run10x for non-model organism

Hello again, I wonder if you can help with this issue. I am trying to obtain a loom file from 10x data. I know the program runs well in human 10x data (tried and tested) but when I try to run it on a non-model organims I am working with, it gives me an error which I think it is to do with the type of annotation. I wonder if you could have a look at the error below? Have you seen this error before? I would be very greateful if you could help with this. Many thanks in advance and apologies for the many questions PS: I am have also attached the velocyto log file. Hope you can see it

Cheers, Carmen

2018-05-16 20:42:58,966 - WARNING - The entry exon_number was not present in the gtf file. It will be infferred from the position. 2018-05-16 20:43:02,117 - DEBUG - Parsing Chromosome SM_V7_1 strand - [line 0] 2018-05-16 20:43:07,784 - DEBUG - Fixing corner cases of transcript models containg intron longer than 1000Kbp /nfs/users/nfs_m/my4/scratch/src/miniconda3/lib/python3.6/site-packages/h5py/init.py:36: FutureWarning: Conversion of the second argument of issubdtype from float to np.floating is deprecated. In future, it will be treated as np.float64 == np.dtype(float).type. from ._conv import register_converters as _register_converters Traceback (most recent call last): File “/nfs/users/nfs_m/my4/scratch/src/miniconda3/bin/velocyto”, line 11, in <module> sys.exit(cli()) File “/nfs/users/nfs_m/my4/scratch/src/miniconda3/lib/python3.6/site-packages/click/core.py”, line 722, in call return self.main(*args, **kwargs) File “/nfs/users/nfs_m/my4/scratch/src/miniconda3/lib/python3.6/site-packages/click/core.py”, line 697, in main rv = self.invoke(ctx) File “/nfs/users/nfs_m/my4/scratch/src/miniconda3/lib/python3.6/site-packages/click/core.py”, line 1066, in invoke return _process_result(sub_ctx.command.invoke(sub_ctx)) File “/nfs/users/nfs_m/my4/scratch/src/miniconda3/lib/python3.6/site-packages/click/core.py”, line 895, in invoke return ctx.invoke(self.callback, **ctx.params) File “/nfs/users/nfs_m/my4/scratch/src/miniconda3/lib/python3.6/site-packages/click/core.py”, line 535, in invoke return callback(*args, **kwargs) File “/nfs/users/nfs_m/my4/scratch/src/miniconda3/lib/python3.6/site-packages/velocyto/commands/run10x.py”, line 104, in run10x samtools_memory=samtools_memory, dump=dump, verbose=verbose, additional_ca=additional_ca) File “/nfs/users/nfs_m/my4/scratch/src/miniconda3/lib/python3.6/site-packages/velocyto/commands/_run.py”, line 180, in _run annotations_by_chrm_strand = exincounter.read_transcriptmodels(gtffile) File “/nfs/users/nfs_m/my4/scratch/src/miniconda3/lib/python3.6/site-packages/velocyto/counter.py”, line 512, in read_transcriptmodels tm.chop_if_long_intron() # Change it in place File “/nfs/users/nfs_m/my4/scratch/src/miniconda3/lib/python3.6/site-packages/velocyto/transcript_model.py”, line 93, in chop_if_long_intron long_feats = [i for i in self.list_features if len(i) > maxlen and i.kind == ord(“i”)] File “/nfs/users/nfs_m/my4/scratch/src/miniconda3/lib/python3.6/site-packages/velocyto/transcript_model.py”, line 93, in <listcomp> long_feats = [i for i in self.list_features if len(i) > maxlen and i.kind == ord(“i”)] ValueError: len() should return >= 0

veloctyo.log

[Bio-MingChen]

I recently encountered the same error in my gtf of non-model organism , the reason is exons’ coordinates have overlap for the same transcript resulting in a intron with length less than 0. just look at the image below:

[wuzhigui]

I have got the best way to solve this error ! sed -n '/exon/p' genes_filter1.gtf | sed -n '/gene_name/p' > GRCh38_rmsk.gtf ‘genes_filter1.gtf’ is the gtf file you have used to run 10X cellranger . Then velocyto run -b /barcode/${i}.txt -@ 10 --samtools-memory 1024 --sampleid ${i} -o ./out ${i}_new.bam GRCh38_rmsk.gtf thanks!