What sequencing coverage is recommended?

  • Updated

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
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