Hello everyone, Sorry for post this naive sequencing question. I aligned a paired DNA sample to hg19 by bowtie2 with parameter "-q -N 1 -X 2000 --no-mixed --no-discordant". I saw some strange read pairs in my bam file like below (I deleted some ATCG for illustration). read1 can either be forward and reverse. This confused me.
（1）read1 is forward, chr1:10033-10110
reference CCCTAACCCAACCCTAACCCAA read1 CCCTAACCCAACCCTAACCC read2 CTACCCCAACCCTAACCCTA
（2）read1 is reverse, chr1:10018-10095
reference TAACCCAACCCTAACC read1 ACCCAACCCTAACC read2 TAACCCAACCCTAA
My question is :
(1) bowtie2 use --fr in alignment procedure by default. Is this means read1 should come from forward strand and read2 from reverse strand?
(2) How the above phenomenon occured?
(3) If this is normal, how to determine the start and end of the DNA fragment? which is 5' and which is 3'?