Coverage is defined as the number of available sequencing reads per cell per amplicon (reads per cell per antibody for protein).
Expected cell throughput for determining total reads required is ~11,000 cells
This throughput assumes loading at the maximum recommended concentration (3,000 cells/µL). Cell throughput will vary depending on many factors such as whole cells vs nuclei, heme vs solid tumor samples, cell size, and viability. Your typical throughput may vary. If initially too few reads are allocated, libraries may be resequenced and both new and old reads can be processed at the same time to achieve desired coverage.
DNA libraries
Mission Bio recommends an average coverage of 80x read pairs per amplicon.** This represents the total number before filtering to allow for adequate read depth post filtering and cell calling.
Recommended reads for the AML panel (example):
- number of expected cells x number of amplicons x recommended coverage
- 11,000 x 127 x 80 = 1112 M read pairs
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.
** 1 R1 read + 1 R2 read = 1 read pair. Note that illumina references single-end reads and paired-end reads specifications for their flow cells. Our coverage requirements align with the single-end read numbers provided by illumina (ie a NovaSeq 6000 SP flow cell can provide 650-800 M Read Pairs).
Protein libraries
Mission Bio recommends an average coverage of 750x read pairs per cell per antibody. This represents the total number before filtering to allow for adequate read depth post filtering and cell calling.
Recommended reads for a 10-AOC panel (example):
- number of expected cells x number of antibodies x recommended coverage
- 11,000 x 10 x 750 = 75 M read pairs
Hashing Antibodies
If using hashing antibodies increase the number of reads required by 750x per cell, regardless of the number of hashing antibodies used. For example, if using the TotalSeq-D Human Heme Oncology Cocktail 45-plex and 3 hashing antibodies, you would need 11,000 x 46 x 750 =380 M read pairs.
RNA libraries
Mission Bio recommends an average coverage of 100x read pairs per cell per gene. This represents the total number before filtering to allow for adequate read depth post filtering and cell calling.
Recommended reads with the 216 core gene panel (example):
- number of expected cells x number of genes x recommended coverage
- 11,000 x 216 x 100 = 238 M read pairs
Tapestri Bulk DNA Sequencing Libraries
We recommend allocating 1 M reads for any bulk library regardless of the number of amplicons. This also helps account for off target peaks that are often seen in bulk libraries. This may require performing a serial dilution of bulk libraries to get in the appropriate range for pooling.