Part 3: Single cell genome assembly

3.5 Gene prediction using Prodigal

Prodigal is a tool that can identify open reading frames (ORFs) in microbial genomes (bacteria or archaea). In this exercise, you will learn how to use Prodigal to predict ORFs and to prepare them for running Blastp later.
Type the following commands to run Prodigal:

For each assembly file run the following command. This command is based on the output file for IDBA assembler, do the same for Ray and SPAdes output.
Hint: use the command ls *.fasta to see all assembly files.
Please, adapt this command according to your file name:

prodigal \
-i G5_${sample}${trim}_IDBA_contig.fasta \
-a G5_${sample}${trim}_IDBA_contig.prodigal.faa \
-o G5_${sample}${trim}_IDBA_contig.prodigal.gff

Take a look at the files produced by Prodigal. For example, type
less G5_${sample}${trim}_IDBA_contig.prodigal.faa and see what the contents look like.
Can you count how many genes are predicted by Prodigal?
Hint: Use the example given in the Part 1 to count the number of genes (look for a repeating pattern in the file and search for that pattern using grep). Tabulate the results in the spreadsheet.

3.6 Running completeness estimates

Of course, you will be interested to know how ‘complete’ is your assembly. In order to determine completeness, we make use of an in-house script that checks for the presence of a number of universally conserved genes. Based on how many of these genes will be identified, an estimation of genome completeness will be made.

From the assembly directory and run the following perl script (repeat the command for all prodigal files):

# Fix dep
module load hmmer/3.1b1-gcc
# End fix
perl /proj/g2015028/nobackup/single_cell_exercises/scripts/micomplete.pl \
-h /proj/g2015028/nobackup/single_cell_exercises/scripts/Bact139.hmm \
-w /proj/g2015028/nobackup/single_cell_exercises/scripts/Bact139.weights \
-p G5_${sample}${trim}_IDBA_contig.prodigal.faa \
-t 8 -e -c 1e-10

If the script completed without any errors, you should see that the script printed something like this:

Completeness: ??
N markers found: ?? out of 139
For all assemblies, report the completeness in the spreadsheet.

The script looks for unique marker genes that are present in single copies in most bacteria and estimates how complete the genome is based on the marker genes found. Completeness is shown as fractions and you should multiply this with 100 to get the percentage completeness.