16S RNA sequencing analysis Bioinformatics and 16S RNA sequencing analysis http://okinbretest.ouhsc.edu/ http://okinbretest.ouhsc.edu/Bioinformatics.aspx

Three 16S rRNA Sequencing Questions to Answer Why would we want to do it? How is it performed? What is 16S rRNA sequencing?

What is bioinformatics? Terms to Define Biology Computer Science Math/Statistics What is bioinformatics?

What is Amplicon Sequencing? When a particular gene or gene fragment is amplified and the sequence determined to achieve insight when studying microbiomes (all microorganisms in a particular environment). http://users.ugent.be/~avierstr/principles/pcr.html

Metagenome-the genomes of the total microbiota in a community. Metagenomics allows us to extract DNA sequences from a microbial community in nature bypassing the need for cultures.

What is the 16S gene and why us it? 16S – named because rate at which it sediments in ultracentrifuge (Svedberg) Protein found in small subunit of ribosome (Present in all species) Ubiquitous - Highly conserved because mutation would probably be deleterious Extreme sequence conservation useful for primers Variable regions for organism identification Well annotated reference databases http://www.alimetrics.net/en/index.php/dna-sequence-analysis http://www.nature.com/nrmicro/journal/v12/n9/fig_tab/nrmicro3330_F1.html

Variable and conserved regions within the 16S rRNA gene Adapted from: Kevin E. Ashelford et al. Appl. Environ. Microbiol. 2005;71:7724-7736 http://www.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf Illustrating variable regions within the 16S rRNA gene and location of chimeric breakpoints. (A) The frequency of occurrence of the most common nucleotide residue at each base position within the 16S rRNA gene, as determined from RDP-listed 4,383 type strains, with E. coli U00096 as a reference. These frequencies are measures of variability within the gene. (B) Smoothing the data, by taking the mean frequency within a window of 50 bases, moving one base at a time along the gene, creates the plot shown in panel B. The locations of the hypervariable regions are labeled, with gray bars on the x axis defining these regions as V1 to V9 (the Comparative RNA Web Site [http://www.rna.icmb.utexas.edu/]). (C) Histogram of all chimera breakpoints identified in this study and that of Hugenholtz and Huber (8). Fry and Ellis, Patent Publication number WO2014138119 A2 Example of primers

Variation in rRNA gene copy number Valdivia-Anistro, J.A., et al., Front. Microbiol. 05 January 2016| http://dx.doi.org/10.3389/fmicb.2015.01486 Most bacteria contain more than one rRNA operon, and copy number varies Can affect abundance estimates And some bacteria have high levels of sequence divergence in the rRNA operons This can inflate diversity estimates Can attempt to correct this (PICRUSt, CopyRighter)

16S pipeline Prepping Sample Extracting DNA Sampling Bioinformatics http://newsexaminer.net/opinion/were-treating-soil-like-dirt-its-a-fatal-mistake-because-all-human-life-depends-on-it/ http://mrdnalab.com/dna-sequencing/illumina-miseq.html http://www.copybook.com/pharmaceutical/companies/anachem/articles/the-new-kapa-express-extract-from-anachem-ltd https://www.neb.com/protocols/2015/01/23/setting-up-the-pcr-reaction-e7600 http://www.clipartbest.com/cartoon-computer-pictures Sampling Extracting DNA Prepping Sample Bioinformatics Sequencing

Which sequencing chemistry to use? Kuczynski et al., Nat. Rev. Genet. 13: 4-58 (2012)

Sequencing by Synthesis and Basecalling http://openwetware.org/wiki/BioMicroCenter:Sequencing

Illumina Inc. Video Illustrating Prepping and Sequencing by Synthesis Illumina Sequencing Technology Intro to Sequencing by Synthesis: Industry-leading Data Quality https://www.youtube.com/watch?v=womKfikWlxM https://www.youtube.com/watch?v=HMyCqWhwB8E

Bioinformatics

Workflow for deep sequencing of 16S rRNA gene amplicons Schematic overview of the workflow involved to analyze the microbiota composition by deep sequencing of 16S rRNA gene amplicons. Ines Yang et al. FEMS Microbiol Rev 2013;37:736-761

Operational Taxonomic Units (OTUs) and Annotation - MiSeq Reporter Operational taxonomic unit-extant taxon Cluster of similar amplicon sequences 97% identity commonly used Pre-clustering to collapse all identical sequences into one category speeds analysis OTU determination De novo methods cluster by similarity with no reference to outside sequences Taxonomy-based methods cluster based on similarity to known sequences-uses ClassifyReads, a proprietary algorithm Combined taxonomy + de novo methods Known sequence databases Ribosomal Database Project (https://rdp.cme.msu.edu/) GreenGenes (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi)

Statistically Speaking and Interpreting the Data

Species Diversity Results Species richness Number of species present in a sample Determined by the number of OTUs present Influenced by sequencing depth Species evenness How close in numbers each species is in a sample Species diversity Composite of species richness and species evenness

α-diversity and Shannon’s diversity index Diversity within a sample Measures the amount of information needed to describe every member of the community Shannon’s diversity index in an information statistic index If pi is the proportion of individuals of species i, then the diversity (H’) is: From this, one can calculate evenness, which is the ratio of the actual H’ to the maximum value (and so ranges from 0 to 1) Adapted from: http://ww2.tnstate.edu/ganter/B412%20L16%20Communities.html

Species Diversity Results Species richness Number of species present in a sample Determined by the number of OTUs present Influenced by sequencing depth Species evenness How close in numbers each species is in a sample Species diversity Composite of species richness and species evenness

Flower species Field 1 Field 2 Daisy 300 20 Dandelion 335 49 https://www.greenthumb.co.uk/help-and-advice/lawn-problems/weeds Flower species Field 1 Field 2 Daisy 300 20 Dandelion 335 49 Buttercup 365 931 Total 1000 1000 Which field is more diverse? Field 1

Hierarchical Clustering Dendrogram Dendrogram: a tree diagram Topology- how closely sample related The two samples (clusters) most similar are clustered together forming a new cluster. At each step, the next closest sample is clustered with the new cluster.

What is a Principle Coordinate Analysis (PCoA) Graphically represents similarities or dissimilarities of data. Begins with a distance matrix ends with computation of Eigen values and vectors. Objects ordinated closer to one another are more similar than those ordinated further away.

Why do 16S rRNA sequencing? http://commonfund.nih.gov/hmp/overview

Scheme for studying the “normal” human microbiome. Bacterial distribution by body site. This figure shows the distribution by body site of bacteria that have been sequenced under the HMP or are in the sequencing pipelines. The NIH HMP Working Group, Peterson J, Garges S, et al. The NIH Human Microbiome Project. Genome Research. 2009;19(12):2317-2323. doi:10.1101/gr.096651.109.

α Diversity rarefaction curves of cutaneous microbiota in psoriasis (lesion), unaffected and control specimens. (A) Taxonomical richness trends towards decreasing α diversity in unaffected and lesion specimens relative to control, with no statistically significant differences between skin types. (B) Shannon index is significantly different (decreases from control to unaffected to lesion) among skin types at all taxonomic levels (P <0.05), except at the operational taxonomical unit (OTU) level. (C) Analysis of taxa sharing. Taxa present in <3 samples excluded from the analysis. Taxa that are only observed in one clinical skin type are denoted as ‘unique’. Taxa that are present in two types of skin are denoted as ‘shared’. The data show that nearly all taxa are represented in all three types of skin. The shading represents the relative distribution (heatmap) for each column number (green = low, yellow = intermediate, red = high). Alekseyenko AV, Perez-Perez GI, De Souza A, et al. Community differentiation of the cutaneous microbiota in psoriasis. Microbiome. 2013;1:31. doi:10.1186/2049-2618-1-31.

Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences Morgan G I Langille, et al. Nature Biotechnology 31, 814–821 (2013) doi:10.1038/nbt.2676 (2013).

Shannon Species Diversity Number of Species Identified Site 1 Site 2 Site 4 Site 3 Site 5 Site 6 Sample Shannon Species Diversity Number of Species Identified St. James Place 2.625 653 53rd Street 2.553 1490 44th Street West 2.571 1478 44th Street East 2.659 1178 38th Street 2.727 1545 Coombs Road 2.826 462

Summary What? How? Why? Bioinformatics Amplicon sequencing and metagenomics 16S gene How? Pipeline and Different Sequencing Platforms Bioinformatic’s Step Operational Taxonomic Units Statistical Terms Alpha Diversity and Shannon Index Species Diversity, Richness and Evenness Hierarchical Clustering Dendrogram and PCoA Why? Human Microbiome Project – Microbiota in Psoriasis Demonstration Project Cameron University’s Wolf Creek Microbial Diversity Project