Methods, systems, and computer readable media for evaluating variant likelihood

A non-transitory machine-readable storage medium comprising instructions which, when executed by a processor, to cause the processor to perform a method comprising:

• exposing a plurality of template polynucleotide strands, sequencing primers, and polymerase to a series of flows of nucleotide species according to a predetermined order to form a plurality of primer extensions, wherein the plurality of template polynucleotide strands, sequencing primers, and polymerase are disposed in a plurality of microwells of a microwell array aligned with a corresponding plurality of sensors of a sensor array, wherein the plurality of microwells is at least 105 microwells;
• measuring, using the sensor array, electrical or chemical changes in the plurality of microwells resulting from incorporation of the nucleotide species into the plurality of primer extensions to obtain an ensemble of sequencing reads corresponding to the plurality of primer extensions;
• converting the electrical or chemical changes measured using the sensor array into output signals;
• assigning measured values, based on the output signals, to the ensemble of sequencing reads;
• calculating model-predicted values for the plurality of template polynucleotide strands utilizing a predictive model of nucleotide incorporations resulting from flows of nucleotide species according to the predetermined order;
• modifying at least some model-predicted values using a first bias for forward strands and a second bias for reverse strands, wherein the modifying is based on variations between model-predicted values for different hypothesized sequences obtained using the predictive model of nucleotide incorporations resulting from the flows of nucleotide species according to the predetermined order;
• calculating a measurement confidence value for each read in the ensemble of sequencing reads, wherein the confidence value represents variations between the measured values and the modified model-predicted values;
• and
• identifying a plurality of the reads in the ensemble as corresponding to a variant sequence based on the measurement confidence value.

A system for identifying sequence variants, including:

• a microwell array comprising a plurality of microwells comprising a plurality of template polynucleotide strands, sequencing primers, and polymerase;
• a sensor array comprising a plurality of sensors aligned with the plurality of microwells, wherein the plurality of microwells is at least 105 microwells;
• a machine-readable memory operably coupled to the sensor array;
• and
• a processor operably coupled to the sensor array and the machine-readable memory, the processor being configured to execute machine-readable instructions, which, when executed by the processor, causes the system to perform a method for improving sensor array accuracy in identifying sequence variants, the method comprising:
• exposing the plurality of template polynucleotide strands, the sequencing primers, and the polymerase to a series of flows of nucleotide species according to a predetermined order to form a plurality of primer extensions;
• measuring, using the sensor array, electrical or chemical changes in the plurality of microwells resulting from incorporation of the nucleotide species into the plurality of primer extensions to obtain an ensemble of sequencing reads corresponding to the plurality of primer extensions;
• converting the electrical or chemical changes measured using the sensor array into output signals;
• assigning measured values, based on the output signals, to the ensemble of sequencing reads;
• calculating model-predicted values for the plurality of template polynucleotide strands utilizing a predictive model of nucleotide incorporations resulting from flows of nucleotide species according to the predetermined order;
• modifying at least some model-predicted values using a first bias for forward strands and a second bias for reverse strands, wherein the modifying is based on variations between model-predicted values for different hypothesized sequences obtained using the predictive model of nucleotide incorporations resulting from the flows of nucleotide species according to the predetermined order;
• calculating a measurement confidence value for each read in the ensemble of sequencing reads, wherein the confidence value represents variations between the measured values and the modified model-predicted values;
• and
• identifying a plurality of the reads in the ensemble as corresponding to a variant sequence based on the measurement confidence value.