US 11,479,807 B2Granted

Methods for targeted nucleic acid sequence enrichment with applications to error corrected nucleic acid sequencing

AssigneeUniversity of Washington

InventorsScott R. Kennedy, Jesse J. Salk, Michael Hipp, Elizabeth Schmidt, Rosa Ana Risques, Daniela Nachmanson

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Abstract

The present technology relates generally to methods and compositions for targeted nucleic acid sequence enrichment, as well as uses of such enrichment for error-corrected nucleic acid sequencing applications. In some embodiments, highly accurate, error corrected and massively parallel sequencing of nucleic acid material is possible using a combination of uniquely labeled strands in a double-stranded nucleic acid complex in such a way that each strand can be informatically related to its complementary strand, but also distinguished from it following sequencing of each strand or an amplified product derived therefrom. In various embodiments, this information can be used for the purpose of error correction of the determined sequence.

Claims38

Dependent claims

A method comprising:

providing double-stranded nucleic acid material comprising one or more double-stranded nucleic acid molecules, wherein at least one of the double-stranded nucleic acid molecules comprises a single molecule identifier on each strand and an adapter molecule on at least one of the ends of the double-stranded nucleic acid molecule, and wherein a first adapter sequence is associated with a first strand and a second adapter sequence is associated with a second strand of the double-stranded nucleic acid molecule;
amplifying the nucleic acid material to generate first strand amplicons and second strand amplicons;
separating the amplified nucleic acid material into a first sample and a second sample, wherein both the first sample and the second sample comprise at least first strand amplicons and second strand amplicons;
for any particular double-stranded nucleic acid molecule:
exponentially amplifying only the first strand or a first strand amplicon thereof in the first sample through use of (a) at least one single-stranded oligonucleotide comprising a region specific to a sequence present in the first adapter sequence and (b) at least one single-stranded oligonucleotide comprising a region specific to a target sequence of interest to provide a first nucleic acid product, wherein the single molecule identifier is at least partially maintained in the first nucleic acid product;
exponentially amplifying only the second strand or a second strand amplicon thereof in the second sample through use of (a) at least one single-stranded oligonucleotide comprising a region specific to a sequence present in the second adapter sequence and (b) at least one single-stranded oligonucleotide comprising a region specific to a target sequence of interest to provide a second nucleic acid product, wherein the single molecule identifier is at least partially maintained;
sequencing the first nucleic acid product and second nucleic acid product;
and
comparing the sequence of the first nucleic acid product to the sequence of the second nucleic acid product.

6 more elements

A method of sequencing double-stranded nucleic acid material, comprising:

providing double-stranded nucleic acid material comprising one or more double-stranded nucleic acid molecules, wherein at least one of the double-stranded nucleic acid molecules comprise a single molecule identifier on each strand and an adapter sequence on at least one of the ends of each strand of the double-stranded nucleic acid molecule, and wherein a first adapter sequence is associated with a first strand and a second adapter sequence is associated with a second strand of the nucleic acid molecule;
amplifying the nucleic acid material to generate first strand copies and second strand copies;
separating the amplified nucleic acid material into a first sample and a second sample, wherein both the first sample and the second sample comprise first strand copies and second strand copies;
for any particular double-stranded nucleic acid molecule:
exponentially amplifying only the first strand or a first strand copy thereof in the first sample through use of a first primer specific to the first adapter sequence and a second primer specific to a non-adaptor portion of the first strand to provide a first nucleic acid product, wherein the single molecule identifier is at least partially maintained;
exponentially amplifying only the second strand or a second strand copy thereof in the second sample through use of a first primer specific to the second adapter sequence and a second primer specific to a non-adapter portion of the second strand to provide a second nucleic acid product, wherein the single molecule identifier is at least partially maintained;
sequencing each of the first nucleic acid product and second nucleic acid product;
relating sequences of the first nucleic acid product and sequences of the second nucleic acid product sharing the same single molecule identifier;
and
comparing the related sequences of the first nucleic acid product to the related sequence of the second nucleic acid product.

7 more elements

Dependencies are inferred from the claim text.

Facts

Application: 16/496,936Filed 23 Mar 2018 · us provisional application
Priority: 23 Mar 2017
Published: US 2020/0131561 A130 Apr 2020
Granted: 25 Oct 2022
Term ends: Not estimatedThe source records don’t support an estimate.

Classification9 codes

C12Q 1/6806
Involving nucleic acids: preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6855
Using modified primers or templates: ligating adaptors
C12Q 1/686
Nucleic acid amplification reactions: polymerase chain reaction [PCR]
C12Q 2521/501
Other enzymatic activities: ligase