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 4 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.

Note: This year's edition will focus on the new RNA004 chemistry.

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 required.

The course will be taught over the course of four days, with the fourth day being dedicated to a hands-on session where the participants will be able to perform analyses of either their own data, on their own server, or data provided by the instructors,  on our server.

Day 1: Intro to Nanopore sequencing, Nanopore data structure and RNA004 chem
Day 2: Basecalling, mapping and transcript quantification analysis
Day 3: Tail length and RNA modification analysis
Day 4: Hands-on session (several groups based on participants’ interests)


Day 1. 2-7 PM Berlin time
 
Introduction to Nanopore Sequencing

- 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
- Direct RNA-Seq chemistries (RNA002 and RNA004)
- Types of dRNA seq experiments

Introduction to Nanopore Data

- Format and structure of Nanopore data (fast5 files, etc.)
- From signal to sequence: the basecalling process (theoretical aspects)
- Bioinformatics for Nanopore

Getting ready for the analysis

-Workflow of a dRNA analysis pipeline
-Quality checks
-Demultiplexing


Day 2. 2-7 PM Berlin time

First steps in the analysis of dRNA

-Basecalling
-Converting the raw signal to fastq files with Dorado

Mapping

-Read mapping with minimap2
-Inspection of the reads in IGV

Transcript analysis

-Isoform discovery and quantification with Isoquant


Day 3. 2-7 PM Berlin time
Exploit the native information of dRNA-Seq: tail length and RNA modifications

The potential of single molecule and direct sequencing

-Measure the length of the poly-A tail
-RNA modification detection strategies per position and per read
-Focus on m6A: the new generation of tools
-Concepts of per read analysis


Day 4. 2-7 PM Berlin time

Individual assignments and conclusions

The participants will either analyze their own data under our guidance, or be given a direct RNA-Seq dataset to analyse independently using various techniques learnt during the course.

The participants will be split into four groups based on their preferences:

1. Analyzing their own data (on their own sever)
2. Analyzing rRNA to find differences in modification levels between two samples
3. Analyzing mRNA to detect m6A-DRACH
4. Analyzing mRNA to detect isoforms of selected genes

Case Studies and future perspectives

We will discuss recent developments in the field (selected publications and technology improvements), as well as future directions the field could develop in.

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.