4ml ultrapure water dilution 1ml 5 * Mock Amplification Mix, the working fluid of 1 * concentration is processed in the vortex concussion.
5) will shake the oil in water emulsion of accomplishing installs in the 1.7ml centrifuge tube by 600 μ L/ centrifuge tube branches.
6) the MockAmplification Mix after the adding 290 μ L dilution in every pipe oil in water emulsion.
7) mixing that turns upside down 3 times is fixed in TissueLyser (production of Qiagen company).
8) the frequency oscillation 5min of 5Hz.
9) take off the Emulsion pipe.
10) preparation emPCR amplification mixed solution: according to Emulsion reacting weight preparation emPCR amplification mixed solution.Add each component by following order, prepare the amplification mixed solution of 937 μ L.Vortex vibration amplification liquid 5s, and be stored on ice.
2, catch in the DNA library
1) add the ultrapure water of 9ml, 10 * Capture Bead Wash Buffer TW of 1ml is diluted to 1 * working fluid.
2) vortex concussion DNA Capture Beads.
3) in the centrifuge tube of 1.7ml, react 80 μ L and add DNA CaptureBeads by each Emulsion.
4) behind the centrifugal 10s, recentrifuge 10s behind the Rotate 180 °, deposition pearl.
5) carefully remove supernatant, note not running into pearl.
6) 1 * Capture Bead Wash Buffer TW of adding 1ml, vortex shakes resuspended pearl and cleans, and this step repeats twice, and centrifugal back is inhaled and is removed supernatant.
7) the Capture Beads after will cleaning is dispensed to the amount of single Emulsion reaction:
A. use the resuspended every part of DNA Capture Beads of Capture Bead Wash Buffer TW of the amount of 40 * Emulsion stoichiometric number.
B. measure the TV of DNA Capture Beads suspension-s, uniformly distributing is to the 1.7ml centrifuge tube then.
C. according in 2 4) method and step deposition pearl, absorb supernatant.
8) the DNA library that thaws and need increase:
The library volume of control dilution between 1～15 μ L, heat denatured DNA library on the PCR appearance, 95 ℃, 2min, 4 ℃ of preservations.
9) calculate the volume in required DNA library, and join among the DNA CaptureBeads after the cleaning.
Equation:
10) vortex concussion 5s, fully mixing gets the library and catches mixed solution.
11) add 215 μ L amplification mixed solution to library and catch in the mixed solution vortex concussion.
3. emulsification
1) drawing the library (2 preparation) of catching joins in the Emulsion pipe.
2) turn upside down behind 3 mixings of pipe support, place TissueLyser.
3) 15Hz vibrations 5min.
4. amplification
4.1 distribution emulsification reagent
1) after the emulsification, the emPCR mixed solution that increases is dispensed in the 96 hole PCR plates every hole 100 μ L.Every pipe emPCR amplification mixed solution can distribute 10 holes.Sealing PCR plate.
2) clean the reaction mixture that spills.
4.2 amplified reaction, its response procedures is:
①94℃，4min；
②94℃，30s；
③58℃，4.5min；
④68℃，30s；
Step is 2. to 4. totally 50 circulations;
5. 10 ℃ of preservations.
5. magnetic bead reclaims
5.1 collecting emulsion cleans with preliminary
1) takes out Enhancing Fluid XT and Annealing Butter XT, keep at room temperature.
2) assembling flush end syringe needle is to disposable sterilized 10ml injector tip, and each sample matees a syringe.
3) emulsion with each sample is drawn onto in the syringe of suitable quantity.
4) add 100 μ L Virahols in each hole, draw mixing up and down.
5) from the hole, draw aqueous isopropanol.
6) secondary is drawn surplus solution in the hole.
7) put upside down syringe and suck the 3ml air.
8) remove the flush end syringe needle.
9) blue filter is filled to injector tip.
10) Kimwipe dust-free cleaning paper (Fisher Scientific Company products, numbering 06-666A) is put to the vortex platform, injector tip places vortex vibrator concussion 5s.
11) through strainer syringe contents is discharged.
5.2 magnetic bead cleans and reclaims
1) puts upside down syringe, suck the 3ml air.
2) suck the 3-5ml Virahol.
3) strainer points to Kimwipe downwards, maximum fast whirlpool syringe 5s.
4) through strainer syringe contents is discharged.
5) repeat 1) to 4) go on foot 2 times.
6) put upside down syringe, suck the 3ml air.
7) suck 3ml Enhancing Fluid XT, do not suck air.
8) strainer points to Kimwipe downwards, maximum fast whirlpool syringe 5s.
9) through strainer syringe contents is discharged.
10) repeat 6) to 9) go on foot 2 times.
11) put upside down syringe, suck the 3ml air.
12) suck Enhancing Fluid XT to 0.5ml scale place, do not suck air.
13) strainer points to Kimwipe downwards, maximum fast whirlpool syringe 5s.
14) promptly put upside down syringe, suck extra 3ml air.
15) remove (not losing) strainer, bead suspension is got in the new 1.5ml EP pipe.
16) centrifugal 1.5ml EP pipe 10s, Rotate 180 °, centrifugal again 5s.Absorb supernatant.
17) put upside down syringe, suck the 3ml air.
18) strainer is reinstalled syringe, suck Enhancing Fluid XT to 0.5ml scale place, do not suck air.
19) strainer points to Kimwipe downwards, maximum fast whirlpool 5s.
20) promptly put upside down syringe, suck extra 3ml air.
21) remove strainer, bead suspension is got in the same 1.5ml EP pipe.
22) abandon syringe and strainer.
6.DNA library enrichment with magnetic bead
6.1 prepare before the enrichment
1) opens block heater (production of Labnet company), be set to 65 ℃.
2) mix 125 μ L10N NaOH (production of Amresco company) and 9.875ml ultrapure water, be mixed with Melt Solution.
3) centrifugal Rotate 180 ° centrifugal again EP pipe is absorbed supernatant.
4) add 1ml Melt Solution, whirlpool in every pipe.Incubated at room 2min.Centrifugal Rotate 180 is ° centrifugal again, absorbs supernatant.
5) repeating 4) step is once.
6) add 1ml Annealing Buffer XT, whirlpool in every pipe.Centrifugal Rotate 180 is ° centrifugal again, absorbs supernatant.
7) repeating 6) step is once.
8) add 30 μ L Annealing Buffer XT, the Enrichment Primer of 12 μ L, whirlpool in every pipe.
9) be positioned over 5min on 65 ℃ of block heater, put rapidly afterwards to 2min on ice.
10) every pipe adds 500 μ L Enhancing Fluid XT, whirlpool.Centrifugal Rotate 180 is ° centrifugal again, absorbs supernatant.
11) repeating step 10) once.
12) add 800 μ L Enhancing Fluid XT, whirlpool in every pipe.
6.2 prepare the enrichment pearl
1) brown " Enrichment Beads " pipe 1min of vortex concussion makes it fully resuspended.
2) pipe is placed on the magnetic force frame (production of Invitrogen company) the enrichment magnetic bead.
3) absorb supernatant, carefully be not drawn onto magnetic bead.Take off.
4) add 500 μ L Enhancing Fluid XT, the vortex concussion.
5) place on the magnetic force frame absorption Enrichment Beads.
6) absorb supernatant, carefully be not drawn onto magnetic bead.
7) repeat the 4-6 step.
8) behind the absorption supernatant, it is taken off from the magnetic force frame.
9) manage 20 μ L by every emulsion oil and add Enhancing Fluid XT, the vortex concussion.
6.3 the magnetic bead of dna fragmentation is carried in enrichment
1) in the Capture Beads pipe that every pipe cleaned, adds the EnrichmentBeads 40 μ L that cleaned.
2) on mixing tank, rotate 5min under the room temperature.
3) with on each Guan Fangzhi magnetic force frame, wait for 3-5min, magnetic bead is adsorbed onto on the magnetic force frame.
4) put upside down the magnetic force frame several times.
5) absorb supernatant carefully with 1000 μ L suction nozzles, do not interfere with magnetic bead.
6) clean magnetic bead by following method with Enhancing Fluid XT, in supernatant, do not have visible pearl:
A. add 1ml Enhancing Fluid XT in every pipe;
B. each pipe is taken off whirlpool from the magnetic force frame;
C. each pipe is put back on the magnetic force frame, made magnetic bead be adsorbed onto tube wall, put upside down the magnetic force frame;
D. absorb the supernatant in every pipe carefully with 1000 μ L suction nozzles;
E. repeat 6-10 time and in supernatant, do not have visible pearl.
6.4 collect enrichment good be connected with the dna fragmentation magnetic bead
1) each pipe is taken off from the magnetic force frame, add the resuspended magnetic bead of Melt Solution of 700 μ L.
2) whirlpool 5s puts back to the magnetic force frame with each pipe and is adsorbed onto tube wall until Enrichment Beads.
3) supernatant in each pipe is transferred in the 1.5ml EP pipe.
4) centrifugal Rotate 180 is ° centrifugal again, absorbs supernatant.
5) add one time 700 μ L Melt Solution to each former Guan Zhongzai.
6) whirlpool 5s puts back to each pipe on the magnetic force frame, is adsorbed onto tube wall until Enrichment Beads.
7) shift supernatant to same 1.5ml EP pipe.
8) abandon former pipe.
9) centrifugal Rotate 180 is ° centrifugal again, absorbs supernatant.
10) in each pipe, add 500 μ LAnnealing Buffer XT, whirlpool 5s.
11) centrifugal Rotate 180 is ° centrifugal again, removes supernatant.
12) repeat 10) to 11) go on foot 2 times.
13) the Annealing Buffer XT of adding 60 μ L in each pipe, whirlpool resuspension pearl.
7. sequencing primer annealing
1) sequencing primer of adding 12 μ L, vortex vibration mixing.
2) in the block heater, 65 ℃ the heating 5min, after be put in 2min on ice rapidly.
3) add 500 μ L Annealing Buffer XT in every pipe, vortex vibration 5s, centrifugal Rotate 180 is ° centrifugal again, removes supernatant.
4) with 500 μ L Annealing Buffer repeating steps 3) twice.
5) add 1ml Annealing Buffer in each pipe, the vortex concussion.
6) counting pearl, calculate the percentage of enrichment:
Promptly get the emPCR product that checks order used through above-mentioned each step.
Embodiment 2
All methods of present embodiment and step are with embodiment 1, and its difference comprises following 2 points:
One, in the step 1 the 1st) described emPCR amplification of step mixed solution comprises following each component:
Two, in the amplified reaction of step 4.2, its response procedures is:
①90℃，3min；
②95℃，28s；
③57℃，5min；
④66℃，32s；
Step is 2. to 4. totally 45 circulations;
5. 4 ℃ of preservations.
Embodiment 3
All methods of present embodiment and step are with embodiment 1, and its difference comprises following 2 points:
One, in the step 1 the 1st) described emPCR amplification of step mixed solution comprises following each component:
Two, in the amplified reaction of step 4.2, its response procedures is:
①94℃，4min；
②93℃，32s；
③59℃，4min；
④70℃，28s；
Step is 2. to 4. totally 55 circulations;
5. 10 ℃ of preservations.
Embodiment 4
All methods of present embodiment and step are with embodiment 1, and its difference comprises following 2 points:
One, in the step 1 the 1st) described emPCR amplification of step mixed solution comprises following each component:
Two, in the amplified reaction of step 4.2, its response procedures is:
①94℃，4min；
②94℃，30s；
③58℃，5min；
④68℃，30s；
Step is 2. to 4. totally 50 circulations;
5. 10 ℃ of preservations.
Embodiment 5
All methods of present embodiment and step are with embodiment 1, and its difference comprises following 2 points:
One, in the step 1 the 1st) described emPCR amplification of step mixed solution comprises following each component:
Two, in step 4.2 amplified reaction, its response procedures is:
①95℃，6min；
②94℃，30s；
③57℃，5min；
④68℃，31s；
Step is 2. to 4. totally 50 circulations;
5. 10 ℃ of preservations.
Effect embodiment 1
Combine effect embodiment that the present invention is further described now.
This effect embodiment has compared respectively through the workbook---" Roche among the test kit RocheGS Titanium LVemPCR Kit of Roche Holding Ag (Lib-L) v2; EmPCR Amp-Lib L SVMethod Manual_XL+_May2011 " on the method put down in writing be that the emPCR product of prior art for preparing and the emPCR product through embodiment 1 said method preparation are used for the effect based on 454 high-flux sequences of tetra-sodium principle, the result is as shown in the table:
Can find out from above table; The emPCR product that adopts the method for the invention preparation is at 454 high-flux sequences that are used for based on the tetra-sodium principle; Its ratio through filtration sequence compared with prior art is greatly increased, and on average reads length and total amount of data and also increases substantially.Therefore, in research and production practice, adopt the method for the invention can effectively reduce the non-specific amplification signal based on 454 high-flux sequences of tetra-sodium principle; Reduce redundant data; Make the output of valid data improve 30%, thereby improved conventional efficient, practiced thrift experimental cost.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition that after having read foregoing of the present invention those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. method that is used for based on 454 high-flux sequence emPCR of tetra-sodium principle, it comprises step:
1) oil in water emulsion, emPCR amplification mixed solution and reagent are prepared;
2) catch in the DNA library;
3) emulsification;
4) amplification;
5) magnetic bead reclaims;
6) DNA library enrichment with magnetic bead;
7) sequencing primer annealing promptly makes the used emPCR product of order-checking;
It is characterized in that the described emPCR amplification of step 1) mixed solution comprises following each component: emPCRAdditive, 30.6%～43.0% (v/v); 5 * amplification liquid, 18.4%～21.5% (v/v); Amplimer, 2.0%～7.5% (v/v); EmPCR enzyme mixed solution, 2.6%～5.9% (v/v); PPiase, 0.1%～0.3% (v/v); Surplus is a water.
2. the method for claim 1 is characterized in that, the described emPCR amplification of step 1) mixed solution comprises following each component: emPCR Additive, 38.4% (v/v); 5 * amplification liquid, 20.3% (v/v); Amplimer, 4.8% (v/v); EmPCR enzyme mixed solution, 5.3% (v/v); PPiase, 0.2% (v/v); Surplus is a water.
3. the method for claim 1 is characterized in that, the TV of described emPCR amplification mixed solution is 930 μ L～980 μ L.
4. the method for claim 1 is characterized in that, the TV of described emPCR amplification mixed solution is 937 μ L, comprises following each component: 360 μ L emPCRAdditive; 180～200 μ L 5 * amplification liquid; 35～55 μ L amplimers; 50 μ L emPCR enzyme mixed solutions; 2 μ L PPiase; Surplus is a water.
5. the method for claim 1 is characterized in that, the TV of described emPCR amplification mixed solution is 937 μ L, comprises following each component: 290 μ L ultrapure waters; 360 μ L emPCRAdditive; 190 μ L5 * amplification liquid; 45 μ L amplimers; 50 μ LemPCR enzyme mixed solutions; 2 μ L PPiase.
6. the method for claim 1 is characterized in that, the response procedures of the said amplification of step 4) is:
①90-95℃，3-6min；
②93-95℃，28-32s；
③57-59℃，4-5min；
④66-70℃，28-32s；
Step is 2. to 4. 45-55 circulation altogether;
5. 4-10 ℃ of preservation.
7. method as claimed in claim 6 is characterized in that, step is 2. to 4. being in the response procedures of said amplification:
②94-95℃，28-32s；
③57-58℃，4-5min；
④67-69℃，28-32s。
8. method as claimed in claim 6 is characterized in that, 1. step is 94 ℃ in the response procedures of said amplification, 4min; 5. step is 10 ℃ of preservations.
9. method as claimed in claim 6 is characterized in that, the response procedures of said amplification is:
①94℃，4min；
②94℃，30s；
③58℃，4.5min；
④68℃，30s；
Step is 2. to 4. totally 50 circulations;
5. 10 ℃ of preservations.
10. the method for claim 1 is characterized in that, described high-flux sequence is for transcribing the group order-checking based on 454 high-throughputs of tetra-sodium principle.
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| CN104774952A (en) * | 2015-04-22 | 2015-07-15 | 福建正源饲料有限公司 | Method for evaluating dynamic change of microbes of aquatic products |
| CN104830841A (en) * | 2015-06-04 | 2015-08-12 | 北京中科紫鑫科技有限责任公司 | Production method of sequencing template |
| CN104928374A (en) * | 2015-06-04 | 2015-09-23 | 北京中科紫鑫科技有限责任公司 | EmPCR (emulsion PCR) method used in pyrosequencing |
| CN104946625A (en) * | 2015-06-04 | 2015-09-30 | 北京中科紫鑫科技有限责任公司 | Emulsion PCR-based sequencing template preparation method |
| EP3161157A4 (en) * | 2014-06-24 | 2017-12-20 | Bio-Rad Laboratories, Inc. | Digital pcr barcoding |
| CN110894526A (en) * | 2019-09-05 | 2020-03-20 | 中国环境科学研究院 | High-throughput sequencing method for benthonic animal COI gene and application thereof |
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| CN110894526A (en) * | 2019-09-05 | 2020-03-20 | 中国环境科学研究院 | High-throughput sequencing method for benthonic animal COI gene and application thereof |
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