Long-read transcript sequencing identifies differential isoform expression in the entorhinal cortex in a transgenic model of tau pathology

Abstract

Increasing evidence suggests that alternative splicing plays an important role in Alzheimer's disease (AD), a devastating neurodegenerative disorder involving the intracellular aggregation of hyperphosphorylated tau. We used long-read cDNA sequencing to profile transcript diversity in the entorhinal cortex of wild-type (WT) and transgenic (TG) mice harboring a mutant form of human tau. Whole transcriptome profiling showed that previously reported gene-level expression differences between WT and TG mice reflect changes in the abundance of specific transcripts. Ultradeep targeted long-read cDNA sequencing of genes implicated in AD revealed hundreds of novel isoforms and identified specific transcripts associated with the development of tau pathology. Our results highlight the importance of differential transcript usage, even in the absence of gene-level expression alterations, as a mechanism underpinning gene regulation in the development of neuropathology. Our transcript annotations and a novel informatics pipeline for the analysis of long-read transcript sequencing data are provided as a resource to the community.

Rapid and Inducible Mislocalization of Endogenous TDP43 in a Novel Human Model of Amyotrophic Lateral Sclerosis

“…Libraries for long read ONT platform were prepared according to the manufacturer’s instructions and sequenced on a PromethION sequencer at the Exeter sequencing centre. Reads were mapped to hg19 using minimap2 (H. Li 2018) and transcript isoforms were analysed using the FICLE pipeline (Leung et al 2023). Small RNA sequencing was performed commercially through Macrogen and the fastq data was mapped and counts generated using the miRDeep2 package (Friedländer et al 2008).…”

“…Our investigation predominantly focused on STMN2 due to its significance in ALS and a substantial number of reads (>100) mapping to this gene. Employing our FICLE pipeline (Leung et al 2023), we identified a total of 476 isoforms related to STMN2 . Out of these, 17 isoforms were congruent with the exonic structure of known reference isoforms.…”

An atlas of expressed transcripts in the prenatal and postnatal human cortex

“…Exome-sequencing data from schizophrenia patients, bipolar disorder patients and controls were obtained as described previously [12][13][14] and variants overlapping identified novel coding sequences were extracted. Re-annotating these to the relevant transcript, we kept variants annotated as missense or more severe, and also synonymous variants as a control.…”

“…AS is known to be a major driver of transcriptional diversity in the central nervous system 13 . To fully describe AS events for individual genes, we developed FICLE , a bespoke analysis pipeline that compares splice junctions between long-read-derived and reference transcripts, enabling a gene-level overview of all detected isoforms 14 . We used FICLE to characterise the frequency of multiple different AS events (exon skipping (ES), alternative first exon use (AF), alternative last exon use (AL), differential A3’ and A5’ splice site use, and intron retention (IR)) associated with transcripts expressed from all multi-exonic protein-coding genes in the human cortex ( Fig.…”