Fermer

Metabarcoding pipeline building

5-9 October

University of Fribourg

Course in collaboration with the CUSO Doctoral Programs in Ecology & Evolution, StarOmics and Microbial Sciences.

Organisers: Rachel Korn (University of Fribourg), Dr Magdalena Steiner (Agroscope, Wädenswil)

Program

Objectives

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.

Speakers

Content

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.

Preliminary Program

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

Requirements for attending and completing the workshop

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.

General information

Dates: 5-9 October 2020 (5 days)

Schedule: 8.55-17.00

Venue:  Boulevard de Pérolles 90, building 21/22, rooms G314 & G514, 1700 Fribourg To be confirmed

ECTS: 2.5 (Research tools)

Evaluation: Full attendance and active participation

Information: Please contact  the organisers or the Doctoral Program Coordinator

Registration fee: free for members of the Interuniversity doctoral program in organismal biology (DPOB)

Travel expenses: For participants of the DPOB: see reimbursement conditions

Make sure to sign the attendance sheet each and every day and to take your certificate of attendance at the end of the course (no certificate will be sent by mail)

Registration

  • Maximum number of participants: 16
  • Limited number of participants: only 4 for DPOB program

Registration through the web only: CUSO EE activity webpage. There are 0 places left for DPOB!

Please note the cancellation policy (CHF 100).

Deadline: please refer to the CUSO EE activity webpage