<p>Hi, I&#39;m wondering what exactly is the meaning of an aligner being &quot;splice aware&quot;. I know it has to do with the mapping of reads spanning splice junctions, but as someone pretty new to RNA-seq and molecular biology, that&#39;s not quite enough for me to grasp the concept.</p>

<p>The following is my best reasoning of its meaning. RNA-seq reads are derived from mature mRNA, so there&#39;s typically no introns in the sequence. But aligners use a reference <em>genome</em> to aid in the process, so a read spans (what in the actual transcript are) two exons, while the reference would have one exon followed by an intron. So the reference genome would find a matching sequence in only one of the exons, while the rest of the read would not match the intron in the reference, so the read can&#39;t be properly aligned. A splice-aware aligner would know not to try to align RNA-seq reads to introns, and would somehow identify possible downstream exons and try to align to those instead, ignoring introns altogether.</p>

<p>Is this anywhere close to the meaning of splice-aware? And if so, would a splice-<em>un</em>aware aligner properly align RNA-seq data, given a reference <em>transcriptome?</em></p>

I think, that pretty much hits the point. :) The major problem is that introns not only vary in length but that they can also be very long. An DNA-DNA aligner (i.e., splice-unaware) would have to introduce a long gap in the mapping of a read to span an intron. This is not desired for DNA read mapping and might lead to false mappings. Without transcript information/restriction it is quite likely that the aligner is able to find a genome-sequence that matches the remaining read sequence. But that can be at any position downstream...

Regarding your second question, the splice-aware <a href="https://ccb.jhu.edu/software/tophat/index.shtml">Tophat</a> internally uses the splice-**unaware** <a href="http://bowtie-bio.sourceforge.net/">Bowtie</a>. So yes, in principle it is possible to use a splice-unaware aligner for mapping RNA-Seq reads to a transcriptome. However, using dedicated *RNA-Seq read mapper* or *RNA-Seq read mapping work flow* might give you better results by taking care of possible caveats etc. (read the papers for this ;-) ).

PS: See also https://www.biostars.org/p/175068/