Feature request : Demultiplexing to process 10x Fixed RNA Profiling kit

[combine-lab]

Feature request : Demultiplexing to process 10x Fixed RNA Profiling kit

[ATpoint]

Thanks @rob-p for opening the issue. Just as a comment, I was wrong to assume that the additional barcode was in R1, and it's called Probe BC, not pCS1, sorry for that. After diving deeper into the protocol, pages 23 and 79 I realize that in fact the additional barcode is called "Probe BC", and it is 8bp long, sequenced as part of Read2, not Read1. Read1 as usual covers the 16bp CB and 12bp UMI, and Read2 covers the ligated gene expression probe (lets call it GExp), followed by a constant linker sequence and the 8bp Probe BC. So in any case I guess one needs to make sure that this constant linker does not end up being quantified together with the gene expression part of the read if Read2. I guess the trick would be to scan Read2 for the constant linker and then consider the part upstream of it for the GExp quantification, and the 8bp downstream for the Probe BC. That probably should then be an additional flag/module in alevin/alevin-fry, something like --chromiumFixedRNA to trigger all this internally, as it seems to be quite different than regular 10x 3' libraries. The GExp size is about 50bp, this constant linker is 16bp.

[laurie-tonon]

Hi,

I was wondering if this functionality has already been implemented in alevin-fry.
I have 10x multiplexed FRP data that I am having trouble analyzing with cellranger, and I would like to try alevin-fry on it.

Thanks