Classification of nucleic acid templates
AssigneePacific Biosciences of California, Inc.
InventorsBenjamin Flusberg, Stephen Turner, Jon Sorenson, Kevin Travers, Cheryl Heiner
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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.
Claims23
-15. (canceled)
A method for identifying one or more modified bases in a nucleic acid template, the method comprising:
- a) providing a circular nucleic acid template that may have one or more modified bases;
- b) providing a polymerase enzyme having strand displacement activity and capable of processing the circular nucleic acid template;
- c) contacting the circular nucleic acid template with the polymerase enzyme;
- d) monitoring processing of the circular nucleic acid template by the polymerase enzyme, wherein the polymerase enzyme makes multiple passes around the circular nucleic acid template, whereby the processing results in the synthesis of a nascent nucleic acid strand, and wherein the monitoring:
- (i) occurs during the processing, and (ii) detects incorporation of single nucleotides into the nascent nucleic acid strand, thereby generating multiple sequence reads complementary to the circular nucleic acid template;
- e) measuring the kinetics of single nucleotide incorporations;
- and
- f) identifying one or more modified bases within the circular nucleic acid template using the measured kinetics of nucleotide incorporations for the multiple sequence reads.
The method of claim 1 wherein the identifying includes kinetic measurements for nucleotides incorporated upstream and downstream of the one or more modified bases in the nucleic acid template.
The method of claim 1 wherein the kinetic measurements include interpulse duration (IPD) or pulse width (PW).
The method of claim 1 wherein the kinetic measurements include interpulse duration (IPD) and pulse width (PW).
The method of claim 1 wherein the kinetic measurements from the multiple sequence reads are averaged.
The method of claim 1 wherein the polymerase makes 3 or more passes around the circular nucleic acid template.
The method of claim 1 wherein the polymerase makes 5 or more passes around the circular nucleic acid template.
The method of claim 1 wherein the circular nucleic acid template comprises a double stranded nucleic acid capped with hairpins or stem-loop structures.
The method of claim 7 wherein the hairpins or stem-loop structures comprise barcodes.
The method of claim 1 wherein the one or more modified bases is a methylated base, a pseudouridine base, an 7,8-dihydro-8-oxoguanine base, or a 2′-O-methyl derivative base
The method of claim 1 wherein the modification is a methylated cytosine or a methylated adenine base.
The method of claim 1 wherein the one or more modified bases is 5-methyl cytosine.
The method of claim 1 wherein the one or more modified bases is 5-hydroxymethyl cytosine.
The method of claim 1 wherein the circular nucleic acid template comprises DNA.
The method of claim 1, wherein the circular nucleic acid template is DNA.
The method of claim 1, wherein the sample is derived from a human.
The method of claim 1 wherein the one or more organisms are bacteria.
The method of claim 1, prior to providing the circular nucleic acid template the nucleic acid template is subjected to a treatment to alter a modification.
The method of claim 1 wherein the polymerase enzyme is immobilized at optically resolvable reaction sites on the substrate during the monitoring.
The method of claim 1 wherein the circular nucleic acid template comprises regions of internal complementarity.
Dependencies are inferred from the claim text.