Pseudogenes limit the identification of novel common transcripts generated by their parent genes

Genomic sequences with high sequence similarity, such as parent-pseudogene pairs, cause short sequencing reads to align to multiple locations, thus complicating genomic analyses. However, their impact on transcriptomic analyses, including the estimation of gene expression and transcript annotation, has been less studied. Here, we investigated the impact of pseudogenes on transcriptomic analyses. Overall design: Human Poly A+ RNA of healthy individuals that passed away from sudden death/trauma derived from frontal lobe and hippocampus were commercially purchased through Clontech. A total of 100ng of Poly A+ RNA per sample was used for initial cDNA synthesis and subsequent library preparation according to the direct cDNA sequencing (SQK-DCS109). Sequencing was performed on the PromethION using one R9.4.1 flow cell per sample and base-called using Guppy (v 4.0.11; Oxford Nanopore Technologies—ONT, Oxford, UK). Resulting fastq files were processed through the “pipeline-nanopore-ref-isoforms” (https://github.com/nanoporetech/pipeline-nanopore-ref-isoforms). Gene abundances was calculated implementing the -A parameter in StringTie (v 2.1.1 RRID:SCR_016323).