Genome comparison and phylogeny

This tutorial will introduce genome comparison techniques and some simple methods to compute phylogeny. It will introduce the following pieces of software:

Mauve, for genome alignment PhyloSift & FastTree, for phylogeny

We’ll analyze some E. coli genome assemblies that were precomputed with techniques previously introduced in the course.

Interactive visual genome comparison with Mauve

Download a copy of the Mauve GUI installer for your platform: Mac OS X Windows Linux

Download the following GenBank format genomes from NCBI: E. coli O157:H7 EDL933 E. coli CFT073

An assembly of the Ribiero et al 2012 E. coli MiSeq data (accession SRR519926) made by the A5-miseq pipeline (paper here):

E. coli A5-miseq assembly

An assembly of the Chitsaz et al 2011 data made by the Velvet diginorm workflow:

E. coli Velvet DN assembly

Running a genome alignment

Launch the Mauve software, either from the start menu on Windows, or by double-clicking the Mauve app on Mac. On newer Mac OS it may be necessary to disable the nanny security features that prohibit opening downloaded software. If you see error messages suggesting the disk image is damaged and can not be opened then you need to disable the security check.

Once Mauve is open, go to the File menu, select “Align with progressiveMauve...” Drag & drop the NCBI genomes in first. Then drag & drop in the draft E. coli assemblies

The assembly will take 20+ minutes to compute. Proceed to the next steps while this process runs.

Booting an Amazon AMI

Start up an Amazon computer (m1.large or m1.xlarge) using an ubuntu 64-bit linux instance (see Start up an EC2 instance and Starting up a custom operating system).

Log in with Windows or from Mac OS X.

Ensure you have dropbox installed on your virtual machine and mounted at /mnt/Dropbox

Logging in & updating the operating system

Copy and paste the following three commands

sudo add-apt-repository ppa:webupd8team/java
sudo apt-get update
sudo apt-get install oracle-java6-installer
sudo apt-get remove openjdk-7-jre openjdk-7-jre-headless

to update the computer with all the bundled software you’ll need. Mauve, when run in command-line mode, depends on the Oracle Java 6 runtime which the above commands install. The above commands will also remove the openjdk if it’s present on the system. If you would prefer to keep this for some reason then look at the update-alternatives system.

Packages to install

Install the latest Mauve snapshot to your home directory:

curl -O
tar xzf mauve_linux_snapshot_2012-06-07.tar.gz
export PATH=$PATH:$HOME/mauve_snapshot_2012-06-07/linux-x64/

as well as the PhyloSift software, for marker-based analysis of genome & metagenome phylogeny:

curl -O
tar xjf phylosift_v1.0.1.tar.bz2

Getting the E. coli genome data

Now, let’s create a working directory:

mkdir draft_genome
cd draft_genome

Download some genome assemblies. The first one is an assembly constructed with A5-miseq. The second is an assembly constructed with Velvet.

curl -O
curl -O

What is the nearest reference genome?

In this step, we will use the PhyloSift software to determine where our newly assembled genome falls in the tree of life, and by extension what closely related genomes might be available for comparative genomics. In principle this would be a first step after sequencing a novel organism

~/phylosift_v1.0.1/bin/phylosift all --debug ecoli_dn_velvet25.fa

When that finishes (probably after a very long time), copy the results to dropbox for local viewing:

cp -r PS_temp/ecoli_dn_velvet25.fa /mnt/Dropbox

once on your local computer, open the .html file in the ecoli_dn_velvet25.fa directory in your web browser. It should be possible to launch the concat marker viewer in java from the link in the bottom left of the page.

Ordering the assembly contigs against a nearby reference

In the previous step we discovered that our genome was similar to E. coli. We will now use an existing finished-quality E. coli genome as a reference for ordering and orienting the contigs in the assembly. This is useful because the genome assembly process usually creates a large number of contigs or scaffolds rather than complete reconstructions of the chromosome(s) and these sequences appear in an arbitrary order. By ordering against a reference we can generate a candidate ordering which could be used for later manual closure efforts (e.g. via PCR) or other analyses. Let’s use the E. coli K12 genome from NCBI as a reference:

curl -O

And now run the Mauve Contig Mover to order the contigs:

java -Xmx500m -Djava.awt.headless=true -cp ~/mauve_linux_snapshot_2012-06-07/Mauve.jar  org.gel.mauve.contigs.ContigOrderer -output reorder_a5 -ref NC_010473.fna -draft
java -Xmx500m -Djava.awt.headless=true -cp ~/mauve_linux_snapshot_2012-06-07/Mauve.jar  org.gel.mauve.contigs.ContigOrderer -output reorder_a5 -ref NC_010473.fna -draft ecoli_dn_velvet25.fa

Let’s copy the newly ordered genome to dropbox:

cp reorder_a5/alignment3/ /mnt/Dropbox/

This could now be aligned with Mauve as demonstrated above to observe the improvement in contig order.

Making a phylogeny of many E. coli assemblies

For this component we will use a collection of related E. coli and Shigella genomes already on NCBI. In practice, you might use your own collection of assemblies of these genomes. Let’s start out in a new directory and download these:

mkdir ~/phylogeny ; cd ~/phylogeny

# download a bunch of genomes from NCBI. Alternatively you can use the approach that
# Adina introduced in the previous lesson to programmatically download many files
curl -O
curl -O
curl -O
curl -O
curl -O
curl -O
curl -O
curl -O
curl -O

# find homologs of the elite marker genes
find . -maxdepth 1 -name "*.fna" -exec ~/phylosift_v1.0.1/bin/phylosift search --isolate --besthit {} \;
~/phylosift_v1.0.1/bin/phylosift search --isolate --besthit

# align to the marker gene profile HMMs
find . -maxdepth 1 -name "*.fna" -exec ~/phylosift_v1.0.1/bin/phylosift align --isolate --besthit {} \;
~/phylosift_v1.0.1/bin/phylosift align --isolate --besthit

# combine the aligned genes into a single file
find . -type f -regex '.*alignDir/concat.codon.updated.1.fasta' -exec cat {} \; | sed -r 's/\.1\..*//'  > codon_alignment.fa

# infer a phylogeny with FastTree
~/phylosift_v1.0.1/bin/FastTree -nt -gtr < codon_alignment.fa > codon_tree.tre

# now copy the tree over to dropbox
cp codon_tree.tre /mnt/Dropbox/

From tree file to figures

At last we have a phylogeny! The last steps are to view it, interpret it, and publish it. There are many phylogeny viewer softwares, here we will use FigTree. You will need to download and install FigTree to your computer. Once installed, either launch by double-click (Mac) or via the start menu (Windows). Now we can open the tree file codon_tree.tre from dropbox.

Once open, enable the node labels which show bootstrap confidence. Optionally midpoint root the tree, adjust the line width, and export a PDF.

This document (c) copyleft 2014 Aaron Darling.

LICENSE: This documentation and all textual/graphic site content is licensed under the Creative Commons - 0 License (CC0) -- fork @ github. Presentations (PPT/PDF) and PDFs are the property of their respective owners and are under the terms indicated within the presentation.
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