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

Basecalling
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)

 

 

 

Saturday 25th- Classes from 9.30 am to 7 pm Berlin time.

 

Session I. Advanced transcript quantification

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

Transcriptome-based genome annotation

Isoform calling with a reference genome

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

Session II. Resquiggling and polyA tail measurement

Resquiggling
You will learn to realign the raw electrical signal to the aligned sequence with Nanopolish (resquiggling) and to visualise the event-aligned signal.

PolyA tail length
We will then present common algorithms to measure the length of the poly-A tail (Nanopolish, Tailfindr)

 

 

Sunday 26th- Classes from 9.30 am to 3 pm Berlin time.

 

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

 

 Theoretical overview

 

 Practical (Tombo? Nanocompore)

Session II. Individual assignments and conclusions

 

Assignments
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.

 

Review & wrap-up