Patent ID: 11970740
Assignee: UNIVERSITY OF WASHINGTON THROUGH ITS CENTER FOR COMMERCIALIZATION
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 18:
19. A method of generating sequence reads from a population of double-stranded target nucleic acid molecules, comprising:
(a) for at least a plurality of double-stranded target nucleic acid molecules in the population, grouping first strand sequence reads and second strand sequence reads into a family of first and second strand sequence reads based on a single molecule identifier (SMI) sequence alone or in combination with sequence from the target nucleic acid molecule ends; wherein prior to grouping:
(i) double-stranded target nucleic acid molecules were obtained;
(ii) an adapter molecule was ligated to both ends of each of the plurality of double-stranded target nucleic acid molecules through enzymatic ligation, to form a population of adapter-target nucleic acid complexes, wherein individual adapter molecules comprise:
a single-stranded 5′ arm, a single-stranded 3′ arm, and a single molecule identifier (SMI) sequence that alone or in combination with a sequence from the target nucleic acid molecule uniquely labels the ligated double-stranded target nucleic acid complex such that each individual ligated double-stranded target nucleic acid complex is distinguishable from other ligated double-stranded target nucleic acid complexes in the population; and
a strand-distinguishing nucleotide sequence that, following the ligation step, provides a region of non-complementarity between a first strand of an individual adapter-target nucleic acid complex and a second strand of the same adapter-target nucleic acid complex,

(iii) for one or more individual adapter-target nucleic acid complexes in the population, each strand of the adapter-target nucleic acid complex was amplified to produce a plurality of first strand adapter-target nucleic acid complex amplicons and a plurality of second strand adapter-target nucleic acid complex amplicons; and
(iv) at least a portion of the adapter-target nucleic acid complex amplicons were sequenced to produce a plurality of first strand sequence reads and a plurality of second strand sequence reads;

(b) within any family, separating the first and second strand sequence reads into a set of first strand sequence reads and a set of second strand sequence reads based on the region of non-complementarity between the first strand and the second strand of the adapter-target nucleic acid complex;
(c) within any family, comparing at least one first strand sequence read with at least one second strand sequence read;
(d) within any family, identifying nucleotide positions where the compared first and second strand sequence reads are non-complementary;
(e) within any family, identifying nucleotide positions where the compared first and second strand sequence reads are complementary; and
(f) providing a base call read at one or more nucleotide positions for one or more of the plurality of double-stranded nucleic acid molecules in the population; wherein the base call reads are at nucleotide positions where the compared first and second strand sequence reads are complementary.