Monday, April 28, 2014

Breaking BAM

It's fun to mess with our bioinformatics tools and laugh at ourselves. The BAM format carries a query name string. In an idle moment I wondered, how long a string can I put here before bad things happen to the BAM?

Generally this string carries information related to the device that produced the read, lane number, run number and so and so forth. It's completely specification free and everyone encodes information here in their own way, so you would think it's an arbitrary length string. Almost.

Try out the short Python snippet below. It generates a dummy fasta file (test.fa) and a dummy BAM file (test.bam) with one 'aligned' read. The only funky thing is that you can make the qname field of the read as long as you want (by varying the lone input parameter to the script).

import sys
import pysam

try:
  qnamelen = int(sys.argv[1])
except:
  qnamelen = 255

with open('test.fa', 'w') as f:
  f.write('>test\n')
  f.write('A'*100)

bam_hdr = {'HD': {'VN': '1.4'},
           'SQ': [{'LN': 100, 'SN': 'test'}]}
bf = pysam.Samfile('test2.bam', 'wb', header=bam_hdr)  # Write binary BAM with header
ar = pysam.AlignedRead()
ar.qname = 'Q'*qnamelen  # Gives Tablet headaches when str len > 254
ar.seq = 'A'*50
ar.qual = '~'*50
ar.mapq = 100
ar.pos = 0
ar.cigarstring = '50M'
bf.write(ar)
bf.close()
pysam.sort('test2.bam', 'test')
pysam.index('test.bam')

Now open the pair of fasta and BAM files in samtools tview, Tablet and IGV (and indeed in anything else). Laugh loudly until your office mates banish you.


Only samtools tview - the least fancy one - loads the alignment without batting an eyelid.

The specs allow an arbitrary length null terminated string.

(Oh, as an aside, try this. Create a .fasta file with this simple corruption: Remove the newline from the sequence id in the fasta file (so that the sequence looks something like 'testAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA'. Load the sequence in IGV. Now fix the sequence and try and convince IGV to load the new corrected sequence. Hint: it won't go away until you use igvtools to reindex the fasta file. running bwa index won't help)

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