Changqing Wang & Dr Shian Su

WORKSHOP: Analysis of long-read sequencing technology

Part 1: Igniting full-length isoform and mutation analysis of single-cell RNA-seq data with FLAMES

Long-read single-cell RNA-sequencing (scRNA-seq) enables accurate determination of novel isoforms in order to assess transcript heterogeneity in health and diseases. In addition, Single-nucleotide variants (SNPs) and small insertions and deletions (INDELs) can be quantified at the single-cell level to investigate cancer heterogeneity.The analysis of long-read scRNA-seq data is currently limited by the scarcity of relevant software. To fill this gap, we have developed the open-source FLAMES software, which covers all major aspects of long-read scRNA-seq data analysis from preprocessing through to differential analyses. This part of the workshop will cover isoform and mutations analysis with FLAMES.

Part 2: Long-read methylation data analysis with NanoMethViz and Bioconductor

In this workshop, we provide a Bioconductor analysis pipeline for DNA methylation. We highlight NanoMethViz, an R package for the analysis of DNA methylation using long-read sequencing data. DNA methylation is a critical epigenetic mechanism involving the addition of methyl groups to DNA, affecting gene expression without altering the genetic sequence. This process plays a pivotal role in development, health, and disease, making its study essential. Starting from modBAM files, which are currently the standard output of ONT-based modification calling pipelines, we will learn to perform exploratory data analysis to uncover high level methylation patterns over genes and across samples. We proceed to delve deeper to find differential methylated regions (DMRs), and associate them with genes to potentially uncover features that are affected by epigenetic regulation. Using NanoMethViz we can plot the methylation signals in the discovered DMRs or other regions of interest in order to generate a high resolution plots of methylation profiles, as well as data from individual long-reads. We will also cover data querying features of NanoMethViz to perform more custom analyses on the raw data, as well as more advanced features of the package for methylation data analysis.

Keywords: Long-read sequencing, methylation

Requirements: Laptop