SAM format flag

[eoh001]

Hello
I have been analysing a SAM file(alinged by Bioscope) with SOLiD transcriptome paired-end sequences, and the FLAGs were kind of confusing.

FLAG(F3, F5) = (99,147) means the paired reads are mapped properly.
FLAG(F3, F5) = (97, 145) means the paired reads are mapped unproperly.

When I filted FLAG(F3,F5)=(97,145), 10% of the paired reads were mapped in the same chromosome with proper insert size(50< < 50000). I wonder, why the reads seemingly properly mapped were flagged as 97 or 145.

Also, I found similar situations with different orientations
FLAG(F3, F5) = (83, 163) : mapped properly
FLAG(F3, F5) = (81, 161) : mapped unproperly

the reads with FLAG (81, 161) included the reads with proper insert size.

What is the meaning of 'properly aligned' for paired reads?

Thank you

[swbarnes2]

Hello
I have been analysing a SAM file(alinged by Bioscope) with SOLiD transcriptome paired-end sequences, and the FLAGs were kind of confusing.

FLAG(F3, F5) = (99,147) means the paired reads are mapped properly.
FLAG(F3, F5) = (97, 145) means the paired reads are mapped unproperly.

When I filted FLAG(F3,F5)=(97,145), 10% of the paired reads were mapped in the same chromosome with proper insert size(50< < 50000). I wonder, why the reads seemingly properly mapped were flagged as 97 or 145.

Also, I found similar situations with different orientations
FLAG(F3, F5) = (83, 163) : mapped properly
FLAG(F3, F5) = (81, 161) : mapped unproperly

the reads with FLAG (81, 161) included the reads with proper insert size.

What is the meaning of 'properly aligned' for paired reads?

Thank you

I don't know bioscope exactly, but "properly paired" usually means that not only is the insert size appropriate, but that the reads have the right orientation with respect to each other. I assume that solid reads are supposed to point in towards each other, if your reads are pointing away from each other, that would explain the flags.