Patent Document ID: 9092401
Application ID: 13665671
Patent Flag: 1

Claim One:
1. A method of detecting genetic variation in a subject's genome comprising: (a) providing a plurality of clusters of polynucleotides, wherein (i) each cluster comprises multiple copies of a nucleic acid duplex attached to a support; (ii) each duplex in a cluster comprises a first molecule comprising sequences A-B-G′-D′-C′ from 5′ to 3′ and a second molecule comprising sequences C-D-G-B′-A′ from 5′ to 3′; (iii) sequence A′ is complementary to sequence A, sequence B′ is complementary to sequence B, sequence C′ is complementary to sequence C, sequence D′ is complementary to sequence D, and sequence G′ is complementary to sequence G; (iv) sequence G is a portion of a target polynucleotide sequence from a subject and is different for each of a plurality of clusters; (v) sequence B′ is located 5′ with respect to sequence G in the corresponding target polynucleotide sequence; and (vi) each first molecule comprises a barcode sequence; (b) sequencing sequence G′ by extension of a first primer comprising sequence D to produce an R1 sequence for each cluster; (c) sequencing sequence B′ by extension of a second primer comprising sequence A to produce R2 sequence for each cluster; (d) performing a first alignment using a first algorithm to align all R1 sequences to a first reference sequence; (e) performing a second alignment using a second algorithm to locally align R1 sequences identified in said first alignment as likely to contain an insertion or deletion with respect to the first reference sequence, to produce a single consensus alignment for each insertion or deletion; (f) performing an R2 alignment by aligning all R2 sequences to a second reference sequence; (g) transmitting a report identifying sequence variation identified by steps (d) to (f) to a receiver; and (h) hybridizing a third primer to sequence C′ and sequencing the barcode sequence by extension of the third primer to produce a barcode sequence for each cluster.