Advice on how much data output to specify for single Cell RNA seq of human PBMCs?

CharlesG · 04-20-2020, 07:44 AM

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, 08:20 AM

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

CharlesG · 04-20-2020, 08:32 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, 09:10 AM

How many cells did you submit?

CharlesG · 04-20-2020, 09:14 AM

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

GenoMax · 04-20-2020, 09:51 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, 10:07 AM

Thank you very much.

04-20-2020, 07:44 AM	#1
CharlesG Junior Member Location: Germany Join Date: Jul 2019 Posts: 8	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.

04-20-2020, 08:32 AM	#3
CharlesG Junior Member Location: Germany Join Date: Jul 2019 Posts: 8	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? Last edited by CharlesG; 04-20-2020 at 08:37 AM.

04-20-2020, 09:51 AM	#6
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,089	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. Last edited by GenoMax; 04-20-2020 at 01:18 PM.

04-20-2020, 08:20 AM	#2
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,089	Are you using 10x? If so take a look at: https://kb.10xgenomics.com/hc/en-us/...ion-libraries-

04-20-2020, 09:10 AM	#4
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,089	How many cells did you submit?

04-20-2020, 09:14 AM	#5
CharlesG Junior Member Location: Germany Join Date: Jul 2019 Posts: 8	Thanks a lot for helping. We submitted approximately 10000 (ten thousand cells) per condition (before and after treatment).

04-20-2020, 10:07 AM	#7
CharlesG Junior Member Location: Germany Join Date: Jul 2019 Posts: 8	Thank you very much.