Hi, all,

When dealing with strand-specific pair-end RNA-Seq data, sequenced from 5' end and mapped with --library-type fr-secondstrand, I got some strang flags and XS:A labeling listed as follows.

H2208:7439:9018 pPr1   chr1    4887061 50      100M    =  4887050 -111 XS:A:+  NH:i:1
H2208:7439:9018 pPR2   chr1    4887050 50      100M    =  4887061 111  XS:A:+  NH:i:1

H16340:40651    pPR2 chr1    6284001 50        100M    =  6284208 307  XS:A:-  NH:i:1
H16340:40651    pPr1 chr1    6284208 50        100M    =  6284001 -307 XS:A:-  NH:i:1

According to my library type, I can guess the reads are from negative strand. Why in the first pair, these reads are assumed to be from positive strand? There are about 1 pair of reads like the first one in every 400 pairded reads with flag pPr1 or 83. I wonder if this is a bug of Tophat or my assumption is wrong?

Thank you!

T Chen