Random array sequencing of low-complexity libraries
First Claim
Patent Images
1. A method of sequencing a template nucleic acid from a library of homologous template nucleic acids, the method comprising the steps of:
- amplifying a library of homologous template nucleic acids from a sample, wherein each template nucleic acid from the library of homologous template nucleic acids is amplified with a set of forward primers and at least one reverse primer, wherein each forward primer in the set of forward primers has a 3′
region comprising a sequence complementary to a template nucleic acid from the library of homologous template nucleic acids and a 5′
tail, wherein the 5′
tail comprises a sequencing primer binding site and a set of wildcard nucleotides at its 3′
terminus comprising at least 1 wildcard nucleotide, and wherein the set of wildcard nucleotides is sandwiched between and located immediately adjacent to both the sequencing primer binding site and the 3′
region comprising the sequence complementary to the template nucleic acid, wherein the set of wildcard nucleotides in each forward primer in the set of forward primers is a different set of wildcard nucleotides from the wildcard nucleotides in each other forward primer in the set of forward primers, wherein the amplifying generates a plurality of amplicons for each template nucleic acid, wherein each amplicon from the plurality of amplicons for each template nucleic acid comprises a different set of wildcard nucleotides located between the sequencing primer binding site and sequence for the template nucleic acid;
randomly arraying the plurality of amplicons for each template nucleic acid of the library of homologous template nucleic acids on a surface, thereby generating a random array of amplicons of homologous template nucleic acids; and
extending in a sequencing by synthesis reaction a sequencing primer annealed to the sequencing primer binding site present in each amplicon on the random array, wherein the extending generates sequence reads from each amplicon from the plurality of amplicons for each template nucleic acid that comprises a set of wildcard nucleotides that differs between sequence reads from each other amplicon from the plurality of amplicons for each template nucleic acid, wherein the set of wildcard nucleotides is the initial nucleotide(s) sequenced in the sequence reads from each amplicon.
3 Assignments
0 Petitions
Accused Products
Abstract
The invention is directed to a method of sequencing low-complexity amplicons randomly arrayed at high density on a surface. Methods of the invention include preparing amplicons for sequencing by a sets of primers that ensure initial signals front different amplicons on the surface will be evenly distributed among the different nucleotides being added in a sequencing by synthesis operation.
333 Citations
8 Claims
-
1. A method of sequencing a template nucleic acid from a library of homologous template nucleic acids, the method comprising the steps of:
-
amplifying a library of homologous template nucleic acids from a sample, wherein each template nucleic acid from the library of homologous template nucleic acids is amplified with a set of forward primers and at least one reverse primer, wherein each forward primer in the set of forward primers has a 3′
region comprising a sequence complementary to a template nucleic acid from the library of homologous template nucleic acids and a 5′
tail, wherein the 5′
tail comprises a sequencing primer binding site and a set of wildcard nucleotides at its 3′
terminus comprising at least 1 wildcard nucleotide, and wherein the set of wildcard nucleotides is sandwiched between and located immediately adjacent to both the sequencing primer binding site and the 3′
region comprising the sequence complementary to the template nucleic acid, wherein the set of wildcard nucleotides in each forward primer in the set of forward primers is a different set of wildcard nucleotides from the wildcard nucleotides in each other forward primer in the set of forward primers, wherein the amplifying generates a plurality of amplicons for each template nucleic acid, wherein each amplicon from the plurality of amplicons for each template nucleic acid comprises a different set of wildcard nucleotides located between the sequencing primer binding site and sequence for the template nucleic acid;randomly arraying the plurality of amplicons for each template nucleic acid of the library of homologous template nucleic acids on a surface, thereby generating a random array of amplicons of homologous template nucleic acids; and extending in a sequencing by synthesis reaction a sequencing primer annealed to the sequencing primer binding site present in each amplicon on the random array, wherein the extending generates sequence reads from each amplicon from the plurality of amplicons for each template nucleic acid that comprises a set of wildcard nucleotides that differs between sequence reads from each other amplicon from the plurality of amplicons for each template nucleic acid, wherein the set of wildcard nucleotides is the initial nucleotide(s) sequenced in the sequence reads from each amplicon. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
-
Specification