Primary analysis in next generation sequencing

A method comprising:

• providing a first plurality of library molecules on a flow cell, wherein each of the first plurality of library molecules comprise:
• a first insert sequence derived from a first sample source and a first sample index sequence, wherein the first sample index sequence comprises a first k-mer sequence and a first universal sample index sequence, the first universal sample index identifying the first sample source of the first insert sequence;
• providing a second plurality of library molecules on a flow cell, wherein each of the second plurality of library molecules comprise:
• a second insert sequence derived from a second sample source and a second sample index sequence, wherein the second sample index sequence comprises a second k-mer sequence and a second universal sample index sequence, the second universal sample index identifying the second sample source of the second insert sequence;
• conducting, by a sequencing system, k cycles of sequencing reactions of the first and second k-mer sequences, thereby generating a first plurality of flow cell images;
• determining, by a processor, for pixels of the first plurality of flow cell images, pixel intensities from multiple color channels and one or more cycles of the k cycles and a respective signal specificity metric for each pixel, the respective signal specificity metric based on comparison of the pixel intensities;
• and
• determining, by the processor, base calling locations based on the pixel intensities and the respective signal specificity metric.

A system comprising:

• one or more hardware processors;
• one or more data storage devices storing instructions executable by the one or more hardware processors that, when executed, cause the one or more hardware processors to perform operations, the operations comprising:
• providing a first plurality of library molecules on a flow cell, wherein each of the first plurality of library molecules comprise:
• a first insert sequence derived from a first sample source and a first sample index sequence, wherein the first sample index sequence comprises a first k-mer sequence and a first universal sample index sequence, the first universal sample index identifying the first sample source of the first insert sequence;
• providing a second plurality of library molecules on the flow cell, wherein each of the second plurality of library molecules comprise:
• a second insert sequence derived from a second sample source and a second sample index sequence, wherein the second sample index sequence comprises a second k-mer sequence and a second universal sample index sequence, the second universal sample index identifying the second sample source of the second insert sequence;
• conducting, by a sequencing system, k cycles of sequencing reactions of the first and second k-mer sequences, thereby generating a first plurality of flow cell images;
• determining, by a processor, for pixels of the first plurality of flow cell images, pixel intensities from multiple color channels of the sequencing system and one or more cycles of the k cycles and a respective signal specificity metric for each pixel, the respective signal specificity metric based on comparison of the pixel intensities;
• and
• determining, by the processor, base calling locations based on the pixel intensities and the respective signal specificity metric.

One or more non-transitory computer storage media encoded with instructions executable by one or more hardware processors that, when executed, cause the one or more hardware processors to perform operations in sequencing data analysis, the operations comprising:

• providing a first plurality of library molecules on a flow cell, wherein each of the first plurality of library molecules comprise:
• a first insert sequence derived from a first sample source and a first sample index sequence, wherein the first sample index sequence comprises a first k-mer sequence and a first universal sample index sequence, the first universal sample index identifying the first sample source of the first insert sequence;
• providing a second plurality of library molecules on the flow cell, wherein each of the second plurality of library molecules comprise:
• a second insert sequence derived from a second sample source and a second sample index sequence, wherein the second sample index sequence comprises a second k-mer sequence and a second universal sample index sequence, the second universal sample index identifying the second sample source of the second insert sequence;
• conducting, by a sequencing system, k cycles of sequencing reactions of the first and second k-mer sequences, thereby generating a first plurality of flow cell images;
• determining, by a processor, for pixels of the first plurality of flow cell images, pixel intensities from multiple color channels of the sequencing system and one or more cycles of the k cycles and a respective signal specificity metric for each pixel, the respective signal specificity metric based on comparison of the pixel intensities;
• and
• determining, by the processor and before conducting one or more cycles of the sequencing reactions of the first or second insert sequence, base calling locations based on the pixel intensities and the respective signal specificity metric.