Task5 - Comparative genomics - gene set comparison

This task is a tutorial on comparative genomics with a focus on gene set comparison.

You are going to download, annotate and compare the genomes of a enterohemorrhagic E. coli (strain O157:H7) versus a non-pathogenic E. coli (strain K12). You are then going to identify genes and gene duplications that are unique to each organism and biologically interpret your results.

Requirements

• Access to a linux-based OS running BASH
• BLAST
• prokka

Getting Started

• Login to your linux environment and create a new folder for your task5
• Work on your assignment in the folder your created

Retrieving the raw data

You will be comparing two genomes of E. coli - strain K12 (non-pathogenic lab strain) and O157H7 (pathogenic E. coli associated with disease outbreaks).

• Download both of these genomes using curl or wget. Check the man pages for curl and wget or use the –help flag to determine how to save the files with the following file names.
  • Name the O157H7 genome O157H7.fna
  • Name the K12 genome K12.fna

ftp://ftp.ncbi.nlm.nih.gov/genomes/archive/old_genbank/Bacteria/Escherichia_coli_O157H7_EDL933_uid259/AE005174.fna
ftp://ftp.ncbi.nlm.nih.gov/genomes/archive/old_genbank/Bacteria/Escherichia_coli_K_12_substr__DH10B_uid20079/CP000948.fna

Q1 - Look within the ftp directories for these bacterial genome projects. i.e., go to: https://ftp.ncbi.nlm.nih.gov/genomes/archive/old_genbank/Bacteria/Escherichia_coli_O157H7_EDL933_uid259

What do the other files contain?

Annotating both genomes

• Next, annotate both genomes using prokka

#this may take a while so be patient... Remember, these are full bacterial genomes as opposed to small mitochondrial contigs
prokka O157H7.fna --outdir O157H7 --norrna --notrna
prokka K12.fna --outdir K12 --norrna --notrna

Generating gene lists

• Next, make text files of the predicted gene lists for each genome. Have a look back at Task1 for how to redirect program output to a file.
  • redirect the output for O157H7 to a file named genelist_O157H7.txt inside the O157H7 folder
  • redirect the output for K12 to a file named genelist_K12.txt inside the K12 folder

#generate a gene list text file by grepping the gene names from the .tbl file
cat PROKKA*.tbl | awk '{if ($1 == "gene") {print $2}}' | awk -F'_' '{print $1}' | sort 

Q2 - How many genes are present in each genome?

Comparing gene lists

Now, let’s compare these lists to find genes that are common to both, duplicated or unique to either

A lot of this work can be done simply using the comm and uniq functions within the shell. Explore the command-line usage and options of these commands using man

Comparison including gene duplicates

We can compare both gene lists like this in your task5 folder:

comm O157H7/genelist_O157H7.txt K12/genelist_K12.txt >geneListComparison.txt

Examine the output of geneListComparison.txt using less

Q3 - What do the genes in column 1, column 2, and column 3 represent?

Now, suppose we want to output the genes in column 1 (ignoring spaces). We can do so like this:

cat geneListComparison.txt | awk -F'\t' '{print $1}' | grep -v -e '^$'

… and we can then count them by piping this command to wc -l

cat geneListComparison.txt | awk -F'\t' '{print $1}' | grep -v -e '^$' | wc -l

• Analyze the core versus variable gene content for these two strains

Q4 - How many genes are only in the O157H7 genome? Only in the K12 genome? In both?

Comparison without gene duplicates (finding unique genes)

Construct a simple Venn diagram illustrating the number of shared vs genome-specific genes. In this Venn diagram, if a gene G is duplicated (2 copies) in genome A and not in genome B (1 copy), then gene G will contribute to the unique genes in genome A. This is not really true, since genome B has a copy of gene G.

Let’s account for this by “removing gene redundancy”. This can be done by first filtering each initial gene list using the tool uniq

cd /O157H7/
uniq genelist_O157H7.txt > unique_genelist_O157H7.txt

cd ../K12
uniq genelist_K12.txt > unique_genelist_K12.txt

cd ..

Now, when we compare these lists using comm, we will only be comparing single copies of each gene. Therefore, the result of comm should show us only those genes that are unique to genome 1, unique to genome 2, or shared between both

comm O157H7/unique_genelist_O157H7.txt K12/unique_genelist_K12.txt > uniqueGeneListComparison.txt

Q5 - How many unique genes are only in the O157H7 genome? Only in the K12 genome? In both?

Going further: inspecting your duplicated and unique genes within each organism

Now, let’s examine the output of the lists above.

Examine geneListComparison.txt to find the gene expansions specific to enterohemorrhagic E. coli O157:H7. The following code will sort the O157:H7-specific genes by their copy number. This will identify those that have undergone the most pathogen-specific duplication.

cat geneListComparison.txt | awk -F'\t' '{print $1}' | sort | uniq -c | sort -n -r | head -20

Examine your result carefully. Column 1 states the copy number and copy 2 states the gene name.

Q6 - Which gene in O157H7 occurs the most times. In K12?

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ASSIGNMENT QUESTIONS

The questions for this task are indicated by the lines starting with above. Please submit your answers using the quiz on LEARN.