Granted Patent B2

US 12,378,605 · App. 17/842,113 · Granted Aug 5, 2025

Inventors: Joel Pel (Vancouver, CA); Andrea Marziali (North Vancouver, CA)

Assignee: NCAN GENOMICS, INC.

C12Q1/6869C12Q1/6806C12Q1/6855C12Q1/6876

Abstract

Claims (32)

1. A method for sequencing a region of interest, the method comprising:

providing adapters wherein each adapter comprises at least a double stranded stem, wherein a first strand of the stem comprises a universal priming site and a barcode that is 3′ of the universal priming site within the first strand and wherein a second strand of the stem comprises a reverse complement of the universal priming site and a reverse complement of the barcode;

ligating the adapters onto a plurality of duplex nucleic acid fragments wherein the plurality of duplex nucleic acid fragments comprise a sense strand and an antisense strand;

denaturing the plurality of ligated duplex nucleic acid fragments to create single-stranded nucleic acid fragments each comprising one of the universal priming site or the reverse complement of the universal priming site;

amplifying the single-stranded nucleic acid fragments using universal primers physically linked to a probe that anneals to a target within the sense strand or a complement of the target to generate a plurality of amplified fragments comprising copies of the sense and antisense strands labelled with the barcode or the reverse complement of the barcode; and

sequencing the plurality of amplified fragments to determine a sequence of the region of interest.

2. The method of claim 1 , further comprising:

determining the sequence of the target by comparing at least two sequencing reads of the plurality of amplified copies comprising copies of the sense strand and the antisense strand; and

generating a consensus sequence by determining a base call for each position that represents a consensus from the two or more sequencing reads.

3. The method of claim 1 , wherein the amplifying step uses a high-fidelity polymerase or a strand displacing polymerase.

4. The method of claim 3 , wherein the amplifying step occurs under conditions that require binding of the probe to the target to permit binding of the universal primer to the universal priming site, and wherein the probe is blocked from extension, wherein amplifying the single-stranded nucleic acid fragments comprises extending the universal primer using a strand displacing polymerase to produce the plurality of amplified fragments.

5. The method of claim 1 , wherein the denaturing and amplifying steps are performed in an emulsion droplet.

6. The method of claim 1 , wherein the barcode comprises a unique molecular identifier.

7. The method of claim 1 , wherein the amplifying step comprises extending the universal primers using a strand displacing polymerase selected from the group consisting of Taq polymerase, BST polymerase, phi29 polymerase, and SD polymerase.

8. A method for generating a sequencing read from a double stranded region of interest, the method comprising:

providing adapters comprising a double-stranded stem portion, the stem portion comprising a universal priming site, a barcode that is 3′ to the universal priming site, and their respective reverse complements;

ligating the adapters onto a plurality of duplex nucleic acid fragments wherein the plurality of duplex nucleic acid fragments comprise a sense strand and an antisense strand;

denaturing the plurality of ligated duplex nucleic acid fragments to create single-stranded nucleic acid fragments each comprising one of the universal priming site or its complement;

amplifying the single-stranded nucleic acid fragments using universal primers physically linked to probes that each anneal to a target within the sense strand or a reverse complement of the target to generate a plurality of amplified fragments comprising copies of the sense and antisense strands labelled with the barcode or its complement;

sequencing the plurality of amplified copies to produce a plurality of sequencing reads; and

comparing at least two sequencing reads that originated from one of the plurality of duplex nucleic acid fragments and generating a consensus sequence by determining a base call for each position that represents a consensus from the two or more sequencing reads.

9. The method of claim 8 , wherein the barcode comprises a unique molecular identifier.

10. The method of claim 8 , wherein the comparing step further comprises creating a consensus sequence for each one of the plurality of duplex nucleic acid fragments by:

grouping sequencing reads comprising a sequence of the barcode into a first group;

grouping sequencing reads comprising a second sequence from a reverse complement of the barcode into a second group; and

determining a base call for each position in the first group and the second group.

11. The method of claim 10 , wherein determining the base call comprises determining the base found at that position in the majority of the first group or the second group.

12. The method of claim 8 , wherein the amplifying step uses a high-fidelity polymerase.

13. The method of claim 8 , wherein the amplifying step uses a strand-displacing polymerase.

14. The method of claim 13 wherein the strand-displacing polymerase is selected from the group consisting of Taq polymerase, BST polymerase, phi29 polymerase, and SD polymerase.

15. The method of claim 8 , wherein the universal primer only primes an extension reaction on a single-stranded nucleic acid fragment during the amplifying step when the probe is annealed to the target on the single-stranded nucleic acid fragment.

16. The method of claim 7 , wherein after the ligating and denaturing steps, the single-stranded nucleic acid fragments each comprises the universal priming site and the reverse complement of the universal priming site.

Assignments (2)

Continuity (3)

References Cited (138)