Hello, I would like to add to others' thanks to the authors for making this very impressive resource available! For an analysis on this data I would like to perform, I would need the Cellranger-produced BAM files for each subject (or tube) separately. As @marina_terekhova had suggested in https://www.synapse.org/Synapse:syn49637038/discussion/threadId=10763?replyid=31433 I have read through the paper's methods and the Github repository, but I am still unsure if there is enough information to generate such files. In the Github repo I see in the Snakefile code such as ``` expand("output/{patients}/patient_subset.bam", patients=PATIENTS_POOL) bam=expand("/storage1/fs1/martyomov/Active/NextGenImmunology/BSL3/{project}/align/GEX+FB/{samples}/outs/possorted_genome_bam.bam", samples=SAMPLE, project=PROJECT) ``` which I think reflects the presence of separate BAM files for each subject somewhere? Would it be possible, if those are what I think they are, to upload them to Synapse? Alternatively, if you would be able to share the Cellranger commands with the associated files with the sample hashtag barcodes (as referenced in https://www.synapse.org/Synapse:syn49637038/discussion/threadId=11362 I think) I would be happy to try re-generating them locally. I apologize that my expertise in using Cellranger is somewhat limited to simple usecases (pretty much just using cellranger count on single-well-single-sample sequencing results), and would be very grateful for your guidance in how to access these subject- or tube-specific BAM files. Thank you! Yours, Amit

Created by Amit Lakhanpal amitlakhanpal
Yes, this one. It can split bam files based on the list of provided barcodes. Kind regards, Marina
Dear Marina, Thank you for your advice! The BAM files are certainly quite large and it makes sense not to keep them around if there doesn't seem to be an imminent use of them. I was not aware of the function to split the BAM file (which I think is this? [https://github.com/10XGenomics/subset-bam](url) ), thank you for suggesting that. I'm running Cell Ranger and will hopefully successfully split the file. Thanks again, Yours, Amit
Dear Amit, The file path you're referring to (output/{patients}/patient_subset.bam) is part of the demultiplexing pipeline. These files were intermediate and were deleted right after the demultiplexing step was completed, as they are not required for any subsequent steps in the pipeline and take up a significant amount of storage space (much more than the FASTQ files). Those files can be easily regenerated by running Cell Ranger on each pool and using the split-bam function with the already prepared list of barcodes per donor. You do not need the hashtag files to split the BAM files by donor, and you do not need those to run the Cellranger. If you want to redo the Demultiplexing from scratch, like in the request you are referring to: https://www.synapse.org/Synapse:syn49637038/discussion/threadId=11362, I can send you the hashtags by email as a personal request, as I did for those authors. Kind regards, Marina

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