**Setup:** For this exercise you need to be logged in to Uppmax. Follow the UPPMAX login instructions.

Organizing data

Before going into the exercises below, you should create in your home folder a specific folder for this practical session and copy a folder with the course data using:

mkdir annotation course

cd annotation_course

ln -s /sw/courses/annotation/2016/course_material/

mkdir practical1

cd practical1

The data we are providing for the course is organized in the following way in the folder annotation_course:

course_material/data/prokka

course_material/data/human

course_material/data/dmel

course_material/data/blastdb/

course_material/git/

course_material/lib/

course_material/script

course_material/augustus_path/

The folder git contains scripts that we will use to analyse or format data. the folder lib contains perl library required by perl script. The Blastdb folder will be used for the functional annotation exercise tomorrow.

All the files needed for this practical will be in course_material/data/prokka and course_material/augustus_path

Bacterial annotation using Prokka

Before running Prokka on genomes assemblies, it is a good step to start with checking the gene content of the assembly

Checking genes in the assembly

BUSCO2 provides measures for quantitative assessment of genome assembly, gene set, and transcriptome completeness. Genes that make up the BUSCO2 sets for each major lineage are selected from orthologous groups with genes present as single-copy orthologs in at least 90% of the species. It includes 1,066 genes for arthropods, 2,586 for vertebrates, 978 for metazoans, 290 for fungi, 303 for eukaryotes and for bacteria 40 universal marker genes.

You will run BUSCO on 3 bacterial assemblies provided (one E coli, one chlamydia and one streptococcus). We will select the lineage set of bacteria.

BUSCO2 is using augustus to run, as we have no administator rights on uppmax we need to copy the config file of augustus in a folder we can write in and set up the environment.

cp -r ~/annotation_course/course_material/augustus_path .

chmod ug+w -R augustus_path

module load bioinfo-tools
module load BUSCO

AUGUSTUS_CONFIG_PATH=augustus_path

BUSCO -i /home/login/annotation_course/course_material/data/prokka/Chlamydia_trachomatis_a_363.fa -o chlamydia_busco -m geno -c 8 -l /sw/apps/bioinfo/BUSCO/v2_lineage_sets/bacteria_odb9

look at the results of busco in short_summary_chlamydia_busco.txt

Do the same for the two other assemblies and answer those questions again.

Prokka

Prokka is a really easy tool to use for bacterial annotation.

You are going to use the same assemblies you used previously for Busco

module load bioinfo-tools
module unload BUSCO

module load prokka

prokka –help

The goal of the exercise is for you to learn how to use prokka and to annotate the 3 assemblies and then visualize them in IGV.

run prokka without any options and then with options of your choices (we encourage you to try at least the options –proteins and –rfam )

Look at the different results obtained :

You could now also visualise all this information using a genome browser, such as IGV. IGV requires a genome fasta file and any number of annotation files in GTF or GFF3 format (note that GFF3 formatted file tend to look a bit weird in IGV sometimes).

Transfer the gff3 files to your computer using scp:
scp login@milou.uppmax.uu.se:/home/login/annotation_course/practical1/prokka/YOURFILE .