Methods and compositions for sequencing modified nucleic acids

• US 10,590,484 B2
• Filed: 01/19/2018
• Issued: 03/17/2020
• Est. Priority Date: 03/30/2012
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Korlach, Jonas, Turner, Stephen, Clark, Tyson A.
• Primary Examiner(s): Woolwine, Samuel C
• Application Number:
  US15/875,652
• Publication Number:
  US 20190024162A1
• Time in Patent Office: 788 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/514 Mismatch repair protein
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, abasic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid.

Claims

1. A method of determining whether a cell sample comprises actively dividing cells, the method comprising:

obtaining a non-amplified, double-stranded genomic DNA library from the cell sample;

performing single-molecule sequencing to generate sequence reads for both strands of a plurality of individual double-stranded nucleic acids in the genomic DNA library, wherein the sequence reads comprise both base sequence data and base modification data; and

analyzing the base sequence and base modification of the plurality of individual double-stranded nucleic acids, wherein identification of individual double-stranded nucleic acids in the genomic DNA library that are hemi-modified indicates that the cell sample comprises actively dividing cells.

2. The method of claim 1, wherein the cell sample comprises one or more type of microorganism.

3. The method of claim 2, wherein the cell sample is a metagenomic sample.

4. The method of claim 2, wherein the one or more type of microorganism is selected from the group consisting of:

one or more type of bacteria, one or more type of archaean, one or more type of protozoan, one or more type of fungus, or any combination thereof.

5. The method of claim 2, wherein the one or more microorganism is a pathogenic microorganism.

6. The method of claim 1, wherein the single-molecule sequencing performed is a sequencing-by-incorporation method.

7. The method of claim 1, wherein the single-molecule sequencing performed is a nanopore sequencing method.

8. The method of claim 1, further comprising assembling one or more contigs from the sequence reads of the plurality of individual double-stranded nucleic acids.

9. The method of claim 8, wherein the assembling is based on both the base sequence data and the base modification data.

10. The method of claim 9, wherein the assembling generates a single contig for a chromosome of a microorganism in the cell sample.

11. The method of claim 1, wherein the base modification is selected from the group consisting of:

methylation, hydroxymethylation, glucosylation, and combinations thereof.

12. The method of claim 1, wherein the base modification is methylation.

13. The method of claim 1, wherein the non-amplified, double-stranded genomic DNA library is not bisulfite-converted.