Faculty of Science

Microbiome analysis using 2nd and 3rd generation DNA sequencing - 2023 - 5.5 ECTS
Provider: Faculty of Science

Activity no.: 5325-23-04-31
	Enrollment deadline: 01/08/2023

Place

Department of Food Science

Date and time

14.08.2023, at: 09:00 - 25.08.2023, at: 16:00

Regular seats

Activity Prices:

- Industry/For-profit

18,000.00 kr.

- PhD student (Open Market Agreement, Nova, VLAG)

6,000.00 kr.

ECTS credits

5.50

Contact person

Henriette Hansen E-mail address: henha@food.ku.dk

Enrolment Handling/Course Organiser

Lukasz Krych E-mail address: krych@food.ku.dk

Written language

English

Teaching language

English

Semester/Block

Summer

Exam form

Course Participation; Written Assignment

Grading scale

Passed / Not passed

Course workload

Course workload category	Hours
Course Preparation	15.00
Laboratory	20.00
Lectures	25.00
Theoretical exercises	30.00
Report writing	60.00

Sum	150.00

Enrolment guidelines

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., fast5/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.

The course is dedicated for PhD students and industry representatives. The course fee is 6000 DKK for students at Danish universities and 18,000 DKK for all other participants. The fee is to cover the cost of reagents and food (breakfast and lunch).

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 of raw data (e.g., fast5/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.
• Reconstruct microbiomes based on Illumina and Oxford Nanopore Technologies based amplicon sequencing; introduction to ONT metagenome analysis and hybrid assembly (Illumina & ONT) approaches.

Formal requirements

Students must bring their own laptops with native UNIX-like based systems only, e.g., Linux (Ubuntu 16.04 or higher) or macOS (Mojave 10.14 or higher). We cannot guarantee support if other systems are used.

Learning outcome

The participant will be able to:

• Assess DNA quality and concentration with spectrophotometric and colorimetric methods.
• 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.
• 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 (introduction to: e.g., Trimmomatic, NanoFilt, (meta)-Spades, Canu, Flye).
• 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)

Teaching and learning methods

The course consists of lectures, practical wet-lab and dry-lab exercises. Students will be able to get hand-on experience with ONT and Illumina libraries preparation techniques. The course includes computer exercise where students will be able to analyze microbiome data generated during the course.

Lecturers

Lukasz Krych
Associate professor, Department of Food Science, University of Copenhagen

Josue L. Castro-Mejia
Assistant Professor, Department of Food Science, University of Copenhagen

Dennis S. Nielsen
Professor, Department of Food Science, University of Copenhagen

Morten A. Rasmussen
Associate professor, Department of Food Science, University of Copenhagen

Jakub D. Rybka
Associate professor, Centre of Advanced Technologies, Adam Mickiewicz University of Poznan, Poland

Grzegorz Nowicki
R&D Manager at GenXone Sequencing, Oxford Nanopore Technologies Certified Service Provider, Poznan, Poland.

Remarks

The course fee is 6,000 DKK for students at Danish universities and VLAG and 18,000 DKK for all other participants. The course fee covers the cost of reagents and food (breakfast and lunch).

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