IP Library › Granted Patent US 12,247,254
Granted Patent B2
US 12,247,254 · App. 17/181,271 · Granted Mar 11, 2025

Compositions and methods for improving sample identification in indexed nucleic acid libraries

Inventors: Eric Hans Vermaas (San Diego, CA); Mahdieh Khosroheidari (San Diego, CA); Claire Bevis-Mott (Cambridge, GB)
Assignees: Illumina, Inc.; Illumina Cambridge Limited
C12Q1/6874C12Q1/6806C12Q1/6809C12Q1/6816C12Q1/6834C12Q1/6855C40B20/04C40B50/16C40B50/18C40B70/00C40B80/00
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Quick Facts
Patent No.
US 12,247,254
App. No.
17/181,271
Granted
Mar 11, 2025
Kind
B2
Abstract

The present invention is concerned with compositions and methods for improving the rate of correct sample identification in indexed nucleic acid library preparations for multiplex next generation sequencing by modifying or blocking 5′ and 3′ ends of pooled indexed polynucleotides from multiple samples, with an optional exonuclease treatment, prior to amplification and sequencing.

Claims (26)

1. A polynucleotide prepared for sequencing, comprising

an adapter-target-adapter sequence,

wherein the adapter sequence comprises a library specific sequence,

wherein the polynucleotide comprises a double stranded region,

wherein the 5′ ends are each modified to prevent digestion by an enzyme having 5′ exonuclease activity,

wherein the 3′ ends are each modified to inhibit digestion by an enzyme having 3′ exonuclease activity, to inhibit addition of nucleotides to the 3′ ends by an enzyme having polymerase activity, or to inhibit digestion by an enzyme having 3′ exonuclease activity and to inhibit addition of nucleotides to the 3′ ends by an enzyme having polymerase activity, and

wherein the 3′ ends each comprise a dideoxynucleotide.

2. The polynucleotide according to claim 1 , wherein the 5′ ends comprise a phosphorothioate bond.

3. The polynucleotide according to claim 1 , wherein the 5′ ends comprise three phosphorothioate bonds.

4. The polynucleotide according to claim 1 , wherein the region that is double stranded comprises the target and at least a portion of the adapter on both ends of the target, and wherein 5′ and 3′ ends of the polynucleotide in a region of the adapter sequence are single stranded.

5. A method comprising:

sequencing at least a portion of the polynucleotide of claim 1 .

6. A method comprising:

hybridizing a portion of the 3′ end of the polynucleotide according to claim 1 to an oligonucleotide attached to a surface of a substrate, the oligonucleotide having a free 3′ end.

7. The method according to claim 6 , comprising extending the oligonucleotide attached to the surface of the substrate from the free 3′ end by incorporating nucleotides complementary to a sequence of the polynucleotide to produce a copy of the polynucleotide such that the copy is attached to the surface of the substrate.

8. The method according to claim 7 , comprising amplifying the copy attached to the surface of the substrate.

9. A composition comprising the polynucleotide according to claim 1 .

10. The composition according to claim 9 , comprising an exonuclease.

11. The composition according to claim 10 , wherein the exonuclease has 3′ exonuclease activity.

12. The composition according to claim 10 , wherein the exonuclease comprises Exonuclease V.

13. The composition according to claim 9 , comprising one or more enzymes having 5′ exonuclease activity and 3′ exonuclease activity.

14. The composition according to claim 9 , comprising amplification reagents.

15. The composition according to claim 9 , comprising sequencing reagents.

16. A method comprising:

incubating an exonuclease with a composition according to claim 9 .

17. The method according to claim 16 , wherein the exonuclease has 3′ exonuclease activity and 5′ exonuclease activity.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 1, 2021
From: VERMAAS, ERIC HANS; KHOSROHEIDARI, MAHDIEH
To: ILLUMINA, INC.
Reel/Frame 057394/0687 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 19, 2021
From: BEVIS-MOTT, CLAIRE
To: ILLUMINA CAMBRIDGE LIMITED
Reel/Frame 056287/0945 →
Continuity (3)
Division 15959984 · Apr 23, 2018
Provisional Application 62488833 · Apr 23, 2017
Related Publication 20210172014A1 · Jun 10, 2021
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