Coverage is defined as the number of available sequencing reads per cell per loci (target for protein).
DNA libraries
Mission Bio recommends an average coverage of 60 – 80x. This represents the total number before filtering.
The total number of reads required for one sample can be calculated as follows:
Total reads with AML panel (example):
- number of expected cells x number of amplicons x recommended coverage
- 7,500 x 127 x 80 = 76.2 M
This 60 – 80X criteria takes the following into account and aims to provide sufficient sequence information needed to perform robust genotyping across all cells and amplicons:
- Not all outputted sequencing reads are associated to cells. Typically, 40 – 70 % of reads are mapped to cells and the rest linked to off-target events, non-incorporated barcodes, compromised cells, etc.
- Not all amplicons are equally well-performing. Amplicons that are amplified by primers in more challenging genomic loci, e.g., GC-rich regions, receive on average a lower number of sequencing reads compared to other amplicons. The 60 – 80X coverage criteria accounts for that imbalance to maximize the read number for low-performing amplicons.
Protein libraries
Mission Bio recommends an average coverage of 500 – 1,000x. This represents the total number before filtering.
The total number of reads required for one sample can be calculated as follows:
Total reads with 10-AOC panel (example):
- number of expected cells x number of amplicons x recommended coverage
- 7,500 x 10 x 1,000 = 75.0 M