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Hi i'm using the human week 10 fetal forebrain dataset(SRP129388) in your paper to do the velocyto. I hava downloaded SRR6470906.sra and SRR6470907.sra and use fastq-dump --split-3 --gzip to get the fastq. And there is one fastq file after fastq-dump named SRR6470906.fastq.gz and SRR6470907.fastq.gz. I renamed them as SRR6470906_S1_L001_R1_001.fastq.gz and SRR6470907_S1_L001_R1_001.fastq.gz. Then i use cellranger to get the output. My code is
cellranger count --localcores=12 --id=SRR6470906_output --transcriptome=/data/cellranger-hg19/refdata-cellranger-hg19-3.0.0 --fastqs=./ --sample=SRR6470906
Finally i use velocyto to run the 10 × data.
/opt/python3.7/bin/velocyto run10x SRR6470906_output /data1/database/GTF/gencode.v27lift37.chr_hg19_annotation.gtf
But the error is like:
Traceback (most recent call last):
File "/opt/python3.7/bin/velocyto", line 11, in <module>
load_entry_point('velocyto==0.17.17', 'console_scripts', 'velocyto')()
File "/opt/python3.7/lib/python3.7/site-packages/click/core.py", line 764, in __call__
return self.main(*args, **kwargs)
File "/opt/python3.7/lib/python3.7/site-packages/click/core.py", line 717, in main
rv = self.invoke(ctx)
File "/opt/python3.7/lib/python3.7/site-packages/click/core.py", line 1137, in invoke
return _process_result(sub_ctx.command.invoke(sub_ctx))
File "/opt/python3.7/lib/python3.7/site-packages/click/core.py", line 956, in invoke
return ctx.invoke(self.callback, **ctx.params)
File "/opt/python3.7/lib/python3.7/site-packages/click/core.py", line 555, in invoke
return callback(*args, **kwargs)
File "/opt/python3.7/lib/python3.7/site-packages/velocyto/commands/run10x.py", line 115, in run10x
samtools_memory=samtools_memory, dump=dump, loom_numeric_dtype=dtype, verbose=verbose, additional_ca=additional_ca)
File "/opt/python3.7/lib/python3.7/site-packages/velocyto/commands/_run.py", line 159, in _run
exincounter.peek(bamfile[0])
File "/opt/python3.7/lib/python3.7/site-packages/velocyto/counter.py", line 158, in peek
raise IOError("The bam file does not contain cell and umi barcodes appropriatelly formatted. If you are runnin UMI-less data you should use the -U flag.")
OSError: The bam file does not contain cell and umi barcodes appropriatelly formatted. If you are runnin UMI-less data you should use the -U flag.
Usage: velocyto run10x [OPTIONS] SAMPLEFOLDER GTFFILE
Try "velocyto run10x --help" for help.
The bam file in SRR6470906_output is here:
SRR6470906.238819510 272 1 10066 1 57M * 0 0 CTACCCCTACCCCTACCCCTACCCCAACCCCTAACCCTAACCCAACCCTAACCCTAA <...A<<..<G<<.<.G<..<.G<..<.G<..<.GG<...<G.GGIIGGGGIGGGGG NH:i:3 HI:i:2 AS:i:44 nM:i:6 RE:A:I CR:Z:AAGCAGTGGTATCAAC CY:Z:GGAAGGGIGGGGGGGG UR:Z:GCAGAGTACA UY:Z:GGIGGGGGGG UB:Z:GCAGAGTACA RG:Z:SRR6470906_output:MissingLibrary:1::
I check my coding and i really don't know what's happening. Why the bam don't have cell and umi barcodes. Can you help me. Thanks a lot!
Activity
gioelelm commentedon Apr 1, 2019
songxiaoning commentedon Apr 1, 2019
@gioelelm Thanks for your reply. But I don't think so because the log shows that velocyto looks for 5000 entrys of the bam file:
I use cellranger --version (2.1.1) and velocyto, version 0.17.17. The output of cell ranger is:
The outs file contains:
And the possorted_genome_bam.bam looks:
Always the 10 ×data contains R1 and R2 fastq but this only contain 1 fastq after fastq-dump. Is this something wrong?
gioelelm commentedon Apr 1, 2019
songxiaoning commentedon Apr 2, 2019
@gioelelm Thanks. I have downloaded the bam file but velocyto requires a output file as this
I still can't run velocyto only have the bam file. Could you please update the sra file?
Thanks a lot for your help.
gioelelm commentedon Apr 2, 2019
songxiaoning commentedon Apr 2, 2019
@gioelelm Thanks! It works with velocyto run. But still i need the counts matrix for pagoda for clustering……
gioelelm commentedon Apr 2, 2019
songxiaoning commentedon Apr 2, 2019
@gioelelm I mean the matirx which contains the UMI counts of every barcode. Then i can use that for clustering.
gioelelm commentedon Apr 2, 2019
songxiaoning commentedon Apr 2, 2019
@gioelelm Yes and thanks!
shaaaarpy commentedon Mar 16, 2020
@songxiaoning can you please tell you machine configurations, as i also have 20gb bam file like yours, and my velocyto run command gets an error message killed while creating loom file.
Also if i sort my bam file, i get umi not found error.
Thank you
denvercal1234GitHub commentedon Nov 15, 2021
@shaaaarpy -- Did you resolve this memory issue?