Metabarcoding Pipeline Building (Mothur & R)

5 - 9 July 2021

Prof. Louis-Felix Bersier, UNIFR
Ms Rachel Korn, UNIFR
Dr Magdalena Steiner, Agroscope, Wädenswil

Prof. Gerhard Thallinger, Technical University of Graz
Ms Rachel Korn, University of Fribourg
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

UNIFR

Full attendance and active participation.

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

When?

to be determined

Where?

University of Fribourg, Pérolles campus

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]

Reimbursement:

Travel:

StarOmics PhD students are eligible for the reimbursement of incurred travel expenses by train (half-fare card, and 2nd class).

The online reimbursement system is now in place, so once the course is over you will be able to request the reimbursement via MyCUSO, without sending the paperwork to me.

Accommodation:

Please contact the coordinator of the doctoral program (staromics(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. In case of overnight stay, please post the original tickets and original bills along with the reimbursement form to:

Corinne Dentan, StarOmics, UNIL, CIG, Le Génopode, 1015 Lausanne

NO reimbursement of meal expenses

To register, please use your MyCUSO account!

REGISTRATION IS NOT OPEN YET

Deadline for registration: to be determined

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

Registration Fees:

Free for participants enrolled in the CUSO StarOmics doctoral program.

In case of cancellations, before the deadline: free

Late cancellations or no-show: 100 CHF administrative fee

Other participants: please contact the program coordinator at ecologie-evolution(at)cuso.ch

16