SAMtools is a suite of commands for dealing with databases of mapped reads. You'll be using it quite a bit throughout the course.

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 Introduction to mapping (bowtie, BWA). You will need the output SAM files from that tutorial to continue here.

Create a new output directory:

mkdir samtools_bowtie

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

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

Index the reference file. (This isn't indexing it for mapping. It's indexing it so that SAMtools can quickly jump to a certain base.)

samtools faidx samtools_bowtie/NC_012967.1.fasta

Take a look at the new file that was created by this command.

SAM is a text file, so it is slow to access information about a read. SAMtools and many of the commands that we will run later work on BAM files (essentially 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 -bS -o samtools_bowtie/SRR030257.bam bowtie/SRR030257.sam |borderStyle=solid}

Sort the BAM file.

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

Output VCF file.

samtools mpileup -uf samtools_bowtie/NC_012967.1.fasta samtools_bowtie/SRR030257.sorted.bam \|bcftools view -vcg - \> samtools_bowtie/output.vcf 

Output - Samtools

Exercise 1

VCF format has Allele Frequency tags denoted by AF1. Try the following command to see what values we have in our files.

cat input.vcf | grep AF1

For the data we are dealing with, predictions with an allele frequency not equal to 1 are not really applicable. (The reference genome is haploid. There aren't any heterozygotes.) How can we remove these lines from the file and continue on?

grep -v *something*

Solutions

Output - Filtering Allele Frequencies

Calling variants in reads mapped by BWA

Follow the same directions to call variants in the BWA-mapped reads.

Just be sure you don't write over your old files. Maybe create another new directory:

mkdir samtools_bwa

Exercise: Determining Differences Between Mappers.

Set up a new output directory and copy the respective VCF files to it.

mkdir comparison
cp samtools_bowtie/output.vcf output/bowtie.vcf
cp samtools_bwa/output.vcf output/bwa.vcf
cd comparison

Bedtools is a suite of utility programs that work on a variety of file formats, one of which is conveniently VCF format. Using intersectBed and subtractBed we can find equal and different predictions between mappers.

Load Bedtools.

module load bedtools

Finding alike mutations.

intersectBed -a bowtie.vcf -b bwa.vcf > intersect.vcf

Finding unique mutations for each mapper.

subtractBed -a bowtie.vcf -b intersect.vcf > unique_bowtie.vcf
subtractBed -a bwa.vcf -b intersect.vcf > unique_bwa.vcf

Exercises

• Which mapper finds more variants?

Next up

We will examine the reads supporting these variants by Using the Integrative Genomics Viewer (IGV)

Output - Determining Differences Between Mappers