In what way should cellranger count be performed , when there are multiple runs for each of the samples ?
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20 days ago

I am trying to analyze single-cell RNA-seq data using cellranger, from publicly available datasets/ studies which have been published. I am getting SRA files from NCBI GEO - the data set currently under consideration is GSE168388 . When I look into this data, I can see that this dataset consists of 5 samples - Healthy donor 1 , Healthy donor 2 , Covid 1, Covid 2 and Covid 3. Within each of these samples there are 4 SRA for 4 runs. So a total of 20 SRA files (5 samples * 4 runs each ) are present.

Now I understand from the cellranger website + tutorials that for every sample, cellranger count needs to be run separately, and then ultimately, the cellranger count outputs can be merged using cellranger aggr. Coming to my problem area -- when I am considering one sample, say for example, Healthy donor 1 - there are 4 SRA files ( for each of the runs) : SRR13870501, SRR13870500, SRR13870499 and SRR13870498 - and therefore I have 4 sets of fastq files for this sample. Where my confusion lies --- when I want to run cellranger count for the Healthy donor 1 sample, do I have to run cellranger count 4 times, for each of the runs , Or can I run cellranger count once instead, specifying all of the above 4 sets of fastq files in the sample id argument of cellranger count command (as shown below) .

So basically, can my command cellranger count command look something like this or not ? cellranger count --id=healthy_donor1 \ --fastqs=/path/to/fastqs \ --sample=SRR13870498,SRR13870499,SRR13870500,SRR13870501 \ --transcriptome=path/to/refdata-gex-GRCh38-2020-A

I would really appreciate any/all help on this matter , cheers :)

count cellranger RNA-seq single-cell • 181 views
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Entering edit mode
20 days ago

If you did one library prep and ran that one library prep multiple times, it is probably easiest to combine the fastqs (make sure to give them a name that perfectly matches the expected Ilumina naming scheme) and give that to cellranger. I am not at all sure that cellranger will willingly combine for different "sample names" into one. But it hardly hurts to try!

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