Direction/Orientation of Illumina read in SAM file

[flobpf]

Documenting my 3 hours spent trying to understand what parameter in SAM file tells read orientation in the hope it will turn out to be helpful to someone. (but if my inference below is wrong!, please post a reply!)

I have been looking up ways to find which strand a read is mapping to and in the SAM file, there seems to be two confusing parameters:

XS:A:+ and XS:A-
and
0 or 16 in the second column

After much researching to confirm my belief of 0 or 16 corresponding to read orientation on strand, I found that the SAM specification PDF says:

Column 2 is bitwise FLAG and a bit value 0x10 means SEQ being reverse complemented

I then ignored this for some time ('coz I didnt know what this meant!!) before realizing that bit value of 0X10 corresponds to a decimal value of 16 (Thanks Google!!)

So if the second column is 0, it means read is mapping to one strand, and a value of 16 means it is mapping to the other strand. Still havent figured out clearly what XS:A stands for...whether it relates to a read mapping across a splice junction or even a completely exonic read is considered...not quite sure...

these posts may be of help:

http://seqanswers.com/forums/showthread.php?t=9930

AND

http://seqanswers.com/forums/showthread.php?t=4704

[nicolallias]

Hi,
Look at the second column, the CIGAR line, set its values in http://picard.sourceforge.net/explain-flags.html

You can also use in command line

smatools view -X

[roll]

Hi,
Look at the second column, the CIGAR line, set its values in http://picard.sourceforge.net/explain-flags.html

You can also use in command line

smatools view -X

what exactly does samtools view -x or -X do?

[dpryan]

what exactly does samtools view -x or -X do?

-x       output FLAG in HEX (samtools-C specific)
-X       output FLAG in string (samtools-C specific)