How to get the gft file to run velocyto for velocity analysis?

Question

[Chris]

Hi Biostars,

I try to do velocity analysis and it needs a loom file so I need to run velocyto. To run velocyto, I need a gtf file and after reading the material below, I still get lost on how to get it. Would you please have a suggestion? Thank you so much!
https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/advanced/references

https://velocyto.org/velocyto.py/tutorial/cli.html#run10x-run-on-10x-chromium-samples

Answer 1

[ATpoint]

You need the same GTF file that was used during mapping of your 10x data. If you used CellRanger and it was mouse (given you used recent CellRanger) you can find it in the CellRanger folder under refdata-gex-mm10-2020-A/genes/genes.gtf.

The input for the analysis is the BAM file from CellRanger, and the output is a loom file. loom can be read into R with LoomExperiment::import().

[Chris]

Thanks ATpoint! I think I can get the gtf file at: https://cf.10xgenomics.com/supp/cell-exp/refdata-gex-GRCh38-2020-A.tar.gz but velocyto also need repeat_msk.gtf which I don't know how to get after reading 10x website. Would you please have a suggestion?

velocyto run10x -m repeat_msk.gtf mypath/sample01 somepath/refdata-cellranger-mm10-1.2.0/genes/genes.gtf

[bk11]

You can get from here- UCSC. Just click on get output botton.

[Chris]

Yes, thank you! I figured it out but I got an error:

velocyto run10x -m Hg38_rmsk.gtf WT/outs /cellranger/reference/refdata-cellranger-GRCh38-3.0.0/genes/genes.gtf

Inside WT/outs I have some folders such as filtered_feature_bc_matrix. Inside filtered_feature_bc_matrix, I have barcodes.tsv.gz

• ERROR - This is an older version of cellranger, cannot check if the output are ready, make sure of this yourself
• ERROR - Can not locate the barcodes.tsv file!
  line 91, in run10x.
  bcfile = bcmatches[0].
  IndexError: list index out of range.
  Would you please have a suggestion?

[e.r.zakiev]

a possible suggestion: the masked repeats you were pointed to in this thread are from UCSC while your alignment was done using Ensembl reference?