BAM parsing with samtools
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Explore BAM files

Capture the flag

As we discussed earlier, the FLAG field in the BAM format encodes several key pieces of information regarding how an alignment aligned to the reference genome. We can exploit this information to isolate specific types of alignments that we want to use in our analysis.

For example, we often want to call variants solely from paired-end sequences that aligned “properly” to the reference genome.

To ask the view command to report solely “proper pairs”, we use the -f option and ask for alignments where the second bit is true (proper pair is true):

samtools view -f 0x2 sample.sorted.bam | head

How many properly paired alignments are there? (use the -c option)

samtools view -c -f 0x2 sample.sorted.bam

Now, let’s ask for alignments that are NOT properly paired. To do this, we use the -F option (note the capitalization to denote “opposite”).

samtools view -c -F 0x2 sample.sorted.bam

How many total alignments?

samtools view -c sample.sorted.bam

Does everything add up?

To get a summary of the flags in our BAM file, we can use samtools flagstats:

samtools flagstats sample.sorted.bam
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