Help demultiplexing SMART-Seq single cell project run on Illumina
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13 months ago


I apologize in advance for my lack of knowledge- I'm very new to bioinformatics and appear to have stumbled on a complex data processing problem. To give you a sense of what I'm working with, I've successfully completed a bulk RNAseq project but this is my first single cell venture.

I have a single cell RNAseq project structured as 4 GEMs with 4 sample barcodes per GEM. The isolation, library creation, and sequencing steps were done by a commercial entity. As an output, I received I1, I2, R1, and R2 files for the GEX and Cellplex components of each GEM. This is a total of 32 files. My initial plan had been to use the cellranger pipeline to process my files. It has since come to my attention that SMART-Seq chemistries were used by the sequencing company. Specifically, they used the SMART-Seq® v4 3’ DE Kit with NovaSeq. This uses in-line cell barcodes per the documentation. The sample barcodes however, are CMOs. My understanding is that I cannot use cellranger with SMART-Seq, but the demultiplexer on Takara's SMART-Seq website will not demultiplex Illumina outputs. At this point, I've attempted to use cellranger, Starsolo, Takara's Cogent platform, and the scruff package within R to demultiplex these files without success. In case it matters, it's mouse data.

Can someone provide guidance on how to demultiplex, align, and count these files? I'd do anything for a count matrix at this point. Thanks in advance for your help.

demultiplex barcodes processing • 552 views
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Have you tried to use this software provided by Takara? Page indicates that this can be used with the kit you have. If this software does not use independent index files then you can simply use the R1/R2 files as inputs.

If the sequencing company helped you choose this kit then it is irresponsible of them that they are not helping you demultiplex the data.


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