Abstract: Provided are a group of phi29 DNA polymerase mutants having increased thermal stability and use thereof. The phi29 DNA polymerase mutants are proteins obtained by performing point mutation A and/or point mutation B and/or point mutation C on phi29 DNA polymerase, the point mutation A meaning that an amino acid residue M at position 97 of the phi29 DNA polymerase is mutated to other amino acid residue, the point mutation B meaning that an amino acid residue L at position 123 of the phi29 DNA polymerase is mutated into other amino acid residue, and the point mutation C meaning that an amino acid residue E at position 515 of the phi29 DNA polymerase is mutated to other amino acid residue. The stability of the phi29 DNA polymerase mutants is higher than that of a wild-type phi29 DNA polymerase.

Abstract: Improved single molecule sequencing methods, compositions, and devices, are provided. In a first aspect, the present invention provides a multi-pass method of sequencing a target sequence using nanopore sequencing, the method comprising: i) providing a non-naturally occurring concatemer nucleic acid molecule comprising a plurality of copies of the target sequence; ii) nanopore sequencing at least three copies of the target sequence in the concatemer, thereby obtaining a multi-pass sequence dataset, wherein the multi-pass sequence dataset comprises target sequence datasets for the at least three copies of the target sequence; and iii) using the multi-pass sequence dataset to determine the target sequence.

Abstract: Provided are a phi29 DNA polymerase mutant with increased thermo stability, a method for preparing the mutant, the use of the mutant, and a method for increasing the stability of the phi29 DNA polymerase.

Abstract: Provided are a vesicular adaptor and a single-chain cyclic library constructed by using the adaptor. The library can be used for RNA sequencing and other sequencing platforms dependent on a single-stranded cyclic library, and has the advantage of high throughput sequencing, high accuracy and simple operations.

Abstract: The present invention provides a primer for nucleic acid random fragmentation and a nucleic acid random fragmentation method. The primer consists of a plurality of upstream random primers and downstream random primers. The sequence composition of the upstream random primers is 5?-X-Y-3?, and the sequence composition of the downstream random primers is 5?-P-Y?-X?-close-3?, wherein Y and Y? are random sequences, X is all or part of sequences of a sequencing platform 5? end adaptor, X? is all or part of sequences of a sequencing platform 3? end adaptor, P is phosphorylation modification, and close is close modification. The primer of the present invention adopts double random anchoring of both the upstream random primers and the downstream random primers, and a DNA sample can be randomly broken.

Abstract: Provided are a phi29 DNA polymerase and an encoding gene and an application thereof. The phi29 DNA polymerase is C1) or C2): C1) is a protein with DNA polymerase activity obtained by substituting at least one of the 58th, 61st, 94th, 96th, 119th, and 155th amino acid residues in the amino acid sequence of a wild type phi29 DNA polymerase as shown in SEQ ID NO: 2 in the sequence listing; and C2) is a fusion protein obtained by linking a label to the N-terminus and/or C-terminus of the protein represented by C1). A 3?-5?exonuclease of the phi29 DNA polymerase has activity lower than that of the wild type phi29 DNA polymerase, and can efficiently and continuously synthesize DNA during amplification and sequencing.

Abstract: Provided are a vesicular linker and a single-chain cyclic library constructed by using the linker. The library can be used for RNA sequencing and other sequencing platforms dependent on a single-stranded cyclic library, and has the advantages of high throughput sequencing, high accuracy and simple operations.

Abstract: Provided is a linker element and a method of using the linker element to construct a sequencing library, wherein the linker element consists of a linker A and a linker B, the linker A is obtained through the complementary pairing of a long nucleic acid strand and a short nucleic acid strand, the 5? end of the long strand has a phosphoric acid modification, and the 3? end of the short strand has an enclosed modification, with enzyme sites in the short strand; and the linker B is a nucleic acid single strand, and the 3? end thereof can be in a complementary pairing with the 5? end of the long strand of the linker A. Using the linker element of the present invention for constructing a sequencing library ensures the linking directionality of the linkers while solving the problems of fragment interlinking, linker self-linking and low linking efficiency, and reducing the purification reaction between steps, shortening the linking time and reducing costs.

Abstract: A method and reagent for constructing a nucleic acid double-linker single-strand cyclic library.

Abstract: Disclosed in the present disclosure is a recombinant DNA polymerase. The recombinant DNA polymerase is any one selected from: A) a protein, having amino acid modifications at positions 408, 409 and 485, and at least one of amino acid modification(s) at positions 53, 59, 199, 243, 526, 558, 613, 641, 671, 673, 674, 692 and 709 compared to the amino acid sequence of a wild-type KOD DNA polymerase; B) a protein derived from the protein in A), formed by deleting amino acids 1 to 29 from a C-terminus of the protein in A) and keeping the remaining amino acids unchanged; and C) a protein derived from the protein in A) or B), formed by connecting a tag to the N-terminus or C-terminus of the amino acid sequence of the protein in A) or B), wherein the protein in A), B) and C) has DNA polymerase activity.

Abstract: Provided is a single fluorescent dye based sequencing method. Moreover, the present invention further provide modified nucleosides and nucleotides, and a kit comprising the nucleoside and/or nucleotide, particularly suitable for the sequencing method of the present invention. Additionally, the present invention further provides uses of the nucleoside, the nucleotide and the kit for sequencing.

Abstract: Provided is a method of constructing a sequencing library. The method includes 1) providing a single-stranded DNA fragment from a biological sample; 2) subjecting the single-stranded DNA fragment to whole genomic amplification to obtain a whole genome amplification product; 3) fragmenting the whole genome amplification product using a transposase embedded with two adaptors to obtain a fragmented product with two adaptors respectively at two ends; and 4) amplifying the fragmented product with two adaptors respectively at two ends using a tag sequence and a pair of primers to obtain said sequencing library.

Abstract: Provided are a method for constructing a nucleic acid single-stranded cyclic library and the reagents used therein. By the combination of interruption via a transposase with a restricted nick translation reaction, the method realizes a simple and rapid nucleic acid single-stranded cyclic library construction.

Abstract: Disclosed are a nucleic acid fragmentation method and a sequence combination. The method comprises the following steps: subjecting a denatured nucleic acid to annealing and an extension reaction by using a single-stranded 5?-end extension primer, wherein the single-stranded 5?-end extension primer comprises a sequencing platform adaptor sequence of a 5? end and a connected random sequence, and the random sequence is subjected to annealing on a random site of the denatured nucleic acid; and directionally connecting a double-stranded 3?-end adaptor sequence to the 3? end of the nucleic acid generated in the extension reaction, and carrying out denaturalization and purification to obtain a fragmented single-stranded nucleic acid with adaptor sequences on two ends.

Abstract: Provided is a method of constructing a sequencing library using an adaptor element in a bubble shape. The bubble-shaped adaptor is ligated to a DNA fragment respectively at the 3?-terminal and the 5?-terminal i.e., two bubble-shaped adaptors with same sequences are ligated in one step. The bubble-shaped adaptor-ligated product is then amplified with a primer complementary to the 3?-terminal of the long-chain nucleic acid of the bubble-shaped adaptor, so as to replace the non-paired sequence in the short-chain nucleic acid.

Abstract: Provided are a method for constructing a nucleic acid single-stranded cyclic library and the reagents used therein. By the combination of interruption via a transposase with a restricted nick translation reaction, the method realizes a simple and rapid nucleic acid single-stranded cyclic library construction.

Abstract: Improved single molecule sequencing methods, compositions, and devices, are provided. In a first aspect, the present invention provides a multi-pass method of sequencing a target sequence using nanopore sequencing, the method comprising: i) providing a non-naturally occurring concatemer nucleic acid molecule comprising a plurality of copies of the target sequence; ii) nanopore sequencing at least three copies of the target sequence in the concatemer, thereby obtaining a multi-pass sequence dataset, wherein the multi-pass sequence dataset comprises target sequence datasets for the at least three copies of the target sequence; and iii) using the multi-pass sequence dataset to determine the target sequence.

Abstract: Disclosed are a nucleic acid fragmentation method and a sequence combination. The method comprises the following steps: subjecting a denatured nucleic acid to annealing and an extension reaction by using a single-stranded 5?-end extension primer, wherein the single-stranded 5?-end extension primer comprises a sequencing platform adaptor sequence of a 5? end and a connected random sequence, and the random sequence is subjected to annealing on a random site of the denatured nucleic acid; and directionally connecting a double-stranded 3?-end adaptor sequence to the 3? end of the nucleic acid generated in the extension reaction, and carrying out denaturalization and purification to obtain a fragmented single-stranded nucleic acid with adaptor sequences on two ends.

Abstract: Provided in the present invention are a method for constructing a nucleic acid single-stranded cyclic library and reagent kit thereof. The method comprises the steps of using a transposase embedding complex to randomly break nucleic acids and connect a first linker; connecting a second linker at a gap; performing a first PCR reaction, wherein the 5? end of one of the primers has a first affinity tag, resulting in a product with two ends connected to different linker sequences; binding the product to a solid vector having a second affinity tag; degenerating and separating single strands having no affinity tag; and cyclizing the single strands.

Abstract: Disclosed is a method for constructing a long fragment DNA library, comprising the following steps: 1) breaking a long fragment DNA into target fragments of 3-10 kb by transposase, then amplifying the target fragments, and obtaining target fragment amplification products containing dUTP; 2) amplifying the dUTP in the products by removing the target fragments, fragmenting the target fragments secondarily into DNA short fragments of 300-1200 bp; 3) connecting both ends of the DNA short fragments with sequencing linker single chains A and sequencing linker single chains B respectively; and obtaining connecting sequencing linker products; and 4) PCR amplifying the connecting sequencing linker products, to obtain amplification products.