An introduction into 16S rrna gene sequencing analysis Stefan Boers
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
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), 11:1736-1751
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):e1002533
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
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
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
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):334-342
Workflow Sampling Extraction Amplification NGS Bioinformatics
Workflow Sampling Extraction Amplification NGS Bioinformatics Every step in this process could have serious impact on the microbiota results Topic of today: Bioinformatics
Data analysis ~ 25.000.000 reads
Data analysis Pre-processing Artefact removal OTU clustering Taxonomy assignment Results Reporting
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!
Chimera formation Chimeric sequences are artefacts resulting in: - Erroneous taxonomic identifications - Overestimated microbiota richness Genome Res (2011), 21: 494-504
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): 2194-2200
Prevent chimera formation Standard PCR 1 compartment Micelle PCR 10 10 compartments Sci Rep (2015), 5: 14181
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
Results of a synthetic community sample Sci Rep (2015), 5: 14181
OTU Clustering Clustering on basis of homology of the reads 97%
Assign taxonomy Homology with reference databases Berkeley lab August 2013 202,421 entries Max-Planc-Institut July 2015 172,418 entries Accuracy depends on quality and completeness of databases used Databases are inevitably incomplete Manual evaluation recommended (e.g. NCBI blast)
Results
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
MYcrobiota User just specifies input files and clicks Execute!
Huh, me?... Questions?...eh!...