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Multimappers are not the only complex situation for read alignment. RNA splicing can create interesting situations when you align your RNA read sequences.

Recall that RNA splicing involves removing pieces of RNA sequence during transcription. Introns are the pieces of RNA that are removed, while exons are the pieces of RNA that remain in the final molecule.

What if we have a sequencing read that comes from one of these splice sites where two exons are connected? When we go to align this read to the genome, part of the read will align to one location, while the remainder of the read can align thousands of base pairs away. This is because the read skips the intron sequence that was removed during splicing.

Spliced alignment