Methods for simultaneous sequencing of DNA sense and antisense strands
DNA nanosphere templates are generated through two rounds of rolling ring amplification and multiple replacement amplification, and synchronous sequencing of DNA sense strands and antisense strands are achieved, solving the problems of low sequencing throughput and high cost in the prior art, and improving sequencing efficiency and quality.
Patent Information
- Application Number
- CN202080087320.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2020-01-17
- Publication Date
- 2025-04-29
- Estimated Expiration
- 2040-01-17
AI Technical Summary
The existing two-ended sequencing methods require sequencing of two strands of DNA in succession, resulting in a decrease in sequencing throughput and increased cost.
Through two rounds of rolling ring amplification and multiple replacement amplification, DNA nanosphere templates that can be sequenced on the machine are generated, so as to achieve synchronous sequencing of DNA sense strands and antisense strands, that is, sequencing the first and second strands simultaneously.
This greatly saves sequencing time and cost, improves sequencing throughput, and realizes synchronous sequencing of DNA sense strands and antisense strands.
Smart Images
Figure CN114846153B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of nucleic acid sequencing, and in particular to a method for simultaneously sequencing a positive strand and an antisense strand of a DNA. Background Art
[0002] The main methods of high-throughput sequencing are: single-end sequencing and paired-end sequencing (PE / MP). PE / MP sequencing is also called bidirectional sequencing, which measures the sequences at both ends of a long sequence. The sequences at both ends form a "pair", and the distance in the middle is the length of the inserted fragment. Sequence assembly and alignment can be performed. For duplications, deletions and insertions of gene fragments, this method is more accurate and has a wider coverage of the genome. The difference between paired-end and mate-paired is that the library construction method is different. Currently, paired-end sequencing has become the mainstream. While it increases the sequencing length, it can also provide a new method for structural variation analysis.
[0003] For the double-end sequencing method, different sequencing platforms currently have different sequencing schemes, but basically they all need to sequence one DNA chain read1 first, and then sequence the complementary chain read2 after sequencing is completed. For example, the double-end sequencing of Illumina, which currently has the highest market share, mainly adopts the bridge amplification method. First, DNA is fragmented, and then adapters are added at both ends. The adapters include sequencing primer binding sites. After the first round of read1 sequencing is completed, the template chain of the first round of sequencing is removed, and the paired sequencing module (Paired-End Module) is used to guide the complementary chain to regenerate and amplify at the original position to reach the template amount used for the second round of sequencing, and the second round of synthetic sequencing of the complementary chain read2 is performed.
[0004] The problem with the current double-end sequencing method is that it is necessary to sequence the two strands of DNA separately, thereby reducing the sequencing throughput and increasing the sequencing cost. Summary of the invention
[0005] The present invention provides a method for simultaneously sequencing the sense strand and antisense strand of DNA, which can realize sequencing from both ends of DNA at the same time, realize synchronous sequencing of the sense strand and antisense strand of DNA, that is, read1 and read2 are sequenced simultaneously, thereby greatly saving sequencing time and sequencing cost and improving sequencing throughput.
[0006] The present invention is achieved through the following technical solutions:
[0007] A method for simultaneously sequencing a positive strand and an antisense strand of a DNA, comprising:
[0008] The first round of rolling circle amplification is performed using the circular DNA molecule to be sequenced as an amplification template to generate the first part of the template chain;
[0009] Hybridizing a blocked or unblocked amplification primer on the first part of the template chain, and then continuing to perform a second round of rolling circle amplification using the circular DNA molecule as an amplification template to generate a second part of the template chain;
[0010] Using any one of the first part of the template chain and the second part of the template chain as a one-strand sequencing template, and using the other part of the template chain as an amplification template to perform multiple displacement amplification to generate a two-strand sequencing template;
[0011] A first-strand sequencing primer and a second-strand sequencing primer are hybridized on the first-strand sequencing template and the second-strand sequencing template, respectively, and first-strand sequencing and second-strand sequencing are performed simultaneously to obtain the sequences of the sense strand and antisense strand of the DNA.
[0012] In a preferred embodiment, the blocked amplification primer is a single-strand sequencing primer with 3'-end phosphorylation; and the unblocked amplification primer is a multiple displacement amplification primer.
[0013] In a preferred embodiment, the above method comprises the following steps:
[0014] Using single-stranded circular DNA molecules as templates, the first round of rolling circle amplification was performed to form DNA nanospheres;
[0015] A strand of sequencing primer with 3'-end phosphorylation is hybridized to the first part of the template strand generated by the first round of rolling circle amplification;
[0016] The DNA nanoball is subjected to a second round of rolling circle amplification to extend the 3' end of the template chain to generate a second portion of the template chain, and the second portion of the template chain is used as a first-strand sequencing template;
[0017] The 3'-end phosphorylated first-strand sequencing primer is dephosphorylated, and multiple displacement amplification is performed to generate a second-strand sequencing template, and the second-strand sequencing primer is hybridized;
[0018] Hybridizing a first-strand sequencing primer on the first-strand sequencing template to obtain a DNA nanoball template that can be sequenced on a machine, wherein the first-strand sequencing primer and the second-strand sequencing primer are hybridized on the DNA nanoball template;
[0019] Under the action of the above-mentioned first-strand sequencing primer and second-strand sequencing primer, first-strand sequencing and second-strand sequencing are performed simultaneously to obtain the sequences of the above-mentioned DNA sense strand and antisense strand.
[0020] In a preferred embodiment, the above method comprises the following steps:
[0021] Using single-stranded circular DNA molecules as templates, the first round of rolling circle amplification was performed to form DNA nanospheres;
[0022] hybridizing a multiple displacement amplification primer to the first portion of the template strand generated by the first round of rolling circle amplification;
[0023] The DNA nanoball is subjected to a second round of rolling circle amplification to extend the 3' end of the template chain to generate a second portion of the template chain, and the second portion of the template chain is used as a first-strand sequencing template;
[0024] Under the action of the above-mentioned multiple displacement amplification primers, multiple displacement amplification is performed to generate a double-stranded sequencing template, and the double-stranded sequencing primer is hybridized;
[0025] Hybridizing a first-strand sequencing primer on the first-strand sequencing template to obtain a DNA nanoball template that can be sequenced on a machine, wherein the first-strand sequencing primer and the second-strand sequencing primer are hybridized on the DNA nanoball template;
[0026] Under the action of the above-mentioned first-strand sequencing primer and second-strand sequencing primer, first-strand sequencing and second-strand sequencing are performed simultaneously to obtain the sequences of the above-mentioned DNA sense strand and antisense strand.
[0027] In a preferred embodiment, the above method comprises the following steps:
[0028] Using single-stranded circular DNA molecules as templates, the first round of rolling circle amplification was performed to form DNA nanospheres;
[0029] A strand of sequencing primer with 3'-end phosphorylation is hybridized to the first part of the template strand generated by the first round of rolling circle amplification;
[0030] The DNA nanoballs are subjected to a second round of rolling circle amplification to extend the 3' end of the template chain, wherein the second round of rolling circle amplification is first extended with a mixture containing dUTP to form a partial template chain with a U base, and then further extended with a dNTPs mixture to generate a second partial template chain;
[0031] hybridizing a multiple displacement amplification primer to the second part of the template strand, and performing multiple displacement amplification to generate a double-stranded sequencing template;
[0032] Hybridizing the second-strand sequencing primer on the second-strand sequencing template, and dephosphorylating the first-strand sequencing primer phosphorylated at the 3' end, to obtain a DNA nanoball template that can be sequenced on a machine, wherein the first-strand sequencing primer and the second-strand sequencing primer are hybridized on the DNA nanoball template;
[0033] Under the action of the above-mentioned first-strand sequencing primer and second-strand sequencing primer, first-strand sequencing and second-strand sequencing are performed simultaneously to obtain the sequences of the above-mentioned DNA sense strand and antisense strand.
[0034] In a preferred embodiment, in the above method, the copy numbers of the first-strand sequencing template and the second-strand sequencing template are controlled by controlling the time of the rolling circle amplification and the multiple displacement amplification.
[0035] In a preferred embodiment, there is a multiple difference between the copy numbers of the first-strand sequencing template and the second-strand sequencing template, so that the sequenced bases can be located during the sequencing process by the signal difference caused by the multiple difference.
[0036] In a preferred embodiment, the above method adds different specific antibodies with different fluorescence during the sequencing process to specifically identify different bases with blocking groups to achieve base signal collection.
[0037] In a preferred embodiment, the sequencing process includes: first adding a base with a blocking group, then adding a specific antibody with different fluorescence that can specifically recognize the base with a blocking group and performing signal collection, then performing antibody elution to remove the signal and then removing the blocking group, thereby entering the next cycle. In a preferred embodiment, the sequencing is performed on an MGISEQ-2000 sequencer.
[0038] The sequencing method of the present invention generates a DNA nanoball template that can be sequenced on a machine through two rounds of rolling circle amplification and multiple displacement amplification. A first-strand sequencing primer and a second-strand sequencing primer are hybridized on the DNA nanoball template, and first-strand sequencing and second-strand sequencing can be performed simultaneously to obtain the sequences of the DNA sense strand and antisense strand, thereby greatly saving sequencing time and sequencing costs and improving sequencing throughput. BRIEF DESCRIPTION OF THE DRAWINGS
[0039] Figure 1 This is a schematic diagram of the principle of the first solution of the method for simultaneously sequencing the sense strand and antisense strand of DNA in an embodiment of the present invention;
[0040] Figure 2 This is a schematic diagram of the principle of the second solution of the method for simultaneously sequencing the sense strand and antisense strand of DNA in an embodiment of the present invention;
[0041] Figure 3 It is a schematic diagram of the principle of the third solution of the method for simultaneously sequencing the positive strand and the antisense strand of DNA in an embodiment of the present invention;
[0042] Figure 4 This is a graph showing sequencing signal results of the first solution of the method for simultaneously sequencing the sense strand and antisense strand of DNA in an embodiment of the present invention;
[0043] Figure 5 This is a graph showing sequencing signal results of the second solution of the method for simultaneously sequencing the sense strand and antisense strand of DNA in an embodiment of the present invention;
[0044] Figure 6This is a graph of sequencing signal results of the third solution of the method for simultaneously sequencing the positive strand and antisense strand of DNA in an embodiment of the present invention. DETAILED DESCRIPTION
[0045] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many details are described to enable the present invention to be better understood. However, those skilled in the art can easily recognize that some of the features can be omitted in different situations, or can be replaced by other materials or methods.
[0046] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementations. At the same time, the steps or actions in the method description can also be interchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the specification and the drawings are only for the purpose of clearly describing a certain embodiment and are not meant to be a required sequence, unless otherwise specified that a certain sequence must be followed.
[0047] The method of the present invention utilizes two rounds of rolling circle amplification and multiple displacement amplification to generate a DNA nanoball template that can be sequenced on a machine, and can generate a template of a two-strand read2 or a one-strand read1 by controlling the time of rolling circle amplification (RCA) and the time of multiple displacement amplification (MDA), which means that the copy number of the two-strand read2 and the copy number of the one-strand read1 are both controllable, and a template having both a two-strand and a one-strand is formed through certain schemes, and then a one-strand sequencing primer and a two-strand sequencing primer are hybridized respectively, and through the signal difference brought about by the different copy numbers of the two-strand read2 and the one-strand read1, for example, the signal of the two-strand read2 is 2 times or other multiples of that of the one-strand read1, the one-strand read1 and the two-strand read2 bases to be sequenced can be positioned, and a one-strand and two-strand synchronous sequencing reaction is performed, thereby realizing the technical solution of synchronous sequencing of the DNA sense chain and the antisense chain.
[0048] Specifically, in an embodiment of the present invention, a method for simultaneously sequencing a positive strand and an antisense strand of DNA comprises the following steps:
[0049] The first round of rolling circle amplification is performed using the circular DNA molecule to be sequenced as an amplification template to generate the first part of the template chain;
[0050] Hybridizing a blocked or unblocked amplification primer on the first part of the template chain, and then continuing to perform a second round of rolling circle amplification using the circular DNA molecule as an amplification template to generate a second part of the template chain;
[0051] Using the first part of the template chain and a part of the template chain in the second part of the template chain as a first-strand sequencing template, and using the other part of the template chain as an amplification template to perform multiple displacement amplification to generate a second-strand sequencing template;
[0052] A first-strand sequencing primer and a second-strand sequencing primer are hybridized on the first-strand sequencing template and the second-strand sequencing template, respectively, and first-strand sequencing and second-strand sequencing are performed simultaneously to obtain the sequences of the sense strand and antisense strand of the DNA.
[0053] In the present invention, the rolling circle amplification includes two stages, namely the first round of rolling circle amplification and the second round of rolling circle amplification, which respectively generate the first part of the template chain and the second part of the template chain. By controlling the time of the rolling circle amplification, the length of the first part of the template chain and the second part of the template chain can be controlled, and accordingly, the copy number of the first chain and the second chain can be controlled. The rolling circle amplification is generally carried out under the action of a DNA polymerase, and the most commonly used is phi29 DNA polymerase.
[0054] In the present invention, any one of the first template strand and the second template strand can be used as a single-strand sequencing template, and accordingly, the other one is used as a template strand for multiple displacement amplification to generate a double-strand sequencing template.
[0055] In the present invention, between the first round of rolling circle amplification and the second round of rolling circle amplification, blocked or unblocked amplification primers are hybridized on the first part of the template chain, which can temporarily block the first part of the template chain on the one hand, and on the other hand, these amplification primers can be used as primer sequences for subsequent multiple displacement amplification, or as primers for subsequent single-chain sequencing.
[0056] In the present invention, these amplification primers are blocked or unblocked, wherein the blocked amplification primer generally refers to a primer whose 3' end is blocked by a blocking group so that it cannot be extended under the action of a polymerase. In one embodiment of the present invention, the blocked amplification primer is a chain sequencing primer with 3' end phosphorylation. Since the 3' end of the chain sequencing primer has a phosphorylation modification and cannot be extended, it remains unchanged during the second round of rolling circle amplification. After the second round of rolling circle amplification, the chain sequencing primer with 3' end phosphorylation can be dephosphorylated, and the 3' end group of the chain sequencing primer becomes a hydroxyl group, which can be extended under the action of a polymerase. Therefore, the chain sequencing primer after the dephosphorylation treatment can be used as a primer for subsequent multiple displacement amplification or a primer for subsequent chain sequencing, and the specific use can be determined according to the design form of the technical solution.
[0057] The unblocked amplification primer is bound to the first part of the template strand. During the second round of rolling circle amplification, the amplification primer will undergo an extension reaction, but will not affect the extension reaction of the rolling circle amplification. After the second round of rolling circle amplification, under appropriate reaction conditions, an MDA primer extension reaction is performed to generate a two-strand sequencing template. Therefore, in one embodiment of the present invention, the unblocked amplification primer is a multiple displacement amplification primer.
[0058] The method of the present invention for simultaneously sequencing the positive and antisense strands of DNA can be implemented by a variety of specific technical solutions. The present invention provides three typical but non-limiting detailed solutions. It should be understood that based on these three technical solutions, those skilled in the art can also make other suitable variant solutions.
[0059] like Figure 1 As shown, a first detailed technical scheme of the method for simultaneously sequencing the positive and antisense strands of DNA includes: using a single-stranded circular DNA molecule as a template, performing a first round of rolling circle amplification (RCA) to form a DNA nanoball (DNB), and not adding a stop solution; then hybridizing a first strand sequencing primer with a 3'-terminal phosphorylation modification on the first part of the template strand generated in the first round of rolling circle amplification; then continuing to perform a second round of rolling circle amplification on the DNB to extend the 3' end of the template strand, and the extended template strand is subsequently used as a sequencing template for a first strand read1; then dephosphorylating the previously hybridized 3'-terminal phosphorylation-modified first strand sequencing primer, performing multiple displacement amplification (MDA) to generate a second strand read2 sequencing template, and hybridizing with a second strand sequencing primer; finally hybridizing a first strand sequencing primer, at which time the DNB template has both a hybridized first strand sequencing primer and a generated second strand sequencing template and a hybridized second strand sequencing primer; under the action of the first strand sequencing primer and the second strand sequencing primer, the first strand sequencing and the second strand sequencing are simultaneously performed to obtain the sequences of the positive and antisense strands of DNA. Since the time of rolling circle amplification (RCA) and the time of multiple displacement amplification (MDA) to generate second-strand read2 can be controlled, this means that the copy number of second-strand read2 and the copy number of first-strand read1 are both controllable. Through the signal difference brought by the copy number of second-strand read2 and first-strand read1, for example, the signal of second-strand read2 is twice or other multiples of that of first-strand read1, the sequencing bases can be located for read1 and read2.
[0060] like Figure 2As shown, a second detailed technical scheme of the method for simultaneously sequencing the sense and antisense strands of DNA includes: using a single-stranded circular DNA molecule as a template, performing a first round of rolling circle amplification (RCA) to form a DNA nanoball (DNB), without adding a stop solution; then hybridizing a multiple displacement amplification (MDA) primer; then continuing to perform a second round of rolling circle amplification on the DNB to extend the 3' end of the template chain, and the extended template is subsequently used as a sequencing template for a single-strand read1, and then performing multiple displacement amplification (MDA) on the portion that has been hybridized with the MDA primer to generate a sequencing template for a second-strand read2, and hybridizing a second-strand sequencing primer; then hybridizing a single-strand sequencing primer on the sequencing template of the single-strand read1, at this time, the DNB template has both a hybridized single-strand sequencing primer and a generated second-strand sequencing template and a hybridized second-strand sequencing primer; finally, under the action of the single-strand sequencing primer and the second-strand sequencing primer, single-strand sequencing and second-strand sequencing are performed simultaneously to obtain the sequences of the sense and antisense strands of DNA. Since the time of rolling circle amplification (RCA) and the time of multiple displacement amplification (MDA) to generate the second-strand read2 template can be controlled, this means that the copy number of the second-strand read2 and the copy number of the first-strand read1 are both controllable. Through the signal difference brought by the copy number of the second-strand read2 and the first-strand read1, for example, the signal of the second-strand read2 is twice or other multiples of the first-strand read1, the sequencing bases can be located for read1 and read2.
[0061] like Figure 3As shown in the figure, the third detailed technical scheme of the method for simultaneously sequencing the positive and antisense strands of DNA includes: using a single-stranded circular DNA molecule as a template, performing a first round of rolling circle amplification (RCA) to form a DNA nanoball (DNB), and not adding a stop solution; hybridizing a strand of sequencing primers with a 3'-end phosphorylation modification on the first part of the template strand generated by the first round of rolling circle amplification, and the primer can be subsequently dephosphorylated for single-strand read1 sequencing; and then continuing to perform a second round of rolling circle amplification on the DNB in two stages to extend the 3' end of the template strand, first extending it with a mixed solution containing dUTP to form a partial template strand with a U base, and the template strand containing the U base cannot be phi29 DNA polymerase recognizes this part, so this part cannot be amplified by MDA, and then it is extended normally with a dNTPs mixture to generate the second part of the template chain, which is subsequently used for MDA amplification; then a first-strand sequencing primer is hybridized on the second part of the template chain, and multiple displacement amplification (MDA) is performed to generate a second-strand read2 sequencing template, and the second-strand read2 sequencing primer is hybridized, and then the first-strand sequencing primer modified by 3' end phosphorylation is dephosphorylated. At this time, the DNB template has both a hybridized first-strand sequencing primer and a generated second-strand template and a hybridized second-strand sequencing primer; finally, under the action of the first-strand sequencing primer and the second-strand sequencing primer, the first-strand sequencing and the second-strand sequencing are performed simultaneously to obtain the sequences of the DNA sense chain and the antisense chain. Since the time of rolling circle amplification (RCA) and the time of multiple displacement amplification (MDA) to generate second-strand read2 can be controlled, this means that the copy number of second-strand read2 and the copy number of first-strand read1 are both controllable. Through the signal difference brought by the copy number of second-strand read2 and first-strand read1, for example, the signal of second-strand read2 is twice or other multiples of that of first-strand read1, the sequencing bases can be located for read1 and read2.
[0062] The method of the present invention is also applicable to other double-stranded DNA or other structures of DNA for simultaneous sequencing of sense strand and antisense strand after appropriate modification and variation. In the variant technical solution, different means are needed to form both a single-strand read1 sequencing template and a double-strand read2 sequencing template, and the copy numbers of the double-strand read2 and the single-strand read1 have a certain difference, thereby forming a difference in the signal intensity of read1 and read1, so as to distinguish and locate the two templates, thereby realizing a method for simultaneous DNA sense strand and antisense strand sequencing.
[0063] In the sequencing process of the method of the present invention, different specific antibodies with different fluorescence can be added to specifically identify different bases with blocking groups to realize base signal collection. For example, in one embodiment, a base with a blocking group is first added, and then a specific antibody with different fluorescence that can specifically identify the A base and T base with a blocking group is added to collect signals, followed by antibody elution to remove the signal, and then a specific antibody with different fluorescence that can specifically identify the C base and G base with a blocking group is added to collect signals. In this way, in each round of reaction, only two fluorescent signals emit light, and then read2 and read1 have signal differences, achieving a better signal recognition effect, thereby improving the sequencing quality of synchronous sequencing.
[0064] The technical solutions and effects of the present invention are described in detail below through examples. It should be understood that the examples are merely illustrative and cannot be construed as limiting the present invention.
[0065] Example 1
[0066] 1. Equipment:
[0067] MGISEQ-2000 sequencer, MGISEQ-2000 sequencing reagent slide (715nm), mini loader, PCR instrument, 8-tube PCR tube, a set of pipettes, and high-speed centrifuge.
[0068] 2. Reagents:
[0069] The reagents used in this example are shown in Table 1 below:
[0070] Table 1
[0071]
[0072]
[0073] 3. Reagent preparation:
[0074] 1) Dissolution of 3' phosphorylated single-strand sequencing primer:
[0075] Place a 1.5 ml centrifuge tube containing 3' phosphorylated first-strand sequencing primer powder in an eppendorf high-speed centrifuge (5415D) at the highest speed for 5 minutes; according to the instructions on the primer label, use 1XTE buffer to dissolve the primer to 100 μM placeholder primer master solution;
[0076] 2) Preparation of 1 μM 3' phosphorylated one-strand sequencing primer working solution, as shown in Table 2 below:
[0077] Table 2
[0078] Reagent name volume 100 μM 3' phosphorylated first strand sequencing primer master mix 100 μl 5XSSC buffer 9.9 ml total 10 ml
[0079] 3) Preparation of PNK enzyme reagent (for dephosphorylation), as shown in Table 3 below:
[0080] Table 3
[0081] Reagent name Final concentration 10U T4PNK(BGI) 0.1U 10X reaction buffer (PH 5.9) 1X
[0082] 4. Operation steps:
[0083] 1) Prepare DNA nanoballs for the E. coli library with reference to the "MGISEQ-2000RS High-throughput Sequencing Reagent Set Instructions", prepare 3 sequencing slides, and load the DNA nanoballs onto the MGISEQ-2000 sequencing reagent slide (715 nm); then form the two-strand read2 and one-strand read1 templates according to the three technical schemes introduced in the present invention.
[0084] 2) Prepare three sets of sequencing kits according to the MGISEQ-2000RS High-throughput Sequencing Reagent Set User Manual.
[0085] 3) According to the "MGISEQ-2000RS High-throughput Sequencing Reagent Set Instructions", place the sequencing kit and chip on the MGI2000-RS sequencer, select the corresponding script, set SE1, and perform sequencing. In order to prove the feasibility of this scheme, this sequencing first hybridized the second-strand read2 sequencing primer and performed the first cycle (cycle1) sequencing of read2. After the sequencing is completed, the sequencing chain of read2 is blocked, and then the first chain read1 sequencing primer is hybridized to perform the first cycle (cycle1) sequencing of read1.
[0086] 5. Results:
[0087] The results are as follows Figures 4 to 6 As shown, Figure 4 is the sequencing signal result of the first solution of the present invention, Figure 5 is the sequencing signal result of the second solution of the present invention, Figure 6 This is the sequencing signal result of the third solution of the present invention. The results show that the above three solutions are all feasible, but the first solution and the second solution are better than the third solution. Specifically, the signal of read2 of the first solution is about 2 times that of read1, the signal of read2 of the second solution is about 2.5 times that of read1, and the signal of read2 of the third solution is about 1.2 times that of read1.
[0088] The above specific examples are used to illustrate the present invention, which is only used to help understand the present invention and is not intended to limit the present invention. For those skilled in the art, according to the concept of the present invention, some simple deductions, modifications or substitutions can be made.
Claims
1. A method for simultaneously sequencing a positive strand and an antisense strand of DNA, characterized in that: The method comprises: The first round of rolling circle amplification is performed using the circular DNA molecule to be sequenced as an amplification template to generate the first part of the template chain; A blocked amplification primer is hybridized on the first partial template chain, and then the circular DNA molecule is used as an amplification template to perform a second round of rolling circle amplification to generate a second partial template chain; any one of the first partial template chain and the second partial template chain is used as a one-strand sequencing template, and the other partial template chain is used as an amplification template to perform multiple displacement amplification to generate a two-strand sequencing template; or, an unblocked amplification primer is hybridized on the first partial template chain, and then the circular DNA molecule is used as an amplification template to perform a second round of rolling circle amplification to generate a second partial template chain; the second partial template chain is used as a one-strand sequencing template, and the first partial template chain is used as an amplification template to perform multiple displacement amplification to generate a two-strand sequencing template; Hybridizing a first-strand sequencing primer and a second-strand sequencing primer on the first-strand sequencing template and the second-strand sequencing template, respectively, and performing first-strand sequencing and second-strand sequencing simultaneously to obtain the sequences of the sense strand and antisense strand of the DNA; In the method, the copy numbers of the first-strand sequencing template and the second-strand sequencing template are controlled by controlling the time of the rolling circle amplification and the multiple displacement amplification; There is a multiple difference between the copy numbers of the first-strand sequencing template and the second-strand sequencing template, so that the bases to be sequenced can be located during the sequencing process by the difference in signal intensity caused by the multiple difference.
2. The method according to claim 1, characterized in that The blocked amplification primer is a single-strand sequencing primer with 3'-end phosphorylation; the unblocked amplification primer is a multiple displacement amplification primer.
3. The method according to claim 1, characterized in that The method comprises the following steps: Using single-stranded circular DNA molecules as templates, the first round of rolling circle amplification was performed to form DNA nanospheres; hybridizing a strand of sequencing primer with 3'-end phosphorylation on the first part of the template strand generated by the first round of rolling circle amplification; The DNA nanoball is subjected to a second round of rolling circle amplification to extend the 3' end of the template chain to generate a second portion of the template chain, and the second portion of the template chain is used as a first-strand sequencing template; Dephosphorylating the 3'-terminal phosphorylated first-strand sequencing primer, performing multiple displacement amplification to generate a second-strand sequencing template, and hybridizing the second-strand sequencing primer; Hybridizing a first-strand sequencing primer on the first-strand sequencing template to obtain a DNA nanoball template that can be sequenced on a machine, wherein the first-strand sequencing primer and the second-strand sequencing primer are hybridized on the DNA nanoball template; Under the action of the first-strand sequencing primer and the second-strand sequencing primer, first-strand sequencing and second-strand sequencing are performed simultaneously to obtain the sequences of the positive strand and the antisense strand of the DNA.
4. The method according to claim 1, characterized in that: The method comprises the following steps: Using single-stranded circular DNA molecules as templates, the first round of rolling circle amplification was performed to form DNA nanospheres; hybridizing a multiple displacement amplification primer on the first portion of the template strand generated by the first round of rolling circle amplification; The DNA nanoball is subjected to a second round of rolling circle amplification to extend the 3' end of the template chain to generate a second portion of the template chain, and the second portion of the template chain is used as a first-strand sequencing template; Under the action of the multiple displacement amplification primers, multiple displacement amplification is performed to generate a double-stranded sequencing template, and the double-stranded sequencing primers are hybridized; Hybridizing a first-strand sequencing primer on the first-strand sequencing template to obtain a DNA nanoball template that can be sequenced on a machine, wherein the first-strand sequencing primer and the second-strand sequencing primer are hybridized on the DNA nanoball template; Under the action of the first-strand sequencing primer and the second-strand sequencing primer, first-strand sequencing and second-strand sequencing are performed simultaneously to obtain the sequences of the positive strand and the antisense strand of the DNA.
5. The method according to claim 1, characterized in that The method comprises the following steps: Using single-stranded circular DNA molecules as templates, the first round of rolling circle amplification was performed to form DNA nanospheres; hybridizing a strand of sequencing primer with 3'-end phosphorylation on the first part of the template strand generated by the first round of rolling circle amplification; The DNA nanoball is subjected to a second round of rolling circle amplification to extend the 3' end of the template chain, wherein the second round of rolling circle amplification is first extended with a mixture containing dUTP to form a partial template chain with a U base, and then further extended with a dNTPs mixture to generate a second partial template chain; hybridizing a multiple displacement amplification primer on the second part of the template strand, and performing multiple displacement amplification to generate a double-stranded sequencing template; Hybridizing the second-strand sequencing primer on the second-strand sequencing template, and dephosphorylating the first-strand sequencing primer phosphorylated at the 3' end, to obtain a DNA nanoball template that can be sequenced on a machine, wherein the first-strand sequencing primer and the second-strand sequencing primer are hybridized on the DNA nanoball template; Under the action of the first-strand sequencing primer and the second-strand sequencing primer, first-strand sequencing and second-strand sequencing are performed simultaneously to obtain the sequences of the positive strand and the antisense strand of the DNA.
6. The method according to any one of claims 1 to 5, characterized in that: The method adds different specific antibodies with different fluorescence during the sequencing process to specifically identify different bases with blocking groups, so as to realize base signal collection.
7. The method according to claim 6, characterized in that The sequencing process includes: first adding bases with blocking groups, then adding specific antibodies with different fluorescence that can specifically recognize bases with blocking groups and collecting signals, then eluting the antibodies to remove the signals, and then removing the blocking groups to enter the next cycle.
8. The method according to any one of claims 1 to 5, characterized in that: The sequencing was performed on an MGISEQ-2000 sequencer.
9. The method according to any one of claims 1 to 5, characterized in that: The signal intensity of the second chain is twice or other multiples of the signal intensity of the first chain.
Citation Information
Patent Citations
Methods and compositions for sequencing double stranded nucleic acids
US20220349002A1