Read normalization in train fails due to unrecognized flag

[isgilman]

Hello Jon, I've run into a small bug in funannotate train

Are you using the latest release?
version: 1.6.0-dfd805f

Describe the bug
When running train:

funannotate train --input ../MASK/Final_MASK/Pamilis_FHR.clean.sort.FINALsoftmask.fasta --out ./final-train-20Feb2020/ --left ~/scratch60/drought-diff-exp/data/P-amilis_concatenated_R1_001.fastq.gz --right ~/scratch60/drought-diff-exp/data/P-amilis_concatenated_R2_001.fastq.gz --max_intronlen 10000 --species "Portulaca amilis" --cpus 20 --memory 50G

read normalization fails:

[08:46 AM]: OS: linux2, 36 cores, ~ 196 GB RAM. Python: 2.7.15
[08:46 AM]: Running funannotate v1.6.0-dfd805f
[08:46 AM]: Adapter and Quality trimming PE reads with Trimmomatic
[04:50 AM]: Running read normalization with Trinity
[04:50 AM]: Read normalization failed, ./final-train-20Feb2020/training/normalize/left.norm.fq does not exist.

which I traced back to Trinity's insilico_read_normalization.pl script using the funannotate-train.log:

[02/12/20 04:50:53]: Running read normalization with Trinity
[02/12/20 04:50:53]: /gpfs/ysm/project/isg4/conda_envs/super_funannotate/opt/trinity-2.5.1/util/insilico_read_normalization.pl --PARALLEL_STATS --JM 50G --min_cov 5 --max_cov 50 --seqType fq --output ./final-train-20Feb2020/training/normalize --CPU 20 --pairs_together --left ./final-train-20Feb2020/training/trimmomatic/trimmed_left.fastq.gz --right ./final-train-20Feb2020/training/trimmomatic/trimmed_right.fastq.gz
[02/12/20 04:50:54]: Normalized reads: ('./final-train-20Feb2020/training/normalize/left.norm.fq', './final-train-20Feb2020/training/normalize/right.norm.fq', None)
[02/12/20 04:50:54]: Read normalization failed, ./final-train-20Feb2020/training/normalize/left.norm.fq does not exist.

that failed after 1 second, and sure enough, checking trinity_normalization.PE.log:

$ cat trinity_normalization.PE.log
Error, do not understand options: --min_cov 5 

there was an unrecognized flag that stopped the command from running. I tried running this command outside of funannotate, and replicated the error,

$ /gpfs/ysm/project/isg4/conda_envs/super_funannotate/opt/trinity-2.5.1/util/insilico_read_normalization.pl --PARALLEL_STATS --JM 50G --min_cov 5 --max_cov 50 --seqType fq --output ./normalize/ --CPU 20 --pairs_together --left ./trimmomatic/trimmed_left.fastq.gz --right ./trimmomatic/trimmed_right.fastq.gz
Error, do not understand options: --min_cov 5

which can be fixed if the --min_cov 5 option is dropped. I can then pass the normalized reads back to train and continue.

Thanks for your time,
Ian

OS/Install Information

-------------------------------------------------------
Checking dependencies for funannotate v1.6.0-dfd805f
-------------------------------------------------------
You are running Python v 2.7.15. Now checking python packages...
biopython: 1.73
goatools: 0.8.12
matplotlib: 2.2.3
natsort: 6.0.0
numpy: 1.16.3
pandas: 0.24.2
psutil: 5.6.2
requests: 2.21.0
scikit-learn: 0.20.3
scipy: 1.2.1
seaborn: 0.9.0
All 11 python packages installed


You are running Perl v 5.026002. Now checking perl modules...
Bio::Perl: 1.007002
Carp: 1.38
Clone: 0.41
DBD::SQLite: 1.60
DBD::mysql: 4.046
DBI: 1.642
DB_File: 1.852
Data::Dumper: 2.173
File::Basename: 2.85
File::Which: 1.23
Getopt::Long: 2.5
Hash::Merge: 0.300
JSON: 4.00
LWP::UserAgent: 6.36
Logger::Simple: 2.0
POSIX: 1.76
Parallel::ForkManager: 2.02
Pod::Usage: 1.69
Scalar::Util::Numeric: 0.40
Storable: 3.11
Text::Soundex: 3.05
Thread::Queue: 3.13
Tie::File: 1.02
URI::Escape: 3.31
YAML: 1.27
threads: 2.21
threads::shared: 1.59
All 27 Perl modules installed


Checking external dependencies...
CodingQuarry: 2.0
RepeatMasker: RepeatMasker 4.0.8
RepeatModeler: RepeatModeler version DEV
Trinity: 2.5.1
augustus: 3.2.3
bamtools: bamtools 2.4.1
bedtools: bedtools v2.28.0
blat: BLAT v36
diamond: diamond 0.9.24
emapper.py: emapper-0.12.7
ete3: 3.1.1
exonerate: exonerate 2.4.0
fasta: no way to determine
gmap: 2018-07-04
gmes_petap.pl: 4.38
hisat2: 2.1.0
hmmscan: HMMER 3.2.1 (June 2018)
hmmsearch: HMMER 3.2.1 (June 2018)
java: 11.0.1
kallisto: 0.45.1
mafft: v7.407 (2018/Jul/23)
makeblastdb: makeblastdb 2.6.0+
minimap2: 2.16-r922
nucmer: 3.1
pslCDnaFilter: no way to determine
rmblastn: rmblastn 2.6.0+
samtools: samtools 1.9
stringtie: 1.3.6
tRNAscan-SE: 2.0 (December 2017)
tbl2asn: unknown, likely 25.3
tblastn: tblastn 2.6.0+
trimal: trimAl v1.4.rev15 build[2013-12-17]
All 32 external dependencies are installed

Checking Environmental Variables...
$FUNANNOTATE_DB=/gpfs/ysm/scratch60/isg4/Pamilis_funannotate/TRAIN/funannotate_database
$PASAHOME=/gpfs/ysm/project/isg4/conda_envs/super_funannotate/funannotate_deps/PASApipeline
$TRINITYHOME=/gpfs/ysm/project/isg4/conda_envs/super_funannotate/opt/trinity-2.5.1
$EVM_HOME=/gpfs/ysm/project/isg4/conda_envs/super_funannotate/funannotate_deps/evidencemodeler
$AUGUSTUS_CONFIG_PATH=/gpfs/ysm/project/isg4/conda_envs/super_funannotate/config/
$GENEMARK_PATH=/gpfs/ysm/project/isg4/conda_envs/super_funannotate/funannotate_deps/gmes_petap
$BAMTOOLS_PATH=/gpfs/ysm/project/isg4/conda_envs/super_funannotate/bin
All 7 environmental variables are set
-------------------------------------------------------

[nextgenusfs]

Your trinity version is quite old, I guess the min cov option must have been added in v2.6. Current version is at least 2.8.5.

[nextgenusfs]

And there is now a conda package for newer versions of funannotate. This may or may not work for your environment but hopefully simplified install on most systems.