US 2023/0407388 A1 · Sequencing Templates Comprising Multiple Inserts and Compositions and Methods for Improving Sequencing Throughput

Abstract

Described herein is a polynucleotide for use as a sequencing template comprising multiple inserts. Also described herein are method of generating and using these polynucleotides and methods of use of such templates, including analysis of contiguity information. Further, sequencing templates comprising an insert sequence and a copy of the insert sequence can be used to correct for random errors generated during sequencing or amplification or to identify nucleobase damage or other mutation that leads to non-canonical base pairing in a double-stranded nucleic acid. Methods of performing methylation analysis are also described herein.

Claims27

Dependent claims

A polynucleotide comprising:

a. a 5′ terminal polynucleotide comprising a first read primer binding sequence;
b. a first insert sequence located 3′ of the 5′ terminal polynucleotide, wherein the first insert sequence is derived from a target nucleic acid;
c. a concatenation sequence located 3′ of the first insert sequence comprising a second read primer binding sequence and a hybridization sequence;
d. a second insert sequence located 3′ of the concatenation sequence, wherein the second insert sequence is derived from a discontiguous sequence of the target nucleic acid or from a different target nucleic acid than the first insert sequence;
and
e. a 3′ terminal polynucleotide sequence.

3 more elements

A polynucleotide comprising:

a. a 3′ terminal polynucleotide comprising a first read primer binding sequence;
b. a first insert sequence 5′ of the 3′ terminal polynucleotide that is derived from a target nucleic acid;
c. a concatenation sequence comprising a second read primer binding sequence that is orthogonal to the first read primer binding sequence, wherein the second read primer binding sequence comprises a hybridization sequence;
d. a second insert sequence 5′ of the concatenation sequence and derived from a discontiguous sequence of the target nucleic acid or from a different target nucleic acid than the first insert sequence;
and
e. an attachment polynucleotide at the 5′ end of the polynucleotide and comprising an attachment sequence,
wherein the 3′ terminal polynucleotide, the concatenation sequence, and the attachment polynucleotide are not derived from the target nucleic acid.

4 more elements

An adapter composition or kit comprising a first forked adapter complex and a second forked adapter complex,

wherein the first forked adapter complex comprises:
a. a complement attachment polynucleotide comprising:
i. a 5′ portion comprising a complement attachment sequence;
and
ii. a 3′ portion comprising an adapter;
and
b. a hybridization polynucleotide comprising:
i. a 5′ portion comprising the complement of a portion of the adapter and hybridized thereto;
and
ii. the complement of a hybridization sequence, wherein the complement of the hybridization sequence is not complementary to the complement attachment polynucleotide;
and
the second forked adapter complex comprises:
a. an attachment polynucleotide comprising:
i. a 5′ portion comprising an attachment sequence;
and
ii. a 3′ portion comprising the adapter;
and
b. a hybridization polynucleotide comprising:
i. a 5′ portion comprising the complement of a portion of the adapter and hybridized thereto;
and
ii. a hybridization sequence, wherein the hybridization sequence is not complementary to the attachment polynucleotide.

18 more elements

A forked adapter comprising two polynucleotide strands comprising:

a. a first strand comprising a sequencing primer sequence;
and
b. a second strand comprising a 3′ hybridization sequence or its complement, wherein the 3′ end of the first strand is fully or partially complementary to the 5′ end of the second strand, and wherein the hybridization sequence or its complement is bound to a blocking oligonucleotide that is fully or partially complementary to the hybridization sequence or its complement.

Dependencies are inferred from the claim text.

Sequencing Templates Comprising Multiple Inserts and Compositions and Methods for Improving Sequencing Throughput

Life of this patent

Sequencing Templates Comprising Multiple Inserts and Compositions and Methods for Improving Sequencing Throughput

Life of this patent

Abstract

Claims27