sam flag from paired end reads

Question

[Varun Gupta]

HI Everyone, This might be a simple question but I am not able to understand it. For paired end reads I see 83,163 and 99,147 as the sam flags.

Looking at the description from here about sam flags When I put it as 83 I get first in pair and when i put it as 147 I get second in pair although in both the cases the read maps to reverse strand.

What do we mean by first in pair and second in pair?

Thanks

Answer 1

[Pierre Lindenbaum]

in paired end experiment, the sequencer produces two files. Something like sample.R1.fastq.gz and sample.R2.fastq.gz which are the reads on 5' and 3' of the sequenced fragment. They both contain the same number of reads.

First in pair means that the read comes from sample.R1.fastq.gz Second in pair means that the read comes from sample.R2.fastq.gz

First and Second in pair flags have no meaning about the mapping of the read .

[Varun Gupta]

HI Pierre,

I understand this but then I think If the read is 147 it's second in pair although its mapped on the reverse strand while 83 would mean read is on reverse strand but it is first in pair. So that's why 83,163 go together and 99,147 go together for a paired end read

[Charles Plessy]

First/second in pair refer to the order in which the reads sequenced; this is independent from alignment: even unmapped reads are first or second in pair.