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Microbiome analysis using 2nd and 3rd generation DNA sequencing - 2020
Provider: Faculty of Science

Activity no.: 5325-20-04-31 
Enrollment deadline: 05/08/2020
Date and time17.08.2020, at: 08:30 - 28.08.2020, at: 16:00
Regular seats28
Activity Prices:
  - Industry/For-profit12,000.00 kr.
  - PhD student (Open Market Agreement, Nova, VLAG)4,500.00 kr.
  - Deltager/Participant Others12,000.00 kr.
ECTS credits5.50
Contact personLukasz Krych    E-mail address: krych@food.ku.dk
Enrolment Handling/Course OrganiserLukasz Krych    E-mail address: krych@food.ku.dk
Written languageEnglish
Teaching languageEnglish
Scheme groupNot included in the scheme group
Scheme group noteDaily teaching hours: 8:30-16:30
Exam formWritten assignment
Exam detailsEvaluation of written project report (passed/not passed). The report must be delivered no later than 3 weeks after the end of the course.
Exam aidsAll aids allowed
Grading scalePassed / Not passed
Exam re-examinationAfter agreement with the course responsible
Course workload
Course workload categoryHours
Theory exercises30.00
Report writing60.00


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. 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.

Aim and content:

The course is designed for for 10 days (2 weeks, Monday-to-Friday) of active participation, week one includes wet lab 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.

• Introduce to 2nd (Illumina) and 3rd (Oxford Nanopore Technologies, PacBio) 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.
• Introduction

Formel requirements
Formal requirements: Students must bring their own laptops with native UNIX-like based systems only, e.g. Linux (Ubuntu 16.04 or 18.04) or macOS (Mojave 10.14 or higher). We cannot guarantee support if other systems are used.

Learning outcome
Learning objectives:

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), Porechop and MinKNOW).
• Run de-novo assembly using Illumina, ONT and hybrid-assembly (introduction to: Trimmomatic, NanoFilt, (meta)-Spades, Centrifuge, Kraken2, 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)
• Perform multivariate analysis for integration of microbiome vs omics data (Canonical Correlation Analysis - CCorA)

Teaching and learning methods
The course consists of lectures, practical wet-lab and dry-lab exercises. Students will be able to get hands-on experience with ONT and Illumina libraries preparation techniques. The course includes a computer exercise where students will be able to analyze data.

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

Josue L. Castro-Mejia
Postdoc, 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

Duccio Cavalieri
Associate professor, Department of Biology, University of Florence

The course fee is 4500 DKK for students at Danish universities and VLAG and 12000 DKK for all other participants. The course fee covers the cost of reagents and food (breakfast and lunch).

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