Whitelist for SMARTSEQ2 technology

[dawe]

Hello, I am processing single cells obtained by SMARTSEQ. I have created a 3-columns tsv file with cell-id and the two read pairs (samples). I launched kb count like

kb count -t 8 -o $OUTDIR --workflow standard -i ${D}/index.idx  -g ${D}/t2g.txt   --parity paired -x SMARTSEQ2 --h5ad --gene-names  samples

I get the h5ad file and it contains less than expected cells, this because filtering has been applied (next time I will apply 0-threshold). However, my issue is that cell names in the AnnData are oligonucleotides, whereas the first column in the samples file contains sample names. I assume the cell order in AnnData is the same specified in the samples file, however it's not clear how to identify sample correspondences and which cells have been filtered.

[Yenaled]

Just look at the unfiltered results to match oligonucleotides to sample names — that’s the easiest way to go. kb count outputs unfiltered results (e.g. there will be a cells_x_genes.barcodes.txt file in your unfiltered results that contains your unfiltered oligonucleotides that you can match to your sample names).

[dawe]

Excellent, thanks

[dawe]

Hello @Yenaled , sorry for reopening the issue. I have checked and

$ wc -l samples 
28 samples
$ wc -l $OUTDIR/counts_unfiltered/cells_x_genes.barcodes.txt  
21 RUN799_783/counts_unfiltered/cells_x_genes.barcodes.txt

so 7 cells are missing. I ran kb count with threshold set to 0, I found a whitelist.txt file which also doesn't match

$ wc -l $OUTDIR/whitelist.txt
29 RUN799_783/whitelist.txt

with an extra cell, matrix.cells file contains 28 cells (with the sample name in the original samples file)

$ wc -l $OUTDIR/matrix.cells
28 RUN799_783/matrix.cells

[dawe]

The file matrix.sample.barcodes should be the appropriate one. Nevertheless, how can I disable bustools filtering?

[Yenaled]

^ah yes, that is correct.

bustools doesn’t do any filtering — what might be happening is that some of your “cells” are having 0 reads being mapped in which case they can’t appear in the final matrix.

[dawe]

I've solved specyfying a fixed barcode whitelist like

AAAAAAAAAAAAAAAT
AAAAAAAAAAAAACCT
AAAAAAAAAAAAAAAA
AAAAAAAAAAAAACGA
AAAAAAAAAAAAACGG
AAAAAAAAAAAAAAAC
AAAAAAAAAAAAAAAT
AAAAAAAAAAAAAAGA
AAAAAAAAAAAAAAGA
AAAAAAAAAAAAACAT
…

That is generated by kb itself