# 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% 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? 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)
* (TSO500 ctDNA v2) NovaSeq X Series(1.5B): 4 libraries per flow cell
* (TSO500 ctDNA v2) NovaSeq X Series (10B): 24 libraries per flow cell

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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