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EP-4735638-A2 - NUCLEIC ACID MOLECULE SEQUENCING SYSTEMS AND METHODS

EP4735638A2EP 4735638 A2EP4735638 A2EP 4735638A2EP-4735638-A2

Abstract

Methods of single molecule sequencing are provided. A primer may be hybridized to a template molecule to form a hybridized template. The sequence of a first region of the template molecule may be determined by extending the primer through the first region using labeled nucleotides. The primer may be extended through a second region of the template using unlabeled nucleotides. The sequence of a third region of the template molecule may be determined by extending the primer through the third region using labeled nucleotides. The sequence of the second region of the template molecule may be determined by comparing the sequence of the first region and the third region to a reference sequence.

Inventors

  • BARAD, Omer
  • OBERSTRASS, Florian
  • HANING, Evan
  • ETZIONI, Yoav
  • RUSINEK, Itai
  • ALMOGY, GILAD
  • LIPSON, DORON
  • DUNBAR, WILLIAM
  • MAZUR, DANIEL
  • FRANCAVILLA, CHARLES
  • ONO, Robert

Assignees

  • Ultima Genomics, Inc.

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 1. A method of sequencing a template molecule, comprising: a. hybridizing a primer to the template molecule to form a hybridized template; b. determining the sequence of a first region of the template molecule by extending the primer through the first region using labeled nucleotides; c. extending the primer through a second region of the template using unlabeled nucleotides; d. determining the sequence of a third region of the template molecule by extending the primer through the third region using labeled nucleotides; e. determining the sequence of the second region of the template molecule by comparing the sequence of the first region and the third region to a reference sequence.
  2. 2. The method of claim 1, wherein the determining (b) the sequence of the first region comprises^ for each nucleotide flow in a first plurality of nucleotide flows: a. providing labeled nucleotides of a respective base type; and b. detecting a signal indicating a presence or absence of incorporation of labeled nucleotides into the extending primer.
  3. 3. The method of claim 1 or claim 2, wherein the extending (c) the primer through the second region comprises, for each nucleotide flow in a second plurality of nucleotide flows: providing unlabeled nucleotides of a respective base type.
  4. 4. The method of claim 1 or claim 2, wherein the extending (c) the primer through the second region comprises, for each nucleotide flow in a second plurality of nucleotide flows: providing unlabeled nucleotides of one or more base types.
  5. 5. The method of any one of claims 1-4, wherein the determining (d) the sequence of the third region comprises, for each nucleotide flow in a third plurality of nucleotide flows: a. providing labeled nucleotides of a respective base type; and b. detecting a signal indicating a presence or absence of incorporation or labeled nucleotides into the extending primer.
  6. 6. The method of claim 1, wherein the determining (e) the sequence of the second region comprises aligning the first and third regions of the template molecule to a first and third -115- SUBSTITUTE SHEET (RULE 26) regions of the reference sequence, respectively, wherein the first and third regions are separated from each other by a second region in the reference sequence.
  7. 7. The method of claim 6, wherein the sequence of the second region of the template molecule is determined as the sequence of the second region of the reference genome.
  8. 8. The method of claim 5, wherein the first plurality of nucleotide flows, and third plurality of nucleotide flows comprise 50 nucleotide flows, respectively.
  9. 9. The method of claim 8, wherein the second plurality of nucleotide flows comprises 10 nucleotide flows.
  10. 10. The method of claim 1, further comprising repeating the determining (b) and extending (c) one or more times prior to determining (d) and determining (e).
  11. 11. The method of claim 1, wherein the template molecule is a nucleic acid.
  12. 12. A method of sequencing a template molecule, comprising: a. hybridizing a primer to the template molecule to provide a hybridized template; and b. performing primer extension by providing: i) one or more nucleotide flows comprising labeled nucleotides of a first base type, and ii) one or more nucleotide flows.
  13. 13. The method of claim 12, wherein the primer extension (b) further comprises, in each of the one or more first nucleotide flows: a. providing labeled nucleotides of the first base type; and b. detecting a signal indicating a presence or absence of incorporation of a labeled nucleotide into the extending primer.
  14. 14. The method of claim 13, further comprising (c) determining the sequence of the template molecule by aligning the detected signals to a reference sequence.
  15. 15. The method of any one of claims 12-14, wherein the proportion of labeled nucleotides in the one or more first nucleotide flows is 100%.
  16. 16. The method of any one of claims 10-15, further comprising: a. removing the extended primer from the template molecule; b. hybridizing a primer to the template molecule to provide a hybridized template; -116- SUBSTITUTE SHEET (RULE 26) c. performing primer extension in a second flow cycle, wherein the second flow cycle comprises one or more second nucleotide flows comprising nucleotides of a second base type different from the first base type; d. removing the extended primer from the template molecule; e. hybridizing a primer to the template molecule to provide a hybridized template; and f. performing primer extension in a third flow cycle, wherein the third flow cycle comprises one or more third nucleotide flows comprising nucleotides of a third base type different from the first and second base types.
  17. 17. The method of claim 16, wherein nucleotides in one or more second nucleotide flows comprise labeled nucleotides, and wherein the proportion of labeled nucleotides in the one or more second nucleotide flows is 100%.
  18. 18. The method of claim 16, wherein nucleotides in the one or more third nucleotide flows comprise labeled nucleotides, and wherein the proportion of labeled nucleotides in the one or more third nucleotide flows is 100%.
  19. 19. The method of any one of claims 16-18, wherein the first flow cycle, the second flow cycle, and the third flow cycle each comprises one or more additional nucleotide flows comprising unlabeled nucleotides.
  20. 20. The method of claim 19, wherein the one or more additional nucleotide flows in the first, second, and third flow cycles do not comprise the first base type, the second base type, or the third base type, respectively.

Description

NUCLEIC ACID MOLECUEE SEQUENCING SYSTEMS AND METHODS CROSS-REFERENCE [1] This application claims the benefit of U.S. Provisional Pat. App. Nos. 63/511,612 filed on June 30, 2023, 63/581,542 filed September 8, 2023, 63/563,265 filed March 8, 2024, and 63/632,508 filed April 10, 2024, each of which is entirely incorporated by reference herein for all purposes. BACKGROUND [2] Biological sample processing has various applications in the fields of molecular biology and medicine (e.g., diagnosis). For example, nucleic acid sequencing may provide information that may be used to diagnose a certain condition in a subject and in some cases tailor a treatment plan. Sequencing is widely used for molecular biology applications, including vector designs, gene therapy, vaccine design, industrial strain design and verification. Biological sample processing may involve a fluidics system and/or a detection system. [3] Single molecule sequencing offers increasingly granular and more accurate information For example, the ability to sequence a single molecule reduces the presence of errors introduced by amplification and makes possible the sequencing of molecules that are difficult to amplify (e g., due to high GC content, secondary structures). Further, amplification can be a lengthy process, and without that requirement overall sequencing throughput can be increased. However, less light may be available from single molecule sequencing for signal readout. There is thus a need in the art for improved single molecule sequencing methods. SUMMARY [4] Provided herein are systems and methods for nucleic acid molecule sequencing. The present disclosure may be advantageous to improve sequencing results. [5] An aspect of the disclosure provides a method of sequencing a template molecule, comprising: hybridizing a primer to the template molecule to form a hybridized template; determining the sequence of a first region of the template molecule by extending the primer through the first region using labeled nucleotides; extending the pnmer through a second region of the template using unlabeled nucleotides; determining the sequence of a third -1- SUBSTITUTE SHEET (RULE 26) region of the template molecule by extending the primer through the third region using labeled nucleotides; determining the sequence of the second region of the template molecule by comparing the sequence of the first region and the third region to a reference sequence. [6] In some embodiments, the determining (b) the sequence of the first region comprises, for each nucleotide flow in a first plurality of nucleotide flows: providing labeled nucleotides of a respective base type; and detecting a signal indicating a presence or absence of incorporation of labeled nucleotides into the extending primer. [7] In some embodiments, the extending (c) the primer through the second region comprises, for each nucleotide flow in a second plurality of nucleotide flows: providing unlabeled nucleotides of a respective base type. [8] In some embodiments, the extending (c) the primer through the second region comprises, for each nucleotide flow in a second plurality of nucleotide flows: providing unlabeled nucleotides of one or more base types. [9] In some embodiments, the determining (d) the sequence of the third region comprises, for each nucleotide flow in a third plurality of nucleotide flows: providing labeled nucleotides of a respective base type; and detecting a signal indicating a presence or absence of incorporation or labeled nucleotides into the extending primer. [10] In some embodiments, the determining (e) the sequence of the second region comprises aligning the first and third regions of the template molecule to a first and third regions of the reference sequence, respectively, wherein the first and third regions are separated from each other by a second region in the reference sequence. [11] In some embodiments, the sequence of the second region of the template molecule is determined as the sequence of the second region of the reference genome. [12] In some embodiments, the first plurality of nucleotide flows and third plurality of nucleotide flows comprise 50 nucleotide flows, respectively. In some embodiments, the second plurality of nucleotide flows comprises 10 nucleotide flows. [13] In some embodiments, the method further comprises repeating the determining (b) and extending (c) one or more times prior to determining (d) and determining (e). [14] In some embodiments, the template molecule is a nucleic acid. [15] In another aspect, this disclosure provides a method of sequencing a template molecule, comprising: hybridizing a primer to the template molecule to provide a hybridized template; and performing primer extension by providing: i) one or more nucleotide flows comprising labeled nucleotides of a first base type, and ii) one or more nucleotide flows. -2- SUBSTITUTE SHEET (RULE 26) [16] In some embodiments, the primer extension (b) further comprises, in each of