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Introduction to Microbiome Analysis


last_modification Published: Jun 22, 2017
last_modification Last Updated: May 29, 2024

Microbiome Analysis


Why study the microbiome?


] .pull-right[

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


Why study the microbiome?

.image-75[ Rhizodeposition: image of a tree converting sun and co2 into fixed carbon used as food for soil microbes. ]

Shotgun vs Amplicon


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

Shotgun Sequencing


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

Amplicon Sequencing

Speaker Notes

puzzle analogy: think of each organism as a jigsaw puzzle


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


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

Speaker Notes V1, V2 etc are the variable regions


Speaker Notes 18S gene also amplifies archaea

Shotgun metagenomics


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


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

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:


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

OTU Clustering / Denoising

.pull-left[ OTU clustering:

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


.pull-right[ ASV Denoising:

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


Speaker Notes ASV stands for “Amplicon Sequence Variants”

TODO: expand on OTU vs ASV a bit more

Chimera Removal

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

Search marker database and taxonomy assignment

Search functional database

Results: OTU table

screenshot of an excel table

Results: Visualizations

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

Results: Visualizations

screenshot of phinch website showing multiple plot types

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.