SAM flag forward/reverse first/second

[nupurgupta]

Hello,
I am a little confused with the interpretation of SAM flags
From http://ppotato.wordpress.com/2010/08...-paired-reads/

...
0×0010 16 strand of the query (0 for forward; 1 for reverse strand)
--
0×0040 64 the read is the first read in a pair
0×0080 128 the read is the second read in a pair

What is the difference? Shouldn't forward correspond to first read in a pair and reverse correspond to second read in a pair?

I have a bam file with paired-unmapped reads- I am trying to extract the reads and mates into separate files. How would I use the SAM flag to figure this out please?

I have read related stuff about this on the forum, but I am new to this, and cannot quite parse the exact information I need from it. Thank you so much!

[swbarnes2]

What is the difference? Shouldn't forward correspond to first read in a pair and reverse correspond to second read in a pair?

No, that's not true at all. The DNA usually gets sheared, and then one adaptor goes on one end, and the other goes on the other end, and the adaptors don't know which end is forward according to the convention of your fasta.

You can use samtools view to filter by flags. All the lines with 64 in the flag came from the first fastq, the ones with 128 came from the second fastq.

[nupurgupta]

Thanks very much for this!

[ahmedalfahad]

thank you for this great chance to learn