Summary

• Goal of the exercise
• Install & configure Artemis
• Get annotation and evidence gff3 files
• Launch Artemis & Load data
• Some Artemis functionalities
• Annotate AT4G32500
• Annotate AT4G32510

Goal of the exercise

• General: Become familiar with the Artemis annotation tool with regard to manual gene curation.
• Specific: Annotate two genes (AT4G32500 and AT4G32510) of the whole Arabidopsis chromosome 4 from the MAKER gene prediction with functional annotation

Install & configure artemis :

Download Artemis from github.io (e.g. v17 20180928).

Increase the memory allocated to Artemis following the FAQ recommendations For instance -Xmx8g (Java max heap size) instead of -Xmx2g in Artemis.cfg if you have 16Gb of RAM on your personal computer

Get annotation and evidence gff3 files :

To facilitate manual annotation, match_part must be linked in Artemis, so gff files need to be reformatted. For your information, from the Eugene-EP IFB appliance:

/root/work_dir/0001/Chr4/
awk 'BEGIN{g=0}{if($3 ~ /EST_match/){g++; print $1"\t"$2"\tgene\t"$4"\t"$5"\t"$6"\t"$7"\t"$8"\tID="g; print  $1"\t"$2"\tmRNA\t"$4"\t"$5"\t"$6"\t"$7"\t"$8"\t"$9";Parent="g}else{if($3 ~ /match_part/){print $0}else{print $0}}}' Chr4.est1.gff3 |sed  's/match_part/exon/' > Chr4.est1_gene.gff3

Reformatted data are centralised on the Slovenian VM, for your information MAKER formatting

awk 'BEGIN{g=0}{if($3 ~ /protein_match/){g++; print $1"\t"$2"\tgene\t"$4"\t"$5"\t"$6"\t"$7"\t"$8"\tID="g; print  $1"\t"$2"\tmRNA\t"$4"\t"$5"\t"$6"\t"$7"\t"$8"\t"$9";Parent="g}else{if($3 ~ /match_part/){print $0}else{print $0}}}' protein_gff\:protein2genome.gff |sed  's/match_part/CDS/' > protein2genome_gene.gff
awk 'BEGIN{g=0}{if($3 ~ /^match$/){g++; print $1"\t"$2"\tgene\t"$4"\t"$5"\t"$6"\t"$7"\t"$8"\tID="g; print  $1"\t"$2"\tmRNA\t"$4"\t"$5"\t"$6"\t"$7"\t"$8"\t"$9";Parent="g}else{if($3 ~ /match_part/){print $0}else{print $0}}}' augustus_masked.gff |sed  's/match_part/exon/' > augustus_masked_gene.gff

Transfer the reformatted annotation files from the Slovenian VM to your personal computer

scp -r -P 65034 gaas23@terminal.mf.uni-lj.si:/home/data/byod/Annotation/ARATH/ARATH04_MAKER/ .
scp -r -P 65034 gaas23@terminal.mf.uni-lj.si:/home/data/byod/Annotation/ARATH/ARATH04_EGN .

Launch artemis & Load data :

• File/Open Tous les fichiers (All files) Chr4

• File/Read An Entry

maker_abinitio_functional.gff

Chr4_egnep.gff3

Chr4.est1_gene.gff3

protein2genome_gene.gff

augustus_masked.gff

Some Artemis functionalities :

• Goto navigator
• To change the feature visusalisation mode Click right & tick ‘One line per feature’ & ‘all features on frame line’ options

• Next methionine: click on a CDS in cyan and type the ‘cmd Y’ (Mac) or ‘ctrl Y’ (Windows / Linux)

• Undo: clicking on a CDS in cyan and type the ‘cmd Y’ (Mac) or ‘ctrl Y’ (Windows / Linux)

Annotate AT4G32500 :

• Shorter an exon and CDS & save CDS(15684277..15684879) -> CDS(15684277..15684843)
• Check the functional annotation Open the Gene Builder by clicking on a feature and then typing the ‘cmd E’ (Mac) or ‘ctrl E’ (Windows / Linux)

Annotate AT4G32510 :

• Set smaller gene as ‘obsolete’

• Set the correct boundaries of the first transcript gene complement(15685825..15688811)

mRNA complement(15685903..15688811)

CDS complement(join(15685903..15686359,15686452..15686777,15686908..15686994,15687072..15687167,15687259..15687432,15687523..15687689,15687772..15687937,15688025..15688163,15688258..15688450,15688544..15688626,15688708..15688811))

• Duplicate the transcript, set the correct boundaries of the second alternative transcript and save mRNA.1 complement(15685825..15688811) CDS.1 complement(join(15685825..15685851,15685927..15686069,15686115..15686359,15686452..15686767,15686874..15686994,15687072..15687167,15687259..15687432,15687523..15687689,15687772..15687937,15688025..15688163,15688258..15688450,15688544..15688626,15688708..15688811))
• Write polypeptide sequence, make blastp on uniprot KB to curate the functional annotation (product, gene_symbol) and save