R course

Daniel Vaulot

2023-01-26

Metabarcode analysis - Introduction

Outline

• Metabarcoding data

• Factors affecting protist communities

• Diversity

Intro to Metabarcoding

Metabarcoding

Sequences

Fastq files

Cluster

Assign

Metabarcoding pipeline

Data tables - ASVs

Data tables - Abundance

Data tables - Metadata

Data tables - Merged

Factors affecting protist communities

Substrate

• Water
• Ice
• Sediment
• Soil
• Microbiome

Ecosystem

• Oceanic
• Coastal
• Rivers
• Lakes
• Terrestrial

Size fraction

• Total (0.2 µm -> 100 µm)
• Pico (0.2 µm -> 2-3 µm)
• Nano (2-3 µm -> 20 µm)
• Micro (20 µm -> 100-200 µm)
• Meso (100 µm -> 1000 µm)

Factors affecting protist communities

Environmental conditions

In oceanic waters:

• temperature
• salinity
• light
• nutrients

… which depend on:

• substrate (water vs.ice)
• latitude
• time of the year
• depth
• oceanic currents
• proximity of coast

Estimating diversity

Diversity

Microbial species in a sample

• species richness: total number of species
• species abundance: proportion of each species

Richness vs. Evenness

Diversity

Alpha diversity - Diversity within a given sample

• Chao 1 is a non-parametric estimator of the number of species in a community.

• Shannon index¹

\(H = - \sum_{i=1}^{S} p_i \cdot \log{p_i}\)

Where:

\(p_i\) = fraction of the entire population made up of species \(i\) (proportion of a species i relative to total number of species present)

\(S\) = numbers of species encountered

A high value of \(H\) would be a representative of a diverse and equally distributed community and lower values represent less diverse community. A value of 0 would represent a community with just one species.

1. Shannon’s diversity index quantifies the uncertainty in predicting the species identity of an individual that is taken at random from the dataset.

Diversity

Alpha diversity - Effect of latitude

Diversity

Beta diversity - Compare diversity between samples

• Compute distance between samples:

  • Bray-Curtis dissimilarity: use abundance information

    • Varies between 0 and 1:
    • 0 means the two samples have the same composition
    • 1 means the two samples do not share any species

    \(BC_{jk} = 1 - \frac{2\sum_{i=1}^{p}min(N_{ij},N_{ik})}{\sum_{i=1}^{p}(N_{ij} + N_{ik})}\)

    where \(N_{ij}\) is the abundance of species \(i\) in sample \(j\) and \(p\) the total number of species

  • Jaccard similarity index

    • Number of common species between samples divided by total number of species in the two samples \(J(A,B) = \frac{|A \cap B|}{|A \cup B|}\)

• Ordinate the samples
  • NMDS: Non-Metric Multidimensional Scaling

Diversity

Beta diversity - Effect of depth on Stramenopiles communities