10.4.4 RNAseq mapping

To map RNA sequence reads to a genome with introns, choose Geneious RNA as the Mapper in the Map to Reference setup dialog. This function can map reads that span existing annotated introns, or discover insertions, novel introns and fusion genes.

This function works in the same way as deletion and structural variant discovery (section 10.4.3 ) for DNA mapping, by analyzing how fragments of each read align to different regions of the reference sequence(s), and creating a junction annotation at the point where the read is split. By default, at least 2 reads must support the discovery of a junction in order for it to be annotated. This threshold can be adjusted under More Options by changing the Minimum support for intron/fusion gene discovery setting.

If Span annotated mRNA introns is checked, junctions will be created from existing annotations on the reference sequence. Reads are still allowed to map anywhere, but will be allowed to freely span these junctions if that produces the best mapping.

To only find introns up to a certain size, check Find novel introns up to...; to find introns of any size, insertions, or structural rearrangements that may indicate a fusion gene, use Find fusion genes and novel introns.

As for deletion and structural variant discovery, junctions are annotated on the reference sequence under a track named after the reads (see Figure 10.5 ). Each junction has the following properties:

Reads spanning junctions may be represented in one of three possible ways.


PIC

Figure 10.5: A single cDNA sequence mapped to a genomic sequence using the Geneious RNA algorithm. In the zoomed out view above, the coverage graph and junction annotation track provides a quick view of where the cDNA maps to the genomic sequence. Five copies of the cDNA sequence appear in the contig, as it maps across 5 exons. The inset shows a zoomed in view of a junction, with the junction annotation properties shown.