Monday

Classes from 2 to 8 pm Berlin time

 

Lecture 1 – Introduction to NGS in microbial ecology

 

•    Key concepts (metabarcoding, metagenomics, single-cell sequencing)

 

•    Sequencing platforms (core concepts, read length, read numbers, error rates)

 

•    In-depth example of sequencing with Illumina platforms (over-and under-loading, sequencing process)

 

•    Genetic markers for metabarcoding (markers, primer selection & evaluation)

 

•    Experimental design (library preparation, replication, multiplexing, coverage, costs) -part1

 

•    Understanding data formats (FASTQ, FASTA, others)

 

•    Core concept of computational pipeline for amplicons

 

•    Introduction of the QIIME2 suite

 

 

 

Lab 1 – Introduction to compute lab

 

 

•    Introduction to the BASH command line (e.g. basic UNIX commands, batch processing)

 

•    Check functionality of computational environment with demo data

 

•    Checking basic characteristics of datasets (number of reads, read length, read quality)

 

 

 

Tuesday

Classes from 2 to 8 pm Berlin time

 

 

Lecture 2 – Quality control of NGS reads

 

 

•    Pre-PCR noise (under-sampling, DNA extraction bias, sample storage, contamination, metadata collection)

 

•    PCR-dependent noise (single nucleotide mis-incorporations, PCR chimeras, primer dimers, unspecific amplification, preferential amplification, template concentrations)

 

•    Sequencing-dependent noise (filtering/trimming poor base calls, dealing with substitution, insertion/deletion errors, index cross-talk, amplicon carry-over)

 

 

 

Lecture 3 – Binning into operational taxonomic units (OTUs) vs Exact Sequence Variants (exact sequence variants)

 

•    Core concept of OTUs and ESV

 

•    OTU binning strategies (de-novo vs. reference-based, impact of alignment strategies, hierarchical clustering algorithms, seed-based clustering algorithms, model-based clustering algorithms)

 

•    OTUs versus ESVs

 

 

 

Lab 2 – Sequence quality control and clustering into operational taxonomic units

 

•    Denoising, OTU binning, and ESV calling (e.g. paired-end merging, sequence filtering, dereplication, OTU clustering, chimera removal, target verification)

 

 

 

Wednesday

Classes from 2 to 8 pm Berlin time

 

 

Lecture 4 – Taxonomic Classification

 

 

•    Core concepts of taxonomic classification

 

•    Reference databases (INSDCs, SILVA, RDP, GREENGENES, UNITE)

 

•    Classification algorithms (similarity-based, composition-based, phylogeny-based)

 

•    Popular assignment approaches (Naïve Bayesian Classifier, BLAST)

 

•    Some additional tips

 

•   ITS sequence analysis

 

 

Lab 3 – Taxonomic classification

 

 

 •    Taxonomic classification using Naïve Bayesian Classifiers and VSEARCH taxonomy implemented in QIIME2

 

 •    Dealing with the preparation of custom databases for any genetic marker from NCBI

 

 •    Popular assignment approaches (Naïve Bayesian Classifier, BLAST)

 

 •    Visualising taxonomy data

 

 

 

Thursday

Classes from 2 to 8 pm Berlin time

 

 

Lecture 5 – Multivariate analysis of ecological communitie

 

•    Experimental design -part2

 

•    Traits of Alpha and Beta Diversity (richness, evenness, dispersion)

 

•    Ordination Techniques (Constrained vs Unconstrained)

 

•    Alpha and Beta Diversity in QIIME2

 

 

 

Lab  4 - Multivariate Statistics

 

 

•    Data import & preparation (normalisations, transformations, metadata)

•    Multivariate tests for differences in community composition (PERMANOVA, PERMDISP)

 

 

Lecture 6 – Differential Abundance Analysis of Microbial Communities

 

Lab 6 - Differential Abundance Analysis with DESeq2

 

 

Friday

Classes from 2 to 8 pm Berlin time

 

Group project

 

Case studies - Practicing what we’ve learned

 

Lecture 7 - Combining Microbiome data and life history data

 

Open discussion - let's discuss about your own data/projects