JP-2022533801-A5 -

Dates

Publication Date

20230511

Application Date

20200501

Description

The Specified Classification also describes a system comprising one or more processors and a non-transient storage medium which includes one or more programs executable by the one or more processors for receiving information relating to one or more coupled sequencing reads and for performing one or more of the above methods. In embodiments of the present invention, for example, the following items are provided. (Item 1) A method for generating coupled sequencing read pairs from polynucleotides, (a) Hybridizing the polynucleotide with a primer to form a hybridized template; (b) A step of generating sequencing data relating to the sequence of the first region of the polynucleotide by extending the primer using a labeled nucleotide and detecting the presence or absence of the incorporated labeled nucleotide; (c) A step of further extending the primer extended in step (b) through a second region using nucleotides provided in a flow sequence of the second region, wherein (i) the primer is extended through the second region without detecting the presence or absence of a label of nucleotides incorporated into the extension primer, (ii) a mixture of at least two different types of nucleotide bases is used in at least one step of the flow sequence of the second region, or (iii) the extension of the primer through the second region proceeds faster than the extension of the primer in step (b); and (d) Sequencing data relating to the sequence of the third region of the polynucleotide is generated by further extending the primer extended in step (c) using the labeled nucleotide and detecting the presence or absence of the incorporated labeled nucleotide. A method that includes this. (Item 2) The method according to item 1, wherein the extension of the primer through the second region proceeds faster than the extension of the primer through the first region. (Item 3) The method according to item 1 or 2, further comprising the step of associating the sequencing data of the first region with the third sequencing data. (Item 4) A method for generating coupled sequencing read pairs from polynucleotides, (a) A step of hybridizing the primer with the first region of the polynucleotide to form a hybridized template; (b) extending the primer through the second region using nucleotides provided in a flow sequence of the second region, wherein (i) the primer is extended through the second region without detecting the presence or absence of a label of nucleotides incorporated into the extension primer, or (ii) a mixture of at least two different types of nucleotide bases is used in at least one step of the flow sequence of the second region; and (c) Sequencing data relating to the sequence of the third region of the polynucleotide is generated by further extending the primer extended in step (b) using the labeled nucleotide, and detecting the presence or absence of the incorporated labeled nucleotide. A method that includes this. (Item 5) The method according to item 4, wherein the first region comprises a naturally occurring sequence that is targeted by the primer. (Item 6) The method according to any one of items 1 to 5, wherein the primer is extended through the second region without detecting the presence or absence of a label of a nucleotide incorporated into the extension primer. (Item 7) The method according to any one of items 1 to 6, wherein at least a portion of the nucleotide used to extend the primer through the second region is an unlabeled nucleotide. (Item 8) The method according to any one of items 1 to 6, wherein the nucleotide used to extend the primer through the second region is an unlabeled nucleotide. (Item 9) The method according to any one of items 1 to 8, wherein a mixture of at least two different types of nucleotide bases is used in at least one step of the flow sequence of the second region. (Item 10) The method according to any one of items 1 to 9, wherein the flow sequence of the second region comprises five or more nucleotide flows. (Item 11) The method according to item 10, wherein each of the nucleotide flows contains a single nucleotide base. (Item 12) The method according to item 10 or 11, wherein the flow sequence of the second region induces signal changes for 50% or more of the possible SNP permutations at 5% or more of the random sequencing start positions at two or more flow positions. (Item 13) The method according to any one of items 10 to 12, wherein the flow sequence of the second region has a base incorporation efficiency of 0.6 or more per flow. (Item 14) The method according to any one of items 1 to 13, further comprising the step of determining expected sequencing data for the second region using a reference sequence and the flow order of the second region. (Item 15) The method according to any one of items 1 to 14, wherein the primer is extended through the third region using nucleotides provided in the flow order of the third region, and the method further comprises the step of dete