Best preset for VASA-seq

[fhartley101]

Hello,

I have single cell RNAseq data produced by VASA-seq using the plate based method (https://www.nature.com/articles/s41587-022-01361-8). This protocol results in reads of very different lengths "VASA-plate samples were sequenced on a NextSeq 500, high-output 150 cycles flow cell (Illumina), with the following parameters: Read1 26 cycles, Index 8 cycles, Read2 135 cycles."

I was planning to choose the "Generic low throughput shotgun single-cell protocol" based on the dataset being single-cell data generated through a plate based method that uses full length fragmented RNA. My only concern is that the differing read lengths will affect alignment. It appears that 10X also produces reads of different lengths so I believe MiXCR can handle this, but is there a different setting I should be using to allow for this, or is the default ok?

Many thanks,
Fiona

[mizraelson]

Hi Fiona,
Are there any UMIs or CELL barcodes in R1 or R2? Different read lengths are not an issue at all.

Sincerely,
Mark

[fhartley101]

Thank you for the quick response!

Good to confirm re the different read lengths. There are barcodes and UMIs

"Read1 starts with a 6-nt-long UFI/UMI, followed by an 8-nt-long cell-specific barcode. There are only 384 cell-specific barcodes, each one corresponding to a well in a 384-well plate"

which I've supplied using --tag-pattern "^(UMI:N{6})(CELL:N{8})(R1:*) \ ^(R2:*)" A test run seems to have successfully separated results based on barcode. I couldn't get the sample table/sheet parameter to work properly, but since my results are low throughput it will be easy to assign barcodes back to cells/wells downstream

Thanks again!
Fiona

[mizraelson]

Great! If you have a list of barcodes per well I can make a preset that will automatically assign a well number instead of the CELL barcode.

[fhartley101]

Thank you for your help! My analysis has already finished running and I've assigned well numbers during downstream processing, so no need to do this for my benefit. I've attached the barcode/well info here in case you want to add a preset for this more generally however :)

barcode_info.csv

[mizraelson]

Hi @fhartley101 , following up on this one, the preset is now available in the MiXCR by default and includes the barcodes:

mixcr analyze vasa-seq-vdj input_R1.fastq.gz input_R2.fastq.gz output