BIOM25: 16S Practical

BIOM25: 16S Practical

In this practical we will analyse datasets from several studies, some very important, others perhaps just a little silly.

At first, we will go through a dataset together, this is from a pioneering paper:

After that, in groups, we will analyse one of three different datasets:

Please watch this video for a useful demonstration of how principal component analysis works: https://www.youtube.com/watch?v=BfTMmoDFXyE

General questions

Q: What is the difference between alpha- and beta-diversity?

Human microbiome in space and time

Paper: http://147.188.173.39/16stutorial/studies/spacetime/nihms245011.pdf

Supplementary material: http://www.sciencemag.org/cgi/content/full/1177486/DC1

Let’s have a look at the results.

Fields of importance: HOST_INDIVIDUAL, SEX, BODY_HABITAT, BODY_SITE, COMMON_NAME

Results:

Alpha diversity: http://147.188.173.39/16stutorial/studies/spacetime/core/arare_max500/alpha_rarefaction_plots/rarefaction_plots.html

Bar plots by sample site: http://147.188.173.39/16stutorial/studies/spacetime/core/taxa_plots_COMMON_SAMPLE_SITE/taxa_summary_plots/bar_charts.html

PCoA analysis: http://147.188.173.39/16stutorial/studies/spacetime/core/bdiv_even500/unweighted_unifrac_emperor_pcoa_plot/index.html

Q: Is there evidence of natural clusters being formed?

Q: Do samples cluster by individual? If not, how do they cluster?

Q: What are the most dominant taxa in stool, skin, urine? Look at different taxonomic levels down to genus.

Q: Are these sites similar or different? What are the major differences in taxonomic profile between these three sites?

##CSI: Microbiome

Original paper: http://147.188.173.39/16stutorial/studies/keyboard/keyboard_paper.pdf

Q: Skim read the introduction of the paper to get a feel for what they are trying to find out.

Q: Look at the Methods section and put the primer selection into TestPrime:

http://www.arb-silva.de/search/testprime

Results: http://147.188.173.39/16stutorial/studies/keyboard/core2/

Important metadata fields for this project: * Description_duplicate - the key from any keyboard * HOST_SUBJECT_ID - the person each keyboard belongs to

Hint: M1, M2 and M9 are the three participants referred to in the paper.

Q: What are the most abundant taxa?

Q: Check the PCA plots, do samples cluster by key, or by subject (hint: HOST_SUBJECT_ID, )

Q: Go back to the taxa barplots, can you figure out which taxa are driving the variation producing grouping?

Q: Which of these taxa are part of the normal skin microbiome? Are any out of plcae? Where might they come from?

Q: Do you think this technique will really be usable for forensics? What are the challenges? What other techniques might work better for studying the microbiome?

Q: Now, read the paper in more detail and prepare a short summary to present the context for the study, the methods employed and the results found.

##Restroom surfaces

Paper: http://147.188.173.39/16stutorial/studies/restrooms/pone.0028132.pdf

Q: Skim read the introduction of the paper to get a feel for what they are trying to find out.

Q: Look at the Methods section and put the primer selection into TestPrime:

Now, look at the output of QIIME:

Results: http://147.188.173.39/16stutorial/studies/restrooms/core/

Fields of importance: Floor, Level, SURFACE, BUILDING

Q: What surfaces have the greatest amount of diversity? Is this expected?

Q: What do the profiles of stool, etc. look like?

Q: Are there any natural looking clusters in the data?

Q: Which sources of samples are most similar to others?

Q: Is there any clustering between different floors of the building?

Q: Compare the weighted vs unweighted Unifrac results, do the clusters look more natural in one or the toher?

Q: Which surfaces have the most diversity? Least?

Q: Now, read the paper in more detail and prepare a short summary to present to the whole group. Consider: the context for the study, the methods that were employed and the results found. What did you think? What are the limitations of the study?

Infant gut metagenome

Paper: http://147.188.173.39/16stutorial/studies/infant_time_series/PNAS-2011-Koenig-4578-85.pdf

Q: Skim read the introduction of the paper to get a feel for what they are trying to find out.

Q: Look at the Methods section and put the primer selection into TestPrime:

Now, look at the output of QIIME:

Results: http://147.188.173.39/16stutorial/studies/infant_time_series/core/

Fields of importance: * SampleID - age in days of infant * SOLIDFOOD * FORMULA * COWMILK * BREASTMILK * COLORDESCRIPTION * HOST_SUBJECT_ID

Q: Is there any evidence of a gradient? (Key: use SampleID and turn gradient colours on)

Q: How do the taxa change over time?

Q: Which infant samples do the maternal stool most look like?

Q: Is the colour of stools associated with their bacterial diversity?

Q: Now, read the paper in more detail and prepare a short summary to present to the whole group. Consider: the context for the study, the methods that were employed and the results found. What did you think? What are the limitations of the study?

##Instructor notes on building this tutorial

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