The most frequently asked questions for my poster presentation at ASM general meeting was the benefits of PE directional RNA-seq over single-end and/or non-directional sequencing.
PE sequencing has the following advantages over SE:
1) Better mapping results.
With PE reads, you know roughly the range of the distance of two reads. This may help you when you map the reads back to a reference genome. For example, with splicing junction events, two exons maybe joined together. If mate 1 and mate 2 are mapped to a genome with 1kb between them, while you expect the distance between them in the transcript is only about 500bp at maximum, then one intron around 500 bp may be moved. You don’t get such information from SE. Similarly, PE may also solve some non-uniq (secondary) mapping problem, if one of the mates come from a repetitive region and another comes from a non-repetitive region.
2) Improved directional sequencing accuracy
Because PE reads point to opposite directions, we can extract all the read pairs of opposite directions to get better confidence in directional RNA-seq. If, for example, you have some read pairs that point to the same direction, then you can ignore those reads for directional information extraction. With SE, you jut cannot tell when there is such a sequencing error.