Help with error running velocyto

Question

[Chris]

Hi Biostars,

I try to get the loom file to do velocity analysis:

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? Thank you so much!

Update: I added the bam file and rename it to: possorted_genome_bam.bam then:
FileNotFoundError: [Errno 2] No such file or directory: 'samtools': 'samtools'

[Ram]

Is there a WT/_versions file? Also, what is the version of velocyto you're using?

[Chris]

No, I don't have _versions inside WT. I. think it is 0.17.17. I also have another folder MT.

[Ram]

Cellranger produces some files with runtime parameters and settings and dumps them in the same directory as the outs directory. Also, please don't guess versions - double check and add what you can verify. I cannot help you with velocyto but any software issue can only benefit from exact version info.

[Chris]

Yes, I checked. It is 0.17.17.

Answer 1

[bk11]

Hey, use Run on any technique (Advanced use) option for your data. You should be able to run velocyto in your multi-ome data.

velocyto run -b barcodes.tsv -o output_dir -m hg38_rmsk.gtf sample_alignments.bam cellranger/refdata-gex-GRCh38-2020-A/genes/genes.gtf

[Chris]

Thank you! I got the same error after gunzip the barcodes.tsv.gz file:

FileNotFoundError: [Errno 2] No such file or directory: 'samtools': 'samtools'

[bk11]

Did you load or have samtools in the path? Also, do not forget to gunzip hg38_rmsk.gtf.gz > hg38_rmsk.gtf before running velocyto.

[Chris]

The tutorial doesn't mention we need to load samtools. Thank you so much! You are awesome. It is running now.

[bk11]

Sure thing! If it worked, please accept the answer.

[Chris]

Unfortunately, I got a new error:

MemoryError: bam file #0 could not be sorted by cells. This is probably related to an old version of samtools, please install samtools >= 1.6. In alternative this could be a memory error, try to set the --samtools_memory option to a value compatible with your system.
Otherwise sort manually by samtools sort -l [compression] -m [mb_to_use]M -t [tagname] -O BAM -@ [threads_to_use] -o cellsorted_[bamfile] [bamfile]

I have samtools 1.9 and ran this:

velocyto run -b MT/outs/filtered_feature_bc_matrix/barcodes.tsv -o output_dir -m Hg38_rmsk.gtf MT/outs/possorted_genome_bam.bam /cellranger/reference/refdata-cellranger-GRCh38
-3.0.0/genes/genes.gtf --samtools-memory 4000 --samtools-threads 8