Methods and processes for calling bases in sequence by incorporation methods

A method of identifying a nucleotide in a nucleic acid sequence, comprising:

• detecting on at least a first location of an optical signal detector, 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, wherein the optical signal detector translates optical signals to optical signal data;
• identifying whether the optical signal data represents an incorporation signal;
• identifying a type of nucleotide associated with the optical signal based upon the location of the detector upon which the optical signal data was derived;
• and
• identifying a base in the nucleic acid sequence from the identification of the nucleotide associated with an optical signal that is also an incorporation 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 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 nucleotide, based upon comparison of the optical signal data to a standard set of optical signal data from different detectable labels, wherein each type of nucleotide has a different detectable label associated therewith;
• calling the base in the template sequence as complementary to the first nucleotide where the optical signal data corresponds to an incorporation event.

A system for determining a sequence of nucleotides in a nucleic acid sequence, comprising:

• a reaction vessel comprising reaction mixture disposed therein, the reaction mixture comprising a nucleic acid polymerase complexed with a template nucleic acid sequence and a primer sequence, and one or more labeled nucleotides, wherein the reaction mixture produces an optical signal when the nucleic acid polymerase reacts with the one or more labeled nucleotides to incorporate the nucleotide into an extended primer;
• an optical train positioned to receive the optical signal from the reaction mixture and image the optical signal onto an imaging detector which translates the optical signal to optical signal data;
• a computer operably coupled to the imaging detector to receive the optical signal data from the detector, and programmed to:
• identify 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;
• correlate the optical signal data to incorporation of a first nucleotide, based upon comparison of the optical signal data 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
• call the base in the template sequence as complementary to the first nucleotide where the optical signal data corresponds to an incorporation event.

A method of identifying a base in a target nucleic acid sequence, comprising:

• receiving a first optical signal pulse from a reaction that produces optical signal pulses in response to incorporation of a nucleotide analog in a template dependent primer extension reaction;
• comparing the first optical signal pulse to a plurality of pulse metrics derived from signal pulses associated with incorporation of one or more different nucleotide analogs in template dependent primer extension reactions;
• and
• identifying the first pulse as a pulse associated with incorporation of specific nucleotide analog.

A method of identifying a nucleotide in a nucleic acid sequence using a computer system comprising:

• at an optical detector pixel array, capturing optical signals from a plurality of locations of sequencing reactions that produce optical signals at incorporation of a labeled nucleotide analog in a template dependent primer extension reaction;
• transmitting captured data to a computer system;
• identifying pixel areas of said optical detector array that correspond to one or more individual reaction locations;
• determining one or more calibration parameters applicable to a plurality of reaction locations;
• determining one or more individualized calibration parameters for one or more individual reaction locations;
• for individual reaction locations, analyzing optical signals captured by corresponding pixels of said optical detector to identify a series of significant pulses;
• for individual reaction locations, classifying pulses in said series of pulses as associated with a type of nucleotide based upon a spectrally distinguishable signal profile of the optical signal pulse;
• for individual reaction locations, calling bases in the nucleic acid sequence from the identification of the nucleotide associated with an optical signal that is also an incorporation signal;
• determining one or more consensus sequences of bases by analyzing called bases and identified pulses from a plurality of locations;
• outputting one or more of pulse characteristics, called bases, or consensus sequences to another computer system or to a user.

A method of identifying a nucleotide in a nucleic acid sequence using a computer system comprising:

• capture pixels from a pixel detector for multiple reaction locations;
• determine correlations (gridding) between pixel sets and reaction locations;
• perform one or more individualized calibrations and store one or more individualized reaction location calibration parameters and/or data sets
• perform one or more collective array calibrations and store one or more collective calibration parameters;
• determine one or more data reduction parameters;
• capture a time sequence of pixel images from a pixel detector for multiple reaction locations of a sequencing array during multiple sequencing reactions;
• perform one or more data reductions on captured pixels;
• for individual reaction locations, extract dye separated spectral traces;
• analyze extracted dye separated traces to determine trace pulse characteristics;
• use trace pulse start and end times (optionally from multiple traces) to identify a pulse in data reduced captured pixel data compare a pulse in data reduced captured pixel data to one or more spectral templates to classify captured pulses as associated with a particular dye-label;
• perform an initial base-calling for individual reaction locations;
• examine pulse data from multiple locations with overlapping sequence data and perform one or more sequence alignments using one or more of initial base calling from individual reaction locations, and probabilities associated with one or more of a classification, a false-positive pulse detection, a false-negative pulse detection;
• and
• output one or more sequences produced by an alignment of pulse data, classification data, and probability data from multiple reaction locations, where output can include presentation to a user on a display or printout, recording sequence data on a tangible medium, or transmitting sequence data to an external computer system.