Sequencing and coverage requirements for TruSight Oncology 500 (TSO500) ctDNA

What is the recommended coverage per sample for a TruSight Oncology 500 (TSO500) ctDNA sequencing run?

• Raw coverage of 35,000X.

• Median Exon Coverage (\*) ≥ 1300X.

• Percent (PCT) Exon 1000X (\*) ≥ 80%.

(\*) coverage after read stitching

How many reads/sample are recommended and what is the depth achieved? In order to reach a high analytical sensitivity and specificity at 0.5% VAF, ~800 million paired end reads and ~35,000X coverage are required before read collapsing.

How is the coverage per sample calculated? Is this done with stitched reads, combining the forward and reverse reads? Coverage is calculated using deduplicated, stitched reads.

How many samples can be sequenced together? Which indexes should be used?

• NovaSeq 6000 S2 run: 8 libraries

• NovaSeq 6000 S4 Standard loading run: 16 libraries

• NovaSeq 6000 S4 Xp loading run: 24 libraries (6 libraries per flow cell lane)

There is not a specific recommendation for index choice at the above pooling numbers, as long as all libraries pooled together are uniquely indexed. Typically, labs will use indexes in order by index number.

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