CN108265111B - Reaction system for double-end sequencing, kit formed by reaction system and application of reaction system - Google Patents
Reaction system for double-end sequencing, kit formed by reaction system and application of reaction system Download PDFInfo
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Abstract
The invention provides a reaction system for double-end sequencing, a kit comprising the reaction system and application of the reaction system, and particularly relates to a reaction system for improving the double-end sequencing quality, a kit comprising the reaction system and a method for improving the double-end sequencing quality, wherein the reaction system comprises 1 x Phi29buffer, dNTP, Phi29 polymerase and a PCR enhancer. According to the invention, the combined PCR enhancer is added in a reaction system for generating the complementary strand, and the synergistic interaction effect is exerted through the mutual promotion among glycerol, DMSO, Triton X-100, trehalose and betaine, so that the complementary strand synthesis efficiency can be obviously improved, the sequencing accuracy is improved, and the average signal value of sequencing is improved by 1.1 times; meanwhile, a complementary strand generated by CTAB treatment is adopted, so that the exoactivity of the Phi29 enzyme is limited, and the Lag & Runon value is obviously reduced.
Description
Technical Field
The invention relates to the technical field of DNA sequencing, relates to a reaction system for double-end sequencing, a kit composed of the reaction system and application of the reaction system, and particularly relates to a reaction system for improving the quality of double-end sequencing, a kit composed of the reaction system and a method for improving the quality of double-end sequencing.
Background
Multiple Displacement Amplification (MDA) is the best single cell genome amplification technology recognized at present, can carry out high-fidelity uniform amplification on a whole genome, can amplify fragments with the size of 10-100 kb, and can provide a large amount of uniform and complete whole genome sequences. The MDA technology is widely used for whole genome amplification, and is a whole genome amplification method which has the widest whole genome coverage and the smallest amplification bias of each site at present. Currently, this method is also used in the field of high throughput sequencing.
The DNA nanoball (DNANOBLL) technology is that genomic DNA is firstly fragmented, then an adaptor sequence is added, and the genomic DNA is cyclized to form single-chain circular DNA, then the single-chain circular DNA is amplified for 2 to 3 orders of magnitude by using Rolling Circle Amplification (RCA), the generated amplification product is called DNB, and finally the nanoball is fixed on an arrayed silicon chip by DNB loading technology.
Rongqin Ke, et al, patent, US 20160237488A 1 discloses a method for sequencing the double ends of DNB by performing DNB complementary strand generation based on the MDA principle, wherein in the double-end sequencing of DNB, after one strand is sequenced, a complementary strand needs to be grown first, and then the complementary strand is sequenced, so that the influence of the good or bad length of the complementary strand on the sequencing quality is great. The existing method is to use the conventional MDA method to generate a complementary strand and then carry out sequencing, and the method can cause lower sequencing signals and poorer sequencing quality.
The current DNB complementary strand generation method has two defects: (1) the complementary strand sequencing signal is low; (2) the complementary strand sequencing indexes Lag and Runon (Lag means that base synthesis is lagged during sequencing, and Runon means that base synthesis is advanced during sequencing) are obviously higher, the sequencing quality is influenced, the sequencing signal of the complementary strand is provided, and the problem that the sequencing indexes Lag and Runon are reduced becomes an urgent need to be solved.
Disclosure of Invention
In order to solve the technical problems, the invention provides a reaction system for double-end sequencing, a kit consisting of the reaction system and application of the reaction system, and particularly relates to a reaction system for improving the double-end sequencing quality, a kit consisting of the reaction system and a method for improving the double-end sequencing quality.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a reaction system for double-ended sequencing, the reaction system comprising a1 x Phi29buffer, dntps, Phi29 polymerase and a PCR enhancer.
In the present invention, the dNTP and the Phi29 polymerase are conventional components in MDA technology in the prior art, and the dNTP and the Phi29 polymerase can adopt conventional concentrations in the prior art, and in the present invention, the molar concentration of the dNTP is 500mM, and the concentration of the Phi29 polymerase is 10U.
According to the invention, the PCR enhancer is any one or combination of at least two of glycerol, DMSO, Triton X-100, trehalose or betaine, preferably the combination of glycerol, DMSO, Triton X-100 and trehalose, or the combination of glycerol, DMSO, Triton X-100 and betaine, or the combination of glycerol, DMSO, Triton X-100, trehalose and betaine, and further preferably the combination of glycerol, DMSO, Triton X-100 and trehalose, or the combination of glycerol, DMSO, Triton X-100 and betaine.
Illustratively, the combination may be, for example, a combination of glycerol, DMSO, a combination of glycerol, Triton X-100, a combination of glycerol, DMSO, trehalose, a combination of glycerol, DMSO, betaine, a combination of glycerol, DMSO, Triton X-100, trehalose, a combination of glycerol, DMSO, Triton X-100, betaine, a combination of glycerol, DMSO, Triton X-100, trehalose, betaine.
According to the invention, through the combination of the PCR enhancers, various enhancers are mutually promoted, the synergistic interaction effect is exerted, and the complementary strand sequencing signal is greatly enhanced.
According to the present invention, the glycerol may account for 15-40% of the reaction system by volume fraction, for example, 15%, 16%, 18%, 20%, 22%, 23%, 25%, 26%, 28%, 30%, 31%, 32%, 33%, 35%, 36%, 38% or 40%, preferably 20-35%, and more preferably 30%, and the specific values therebetween are not exhaustive, and for the sake of brevity and conciseness, the present invention does not list the specific values included in the range.
According to the invention, the DMSO is dimethyl sulfoxide and may represent, for example, 5%, 6%, 7%, 8%, 9% or 10%, preferably 7% of the reaction system in volume fraction, and the specific values between the above values are not exhaustive for the sake of brevity and simplicity.
According to the invention, the Triton X-100 is polyethylene glycol octyl phenyl ether, and is 0.2 to 1% by volume of the reaction system, for example, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%, preferably 0.4 to 0.9%, and further preferably 0.8%, and the specific values between the above values are limited by space and for the sake of brevity, and the invention is not exhaustive of the specific values included in the range.
According to the invention, the betaine has a molar concentration of 0.1-0.6M, which may be, for example, 0.1M, 0.12M, 0.15M, 0.16M, 0.18M, 0.2M, 0.21M, 0.23M, 0.25M, 0.28M, 0.3M, 0.32M, 0.35M, 0.36M, 0.38M, 0.4M, 0.42M, 0.45M, 0.46M, 0.48M, 0.5M, 0.52M, 0.53M, 0.55M, 0.56M, 0.58M or 0.6M, preferably 0.2-0.6M, more preferably 0.5M, and the particular values between the above values are not intended to be exhaustive and for the sake of brevity, and the invention is not intended to include the particular values between the recited ranges.
According to the invention, the trehalose is present in a molar concentration of 0.02-0.2M, such as 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.11M, 0.12M, 0.13M, 0.14M, 0.15M, 0.16M, 0.17M, 0.18M, 0.19M or 0.2M, preferably 0.08-0.2M, more preferably 0.1M, and the values between these values are specific, and for reasons of brevity and clarity, no further recitation of the range of values included is intended.
In the present invention, the concentrations of the respective reagents are final concentrations at the time of use.
In a second aspect, the present invention provides a kit for double-ended sequencing, the kit comprising a reaction system according to the first aspect.
According to the invention, the kit further comprises CTAB.
As a preferred technical scheme, the kit comprises 1 XPhi 29buffer,500 mM dNTP,10U Phi29 polymerase, a PCR enhancer, an excision reagent, a blocking reagent, CTAB and deionized water.
In the present invention, the concentration of each reagent in the kit is the final concentration in use, and the concentration of each reagent in the kit can be selected by those skilled in the art according to the final concentration in use, and is not particularly limited herein.
Preferably, when the kit is used, the PCR enhancer is present in a final concentration of 15-40% by volume of glycerol, 5-10% by volume of DMSO, 0.2-1% by volume of Triton X-100 and 0.1-0.6M by mole, or 15-40% by volume of glycerol, 5-10% by volume of DMSO, 0.2-1% by volume of Triton X-100 and 0.02-0.2M by mole of trehalose, or 15-40% by volume of glycerol, 5-10% by volume of DMSO, 0.2-1% by volume of Triton X-100, 0.1-0.6M by mole of betaine and 0.02-0.2M by mole of trehalose, preferably 15-40% by volume of glycerol, 5-10% by volume of DMSO, 0.2-1% by volume of Triton X-100 by mole of betaine and 0.2-1% by mole of betaine and 0.1-0.6M by mole of betaine, preferably in a final concentration of 15-40% by volume of glycerol, 5-10% by volume of DMSO, 0.2-1% by volume of DMSO, 0.1-1% by mole of betaine and 0.6M by mole of betaine Or 15-40% by volume of glycerol, 5-10% by volume of DMSO, 0.2-1% by volume of Triton X-100 and trehalose at a molar concentration of 0.02-0.2M.
In the present invention, the cleavage reagent and the blocking reagent are not particularly limited, and any reagent capable of base cleavage and base blocking is available in the art, and may be added by a person skilled in the art according to the actual need. Specifically, the excision Reagent adopted by the invention is CPAS Regeneration Reagent (Regeneration Reagent) in BGISEQ-500RS high-throughput kit (PE50V2.0), and the blocking Reagent is CPAS blocking Reagent V3.0(Block Reagent V3.0) in BGISEQ-500RS high-throughput kit (PE50V 2.0). .
Preferably, the CTAB is cetyltrimethylammonium bromide, the molar concentration is 0.5-15mM, and may be, for example, 0.5mM, 0.6mM, 0.8mM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM or 15mM, preferably 1-10mM, and further preferably 1mM, and the specific points between the above values are limited to the space and for the sake of brevity, and the invention is not exhaustive of the specific points included in the range.
In a third aspect, the present invention provides a method of generating complementary strands in double-ended sequencing, comprising the steps of:
(1) loading the DNA nanospheres on a chip, and sequencing the 5' ends of the DNA nanospheres by using a reagent containing a sequencing primer;
(2) after sequencing of the 5' end is completed, cutting off the fluorescence and blocking group carried by the last base by using a cutting reagent, and washing by using an elution reagent;
(3) adding the reaction system as described in the first aspect to generate a complementary strand.
As known to those skilled in the art, before "loading DNA nanospheres on a chip", the DNA nanospheres need to be prepared, specifically, the DNA nanospheres are prepared by firstly fragmenting genomic DNA, adding a linker sequence, cyclizing to form a single-stranded circular DNA, and then amplifying the single-stranded circular DNA by 2-3 orders of magnitude using RCA, and those skilled in the art can prepare the DNA nanospheres according to actual needs by using techniques well known in the art.
In the present invention, any chip suitable for loading DNB nanospheres may be used, and those skilled in the art may select the chip according to actual needs, and the chip is not particularly limited herein, and specifically, a chip of Complete Genome sequencing platform or BGISEQ sequencing platform may be selected, such as: and a chip such as CL300002905, CL300002231, or CL 300002669.
In the present invention, the cleavage reagent is not particularly limited as long as it is a reagent capable of cleaving a base in the art, and the skilled person can select the cleavage reagent according to the actual need. Specifically, the excision Reagent adopted by the invention is CPAS Regeneration Reagent (Regeneration Reagent) in BGISEQ-500RS high-throughput kit (PE50V 2.0).
In the present invention, the elution reagent is a reagent capable of performing elution in the art, and the skilled person selects the elution reagent according to actual needs, and the elution reagent is not particularly limited herein. Specifically, the elution reagent adopted by the invention is from BGISEQ-500RS high-throughput kit PE50V 2.0.
Preferably, the nucleotide sequence of the sequencing primer in the step (1) is shown as SEQ ID No.1, and the nucleotide sequence shown as SEQ ID No.1 is as follows: 5'-GCTCACATCAGGCCATTAGGCTACGAGACTT-3' are provided.
According to the present invention, the complementary strand generation in step (3) specifically comprises the following steps:
and uniformly mixing the reaction system, adding the mixture to the surface of the chip, and generating a complementary strand at 35-37 ℃ for 30-40 min.
According to the invention, the reaction system comprises 1 XPhi 29buffer,500 mM dNTP in molar concentration, 10U Phi29 polymerase, 15-40% glycerol in volume fraction, 5-10% DMSO in volume fraction, 0.2-1% Triton X-100 in volume fraction, and 0.05-0.5M trehalose in molar concentration, or 1 XPhi 29buffer,500 mM dNTP in molar concentration, 10U Phi29 polymerase, 15-40% glycerol in volume fraction, 5-10% DMSO in volume fraction, 0.2-1% Triton X-100 in volume fraction, and 0.1-0.6M betaine in molar concentration, or 1 XPhi 29buffer,500 mM dNTP in molar concentration, 10U Phi29 polymerase, 15-40% glycerol in volume fraction, 5-10% DMSO in volume fraction, 0.2-1% Triton X-100 in molar concentration, 0.02-0.0.02M trehalose in molar concentration, preferably 1 XPHI 29buffer,500 mM dNTP molarity, 10U Phi29 polymerase, 15-40% glycerol volume fraction, 5-10% DMSO volume fraction, 0.2-1% Triton X-100 volume fraction and 0.1-0.6M betaine molarity, or 1 XPHI 29buffer,500 mM dNTP molarity, 10U Phi29 polymerase, 15-40% glycerol volume fraction, 5-10% DMSO volume fraction, 0.2-1% Triton X-100 volume fraction and 0.02-0.2M trehalose molarity.
According to the present invention, the glycerol may account for 15-40% of the reaction system by volume fraction, for example, 15%, 16%, 18%, 20%, 22%, 23%, 25%, 26%, 28%, 30%, 31%, 32%, 33%, 35%, 36%, 38% or 40%, preferably 20-35%, and more preferably 30%, and the specific values therebetween are not exhaustive, and for the sake of brevity and conciseness, the present invention does not list the specific values included in the range.
According to the invention, the DMSO is dimethyl sulfoxide and may represent, for example, 5%, 6%, 7%, 8%, 9% or 10%, preferably 7% of the reaction system in volume fraction, and the specific values between the above values are not exhaustive for the sake of brevity and simplicity.
According to the invention, the Triton X-100 is polyethylene glycol octyl phenyl ether, and is 0.2 to 1% by volume of the reaction system, for example, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%, preferably 0.4 to 0.9%, and further preferably 0.8%, and the specific values between the above values are limited by space and for the sake of brevity, and the invention is not exhaustive of the specific values included in the range.
According to the invention, the betaine has a molar concentration of 0.05-0.6M, such as 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.12M, 0.15M, 0.16M, 0.18M, 0.2M, 0.21M, 0.23M, 0.25M, 0.28M, 0.3M, 0.32M, 0.35M, 0.36M, 0.38M, 0.4M, 0.42M, 0.45M, 0.46M, 0.48M, 0.5M, 0.52M, 0.53M, 0.55M, 0.56M, 0.58M or 0.6M, preferably 0.2-0.6M, further preferably 0.5M, and the specific values therebetween are not included for brevity and the scope of the invention is not limited to the specific values.
According to the invention, the trehalose is present in a molar concentration of 0.02-0.2M, such as 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.11M, 0.12M, 0.13M, 0.14M, 0.15M, 0.16M, 0.17M, 0.18M, 0.19M or 0.2M, preferably 0.04-0.15M, more preferably 0.1M, and the values between these values are specific, and for reasons of brevity and clarity, no further recitation of the range of values is intended to be implied by the present invention.
In the present invention, the concentrations of the respective reagents are final concentrations at the time of use.
In a fourth aspect, the present invention provides a method of double-ended sequencing, comprising the steps of:
(1) performing the method of the third aspect, generating a complementary strand;
(2) adding a blocking reagent to carry out terminal blocking;
(3) then, a reagent containing a complementary strand sequencing primer is added to perform sequencing.
Preferably, the reagent comprising the complementary strand sequencing primer is a mixture comprising the complementary strand sequencing primer and CTAB.
Preferably, the nucleotide sequence of the complementary strand sequencing primer is shown as SEQ ID NO.2, and the nucleotide sequence shown as SEQ ID NO.2 is as follows: 5'-CCTAAGACCAAGCTAGGTCCGACTT-3' are provided.
Preferably, the complementary strand sequencing primer has a molar concentration of 3-6 μ M, such as 3 μ M, 3.2 μ M, 3.5 μ M, 3.8 μ M, 4 μ M, 4.2 μ M, 4.5 μ M, 4.8 μ M, 5 μ M, 5.2 μ M, 5.5 μ M, 5.8 μ M or 6 μ M, preferably 4 μ M, and the specific points between the above values are not exhaustive and are not intended to limit the scope of the invention to the specific points included in the range for brevity and clarity.
Preferably, the CTAB is cetyltrimethylammonium bromide, the molar concentration is 0.5-15mM, and may be, for example, 0.5mM, 0.6mM, 0.8mM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM or 15mM, preferably 1-10mM, and further preferably 1mM, and the specific points between the above values are limited to the space and for the sake of brevity, and the invention is not exhaustive of the specific points included in the range.
The inventors found that washing the complementary strand produced by the CTAB treatment can limit the exoactivity of the Phi29 enzyme and reduce the Lag & Runon value.
After the method is adopted to complete sequencing, the average signal value of four bases can reach 14637.79, compared with the average signal value of four bases 6978.96 in the prior art, the average signal value is improved by 1.1 times, and the Lag and Runon values are also obviously reduced.
In the present invention, the concentrations of the respective reagents are final concentrations at the time of use.
In a fifth aspect, the present invention provides use of the reaction system of the first aspect, the kit of the second aspect, the method of generating complementary strands in double-ended sequencing of the third aspect, or the method of double-ended sequencing of the fourth aspect in high-throughput sequencing.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) according to the invention, the combined PCR enhancer is added in a reaction system for generating the complementary strand, and the synergistic interaction effect is exerted through the mutual promotion among glycerol, DMSO, Triton X-100, trehalose and betaine, so that the complementary strand synthesis efficiency can be obviously improved, the sequencing accuracy is improved, and the average signal value of sequencing is improved by 1.1 times;
(2) according to the invention, the complementary strand generated by CTAB washing treatment is used, so that the exoactivity of phi29 enzyme is limited, and the Lag & Runon value is obviously reduced.
Drawings
FIG. 1 shows the Rho values obtained by performing first base sequencing of complementary strands after MDA is performed after PCR enhancers of different combinations are added to a reaction system according to the present invention;
FIG. 2 is a comparison of the LAg values obtained by adding CTAB to the complementary strand sequencing primer solution of the present invention for sequencing;
FIG. 3 is a comparison of Runon values obtained by adding CTAB to the complementary strand sequencing primer solution of the present invention for sequencing.
Detailed Description
For the purpose of facilitating an understanding of the present invention, the present invention will now be described by way of examples. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1: taking BGISEQ500 platform as an example, the effect of adding different PCR enhancers to the reaction system is shown
The method for sequencing double ends comprises the following steps:
(1) loading the DNA nanospheres on a chip according to the operation instruction of a BGISEQ-500 sequencer, and sequencing the 5' ends of the DNA nanospheres by using a single-ended sequencing primer (SEQ ID NO.1) and a matched reagent (BGISEQ-500RS high-throughput kit PE50V 2.0);
specifically, the nucleotide sequence of the sequencing primer SEQ ID NO.1 is as follows: 5'-GCTCACATCAGGCCATTAGGCTACGAGACTT-3', respectively;
(2) after 5' end sequencing is completed, a fluorescence and blocking group carried by the last base is cut off by using a cutting Reagent (CPAS Regeneration Reagent (from BGISEQ-500RS high-throughput kit PE50V2.0), and then the cut-off group and a Reagent remained in reaction are removed by using an elution Reagent (from BGISEQ-500RS high-throughput kit PE50V 2.0);
(3) respectively and uniformly mixing reaction systems of a control group and an experimental group, adding the reaction systems to the surface of a chip, generating a complementary strand at 35-37 ℃, wherein the generation time is 20min, and adding a blocking Reagent (CPAS blocking Reagent V3.0(Block Reagent V3.0) from BGISEQ-500RS high-throughput kit PE50V2.0) to perform terminal blocking after the complementary strand is generated;
(4) then adding a sequencing primer SEQ ID NO.2 and a matched reagent (BGISEQ-500RS high-throughput kit PE50V2.0) of the complementary strand for sequencing.
Specifically, the nucleotide sequence of the sequencing primer SEQ ID NO.2 is as follows: 5'-CCTAAGACCAAGCTAGGTCCGACTT-3', respectively;
the method is adopted in the subsequent concentration experiment of the PCR enhancer.
(I) Adding glycerol of different concentrations
The experimental conditions are as follows: the control group is the commonly used MDA reaction system conditions (1 XPhi 29buffer,500 MdNTP,10U Phi29 polymerase), the experimental group is that glycerol with different volume fractions is added on the basis of the control group, and the experimental results are shown in the following table 1.
TABLE 1
| Conditions of the experiment | Average signal value of four bases |
| Control (without glycerol) | 4800.74 |
| Adding 5% glycerol | 4859.32 |
| Adding 10% glycerol | 5300.12 |
| Adding 15% glycerol | 5608.71 |
| Adding 20% glycerol | 6590.92 |
| Adding 25% glycerol | 7005.66 |
| Adding 30% glycerol | 7299.23 |
| Adding 32.5% glycerol | 7200.55 |
| Adding 40% glycerol | 5903.37 |
As can be seen from Table 1, according to the BGISEQ500 platform sequencing results, better signals can be obtained by adding glycerol with the final concentration of 15-40%, and the glycerol with the best result is added with 30% for subsequent experiments.
(II) addition of different concentrations of DMSO
The experimental conditions are as follows: the control group was MDA reaction system conditions (1 XPhi 29buffer, 500. mu.M dNTP,10U Phi29 polymerase, 30% glycerol) added with 30% glycerol, the experimental group was DMSO added with different volume fractions based on the control group, and the experimental results are shown in the following Table 2.
TABLE 2
| Conditions of the experiment | Average signal value of four bases |
| Control (without DMSO) | 7206.52 |
| 2% DMSO was added | 7231.61 |
| Adding 5% DMSO | 7500.12 |
| Adding 7% DMSO | 8107.93 |
| Adding 10% DMSO | 8089.34 |
| Adding 15% DMSO | 6605.78 |
As can be seen from Table 2, according to the sequencing results of the BGISEQ500 platform, a better signal can be obtained by adding DMSO with a final concentration of 5-10%, and an optimal condition of 7% DMSO is selected for subsequent experiments.
(III) adding TritonX-100 with different concentrations
The experimental conditions are as follows: the control group was MDA reaction system conditions (1 XPhi 29buffer, 500. mu.M dNTP,10U Phi29 polymerase, 30% glycerol, 7% DMSO) with 30% glycerol, 7% DMSO, and the experimental group was TritonX-100 added in different volume fractions based on the control group, and the experimental results are shown in Table 3 below.
TABLE 3
As can be seen from Table 3, according to the BGISEQ500 platform sequencing results, a better signal can be obtained by adding Triton100 with the final concentration of 0.2-1%, and the optimal condition of 0.8% Triton100 is selected for subsequent experiments. .
(IV) adding betaine of different concentrations
The experimental conditions are as follows: the control group was MDA reaction system with 30% glycerol, 7% DMSO, and 0.8% Triton100 (1 XPhi 29buffer, 500. mu.M dNTP,10U Phi29 polymerase, 30% glycerol, 7% DMSO, and 0.8% Triton100), and the experimental group was betaine added at different molar concentrations based on the control group, and the experimental results are shown in Table 4 below.
TABLE 4
| Conditions of the experiment | Average signal value of four bases |
| Control (without betaine) | 8996.98 |
| Adding 0.05M betaine | 9000.22 |
| Adding 0.1M betaine | 9010.26 |
| Adding 0.2M betaine | 9276.33 |
| Adding 0.5M betaine | 9998.21 |
| Adding 0.8M betaine | 7800.31 |
As can be seen from Table 4, the BGISEQ500 platform sequencing results show that a better signal can be obtained by adding betaine with the final concentration of 0.1-0.5M, and the optimal condition of 0.5M betaine is selected.
(V) addition of trehalose at different concentrations
The experimental conditions are as follows: the control group was MDA reaction system with 30% glycerol, 7% DMSO, and 0.8% Triton100 (1 XPhi 29buffer, 500. mu.M dNTP,10U Phi29 polymerase, 30% glycerol, 7% DMSO, and 0.8% Triton100), and the experimental group was trehalose added to the control group at different molar concentrations, and the experimental results are shown in Table 5 below.
TABLE 5
| Conditions of the experiment | Average signal value of four bases |
| Control (without trehalose) | 8800.23 |
| Adding 0.02M trehalose | 9015.77 |
| Adding 0.04M trehalose | 9231.24 |
| Adding 0.08M trehalose | 9630.85 |
| Adding 0.1M trehalose | 10004.29 |
| Adding 0.2M trehalose | 9761.81 |
| Adding 0.4M trehalose | 8685.53 |
As can be seen from Table 5, the BGISEQ500 platform sequencing results show that a better signal can be obtained by adding trehalose at a final concentration of 0.04-0.2M, and the optimal condition of 0.1M trehalose is selected.
(VI) comparison of the additive combinations
The experimental conditions are as follows: the control group (condition 1, condition 5) was the commonly used MDA reaction system conditions (1 XPhi 29buffer, 500. mu.M dNTP,10U Phi29 polymerase), the experimental conditions of the experimental group (conditions 2-4, conditions 6-8) were as shown in Table 6 below, sequencing was performed for one cycle, and the experimental results were as shown in Table 7 below and in FIG. 1 below.
TABLE 6
TABLE 7
As can be seen from table 7 and fig. 1, the BGISEQ500 platform sequencing results show that the conditions 2, 3, 6, and 7 can obtain better signals, the average signal value of four bases can reach 14000 or more, and the conditions 4 and 8 can obtain next-best signals. It can be seen that the use of the combination of 0.5M betaine + 7% DMSO + 0.8% Triton100+ 30% glycerol, or the combination of 0.1M trehalose + 7% DMSO + 0.8% Triton100+ 30% glycerol, or the combination of 0.5M betaine +0.1M trehalose + 7% DMSO + 0.8% Triton100+ 30% glycerol can significantly improve the sequencing signal value, especially the combination of 0.5M betaine + 7% DMSO + 0.8% Triton100+ 30% glycerol, or the combination of 0.1M trehalose + 7% DMSO + 0.8% Triton100+ 30% glycerol can maximally improve the sequencing signal value.
Example 2: taking BGISEQ500 platform as an example, it is shown that adding 1mM CTAB into complementary strand sequencing primer solution can reduce sequencing Lag & Runon value.
The method for sequencing double ends comprises the following steps:
(1) loading the DNA nanospheres on a chip according to the operation instruction of a BGISEQ500 sequencer, and sequencing the 5' ends of the DNA nanospheres by using a single-ended sequencing primer (SEQ ID NO.1) and a matched reagent (BGISEQ-500RS high-throughput kit PE50V 2.0);
specifically, the nucleotide sequence of the sequencing primer SEQ ID NO.1 is as follows: 5'-GCTCACATCAGGCCATTAGGCTACGAGACTT-3', respectively;
(2) after 5' end sequencing is completed, a fluorescence and blocking group carried by the last base is cut off by using a cutting Reagent (CPAS Regeneration Reagent (from BGISEQ-500RS high-throughput kit PE50V2.0), and then the cut-off group and a Reagent remained in reaction are removed by using an elution Reagent (from BGISEQ-500RS high-throughput kit PE50V 2.0);
(3) uniformly mixing a reaction system, adding the reaction system to the surface of a chip, generating a complementary strand at 35-37 ℃, wherein the generation time is 20min, and adding a blocking Reagent (CPAS blocking Reagent V3.0(Block Reagent V3.0) from BGISEQ-500RS high-throughput kit PE50V2.0) to perform end blocking after the complementary strand is generated;
wherein the reaction system is as follows: 1 XPhi 29buffer, dNTP, Phi29 polymerase, 15-40% glycerol, 5-10% DMSO, 0.2-1% Triton X-100, 0.1-0.6M betaine;
(4) then adding a mixture of a sequencing primer SEQ ID NO.2 of a complementary chain and a matched reagent (BGISEQ-500RS high-throughput kit PE50V2.0) for sequencing.
Specifically, the nucleotide sequence of the sequencing primer SEQ ID NO.2 is as follows: 5'-CCTAAGACCAAGCTAGGTCCGACTT-3', respectively;
the experimental conditions are as follows: CTAB was not added to the complementary strand sequencing primers in the control group, 1mM CTAB, 5mM CTAB, and 10mM CTAB were added to the complementary strand sequencing primers in the experimental group, the specific components of the mixture are shown in Table 8 below, and the experimental results are shown in Table 9 and FIGS. 2 to 3.
TABLE 8
| Reagent composition | Final concentration |
| Complementary strand sequencing primer | 4μM |
| SSC (sodium chloride-sodium citrate solution) | 5× |
| CTAB | 1mM,5mM,10mM |
TABLE 9
As can be seen from FIGS. 2 to 3 and Table 9, 1mM, 5mM and 10mM CTAB were able to lower the Lang & Runon values, and 1mM CTAB was more effective in that it was considered that CTAB was less soluble at low temperature and was likely to be crystallized out.
In conclusion, the combined PCR enhancer is added into a reaction system for generating a complementary strand, and the synergistic interaction effect is exerted through the mutual promotion among glycerol, DMSO, Triton X-100, trehalose and betaine, and the best effect is achieved by adopting the combination of 0.5M betaine, 7% DMSO, 0.8% Triton100 and 30% glycerol or the combination of 0.1M trehalose, 7% DMSO, 0.8% Triton100 and 30% glycerol, so that the complementary strand synthesis efficiency is obviously improved, the sequencing accuracy is improved, and the average signal value of sequencing is improved by 1.1 times; according to the invention, the complementary strand generated by CTAB washing treatment is used, so that the exoactivity of phi29 enzyme is limited, and the Lag & Runon value is obviously reduced.
The applicant states that the present invention is illustrated by the above examples to show the detailed process equipment and process flow of the present invention, but the present invention is not limited to the above detailed process equipment and process flow, i.e. it does not mean that the present invention must rely on the above detailed process equipment and process flow to be implemented. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (12)
1. A reaction system for double-ended sequencing, wherein the reaction system comprises 1 x Phi29buffer, dntps, Phi29 polymerase and PCR enhancers;
the PCR enhancer is a combination of glycerol, DMSO, Triton X-100 and trehalose, or a combination of glycerol, DMSO, Triton X-100 and betaine;
the glycerol accounts for 30% of the reaction system in volume fraction;
the DMSO accounts for 7% of the reaction system in volume fraction;
the Triton X-100 accounts for 0.8% of the reaction system in volume fraction;
the molar concentration of the betaine is 0.1-0.6M;
the molar concentration of the trehalose is 0.02-0.2M;
the reaction system further comprises CTAB;
the molar concentration of CTAB is 1-5 mM.
2. The reaction system for double-ended sequencing according to claim 1,
the molar concentration of the betaine is 0.2-0.6M;
the molar concentration of the trehalose is 0.08-0.2M.
3. The reaction system for double-ended sequencing according to claim 2,
the molar concentration of the betaine is 0.5M;
the molar concentration of the trehalose is 0.1M;
the molar concentration of CTAB is 1 mM.
4. A kit for double-ended sequencing, comprising the reaction system of any one of claims 1-3.
5. A method of generating complementary strands in double-ended sequencing, comprising the steps of:
(1) loading the DNA nanospheres on a chip, and sequencing the 5' ends of the DNA nanospheres by using a reagent containing a sequencing primer;
(2) after sequencing of the 5' end is completed, cutting off the fluorescence and blocking group carried by the last base by using a cutting reagent, and washing by using an elution reagent;
(3) adding the reaction system of any one of claims 1-3 to produce a complementary strand.
6. The method of claim 5, wherein the nucleotide sequence of the sequencing primer of step (1) is shown as SEQ ID No. 1;
the complementary strand generation in the step (3) specifically comprises the following steps:
uniformly mixing the reaction system, adding the mixture to the surface of a chip, and generating a complementary strand at 35-37 ℃ for 30-40 min;
the reaction system comprises: 1 XPhi 29buffer,500 mM dNTP molarity, 10U Phi29 polymerase, 30% volume fraction glycerol, 7% volume fraction DMSO, 0.8% volume fraction Triton X-100 and 0.1-0.6M molarity betaine, or 1 XPhi 29buffer,500 mM dNTP molarity, 10U Phi29 polymerase, 30% volume fraction glycerol, 7% volume fraction DMSO, 0.8% volume fraction Triton X-100 and 0.02-0.2M molarity trehalose.
7. The method of claim 6,
the molar concentration of the betaine is 0.2-0.6M;
the molar concentration of the trehalose is 0.04-0.15M.
8. The method of claim 7,
the molar concentration of the betaine is 0.5M;
the molar concentration of trehalose is 0.1M.
9. A method of double-ended sequencing comprising the steps of:
(1) performing the method of any one of claims 5-8, generating a complementary strand;
(2) adding a blocking reagent to carry out terminal blocking;
(3) then, a reagent containing a complementary strand sequencing primer is added to perform sequencing.
10. The method of claim 9, wherein the reagent comprising the complementary strand sequencing primer is a mixture comprising the complementary strand sequencing primer and CTAB;
the nucleotide sequence of the complementary strand sequencing primer is shown as SEQ ID NO. 2;
the molar concentration of the complementary strand sequencing primer is 3-6 mu M;
the molar concentration of CTAB is 1-5 mM.
11. The method of claim 10,
the molar concentration of the complementary strand sequencing primer is 4 mu M;
the molar concentration of CTAB is 1 mM.
12. Use of the reaction system of any one of claims 1-3, the kit of claim 4, the method of generating complementary strands in double-ended sequencing of any one of claims 5-8, or the method of double-ended sequencing of any one of claims 9-11 in high-throughput sequencing.
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