HI,
I am folloowing Funannotate pipeline for annotation of fungal-genome for which i also have RNA-Seq data. I followed this tutorial on Funannotate-Docs.
I am just confused between some steps because some tools are coming up again and again and i cannot understand if i should run them over again.
So this it the pipeline i followed.
- Funannotate Clean: to remove conbtigs < 1000bp
- Funannotate sort: to sort contigs (big -> small)
- Repeats Annotation using Repeat Modeler/ Repeat Masker
- Funannotate-Train with
genome.fasta RNASeq_1.fq RNASeq_2.fq --jaccard-clip --no-trimmomatic
- Funannotate Predict
- Funannotate Update with
--jaccard_clip --no_trimmomatic
This is where i am confused.
- Interproscan:
At this point tutorial says to run funannotate iprscan which was generating an empty file for me without any log to identify issue, mentioned in one of my old unanswered post here.
So I installed **Interproscan-v5.73-104.0** locally and also installed SignalP-4.1, Phobius-1.0.1 and TmHMM-2.0c as they were showing up as deprecated analysis and i wanted to include them. the resulting .gff3 file shows records for the following
interproscan \
--input $Fanno_out/$prefix/update_results/proteins.fa \
--output-file-base /interproscan_results/proteins.fa.interproscan \
--cpu 90 --disable-precalc --goterms --iprlookup --pathways --seqtype p \
--formats XML,TSV,GFF3 \
--excl-applications SignalP_GRAM_NEGATIVE, SignalP_GRAM_POSITIVE \
--tempdir /interproscan_results/ --verbose
Command:
grep -v "#" proteins.fa.interproscan.gff3 | awk '{print $2}' | sort | uniq
Results:
CDD, Coils, FunFam, Gene3D, Hamap, MobiDBLite, NCBIfam, PANTHER, Pfam, Phobius, PIRSF, PIRSR, PRINTS, ProSitePatterns, ProSiteProfiles, SFLD, SignalP_EUK, SMART, SUPERFAM, LY, TMHMM
- antiSMASH Fungi:
Next i ran **antiSMASH-v7.1.0** which i also installed locally and ran following this command
antismash \
--taxon fungi --cpus 94 --verbose --debug --genefinding-tool none --no-abort-on-invalid-records \
--fullhmmer --cassis --clusterhmmer --tigrfam --asf --cc-mibig --cb-general --cb-subclusters --cb-knownclusters --pfam2go --rre --smcog-trees --tfbs \
--output-basename antismash --output-dir $Fanno_out/$prefix/antismash_results \
$Fanno_out/$prefix/update_results/resulting.gbk
Although, antiSMASH is giving me errors of multiple CDS with same coordinates (because i used RNA-seq data, my .gbk files have multiple transcripts) and i am still looking for a way to solve this.
- Phobius:
At this stage in tutorial, it shows to Optionally run Phobius and i am confused if i should run it or not as it already ran during the Interproscan step.
- SignalP:
Should i also run SignalP individually and then pass the resulting file to Funannotate annotate step or let annotate step itselp run SignalP
- Funannotate annotate:
funannotate annotate \
--input $Fanno_out/$prefix/ \
--antismash $Fanno_out/$prefix/antismash_results/antismash.gbk \
--iprscan $Fanno_out/$prefix/interproscan_results/proteins.fa.xml \
--cpus 94
Questiuons:
- SHould i run SignalP individually ?
- should i run Phobius individually or one which ran with Interproscan is enough ?
- Is this complete approach to annotate assemblies correct or i am missing anything ?
Thank you.
