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US 9,175,343 B2Granted
Methods and processes for calling bases in sequence by incorporation methods
AssigneePacific Biosciences of California, Inc.
InventorsAustin B. Tomaney, Kenneth Mark Maxham, David Holden, Kevin Hester, Devon Murphy, Patrick Marks
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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.
Claims22
A method of identifying a nucleotide in a nucleic acid sequence, comprising:
- receiving an optical signal from a reaction that produces optical signals during a template dependent primer extension reaction comprising a set of differentially labeled nucleotide analogs, the analogs comprising different labels in a plurality of different labels, wherein each respective different label in the plurality of different labels is associated with a unique reference spectral signature in a plurality of reference spectral signatures, and wherein a first reference spectral signature in the plurality of reference spectral signatures is uniquely associated with a first portion of a two-dimensional detector and a second reference spectral signature in the plurality of reference spectral signatures is uniquely associated with a second portion of the two-dimensional detector, wherein the first portion is other than the second portion;
- identifying the optical signal as an incorporation signal associated with incorporation of a base from one of the differentially labeled nucleotide analogs into a nascent strand, wherein the base is unlabeled following the incorporation;
- classifying the incorporation signal as corresponding to one of the labels on one of the differentially labeled nucleotide analogs based upon a plurality of pulse metrics for the incorporation signal, wherein the plurality of pulse metrics include both an intensity and a measured spectral signature, and wherein the classifying comprises matching the measured spectral signature to a reference spectral signature in the plurality of reference spectral signatures thereby providing a pulse classification of the incorporation signal that is correlated to a particular one of the differentially labeled nucleotide analogs;
- and
- once the pulse classification has been provided for the incorporation signal, calling the base in the nucleic acid sequence using the pulse classification and at least one additional signal characteristic.
2 more elements
A method for calling bases comprising:
- receiving optical signal data detected from a reaction of a polymerase/template/primer sequence complex and a detectably labeled nucleotide;
- identifying the optical signal data as corresponding to an incorporation event, if the optical signal data exceeds one or more of a signal intensity threshold and a signal duration threshold;
- correlating the optical signal data to incorporation of a first base from a first nucleotide, based upon comparison of the optical signal data to a standard data set comprising data from a plurality of different detectable labels, wherein each type of nucleotide in a plurality of nucleotide types has a different detectable label in the plurality of different detectable labels associated therewith, and further wherein the optical signal data comprises at least intensity and measured spectral data, wherein
- each respective different label in the plurality of different labels is associated with a unique reference spectral signature in a plurality of reference spectral signatures,
- a first reference spectral signature in the plurality of reference spectral signatures is uniquely associated with a first portion of a two-dimensional detector and a second reference spectral signature in the plurality of reference spectral signatures is uniquely associated with a second portion of the two-dimensional detector, wherein the first portion is other than the second portion, and
- the correlating comprises matching the measured spectral data to a reference spectral signature in the plurality of reference spectral signatures;
- and
- once the optical signal data has been correlated to incorporation of the first base from the first nucleotide, calling the base in the template sequence as complementary to the first nucleotide using a plurality of signal characteristics.
5 more elements
Dependencies are inferred from the claim text.