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ablab / IsoQuant

Transcript discovery and quantification with long RNA reads (Nanopores and PacBio)
https://ablab.github.io/IsoQuant/
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TEST FAILED after installation of 3.4.2 #217

Closed giuliafois closed 2 months ago

giuliafois commented 2 months ago

Hi,

I have downloaded the latest version of this tool, and I ran isoquant.py --test as indicated in the README to verify the installation has gone smoothly.

However, this is the result I obtain:

./isoquant.py --test
=== Running in test mode === 
Any other option is ignored 
2024-07-29 10:53:57,485 - INFO - Running IsoQuant version 3.4.2
2024-07-29 10:53:57,491 - INFO - Novel unspliced transcripts will not be reported, set --report_novel_unspliced true to discover them
2024-07-29 10:53:57,491 - INFO -  === IsoQuant pipeline started === 
2024-07-29 10:53:57,491 - INFO - gffutils version: 0.13
2024-07-29 10:53:57,492 - INFO - pysam version: 0.22.1
2024-07-29 10:53:57,492 - INFO - pyfaidx version: 0.8.1.1
2024-07-29 10:53:57,493 - INFO - Checking input gene annotation
2024-07-29 10:53:57,521 - INFO - Gene annotation seems to be correct
2024-07-29 10:53:57,522 - INFO - Converting gene annotation file to .db format (takes a while)...
2024-07-29 10:53:57,706 - INFO - Gene database written to /home/giulia.f/tools/IsoQuant/isoquant_test/chr9.4M.gtf.db
2024-07-29 10:53:57,707 - INFO - Provide this database next time to avoid excessive conversion
2024-07-29 10:53:57,709 - INFO - Indexing reference
2024-07-29 10:53:57,902 - INFO - Converting gene annotation file /home/giulia.f/tools/IsoQuant/isoquant_test/chr9.4M.gtf.db to .bed format
2024-07-29 10:53:57,954 - INFO - Gene database BED written to isoquant_test/chr9.4M.gtf.bed
2024-07-29 10:53:57,957 - INFO - Aligning /home/giulia.f/tools/IsoQuant/tests/simple_data/chr9.4M.ont.sim.fq.gz to the reference, alignments will be saved to /home/giulia.f/tools/IsoQuant/isoquant_test/TEST_DATA/aux/TEST_DATA_chr9.4M.ont.sim_3ba74a_d10dc8_b4cece.bam
2024-07-29 10:53:57,963 - INFO - Running minimap2 version 2.17-r941 (takes a while)
2024-07-29 10:54:01,552 - INFO - Sorting alignments
2024-07-29 10:54:01,667 - INFO - Indexing alignments
2024-07-29 10:54:01,695 - INFO - Loading gene database from /home/giulia.f/tools/IsoQuant/isoquant_test/chr9.4M.gtf.db
2024-07-29 10:54:01,703 - INFO - Loading reference genome from /home/giulia.f/tools/IsoQuant/tests/simple_data/chr9.4M.fa.gz
2024-07-29 10:54:01,722 - INFO - Processing 1 experiment
2024-07-29 10:54:01,722 - INFO - Processing experiment TEST_DATA
2024-07-29 10:54:01,723 - INFO - Experiment has 1 BAM file: isoquant_test/TEST_DATA/aux/TEST_DATA_chr9.4M.ont.sim_3ba74a_d10dc8_b4cece.bam
2024-07-29 10:54:01,724 - INFO - Collecting read alignments
2024-07-29 10:54:01,748 - INFO - Processing chromosome chr9
2024-07-29 10:54:10,320 - INFO - Finished processing chromosome chr9
2024-07-29 10:54:10,329 - INFO - Counting multimapped reads
2024-07-29 10:54:10,330 - INFO - Loading read assignments from isoquant_test/TEST_DATA/aux/TEST_DATA.save_chr9
2024-07-29 10:54:10,376 - INFO - Resolving multimappers
2024-07-29 10:54:10,380 - INFO - Multimappers resolved
2024-07-29 10:54:10,381 - INFO - Alignments collected, overall alignment statistics:
2024-07-29 10:54:10,381 - INFO - primary: 439
2024-07-29 10:54:10,381 - INFO - secondary: 919
2024-07-29 10:54:10,382 - INFO - unaligned: 0
2024-07-29 10:54:10,384 - INFO - Finishing read assignment, total assignments 38, polyA percentage 47.4
2024-07-29 10:54:10,384 - INFO - Read assignments files saved to isoquant_test/TEST_DATA/aux/TEST_DATA.save*. 
2024-07-29 10:54:10,384 - INFO - To keep these intermediate files for debug purposes use --keep_tmp flag
2024-07-29 10:54:10,385 - INFO - Total alignments used for analysis: 38, polyA tail detected in 18 (47.4%)
2024-07-29 10:54:10,385 - INFO - Processing assigned reads TEST_DATA
2024-07-29 10:54:10,385 - INFO - Transcript models construction is turned on
2024-07-29 10:54:10,389 - INFO - Transcript construction options:
2024-07-29 10:54:10,389 - INFO -   Novel monoexonic transcripts will be reported: no
2024-07-29 10:54:10,389 - INFO -   PolyA tails are required for multi-exon transcripts to be reported: no
2024-07-29 10:54:10,390 - INFO -   PolyA tails are required for 2-exon transcripts to be reported: yes
2024-07-29 10:54:10,390 - INFO -   PolyA tails are required for known monoexon transcripts to be reported: yes
2024-07-29 10:54:10,390 - INFO -   PolyA tails are required for novel monoexon transcripts to be reported: yes
2024-07-29 10:54:10,390 - INFO -   Splice site reporting level: only_stranded
2024-07-29 10:54:10,399 - INFO - Processing chromosome chr9
2024-07-29 10:54:10,425 - INFO - Loading read assignments from isoquant_test/TEST_DATA/aux/TEST_DATA.save_chr9
2024-07-29 10:54:14,098 - INFO - Finished processing chromosome chr9
2024-07-29 10:54:14,126 - INFO - Transcript model file isoquant_test/TEST_DATA/TEST_DATA.transcript_models.gtf
2024-07-29 10:54:14,127 - INFO - Extended annotation is saved to isoquant_test/TEST_DATA/TEST_DATA.extended_annotation.gtf
2024-07-29 10:54:14,128 - INFO - Transcript model statistics
2024-07-29 10:54:14,128 - INFO - known: 28
2024-07-29 10:54:14,128 - INFO - novel_in_catalog: 1
2024-07-29 10:54:14,158 - INFO - Gene counts are stored in isoquant_test/TEST_DATA/TEST_DATA.gene_counts.tsv
2024-07-29 10:54:14,158 - INFO - Transcript counts are stored in isoquant_test/TEST_DATA/TEST_DATA.transcript_counts.tsv
2024-07-29 10:54:14,158 - INFO - Read assignments are stored in isoquant_test/TEST_DATA/TEST_DATA.read_assignments.tsv.gz
2024-07-29 10:54:14,158 - INFO - Read assignment statistics
2024-07-29 10:54:14,159 - INFO - ambiguous: 30
2024-07-29 10:54:14,159 - INFO - inconsistent: 79
2024-07-29 10:54:14,159 - INFO - inconsistent_ambiguous: 8
2024-07-29 10:54:14,159 - INFO - noninformative: 2
2024-07-29 10:54:14,159 - INFO - unique: 140
2024-07-29 10:54:14,159 - INFO - unique_minor_difference: 151
2024-07-29 10:54:14,171 - INFO - Processed experiment TEST_DATA
2024-07-29 10:54:14,171 - INFO - Processed 1 experiment
2024-07-29 10:54:14,171 - INFO -  === IsoQuant pipeline finished === 
2024-07-29 10:54:14,174 - ERROR -  === TEST FAILED ===

All dependencies are installed on the vent through the requirements file. The following are my versions of python, samtools and minimap2:

python 3.9.2
samtools 1.20
minimap2 2.17-r941
andrewprzh commented 2 months ago

Dear @giuliafois

Overall, there is no problem here. The tests checks some numbers in the log, which, indeed, depend on the aligner. I think there is a big difference between minimap 2.17 and 2.18 and higher. Could you check if the test passes with higher minimap version?

I'll fix the test anyway.

Best Andrey

giuliafois commented 2 months ago

Dear @andrewprzh,

I tried with the latest version (2.28) and now the test is successful.

Thanks, Giulia