RNA splicing analysis using heterogeneous and large RNA-seq datasets

Abstract

The ubiquity of RNA-seq has led to many methods that use RNA-seq data to analyze variations in RNA splicing. However, available methods are not well suited for handling heterogeneous and large datasets. Such datasets scale to thousands of samples across dozens of experimental conditions, exhibit increased variability compared to biological replicates, and involve thousands of unannotated splice variants resulting in increased transcriptome complexity. We describe here a suite of algorithms and tools implemented in the MAJIQ v2 package to address challenges in detection, quantification, and visualization of splicing variations from such datasets. Using both large scale synthetic data and GTEx v8 as benchmark datasets, we demonstrate that the approaches in MAJIQ v2 outperform existing methods. We then apply MAJIQ v2 package to analyze differential splicing across 2,335 samples from 13 brain subregions, demonstrating its ability to offer new insights into brain subregion-specific splicing regulation.

Meta-analysis of the amyotrophic lateral sclerosis spectrum uncovers genome instability

“…A proposed molecular mechanism of disease in ALS motor neurons is dysregulated alternative splicing, which may contribute to genomic instability underlying DNA damage response activation 32,33 . To identify alternative splicing changes in ALS iPSNs, we utilised the splice graph tool MAJIQ 34,35 , which quantifies local splicing variations from large heterogeneous RNA-seq datasets and corrects for dataset batch effects (Fig. 4a) 36 .…”

“… a , Schematic of the steps in splicing analysis of ALS and control iPSNs with MAJIQ 35 . b , Differential alternative splicing in poly(A) selected ALS versus control iPSNs.…”

“…All modes of alternative splicing were analysed using MAJIQ v2.4 34,35 on poly(A) selected RNA library samples. STAR aligned BAMs were used as input to the MAJIQ splice graph builder using Ensembl GRCh38.99 transcript annotation.…”

UBQLN2 restrains the domesticated retrotransposon PEG10 to maintain neuronal health in ALS

“…Splicing analysis was performed using the MAJIQ Quantifier followed by the MAJIQ Builder to determine differentially spliced genes ( Vaquero-Garcia et al, 2016 ), and visualized using the MAJIQ Voila Viewer with a Δψ threshold of 0.1 and significance of 0.05. Splice variant classification analysis was performed using the MAJIQ classifier ( Vaquero-Garcia et al, 2021 ) with permission and assistance from Dr. Yoseph Barash.…”

UBQLN2 restrains the domesticated retrotransposon PEG10 to maintain neuronal health in ALS

“…Splicing analysis was performed using the MAJIQ Quantifier followed by the MAJIQ Builder to determine differentially spliced genes (Vaquero-Garcia et al, 2016), and visualized using the MAJIQ Voila Viewer with a Dy threshold of 0.1 and significance of 0.05. Splice variant classification analysis was performed using the MAJIQ classifier (Vaquero-Garcia et al, 2021) with permission and assistance from Dr. Yoseph Barash.…”