Funannotate pipeline steps Confusion

[ufaroooq]

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.

1. Funannotate Clean: to remove conbtigs < 1000bp

2. Funannotate sort: to sort contigs (big -> small)

3. Repeats Annotation using Repeat Modeler/ Repeat Masker

4. Funannotate-Train

funannotate train \
    --input $assembly_in --out $Fanno_out/$prefix/ --left $f1_32931_R1 --right $f1_32931_R2 \
    --jaccard_clip --no_trimmomatic --memory 200G --species "Fo" --isolate 33921 --cpus 94

5. Funannotate Predict

funannotate predict --input $assembly_in -o $Fanno_out/$prefix/ \
    --species "Fo" --isolate 33921 --augustus_species fusarium --busco_seed_species fusarium --cpus 94

6. Funannotate Update

funannotate update --input $Fanno_out/$prefix/ --jaccard_clip --no_trimmomatic \
    --species "Fo" --isolate 33921 --memory 200G --cpus 94

This is where i am confused.

7. 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 #Interproscan creates empty iprscan.xml file Interproscan creates empty iprscan.xml file #987

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.

interproscan \
    --input $Fanno_out/update_results/proteins.fa \
    --output-file-base $Fanno_out/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 $Fanno_out/interproscan_results/ --verbose

#Command:
grep -v "#" proteins.fa.interproscan.gff3 | awk '{print $2}' | sort | uniq

#Results: (what type of peatures where predicted in interproscan resulting file)
CDD, Coils, FunFam, Gene3D, Hamap, MobiDBLite, NCBIfam, PANTHER, Pfam, Phobius, PIRSF, PIRSR, PRINTS, ProSitePatterns, ProSiteProfiles, SFLD, SignalP_EUK, SMART, SUPERFAM, LY, TMHMM

8. antiSMASH Fungi:
   I tried to run antismash via funannotate remote -i fun -m antismash -e your-email@domain.edu but it gave me errors because of the presenvce of multiple CDS with similar coordinates.

So 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, this also generated similar errors but runnign it with --no-abort-on-invalid-records ignored those scaffolds with multiple CDS. (I will look back into it for a solution but for now it worked)

9. Phobius:
   NOw tutorial suggestes to run Phobius, and i am confused if i should run it or not as it already ran during the Interproscan step.

phobius -png -gp -plp \
    $Fanno_out/proteins.fa > $Fanno_out/phobius_results/phobius.txt

## this is still runnig and i am not sure if ir ir running or stuck

10. SignalP:
    Should i also run SignalP individually and then pass the resulting file to Funannotate annotate step or let annotate step itselp run SignalP itself. it is installed.

11. Funannotate annotate:
    Finally i ran withoput phobius results passed

funannotate annotate \
        --input $Fanno_out/ \
        --antismash $Fanno_out/antismash_results/antismash.gbk \
        --iprscan $Fanno_out/interproscan_results/proteins.fa.xml \
        --cpus 94

Here are the results from funannotate check --show-versions

funannotate_dependencies.txt

Questiuons:

1. SHould i run SignalP individually ?
2. should i run Phobius individually or one which ran with Interproscan is enough ?
3. Is this complete approach to annotate assemblies correct or i am missing anything ?

Thank you.