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Microbiome analysis using 2nd and 3rd generation DNA sequencing - 2025 - 5 ECTS
Provider: Faculty of Science

Activity no.: 5325-25-04-31There are 55 available seats 
Enrollment deadline: 01/08/2025
Date and time11.08.2025, at: 09:00 - 22.08.2025, at: 16:00
Regular seats60
Activity Prices:
  - Industry/For-profit18,000.00 kr.
  - PhD student (Open Market Agreement, Nova, VLAG)6,000.00 kr.
ECTS credits5.00
Contact personHenriette Hansen    E-mail address: henha@food.ku.dk
Enrolment Handling/Course OrganiserLukasz Krych    E-mail address: krych@food.ku.dk
Written languageEnglish
Teaching languageEnglish
Semester/BlockSummer
Exam formCourse Participation; Written Assignment
Exam detailsAssessment will include active participation and submission of a written report. The report will involve topics such as data analysis workflows, interpretation of microbiome datasets, and integration of course content. Participants may incorporate their own PhD project data into the report to demonstrate personalized application of course concepts.
Grading scalePassed / Not passed
Course workload
Course workload categoryHours
Course Preparation15.00
Laboratory20.00
Lectures25.00
Theoretical exercises30.00
Report writing47.50
Sum137.50
Aim and content
Adequate generation and analysis of DNA sequencing data should be a part of every (medicine-, research-, and industry-) scientist’s toolbox. In this short course, theory and hands-on sample preparation and DNA sequencing using 2nd (Illumina) and 3rd (Oxford Nanopores Technologies) generation sequencing technologies will be provided. As a core content of the course, participants will be able to process raw data (e.g., pod5/fastq) and use downstream software for mapping amplicons (e.g., 16S rRNA gene), re-constructing genomes and estimating abundance tables (e.g., OTU tables) using state-of-the-art and open access bioinformatic algorithms.

Furthermore, participants will be able to apply exploratory analysis (e.g., alpha- and beta-diversity) for factor/treatment associations, as well as several univariate and multivariate analytical tools for variable/feature selection. These tools are typically needed to gain deeper understanding on microbiome (e.g., variable selection, size effect), and the linking of microbiome with other omics and environmental data.

Formal requirements
Basic Knowledge in molecular biology, DNA extraction, PCR assembly.

Learning outcome
Knowledge:

By the end of this course, participants will be able to
• Assess DNA quality and concentration using spectrophotometric and colorimetric methods.
• Prepare barcoded 16S rRNA gene amplicon libraries for Illumina and ONT sequencing.
• Generate sequencing libraries for near full-length 16S rRNA genes and full genomes using ONT.
• Load and operate sequencing platforms (e.g., MinION, PromethION, flongle, Illumina iSeq).
• Analyze raw sequencing data (Illumina fastq and ONT pod5) to generate OTU tables using R based tools.
• Perform alpha- and beta-diversity analyses and exploratory statistics for microbiome profiling.
• Conduct discriminant analysis (DA) and differential abundance testing using advanced statistical tools.

Skills:
• Build the sequencing libraries for barcoded 16S rRNA gene amplicon (v3 region) sequencing with Illumina
• Build the sequencing libraries for barcoded 16S rRNA gene amplicon (near full: v1-v9) and full genome sequencing with ONT.
• Load and flash MinION flow cells; load flongle. Prepare and load Illumina iSeq flow cells.
Perform alpha-diversity (e.g., Shannon index, Observed Species), beta-diversity analysis (e.g., PCoA, NMDS) and statistical tests (e.g., Adonis, ANOSIM) for exploratory approaches.
• Perform Discriminant Analysis (DA) with taxonomy agglomeration, multiple-testing in DA and multivariate DA (PLS-DA).
• Perform differential abundance testing for differences in taxa between two or more categories (ANCOM, ANOVA, metagenomeSeq)

Competences:
• Analyse data from raw fastq (Illumina) to generation of OTU-table (introduction to basic unix commands, QIIME2, and R).
• Analyse data from raw fast5 (ONT) to generation of OTU-table (introduction to Guppy (GPU based basecalling), MinKNOW).
• Run de-novo assembly using Illumina, ONT and hybrid-assembly

Target group
PhD students, Industry/For-profit

Teaching and learning methods
The course is designed for 10 days (2 weeks, Monday-to-Friday) of active participation. The first week focuses on wet lab procedures and generation and handling of raw data (e.g., pod5/fastq files), while the second week focuses on introduction to basic data analysis and extended practical exercises on microbiome data analysis.

• Introduction to 2nd (Illumina) and 3rd (Oxford Nanopore Technologies) generation DNA sequencing strategies.
• Perform library preparation for amplicon sequencing with Illumina and Oxford Nanopore Technologies (ONT) sequencing, and introduction to metagenome sequencing strategies.

Lecturers
Dr. Lakmal Jayasinghe leads the biological nanopore research team at Oxford Nanopore Technologies.

Lakmal joined Oxford Nanopore Technologies in 2006 after finishing his PhD in chemical biology in the University of Oxford. During his PhD in the Hagan Bayley group, Lakmal has studied different nanopores and has gained a wealth of knowledge in engineering nanopores using genetic and chemical approaches. His responsibilities at ONT include improving the readout signal of the ONT platform by upgrading its current nanopore reader and discovering and engineering new nanopores to suit various ONT applications and platforms. Lakmal also works with many academic nanopore collaborators across the world to make sure Oxford Nanopore uses the best possible nanopore reader in its platforms.

Dr. Michal Kaszuba is a CEO at GenXone sequencing.
GenXone company is active in the biotechnology industry and offers nucleic acid (DNA and RNA) sequencing services based on the innovative nanopore sequencing technology, Next-Generation Sequencing (NGS), also known as third-generation sequencing. The company is the first in Poland and one of 32 companies in the world to be accredited and licensed for commercial use of the nanopore sequencing technology developed by Oxford Nanopore Technologies. Based on the access to ONT technology, the company creates its own, new solutions in the form of laboratory protocols as well as bioinformatics tools, scripts and algorithms, enabling the use of nanopore sequencing in many areas of science and business, including in genetic diagnostics, food industry, veterinary medicine and agriculture. The experienced team of GenXone under the lead of Dr Michal Kaszuba consists of specialists in the field of molecular genetics and bioinformatics, works on modern solutions.

Prof. Jakub Dalibor Rybka is a vice-director at the Centre for Advanced Technologies, Adam Mickiewicz University in Poznan.
Jakub does research in Biotechnology, Solid State Physics and Materials Physics and single cell sequencing. Their current projects revolve around tissue engineering, 3D bioprinting, meniscus regeneration, single cell sequencing and most recently, COVID-19 diagnostics. He is also actively participating in efforts directed towards automation and robotization of biotechnological laboratories. Professor Rybka is giving updates on single cell sequencing and broad perspective view on the future of nucleic acids sequinning using solid-state nanopore systems.

Remarks
Microbiome Analysis Course offered by University of Copenhagen has been designed and organized by Lukasz Krych to meet a high demand from PhD students and a private sector filing the gap in courses portfolio. Microbiome analysis using nanopore sequencing is highly unique, novel, and narrow field of research. Very few units offer comprehensive training on:
1. wet lab molecular solutions
2. nanopore segueing or
3. raw data treatment
4. multivariate data analysis.
To the best of our knowledge our course is the only one that offers all these four points in one, carefully designed course. In 2020 we started with 20 students and each year we receive very positive feedback, and the number of participants is constantly growing. In 2023 and 2024 we have hosted all together over 80 international students.

Course Fee:
o 6,000 DKK for students enrolled at Danish universities.
o 18,000 DKK for all other participants.

The fee covers reagents, food (breakfast and lunch), and access to sequencing instruments.

Students must bring laptops with native UNIX-like systems (e.g., Linux or macOS). Support for other systems cannot be guaranteed.

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