<p>Hello everyone!<br />
I am trying to learn more about the SAM/BAM format and how reads are mapped to the genome.<br />
I understand that reads can be mapped to the forward (sense) and reverse (antisense) strand.<br />
I understand that in every read pair there is a first-in-pair and a second-in-pair read - and that this probably has something to do with the order in which they came off the sequencer and not the position in the genome? (but maybe im wrong)<br />
I understand that weird things can happen. A pair of reads can map millions of base pairs apart. Both reads can map to the same strand. But can reads map in both upstream and downstream direction - i.e. the read sequence once reversed (but not reversed complimented) was mapped.<br />
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With three variables thats 32 different combinations for paired reads (and another 4 for singletons).<br />
With two variables, there&#39;s only 8 different combinations (and 2 for singletons)<br />
Regardless, does anyone know of any tool available which will give you the statistics for the 32+4 or 8+2 combinations in your BAM file? If not, i will add it to the new version of metaflagstat.<br />
<br />
Here&#39;s a picture of what i&#39;m trying to explain :)</p>

<p><img alt="" src="http://ac.gt/example.png" /></p>

Using proper nomenclature, a given read can map to the + strand or reverse complemented to the + strand...which is the same as mapping to the - strand. It will never be mapped reverse to anything. Things a bit more complicated if you're talking about bisulfite converted reads, but aside from that those are the two possibilities. This is at least true with current technologies, but will likely remain so in the future due to the energetics of 3'->5' base addition.

With PE reads, any relative orientation of the mates to each other is possible, though some are vastly more likely than the others.