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Microbial community / environmental DNA analysis with Chipster

This online hands-on course focuses on amplicon based community analysis methods. They are introduced using microbial communities as an example, but the methods are applicable to environmental DNA (eDNA) studies of other organisms as well.

This course covers the whole workflow from quality control and filtering to quantification and statistical analysis using Mothur and Phyloseq tools integrated in the user-friendly Chipster software. Note that while this course focuses on OTU-based analysis, Chipster contains also DADA2 tools for ASV-based analysis. A separate session on ASV analysis will be organized later this year.
The course consists of lectures and practical exercises. The lectures will be available as short videos, and the participants are requested to view them prior to the course. This gives you more time to reflect on the concepts so that you can use the course days more efficiently. The lectures are summarized and questions answered during the course.

Learning goals

After this course, you will be able to
– preprocess amplicon sequencing data for community analysis
– compare the structure of communities using ordinations and multivariate statistics

Prerequisites and target audience

The free and user-friendly Chipster software is used in the exercises, so no previous knowledge of Unix or R is required.
The course is intended for life scientists who are planning to use 16S or other amplicon sequencing in their research. This course is suitable also for those researchers who do not plan to analyze data themselves, but who need to understand the concepts in order to discuss with bioinformaticians.

Agenda

Day 1

  • Checking the quality of reads with MultiQC
  • Preprocessing
    • combining paired reads to contigs
    • screening sequences for length and ambiguous bases
    • removing identical sequences
  • Alignment
    • aligning sequences to the Silva reference alignment
    • screening aligned sequences for alignment position and homopolymers
    • filtering alignment for empty columns and overhangs
    • removing new identical sequences
  • Preclustering, removing chimeric sequences

Day 2

  • Data tidying and analysis
  • Creating and importing phyloseq input files
    • Including taxonomic assignments and OTU clustering
  • Data inspection and tidying using phyloseq
    • Removing unwanted taxa
    • Filtering singletons and doubletons
    • Prevalence filtering
  • Data transformation, ordination and conversion
    • CLR, Hellinger and % relative abundance
    • nMDS and db-RDA
    • DESeq2 format conversion and variance-stabilising transformation
  • Relative abundance plots
  • Community comparisons using PERMANOVA, PERMDISP and DESeq2

Trainers

Dr Heli Juottonen (CSC), Dr Eija Korpelainen (CSC)