Methods and processes for calling bases in sequence by incorporation methods
AssigneePacific Biosciences of California, Inc.
InventorsAustin Tomaney, Kenneth Mark Maxham, David Holden, Kevin Hester, Devon Murphy, Patrick Marks
Life of this patent
Estimating the term…
Abstract
Computer implemented methods, and systems performing such methods for processing signal data from analytical operations and systems, and particularly in processing signal data from sequence-by-incorporation processes to identify nucleotide sequences of template nucleic acids and larger nucleic acid molecules, e.g., genomes or fragments thereof.
Claims24
A method of identifying a nucleotide in a nucleic acid sequence, comprising:
- receiving an optical signal from a reaction that produces optical signals in response to incorporation of a nucleotide analog in a template dependent primer extension reaction;
- identifying the optical signal as an incorporation signal;
- identifying a type of nucleotide associated with the optical signal based upon a spectrally distinguishable signal profile of the optical signal;
- identifying a base in the nucleic acid sequence from both the identification of the nucleotide associated with an optical signal that is also an incorporation signal and a signal environment around the optical signal.
A computer implemented process for calling bases in a nucleic acid sequencing process, comprising:
- receiving optical signal data detected from a reaction of a polymerase/template/primer sequence complex and a plurality of detectably labeled nucleotides;
- identifying the optical signal data as corresponding to a series of incorporation events if a set of optical signals in the optical signal data exceed one or more of a signal intensity threshold and a signal duration threshold;
- correlating a first optical signals in the optical signal data to incorporation of a first nucleotide, based upon a) comparison of the first optical signal to a standard set of optical signal data from different detectable labels, wherein each type of nucleotide has a different detectable label associated therewith, and b) characteristics of a subset of optical signals in the optical signal data that neighbor the first optical signal;
- and
- calling the base in the template sequence as complementary to the first nucleotide where the first optical signal data corresponds to an incorporation event.
A method of identifying a base in a target nucleic acid sequence, comprising:
- receiving a plurality of optical signal pulses from a reaction that produces the optical signal pulses in response to incorporation of a plurality of nucleotide analogs in a template dependent primer extension reaction;
- comparing a first optical signal pulse in the plurality to a set of pulse metrics derived from optical signal pulses associated with incorporation of one or more different nucleotide analogs in template dependent primer extension reactions, where the set of pulse metrics for a given nucleotide analog includes metrics corresponding to the incorporation of the given nucleotide analog and incorporation of additional nucleotide analogs that neighbor the given nucleotide analog;
- and
- identifying the first optical signal pulse as a pulse associated with incorporation of specific nucleotide analog.
A method of base classification comprising:
- providing a classified pulse list, where each pulse in the list has been classified as belonging to a particular dye spectrum;
- providing a machine learning algorithm;
- using the machine learning algorithm to classify a first classified pulse in the classified pulse list as a true base incorporation event or a false base incorporation event based upon one or more pulse metrics for the first classified pulse and at least one adjacent classified pulse in the pulse list, thereby generating a base classification for the first classified pulse.
Dependencies are inferred from the claim text.