Variant calling tutorial

SAMtools is a suite of commands for dealing with databases of mapped reads. You'll be using it quite a bit throughout the course. It includes programs for performing variant calling (mpileup-bcftools).

Calling variants in reads mapped by bowtie

Right now, we'll be using it to call variants (find mutations) in the re-sequenced E. coli genome from the Mapping tutorial. You will need the output SAM files from that tutorial to continue here.

cds
mkdir intro_to_mapping
cd intro_to_mapping
cp -r $BI/ngs_course/intro_to_mapping/bowtie .
cp -r $BI/ngs_course/intro_to_mapping/bwa . 
cp -r $BI/ngs_course/intro_to_mapping/bowtie2 .

We assume that you are still working in the main directory called intro_to_mapping data that you created on $SCRATCH.

Load SAMtools

Load the SAMtools module (if not already loaded).

module load samtools

Can you figure out what version of samtools is loaded on TACC and where it is installed?

samtools
which samtools

Prepare your directories

Create a new output directory called samtools_bowtie or whatever makes sense to you.

Let's copy over just the read alignment file in the SAM format and the reference genome in FASTA format to this new directory, so that we don't have so many files cluttering our space.

mkdir samtools_bowtie
cp bowtie/SRR030257.sam samtools_bowtie
cp bowtie/NC_012967.1.fasta samtools_bowtie

Index the FASTA reference file

First, you need to index the reference file. (This isn't indexing it for read mapping. It's indexing it so that SAMtools can quickly jump to a certain base in the reference.)

Then run this command to index the reference file.

samtools faidx samtools_bowtie/NC_012967.1.fasta

Take a look at the new *.fai file that was created by this command. Any idea what some of the numbers mean?

less samtools_bowtie/NC_012967.1.fasta.fai

Convert mapped reads from SAM to BAM, sort, and index

SAM is a text file, so it is slow to access information about how any given read was mapped. SAMtools and many of the commands that we will run later work on BAM files (essentially GZIP compressed binary forms of the text SAM files). These can be loaded much more quickly. Typically, they also need to be sorted, so that when the program wants to look at all reads overlapping position 4,129,888, it can easily find them all at once without having to search through the entire BAM file.

Convert from SAM to BAM format.

samtools view -b -S -o samtools_bowtie/SRR030257.bam samtools_bowtie/SRR030257.sam

Sort and index the BAM file.

samtools sort samtools_bowtie/SRR030257.bam samtools_bowtie/SRR030257.sorted
samtools index samtools_bowtie/SRR030257.sorted.bam

This is a really common sequence of commands, so you might want to add it to your personal cheat sheet.

• What new files were created by these commands?