Nucleic acid sequencing adapters and uses thereof

Patent metadata

Field	Value
Publication number	US-12534719-B2
Application number	US-202318208770-A
Country	US
Kind code	B2
Filing date	Jun 12, 2023
Priority date	Jun 10, 2016
Publication date	Jan 27, 2026
Grant date	Jan 27, 2026

Abstract

High-fidelity, high-throughput nucleic acid sequencing enables healthcare practitioners and patients to gain insight into genetic variants and potential health risks. However, previous methods of nucleic acid sequencing often introduces sequencing errors (for example, mutations that arise during the preparation of a nucleic acid library, during amplification, or sequencing). Provided herein are sequencing adapters comprising a nondegenerate or variable length molecular barcode and compositions comprising a plurality of sequencing adapters, which can be useful for sequencing nucleic acids. Further provided are methods of using the sequencing adapters, including methods of sequencing nucleic acids, methods of identifying an error in a nucleic acid sequence, and methods of determining the number of nucleic acid molecules in a library.

First claim

What is claimed is:

1 . A method of constructing an error-corrected duplex nucleic consensus sequence, comprising:
ligating a first sequencing adapter and a second sequencing adaptor to a duplex nucleic acid molecule, wherein the first sequencing adaptor comprises a first duplex molecular barcode comprising n base positions, wherein the second sequencing adaptor comprises a second duplex molecular barcode comprising n+x base positions, wherein the second duplex molecular barcode has more base positions than the first duplex molecular barcode, and wherein x is not zero; amplifying a first strand of the duplex nucleic acid molecule and a second stand of the duplex nucleic acid molecule thereby producing a set of amplified first strands and a set of amplified second strands; sequencing the set of amplified first strands thereby producing a set of first strand reads; sequencing the set of amplified second strands thereby producing a set of second strand reads; constructing a first strand consensus sequence using the set of first strand reads; constructing a second strand consensus sequence using the set of second strand reads; comparing the first strand consensus sequence to the second strand consensus sequence; removing errors in the set of first strand reads and the set of second strand reads; and constructing an error-corrected duplex nucleic consensus sequence.

2 . The method of claim 1 , wherein the first sequencing adapter is a U-shaped sequence adapter or a Y-shaped sequence adapter,
wherein the first duplex molecular barcode further comprises a predetermined base fraction at one or more base positions across a plurality of duplex molecular barcodes, wherein the n of the first duplex molecular barcode is between 8 and 16, wherein the second sequencing adapter is a U-shaped sequence adapter or a Y-shaped sequence adapter, and wherein the second duplex molecular barcode further comprises a predetermined base fraction at one or more base positions across the plurality of molecular barcodes, wherein the n of the second duplex molecular barcode is between 8 and 16 and the x is 1, wherein the second duplex molecular barcode has more base positions than the first duplex molecular barcode, wherein the first sequence adapter and the second sequence adaptor comprise a first constant 3′-overhang comprising a thymine residue directly adjacent to the first and second duplex molecular barcode, and wherein for each duplex molecular barcode other than the first duplex molecular barcode a thymine residue does not immediately precede the first constant 3′-overhang of each sequence adapter.

3 . The method of claim 1 , wherein the first strand is sequenced in a first direction and a second direction, and wherein the second strand is sequenced in a first direction and a second direction.

4 . The method of claim 1 , wherein the duplex nucleic acid molecule is a cell-free DNA molecule selected from a cell-free tumor DNA molecule or a cell-free fetal DNA molecule.

5 . The method of claim 1 , further comprising enriching the duplex nucleic acid molecule from a nucleic acid library using a set of capture probes for a region of interest.

6 . The method of claim 5 , wherein the set of capture probes are balanced, wherein a balanced set of capture probes provide a reduced sequencing depth variance relative to a set of capture probes comprising a plurality of approximately equally represented capture probes.

7 . The method of claim 6 , further comprising preparing the balanced set of capture probes, wherein the preparing comprises:
sequencing a reference sequencing library comprising a plurality of nucleic acid molecules enriched using a first set of capture probes, wherein each capture probe comprises a sequence that is substantially complementary to a portion of or adjacent to a region of interest included in the reference sequencing library, and wherein an initial known amount of each capture probe is used to form the first set of capture probes; determining a sequencing depth attributable to each capture probe in the set of capture probes; selecting a subsequent known amount of each capture probe based on the initial known amount and the sequencing depth attributable to each capture probe, wherein the subsequent known amount of each capture probe is selected thereby minimizing a difference between a predicted sequencing depth profile and a desired sequencing depth profile; and constructing the balanced set of capture probes by combining at least a fraction of the capture probes at the subsequent known amount of each capture probe in the balanced set of capture probes.

8 . The method of claim 7 , wherein the minimizing a difference is constrained by a minimum subsequent amount or a maximum subsequent amount of each capture probe.

9 . The method of claim 7 , wherein the desired sequencing depth profile is uniform.

10 . The method of claim 7 , wherein the desired sequencing depth profile is non-uniform.

11 . The method of claim 7 , wherein the difference between the predicted sequencing depth profile and the desired sequencing depth profile is defined or implied by an objective function.

12 . The method of claim 7 , wherein the initial known amount is selected from an initial known volume an initial known mass or an initial known number of moles, and the subsequent known amount is selected from a subsequent known volume, a subsequent known mass or a subsequent known number of moles.

13 . The method of any one of claim 7 , wherein the initial known amount is an initial known relative amount, and the subsequent known amount is a subsequent known relative amount.

14 . The method of claim 13 , wherein each capture probe is not substantially complementary to overlapping portions of the region of interest.

15 . The method of claim 13 , wherein at least two of each capture probe in the plurality of capture probes are substantially complementary to overlapping portions of the region of interest.

16 . The method of claim 13 , further comprising obtaining for each capture probe a binding fraction based on the sequencing depth attributable to the capture probe; and wherein the subsequent known amount is based on the initial known amount and the binding fraction.

17 . The method of claim 16 , wherein obtaining the binding fraction further comprises approximating the sequencing depth for a given capture probe i as a Poisson distribution:
d is =Poisson( N s π i )
wherein:
d is the determined sequencing depth attributable to the capture probe i;
N s is the number of nucleic acid molecules in reference sequencing library s; and
π i is the binding fraction for capture probe i.

18 . The method of claim 17 , further comprising determining π i by fitting a maximum likelihood model or a Markov chain Monte Carlo model.

19 . The method of claim 13 , further comprising obtaining for each capture probe a binding fraction, wherein the binding fraction is determined for the fraction of nucleic acid molecules comprising a segment substantially complementary to at least half of the each capture probe based on the sequencing depth attributable to the capture probe; and
wherein the subsequent known amount is based on the initial known amount and the fraction.

20 . The method of claim 13 , further comprising defining the difference by an objective function of the subsequent amounts of each capture probes in the balanced set of capture probes, according to:

Assignees

• Myriad Womens Health Inc

Inventors

• Evans Eric Andrew
• Haque Imran Saeedul
• Beauchamp Kyle
• Chu Clement
• Artieri Carlo G
• Welker Noah

Classifications

• C12Q2563/185

  Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals · CPC title

• C12Q2527/107

  Temperature of melting, i.e. Tm · CPC title

• C12Q2525/191

  incorporating an adaptor · CPC title

• C12Q1/6832

  Enhancement of hybridisation reaction · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title