Methods and Processes for Calling Bases in Sequence by Incorporation Methods

A system for identifying a nucleotide in a nucleic acid sequence, comprising:

• an array comprising a plurality of reaction locations, wherein each reaction location contains a respective sequencing reaction, in a plurality of sequencing reactions, that produces optical signals during template dependent primer extension 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;
• an excitation light source providing illumination to the plurality of reaction locations;
• a first detector configured for receiving an optical signal from the sequencing reaction, wherein a first reference spectral signature in the plurality of reference spectral signatures is uniquely associated with a first portion of the first detector and a second reference spectral signature in the plurality of reference spectral signatures is uniquely associated with a second portion of the first detector, wherein the first portion is other than the second portion;
• and
• a data processing unit comprising software for:
• a) identifying the optical signal as an incorporation signal associated with incorporation of a base from one of the differentially labeled nucleotide analogs in the set of differentially labeled nucleotide analogs into a nascent strand, wherein the base is unlabeled following the incorporation;
• b) classifying the incorporation signal as corresponding to one of the labels on a differentially labeled nucleotide analog in the set of 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 in the set of differentially labeled nucleotide analogs;
• and
• c) calling the base in the nucleic acid sequence using the pulse classification and at least one additional signal characteristic after the pulse classification has been provided for the incorporation signal.