theislab / 2019_strunz Goto Github PK

Hi, I tried to reproduce your script, but the download.sh doesn't seem to work, can you send a new download link?

Hello, I try to reproduce the figures which you have published according to your code. But, I get the following error. Could you help me figure out? Thank you so much.

Hi All,

Thank you for the fantastic analysis and data.
I think both whole-lung data and high-resolution data are very good resources for me to get familiar with the scanpy and scVelo packages

As you provided EpiHiRes.h5ad data, the reproduction of Fig. 6 went smoothly. For whole-lung data, I did not find a related h5ad file. To solve this issue, I have tried two methods while both failed.

Method 1. Convert provided whole-lung Seurat object to h5ad by Seurat Tutorial

Run:
SaveH5Seurat(seu.ica, filename = "seu.ica.h5Seurat")

Output:

Creating h5Seurat file for version 3.1.5.9900
Adding counts for RNA
Adding data for RNA
Adding scale.data for RNA
Adding variable features for RNA
Adding feature-level metadata for RNA
Adding cell embeddings for ica
Adding loadings for ica
No projected loadings for ica
Error in attr$write(robj) : HDF5-API Errors:
error #000: ../../src/hdf5-1.12.0/src/H5A.c in H5Awrite(): line 676: buf parameter can't be NULL
class: HDF5
major: Invalid arguments to routine
minor: Bad value

Method 2. Generate loom file from bam file by running velocyto.

At first, I processed SRA files to bam files followed by Drop-seq protocol. I have successfully generated the final clean bam and the expression matrix file. The count value is as same as your GSE141259_WholeLung_rawcounts.mtx.gz.

Then, I ran:
velocyto run -o /scratch/2019_Strunz/Velocity -@ 8 /scratch/2019_Strunz/Bam/SRR10574069_merged_exon_tagged_substitution_repaired_clean.bam /scratch/reference/star/2019_Strunz_STAR_reference/hg19_mm10_transgenes.gtf

Output:

Traceback (most recent call last):
File "/home/.conda/envs/RNAseqPython3/bin/velocyto", line 11, in
sys.exit(cli())
File "/home/.conda/envs/RNAseqPython3/lib/python3.9/site-packages/click/core.py", line 1137, in call
return self.main(*args, **kwargs)
File "/home/.conda/envs/RNAseqPython3/lib/python3.9/site-packages/click/core.py", line 1062, in main
rv = self.invoke(ctx)
File "/home/.conda/envs/RNAseqPython3/lib/python3.9/site-packages/click/core.py", line 1668, in invoke
return _process_result(sub_ctx.command.invoke(sub_ctx))
File "/home/.conda/envs/RNAseqPython3/lib/python3.9/site-packages/click/core.py", line 1404, in invoke
return ctx.invoke(self.callback, **ctx.params)
File "/home/.conda/envs/RNAseqPython3/lib/python3.9/site-packages/click/core.py", line 763, in invoke
return __callback(*args, **kwargs)
File "/home/.conda/envs/RNAseqPython3/lib/python3.9/site-packages/velocyto/commands/run.py", line 113, in run
return _run(bamfile=bamfile, gtffile=gtffile, bcfile=bcfile, outputfolder=outputfolder,
File "/home/.conda/envs/RNAseqPython3/lib/python3.9/site-packages/velocyto/commands/_run.py", line 186, in _run
annotations_by_chrm_strand = exincounter.read_transcriptmodels(gtffile)
File "/home/.conda/envs/RNAseqPython3/lib/python3.9/site-packages/velocyto/counter.py", line 508, in read_transcriptmodels
trid = regex_trid.search(tags).group(1)
AttributeError: 'NoneType' object has no attribute 'group'

As you mentioned in your paper, whole-lung data set were re-analysed to validate findings derived from the high
resolution data set by using Scanpy and scvelo.
Would you mind giving me some clues to get the h5ad file for whoel-lung data?

I appreciate anyone that takes the time to read. Thanks !

Kind regards,
Lu

https://theislab.github.io/LungInjuryRegeneration/
links to http://146.107.176.18:3838/Bleo_webtool_v2 which is down

Very low priority, clearly, but this would avoid having to copy/paste the contents of the sh file or forking