Errno 2 and CMD error in Pasa step in funannotate test

[hassantarabai]

Hello,

I installed an updated funannotate buildup with conda and when testing it, am getting the following error:

#########################################################
Now running funannotate update to run PASA-mediated UTR addition and multiple transcripts
CMD: funannotate update -i rna-seq --cpus 32
#########################################################

[May 07 02:49 PM]: OS: Ubuntu 24.04, 128 cores, ~ 264 GB RAM. Python: 3.8.15
[May 07 02:49 PM]: Running 1.8.17
[May 07 02:49 PM]: No NCBI SBT file given, will use default, for NCBI submissions pass one here '--sbt'
[May 07 02:49 PM]: Found relevant files in rna-seq/training, will re-use them:
GFF3: rna-seq/predict_results/Awesome_rna.gff3
Genome: rna-seq/predict_results/Awesome_rna.scaffolds.fa
Single reads: rna-seq/training/single.fq.gz
Single Q-trimmed reads: rna-seq/training/trimmomatic/trimmed_single.fastq.gz
Single normalized reads: rna-seq/training/normalize/single.norm.fq
Trinity results: rna-seq/training/funannotate_train.trinity-GG.fasta
Long-read results: rna-seq/training/funannotate_long-reads.fasta
PASA config file: rna-seq/training/pasa/alignAssembly.txt
BAM alignments: rna-seq/training/funannotate_train.coordSorted.bam
StringTie GTF: rna-seq/training/funannotate_train.stringtie.gtf
[May 07 02:49 PM]: Reannotating Awesome rna, NCBI accession: None
[May 07 02:49 PM]: Previous annotation consists of: 1,631 protein coding gene models and 112 non-coding gene models
[May 07 02:49 PM]: Existing annotation: locustag=FUN_ genenumber=1743
[May 07 02:49 PM]: Aligning long reads to genome with minimap2
[May 07 02:49 PM]: Adding 42 unique long-reads to Trinity assemblies
[May 07 02:49 PM]: Merging BAM files: rna-seq/update_misc/nano_mRNA.coordSorted.bam, rna-seq/update_misc/trinity.alignments.bam
[May 07 02:49 PM]: Converting transcript alignments to GFF3 format
[May 07 02:49 PM]: Converting Trinity transcript alignments to GFF3 format
[May 07 02:49 PM]: PASA database is SQLite: /home/user/test-rna_seq_8ee2e716-e4af-4b04-9378-38e27a06854e/rna-seq/training/pasa/Awesome_rna_pasa
[May 07 02:49 PM]: Running PASA annotation comparison step 1
[May 07 02:49 PM]: CMD ERROR: /usr/local/software/miniconda3/envs/funannotate/opt/pasa-2.5.3/Launch_PASA_pipeline.pl -c /home/user/test-rna_seq_8ee2e716-e4af-4b04-9378-38e27a06854e/rna-seq/update_misc/pasa/annotCompare.txt -g /home/user/test-rna_seq_8ee2e716-e4af-4b04-9378-38e27a06854e/rna-seq/update_misc/genome.fa -t /home/user/test-rna_seq_8ee2e716-e4af-4b04-9378-38e27a06854e/rna-seq/update_misc/trinity.long-reads.fasta.clean -A -L --CPU 32 --annots /home/user/test-rna_seq_8ee2e716-e4af-4b04-9378-38e27a06854e/rna-seq/update_misc/genome.gff3
#########################################################
Traceback (most recent call last):
File "/usr/local/software/miniconda3/envs/funannotate/bin/funannotate", line 8, in
sys.exit(main())
File "/usr/local/software/miniconda3/envs/funannotate/lib/python3.8/site-packages/funannotate/funannotate.py", line 717, in main
mod.main(arguments)
File "/usr/local/software/miniconda3/envs/funannotate/lib/python3.8/site-packages/funannotate/test.py", line 409, in main
runRNAseqTest(args)
File "/usr/local/software/miniconda3/envs/funannotate/lib/python3.8/site-packages/funannotate/test.py", line 357, in runRNAseqTest
assert 1630 <= countGFFgenes(os.path.join(
File "/usr/local/software/miniconda3/envs/funannotate/lib/python3.8/site-packages/funannotate/test.py", line 45, in countGFFgenes
with open(input, 'r') as f:
FileNotFoundError: [Errno 2] No such file or directory: 'test-rna_seq_8ee2e716-e4af-4b04-9378-38e27a06854e/rna-seq/update_results/Awesome_rna.gff3'

Funannotate build up:

Checking dependencies for 1.8.17

You are running Python v 3.8.15. Now checking python packages...
biopython: 1.79
goatools: 1.4.12
matplotlib: 3.7.3
natsort: 8.4.0
numpy: 1.24.4
pandas: 2.0.3
psutil: 6.0.0
requests: 2.32.3
scikit-learn: 1.3.2
scipy: 1.10.1
seaborn: 0.13.2
All 11 python packages installed

You are running Perl v b'5.032001'. Now checking perl modules...
Carp: 1.50
Clone: 0.46
DBD::SQLite: 1.76
DBD::mysql: 4.046
DBI: 1.643
DB_File: 1.858
Data::Dumper: 2.183
File::Basename: 2.85
File::Which: 1.24
Getopt::Long: 2.58
Hash::Merge: 0.302
JSON: 4.10
LWP::UserAgent: 6.68
Logger::Simple: 2.0
POSIX: 1.94
Parallel::ForkManager: 2.03
Pod::Usage: 1.69
Scalar::Util::Numeric: 0.40
Storable: 3.15
Text::Soundex: 3.05
Thread::Queue: 3.14
Tie::File: 1.06
URI::Escape: 5.17
YAML: 1.30
local::lib: 2.000029
threads: 2.25
threads::shared: 1.61
All 27 Perl modules installed

Checking Environmental Variables...
$FUNANNOTATE_DB=/mnt/data01/databases/funannotate/
$PASAHOME=/usr/local/software/miniconda3/envs/funannotate/opt/pasa-2.5.3
$TRINITY_HOME=/usr/local/software/miniconda3/envs/funannotate/opt/trinity-2.9.1
$EVM_HOME=/usr/local/software/miniconda3/envs/funannotate/opt/evidencemodeler-1.1.1
$AUGUSTUS_CONFIG_PATH=/usr/local/software/miniconda3/envs/funannotate/config/
$GENEMARK_PATH=/mnt/data01/databases/gmes_linux_64_4
All 6 environmental variables are set

Checking external dependencies...
/usr/local/bin/signalp6: line 3: /usr/local/software/signalp6_slow_sequential/signalp-6-package/signalp: Is a directory
PASA: 2.5.3
CodingQuarry: 2.0
Trinity: 2.9.1
augustus: 3.5.0
bamtools: bamtools 2.5.1
bedtools: bedtools v2.31.1
blat: BLAT v39x1
diamond: 2.1.10
emapper.py: 2.1.12
ete3: 3.1.3
exonerate: exonerate 2.4.0
fasta: 36.3.8g
glimmerhmm: 3.0.4
gmap: 2024-11-20
gmes_petap.pl: 4.71_lic
hisat2: 2.2.1
hmmscan: HMMER 3.4 (Aug 2023)
hmmsearch: HMMER 3.3.2 (Nov 2020)
java: 17.0.3-internal
kallisto: 0.46.1
mafft: v7.526 (2024/Apr/26)
makeblastdb: makeblastdb 2.16.0+
minimap2: 2.28-r1209
pigz: 2.8
proteinortho: 6.3.5
pslCDnaFilter: no way to determine
salmon: salmon 0.14.1
samtools: samtools 1.16.1
snap: 2006-07-28
stringtie: 2.2.1
tRNAscan-SE: 2.0.12 (Nov 2022)
tantan: tantan 51
tbl2asn: 25.8
tblastn: tblastn 2.16.0+
trimal: trimAl v1.5.rev0 build[2024-05-27]
trimmomatic: 0.39
ERROR: signalp not installed

Kindly, any idea how to resolve it?

Thank you

[hassantarabai]

Resolved by redoing the buildup.