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CN115747301B - Method for constructing sequencing library, kit for constructing sequencing library and gene sequencing method - Google Patents

Method for constructing sequencing library, kit for constructing sequencing library and gene sequencing method Download PDF

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CN115747301B
CN115747301B CN202210916238.3A CN202210916238A CN115747301B CN 115747301 B CN115747301 B CN 115747301B CN 202210916238 A CN202210916238 A CN 202210916238A CN 115747301 B CN115747301 B CN 115747301B
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sequencing
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CN115747301A (en
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甘广丽
李改玲
刘二凯
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Shenzhen Sailu Medical Technology Co ltd
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Shenzhen Sailu Medical Technology Co ltd
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Abstract

The invention belongs to the field of gene sequencing, and discloses a construction method of a sequencing library, a kit for constructing the sequencing library and a gene sequencing method. The construction method of the sequencing library comprises the following steps: fixing the first segment in the microwell of the chip; hybridizing the second fragment with the first fragment, extending and amplifying to obtain a first capture fragment; hybridizing the third fragment with the first capture fragment, extending and amplifying to obtain a second capture fragment; capturing a complex with the second capture fragment, the complex comprising the target DNA and the transposome; removing the transposase to obtain a composite sequence, and extending to obtain a sequencing library. The kit comprises: a chip, a second capture fragment immobilized in a microwell of the chip; transposases and transposons. Sequencing method: the sequencing library is constructed, amplified and sequenced by the method. The construction method of the sequencing library is simple to operate, greatly reduces the cost and is easy to combine with an automatic platform in the later period.

Description

Method for constructing sequencing library, kit for constructing sequencing library and gene sequencing method
Technical Field
The invention belongs to the field of gene sequencing, and particularly relates to a construction method of a sequencing library, a kit for constructing the sequencing library and a gene sequencing method.
Background
The second generation sequencing technology has the characteristics of high throughput, high accuracy and low cost, so that the research and clinical application of gene sequencing in the fields of biology and medicine are greatly changed, however, the second generation sequencing technology can read 300-500 bp at maximum generally because of the relatively short reading length, and cannot be well developed in the fields of detecting genome structural variation, genome sequencing and the like. The sequencing protocols currently commonly employed are: a long fragment DNA with 10-100 ten thousand base is broken and added with a single kind of DNA label and linker in a limited area, and different long fragment DNA is broken and added with different kinds of labels and same linker in different limited areas respectively, and then sequencing is carried out. If the labels of the sequences of a certain part of DNA are identical, a certain region can be automatically judged in the sequencing result, a small amount of DNA can be assembled in a small region, and then the whole assembly is carried out, so that longer assembly sequences can be generated, and the problems of genome phasing and structural variation of second-generation sequencing are solved. Such gene sequences carrying the same DNA tag are called associated short fragments (linked reads).
Based on the above scheme, there are mainly the following methods for constructing sequencing libraries: 1.10 Xgenomics's Chromium adopts microfluidics to generate droplets, and simultaneously controls a long fragment DNA, the same DNA label, breaking, grafting and other reagents into a water-in-oil droplet, breaks the long fragment DNA in the droplet, tags the DNA and splices, and finally breaks the water-in-oil droplet to collect DNA. The method is convenient to use, but the time and flow for generating liquid drops are complex, and the method gradually exits from the market at present. 2. Single tube long fragment reads (st-lfr) and universal Sequencing technology Transposase Enzyme Linked Long-read Sequencing (TELL-Seq) of Huada genes TM ) The method of disruption by transposase is adopted, firstly, microspheres containing different tag sequences are prepared, then, the transposase containing transposon DNA and long fragment DNA are mixed, the transposon is captured through the tag sequences on the microspheres, thus capturing long fragment DNA, generally, one microsphere captures at most one long fragment DNA, and finally, the tag sequences on the microspheres are added to the DNA sequences disrupted by the transposase through extension reaction. The two methods do not need physical space separation of water-in-oil, the homogeneous reaction time is relatively quick, but more manual operation flow is needed, which is unfavorable for later automation.
In addition, the three methods described above all require separate synthesis of a wide variety of primers to modify the capture microspheres or to produce hydrogel microspheres, respectively, which is a core cost source for the three methods. Therefore, it is necessary to provide a method for constructing a sequencing library which is simple to operate, low in cost and easy to operate automatically in a later period.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a construction method of a sequencing library, which is simple to operate, greatly reduces the cost and is easy to combine with an automatic platform in the later period.
The invention also provides a kit for constructing the sequencing library.
The invention also provides a gene sequencing method.
According to one aspect of the present invention, a method for constructing a sequencing library is provided, comprising the steps of:
s1: providing a chip and a first segment, wherein the chip is provided with a plurality of micropores, the first segment comprises a P1 sequence, and the first segment is fixed in the micropores;
s2: providing a second fragment comprising a P1 complement, a first random sequence N1 and a P2 sequence, hybridizing the second fragment to the first fragment; extending and amplifying the first fragment to obtain a first capture fragment immobilized in the microwell;
S3: providing a third fragment comprising a P2 sequence, a second random sequence N2 and a P3 sequence, hybridizing the third fragment to the first capture fragment; extending and amplifying the first capture fragment to obtain a second capture fragment immobilized in the microwell;
s4: capturing a complex with the second capture fragment, the complex comprising a target DNA and a transposome bound to the target DNA, the transposome comprising a transposase and a transposon, the transposon comprising any one of a partial P3 sequence, a full P3 sequence, a partial P3 complement;
s5: removing the transposase to obtain a composite sequence, wherein the composite sequence comprises a part of transposon sequence and fragmented target DNA, and extending the composite sequence to obtain an extension product, wherein the extension product is the sequencing library.
According to a preferred embodiment of the invention, there is at least the following advantageous effect:
the second capture fragment of the invention introduces two random sequences, which are generated by amplifying a large number of random libraries synthesized at one time on a chip, and do not need to separately synthesize a plurality of primers to respectively modify the capture microspheres or produce hydrogel microspheres as in the prior art, thereby remarkably reducing the core cost. In addition, the invention has simple and convenient operation for constructing the sequencing library, and is easy to be combined with an automatic platform for sequencing long fragment DNA in the later period.
In some embodiments of the invention, the chip comprises 2 flow channels, each flow channel comprising a number of the microwells.
In some embodiments of the invention, the chip comprises 800 to 1500 tens of thousands of said microwells.
In some preferred embodiments of the invention, the chip comprises 1000 ten thousand of the microwells.
In some embodiments of the invention, the micropores have a diameter of 2 to 8 μm.
In some preferred embodiments of the invention, the micropores have a diameter of 5 μm.
In some embodiments of the invention, the microwells have a depth of 5 to 15 μm.
In some preferred embodiments of the invention, the microwells are 10 μm deep.
In some embodiments of the invention, the center distance of two adjacent micro-holes is 8-15 μm.
In some preferred embodiments of the present invention, the center distance between two adjacent micro-holes is 12 μm.
In some preferred embodiments of the present invention, the micropores have a diameter of 5 μm and a depth of 10 μm, and the center distance between two adjacent micropores is 12 μm.
In some embodiments of the invention, step S1 further comprises immobilizing a sequencing primer in the microwell.
In some embodiments of the invention, the bottom of the microwells is modified with a silane coupling agent.
In some embodiments of the invention, the bottom of the microwells is modified with either an aminosilane or a chlorosilane-containing coupling agent.
In some embodiments of the invention, the bottom of the microwells is modified with any one of gamma-aminopropyl triethoxysilane (APTES), aminopropyl trimethoxysilane, or trichlorosilaethane.
In some preferred embodiments of the invention, the bottom of the microwells is modified with gamma-aminopropyl triethoxysilane.
The purpose of silane modification of the bottom of the microwells is to immobilize the first fragment or the sequencing primer.
Specifically, the method of immobilizing the first fragment or the sequencing primer in a microwell of the chip comprises: introducing an azide group into the microwell modified with the aminosilane, adding a Dibenzocyclooctyne (DBCO) -modified first fragment or a sequencing primer to the microwell, and immobilizing the first fragment or the sequencing primer in the microwell through a covalent bond formed by the DBCO and the azide group.
Alternatively, 1, 4-benzenediisoisothiocyanate or glutaraldehyde is introduced into the microwell modified with the aminosilane, and the first fragment or the sequencing primer is immobilized in the microwell by covalent bonds formed by the 1, 4-benzenediisoisothiocyanate or glutaraldehyde with the aminosilane and the amino-modified first fragment or sequencing primer, respectively.
Alternatively, a bifunctional SMBP is introduced into the microwell modified with the aminosilane, the first fragment or the sequencing primer being immobilized in the microwell by covalent bonds formed by the bifunctional SMBP with the aminosilane and the sulfhydryl modified first fragment or sequencing primer, respectively.
Alternatively, the modified trichloro-silico ethane in the microwells is first converted to Lvji silane, then sodium iodide (NaI) is added to activate to Dianji, and then a first fragment or sequencing primer modified with a thiol group is added to fix the microwells by coupling with Dianji.
In some embodiments of the invention, the edges of the micropores are modified with at least one of trimethylsilane, isobutylene triethoxysilane, disiloxane, or cyclobutane.
In some preferred embodiments of the invention, the edges of the micropores are modified with the trimethylsilane.
In some embodiments of the invention, the regions between adjacent ones of the microwells are modified with at least one of trimethylsilane, isobutylene triethoxysilane, disiloxane, or cyclobutanesiloxane.
In some preferred embodiments of the present invention, the regions between adjacent ones of the micropores are modified with the trimethylsilane.
The modification is performed at the edge of the microwell and the region between adjacent microwells, and mainly plays a role of hydrophobic property, so that reagents in the microwells, such as enzymes, DNA and the like, are prevented from non-specifically adsorbing or spreading into the adjacent microwells to cause cross reaction.
In some embodiments of the invention, the P1 sequence is a fixed sequence.
In some embodiments of the invention, the P1 sequence is 25 to 100nt in length.
In some preferred embodiments of the invention, the P1 sequence is shown in SEQ ID NO. 1.
In some embodiments of the invention, the sequencing primer is an immobilized sequence.
In some embodiments of the invention, the sequencing primer is partially or fully complementary to the P1 sequence.
In some embodiments of the invention, the P1 complement is a fixed sequence.
In some embodiments of the invention, the P1 complementary sequence is 25 to 100nt in length.
In some preferred embodiments of the invention, the P1 complement is shown in SEQ ID NO. 2.
In some embodiments of the invention, the P2 sequence is a fixed sequence.
In some embodiments of the invention, the P2 sequence is 25 to 100nt in length.
In some preferred embodiments of the invention, the P2 sequence is shown in SEQ ID NO. 3.
In some embodiments of the invention, the P1 sequence and the P2 sequence are not identical and are not complementary.
In some embodiments of the invention, the first random sequence N1 has a length of 10 to 15nt.
In some embodiments of the invention, the concentration of the second fragment is 2 to 15pM.
In some preferred embodiments of the invention, the concentration of the second fragment is 5pM.
Controlling the concentration of the second fragment to 5pM ensures that at least one of the second fragments in each of the microwells hybridizes.
In some embodiments of the invention, after the hybridization, the unhybridized solution is washed off by adding sodium citrate buffer, which does not affect the completed hybridized strand.
In some embodiments of the invention, the amplification described in step S2 requires the addition of a P2 primer.
In some embodiments of the invention, the P2 primer is an immobilized sequence.
In some embodiments of the invention, the sequence of the P2 primer is identical to the P2 sequence.
In some preferred embodiments of the invention, the P2 primer is set forth in SEQ ID NO. 4.
The amplification in step S2 is performed with two primers: the P2 primer and the first fragment.
In some embodiments of the invention, after the P2 primer is added in step S2, oil is introduced to seal the microwells.
In some embodiments of the invention, the oil comprises any one of liquid paraffin oil, silicone oil, or petrolatum.
In some embodiments of the invention, the amplification described in step S2 employs any one of rapid cycle PCR, solid phase PCR, or isothermal PCR amplification methods.
In some embodiments of the invention, the amplification described in step S2 employs an isothermal amplification method.
In some embodiments of the invention, the amplification of step S2 employs any one of a helicase-dependent amplification method or a recombinase polymerase amplification method (RPA).
In some preferred embodiments of the invention, the amplification described in step S2 employs a recombinase polymerase amplification method.
In some embodiments of the invention, in step S2, the enzyme and dNTP required for the amplification are also added when the P2 primer is added.
In some preferred embodiments of the invention, the enzymes required for amplification include recombinant enzymes and DNA polymerases.
In some embodiments of the invention, the time of the amplification in step S2 is 50 to 70min.
In some preferred embodiments of the invention, the time of amplification in step S2 is 60min.
In some embodiments of the invention, step S2 is performed after the amplification is completed by first introducing a solution that disrupts the oil phase system.
In some embodiments of the invention, the solution that disrupts the oil phase system comprises at least one of ethanol, sodium citrate buffer (SSC solution), formamide, sodium hydroxide, detergent, or banana oil.
In some embodiments of the invention, step S2 is performed by adding a washing solution to the microwells to wash the amplified products after the amplification is completed.
In some embodiments of the invention, the wash liquor comprises at least one of formamide, sodium hydroxide, urea, or a helicase.
In some preferred embodiments of the invention, the wash liquor selects the formamide.
Because one strand (i.e., the first capture fragment) of the amplified nucleic acid sequence of step S2 is chemically linked to the microwell and the other strand is complementary to the first strand via hydrogen bonding, the other strand needs to be removed to facilitate the subsequent hybridization step. Washing with the above-described detergent removes the other strand but does not break the chemical bond, i.e., the first capture fragment is retained.
In some embodiments of the invention, the first capture fragment comprises a P1 sequence, a first random sequence complement, a P2 complement.
In some embodiments of the invention, the first random sequence complement is 10 to 15nt in length.
In some embodiments of the invention, the P2 complement is a fixed sequence.
In some embodiments of the invention, the P2 complement is 25 to 100nt in length.
In some preferred embodiments of the invention, the P2 complement is shown in SEQ ID NO. 5.
In some embodiments of the invention, at the completion of step S2, 85-99% of the microwells of the chip include the first capture fragments, and 1-15% of the microwells include only the first fragments.
In some preferred embodiments of the invention, 90 to 99% of the microwells of the chip contain the first capture fragments, and 1 to 10% of the microwells contain only the first fragments.
In some more preferred embodiments of the invention, 95-99% of the microwells of the chip contain the first capture fragments, and 1-5% of the microwells contain only the first fragments.
In some embodiments of the invention, 1 to 5 of the first capture fragments are included in each microwell of the chip.
The inclusion of 1 to 5 first capture fragments in each microwell is the result of multiple experiments by the inventors.
In some embodiments of the invention, the second random sequence N2 has a length of 10 to 15nt.
In some embodiments of the invention, the P3 sequence is a fixed sequence.
In some embodiments of the invention, the P3 sequence is 25 to 100nt in length.
In some preferred embodiments of the invention, the P3 sequence is shown in SEQ ID NO. 6.
In some embodiments of the invention, the P1 sequence, the P2 sequence, and the P3 sequence are different from each other and are not complementary to each other.
In some embodiments of the invention, the concentration of the third fragment is 2 to 15pM.
In some preferred embodiments of the invention, the concentration of the third fragment is 5pM.
Maintaining the concentration of the third fragment at 5pM ensures that at least one of the third fragments in each of the microwells hybridizes.
In some embodiments of the invention, the amplification described in step S3 requires the addition of a P3 primer.
In some embodiments of the invention, the P3 primer is an immobilized sequence.
In some embodiments of the invention, the sequence of the P3 primer is identical to the P3 sequence.
In some preferred embodiments of the invention, the P3 primer is set forth in SEQ ID NO. 7.
The amplification in step S3 is performed with two primers: the P3 primer and the first fragment.
In some embodiments of the invention, when step S1 immobilizes both the first fragment and the sequencing primer in the microwell, part of the P3 primer is added in step S3 for the amplification.
In some preferred embodiments of the invention, the partial P3 primer is shown in SEQ ID NO. 16.
In some embodiments of the invention, the method of amplification of step S3 is the same as that of step S2.
In some embodiments of the invention, after the amplification in step S3 is completed, a solution for destroying the oil phase system is introduced.
In some embodiments of the invention, the solution that disrupts the oil phase system comprises at least one of ethanol, sodium citrate buffer, formamide, sodium hydroxide, detergent, or banana oil.
In some embodiments of the invention, step S3 requires the addition of ddNTP to bind to the 3' end of the amplified product after disruption of the oil phase system.
In some embodiments of the invention, the ddNTP is added by binding the ddNTP to the 3' end of the amplification product by tdt terminal transferase.
The ddNTP is combined with the 3' end of the amplified product to prevent the second capturing fragment from continuing to extend when the target DNA is captured in a later period, so that the effect of recycling the chip can be achieved.
In some embodiments of the invention, the ddNTP need not be added when step S1 immobilizes both the first fragment and the sequencing primer in the microwell.
In some embodiments of the invention, step S3 is performed by adding a washing solution to the microwells after adding ddNTP to the 3' end of the amplified product.
In some embodiments of the invention, the wash liquor comprises at least one of formamide, sodium hydroxide, urea, or a helicase.
In some preferred embodiments of the invention, the wash liquor selects the formamide.
Likewise, after the washing of step S3, the second capture fragment is retained.
In some embodiments of the invention, the second capture fragment comprises a P1 sequence, a first random sequence complement, a P2 complement, a second random sequence complement, a P3 complement.
In some embodiments of the invention, the second random sequence complement is 10 to 15nt in length.
In some embodiments of the invention, the P3 complement is a fixed sequence.
In some embodiments of the invention, the P3 complement is 25 to 100nt in length.
In some preferred embodiments of the invention, the P3 complement is shown in SEQ ID NO. 8.
In some embodiments of the invention, when step S1 immobilizes both the first fragment and the sequencing primer in the microwell, the second capture fragment comprises a P1 sequence, a first random sequence complement, a P2 complement, a second random sequence complement, a partial P3 complement.
The P1 sequence, the P2 sequence, the P3 sequence, the first random sequence N1 and the second random sequence N2 are introduced in the steps S1 to S3, so that the requirement of sequencing at the current stage can be basically met.
Wherein the first random sequence N1 and the second random sequence N2 are introduced as tag sequences to distinguish between different target DNAs captured subsequently; combining the two random sequences can determine whether a single or multiple fragments comprising the P2 or P3 sequence are added to a microwell in the preparation of the second capture fragment; and a combination of two random sequences can label more target DNA.
The P1 sequence is introduced to immobilize the first capture fragment and the second capture fragment and can serve as amplification primers for the first capture fragment and the second capture fragment.
The P2 sequence and the P3 sequence are introduced to serve as primers for measuring the base information of the first random sequence N1 and the second random sequence N2 when sequencing, and the P3 sequence is also used for hybridization with target DNA so as to capture the target DNA.
In the above step, the P1 sequence and the second fragment cannot be introduced at the same time because the P1 sequence is to be immobilized in the microwells of the chip, and the immobilization conditions are different from those at the time of introduction of the second fragment; while the P1 sequence and the second fragment contain complementary sequences (i.e., P1 complementary sequences), if added simultaneously, the binding of the P1 complementary sequences to the P1 sequence can interfere with the immobilization efficiency of the P1 sequence and the extension efficiency of the second fragment within the microwells.
In some embodiments of the invention, the transposon comprises a first double stranded DNA and a second double stranded DNA.
In some embodiments of the invention, the first double-stranded DNA comprises an a-strand and a b-strand.
In some embodiments of the invention, the a-strand comprises a P4 sequence, a coding sequence, an immobilization sequence, and an immobilization sequence complement (ME complement) that is specifically recognized by a transposase.
In some embodiments of the invention, the b-strand comprises a fixed sequence (ME sequence) specifically recognized by a transposase.
In some embodiments of the invention, the a-strand and the b-strand are bound to the transposase by complementary binding of the ME complementary sequence and the ME sequence to form a duplex that is recognized by the transposase.
In some embodiments of the invention, the second double stranded DNA comprises a c-strand and a d-strand.
In some embodiments of the invention, the c-strand comprises the ME complement.
In some embodiments of the invention, the d-chain comprises the partial or complete P3 sequence, as well as the ME sequence.
In some embodiments of the invention, the c-strand and the d-strand are bound to the transposase by complementary binding of the ME complementary sequence and the ME sequence to form a double strand that is recognized by the transposase.
In some embodiments of the invention, the P4 sequence is a fixed sequence.
In some embodiments of the invention, the P4 sequence is 25 to 100nt in length.
In some preferred embodiments of the invention, the P4 sequence is shown in SEQ ID NO. 9.
In some embodiments of the invention, the P1 sequence, the P2 sequence, the P3 sequence, and the P4 sequence are different from each other and are not complementary to each other.
In some embodiments of the invention, the coding sequence is a random sequence.
In some embodiments of the invention, the coding sequence is 6 to 8nt in length.
In some preferred embodiments of the invention, the transposon comprises a partial P3 sequence as shown in SEQ ID NO. 14.
In some embodiments of the invention, when step S1 immobilizes both the first fragment and the sequencing primer in the microwell, the transposon comprises a third double stranded DNA and a fourth double stranded DNA.
In some embodiments of the invention, the third double-stranded DNA is identical to the first double-stranded DNA.
In some embodiments of the invention, the fourth double-stranded DNA comprises an e-strand and an f-strand.
In some embodiments of the invention, the e-chain comprises an ME sequence.
In some embodiments of the invention, the f-strand comprises the partial P3 complement and the ME complement.
In some embodiments of the invention, the e-strand and the f-strand are bound to the transposase by complementary binding of the ME sequence and the ME complement forming a double strand that is recognized by the transposase.
In some embodiments of the invention, the transposase acts to disrupt the target DNA, form the fragmented target DNA, and add adaptors to both ends of the fragmented target DNA.
The enzymatic activity of the transposase and the ratio of the transposase to the target DNA reaction both affect the capture and sequencing effect. The low enzyme activity may result in poor disruption of the target DNA, and the unsuitable ratio of the transposase to the target DNA may result in too long or too short a sequencing library length, thereby degrading sequencing quality.
In some embodiments of the invention, the transposase has an enzymatic activity of 1 to 20U/. Mu.L.
In some embodiments of the invention, 0.01 to 20U of the transposase can bind to 0.1 to 500ng of the target DNA.
In some embodiments of the invention, the second capture fragment in step S4 captures the complex by its P3 complement complementary to part or all of the P3 sequence of the transposon.
In some embodiments of the invention, the second capture fragment in step S4 captures the complex by complementing part or all of its P3 complement to part or all of the P3 sequence of the transposon.
In some embodiments of the invention, when step S1 immobilizes both the first fragment and the sequencing primer in the microwell, step S4 entails providing a fourth fragment, the fourth fragment comprising a P3 sequence, hybridizing the fourth fragment to the second capture fragment; the second capture fragment captures the complex through the fourth fragment.
Specifically, the fourth fragment hybridizes to a portion of the P3 complement in the second capture fragment.
Specifically, sequences in the fourth fragment that do not hybridize to the second capture fragment are used to capture the complex.
More specifically, the sequence of the fourth fragment that is not hybridized to the second capture fragment is complementary to the partial P3 complement of the transposon to capture the complex.
In some embodiments of the invention, the concentration of the fourth fragment is 2 to 15pM.
In some preferred embodiments of the invention, the concentration of the fourth fragment is 5pM.
Maintaining the concentration of the fourth fragment at 5pM ensures that at least one of the fourth fragments in each of the microwells hybridizes.
In some embodiments of the invention, after the extension product is obtained in step S5, the extension product is isolated and collected.
In some embodiments of the invention, the extension products are isolated and collected by adding a wash solution to elute the extension products and collecting.
In some embodiments of the invention, the wash liquor comprises at least one of sodium hydroxide, formamide or urea.
In some preferred embodiments of the invention, the washing liquid selects the sodium hydroxide.
In some embodiments of the invention, the partial transposon sequences of step S5 include part or all of the P3 sequence, ME sequence, a-strand (ME complement, fixed sequence, coding sequence and P4 sequence) of the transposon.
In some embodiments of the invention, the sequence of the sequencing library in step S5 comprises a P1 complement, a first random sequence N1, a P2 sequence, a second random sequence N2, a P3 sequence, an ME sequence, a fragmented target DNA, an a-strand (ME complement, fixed sequence, coding sequence, and P4 sequence).
In some embodiments of the invention, when step S1 immobilizes the first fragment and the sequencing primer in the microwell, step S5 comprises: removing the transposase to obtain the composite sequence, wherein the composite sequence is connected with the second capture fragment to form the sequencing library.
Specifically, after the sequencing library is obtained, a washing solution is added to the microwells to wash the sequencing library.
Specifically, the washing liquid comprises at least one of formamide, sodium hydroxide, urea or helicase.
Specifically, the partial transposon sequences of step S5 include a partial P3 complement, an ME complement, an a-strand complement (P4 complement, a coding sequence complement, a fixed sequence complement, and an ME sequence) of the transposon.
At this time, the sequence of the sequencing library in step S5 includes a P1 sequence, a first random sequence complementary sequence, a P2 complementary sequence, a second random sequence complementary sequence, a P3 complementary sequence, an ME complementary sequence, a fragmented target DNA, an a-strand complementary sequence (P4 complementary sequence, a coding sequence complementary sequence, a fixed sequence complementary sequence, and an ME sequence).
According to a second aspect of the present invention, there is provided a kit for constructing a sequencing library, comprising:
the chip is provided with a plurality of micropores, and a second capturing fragment is fixed in each micropore; the second capture fragment comprises a P1 sequence, a first random sequence complementary sequence, a P2 complementary sequence, a second random sequence complementary sequence and a P3 complementary sequence;
the kit further comprises a transposase and a transposon; the transposon comprises any one of a partial P3 sequence, a full P3 sequence and a partial P3 complementary sequence.
In some embodiments of the invention, a second capture fragment and a sequencing primer are immobilized in the microwell; the second capture fragment comprises a P1 sequence, a first random sequence complementary sequence, a P2 complementary sequence, a second random sequence complementary sequence and a partial P3 complementary sequence;
The kit further comprises a transposase, a transposon and a fourth fragment; the transposon comprises the partial P3 complement and the fourth fragment comprises the P3 sequence.
In some embodiments of the invention, the transposase and the transposon are used to bind target DNA to form a complex.
In some embodiments of the invention, the second capture fragment is used to capture the complex.
Specifically, the second capture fragment captures the complex by complementing part or all of its P3 complementary sequence with part or all of the P3 sequence of the transposon.
In some embodiments of the invention, after the second capture fragment captures the complex, the transposase breaks the target DNA to form fragmented target DNA; and then removing the transposase to obtain a composite sequence.
In some embodiments of the invention, when a second capture fragment and sequencing primer are immobilized in the microwell, the second capture fragment captures the complex through a fourth fragment.
Specifically, the fourth fragment captures the complex by complementing its partial P3 sequence not hybridised to the second capture fragment with the partial P3 complement of the transposon.
In some embodiments of the invention, the composite sequence comprises a partial transposon sequence and fragmented target DNA.
In some embodiments of the invention, the partial transposon sequences include part or all of the P3 sequence, ME sequence, fragmented target DNA, a-strand (ME complement, fixed sequence, coding sequence, and P4 sequence) of the transposon.
In some embodiments of the invention, when the second capture fragment and sequencing primer are immobilized in the microwell, the partial transposon sequence comprises a partial P3 complement, an ME complement, a fragmented target DNA, an a-strand complement (P4 complement, coding sequence complement, fixed sequence complement, and ME sequence) of the transposon.
In some embodiments of the invention, the complex sequence is extended to provide an extension product, which is the sequencing library.
In some embodiments of the invention, the sequencing library is constructed by ligating the complex sequence obtained by removing the transposase with the second capture fragment after the fourth fragment captures the complex.
In some preferred embodiments of the invention, the kit comprises a chip having immobilized in its microwells a second capture fragment; the kit also comprises transposase, transposon, dNTP, DNA polymerase and washing liquid.
In some embodiments of the invention, the dntps and the DNA polymerase are used to extend the composite sequence.
In some embodiments of the invention, the wash liquor comprises at least one of sodium hydroxide, formamide or urea.
The wash is used to elute the extension product.
In some preferred embodiments of the invention, the kit comprises a chip having immobilized in its microwells a second capture fragment and a sequencing primer; the kit also comprises transposase, transposon, fourth fragment, dNTP, DNA polymerase and washing liquid.
According to a third aspect of the present invention, there is provided a method of gene sequencing comprising the steps of: and constructing a sequencing library of the sample to be tested by using the construction method of the sequencing library, and then amplifying and sequencing the sequencing library.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a chip prepared in example 1 of the present invention;
FIG. 2 is a schematic diagram showing the immobilization of a first fragment in a microwell of a chip according to example 1 of the present invention;
FIG. 3 is a schematic representation of a first capture fragment obtained in example 1 of the present invention; wherein the drawing on the left side represents a second fragment, and the second fragment is a P1 complementary sequence, a first random sequence N1 and a P2 sequence from bottom to top in sequence;
FIG. 4 is a schematic diagram of a second capture fragment obtained in example 1 of the present invention; wherein the left drawing represents a third segment, which is a P2 sequence, a second random sequence N2 and a P3 sequence from bottom to top in sequence;
FIG. 5 is a schematic diagram showing the structure of a transposon in example 1 of the present invention; wherein, the immobilized sequence specifically recognized by the ME-transposase and the immobilized sequence complementary sequence specifically recognized by the ME' -transposase;
FIG. 6 is a schematic representation of a second capture fragment capture complex of example 1 of the present invention; the complex comprises target DNA and a transposome bound to the target DNA, the transposome comprises a transposase and a transposon, the transposon comprises a partial P3 sequence;
FIG. 7 is a schematic diagram of the extension product obtained in step (5) of example 1 of the present invention; wherein, the a chain is ME complementary sequence, fixed sequence, coding sequence and P4 sequence from bottom to top;
FIG. 8 is a schematic representation of the eluted sequencing library of example 1 of the present invention; specific labels are as in fig. 7;
FIG. 9 is a schematic diagram of immobilization of a first fragment and sequencing primer in a microwell of a chip in example 2 of the present invention;
FIG. 10 is a schematic diagram of a second capture fragment obtained in example 2 of the present invention; wherein the left drawing represents a third segment, which is a P2 sequence, a second random sequence N2 and a P3 sequence from bottom to top in sequence;
FIG. 11 is a schematic diagram of a transposon in embodiment 2 of the present invention; wherein, the immobilized sequence specifically recognized by the ME-transposase and the immobilized sequence complementary sequence specifically recognized by the ME' -transposase;
FIG. 12 is a schematic representation of capture of a complex by a fourth fragment in example 2 of the present invention; the complex comprises target DNA and a transposome bound to the target DNA, the transposome comprises a transposase and a transposon, and the transposon comprises a partial P3 complementary sequence;
FIG. 13 is a schematic of a sequencing library obtained in example 2 of the present invention; wherein the complementary sequence of the a-strand is a P4 complementary sequence, a coding sequence complementary sequence, a fixed sequence complementary sequence and an ME sequence from bottom to top.
Detailed Description
The following detailed description of embodiments of the invention, examples of which are illustrated in the accompanying drawings, and the embodiments described by reference to the drawings are illustrative only and are not to be construed as limiting the invention.
In the description of the present invention, the description of the first and second is only for the purpose of distinguishing technical features, and should not be construed as indicating or implying relative importance or implying the number of technical features indicated or the precedence of the technical features indicated.
In the description of the present invention, unless explicitly defined otherwise, terms such as amplification, washing, etc. should be construed broadly, and those skilled in the art can reasonably ascertain the specific meaning of the terms in the present invention by combining the specific contents of the technical proposal.
In the description of the present invention, reference to the term "one embodiment," "some embodiments," etc., means that a particular feature, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment. Furthermore, the particular features, materials, or characteristics may be combined in any suitable manner in any one or more embodiments.
The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1
The embodiment constructs an escherichia coli Ecoli genome sequencing library, which comprises the following specific processes:
(1) Through the micro-nano processing and packaging service of the outer package, a chip is prepared, the size of the chip is 25mm 75mm, the chip comprises two flow channels, each flow channel comprises a plurality of micropores, and the whole chip comprises 1000 ten thousand micropores. As shown in FIG. 1, the micropores have a diameter of 5 μm and a depth of 10. Mu.m, and the centers of two adjacent micropores are spaced apart by 12. Mu.m.
The bottom of the microwells was modified with gamma-aminopropyl triethoxysilane (APTES) to immobilize the first fragment (i.e. P1 sequence); the edge of the microwell and the area between the microwells are modified with trimethylsilane to play a hydrophobic role, so as to prevent reagents in the microwells, such as enzymes, DNA and the like, from being nonspecifically adsorbed or spreading into adjacent microwells to cause cross reaction.
In the micropores, the amino group of APTES is modified with an azide group, meanwhile, a Dibenzocyclooctyne (DBCO) is used for modifying a P1 sequence, a DBCO-P1 sequence complex is added into the micropores, a first segment is fixed at the bottom of the micropores of the chip through covalent bonds formed by DBCO and the azide group (figure 2), and the P1 sequence is shown as SEQ ID NO:1 (GTTAACCCTTAGCGGGGCGTTGTAGTGCGTAAAGA).
(2) Providing a second fragment comprising a P1 complementary sequence, namely a first random sequence N1-P2 sequence, wherein the P1 complementary sequence is shown as SEQ ID NO. 2 (TCTTTACGCACTACAACGCCCCGCTAAGGGTTAAC), the length of the first random sequence N1 is 15nt, and the P2 sequence is shown as SEQ ID NO. 3 (GTCCTAACCGAATATGAACCGTCGTCAATCCAGGT);
adding the second fragment to the microwell to hybridize to the first fragment; the concentration of the second fragment added is controlled to be 5pM so as to ensure that at least one second fragment in each microwell hybridizes; adding sodium citrate buffer solution to wash out unhybridized solution, adding recombinase, DNA polymerase, dNTP and P2 primer (the sequence of the P2 primer is the same as that of the P2 primer shown as SEQ ID NO:4 (GTCCTAACCGAATATGAACCGTCGTCAATCCAGGT)) used for isothermal amplification, introducing paraffin oil to seal the micropores, and performing isothermal amplification at 37 ℃ for 1h (using the P2 primer and the first segment as primers for amplification), so that all the P2 primers react repeatedly. After amplification, the oil phase system is destroyed by introducing sodium citrate buffer solution, formamide is added for washing, DNA single chains which are non-covalently bound with micropores in double-stranded DNA obtained by amplification are washed away, the chains which are covalently bound with the micropores are reserved, the chains are called a first capture fragment (P1 sequence-first random sequence complementary sequence-P2 complementary sequence, the length of the first random sequence complementary sequence is 15nt, and the P2 complementary sequence is shown as SEQ ID NO:5 (ACCTGGATTGACGACGGTTCATATTCGGTTAGGAC), and the sequence is shown as a figure 3. At this time, the first capturing fragments were included in 97% of the microwells of the chip, and 1 to 5 first capturing fragments were included in each microwell, whereas only the first fragments were included in 3% of the microwells.
(3) Providing a third fragment comprising a P2 sequence-a second random sequence N2-P3 sequence, the second random sequence being 15nt in length, the P3 sequence being as shown in SEQ ID NO. 6 (GATTACCTATAGAACCTAATACATGATCTACTGCTGGAATGGAATGGAGAATGATTACGCTCCCTAACAGCTAATATGACAGACAGCGAAAGTTACATTG);
adding the third fragment into the microwells of the chip to hybridize with the first capture fragment; the concentration of the third fragment added is controlled to be 5pM so as to ensure that at least one third fragment in each microwell hybridizes; adding sodium citrate buffer solution to wash out unhybridized solution, adding recombinase, DNA polymerase, dNTP and P3 primer (the sequence of the P3 primer is the same as that of the P3 primer shown in SEQ ID NO:7 (GATTACCTATAGAACCTAATACATGATCTACTGCTGGAATGGAATGGAGAATGATTACGCTCCCTAACAGCTAATATGACAGACAGCGAAAGTTACATTG)) used for isothermal amplification, introducing paraffin oil to seal the micropores, and performing isothermal amplification at 37 ℃ for 1h (using the P3 primer and the first fragment as primers for amplification), so that all the P3 primers react repeatedly. After amplification is finished, a sodium citrate buffer solution is introduced to destroy an oil phase system, and then tdt terminal transferase is added to combine ddATP at the 3' end of an amplified product so as to prevent the amplified product from continuing to extend; and then adding formamide for washing, washing out DNA single chains which are non-covalently bound with the micropores in the amplified double-stranded DNA, and reserving the chains which are covalently bound with the micropores, wherein the chains are called a second capture fragment (P1 sequence-first random sequence complementary sequence-P2 complementary sequence-second random sequence complementary sequence-P3 complementary sequence, the length of the second random sequence complementary sequence is 15nt, and the P3 complementary sequence is shown as SEQ ID NO:8 (CAATGTAACTTTCGCTGTCTGTCATATTAGCTGTTAGGGAGCGTAATCATTCTCCATTCCATTCCAGCAGTAGATCATGTATTAGGTTCTATAGGTAATC), and the sequence is shown in figure 4.
(4) Providing a transposon, wherein the transposon comprises a first double-stranded DNA and a second double-stranded DNA, the specific structure diagram is shown in FIG. 5, and two double-stranded DNAs connected on a transposase in FIG. 5 are the first double-stranded DNA and the second double-stranded DNA; the first double-stranded DNA includes a strand (including P4 sequence (SEQ ID NO:9: GCTATATATGAGTTCCATGGCAGATGTCGAT), coding sequence (random sequence of 8 nt), fixed sequence (SEQ ID NO:10: GAGTATTTGTCCGCAGA) and fixed sequence complement recognized specifically by transposase (ME complement shown as SEQ ID NO: 11: AGATGTGTATAAGAGACAG)) and b strand (ME sequence shown as SEQ ID NO: 12: CTGTCTCTCTCTCTCTTACATACATCT), and the second double-stranded DNA includes c strand (including ME complement (SEQ ID NO:13: AGATGTATAAGAGACAG)) and d strand (including partial P3 sequence (SEQ ID NO:14: GATTACCTTATAGATAGATAGACTATACATTACTATCTATGTACGCGAATGGAATGGA) and ME sequence (SEQ ID NO:15: CTGTCTCTCTCTCTTTTAACACATACATCT)). Combining the transposon with a transposase to form a transposome, and incubating the transposome with target DNA in the genome of the prepared escherichia coli (wherein the enzyme activity of the transposase is 10U/. Mu.L, and 0.01-20U of transposase can be combined with 0.1-500 ng of target DNA) to obtain a complex; adding the complex to the microwell of step (3) and capturing the complex by complementing a partial P3 sequence of the second capturing fragment with a partial P3 sequence of the transposon, see FIG. 6;
(5) After capturing, introducing SDS solution to remove transposase to obtain a composite sequence (part of transposon sequences and fragmented target DNA, part of transposon sequences comprise part of P3 sequences, ME sequences and a chains (ME complementary sequences, fixed sequences, coding sequences and P4 sequences) of transposons), adding DNA polymerase and DNA ligase, heating to 37 ℃ for reaction for 10min, and extending the composite sequence to obtain an extension product, wherein the extension product is shown in figure 7; then adding sodium hydroxide solution to elute the extension product, and collecting to obtain a sequencing library (P1 complementary sequence-first random sequence N1-P2 sequence-second random sequence N2-P3 sequence-ME sequence-fragmented target DNA-a chain (ME complementary sequence, fixed sequence, coding sequence, P4 sequence)), as shown in FIG. 8.
Example 2
The embodiment constructs an escherichia coli Ecoli genome sequencing library, which comprises the following specific processes:
(1) A chip was prepared as described in example 1.
The bottom of the microwells was modified with gamma-aminopropyl triethoxysilane (APTES) for immobilization of the first fragment (i.e. P1 sequence) and sequencing primers; the modification of the edges of the microwells and the regions between the microwells was the same as in example 1.
In the above microwells, the amino groups of APTES were modified with azide groups, and simultaneously with Dibenzocyclooctyne (DBCO) to modify the P1 sequence and sequencing primer, a DBCO-P1 sequence complex was added to the microwells, and the first fragment and sequencing primer were immobilized at the bottom of the microwells of the chip through covalent bonds formed by DBCO and azide groups (FIG. 9), and the P1 sequence was shown in SEQ ID NO: 1.
(2) As in example 1.
(3) As in example 1, part of the P3 primer (shown as SEQ ID NO: 16: GAATGATTACGCTCCCTAACAGCTAATATGACAGACAGCGAAAGTTACATTG) was added at the time of isothermal amplification; and after disruption of the oil phase system, it is not necessary to bind ddNTP to the 3' end of the amplified product.
Finally, a second capturing fragment (P1 sequence-first random sequence complementary sequence-P2 complementary sequence-second random sequence complementary sequence-partial P3 complementary sequence, the length of the second random sequence complementary sequence is 15nt, and the partial P3 complementary sequence is shown as SEQ ID NO: 17:CAATGTAACTTTTCTGTCTGTCTATATTACTAGTTAGGGAGTAATAATTC) is obtained, which is shown in figure 10.
(4) Providing a fourth fragment comprising a P3 sequence, wherein the P3 sequence is shown as SEQ ID NO. 6; the fourth fragment was added to the microwell of the chip and hybridized to the partial P3 complement of the second capture fragment.
Preparing a complex: providing a transposon, wherein the transposon comprises a third double-stranded DNA and a fourth double-stranded DNA, the specific structure diagram is shown in FIG. 11, and two double-stranded DNAs connected on a transposase in FIG. 11 are the third double-stranded DNA and the fourth double-stranded DNA; the third double-stranded DNA is identical to the first double-stranded DNA; the fourth double-stranded DNA includes the e-strand (comprising the ME sequence (SEQ ID NO:18: CTGTCTCTCTTTTAACACATCT)) and the f-strand (comprising the partial P3 complement (SEQ ID NO:19: TCCATTCATTCCAGCAAGTAGATATATATATAGTTAAGTAATC) and the ME complement (SEQ ID NO:20: AGATGTGTATAAGAGACAG)). Combining the transposon with a transposase to form a transposome, and incubating the transposome with target DNA in the genome of the prepared escherichia coli (wherein the enzyme activity of the transposase is 10U/. Mu.L, and 0.01-20U of transposase can be combined with 0.1-500 ng of target DNA) to obtain a complex; adding the complex to the microwell, and capturing the complex by complementation of the partial P3 sequence of the fourth fragment, which is not hybridized with the second capturing fragment, with the partial P3 complement sequence of the transposon, see FIG. 12;
(5) After capturing, introducing SDS solution to remove the transposase to obtain a composite sequence (partial transposon sequence and fragmented target DNA), wherein the partial transposon sequence comprises partial P3 complementary sequence, ME complementary sequence and a-strand complementary sequence (P4 complementary sequence, coding sequence complementary sequence, fixed sequence complementary sequence and ME sequence) of the transposon. The composite sequence is connected with a second capture fragment to form a sequencing library (P1 sequence-first random sequence complementary sequence-P2 complementary sequence-second random sequence complementary sequence-P3 complementary sequence-ME complementary sequence-fragmented target DNA-a strand complementary sequence (P4 complementary sequence, coding sequence complementary sequence, fixed sequence complementary sequence, ME sequence)), which is shown in figure 13.
Example 3
The present example was sequenced using the sequencing library of E.coli Ecoli genome constructed in example 1, the specific procedure being:
(1) Sequencing library purification: adding 2 times of DNA purified magnetic beads into the eluted sequencing library according to the volume ratio, uniformly mixing, incubating for 5min at room temperature, placing the mixture on a magnetic rack until the mixture is clear, discarding the supernatant, lightly blowing the magnetic beads with 80% ethanol to wash salt ions, and washing twice. And (3) performing instantaneous centrifugation, discarding the residual ethanol, uncovering and airing until the ethanol is cracked, adding 23 mu L of clean nuclease-free water to elute the sequencing library, placing the sequencing library on a magnetic rack until the sequencing library is clear, and sucking 22 mu L of supernatant into a clean PCR tube for later use.
(2) Sequencing library quality control: 1 μl of supernatant was quantified using the qubit ssDNA quantification kit. If the concentration is greater than or equal to 4nM, the on-machine sequencing is satisfied, the Salus Pro sequencing kit can be directly used for sequencing, and the on-machine step is operated by referring to the Salus Pro sequencing kit, specification version V1.1. If the library concentration is below 4nM, a round of PCR amplification enrichment is required before the next sequencing operation can be performed.
(3) Sequencing library enrichment (optional): to the eluted 20. Mu.L of sequencing library, a total volume of 30. Mu.L (polymerase, dNTP, polymerase reaction buffer, sequencing primer, P1 sequence) was added to perform a three-step PCR reaction on the library, the reaction procedure being: 98℃1min, 4-10 cycles (98℃10s,60℃30s,72℃30 s), 72℃5min,4 ℃. After the reaction is finished, adding 1.2x purified magnetic beads, uniformly mixing, incubating for 5min at room temperature, placing on a magnetic rack until the magnetic beads are clear, discarding the supernatant, lightly blowing the magnetic beads with 80% ethanol to wash salt ions, and co-washing twice. And (3) carrying out instantaneous centrifugation, discarding the residual ethanol, uncovering and airing until the ethanol is cracked, adding 22 mu L of clean nuclease-free water to elute the sequencing library, placing the sequencing library on a magnetic rack until the sequencing library is clear, sucking 20 mu L of supernatant into a clean PCR tube, and taking 1 mu L of supernatant to be quantified by using a qubit ssDNA quantification kit. After the library is qualified, the Salus Pro sequencing kit is used for sequencing, and the step of starting the machine is operated by referring to the Salus Pro sequencing kit and the specification version V1.1.
While the embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims (11)

1. A method of constructing a sequencing library, comprising the steps of:
s1: providing a chip and a first segment, wherein the chip is provided with a plurality of micropores, the first segment comprises a P1 sequence, and the first segment is fixed in the micropores;
s2: providing a second fragment comprising a P1 complement, a first random sequence N1 and a P2 sequence, hybridizing the second fragment to the first fragment; extending and amplifying the first fragment to obtain a first capture fragment fixed in the microwell, wherein the first capture fragment sequentially comprises a P1 sequence, a first random sequence complementary sequence and a P2 complementary sequence;
s3: providing a third fragment comprising a P2 sequence, a second random sequence N2 and a P3 sequence, hybridizing the third fragment to the first capture fragment; extending and amplifying the first capture fragment to obtain a second capture fragment fixed in the microwell, wherein the second capture fragment sequentially comprises a P1 sequence, a first random sequence complementary sequence, a P2 complementary sequence, a second random sequence complementary sequence and a P3 complementary sequence;
S4: capturing a complex with the second capture fragment, the complex comprising a target DNA and a transposome bound to the target DNA, the transposome comprising a transposase and a transposon, the transposon comprising any one of a partial P3 sequence, a complete P3 sequence;
s5: removing the transposase to obtain a composite sequence, wherein the composite sequence comprises a part of transposon sequences and fragmented target DNA, and extending the composite sequence to obtain an extension product, wherein the extension product is the sequencing library;
the P1 sequence, the P2 sequence, and the P3 sequence are not identical and are not complementary to each other.
2. A method of constructing a sequencing library, comprising the steps of:
s1: providing a chip and a first segment, wherein the chip is provided with a plurality of micropores, the first segment comprises a P1 sequence, and the first segment is fixed in the micropores;
s2: providing a second fragment comprising a P1 complement, a first random sequence N1 and a P2 sequence, hybridizing the second fragment to the first fragment; extending and amplifying the first fragment to obtain a first capture fragment fixed in the microwell, wherein the first capture fragment sequentially comprises a P1 sequence, a first random sequence complementary sequence and a P2 complementary sequence;
S3: providing a third fragment comprising a P2 sequence, a second random sequence N2 and a P3 sequence, hybridizing the third fragment to the first capture fragment; extending and amplifying the first capture fragment to obtain a second capture fragment fixed in the microwell, wherein the second capture fragment sequentially comprises a P1 sequence, a first random sequence complementary sequence, a P2 complementary sequence, a second random sequence complementary sequence and a partial P3 complementary sequence;
s4: providing a fourth fragment, said fourth fragment comprising a P3 sequence, hybridizing said fourth fragment to said second capture fragment; the second capture fragment captures a complex comprising a target DNA and a transposome bound to the target DNA, the transposome comprising a transposase and a transposon, the transposon comprising a partial P3 complement, the partial P3 sequence of the fourth fragment not hybridized to the second capture fragment being complementary to the partial P3 complement of the transposon to capture the complex;
s5: removing the transposase to obtain a composite sequence, wherein the composite sequence is connected with the second capture fragment to form the sequencing library;
the P1 sequence, the P2 sequence, and the P3 sequence are not identical and are not complementary to each other.
3. The method according to claim 1 or 2, wherein step S1 further comprises immobilizing sequencing primers in the microwells.
4. The method according to claim 1 or 2, wherein the P1 sequence has a length of 25-100 nt.
5. The method according to claim 1 or 2, wherein in the second fragment the P1 complementary sequence is 25-100 nt in length.
6. The method of claim 5, wherein the first random sequence N1 has a length of 10-15 nt in the second segment.
7. The method of claim 5, wherein the length of the P2 sequence in the second fragment is 25-100 nt.
8. The method according to claim 1 or 2, wherein the concentration of the second fraction is 2 to 15pm.
9. The method according to claim 1 or 2, wherein in the third segment, the length of the second random sequence N2 is 10-15 nt.
10. The method of claim 9, wherein in the third fragment, the P3 sequence is 25-100 nt in length.
11. A method of gene sequencing comprising the steps of: a sequencing library of a sample to be tested is constructed by the construction method according to any one of claims 1 to 10, and then the sequencing library is amplified and sequenced.
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