Metabarcoding in�Microbial Ecology

Overview

1. Background
2. Microbial Community Analysis
3. Case Studies

Background

Microbial ecology (or environmental microbiology) explores the biodiversity, distribution, and abundance of microorganisms, their abiotic and biotic interactions, and their effect on the ecosystem.

Microscopes to sequencers

Pasteur, Koch, Beijerinck, and Winogradsky created the basis of modern microbiology. Kluyver and van Niel brought it into the molecular biology era.

• Medical microbiology
• Immunology
• Industrial microbiology
• Microbial ecology

20/21st century molecular techniques have sped up the investigation of the microbial world astronomically

• The difference between the number of cells countable by microscopic examination and those forming colonies on agar media.
• Growth on media defines very specific conditions
• Only 0.1 - 0.01% of marine bacteria grow on standard plates!

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• Till the last decades of the 20th century, Microbial Ecology was principally based on culture-dependent methods.
• rRNA genes directly captured & sequenced changed everything! - Norman Pace

“The great plate count anomaly” (1985 Staley and Konopka)

“The great plate count anomaly” (1985 Staley and Konopka)

The evolution of sequencing

Illumina: 500,000,000 reads/run

454: 1,000,000 reads/run

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Solexa / Illumina

Ion Torrent

ONT

Pac Bio

Is cost everything? Solving complexity

454

2nd Gen. (AKA NGS)

1st Gen.

3rd Gen. (AKA LRS)

Sanger

Microbial�Community �Analysis

The biodiversity of each environment (all living organisms) can be fully represented by their genetic material.

Microbial’omics

Metabarcoding

Metatranscriptomics

&

Proteomics

Metagenomics

Metagenomics vs Metabarcoding

Metagenomics

• Identify species, genes, and functional capabilities of mixed communities
• Much more expensive in terms of sequencing and computational analysis
• Qualitative

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Metabarcoding

• High sensitivity in species resolution and identification
• Less expensive in terms of sequencing and computational analysis
• Quantitative (or semi-quantitative!?)
• May be biased - Different efficiency of marker amplification
• No functional information

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Metabarcoding / Amplicon Sequencing / Metagenetics

Before High Throughput Sequencing DNA barcoding was used as a method for species identification based on one feature:

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Use a genomic region variable enough to act as a DNA signature.

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Analysis based upon:

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• Raw sequences for direct phylogenetic comparison
• Compare sequences to a reference database for annotation

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Metabarcoding

Burki et al. Current Biology 2021

16S rRNA gene

18S rRNA gene

Dimension fitting with the sequencing platforms read length

Ubiquity in taxonomic range of interest

Reliable discrimination capacity at species level

Hyper-variable regions flanked by highly conserved ones in the taxonomic range under study

Important Features of Barcode Genes

Barcode Gene Choice

Prokaryotes:

• 16S rRNA gene: is widely accepted as marker gene
• 23S rRNA gene

Eukaryotes:

• ITS for fungi
• rbcL, matK, trnL, trnH-psbA for plants
• COX1/12S for animals
• 18S rRNA gene for protists

The ribosomal operon

Case Studies

The Human Microbiome Project

Tara Oceans

Earth Microbiome Project

• Some microbes are native, normally found in the body, Some other microbes are introduced, suddenly arriving at a new residence in the body.
• Development of a reference set of 3,000 isolate microbial genome sequences
• Initial 16S & WGS metagenomic studies to generate an estimate of the complexity of the microbial community at nasal passages, oral cavity, skin, gastrointestinal tract, and urogenital tract.

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Case Study: The Human Microbiome

The microbiome is shaped by:

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• Age
• Diet
• Geographical origin
• Gender
• Drugs

Case Study: The Human Microbiome

The Tara Oceans expedition spanned three years and covered six oceanic regions, representing a monumental effort in marine exploration. The campaign sampled 154 stations, deploying 720 rosettes and 1,360 nets, acquiring 28,420 samples.

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This project revealed more than a hundred thousand single-celled plankton species and most of the marine viruses currently known.

Case Study: Tara Oceans

The Earth Microbiome Project (EMP), launched in 2010, was a global initiative to analyze microbial communities within their environmental contexts. By studying 200,000 samples they aimed to provide new insights into microbial diversity and function.

Developed and curated completely standardi z ed protocols for sampling, metadata collection, sample treatment, storage, and sequencing.

Case Study: The Earth Microbiome Project

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