Visualisation and analysis of RNA-Seq assembly graphs

RNA-sequencing (RNA-Seq) is a powerful transcriptome profiling technology enabling transcript discovery and quantification. RNA-Seq data are large, and most commonly used as a source of genelevel quantification measurements, whilst the underlying assemblies of reads, if inspected, are usually viewed as sequence reads mapped on to a reference genome. Whilst sufficient for many needs, when the underlying transcript assemblies are complex, this visualisation approach can be limiting; errors in assembly can be difficult to spot and interpretation of splicing events is challenging. Here we report on the development of a graph-based visualisation method as a complementary approach to understanding transcript diversity and read assembly from short-read RNA-Seq data. Following the mapping of reads to the reference genome, read-to-read comparison is performed on all reads mapping to a given gene, producing a matrix of weighted similarity scores between reads. This is used to produce an RNA assembly graph where nodes represent reads derived from a cDNA and edges similarity scores between reads, above a defined threshold. Visualisation of resulting graphs is performed using Graphia Professional. This tool can render the often large and complex graph topologies that result from DNA/RNA sequence assembly in 3D space and supports info rmatio no verlay on to nodes, e.g. transcript models. We have also implemented an analysis pipeline for the creation of RNA assembly graphs with both a command-line and web-based interface that allows users to create and visualise these data. Here we demonstrate the utility of this approach on RNA-Seq data, including the unusual structure of these graphs and how they can be used to identify issues in assembly, repetitive sequences within transcripts and splice variants. We believe this approach has the potential to significantly improve our understanding of transcript complexity.