I found in the .bam files, in any region (even though known as non-inverted region), the following bitwise FLAG number, 83,163, 99,147, are almost equally distributed (close to 25% each), implying that there are almost equal numbers of Forward-Reverse and Reverse-Forward pairs. I expected to observe this distribution for an inversion heterozygous individual inside a long inversion region, such as 17p21.31. However, I saw it for individuals with inverted/non-inverted homozygote and even outside this inversion. This seems to indicate that the strand label for the reads was simply randomly chosen.
illumina library prep method captures sequences from both the strands ... so unless you made a strand-specific library you will get reads from both the strands in both read 1 as well as read 2 ... this link may help explain it better ... do note both the strands contain green and orange adapter so both will be sequenced ... https://www.youtube.com/watch?v=womKfikWlxM
Thank you so much. This explains what I saw from the data. However, is it practical in the wet-lab to make a strand-specific library for DNA sequencing (maybe on the genome scale)? I saw strand -specific sequencing only for RNA.
Since there will be coding regions on both the strands of the dna it is not possible to prepare strand specific DNA library. While it is possible for RNA as majority of functional transcripts contain poly-A tails which makes it possible to select only those sequences and subsequently retain the strand information using dUTP or similar methods
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