METHODS AND COMPOSITIONS FOR SEQUENCING DOUBLE STRANDED NUCLEIC ACIDS
A method for determining sequences from sense and antisense strands of a nucleic acid, including (a) providing a nucleic acid cluster attached to a solid support, wherein the nucleic acid cluster includes a sense strand and an antisense strand of a concatemer, the concatemer including multiple copies of a sequence unit, the sequence unit including a target sequence and a primer binding site; (b) hybridizing a primer to a primer binding site in the antisense strand; (c) extending the primer along the antisense strand to determine the sequence from at least a portion of the target sequence in the antisense strand; (d) hybridizing a second primer to a primer binding site in the sense strand; and (e) extending the second primer along the sense strand to determine the sequence from at least a portion of the target sequence in the sense strand.
1 .- 154 . (canceled)
155 . A composition comprising
a solid support having attached thereto a nucleic acid cluster comprising a sense strand of a concatemer and an antisense strand of the concatemer, wherein the concatemer comprises multiple copies of a sequence unit linked in series, wherein the sequence unit comprises a target sequence and a primer binding site, wherein the sense strand is covalently attached to the solid support, and wherein the antisense strand does not have a covalent bond to the solid support but is attached to the nucleic acid cluster by Watson-Crick base pairing to the sense strand.
156 . The composition of claim 155 , further comprising a first primer hybridized to the primer binding site in a sequence unit of the antisense strand.
157 . The composition of claim 156 , comprising an extended first primer hybridized to the primer binding site in a sequence unit of the antisense strand.
158 . The composition of claim 155 , further comprising a second primer hybridized to the primer binding site in a sequence unit of the sense strand.
159 . The composition of claim 158 , comprising an extended second primer hybridized to the primer binding site in a sequence unit of the sense strand.
160 . The composition of claim 155 , wherein the antisense strand is synthesized from the sense strand using an amplification primer that binds to the primer binding site in a sequence unit of the sense strand.
161 . The composition of claim 155 , wherein the solid support comprises a capture primer, wherein the sense strand is synthesized by extending the capture primer hybridized to a nucleic acid template along the nucleic acid template by rolling circle amplification, and wherein the antisense strand is synthesized by extending an amplification primer that binds to the primer binding site in a sequence unit of the sense strand.
162 . The composition of claim 161 , wherein the nucleic acid template is a circular nucleic acid template, wherein the nucleic acid template is circularized from a linear nucleic acid template, wherein the capture primer and the amplification primer are extended using a strand displacing polymerase, or, wherein the amplification primer is non-covalently attached to the primer binding site in the sequence unit of the sense strand.
163 . The composition of claim 155 , wherein the cluster comprises multiple antisense strands of the concatemer.
164 . The composition of claim 163 , wherein the multiple antisense strands outnumber the sense strands in the cluster, or wherein the cluster comprises multiple sense strands of the concatemer.
165 . The composition of claim 155 , wherein the antisense strand of the concatemer has fewer copies of the sequence unit than the sense strand.
166 . The composition of claim 155 , wherein the antisense strand comprises a first portion of the target sequence, or a reverse complement thereof, and wherein the sense strand comprises a second portion of the target sequence, or a reverse complement thereof.
167 . The composition of claim 166 , wherein the target sequence comprises a gap between the first portion of the target sequence and the second portion of the target sequence, wherein the first portion of the target sequence is partially complementary to the second portion of the target sequence, wherein the first portion of the target sequence is complementary to the full length of the second portion of the target sequence, or wherein the second portion of the target sequence is complementary to the full length of the first portion of the target sequence.
168 . The composition of claim 155 , wherein the target sequence comprises at least 100 base pairs.
169 . The composition of claim 155 , wherein the sense strand is generated from a nucleic acid template with isothermal amplification.
170 . The composition of claim 169 , wherein the isothermal amplification is rolling circle amplification, wherein the nucleic acid template is derived or generated from a sample without performing polymerase chain reaction, or wherein the nucleic acid template is derived or generated from a sample by performing at most five cycles of polymerase chain reaction.
171 . The composition of claim 155 , wherein the antisense strand comprises one or more nucleotides that are modified or non-canonical.
172 . The composition of claim 155 , wherein the antisense strand comprises one or more nucleotides with bases that are modified or non-canonical, wherein the bases are uracil.
173 . The composition of claim 172 , wherein a ratio of the number of bases that are uracil to the number of bases that are thymine, or are not uracil, in the antisense strand is from 1:1000 to 1:10, or wherein a percentage of bases in the antisense strands that are uracil is 0.001% to 1%.
174 . The composition of claim 155 , wherein the antisense strand is synthesized by extending a primer in the presence of deoxyribonucleotide triphosphates comprising dATP, dTTP, dGTP, dCTP, and dUTP.