I am new to bioinformatics and currently learning how to use Bowtie 2. As written in the manual:

> A pair that aligns with **the expected relative mate orientation** and with the expected range of distances between mates is said to align "concordantly". If both mates have unique alignments, but the alignments do not match paired-end expectations (i.e. the mates aren't **in the expected relative orientation**, or aren't within the expected distance range, or both), the pair is said to align "discordantly".

I have read about the basics of paired-end sequencing and orientation (in the molecular biology sense). In summary, my understanding is that in paired-end sequencing we sequence both ends of a DNA fragment at the 5' end and the 3' end (we call them mate 1 and mate 2) and by knowing the expected length between the mates we can better align the fragment to a reference genome.

My question is, what is it meant by two mates having an expected relative orientation? If am using Bowtie 2 and giving it a file with all the first mates and another one with all the corresponding second mates, how can two mates align without first having the expected orientation?
In Illumina paired-end sequencing the two two mates will be sequenced in the opposite direction to one another (see my ASCII art below), so what you expect from this is that the reads should be pointing into each other, which would be the expected orientation. The reads can be in other orientations if:

1. say you were mapping to a reference from a related organism which had an inversion in its genome relative to what you sequenced;
2. The reads mapped to a palindromic region and the read mapper could not determine the correct orientation
3. There is a problem with the assembly that you are mapping to (for example errors in a de novo assembly)

</ol>

    ------->                     Read 1
    =========================    DNA fragment
                   <---------    Read 2

Also note that other sequencing technologies produce reads in different orientations. For example, Illumina mate-pair creates reads that point away from each other rather than into each other. I don't know how bowtie handles these cases where other orientations are expected