Samtools

Setup output directory.

mkdir bowtie_samtools

If you do not have an alignment file in the SAM format you may want to start with Introduction to mapping.

cp bowtie/REL606.5.sam bowtie_samtools/ 
cp bowtie/REL606.5.fasta bowtie_samtools/

Index the reference file.

samtools faidx bowtie_samtools/REL606.5.fasta

Convert from SAM to BAM format.

samtools view -bS -o bowtie_samtools/REL606.5.bam bowtie/REL606.5.sam |borderStyle=solid}

Sort the BAM file.

samtools sort bowtie_samtools/REL606.5.bam bowtie_samtools/sorted_REL606.5

Output VCF file.

samtools mpileup -uf bowtie_samtools/REL606.5.fasta bowtie_samtools/sorted_REL606.5.bam \|bcftools view -vcg - \> bowtie_samtools/output.vcf

Produces output.vcf from Bowtie and output.vcf from BWA.
Move all 3(question) bam files and all 3(question) vcf files to lonestar
introduce bedtools.

Exercise.

VCF format has Allele Frequency tags denoted by AF1. Try the following command to see what value 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. How can we remove these lines from the file and continue on?

What does the -v flag do in grep?

grep -v *something*

cat input.vcf | grep AF1=1 > output.vcf

Is not practical, since we will lose vital VCF formatting and may not be able to use this file in the futre.

cat input.vcf | grep -v AF1=0 > output.vcf

Will preserve all lines that don't have an AF1=0 value and is one way of doing this.

sed -i '/AF1=0/ d' input.vcf

Is a better way of doing it inline and not requiring you to make another file.

Determining Differences Between Aligners.

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_bowtie.vcf