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Introduction to metagenomics

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last_modification Last modification: Dec 7, 2022

Metagenomics


Overview


Why study the microbiome?

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.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. ]


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

Speaker Notes

puzzle analogy: think of each organism as a jigsaw puzzle


Amplicon

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


Amplicon

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


Amplicon

Speaker Notes 18S gene also amplifies archaea


Shotgun metagenomics


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


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

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:


Pre-processing

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 ]

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.pull-right[ ASV Denoising:

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

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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 This material is licensed under the Creative Commons Attribution 4.0 International License.