CN111286527B - Method for synthesizing complementary strand of DNA nanosphere and sequencing method - Google Patents
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Abstract
A method of synthesizing a complementary strand of a DNA nanosphere and a sequencing method, wherein the method of synthesizing a complementary strand of a DNA nanosphere comprises: treating a product of one-strand sequencing of the DNA nanospheres with an enzyme, wherein the primer of one-strand sequencing has a modification, and after the enzyme treatment, the modification is destroyed by the enzyme so that the primer is digested; hybridizing to the product of the previous step using a multiplex displacement amplification primer, wherein the multiplex displacement amplification primer comprises at least a first primer that is fully or partially complementary to a primer binding region of a strand sequencing; and under the action of polymerase with a strand displacement function, performing a strand displacement reaction by using the multiplex displacement amplification primer and a strand as primers to generate a complementary strand of the DNA nanosphere. The method of the present invention removes the occupied one-stranded primer prior to MDA, freeing the site to hybridize the MDA primer, thereby increasing the amount of hybridized primer and thus the DNB copy number.
Description
Technical Field
The invention relates to the technical field of sequencing, in particular to a method for synthesizing a complementary strand of a DNA nanosphere and a sequencing method.
Background
Multiple Displacement Amplification (MDA) technology is widely used for whole genome amplification, and is a whole genome amplification method with the widest coverage of the whole genome and the smallest amplification bias at each position. At present, the method is also used in the field of high-throughput sequencing, and DNA Nanosphere (DNB) complementary strand generation is performed based on the MDA principle (Rongqin Ke, et al, US 20160237488A 1) to realize the sequencing of the two ends of the DNB.
In the case of DNB, after the first strand is sequenced, its complementary strand needs to be generated and then sequenced. Thus, the quality of sequencing is greatly affected by the quality of complementary strand production. As shown in FIG. 1, the existing method uses a strand of sequencing strand and a newly hybridized tag (Barcode) primer to generate complementary strand, and after a strand is sequenced by long cycle, one strand of sequencing primer occupies a position, so that only one strand of MDA primer (namely Barcode primer) can be fully hybridized to a joint, the other strand of primer is difficult to hybridize, only two strands of primer (namely Barcode primer and one strand of sequencing strand) are amplified when MDA is performed, and the replacement efficiency of the sequencing strand primer is low, which affects DNB copy number, so that the sequencing signal is low. In addition, after a strand is sequenced through long cycles (cycles), some sequencing enzymes bind to the sequencing strand and residual scars (scars) after cleavage of fluorescent blocked dNTPs affect the displacement effect of Phi29DNA polymerase, thereby affecting the copy number of DNB, making the fluorescent signal lower, and also affecting the sequencing quality.
Disclosure of Invention
The invention provides a method for synthesizing complementary strand of DNA nanospheres and a sequencing method, wherein before MDA, a occupying one-strand primer is removed, and the MDA primer hybridization is carried out by leaving a position, so that the amount of hybridized primer is increased, and the DNB copy number is increased.
According to a first aspect, there is provided in one embodiment a method of synthesizing complementary strands of a DNA nanosphere, comprising:
treating a product of one-strand sequencing of a DNA nanosphere with an enzyme, wherein said one-strand sequenced primer has a modification thereon, wherein said modification is destroyed by said enzyme after said enzyme treatment such that said primer is digested;
hybridizing to the product of the previous step using a multiplex displacement amplification primer, wherein the multiplex displacement amplification primer comprises at least a first primer that is wholly or partially complementary to the primer binding region of the one strand sequencing; and
and (3) under the action of polymerase with a strand displacement function, performing a strand displacement reaction by using the multiple displacement amplification primer and the one strand as primers to generate complementary strands of the DNA nanospheres.
In a preferred embodiment, the multiplex displacement amplification primer comprises the first primer and a second primer, wherein the second primer has a tag sequence between the binding region on the DNA nanosphere and the binding region of the first primer, the tag sequence being used to distinguish between different sample sources and/or molecular sources of the DNA nanosphere.
In a preferred embodiment, the one-strand sequencing primer is modified with uracil bases, and the enzyme is a USER enzyme.
In a preferred embodiment, after the uracil base is digested by the USER enzyme, the product after the USER enzyme treatment is denatured and washed to remove the cleaved primer.
In a preferred embodiment, the number of uracil bases on the primer for one strand sequencing is equal to or less than the number of adenine bases of the template region to which it is bound, preferably equal to the number of adenine bases of the template region to which it is bound.
In a preferred embodiment, the distance between two adjacent uracil bases on the one-strand sequenced primer is 1 to 20 bases, preferably 5-20 bases.
In a preferred embodiment, the denaturation treatment is thermal denaturation; preferably, the above thermal denaturation temperature is 50 ℃ to 70 ℃, preferably 55 ℃ to 60 ℃.
In a preferred embodiment, the polymerase having a strand displacement function is Phi29DNA polymerase.
According to a second aspect, in one embodiment there is provided a complementary strand of a DNA nanosphere synthesized by the method of the first aspect or a complex comprising the complementary strand and the DNA nanosphere.
According to a third aspect, there is provided in one embodiment a sequencing method comprising:
generating complementary strands of the DNA nanospheres by the method of the first aspect; and
hybridization is performed using a sequencing primer that hybridizes to the complementary strand to sequence the complementary strand.
According to a fourth aspect, there is provided in one embodiment a method of sequencing comprising:
loading the DNA nanospheres to be sequenced onto a sequencing chip;
hybridizing a primer for sequencing a strand with the DNA nanospheres, and performing sequencing a strand, wherein the primer for sequencing a strand is modified;
treating the one-strand sequenced product with an enzyme such that the modification is disrupted by the enzyme such that the one-strand sequenced primer is digested;
hybridizing to the product of the previous step using a multiplex displacement amplification primer, wherein the multiplex displacement amplification primer comprises at least a first primer that is wholly or partially complementary to the primer binding region of the one strand sequencing;
under the action of polymerase with a strand displacement function, carrying out a strand displacement reaction by using the multiple displacement amplification primer and the one strand as primers to generate a complementary strand of the DNA nanosphere; and
hybridization is performed using a sequencing primer that hybridizes to the complementary strand to sequence the complementary strand.
In a preferred embodiment, the one-strand sequencing primer is modified with uracil bases, and the enzyme is a USER enzyme.
According to a fifth aspect, there is provided in an embodiment a method of improving the quality of double ended sequencing comprising: after one-strand sequencing of the DNA nanospheres, the one-strand sequenced product of the DNA nanospheres is treated with an enzyme, wherein the one-strand sequenced primer has a modification, and wherein after the enzyme treatment the modification is destroyed by the enzyme such that the primer is digested.
In a preferred embodiment, the one-strand sequencing primer is modified with uracil bases, and the enzyme is a USER enzyme.
According to the method, under the condition that the quality of one-strand sequencing is not affected, one-strand sequencing primer is subjected to modification (for example, uracil base modification), enzyme (for example, USER enzyme) treatment is used for removing the modification (for example, uracil base modification) after one-strand sequencing is finished, and MDA reaction is carried out to achieve the purpose of increasing DNB copy number, so that the complementary strand sequencing signal and quality are improved.
Drawings
FIG. 1 is a schematic diagram showing the principle of complementary strand synthesis by MDA after DNB one-strand sequencing is completed in the prior art;
FIG. 2 is a schematic diagram showing the principle of complementary strand synthesis by MDA after DNB one-strand sequencing is completed in the embodiment of the present invention;
FIG. 3 is a schematic diagram showing a flow chart of DNB one-strand sequencing and complementary strand sequencing in the embodiment of the present invention;
FIG. 4 is a comparison of signal values (RHO) of four bases for PE100 sequencing in two different methods according to the invention and prior art;
FIG. 5 is a comparison of the quality value (Q30) per base for PE100 sequencing by two different methods of the invention and prior art.
Detailed Description
The invention will be described in further detail below with reference to the drawings by means of specific embodiments. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present invention. However, one skilled in the art will readily recognize that some of the features may be omitted in various situations, or replaced by other materials, methods.
Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.
The numbering of the components itself, e.g. "first", "second", etc., is used herein merely to distinguish between the described objects and does not have any sequential or technical meaning.
As shown in FIG. 2, one embodiment of the present invention provides a method for synthesizing complementary strands of a DNA nanosphere by removing one strand of primers occupying a site on the DNA nanosphere prior to Multiple Displacement Amplification (MDA) and freeing the site for MDA primer hybridization, thereby increasing the amount of hybridized primers and thus increasing the DNA nanosphere copy number.
In the embodiment of the invention, a method for synthesizing complementary strands of DNA nanospheres comprises the following steps:
treating a product of one-strand sequencing of a DNA nanosphere with an enzyme, wherein said one-strand sequenced primer has a modification thereon, wherein said modification is destroyed by said enzyme after said enzyme treatment such that said primer is digested;
hybridizing to the product of the previous step using a multiplex displacement amplification primer, wherein the multiplex displacement amplification primer comprises at least a first primer that is wholly or partially complementary to the primer binding region of the one strand sequencing; and
and (3) under the action of polymerase with a strand displacement function, performing a strand displacement reaction by using the multiple displacement amplification primer and the one strand as primers to generate complementary strands of the DNA nanospheres.
In the present invention, there are various modifications and corresponding enzyme treatments that achieve the goal of digesting a primer for sequencing a strand, and in one embodiment of the present invention the enzyme is the USER enzyme and the modifications are uracil base modifications, i.e., a primer for sequencing a strand contains uracil bases.
Thus, the method of one embodiment of the present invention comprises:
treating a product of one-strand sequencing of the DNA nanospheres by using a USER enzyme, wherein a uracil base is carried on a primer of one-strand sequencing, and after the USER enzyme treatment, the uracil base is excised to generate a notch so as to break the primer of one-strand sequencing;
carrying out denaturation treatment on the product treated by the USER enzyme, and washing to remove broken primers;
hybridizing to the product of the previous step using a multiplex displacement amplification primer, wherein the multiplex displacement amplification primer comprises at least a first primer that is fully or partially complementary to a primer binding region of a strand sequencing; and
under the action of polymerase with strand displacement function, the complementary strand of the DNA nanosphere is generated by strand displacement reaction with multiple displacement amplification primers and a strand as primers.
In the examples of the present invention, the USER enzyme refers to uracil-specific excision reagents capable of creating a single nucleotide gap at the uracil position. The USER enzyme is a mixture of Uracil DNA Glycosylase (UDG) and DNA glycosylase-lyase Endo VIII. UDG catalyzes the cleavage of uracil bases to form an abasic (apyrimidinic) site, but maintains the integrity of the phosphodiester backbone structure. The cleaving enzyme activity of Endo VIII breaks the phosphodiester bonds at the 3 'and 5' ends of the abasic site, releasing abasic deoxyribose. The Endo VIII and uracil DNA glycosylase can be purified from E.coli K-12 strain, respectively, to make the USER enzyme. The strain contains genes encoding these two enzymes on its plasmid. The reaction conditions for the USER enzyme are typically CutSmart reaction buffers, which are typically incubated at 37 ℃.
In the embodiment of the invention, the DNA nanospheres are generally multi-copy tandem nucleic acid molecules obtained by rolling circle amplification of circular DNA as a sequencing template, and can be prepared according to a method commonly used in the field, for example, the DNA nanospheres can be generated according to the method invented by Drmanac et al. Such DNA nanospheres are commonly used for sequencing on some sequencing platforms, e.g., MGI, BGI sequencing platforms of the typical hua major gene company, etc. The DNA nanospheres are loaded on a sequencing chip by a suitable loading method, i.e. can be sequenced on-machine. The sequencing chip is fabricated from a variety of suitable substrates for nucleic acid immobilization, such as glass, ceramic, silica, silicon, metal, silicone elastomer, and the like.
In the embodiment of the invention, the "one-strand" is also called as a "first strand" and refers to a synthesized sequence obtained in the process of sequencing while synthesizing under the guidance of a one-strand sequencing primer by taking a DNA nanosphere as a template. Unlike the prior art, the primers of the present invention have uracil (U) bases on them, which are typically thymine (T) bases, which can be cleaved by the USER enzyme resulting in cleavage of the primer of the one-strand sequence and further removal by denaturation.
In embodiments of the invention, the number of uracil bases on a primer for one-strand sequencing is generally equal to or less than the number of adenine bases in the template region to which it is attached, i.e., all or part of the thymine (T) base sites on a primer for one-strand sequencing in the prior art are replaced with uracil (U) bases. In the embodiments of the present invention, there are a minimum of 1 uracil (U) base on a primer for one-strand sequencing, and in the preferred embodiment, the number of uracil bases on a primer for one-strand sequencing is equal to the number of adenine bases in the template region to which it is bound, i.e., all thymine (T) base sites on a primer for one-strand sequencing in the prior art are replaced with uracil (U) bases. The shorter the number of uracil (U) bases, the more advantageous the primer sequenced in a strand will be broken into fragments by USER cleavage. In general, the distance between adjacent two uracil bases on a strand sequenced primer is 1 to 20 bases, preferably 5-20 bases. The shorter the distance between two adjacent uracil bases is, the more advantageous is that uracil bases are uniformly distributed on a primer for sequencing a chain as much as possible, so that the sizes of fragments broken by enzyme digestion of the USER are basically equivalent, removal by denaturation is facilitated, and the removal efficiency is ensured.
Since a primer for sequencing a strand is designed based on the linker sequence on the DNA nanosphere, the primer is at least partially complementary to the linker sequence, the number and distribution of uracil (U) bases on the primer is also primarily determined by the linker sequence.
In embodiments of the present invention, the denaturation step is generally thermal denaturation, although other suitable denaturation methods, such as alkali denaturation, are not excluded. The thermal denaturation temperature can be determined based on the annealing temperature of the primer for one strand sequencing, and the annealing temperature of the fragments after cleavage (depending on the fragment length), and generally, the thermal denaturation temperature is 50℃to 70℃and preferably 55℃to 60 ℃. The thermal denaturation temperature can enable fragments of the one-strand sequencing primer after being broken to be released from the DNA nanospheres in a unwinding mode, and other parts of the one-strand are not denatured and unwound, so that the primer is favorable for being used as a primer for Multiple Displacement Amplification (MDA).
As shown in FIG. 2, the primers used in the multiplex displacement amplification (i.e., MDA primers) include a first primer (short insert primer) that is fully or partially complementary to the primer binding region of a strand of sequencing, i.e., after the strand of sequencing is removed, its occupied position is left empty and the MDA primers hybridize, thus increasing the amount of hybridized primers and thus increasing DNB copy number. In the prior art, MDA primers cannot hybridize and bind because the primer for one-strand sequencing is not removed. In embodiments of the invention, the MDA primer further comprises a second primer (Barcode primer) having a tag sequence between the binding region on the DNA nanosphere and the binding region of the first primer, the tag sequence being used to distinguish between different sample sources and/or molecular sources of the DNA nanosphere. For DNA nanosphere sequencing techniques, the adaptor comprises three parts, namely a region that binds to the first primer, a tag sequence, and a region that binds to the second primer, wherein the complementary pairing relationship of the first primer and/or the second primer to the adaptor may be wholly or partially complementary. The tag sequence (i.e. the Barcode sequence) may be of any suitable length, for example a sequence of 6 to 16 bases (preferably 10 bases), may be a random sequence, with 4 selectable bases at each base position, such tag sequences being able to distinguish between sample sources or molecular sources, so-called "sample sources" for example from different individuals of the same or different species, so-called "molecular sources" for example from different nucleic acid regions of the same sample, etc.
In the embodiment of the present invention, the polymerase having a strand displacement function for multiplex displacement amplification may be any suitable enzyme having such a function, for example, a polymerase having a strand displacement ability such as Phi29DNA polymerase, bst polymerase, etc. In the multiple displacement amplification process, the first primer, the second primer and a strand bound on the DNA nanospheres are used as primers for guiding the amplification, so that the DNB copy number is increased, and the complementary strand sequencing signal and quality are improved.
In a preferred embodiment of the present invention, the above method further comprises: the fluorescent and/or blocking groups carried by the last base after sequencing of the DNA nanosphere strand are excised using an excision reagent. These fluorophores are signal groups in one-strand sequencing whose presence can interfere with the fluorescent signal in complementary strand sequencing; a blocking group is a group that blocks the continuation of sequencing, the presence of which makes this strand difficult as a primer for guiding extension in complementary strand synthesis. The use of a cleavage reagent to cleave these fluorescent groups and blocking groups can avoid the above-described problems. .
In a preferred embodiment of the present invention, after the complementary strand of the DNA nanospheres is generated, it is also necessary to block the ends of the complementary strand with a blocking reagent.
By the method for synthesizing a complementary strand of a DNA nanosphere of the present invention, a complementary strand useful for sequencing can be formed on the DNA nanosphere, and therefore, the complementary strand of the DNA nanosphere or a complex comprising the complementary strand and the DNA nanosphere is also within the scope of the present invention.
Another embodiment of the present invention provides a sequencing method comprising: the complementary strand of the DNA nanosphere is generated by the method for synthesizing the complementary strand of the DNA nanosphere; and hybridizing using the sequencing primer hybridized to the complementary strand to sequence the complementary strand.
As shown in fig. 3, another embodiment of the present invention provides a sequencing method comprising:
loading the DNA nanospheres to be sequenced onto a sequencing chip;
hybridizing a primer for sequencing a strand with the DNA nanospheres, and performing sequencing a strand, wherein the primer for sequencing a strand is modified;
treating the one-strand sequenced product with an enzyme such that the modification is disrupted by the enzyme such that the one-strand sequenced primer is digested;
hybridizing to the product of the previous step using a multiplex displacement amplification primer, wherein the multiplex displacement amplification primer comprises at least a first primer that is wholly or partially complementary to the primer binding region of the one strand sequencing;
under the action of polymerase with a strand displacement function, carrying out a strand displacement reaction by using the multiple displacement amplification primer and the one strand as primers to generate a complementary strand of the DNA nanosphere; and
hybridization is performed using a sequencing primer that hybridizes to the complementary strand to sequence the complementary strand.
In a preferred embodiment, the one-strand sequencing primer is modified with uracil bases, and the enzyme is a USER enzyme.
Another embodiment of the invention provides a method of improving the quality of double ended sequencing comprising: after one-strand sequencing of the DNA nanospheres, the one-strand sequenced product of the DNA nanospheres is treated with an enzyme, wherein the one-strand sequenced primer has a modification, and wherein after the enzyme treatment the modification is destroyed by the enzyme such that the primer is digested.
In a preferred embodiment, the one-strand sequencing primer is modified with uracil bases, and the enzyme is a USER enzyme.
According to the method, under the condition that the quality of one-strand sequencing is not affected, uracil base modification treatment is carried out on one-strand sequencing primer, after one-strand sequencing is finished, uracil base is removed by using USER enzyme treatment, and MDA reaction is carried out to achieve the purpose of increasing DNB copy number, so that the signal and quality of complementary strand sequencing are improved.
The following detailed description of the present invention is provided by way of example only, and should not be construed as limiting the scope of the invention.
Examples
Primers and reagents used in the examples:
a strand sequencing primer:
CAACTCCUTGGCUCACAGAACGACAUGGCTACGAUCCGACTT(SEQ ID NO：1)；
MDA primer 1: AAGTCGGAGGCCAAGCGGTCTTAGAAGACAA (SEQ ID NO: 2);
MDA primer 2: CAACTCCTTGGCTCACAGAACGACATGGCT (SEQ ID NO: 3);
USER enzyme (NEB Co., USA, cat# M5505L);
BGISEQ-500DNB preparation and loading kit (Huada gene company, cat# 85-05531-00);
BGISEQ-500 sequencing kit (PE 100) (Huada Gene Co.);
BGISEQ-500 chip (Huada gene).
Taking BGISEQ-500 platform as an example, the PE100 sequencing effect of the invention is shown:
(1) A one-strand sequencing primer with U base modification was synthesized and diluted with a 5 XSSC solution to a working solution concentration of 1. Mu.M, and the system was as shown in Table 1 below:
TABLE 1
(2) DNB is prepared by using BGISEQ-500DNB preparation loading kit.
(3) DNB was loaded onto the surface of BGISEQ-500 chip and a U base modified one-strand sequencing primer was hybridized to DNB.
(4) One strand of SE100 sequencing was performed on BGISEQ-500 according to the instructions of the BGISEQ-500 sequencing kit (PE 100).
(5) After completion of one strand sequencing, cleavage was still performed on BGISEQ-500, and the fluorescence and blocking groups of the last cycle were excised.
(6) On a BGISEQ-500 sequencer, 0.1U of USER enzyme was pumped in at 37℃to cleave the U-base modified one-strand sequencing primer for 10min.
(7) Heating to 55deg.C, pumping WB1 cleaning solution (component in BGISEQ-500 sequencing kit) for 3 times, and washing out one-strand sequencing primer and USER enzyme.
(8) The MDA primer 1 and the MDA primer 2 were hybridized, and MDA reaction was performed according to a conventional method.
(9) The synthesized complementary strand was sequenced for 100 cycles.
(10) The results of the machine-down data are shown in table 2 below. The overall Q30 and ESR improvement of PE100 is shown to be about 2-4% (Table 2).
As shown in fig. 4 and 5, one strand sequencing was performed using one strand sequencing primer (test set) with U base modification and one strand sequencing primer (control set) without U base modification, with no effect on one strand sequencing quality; q30 in the test group is improved by about 3-4% compared with the complementary strand sequencing quality of the control group (figure 5), and is obviously improved compared with the control group; the complementary strand back rise signal value in the test group was about 30% higher than that in the control group (fig. 4).
TABLE 2 PE100 sequencing results performed under two different conditions
The foregoing description of the invention has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the invention pertains, based on the idea of the invention.
SEQUENCE LISTING
<110> Shenzhen Hua Dazhi manufactured technology Co., ltd
<120> method for synthesizing complementary strand of DNA nanosphere and sequencing method
<130> 18I27304
<160> 3
<170> PatentIn version 3.3
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<211> 42
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<210> 3
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Claims (12)
1. A method of synthesizing complementary strands of a DNA nanosphere, the method comprising:
treating a product of one-strand sequencing of a DNA nanosphere with an enzyme, wherein the primer of one-strand sequencing has a modification, the modification of the primer of one-strand sequencing is uracil base modification, the primer is digested after the enzyme treatment, the enzyme is a USER enzyme, the product after the USER enzyme treatment is denatured after the uracil base is digested by the USER enzyme, and the digested primer is washed away, the denaturation treatment is thermal denaturation;
hybridizing to the product of the previous step using a multiplex displacement amplification primer, wherein the multiplex displacement amplification primer comprises at least a first primer that is wholly or partially complementary to the primer binding region of the one strand sequencing; and
and under the action of polymerase with a strand displacement function, carrying out a strand displacement reaction by taking the multiple displacement amplification primer and the one strand as primers to generate the complementary strand of the DNA nanosphere.
2. The method of claim 1, wherein the multiplex displacement amplification primer comprises the first primer and a second primer, wherein the second primer has a tag sequence between a binding region on the DNA nanosphere and a binding region of the first primer, the tag sequence being used to distinguish between different sample sources and/or molecular sources of the DNA nanosphere.
3. The method of claim 1, wherein the number of uracil bases on the one-stranded sequenced primer is equal to or less than the number of adenine bases of the template region to which it binds.
4. The method of claim 3, wherein the number of uracil bases on the primer for one strand sequencing is equal to the number of adenine bases of the template region to which it binds.
5. The method of claim 1, wherein the distance between two adjacent uracil bases on the primer for one strand sequencing is 1 to 20 bases.
6. The method of claim 5, wherein the distance between two adjacent uracil bases on the primer for one strand sequencing is 5-20 bases.
7. The method of claim 1, wherein the thermal denaturation temperature is 50 ℃ to 70 ℃.
8. The method of claim 7, wherein the thermal denaturation temperature is 55 ℃ to 60 ℃.
9. The method of claim 1, wherein the polymerase having a strand displacement function is Phi29DNA polymerase.
10. A method of sequencing, the method comprising:
generating complementary strands of the DNA nanospheres by the method of any one of claims 1 to 9; and
hybridization is performed using sequencing primers that hybridize to the complementary strands to sequence the complementary strands.
11. A method of sequencing, the method comprising:
loading the DNA nanospheres to be sequenced onto a sequencing chip;
hybridizing a primer for one-strand sequencing with the DNA nanospheres, and performing one-strand sequencing, wherein the primer for one-strand sequencing is provided with a modification, and the modification of the primer for one-strand sequencing is uracil base modification;
treating the product after the one-strand sequencing by using enzyme, wherein the primer after the one-strand sequencing is digested, the enzyme is USER enzyme, and after the uracil base is digested by the USER enzyme, carrying out denaturation treatment on the product after the USER enzyme treatment, and washing to remove the digested primer, wherein the denaturation treatment is thermal denaturation;
hybridizing to the product of the previous step using a multiplex displacement amplification primer, wherein the multiplex displacement amplification primer comprises at least a first primer that is wholly or partially complementary to the primer binding region of the one strand sequencing;
under the action of polymerase with a strand displacement function, carrying out a strand displacement reaction by taking the multiple displacement amplification primer and the one strand as primers to generate a complementary strand of the DNA nanosphere; and
hybridization is performed using sequencing primers that hybridize to the complementary strands to sequence the complementary strands.
12. A method of improving the quality of double ended sequencing, the method comprising: after one-strand sequencing of the DNA nanospheres, treating the one-strand sequenced product of the DNA nanospheres with an enzyme, wherein the one-strand sequenced primer has a modification, the primer is digested after the enzyme treatment, the modification of the one-strand sequenced primer is uracil base modification, the enzyme is a USER enzyme, the uracil base is digested by the USER enzyme, the USER enzyme treated product is denatured, and the digested primer is rinsed and removed, wherein the denaturation treatment is thermal denaturation.
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| CN111910258B (en) * | 2020-08-19 | 2021-06-15 | 纳昂达(南京)生物科技有限公司 | Paired-end library tag composition and application thereof in MGI sequencing platform |
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| CN108265111A (en) * | 2016-12-29 | 2018-07-10 | 深圳华大智造科技有限公司 | A kind of reaction system for being used for double end sequencings, kit of its composition and application |
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| CN108265111A (en) * | 2016-12-29 | 2018-07-10 | 深圳华大智造科技有限公司 | A kind of reaction system for being used for double end sequencings, kit of its composition and application |
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