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aertslab / pySCENIC

pySCENIC is a lightning-fast python implementation of the SCENIC pipeline (Single-Cell rEgulatory Network Inference and Clustering) which enables biologists to infer transcription factors, gene regulatory networks and cell types from single-cell RNA-seq data.
http://scenic.aertslab.org
GNU General Public License v3.0
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[BUG] early termination of pyscenic ctx with no error message #528

Open Mira0507 opened 5 months ago

Mira0507 commented 5 months ago

I've been seeing my pyscenic ctx gets terminated in the middle of the run. My mystery is that it doesn't throw any error message as shown below:


  $ singularity shell -B path/to/working/directory aertslab-pyscenic-0.12.1.sif
  singularity> bash pyscenic_23.sh
  # running..
  # running..
  # running..
  2024-02-09 12:16:56,692 - pyscenic.prune - INFO - Worker mm10_500bp_up_100bp_down_full_tx_v10_clust.genes_vs_motifs.rankings(1): All regulons derived.

  2024-02-09 12:16:56,692 - pyscenic.prune - INFO - Worker mm10_500bp_up_100bp_down_full_tx_v10_clust.genes_vs_motifs.rankings(1): All regulons derived.

  2024-02-09 12:16:56,696 - pyscenic.prune - INFO - Worker mm10_500bp_up_100bp_down_full_tx_v10_clust.genes_vs_motifs.rankings(1): Done.

  2024-02-09 12:16:56,696 - pyscenic.prune - INFO - Worker mm10_500bp_up_100bp_down_full_tx_v10_clust.genes_vs_motifs.rankings(1): Done.
  Singularity>

It comes back to my terminal without the Writing an output file message, which I'm expecting to see if it was successful.

My script is following:

$ cat pyscenic_23.sh
#!/bin/bash

pyscenic ctx \
    scrnaseq-pyscenic-tac1-chat/WT_all_adj.csv \
    cistarget-db/mm10_500bp_up_100bp_down_full_tx_v10_clust.genes_vs_motifs.rankings.feather \
    cistarget-db/mm10_10kbp_up_10kbp_down_full_tx_v10_clust.genes_vs_motifs.rankings.feather \
    --annotations_fname cistarget-db/motifs-v10nr_clust-nr.mgi-m0.001-o0.0.tbl \
    --expression_mtx_fname scrnaseq-pyscenic-tac1-chat/WT_all.loom \
    -o scrnaseq-pyscenic-tac1-chat/WT_all_reg.csv \
    --mask_dropouts \
    --num_workers 32 \

This run ended up creating an empty (zero byte) output file shown here:

$ la | grep WT_all_reg.csv
-rw-rw----. 1 username xxxx   0 Feb  9 15:47 WT_all_reg.csv

I'm assuming that my issue is unrelated to unmatched gene symbols across the input files as I found that over 1000 gene symbols intersected across the ranking (.feather), adjacency (adj.csv), TFs (allTFs_mm.txt), and motif (.tbl) files.

# Load ranking databases
feather = f_db_names['raw'][0]
feather = pd.read_feather(feather)

# Unique gene symbols
feather_g = list(set(list(feather.columns)))  # from feather file
tfs_g = list(pd.read_csv(tfs, header=None).iloc[:,0])  # from TF list
adj_g = list(set(pd.read_csv(adj_csv).loc[:, 'target'].to_list()))  # from adjacency
anno_g = list(set(pd.read_csv(f_motif_path, sep="\t").loc[:,'gene_name']))

# All gene symbols (w duplicates)
all_g = tfs_g + adj_g + feather_g + anno_g

# Retrieve gene symbols intersecting across the feather, tfs, adj, and tbl files
count = pd.Series(all_g).value_counts().to_dict()

# Save gene symbols found in every file to a list
g4 = []
for key, value in count.items():
    if value == 4:
        g4.append(key)

# >>> len(tfs_g)
# 1860

# >>> len(adj_g)
# 20746

# >>> len(feather_g)
# 24069

# >>> len(anno_g)
# 1412

# >>> g4[:6]
# ['Cpeb1', 'Pml', 'Rcor1', 'Rad21', 'Ascc1', 'Prox1']

# >>> len(g4)
# 1054

assert len(g4) > 0, "Ensure to have gene symbols matched across all input files."

Some testings done so far:

I saw a few issue reports on empty output but at least they got Writing an output file message.

My environment was HPC being summarized here:

Single-Cell rEgulatory Network Inference and Clustering (0.12.1+0.gce41b61.dirty)


* Installation method: `singularity build aertslab-pyscenic-0.12.1.sif docker://aertslab/pyscenic:0.12.1`
* Run environment: CLI in singularity
* Package versions: `singularity-ce version 4.0.1`

I think my CLI code is relatively straightforward. I've been also trying with R and python but none of them are successful. I would like to get some hints or suggestions. Thank you very much for your time!
ghuls commented 5 months ago

How much memory did you assign to the job? Try lowering the number of workers.

Mira0507 commented 5 months ago

Interesting. I got it worked out after changing the number of cpus from 32 to 8. (memory was 100g)

Singularity> cat pyscenic_ctx_test.sh
#!/bin/bash

pyscenic ctx \
    scrnaseq-pyscenic-tac1-chat/WT_all_adj.csv \
    cistarget-db/mm10_500bp_up_100bp_down_full_tx_v10_clust.genes_vs_motifs.rankings.feather \
    cistarget-db/mm10_10kbp_up_10kbp_down_full_tx_v10_clust.genes_vs_motifs.rankings.feather \
    --annotations_fname cistarget-db/motifs-v10nr_clust-nr.mgi-m0.001-o0.0.tbl \
    --expression_mtx_fname scrnaseq-pyscenic-tac1-chat/WT_all.loom \
    -o scrnaseq-pyscenic-tac1-chat/WT_all_reg.csv \
    --mask_dropouts \
    --num_workers 8 

ingularity> bash pyscenic_ctx_test.sh
(running...)
2024-02-14 15:44:04,062 - pyscenic.prune - INFO - Worker mm10_10kbp_up_10kbp_down_full_tx_v10_clust.genes_vs_motifs.rankings(3): All regulons derived.

2024-02-14 15:44:04,062 - pyscenic.prune - INFO - Worker mm10_10kbp_up_10kbp_down_full_tx_v10_clust.genes_vs_motifs.rankings(3): All regulons derived.

2024-02-14 15:44:04,081 - pyscenic.prune - INFO - Worker mm10_10kbp_up_10kbp_down_full_tx_v10_clust.genes_vs_motifs.rankings(3): Done.

2024-02-14 15:44:04,081 - pyscenic.prune - INFO - Worker mm10_10kbp_up_10kbp_down_full_tx_v10_clust.genes_vs_motifs.rankings(3): Done.

2024-02-14 15:44:04,147 - pyscenic.cli.pyscenic - INFO - Writing results to file.

$ la | grep WT_all_reg.csv
-rw-rw----. 1 username xxxx 2.5M Feb 14 15:44 WT_all_reg.csv

I've never thought about more cpus causing problems. Do you have any guess about this situation?

Thank you so much for the discussion, @ghuls!

ghuls commented 5 months ago

Each worker loads the databases again, so you need memory for this.