[REPORT À 2021] Metabarcoding Pipeline Building - reported

the course has been postponed to a later date

Rachel Korn, UNIFR

Dr Magdalena Steiner, Agroscope, Wädenswil

Prof. Gerhard Thallinger, Graz University of Technology

Rachel Korn, UNIFR

Dr Magdalena Steiner, Agroscope Wädenswil

Metabarcoding is a rapidly evolving technique that identifies DNA from a set of often unknown organisms and reveals microbial communities in unprecedented taxonomical resolution by using universal PCR primers and high-throughput sequencing. It increasingly gains importance in almost all fields of biological and medical sciences. Its wide application range includes biodiversity assessment and monitoring, diet analysis, reconstruction of paleocommunities and more. The processing of metabarcoding data requires a broad range of skills at the interface of molecular biology, bioinformatics, biostatistics and biology/ecology.

In this workshop the participants will be introduced to the workflow of microbiome research targeting the 16S and 18S rRNA gene: preceded by an overview of the full process, this course is aimed at the analysis of high-throughput sequencing data with open-source and cross-platform software (mothur and R/bioconductor packages) from the raw reads to the visualization of results. Theoretical content will be visualized and strengthened by practical exercises. Participants will practice during the workshop on prepared data sets.

Prior requirements

A basic statistic knowledge as well as basic skills in R are a precondition for this workshop. The participants are required to bring their own notebooks with the software already installed. For this, a Docker image containing all necessary software will be provided to guarantee architecture and version control.

Preliminary Schedule

Introduction to metabarcoding

• DNA extraction
• Internal controls (positive, negative)
•  Primer choice (16S, 18S, ITS…)
• NGS technologies (Illumina, IonTorrent, PacBio)
• Sequencing variants, data formats
• FASTQ format
• Reference databases (SILVA, greengenes, PR2, …)
• Amplicon sequence variants (ASV/ESV) vs. operational taxonomic units (OTU)

Quality control

• Visualization, trimming and filtering (dada2)

Pipeline: OTU construction (mothur)

• Read merging/contigs assembly
• Alignment to reference database (SILVA)
• Pre-clustering
• Chimera detection
• Singleton filtering
• Distance matrix
• OTU clustering, generation and classification (taxonomy, methods)

Pipeline: ASV construction (dada2)

• Denoising (sample-wise error rate)
• Read merging/contigs assembly
• Chimera detection
• Taxonomy assignment (SILVA, DECIPHER)

Analysis (R, main packages: phyloseq & vegan)

• BIOM format
• Analysis approaches: OTU/ASV vs. phylogeny
• Preprocessing & vizualisation (absolute, relative, rank abundance)
• Taxonomic filtering
• Rarefaction
• Community composition (α-, β- and γ-diversity, diversity indices...), multivariate analysis (ordination)
• Differentially abundant OTUs/ASVs
• LefSe (Linear discriminant Effect Size analysis)
• Procrustes 

University of Fribourg

Full attendance and active participation.

Make sure to sign the attendance list each and every day!

When?

The course has been postponed to a later (not yet known) date

Where?

University of Fribourg, Perolles campus: Mo 09:00 - 12:00 & 13:00 - 15:00 PER 21 B207

Tu 09:00 - 12:00 & 13:30 - 18:00 Pavillion Vert (inside botanical garden)

We 09:00 - 12:00 & 13:30 - 18:00 Pavillion Vert

Th 09:00 - 12:00 & 13:30 - 18:00 Pavillion Vert Social evening

Fr 09:00 - 12:00 Pavilion Vert

The course is organized in collaboration with the CUSO Microbiology, CUSO StarOmics and UNINE Organismal Biology Doctoral Programs.

16 places in total, equally shared among the co-organising programs (4 for each program)

Questions?

Marta Bellone

@: [email protected]

Phone: +41 (0)26 300 88 91

Reimbursement:

Participants should request their reimbursement to their own program coordinator.

PhD candidate of DPEE are eligible for reimbursement of incurred travel expenses by train (half-fare card, and 2nd class). Please send the original tickets along with the reimbursement form to:

Marta Bellone Department of Biology

University of Fribourg

Chemin du Musée 10

CH-1700 Fribourg

NO reimbursement of your meal expenses

Regarding reimbursement of accommodation, please contact the coordinator of the doctoral program (ecologie-evolution(at)cuso.ch) BEFORE the beginning of the course. NO reimbursement of accommodation without the coordinator of the doctoral program agreement prior of the course.

Registration is open until 21 September 2020.

Priority is given to PhD students of the co-organising programs (each program has 4 places reserved) unitl 13 September 2020 - after this deadline places will be assigned on a first come, first serve basis, starting from the people in waiting list.

To register, please use your MyCUSO account!

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