Visualizing the pan-genome

ASM Meeting - Melbourne - Tuesday 8 July 2014

Dr Torsten Seemann

Introduction

The “genome”

Two historical definitions:

● The set of “genes” in an organism○ traditionally just protein coding genes○ (should) now include non-coding RNAs

● The DNA sequence of all replicons○ ignore epigenetic decorations

The “pan-genome”

For a given set of strains:

● The full complement of genes○ orthologous genes only count once○ ignoring paralogs with identical function

● The set of unique DNA segments○ ignore epigenetic decorations○ will include intergenic regions like promoters etc.

Core

Disp.

Disp.Disp.

UniqueUnique

Unique

Three genomesCoreIn all 3 strains (∈ N strains)

DispensableIn 2 strains(∈ [2,N-1] strains)

UniqueIn only 1 strain(∈ 1 strain)

Open vs. closed pan-genomes

How to compute it

Whole genome alignment

● Align whole genomes○ typically at DNA level○ can use AA level if genomes are divergent

● Software○ Mauve, MuMmer, ...

A ================================ ==== ==== B ======== =================== ================ ====C ========================== ============ =====

Pan ======================================================Core ===== =================== ===== = Uniq ====

Ortholog clustering

● Group orthologous proteins together○ all versus all protein comparison (not scalable)

○ information used: sequence similarity, synteny

○ difficulty increases with taxa distance

○ depends on annotation quality!

● Software○ ProteinOrtho5, OrthoMCL, cd-hit, uclust, blastclust

● Databases○ COG, NOG, EggNOG, ... mostly uncurated

Another giant spreadsheet CLUSTER STRAIN1 STRAIN2 STRAIN3

00001 DNO1000 EHEC1000 MRSA_1000

00002 DNO1001 EHEC1002 MRSA_1001

00003 DNO1002 EHEC1003 MRSA_1002

00004 DNO1003 EHEC1004 MRSA_1003

00005 DNO1004 EHEC1005 MRSA_1022

: : : :

02314 DNO1005 na MRSA_1023

02315 DNO1451 EHEC3215 na

02316 na EHEC3216 MRSA_1923

: : : :

04197 DNO1456 na na

04198 na EHEC3877 na

04199 na na MRSA_0533

Core

Dispensable

Strain-specific

Venn will it end?

But we want to explore!

FriPan

Pan-genome map

One row per strain

Mini-map of whole pan-genome

One column per geneGrey means not present

Zooming: brush along the mini-map

Where we are in within whole pan-genome

Each block is a gene:Green=presentGrey=absent

Strains

Dendrogram

This uses gene absence and presence.This is not a SNP based phylogeny.

Dendrogram: Horizontal

Radial phylogram

Radial dendrograms work well for >100 taxa

Dendrogram: Radial

MDS plot: multi-dimensional scaling

Reduces 1000s of dimensions to the 2 biggestRelated to principal component analysis (PCA)

MDS plot

Shows 4 main groups or “clades”

~ no. of unshared genes

Identify outliers: Really different, or bad annotation?

Colour & Order

What each colour means

Choose attributes to sort and colour on

Annotation

Hover over a gene to see its annotation

Key features

● Interactive

○ can select, re-arrange, zoom

● Dynamic○ pan-genome chart, MDS plot, dendrogram

○ update/change depending on current selection

What’s next?

More features

● Proteins○ width proportional to protein length

○ retrieve sequence of one, or whole cluster

○ “Send to BLAST, Pfam, ...”

● Not just proteins○ non-coding RNAs

○ even variants! (SNPs, indels)

More features

● Customise○ colours at the gene level (AbR, PaLOC, COG)

○ add/remove taxa

● Export○ save diagram as SVG for publication

○ deploy as a single HTML file for offline viewing

● Suggestions○ happy to hear ideas from the audience!

Acknowledgements

Dr David Powell

Jason KwongAndrew Buultjens

Tim StinearDieter Bulach

Simon GladmanSarah Baines

Did most of the implementation Beta testers!

1. Go to: http://bioinformatics.net.au

2. Click on the left menu bar:

3. Click on FriPan:

FriPan is open-source