Find pan-genomes using Roary (using the commandline)

A concept in comparative microbial genomics is core and pan genomes. If we analyse DNA from several bacterial strains, we may want to know which genes they have in common and which are unique to some strains.

  • The core genome is the group of genes shared by all strains in the clade of interest. Gene sequences are similar but not necessarily identical. “Core genome SNPs” are those SNPs found in the genes in the core genome; i.e. at a particular site, the nucleotide varies. We can use these SNPs to infer relationships between the strains.

  • The accessory genome is the group of genes that are not in all the strains. These genes may be in one or more strain.

  • The pan genome is the sum of the core and accessory genomes. That is, a combination of all the genes that are found in the clade of interest.

Bacteria can horizontally-transfer genes to other bacteria via plasmids, and so their accessory genome can be large relative to those of eurkaryotes. Bacterial accessory genomes often house genes for drug resistance.

This tutorial demonstrates how to calculate the pan and core genomes of a set of input bacterial samples, using the tool Roary.

Get data

Input:

  • annotated genome in GFF3 format, per sample (e.g. output from Prokka)
  • samples must be from same species

Our data:

  • Five strains of Staphylococcus aureus, in .gff format.

Run

Run roary

roary -e --mafft -p 8 *.gff
  • -e --mafft aligns the core genes using the tool MAFFT
  • -p 8 uses 8 threads

What does Roary do

  • converts coding sequences into protein sequences
  • clustered these protein sequences by several methods
  • further refines clusters into orthologous genes
  • for each sample, determines if gene is present/absent: produces gene_presence_absence.csv
  • uses this gene p/a information to build a tree, using FastTree: produces accessory_binary_genes.fa.newick
  • overall, calculates number of genes that are shared, and unique: produces summary_statistics.txt
  • aligns the core genes (if option used, as above) for downstream analyses

Output

Core and pan genes

Open the summary file:

less summary_statistics.txt

This shows a table of counts of shared genes (core genome) and total genes (pan genome).

stats

What are the core genes?

query_pan_genome -a intersection -o core_genome_results *.gff

What are the accessory genes?

query_pan_genome -a complement -o accessory_genome_results *.gff

What are the pan genes?

query_pan_genome -a union -o pan_genome_results *.gff

View: e.g.

less core_genome_results | column -t

Gene presence/absence

Transfer the file gene_presence_absence.csv to your local computer and view in spreadsheet software.

  • Search for the gene that confers methicillin resistance. It is only found in some of the strains.

Vizualize with Phandango

Copy these files to your local computer:

  • accessory_binary_genes.fa.newick
  • gene_presence_absence.csv

Go to http://phandango.net

  • drag and drop the two files onto the landing page.
  • view the tree of samples and their core and pan genomes

phandango