Querying Bam Files With Samtools/Awk

Question

[Jneary1978]

Hello,

I am trying to generate four column files for methylation analysis from my NGS alignments against the human genome (single and paired-end). The file needs to contain the following information for any read that aligns:

1 - chr#
2 - start position of alignment 
3 - stop position of alignment 
4 - strand (+/-)

And nothing else.

Currently I am trying to awk the bam files to obtain the info, and it seems to work well enough except for strand info. I am either obtaining all positive or all negative strand reads, even though I can see I have alignments to both. I don't know if this is a problem with my awk command or what. If I recall correctly, the PE reads all came out positive strand and the single reads in another run all came out negative. (possibly it was the reverse pattern)

So I have two questions. First, is my awk command roughly correct? Do you have better suggestions or any idea what I am doing wrong. Second, can you think of any other way to generate this 4 column file? (comma separated preferably)

Here is the awk for the PE reads:

samtools view -f 0x0002 -X yourbamfilehere | gawk '{ printf "%s,%s,%s,%s\n", $3, $4, $4 + length($10), (index($2,"r") == 0) }' > yourcsvfilenamehere

Thanks so much! JN

Answer 1

[brentp]

You can simplify (or change) your awk to something like:

samtools view -f 0x02  /home/brentp/work/samtools-snp-test/data/F3-R3-Paired.bam \
  | awk '{ if ( and($2, 16) == 0 ) { strand="+" } 
            else { strand="-" }; 
         print $3,$4,$4 + length($10),strand 
  }'

since awk supports and as a bitwise operation. Also, if you pre-filter your BAM to only contain proper pairs, then you can use bedtools bamToBed for this.

[Jneary1978]

Thank you so much!

Answer 2

[Farhat]

The awk looks alright except for (index($2,"r") == 0). You could try changing that to and($2, 0x10) to get whether the read is reverse complemented or not.

[Jneary1978]

Thank you so much!