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.

 

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.

 

 

Monday 7th - 2-7 PM Berlin time - 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
* Direct RNA-Seq chemistries (RNA002 and RNA004)

Introduction to Nanopore Data
* From signal to sequence: the basecalling process (theoretical aspects)
* Format and structure of Nanopore data (fast5 files, etc.)
* Bioinformatics for Nanopore: an introduction
 
This session will start from an introduction on how a sequencing run is started, monitored and stopped. Importance of knowing the underlying wet lab experiment.

Getting ready for the analysis

• Quality checks (Nanoplot)

• One pipeline to run them all: Master of Pores

• Installing the pipeline and overview on the structure

 
Tuesday 8th - 2-7 PM Berlin time - First steps in the analysis of dRNA: mop_preprocess

Demultiplexing
Did you know that you can barcode your dRNA run?

• demultiplexing with deeplexicon

Basecalling

Converting the raw signal to fastq files and basecalled fast5 files.

• basecalling with guppy and with Dorado

Read Mapping
We will run common strategies to map the reads to the reference genome and transcriptome and evaluate the mapping.

• genome mapping with minimap2

• transcriptome mapping with graphmap

• inspection of the reads in IGV

Quantification and Transcript analysis
Perform a simple quantification of gene and transcript expression

• HTSeq count

• nanocount

Assign your reads to isoforms for splicing analysis and isoform expression

• Isoquant

Wednesday 9th - 2-7 PM Berlin time - Exploit the single molecule resolution of dRNA-Seq: tail length and RNA modifications

Resquiggling and polyA tail measurement
Common algorithms to measure the length of the poly-A tail

• Nanopolish

• Tailfindr

RNA modification detection - per position
Strategies and algorithms to detect the presence of RNA modifications

• Epinano

• Nanocompore

• Nanopolish

• Tombo

• all at once: mop_mod and mop_consensus

RNA modification detection - per read
Strategies and algorithms to call a modification at the basecalling step (focus on m6A)

• m6A basecaller

• Dorado

Thursday 10th - 2-7 PM Berlin time - 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.
 
Review & wrap-up

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.