Sequencing and coverage recommendations for TruSight Oncology 500 (TSO500) ctDNA v2

What is the recommended coverage per sample for a TruSight Oncology 500 ctDNA v2 (TSO500 ctDNA v2) sequencing run?

• Raw coverage of 35,000X

• Median Exon Coverage (*) ≥ 1300X

• Percent (PCT) Exon 1000X (*) ≥ 80%

(*) coverage after UMI Collapsing

How many reads/sample are recommended and what is the depth achieved? In order to reach a high analytical sensitivity and specificity at 0.2% Variant Allele Frequency (VAF), ~800 million paired-end reads and ~35,000X coverage are required before read collapsing.

How is the coverage per sample calculated? Coverage is calculated using deduplicated reads.

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

• NextSeq 2000 P4 run: 4 libraries

• NovaSeq 6000 S1 run: 4 libraries

• NovaSeq 6000 S2 run: 8 libraries

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

• NovaSeq X Series 1.5B run: 4 libraries per flow cell

• NovaSeq X Series 10B run: 24 libraries per flow cell

• NovaSeq X Series 25B run: 64 libraries (8 libraries per flow cell lane)

There is no specific recommendation for index selection at the above pooling numbers, as long as all pooled libraries have unique indexes. Typically, labs use indexes in numerical order.

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