An introduction to Nanopore direct RNA Sequencing


24-26 June 2022

Due to the COVID-19 outbreak, this course will be held online




The recent advent of Nanopore direct RNA Sequencing allows for the first time to directly sequence full-length, native RNA molecules without the need for retrotranscription or amplification. This new technology offers remarkable advantages compared to short-read sequencing methods, for example it greatly simplifies the accurate detection and quantification of RNA isoforms, it allows the measurement of the polyA tail length and also allows the detection of RNA modifications. At the same time, the unique nature of Nanopore data poses various analytical challenges, thus requiring dedicated tools and new expertise.
This course is structured over 3 days of theoretical and hands-on training and covers the majority of the concepts and challenges commonly faced when analysing direct RNA-Seq data. It will start from common tasks such as data QC and gene expression quantification and then move on to more advanced topics such as transcriptome assembly, polyA-tail length measurements and RNA modifications detection.


Target audience and assumed background


This course is intended for an audience of researchers with a certain degree of familiarity with RNA sequencing concepts. While not exclusively directed to attendees with bioinformatics training, the majority of the practicals will make use of command-line tools. Therefore a good level of experience with a *nix environment (e.g. Linux or MacOS) and the shell (e.g. Bash) are highly desirable. Some familiarity with R will also be an advantage.




Friday 24th- Classes from 9.30 am to 7 pm Berlin time.


Session I. Theoretical introduction to Nanopore direct RNA-Sequencing

 Theoretical Background
 * 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 SBS
* Advanced applications (e.g. targeted sequencing, metabolic labelling)
* Direct RNA-Seq chemistry

 Introduction to Nanopore Data
 * From signal to sequence: the basecalling process (theoretical aspects)
* Format and structure of Nanopore data (fast5 files, etc.)
* Bioinformatics ecosystem for Nanopore: an introduction


 Mission Control
This session will start from an introduction on how a sequencing run is started, monitored and stopped. Run diagnostics parameters.

Session II. Quality Controls and basic analysis

We will basecall the raw data (Guppy) and look at how they are stored and organised on the filesystem (HDFview, ONT APIs)

Quality Checks
In this session you will learn to run and interpret standard quality checks performed on the basecalled data (pycoQC).

Read Mapping
We will run common strategies to map the reads to the reference genome and transcriptome (minimap2)

Basic Quantification
Perform a simple quantification of gene and transcript expression (Nanocount)




Dr. Tommaso Leonardi






COst overview

Cancellation Policy:




> 30  days before the start date = 30% cancellation fee


< 30 days before the start date= No Refund.




Physalia-courses cannot be held responsible for any travel fees, accommodation or other expenses incurred to you as a result of the cancellation.