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Hey @rob-p and @Gaura -
I've started working with 10x 5' V2 data, and wanted to utilize alevin-fry for processing of the raw data. I initially found some discussions from @k3yavi on the alevin repo, describing how the only change needed to handle this different data type was to switch alevin's -l ISR to -l ISF (e.g. here). However, I ended up getting far fewer cells detected for each sample, and the results didn't seem to make sense nearly as much as the same data processed via cellranger.
Digging deeper, I discovered a very fruitful and helpful discussion here, where effectively the conclusion was to run alevin with -l ISR as normal, but switch alevin-fry generate-permit-list's expected orientation from -d fw to -d rc. This made a huge difference both in the example data analyzed by @allyhawkinshere and in my own data; for me I detected almost 60% more cells that passed QC filters, and a totally different (and much more sensible) set of clusters and markers characterizing them after making this change.
For others interested in processing data of this type, and assuming it isn't somewhere already that I've missed, it might be helpful to elevate the cellranger vs alevin-fry comparison doc linked above to a polished vignette here and/or mention this more clearly on the main alevin-fry docs or within the generate-permit-list page. I'm curious as well regarding how this approach would/could get handled within the simpleaf and nf-core/scrnaseq frameworks.
Thanks as always!
The text was updated successfully, but these errors were encountered:
hey @jeremymsimon@rob-p I was processing some 10x5' V2 data last week and the number of reads per cell is much fewer than the cellranger output. I then found this issue and indeed changing to -d rc made a difference.
Thanks, @crazyhottommy! @DongzeHE : We should figure out the best way to add this information to the documentation — something like a table of protocols with notes or some such.
Hey @rob-p and @Gaura -
I've started working with 10x 5' V2 data, and wanted to utilize
alevin-fry
for processing of the raw data. I initially found some discussions from @k3yavi on thealevin
repo, describing how the only change needed to handle this different data type was to switchalevin
's-l ISR
to-l ISF
(e.g. here). However, I ended up getting far fewer cells detected for each sample, and the results didn't seem to make sense nearly as much as the same data processed viacellranger
.Digging deeper, I discovered a very fruitful and helpful discussion here, where effectively the conclusion was to run
alevin
with-l ISR
as normal, but switchalevin-fry generate-permit-list
's expected orientation from-d fw
to-d rc
. This made a huge difference both in the example data analyzed by @allyhawkins here and in my own data; for me I detected almost 60% more cells that passed QC filters, and a totally different (and much more sensible) set of clusters and markers characterizing them after making this change.For others interested in processing data of this type, and assuming it isn't somewhere already that I've missed, it might be helpful to elevate the
cellranger
vsalevin-fry
comparison doc linked above to a polished vignette here and/or mention this more clearly on the mainalevin-fry
docs or within thegenerate-permit-list
page. I'm curious as well regarding how this approach would/could get handled within thesimpleaf
andnf-core/scrnaseq
frameworks.Thanks as always!
The text was updated successfully, but these errors were encountered: