QuestionsCategory: Single-Cell RNA-SeqWhat sequencing depth do I need for scRNA-Seq samples?
kellymc@nih.gov asked 3 months ago

What are the considerations for deciding how deeply to sequence a scRNA-Seq sample?

2 Answers
kellymc@nih.gov answered 3 months ago

For droplet-based end-counting libraries we find that about 50,000 reads will cover most use cases for data analysis. You achieve surprising decent separation of diverse cell types even with as low as 5,000 reads per cell (see attached example), but something about 20,000 per cell is more reasonable. If you are trying to discriminate difference between more fine-grained subtypes, more reads should help. Also, if you are looking to run something like a variant detection or something like velocity inference, the additional reads will also help.

Attachments
dadkhahe@nih.gov answered 3 months ago

Minimum Sequencing depth for any type of Library:

 

Gene Expression

20,000 read pairs/ cell

Immune profiling (VDJ)

5,000 read pairs/ targeted cell

Gene Expression with Feature Barcoding technology

Minimum 5,000 read pairs/cell

ATACSeq

25,000 read pairs per nucleus (50,000 individual reads. 25,000 from R1, 25,000 from R2

CNV

750,000 read pairs per cell (for human) enables accurate detection of 2 Mb events per single cell