LINKED PAIRED STRAND SEQUENCING
Provided herein are methods for sequencing both strands of a double stranded nucleic acid fragment that improves fidelity and accuracy of a sequence determination compared to traditional next generation sequencing methods. Compositions and kits for use in the methods are also provided.
1 - 84 . (canceled)
85 . A solid support comprising:
a template nucleic acid molecule comprising, from 3′ to 5′, a first primer binding sequence, a first strand, a second primer binding sequence, and a second strand, wherein the first strand is complementary to the second strand; and
a blocking strand hybridized to the first strand or the second strand;
wherein said template nucleic acid molecule is attached to the solid support.
86 . The solid support of claim 85 , wherein the blocking strand is not attached to the solid support.
87 . The solid support of claim 85 , comprising a plurality of capture oligonucleotides covalently attached to the solid support.
88 . The solid support of claim 87 , wherein at least a portion of the first primer binding sequence is hybridized to a first capture oligonucleotide.
89 . The solid support of claim 87 , wherein at least a portion of the second primer binding sequence is hybridized to a second capture oligonucleotide.
90 . The solid support of claim 87 , wherein the template nucleic acid molecule further comprises a third primer binding sequence 5′ of the first strand.
91 . The solid support of claim 90 , wherein at least a portion of the third primer binding sequence is hybridized to a third capture oligonucleotide.
92 . The solid support of claim 85 , wherein the solid support comprises a surface of a flow cell.
93 . The solid support of claim 85 , wherein the solid support comprises a plurality of wells.
94 . The solid support of claim 85 , wherein a plurality of template nucleic acid molecules is attached to the solid support at addressable locations.
95 . The solid support of claim 85 , wherein the template nucleic acid molecule further comprises a sample barcode sequence, a molecular identifier sequence, or a sample barcode sequence and a molecular identifier sequence.
96 . The solid support of claim 85 , wherein the template nucleic acid molecule is about 100 to 1000 nucleotides in length.
97 . The solid support of claim 85 , further comprising a polymerase bound to the blocking strand.
98 . The solid support of claim 85 , wherein the template nucleic acid molecule comprises a temporary stopper moiety between the first strand and the second strand.
99 . The solid support of claim 98 , wherein the temporary stopper comprises a modified nucleotide, wherein said modified nucleotide comprises a PEG moiety, a cleavable linker, an iso dGTP or an iso dCTP.
100 . The solid support of claim 98 , wherein the temporary stopper is bound to a single-stranded DNA-binding protein.
101 . The solid support of claim 85 , wherein the template nucleic acid molecule is a cfDNA molecule.
102 . The solid support of claim 85 , wherein the first primer binding sequence comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.
103 . The solid support of claim 90 , wherein the third primer binding sequence comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.
104 . The solid support of claim 85 , wherein the second primer binding sequence comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19.