Microbes

Week 11 - Analyzing protist communities

MetaPR2

A database of metabarcodes

Daniel Vaulot

2023-01-17

Outline

  • Metabarcoding data

  • Factors affecting protist communities

  • Diversity

  • Visualization/Analysis

  • MetaPR2 in practice

  • Final presentation

Metabarcoding

Metabarcoding

Fastq files

Metabarcoding

Metabarcoding

Data tables

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

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.

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

MetaPR2 - Datasets

MetaPR2 - Taxonomy

Eight levels:

  • Kingdom: Eukaryota
  • Supergroup: Archaeplastida
  • Division: Chlorophyta
  • Class: Mamiellophyceae
  • Order: Mamielliales
  • Family: Bathycoccaceae
  • Genus: Bathycococcus
  • Species: B. prasinos

MetaPR2 - Visualization

MetaPR2 - In practice

Help

  • Read in detail

Sample table

  • dataset_name
  • paper (can be useful to read)
  • number of samples
  • number of ASVs
  • number of reads per sample (coverage)

Sample selection

  • Major datasets: OSD, Tara, Malaspina
  • By habitat: oceanic, coastal etc…
    • Start by “marine global V4”
    • Extend to other habitats/datasets
  • V4 vs V9
  • DNA vs. RNA
  • Ecosystems
  • Sustrate: water, ice, soil…
  • Size fractions: total, pico…
  • Depth level: surface, euphotic…
  • Minimum ASV: will filter out rare ASVs (e.g. 1000)
  • Selection can be saved (yaml file)

MetaPR2 - In practice

Taxonomy

  • Can select several taxa within one level
  • Press validate every time you need to refresh
  • Can exclude taxa to remove fungi, metazoa…
  • Can save taxonomy and reload taxonomy (yaml file)

MetaPR2 - In practice

Treemaps

  • Left panel: abundance (number of reads)
    • Reads are “normalized” to 100
  • Right panel: diversity (number of ASVs)

Maps

  • Read information at top
    • Taxo level
    • Number of samples with/without taxa
  • Crosses where taxa absent
  • Map types
    • Dominant
    • Pie chart
  • Circle scale
    • Moving right increases size

Barplots

  • taxonomy vs. function
  • variables to use (but this depends on samples selected !)
    • fraction name
    • ecosystem
    • substrate
    • depth level
    • DNA_RNA
    • latitude
    • temperature
    • salinity
    • year, month, day for time series

MetaPR2 - In practice

Diversity

  • Hit “Compute…” after refreshing taxonomy
  • Time proportional to N samples and taxa
  • Information about
    • Number of samples
    • Number of taxa (ASVs)

Alpha diversity

  • X: Chao1, Shannon, Simpson (compare)
  • Discretize continuous Y
  • Change Y (see barplots)
  • Change shape
  • Change color

Beta diversity

  • Ordination method (difference ?)
  • Ordination distance (Bray, Jaccard…)
  • Change color and shape

MetaPR2 - In practice

Download

  • Download
    • datasets
    • samples
    • asv list with taxonomy
    • asv sequences

Only for those with extensive experience with data processing.

Final presentation

Taxonomic groups

Green algae

  • Prasinoderma

  • Ostreococcus

Ochrophyta (Stramenopiles)

  • Pelagomonas, Aureococcus

  • Florenciella

  • Pinguiophyceae

Final presentation

Taxonomic groups

Diatoms

  • Pseudo-nitzschia

  • Fragiliaropsis

  • Minidiscus

  • Rhizosolenia

Dinoflagellates

  • Dinophysis

  • Ceratium, Tripos

Final presentation

Key points

  • Look for key papers on this group
  • What are the dominant species?
  • What is the microdiversity [diversity within dominant species (ASVs)]?
  • What is distribution ?
    • Substrate (water, ice…)
    • Ecosystems (marine, freshwater, terrestrial)
    • Size fraction
    • Depth layers (euphotic zone vs. meso and bathypelagic)
    • Latitudinal bands (polar, temperate, tropical)
    • Coastal vs Pelagic
  • Alpha diversity
  • Beta diversity

Final presentation

In practice

  • Each group will have max of 15’ to present their results. Your time will be cut after 15’.
  • Don’t overload your presentation and run when talking. This will decrease the clarity of your presentation.
  • Share equally time between group members.
  • Introduce very briefly the main biological characteristics and ecological importance of your taxonomic group.
  • Explain which hypotheses/questions your group were interested in.
  • Explain the results you have observed. Focus on main points.
  • Each group will have 5’ to answer questions.

Final presentation

Evaluation

  • Profs, TAs and PhD’s students will be judging your presentation (Only Profs will grade!):
    • Grade scale: 0 = unacceptable; 1 = poor; 2 = fair; 3 = good; 4 = outstanding

Criteria

  • Quality of presentation
    • Slides (font size, amount on slide, legible and clear, references, no errors, etc).
    • Organization of presentation (outline, logical sequence, good transitions, easy to follow, etc).
    • Quality of oral presentation (well paced, projected voice, face audience, eye contact, confident, etc).
    • Did the group keep the audience interested? (show enthusiasm, command attention, did you learn something new?)
    • Was the presentation within the 15 minutes in length?
  • Content of presentation
    • Was the presentation well structured ?
    • Did the group show an overall understanding of the topic? (background, objectives and significance thoroughly explained?).
    • Did the presentation cited the relevant material from the litterature?
    • Did the group answered questions accurately? Did the group possess good understanding of topic based on answers?