Isoform characterisation & splicing signatures of AD‐risk genes using long‐read sequencing.

Background: An increasing number of studies implicate a role for alternative splicing in development and neuropathology of Alzheimer's disease (AD). However, it has been historically challenging to characterise splicing events, due to the inherent limitations of traditional RNA‐sequencing (RNA‐Seq) to capture full‐length transcripts critical for transcriptome assembly. In this study, we use two complementary targeted long‐read sequencing approaches, Pacific Biosciences (PacBio) isoform sequencing (Iso‐Seq) and Oxford Nanopore Technologies (ONT) nanopore cDNA sequencing, to profile the cortex of a mouse model of tau pathology (rTg4510) with ultra‐deep sequencing of a panel of 20 genes robustly implicated in AD. Method: Targeted PacBio Iso‐Seq and targeted ONT profiling were performed on RNA isolated from 24 female rTg4510 transgenic and wild‐type mice, aged 2, 4, 6 and 8 months. Following successful enrichment for target genes using custom‐designed probes (IDT), library preparation were performed for subsequent sequencing on Sequel and MinION. The panel of genes selected for enrichment included genes (i.e. App, Mapt, Snca) and dementia risk genes identified from GWAS analyses. Raw sequence data was processed using a customised bioinformatics, and full‐length reads were merged and annotated with our developed custom scripts (FICLE). Result: We identify thousands of novel transcripts across the panel of 20 AD‐ and dementia‐associated genes. We reveal unprecedented diversity of alternatively‐spliced isoforms with widespread usage of alternative novel 5' and 3' splice sites. We further robust transcriptional and splicing differences in these AD‐risk genes associated with the development of tau pathology. Among these changes, we observed global up‐regulation of Trem2‐associated isoforms and isoform switches in Bin1, further supporting a role for the dysregulation of the immune response in the development of AD pathology. Conclusion: This represents the first study to comprehensively assess variable AS associated with the development of tau pathology using both PacBio Iso‐Seq and ONT nanopore sequencing. Our analyses confirm the importance of alternative RNA splicing in AD and identify evidence of differential transcript usage, even in the absence of gene‐level expression alterations. Further work will be undertaken to validate and functionally characterise these novel isoforms, and to extend these analyses to human post‐mortem AD brain samples. [ABSTRACT FROM AUTHOR]