Content Comparison

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

Load SAMtools

Check if SAMtools is loaded and if not Load the SAMtools module (if not already loaded).

module list samtools
module avail samtools
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

From inside your main mapping directory, create a new output directory called samtools_bowtie2 or whatever makes sense to you.

LetSince the $SCRATCH directory on lonestar is effectively infinite for our purposes, we're going to copy the relevant files from our mapping tutorial into a new directory for this tutorial. This should help you identify what files came from what tutorial if you look back at it in the future. 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.

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a new directory called samtools_tutorial.

cds
mkdir samtools_tutorial
cd samtools_tutorial
cp $SCRATCH/bowtie2MappingTutorial/bowtie2/SRR030257.sam 

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cp $SCRATCH/bowtie2MappingTutorial/bowtie2/NC_012967.1.fasta 

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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

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samtools faidx samtools_bowtie2/NC_012967.1.fasta

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

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less NC_012967.1.fasta.fai 

 # can exit with "q"

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.

The following 3 commands are used to convert from SAM to BAM format, sort the BAM file, and index the BAM file. As each command requires the previous command to have been completed it makes more sense to run them on an iDEV node. If you want to submit them to the queue, separate them with a ";" to ensure that they run sequentially on the same node. Under no circumstances should you run this on the head node.

idev -m 60 -q development -A CCBB

Convert from SAM to BAM format.

samtools view -b -S -o 

SRR030257.bam 

SRR030257.sam

Sort and index the BAM file.

samtools sort 

SRR030257.bam 

SRR030257.sorted
samtools index 

SRR030257.sorted.bam

Examine the output of the previous commands to get an idea of whats going on. Here are some prompts of how to do that:

• expandCheck that Check that

  Expand
  title What new files were created by these commands?
  Code Block	language	bash Code Block title List the contents of the output directory ls samtools_bowtie2 Code Block title Expected output NC_012967.1.fasta SRR030257.sorted.bam.bai NC_012967.1.fasta.fai SRR030257.sam SRR030257.bam SRR030257.sorted.bam

• Expand
  title Why didn't we name the

  output 

  output SRR030257.sorted.bam

   in the 

  in the samtools sort

   command

  command?
  Expand
  Answer...	Answer...	Samtools appends an extra .bam to whatever we put here, so it would have created SRR030257.sorted.bam.bam, and then we would have had to make a joke about the Flintstones.

• Expand
  title Can you guess what a *.bai file is?

  expand

  Answer... Answer...

  Sure enough, it's the index file for the BAM file.

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Call genome variants

Now we use the mpileup command from samtools to compile information about the bases mapped to each reference position. Output BCF The output is a BCF file. This is a binary form of the text Variant Call Format (VCF).

samtools mpileup -u -f samtools_bowtie2/NC_012967.1.fasta samtools_bowtie2/SRR030257.sorted.bam > samtools_bowtie2/SRR030257.bcf

What are all the options doing? 

The samtools mpileup command will take a few minutes to run. You might consider putting it in the background by pressing control-z and then typing the command bg so that you can do some other things in this terminal window at the same time – like starting to run variant calling on the BWA or bowtie mapping results. Remember, there are still many other processors available on this node!

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bcftools view -v -c -g samtools_bowtie2/SRR030257.bcf > samtools_bowtie2/SRR030257.vcf

What are these options doing?

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