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-   -   Advice on how much data output to specify for single Cell RNA seq of human PBMCs? (http://seqanswers.com/forums/showthread.php?t=93954)

CharlesG 04-20-2020 06:44 AM

Advice on how much data output to specify for single Cell RNA seq of human PBMCs?
 
Hi everyone, I am a novice in to single cell RNA seq and my colleagues and I have sent PBMCs of one patient (before and after treatment) to Novogene for paired-end single cell RNA sequencing. We are interested in obtaining as much biological information as possible from the sequenced PBMCs including sepration of sub-populations, differential expression analysis, gene-gene correlation or covariation, possible discovery of novel cell types.

Novogene has sent us an urgent email asking us how much data to output from the PBMCs and also if we need information about the sequencing saturation (we will be charged more for this). They can output between 12 million bp and 50 million bp. My colleagues and I have searched the internet but cannot find exactly which information to provide to Novogene in this regard. Please can any one help us with some advice? So far all we know is that 30000 reads per cell is suffifient to seprate different populations of PBMCs.Thank you in advance for your kind help.

GenoMax 04-20-2020 07:20 AM

Are you using 10x? If so take a look at: https://kb.10xgenomics.com/hc/en-us/...ion-libraries-

CharlesG 04-20-2020 07:32 AM

Thank you very much for the very helpful suggestion. However I still wish to know how much data output in Giga bases should I specify to Novogene for a 90% sequencing saturation level and 30000 reads per cell?

GenoMax 04-20-2020 08:10 AM

How many cells did you submit?

CharlesG 04-20-2020 08:14 AM

Thanks a lot for helping. We submitted approximately 10000 (ten thousand cells) per condition (before and after treatment).

GenoMax 04-20-2020 08:51 AM

If we assume all 10000 cells survived. You want a minimum 50000 reads per cell so that is 500M reads * 2 conditions = 1 Billion clusters/single-end reads total. Sequencing length if fixed based on the kit used so you should just worry about reads than basepairs.

According to 10x this will give you 30-50% saturation. If you need more saturation then you will need to sequence more. This assumes that libraries work/sequence well.

CharlesG 04-20-2020 09:07 AM

Thank you very much.


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