As Nathan showed you yesterday, the main type of output from aligning reads to a databases is a binary alignment file, or BAM file. These files are compressed, so they can't be viewed using standard unix file viewers such as more, less and head. Samtools allows you to manipulate the bam files - they can be converted into a non-binary format (SAM format specification here) and can also be ordered and sorted based on the quality of the alignment. This is a good way to remove low quality reads, or make a bam file restricted to a single chromosome.
We'll be focusing on just a few of samtools functions in this series or exercises:
Since most aligners produce a bam file, we'll work on some basic manipulations of the bam files we produced from our alignments yesterday. Most functionality while using bam files can be described as such:
- SAM files are converted into BAM files (samstools view)
- BAM files are sorted by reference coordinates (samtools sort)
- Sorted BAM files are indexed (samtools index)
- Sorted, indexed bam files are filtered (based on location, flags, mapping quality)
These steps presume that you are using a mapper/aligners such as bwa, which records mapped vs unmapped reads - make sure you check how the aligner writes it's output to sam/bam format, or you may get a strange surprise!
The code block below details some basic samtools functionality:
samtools view -bH -o outfile_view.bam infile.bam #use the -c option to just count alignments samtools sort infile.bam outfile.sorted.bam samtools index aln.sorted.bam
First, logon to stampede and copy the file yeast_pairedend.bam to your scratch directory:
cds mkdir samtools cd samtools cp /scratch/02423/nsabell/core_ngs/bam/yeast_pairedend.bam .
Now that we have a bam file, we need to index it. All bam files need an index, as the tend to be large and the index allows us to perform computationally complex operations on these files without it taking days to complete.
Exercise 1: sort and index the file "yeast_pairedend.bam"
We have a sorted, indexed bam file. Now we can use other samtools functionality to filter this file and count mapped vs unmapped reads in a given region. Samtools allows you to sort based on certain flags that are specified on page 4 on the sam format specification. We'll focus on a couple, below.
Below are three useful flags to sort on, we'll be using the unmapped flag.
sam specifications and common flag usage (from p4) 0x04 = unmapped 0x02 = properly aligned 0x40 = optical duplicate
Exercise: count unmapped reads vs total reads on chromosome III for the yeastpairedend_sort.bam file you created above
What proportion of the reads are mapped?
Exercise 3: Making a sorted bam file with reads that map to multiple places removed