Classes from 2 to 8 pm Berlin time.



Theoretical introduction to Nanopore direct RNA-Sequencing and its applications



This first session will introduce the course and provide various concepts related to the execution and analysis of direct RNA-Seq experiments:


* Nanopore sequencing in general: what it is and how it works


* Application of Nanopore sequencing: DNA, cDNA and direct RNA (dRNA-Seq)


* Advantages and disadvantages of dRNA-Seq: comparison with Sequencing by Synthesis


* Direct RNA-Seq chemistry


* Format and structure of Nanopore data (fast5 files, etc.)


* From signal to sequence: the basecalling process


* Bioinformatics ecosystem for Nanopore: an introduction


* Advanced applications (e.g. targeted sequencing, metabolic labelling)







This session will start from an introduction on how a sequencing run is started, monitored and stopped. We will then basecall the raw data (Guppy) and look at how it is stored and organised on the filesystem (HDFview, ONT APIs)







Quality Controls



In this session you will learn to run and interpret standard quality checks performed on the basecalled data (pycoQC). We will then run common strategies to map the reads to the reference genome and transcriptome (minimap2) and perform a simple quantification of gene and transcript expression (Nanocount).




Advanced transcript quantification



We will look at various more advanced strategies to quantify alternative isoforms and perform splicing analyses using various algorithms (e.g. Flair, Sqanti, Pinfish, StringTie2)






De novo assembly of a transcriptome



This session will cover the task of de novo transcriptome assembly with isONclust2




Resquiggling and polyA tail measurement



You will learn to realign the raw electrical signal to the aligned sequence with Nanopolish (resquiggling) and to visualise the event-aligned signal. We will then present common algorithms to measure the length of the poly-A tail (Nanopolish, Tailfindr)



RNA modifications detection


In this session we will cover alternative strategies and algorithms to detect the presence of RNA modifications (e.g. Nanocompore, Epinano, Tombo, MINES)


Good practices in bioinformatics


In this session we will cover various modern technologies and best practices in bioinformatics. The concepts covered, while not necessarily specific for the analysis of Nanopore data, will provide a useful foundation for structuring analyses in an organised, clean and reproducible manner. The topics will include the use of containers (e.g. Docker and Singularity), environment management systems (e.g. conda) and workflow management (e.g. Snakemake and Nextflow)






Individual assignments and conclusions


The participants will be given a direct RNA-Seq dataset to analyse independently using various techniques learnt during the course. We also encourage participants to bring their own data to analyse under our guidance.