Question: about the use of CellRanger aggr
0
gravatar for Bogdan
8 weeks ago by
Bogdan850
Palo Alto, CA, USA
Bogdan850 wrote:

Dear all,

i am writing to ask please for your suggestions about the appropriate use of CELLRANGER AGGR function in the following context :

we do have 2 experimental replicates for a WT and DISEASE mice :

-- WT_replicate1

-- WT_replicate2

-- DIS_replicate1

-- DIS_replicate2

in order to combine the replicates, shall we just use CELLRANGER AGGR, or shall we just put the fastq files in the same folders i.e.

the fastq files of WT_replicate1, WT_replicate2 in a folder,

and

the fastq files of DIS_replicate1, DIS_replicate2 in another folder ?

thanks a lot,

bogdan

cellranger scrna-seq • 196 views
ADD COMMENTlink modified 8 weeks ago • written 8 weeks ago by Bogdan850

that is a great suggestion, thank you very much !

ADD REPLYlink written 8 weeks ago by Bogdan850

Dear Lior, on a side note, may I ask you please for another suggestion :

what batch-correction method would you recommend for scRNA-seq ? CCA, MNNCORRECT, other methods ?

thanks again !

ADD REPLYlink written 8 weeks ago by Bogdan850
3
gravatar for Lior Pachter
8 weeks ago by
Lior Pachter370
United States
Lior Pachter370 wrote:

You should not process the FASTQs together, because cell barcodes will clash. So you should process them separately. I would not recommend merging the replicates after processing, information about variance within condition will be useful and important for downstream analysis. If you do aggregate the datasets, you should be aware that the aggr function performs a normalization of the data. In the kallisto|bustools workflow we have a tutorial for aggregation that allows you to explore other normalizations.

ADD COMMENTlink written 8 weeks ago by Lior Pachter370
1
gravatar for swbarnes2
8 weeks ago by
swbarnes26.5k
United States
swbarnes26.5k wrote:

cellranger aggr aggregates outputs from multiple runs of cellranger count, normalizing those runs to the same sequencing depth and then recomputing the feature-barcode matrices and analysis on the combined data. The aggr pipeline can be used to combine data from multiple samples into an experiment-wide feature-barcode matrix and analysis.

https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger

ADD COMMENTlink written 8 weeks ago by swbarnes26.5k

Thank you. I have received also an answer from 10X genomics that say :

  1. First you will need to run cellranger count separately four times on each of your 4 libraries:

https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/count:

  1. Then to combine them with cellranger aggr​ on all four libraries at the same time, I would set up a CSV like this:

library_id,molecule_h5,treatment

WT_replicate1,/path/to/molecule_info.h5,WT

WT_replicate2,/path/to/molecule_info.h5,WT

DIS_replicate1,/path/to/molecule_info.h5,DIS

DIS_replicate2,/path/to/molecule_info.h5,DIS

This way you can combine all four libraries in Loupe browser and combine the WT and DIS treatments if you like.

Please see: https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/aggregate

ADD REPLYlink modified 8 weeks ago • written 8 weeks ago by Bogdan850
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