BioLegend Recommended Sequencing Depth (reads/cell)
TotalSeq™-A, B, and C Libraries
Library Type
|
Minimum Sequencing Depth (reads/cell)
|
3’ and 5’ Gene Expression Library
|
Follow platform recommended guidance
|
Cell Surface Protein Library <100 ADT panel
|
5,000
|
Cell Surface Protein Library ≥100 ADT panel
|
10,000
|
Cell Hashing Libraries*
|
500
|
*Note: If using TotalSeq-A-independent cDNA, ADT and HTO libraries are made allowing the user to attribute reads/cell to each library. This is not possible for TotalSeq-B and -C ADT and HTO libraries as they are constructed in the same cDNA pool and standard reads/cell should be followed based on the total number of antibodies used.
TotalSeq™-D Protein Libraries
Mission Bio recommends an average coverage of 750x per cell per antibody. This represents the total number before filtering to allow for adequate read depth post-filtering and cell calling. 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
V2 5,000 x 10 x 750 = 37.5 M
V3 11,000 x 10 x 750 = 75 M
Sequencing Run Parameters, Library Loading, and Pooling
Refer to the platform-specific user guides for guidance on run parameters, library loading, and pooling. For additional information regarding denaturing and diluting libraries, refer to the corresponding documentation for the sequencer you are using.
If using TotalSeq-A, you can find run parameter and pooling guidance in our TotalSeq-A Antibodies and Cell Hashing with 10x Single Cell 3' Reagent Kit v3.1 (Dual Index) Protocol.
Illumina Knowledge contains additional information, FAQs, troubleshooting articles, and reference material for Illumina products and workflows, covering all Illumina microarrays and every stage of sequencing.
Follow Us