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:
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
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?
What does the -v flag do in grep?
grep -v *something*
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:
Comparing the results of different 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.
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
- Which mapper finds more variants?
We will examine the reads supporting these variants by Using the Integrative Genomics Viewer (IGV)