RNAseq, reads outside exons

[julieta]

Hello. Why do readings outside of genes appear in an RNAseq experiment in IGV? The readings are supposed to match exons, right?
Thank you

[jennaj]

It is possible to have reads align to regions of the genome not specifically targeted by the library construction/sequencing method.

BAM datasets can filtered to reduce spurious hits, for example, by mapping quality, concordantly aligned paired reads. And can be ignored in some protocols as they are excluded by the analysis tools in various ways.

Some Galaxy tutorials that might be helpful if you are just getting started: Galaxy Training!

With all here, including those that cover RNA-seq:

Galaxy Training Network Tutorials : Some GTN tutorials are appropriate for Galaxy Main and some are not. Where you can run each is noted per tutorial – click on the Galaxy instances gear icon to review the Public Galaxy server choices. If a tutorial is supported by a pre-configured Galaxy Docker training image, instructions for how to get it will be listed below the tutorial listings, per category.

[julieta]

Yes, but what is the cause of these alignments in the intergenic regions.
Thank you.

[jxtx]

At least two possibilities:

1. These are real RNA molecules that were in your library. poly-a selection in RNA-seq is not 100% efficient, so these could be ncRNAs, or just spurious transcription

2. Alignment error: alignment is also not perfect, some reads may be placed in intergenic regions incorrectly.

This is typically not something to worry about. Downstream, if you assemble transcripts or quantify using an existing transcriptome, these will be ignored.

[julieta]

Ok Jxtx, I understand.
Thank you very much for your answer.