An introduction into 16S rrna gene sequencing analysis. Stefan Boers

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1 An introduction into 16S rrna gene sequencing analysis Stefan Boers

2 Microbiome, microbiota or metagenomics? Microbiome The entire habitat, including the microorganisms, their genomes (i.e., genes) and the surrounding environmental conditions. Microbiota The assemblage of microorganisms that reside in a defined environment. Metagenome (and metagenomics) The collection of genomes and genes from the members of a microbiota. Microbiome (2015), 3:31

3 Microbiota profiling Greatly enhancing our insights into the microbial diversity and taxonomy of many different types of environments and ecosystems WHO IS THERE? 16S rrna gene - Universal gene in bacteria and archaea - Conserved regions - species-independent PCR amplification - Variable regions identify and compare bacteria/archaea Environ Microbiol (2009), 11:

4 The human microbiota Even odds - Bacteria were once thought to outnumber human cells by 10-to-1 - New calculations show roughly equal numbers of each (1.3-to-1) The microbiome is essential for human development, immunity and nutrition - Help to digest food - Protect against other bacteria - Produce vitamins - Regulate our immune system PLoS Biol. (2016), 14(8):e

5 Fecal microbiota transplantation (FMT) Restore the healthy complement of gut bacteria - First described in the 4 th century China ( yellow soup ) FMT was significantly more effective for the treatment of recurrent C. difficile infection: - FMT cured 15/16 patients (94%) - Vancomycin cured only 7/26 patients (27%) Also promising results with Irritable Bowel Syndrome and Crohn s Disease N Engl J Med (2013), 368;5

6 Requirement for scepticism Microbiota research is currently a hot topic - Many claims of associations between the human microbiota and disease Overweight? - Wrong balance of bacteria in the digestive tract Liver disease among alcoholics? - Overgrowth of intestinal bacteria of which their toxic breakdown products damage the liver NRC.nl, 16 April 2016

7 Requirement for scepticism We see disease alter the intestinal flora, but is that cause or effect? Willem de Vos, microbiologist Microbiota results are often obtained using small cohort-sized studies - Many studies lack the statistical power to test microbiota-based hypotheses What about the quality of microbiota research - Reproducibility? NRC.nl, 16 April 2016

8 Inter-laboratory quality assessment High inter-laboratory deviations were observed Standardization and development of methods to increase cross-study comparability is urgently needed Int J Med Microbiol (2016), 306(5):

9 Workflow Sampling Extraction Amplification NGS Bioinformatics

10 Workflow Sampling Extraction Amplification NGS Bioinformatics Every step in this process could have serious impact on the microbiota results Topic of today: Bioinformatics

11 Data analysis ~ reads

12 Data analysis Pre-processing Artefact removal OTU clustering Taxonomy assignment Results Reporting

13 Pre-processing Good Okay Bad There are a lot of options to filter and trim your data However, this does not always improve things as we lose information!

14 Chimera formation Chimeric sequences are artefacts resulting in: - Erroneous taxonomic identifications - Overestimated microbiota richness Genome Res (2011), 21:

15 Chimera removal Query Chunk Chunk Chunk Chunk Hits Chimera A A Normal Query Query B No bioinformatics method has been shown to eliminate these artefacts entirely Bioinformatics (2011), 27(16):

16 Prevent chimera formation Standard PCR 1 compartment Micelle PCR compartments Sci Rep (2015), 5: 14181

17 Micelle PCR Clonal-based amplification strategy - Lower susceptibility to chimera formation - Lower susceptibility to variations in PCR amplicon efficiencies Addition of an unique internal calibrator - Express the results as a measure of 16S rrna gene copies - Subtract 16S rrna gene copies that were also quantified in a negative extraction control Sci Rep (2015), 5: 14181

18 Results of a synthetic community sample Sci Rep (2015), 5: 14181

19 OTU Clustering Clustering on basis of homology of the reads 97%

20 Assign taxonomy Homology with reference databases Berkeley lab August ,421 entries Max-Planc-Institut July ,418 entries Accuracy depends on quality and completeness of databases used Databases are inevitably incomplete Manual evaluation recommended (e.g. NCBI blast)

21 Results

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23 How to do all that? Two pieces of very sophisticated open source software: mothur QIIME Both programs are driven from the command line Requires bioinformatics skills Results depend on algorithms used and their settings Development of MYcrobiota: Translation of a validated workflow in a user-friendly (automated) pipeline Plug and Play standardized algorithms and settings Quality control where did I lose reads and the ability to download them to check! User defined reporting based on ireport functionality Dynamic visualization of bacterial taxonomies and alpha/beta diversities

24 MYcrobiota User just specifies input files and clicks Execute!

25 Huh, me?... Questions?...eh!...