Filtering a SAM file generated by TopHat to find uniquely mapped, concordant read pairs: AWK vs SAMtools

Software suites like SAMtools (or should that be [SAMsam]tools?) offer a powerful way to slice and dice files in the SAM, BAM, and CRAM file formats. But sometimes other approaches work just as well.

If you have aligned paired RNA-Seq read data to a genome or transcriptome using TopHat you may be interested in filtering the resulting SAM/BAM file to keep reads that are:

  • a) uniquely aligned (only match one place in target sequence)
  • b) mapped as concordant read pairs (both pairs map to same sequence in correct orientation with suitable distance between them)

TopHat has a --no-discordant command-line option which only report read alignments if both reads in a pair can be mapped, but the name of option is somewhat misleading, as you still end up with discordantly mapped read pairs in the final output (always good to check what difference a command-line option actually makes to your output!.

So if you have a TopHat SAM file, and you wanted to filter it to only keep uniqueley mapped, concordant read pairs, you could use two of the options that the samtools view command provides:

  • -q 50 — This filters on the MAPQ field (5th column of SAM file). TopHat uses a value of 50 in this field to denote unique mappings (this important piece of information is not in the TopHat manual).
  • -f 0x2 — This option filters on the bitwise FLAG score (column 2 of SAM file), and will extract only those mappings where the 2nd bit is set. From the SAM documentation, this is described as each segment properly aligned according to the aligner. In practice this means looking for values that are either 83, 89, 147, or 163 (see this helpful Biobeat blog post for more information about this).

So if you have a SAM file, in essence you just need to filter that file based on matching certain numbers in two different columns. This is something that the Unix AWK tool excels at, and unlike SAMtools, AWK is installed on just about every Unix/Linux system by default. So do both tools give ou the same result? Only one way to find out:

Using SAMtools

The 'unfiltered.sam' file is the result of a TopHat run that used the --no-discordant and --no-mixed options. The SAM file contained 34,340,754 lines of mapping information:

time samtools  view -q 50 -f 0x2 unfiltered.sam > filtered_by_samtools.sam

real    1m57.068s
user    1m18.705s
sys     0m13.712s

Using AWK

time awk '$5 == 50 && ($2 == 163 || $2 == 147 || $2 == 83 || $2 == 99) {print}' unfiltered.sam  > filtered_by_awk.sam

real    1m31.734s
user    0m46.855s
sys     0m15.775s

Does it make a difference?

wc -l filtered_by_*.sam
31710476 filtered_by_awk.sam
31710476 filtered_by_samtools.sam

diff filtered_by_samtools.sam filtered_by_awk.sam

No difference in the final output files, with AWK running quite a bit quicker than SAMtools. In this situation, filtering throws away about 8% of the mapped reads.