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Introduction to Microbiome Analysis

Contributors

last_modification Published: Jun 22, 2017
last_modification Last Updated: Aug 8, 2024

Microbiome Analysis

Speaker Notes


Overview

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Why study the microbiome?

.pull-left[

] .pull-right[

.image-100[ image of a human with lines leading from various points on the body to pie charts of bacteria distribution ]

]

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Why study the microbiome?

.image-75[ Rhizodeposition: image of a tree converting sun and CO2 into fixed carbon used as food for soil microbes. ]

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Types of microbiome analysis

.image-75[ Experimental-workflows-for-mainstream-omics ]

.footnote[Image source: 10.1039/D3MO00089C]

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Shotgun vs Amplicon

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.image-75[ jigsaw puzzle image illustrating shotgun sequencing approach ]

Shotgun Sequencing

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.image-75[ jigsaw puzzle image illustrating amplicon sequencing approach ]

Amplicon Sequencing

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Amplicon

left is picture of e. coli ribosome with "so much rna" written, right is the RNA structure in a diagram

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Amplicon

line graph showing V regions as peaks along x axis of alignment position, and y index of shannon-index/alignment-index.

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Amplicon

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Shotgun metagenomics

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End-to-End

graphi with an arrow going from sampling (nose picture) to extraction to amplification to ngs to bioinformatics (computer picture)

Every step in this process can have serious impact on the results

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Bioinformatics

cartoon titled drowned in NGS data with a man covered in messy data.

Roche 454 GS:    ~ 100.000 reads
Illumina MiSeq:  ~ 25.000.000 reads
Shotgun:             ~ ? reads

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Analysis pipelines

comparison of amplicon and shotgun pipelines. amplicon pipeline goes to community structure viz, shotgun pipeline goes to community structure in addition to functional assignment.

Amplicon analysis variants:

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Pre-processing

fastqc plot with regions labelled good, okay, and bad based on the plot's colour

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OTU Clustering / Denoising

.pull-left[ OTU clustering:

.image-75[ tree with clusters around the leaves labelled OTUs and 97% identity threshold ]

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.pull-right[ ASV Denoising:

.image-90[ DNA sequences with hierarchical clustering shown ]

]

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Chimera Removal

aborted extension leads to mispriming, which gets extended with the wrong sequence, and results in a chimera

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Search marker database and taxonomy assignment

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Search functional database

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Results: OTU table

screenshot of an excel table

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Results: Visualizations

doughnut plot with bands for each kingdom, down to genus, species

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Results: Visualizations

screenshot of phinch website showing multiple plot types

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Differential analyses

Maaslin2 Example

.footnote[Figure taken from: https://doi.org/10.1101/2021.01.20.427420] Speaker Notes


Diversity

Diversity Example

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Assembly

Assembly Example

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Binning

Binning Example

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Thank you!

This material is the result of a collaborative work. Thanks to the Galaxy Training Network and all the contributors! Galaxy Training Network Tutorial Content is licensed under Creative Commons Attribution 4.0 International License.