Not using a valid initiator codon and genetic code for virus

[MonicaSteffi]

Dear Developer,

I am trying prokka for viral genome annotation. I got my assembled contigs from spade assembler. When I tried prokka, I got the following warning. Have attached my logfile for your reference. Pls check `[10:55:47] This is prokka 1.12 [10:55:47] Written by Torsten Seemann [email protected] [10:55:47] Homepage is https://github.com/tseemann/prokka [10:55:47] Local time is Mon Feb 27 10:55:47 2023 [10:55:47] You are ra78zut [10:55:47] Operating system is linux [10:55:47] You have BioPerl 1.006924 [10:55:47] System has 56 cores. [10:55:47] Will use maximum of 8 cores. [10:55:47] Annotating as >>> Viruses <<< [10:55:47] Generating locus_tag from 'spade_lim3_rRNA_vul/contigs.fasta' contents. [10:55:47] Setting --locustag MBDGPOCJ from MD5 6bd098c35b7d372b86b01ecd1e9db471 [10:55:47] Re-using existing --outdir prokka_lim3 [10:55:47] Using filename prefix: PROKKA_02272023.XXX [10:55:47] Setting HMMER_NCPU=1 [10:55:47] Writing log to: prokka_lim3/PROKKA_02272023.log [10:55:47] Command: /dss/dsshome1/0F/ra78zut/miniconda3/bin/prokka --outdir prokka_lim3 --addgenes spade_lim3_rRNA_vul/contigs.fasta --kingdom Viruses --force --centre X [10:55:47] Appending to PATH: /dss/dsshome1/0F/ra78zut/miniconda3/bin [10:55:47] Looking for 'aragorn' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/aragorn [10:55:47] Determined aragorn version is 1.2 [10:55:47] Looking for 'barrnap' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/barrnap [10:55:47] Determined barrnap version is 0.9 [10:55:47] Looking for 'blastp' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/blastp [10:55:47] Determined blastp version is 2.5 [10:55:47] Looking for 'cmpress' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/cmpress [10:55:47] Determined cmpress version is 1.1 [10:55:47] Looking for 'cmscan' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/cmscan [10:55:47] Determined cmscan version is 1.1 [10:55:47] Looking for 'egrep' - found /usr/bin/egrep [10:55:47] Looking for 'find' - found /usr/bin/find [10:55:47] Looking for 'grep' - found /usr/bin/grep [10:55:47] Looking for 'hmmpress' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/hmmpress [10:55:47] Determined hmmpress version is 3.3 [10:55:47] Looking for 'hmmscan' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/hmmscan [10:55:47] Determined hmmscan version is 3.3 [10:55:47] Looking for 'java' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/java [10:55:47] Looking for 'less' - found /usr/bin/less [10:55:47] Looking for 'makeblastdb' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/makeblastdb [10:55:47] Determined makeblastdb version is 2.5 [10:55:47] Looking for 'minced' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/minced [10:55:48] Determined minced version is 4.2 [10:55:48] Looking for 'parallel' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/parallel [10:55:48] Determined parallel version is 20230122 [10:55:48] Looking for 'prodigal' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/prodigal [10:55:48] Determined prodigal version is 2.6 [10:55:48] Looking for 'prokka-genbank_to_fasta_db' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/prokka-genbank_to_fasta_db [10:55:48] Looking for 'sed' - found /usr/bin/sed [10:55:48] Looking for 'tbl2asn' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/tbl2asn [10:55:48] Determined tbl2asn version is 25.8 [10:55:48] Using genetic code table 1. [10:55:48] Loading and checking input file: spade_lim3_rRNA_vul/contigs.fasta [10:55:48] Wrote 2 contigs totalling 92486 bp. [10:55:48] Predicting tRNAs and tmRNAs [10:55:48] Running: aragorn -l -gc1 -w prokka_lim3/PROKKA_02272023.fna [10:55:48] Found 0 tRNAs [10:55:48] Disabling rRNA search: --kingdom=Viruses or --norrna=0 [10:55:48] Skipping ncRNA search, enable with --rfam if desired. [10:55:48] Total of 0 tRNA + rRNA features [10:55:48] Searching for CRISPR repeats [10:55:48] Found 0 CRISPRs [10:55:48] Predicting coding sequences [10:55:48] Contigs total 92486 bp, so using meta mode [10:55:48] Running: prodigal -i prokka_lim3/PROKKA_02272023.fna -c -m -g 1 -p meta -f sco -q [10:55:49] Found 118 CDS [10:55:49] Connecting features back to sequences [10:55:49] Not using genus-specific database. Try --usegenus to enable it. [10:55:49] Annotating CDS, please be patient. [10:55:49] Will use 8 CPUs for similarity searching.

--------------------- WARNING --------------------- MSG: Seq [gnl|X|MBDGPOCJ_1]: Not using a valid initiator codon!

--------------------- WARNING --------------------- MSG: Seq [gnl|X|MBDGPOCJ_1]: Not using a valid initiator codon!

[10:55:49] There are still 118 unannotated CDS left (started with 118) [10:55:49] Will use blast to search against /dss/dsshome1/0F/ra78zut/miniconda3/bin/../db/kingdom/Viruses/sprot with 8 CPUs [10:55:49] Running: cat prokka_lim3/sprot.faa | parallel --gnu --plain -j 8 --block 1811 --recstart '>' --pipe blastp -query - -db /dss/dsshome1/0F/ra78zut/miniconda3/bin/../db/kingdom/Viruses/sprot -evalue 1e-06 -num_threads 1 -num_descriptions 1 -num_alignments 1 -seg no > prokka_lim3/sprot.blast 2> /dev/null [10:55:50] Deleting unwanted file: prokka_lim3/sprot.faa [10:55:50] Deleting unwanted file: prokka_lim3/sprot.blast [10:55:50] Labelling remaining 115 proteins as 'hypothetical protein' [10:55:50] Found 0 unique /gene codes. [10:55:50] Fixed 0 colliding /gene names. [10:55:50] Adding /locus_tag identifiers [10:55:50] Assigned 118 locus_tags to CDS and RNA features. [10:55:50] Writing outputs to prokka_lim3/

--------------------- WARNING --------------------- MSG: Seq [MBDGPOCJ_00010]: Not using a valid initiator codon!

--------------------- WARNING --------------------- MSG: Seq [MBDGPOCJ_00064]: Not using a valid initiator codon!

[10:55:50] Generating annotation statistics file [10:55:50] Generating Genbank and Sequin files [10:55:50] Running: tbl2asn -V b -a r10k -l paired-ends -M n -N 1 -y 'Annotated using prokka 1.12 from https://github.com/tseemann/prokka' -Z prokka_lim3/PROKKA_02272023.err -i prokka_lim3/PROKKA_02272023.fsa 2> /dev/null [10:55:50] Deleting unwanted file: prokka_lim3/errorsummary.val [10:55:50] Deleting unwanted file: prokka_lim3/PROKKA_02272023.dr [10:55:50] Deleting unwanted file: prokka_lim3/PROKKA_02272023.fixedproducts [10:55:50] Deleting unwanted file: prokka_lim3/PROKKA_02272023.ecn [10:55:50] Deleting unwanted file: prokka_lim3/PROKKA_02272023.val [10:55:50] Repairing broken .GBK output that tbl2asn produces... [10:55:50] Running: sed 's/COORDINATES: profile/COORDINATES:profile/' < prokka_lim3/PROKKA_02272023.gbf > prokka_lim3/PROKKA_02272023.gbk [10:55:50] Deleting unwanted file: prokka_lim3/PROKKA_02272023.gbf [10:55:50] Output files: [10:55:50] prokka_lim3/PROKKA_02272023.tbl [10:55:50] prokka_lim3/PROKKA_02272023.err [10:55:50] prokka_lim3/PROKKA_02272023.faa [10:55:50] prokka_lim3/PROKKA_02272023.fna [10:55:50] prokka_lim3/PROKKA_02272023.txt [10:55:50] prokka_lim3/PROKKA_02272023.sqn [10:55:50] prokka_lim3/PROKKA_02272023.log [10:55:50] prokka_lim3/PROKKA_02272023.ffn [10:55:50] prokka_lim3/PROKKA_02272023.fsa [10:55:50] prokka_lim3/PROKKA_02272023.tsv [10:55:50] prokka_lim3/PROKKA_02272023.gbk [10:55:50] prokka_lim3/PROKKA_02272023.gff [10:55:50] Annotation finished successfully. [10:55:50] Walltime used: 0.05 minutes [10:55:50] If you use this result please cite the Prokka paper: [10:55:50] Seemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics. 30(14):2068-9. [10:55:50] Type 'prokka --citation' for more details. [10:55:50] Share and enjoy!`

And also when I include gcode as 11, I didn't get any warning as above. Is using -gcode 11 right for viral genome

prokka --outdir prokka_lim3 --addgenes spade_lim3_rRNA_vul/contigs.fasta --kingdom Viruses --force --centre X -gcode 11

[10:58:57] This is prokka 1.12 [10:58:57] Written by Torsten Seemann <[email protected]> [10:58:57] Homepage is https://github.com/tseemann/prokka [10:58:57] Local time is Mon Feb 27 10:58:57 2023 [10:58:57] You are ra78zut [10:58:57] Operating system is linux [10:58:57] You have BioPerl 1.006924 [10:58:57] System has 56 cores. [10:58:57] Will use maximum of 8 cores. [10:58:57] Annotating as >>> Viruses <<< [10:58:57] Generating locus_tag from 'spade_lim3_rRNA_vul/contigs.fasta' contents. [10:58:57] Setting --locustag MBDGPOCJ from MD5 6bd098c35b7d372b86b01ecd1e9db471 [10:58:57] Re-using existing --outdir prokka_lim3 [10:58:57] Using filename prefix: PROKKA_02272023.XXX [10:58:57] Setting HMMER_NCPU=1 [10:58:57] Writing log to: prokka_lim3/PROKKA_02272023.log [10:58:57] Command: /dss/dsshome1/0F/ra78zut/miniconda3/bin/prokka --outdir prokka_lim3 --addgenes spade_lim3_rRNA_vul/contigs.fasta --kingdom Viruses --force --centre X -gcode 11 [10:58:57] Appending to PATH: /dss/dsshome1/0F/ra78zut/miniconda3/bin [10:58:57] Looking for 'aragorn' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/aragorn [10:58:57] Determined aragorn version is 1.2 [10:58:57] Looking for 'barrnap' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/barrnap [10:58:57] Determined barrnap version is 0.9 [10:58:57] Looking for 'blastp' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/blastp [10:58:57] Determined blastp version is 2.5 [10:58:57] Looking for 'cmpress' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/cmpress [10:58:57] Determined cmpress version is 1.1 [10:58:57] Looking for 'cmscan' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/cmscan [10:58:57] Determined cmscan version is 1.1 [10:58:57] Looking for 'egrep' - found /usr/bin/egrep [10:58:57] Looking for 'find' - found /usr/bin/find [10:58:57] Looking for 'grep' - found /usr/bin/grep [10:58:57] Looking for 'hmmpress' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/hmmpress [10:58:57] Determined hmmpress version is 3.3 [10:58:57] Looking for 'hmmscan' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/hmmscan [10:58:57] Determined hmmscan version is 3.3 [10:58:57] Looking for 'java' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/java [10:58:57] Looking for 'less' - found /usr/bin/less [10:58:57] Looking for 'makeblastdb' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/makeblastdb [10:58:57] Determined makeblastdb version is 2.5 [10:58:57] Looking for 'minced' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/minced [10:58:57] Determined minced version is 4.2 [10:58:57] Looking for 'parallel' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/parallel [10:58:57] Determined parallel version is 20230122 [10:58:57] Looking for 'prodigal' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/prodigal [10:58:57] Determined prodigal version is 2.6 [10:58:57] Looking for 'prokka-genbank_to_fasta_db' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/prokka-genbank_to_fasta_db [10:58:57] Looking for 'sed' - found /usr/bin/sed [10:58:57] Looking for 'tbl2asn' - found /dss/dsshome1/0F/ra78zut/miniconda3/bin/tbl2asn [10:58:57] Determined tbl2asn version is 25.8 [10:58:57] Using genetic code table 11. [10:58:57] Loading and checking input file: spade_lim3_rRNA_vul/contigs.fasta [10:58:57] Wrote 2 contigs totalling 92486 bp. [10:58:57] Predicting tRNAs and tmRNAs [10:58:57] Running: aragorn -l -gc11 -w prokka_lim3\/PROKKA_02272023\.fna [10:58:57] Found 0 tRNAs [10:58:57] Disabling rRNA search: --kingdom=Viruses or --norrna=0 [10:58:57] Skipping ncRNA search, enable with --rfam if desired. [10:58:57] Total of 0 tRNA + rRNA features [10:58:57] Searching for CRISPR repeats [10:58:58] Found 0 CRISPRs [10:58:58] Predicting coding sequences [10:58:58] Contigs total 92486 bp, so using meta mode [10:58:58] Running: prodigal -i prokka_lim3\/PROKKA_02272023\.fna -c -m -g 11 -p meta -f sco -q [10:58:58] Found 118 CDS [10:58:58] Connecting features back to sequences [10:58:58] Not using genus-specific database. Try --usegenus to enable it. [10:58:58] Annotating CDS, please be patient. [10:58:58] Will use 8 CPUs for similarity searching. [10:58:58] There are still 118 unannotated CDS left (started with 118) [10:58:58] Will use blast to search against /dss/dsshome1/0F/ra78zut/miniconda3/bin/../db/kingdom/Viruses/sprot with 8 CPUs [10:58:58] Running: cat prokka_lim3\/sprot\.faa | parallel --gnu --plain -j 8 --block 1811 --recstart '>' --pipe blastp -query - -db /dss/dsshome1/0F/ra78zut/miniconda3/bin/../db/kingdom/Viruses/sprot -evalue 1e-06 -num_threads 1 -num_descriptions 1 -num_alignments 1 -seg no > prokka_lim3\/sprot\.blast 2> /dev/null [10:58:59] Deleting unwanted file: prokka_lim3/sprot.faa [10:58:59] Deleting unwanted file: prokka_lim3/sprot.blast [10:58:59] Labelling remaining 115 proteins as 'hypothetical protein' [10:58:59] Found 0 unique /gene codes. [10:58:59] Fixed 0 colliding /gene names. [10:58:59] Adding /locus_tag identifiers [10:58:59] Assigned 118 locus_tags to CDS and RNA features. [10:58:59] Writing outputs to prokka_lim3/ [10:58:59] Generating annotation statistics file [10:58:59] Generating Genbank and Sequin files [10:58:59] Running: tbl2asn -V b -a r10k -l paired-ends -M n -N 1 -y 'Annotated using prokka 1.12 from https://github.com/tseemann/prokka' -Z prokka_lim3\/PROKKA_02272023\.err -i prokka_lim3\/PROKKA_02272023\.fsa 2> /dev/null [10:59:00] Deleting unwanted file: prokka_lim3/errorsummary.val [10:59:00] Deleting unwanted file: prokka_lim3/PROKKA_02272023.dr [10:59:00] Deleting unwanted file: prokka_lim3/PROKKA_02272023.fixedproducts [10:59:00] Deleting unwanted file: prokka_lim3/PROKKA_02272023.ecn [10:59:00] Deleting unwanted file: prokka_lim3/PROKKA_02272023.val [10:59:00] Repairing broken .GBK output that tbl2asn produces... [10:59:00] Running: sed 's/COORDINATES: profile/COORDINATES:profile/' < prokka_lim3\/PROKKA_02272023\.gbf > prokka_lim3\/PROKKA_02272023\.gbk [10:59:00] Deleting unwanted file: prokka_lim3/PROKKA_02272023.gbf [10:59:00] Output files: [10:59:00] prokka_lim3/PROKKA_02272023.tbl [10:59:00] prokka_lim3/PROKKA_02272023.err [10:59:00] prokka_lim3/PROKKA_02272023.faa [10:59:00] prokka_lim3/PROKKA_02272023.fna [10:59:00] prokka_lim3/PROKKA_02272023.txt [10:59:00] prokka_lim3/PROKKA_02272023.sqn [10:59:00] prokka_lim3/PROKKA_02272023.log [10:59:00] prokka_lim3/PROKKA_02272023.ffn [10:59:00] prokka_lim3/PROKKA_02272023.fsa [10:59:00] prokka_lim3/PROKKA_02272023.tsv [10:59:00] prokka_lim3/PROKKA_02272023.gbk [10:59:00] prokka_lim3/PROKKA_02272023.gff [10:59:00] Annotation finished successfully. [10:59:00] Walltime used: 0.05 minutes [10:59:00] If you use this result please cite the Prokka paper: [10:59:00] Seemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics. 30(14):2068-9. [10:59:00] Type 'prokka --citation' for more details. [10:59:00] Share and enjoy!

Thank you in advance