FI:IV110/IV114 Projekt z bioinformatiky

a systémové biologie
Monika Čechová, Matej Lexa

@biomonika, @matej_lexa
My motivation:


Answering biological questions using next-generation sequencing data
● Course requirements


● Project proposals

● Nanopore sequencing: collaboration with the Institute of Biophysics of the Czech
Academy of Sciences

● Introduction to the Nanopore sequencing and its applications


● Hands-on exercise: species identification
Detekce kontaminací
● Dr. Lucie Grodecká a Dr. Roman Hobza
Nanopore sequencing
https://www.gatc-biotech.com/fileadmin/_processed_/


csm_Sanger_sequencing_illustration_small_898122494c.jpg
Nanopore
https://www.scienceinschool.org/content/
decoding-dna-pocket-sized-sequencer
Nanopore
●Introduction (3 mins)


○ https://www.youtube.com/watch?
v=GUb1TZvMWsw
https://twitter.com/MakovaLab
Real-time surveillance
Sequencing the station: Investigation aims to
identify unknown microbes in space
https://phys.org/news/2017-04-sequencing-station-aims-unknown-
microbes.html
https://nanoporetech.com/resource-centre/posters/dna-sequencing-microgravity-international-space-
station-iss-using-minion
https://flxlexblog.wordpress.com/2016/07/08/
developments-in-high-throughput-sequencing-july-2016-
edition/
Long-read sequencing
● Sample quality

● Library preparation (size selection, repair)


● Protocols


● Sequencing throughput


● Multiplexing


Budgeting is important: get yourself familiar with the cost of reagents and sequencing!


There’s always a trade-off.
Long-read applications
Long reads
1. Transcriptome (isoforms)


2. Assembly (PacBio HiFi + ultra-long Nanopore reads)


3. Epigenetic modifications
1. RNA sequencing with Nanopore
https://nanoporetech.com/applications/rna-sequencing
2. Linear assembly of the RP11 Y centromere
3. Nanopore sequencing meets epigenetics


Figure 1 | DNA methylation can be read out
directly by nanopore sequencing. Single-stranded
DNA alters ionic current in a sequence-dependent
manner as it passes through a pore (left), with
methylated bases highlighted with red and blue
tags. The raw current signal (right) indicates small
changes due to methylation that a new set of
algorithms can robustly interpret.
Schatz, M. Nanopore sequencing meets epigenetics. Nat
Methods 14, 347–348 (2017). https://doi.org/10.1038/
nmeth.4240
Error profiles
	
	
	
	
	
	


	
	
	


	
	
	
	


	
	
	
	
	


RS Harris, M Cechova, KD Makova. Noise-Cancelling Repeat Finder: Uncovering tandem
repeats in error-prone long-read sequencing data. BIOINFORMATICS, 2019.

	
	
	
	


Cechovaetal.,2019


Harrisetal.,2019
We demonstrated that
orthogonal
technologies (such as
Illumina, Nanopore,
and PacBio) are
generally concordant
in distinguishing
between highly and
lowly abundant
repeated motifs.
The visualization of sequencing errors
https://www.pacb.com/blog/igv-3-improves-support-pacbio-long-reads/
Metagenomics
● https://nanoporetech.com/resource-centre/snow-sledges-and-sequencing-
grid-metagenomics-polar-expedition
Read until
https://nanoporetech.com/resource-centre/read-until-adaptive-sampling
Hands-on exercise: species identification
● Go to the folder: https://is.muni.cz/auth/el/fi/podzim2021/IV110/data/fast5/


● Download three files in .fast5 format


● Identify the species(s) of origin


● Upload your results into Odevzdávarna


Fast5 format: https://medium.com/@shiansu/a-look-at-the-nanopore-fast5-
format-f711999e2ff6