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WO-2026093331-A1 - HAIRPIN ADAPTER CONSTRUCTS AND PRIMERS TO GENERATE NEXT-GENERATION SEQUENCING LIBRARY MOLECULES

WO2026093331A1WO 2026093331 A1WO2026093331 A1WO 2026093331A1WO-2026093331-A1

Abstract

The present disclosure is directed to adapters suitable for use in generating a library of nucleic acid molecules suitable for sequencing (e.g., with a next-generation sequencing technique), wherein the nucleic acid molecules suitable for sequencing including a hairpin, thereby facilitating two-pass sequencing.

Inventors

  • FURTADO, Nitya Margaret
  • SNYDER, Jordan
  • WU, Beijing
  • GODWIN, BRIAN CHRISTOPHER
  • LI, JINGCHUAN
  • LI, YUHANG
  • LI, ZHI
  • LI, ZI
  • NGO, Steven Minhthong
  • NGUYEN, Duylinh
  • RISTOW, Peter Gustav

Assignees

  • F. HOFFMANN-LA ROCHE AG
  • Roche Sequencing Solutions, Inc.
  • KAPA BIOSYSTEMS, INC.

Dates

Publication Date
20260507
Application Date
20251028
Priority Date
20241028

Claims (20)

  1. 1. An oligonucleotide adapter comprising a first strand and a second strand, wherein a 3' portion of the first strand and a 5' portion of the second strand form a substantially double-stranded stem region by sequence complementarity; wherein a 5' portion of the first strand and a 3' portion of the second strand are each single stranded and non-complementary; and wherein the 5' portion of the first strand comprises a first hairpin region including two first duplex sequences that are substantially complementary to each other, wherein the two first duplex sequences are separated by an optional first loop sequence.
  2. 2. The oligonucleotide adapter of claim 1, wherein the substantially double-stranded stem region includes one or more modifications to prevent digestion from an exonuclease.
  3. 3. The oligonucleotide adapter of claim 1, wherein the substantially double-stranded stem region includes one or more phosphorothioate bonds.
  4. 4. The oligonucleotide adapter of claim 1, wherein the 3' portion of the second strand comprises a region including a primer binding sequence.
  5. 5. The oligonucleotide adapter of claim 4, wherein the region including the primer binding sequence comprises a recognition site for a nicking enzyme.
  6. 6. The oligonucleotide adapter of claim 4, wherein the first hairpin region forms a first loop-like structure at temperatures below or above about 50°C.
  7. 7. The oligonucleotide adapter of claim 1, wherein the 3' portion of the second strand comprises a second hairpin region including two second duplex sequences, wherein each of the two second duplex sequences are separated by an optional second loop sequence.
  8. 8. The oligonucleotide adapter of claim 7, wherein the two first duplex sequences have different nucleotide sequences than the two second duplex sequences.
  9. 9. The oligonucleotide adapter of claim 7, wherein each of the first and second hairpin regions form independent loop-like structures at a temperature below or above about 50°C.
  10. 10. The oligonucleotide adapter of any one of the preceding claims, wherein the adapter further includes one or more barcodes.
  11. 11. The oligonucleotide adapter of claim 10, wherein the one or more barcodes are located within the substantially double-stranded stem region.
  12. 12. The oligonucleotide adapter of any one of claims 10 and 11, wherein the one or more barcodes are located within either the first or second hairpin regions.
  13. 13. A double stranded nucleic acid molecule ligated to the oligonucleotide adapter of any one of the preceding claims.
  14. 14. An oligonucleotide adapter comprising a first strand and a second strand, wherein a 5' portion of the first strand and a 3' portion of the second strand form a substantially double-stranded - 66 - stem region by sequence complementarity; wherein a 3' portion of the first strand and a 5' portion of the second strand are each single stranded and non-complementary; and wherein the 3' portion of the first strand comprises a first hairpin region including two first duplex sequences that are substantially complementary to each other, wherein the two first duplex sequences are separated by an optional first loop sequence.
  15. 15. The oligonucleotide adapter of claim 14, wherein the substantially double-stranded stem region includes one or more modifications to prevent digestion from an exonuclease.
  16. 16. The oligonucleotide adapter of claim 14, wherein the substantially double-stranded stem region includes one or more phosphorothioate bonds.
  17. 17. The oligonucleotide adapter of claim 14, wherein the 5' portion of the second strand comprises a region including a primer binding sequence.
  18. 18. The oligonucleotide adapter of claim 17, wherein the region including the primer binding sequence comprises a recognition site for a nicking enzyme.
  19. 19. The oligonucleotide adapter of claim 17, wherein the first hairpin region forms a first looplike structure at temperatures below or above about 50°C.
  20. 20. The oligonucleotide adapter of claim 14, wherein the 5' portion of the second strand comprises a second hairpin region including two second duplex sequences separated by an optional second loop sequence.

Description

HAIRPIN ADAPTER CONSTRUCTS AND PRIMERS TO GENERATE NEXTGENERATION SEQUENCING LIBRARY MOLECULES SEQUENCE LISTING INCORPORATION BY REFERENCE BACKGROUND OF THE DISCLOSURE [0001] DNA sequencing is a fundamental tool in biological and medical research. The importance of DNA sequencing has increased dramatically from its inception four decades ago. It is recognized as a crucial technology for most areas of biology and medicine and as the underpinning for the new paradigm of personalized and precision medicine. Information on individuals' genomes and epigenomes can help reveal their propensity for disease, clinical prognosis, and response to therapeutics, but routine application of genome sequencing in medicine requires comprehensive data delivered in a timely and cost-effective manner. [0002] Compiling the sequential reads during sequencing allows one to construct the sequence of the sample. For single molecule sequencing methods, the sequencing accuracy is diminished because of the brief time that a signal representing a base is present in the detector. To improve accuracy, the sample DNA is often read multiple times at the same sensor, and consensus reads are compiled for each sample fragment. Indeed, one way to reduce sequencing error is to determine a consensus sequence by sequencing the target many times, thereby achieving a desired consensus sequence accuracy. BRIEF SUMMARY OF THE DISCLOSURE [0003] Applicant has developed methods for preparing libraries including nucleic acid molecules suitable for two-pass sequencing. In particular, the disclosed methods facilitate the formation of a library including one or more hairpin duplex molecules which are suitable for two- pass sequencing. The disclosed methods use current state-of-the art library preparation steps while permitting for standard PCR amplification and target enrichment methods prior to conversion to into hairpin duplex molecules for two-pass sequencing. [0004] The methods of the present disclosure facilitate the generation of consensus sequencing reads using the sequencing information from the original template and the copied product. As discussed herein, this facilitates the retention of methylation information from the original template strand while assessing the copied strand for additional sequence information. [0005] A first aspect of the present disclosure is an oligonucleotide adapter comprising a first strand and a second strand, wherein a 3' portion of the first strand and a 5' portion of the second strand form a substantially double-stranded stem region by sequence complementarity; wherein a 5' portion of the first strand and a 3' portion of the second strand are each single stranded and non-complementary; and wherein the 5' portion of the first strand comprises a first hairpin region including two first duplex sequences that are substantially complementary to each other, wherein the two first duplex sequences are separated by an optional first loop sequence. [0006] In some embodiments, the substantially double-stranded stem region includes one or more modifications to prevent digestion from an exonuclease. In some embodiments, the substantially double-stranded stem region includes one or more phosphorothioate bonds. In some embodiments, the substantially double-stranded stem region includes one or more phosphorothioate bonds, wherein the one or more phosphorothioate bonds are positioned at end of the first strand and/or the second strand of the double-stranded region. In some embodiments, the substantially double-stranded stem region includes two or more phosphorothioate bonds. [0007] In some embodiments, the 3' portion of the second strand comprises a region including a primer binding sequence. In some embodiments, the region including the primer binding sequence comprises a recognition site for a nicking enzyme. In some embodiments, the optional first loop sequence includes a primer binding sequence, which may optionally include a recognition site for a nicking enzyme. [0008] In some embodiments, the first hairpin region forms a first loop-like structure at temperatures below about 50°C. In some embodiments, the first hairpin region forms a first looplike structure at temperatures above about 50°C. In some embodiments, the first hairpin region forms a first loop-like structure at temperatures below about 45°C. In some embodiments, the first hairpin region forms a first loop-like structure at temperatures below about 40°C. In some embodiments, the first hairpin region forms a first loop-like structure at temperatures below about 35°C. In some embodiments, the first hairpin region forms a first loop-like structure at temperatures of about 58°C. [0010] In some embodiments, the 3' portion of the second strand comprises a second hairpin region including two second duplex sequences, wherein each of the two second duplex sequences are separated by an optional second loop sequence. In some embodiments, the two first duplex sequences have diff