METAGENOMICS. Aina Maria Mas Calafell Genomics

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1 METAGENOMICS Aina Maria Mas Calafell Genomics

2 Introduction Microbial communities Primary role in biogeochemical systems

3 Study of microbial communities 1.- Culture-based methodologies Only isolated microbes Phenotypic, biochemical and physiological tests Molecular techniques o G+C content o DNA-DNA pairing o Targeted sequencing (Rosselló-Mora & Amann, 2001)

4 Study of microbial communities 2.- Culture-independent DNA sequence-based methodologies Metagenomics Culture-independent and genomic analysis of microbial communities (Tringe & Rubin, 2005)

5 2.1.- Sanger sequencing Low-throughput targeted sequencing Sequencing of 16S rrna genes (Janda & Abott, 2007) Well-conserved Large enough No horizontal transfer Molecular clock Community structure

from the uncultured shotgun libraries with a

6 2.1.- Sanger sequencing Low-throughput shotgun sequencing Community structure A) Number of sequences from the uncultured shotgun libraries with a significant hit (E < 0.001) to GenBank Functionallity (Breitbart et al., 2002)

Study of microbial communities 2.- Culture-independent DNA sequence-based methodologies 2.1.

and bacteriophague sequences are not carried stably by E.coli 2.

7 Study of microbial communities 2.- Culture-independent DNA sequence-based methodologies Sanger sequencing Cost Prohibitive to sequence highly complex communities Certain microbial and bacteriophague sequences are not carried stably by E.coli SGS and TGS Higher troughput (Mardis, 2008) Lower cost Faster In vitro amplification (SGS) or lack of amplification (TGS)

8 2.2.- SGS and TGS Expansion of microbiome studies (Segata et al., 2013) High-throughput shotgun sequencing Whole-metagenome shotgun Sequence of thousands of organisms at the same time High-throughput targeted sequencing Detection of very low abundance members Increased taxonomic resolution (Petrosino et al., 2009)

9 2.2.- SGS and TGS High-throughput shotgun sequencing Comparison of technologies (Luo et al., 2012) (Mardis, 2008) (Barzon et al., 2011)

10 2.2.- SGS and TGS High-throughput shotgun sequencing (Bragg & Tyson 2014)

11 New view Metagenomics Human microbiome Honey bee colonies Sea Deep mine (Venter et al., 2004) Soil (Fierer et al., 2007)

12 Human microbiome

13 Human microbiome (Bäckhed et al., 2006) (Qin et al., 2010) Humans ~ Superorganisms composed of human and microbial components

highlight how they contribute to our normal physiology and

14 Human Microbiome Project To understand the microbial components of our genetic and metabolic landscape To highlight how they contribute to our normal physiology and disease predisposition (Turnbaugh et al., 2007) (NIH HMP Working group, 2009)

15 Human Microbiome Project (The Human Microbiome Jumpstart Reference Strains Consortium, 2010) (Cuomo et al., 2014) (The Human Microbiome Project Consortium, 2012)

16 Future insights Deeper coverage of the microbiome Effects of age and diet? Effects of pathologic states? Effect of different living environments Human Microbiome

17 Future insights Human Microbiome Deeper coverage of the microbiome Effects of age and diet? Effects of pathologic states? Effect of different living environments New biomarkers for defining our health New ways for optimizing our personal nutrition New ways to forecast our predispositions to disorders

18 A wide view of microbial communities Meta omics (Segata et al., 2013)

19 Conclusions The understanding of complex microbial communities can have several applications. DNA sequence-based methods circumvented the obstacles of culture-based approaches. Next-generation sequencing technologies have expanded the experimental tools available for studying microbiomes. The current bottleneck is the downstream analysis of data. Metagenomic studies are ongoing in several important ecosystems, specially the human body in health and disease. Metagenomics is complemeted with different approaches to have an overall view of microbial communities, including their functionallity.

20 References Bäckhed et al. «Host-bacterial mutualism in the human intestine.» Science, 2006 Mar 25; 307(5717): Barzon et al. «Applications of Next-Generation Sequencing Technologies to.» Int. J. Mol. Sci., 2011; 12, Bragg andtyson. «Metagenomics using Next-Generation Sequencing.» Methods Mol Biol, 2014; 1096: Breitbart et al. «Genomic analysis of uncultured marine viral communities.» Proceedings of the National Academy of Science, 2002 Oct 29; 99(22): Cuomo, y otros. «Genome Sequence of the Pathogenic Fungus Sporothrix schenckii (ATCC 58251).» Genome Announc., 201; May 22;2(3). Fierrer, y otros. «Metagenomic and small-subunit rrna analyses of the genetic diversity of bacteria, archaea, fungi and viruses in soil.» Appl Environ Microbiol, 2007; Nov; 73(21): Janda, y Abbott. «16S rrna Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls.» J Clin Microbiol, 2007; Sep 45(9): Luo, Tsementzi, Kyrpides, Read, y Konstantinidis. «Direct Comparisons of Illumina vs. Roche 454 Sequencing Technologies on the Same Microbial Community DNA Sample.» PLoS One, 2012; Feb 7(2). Mardis. «The impact of next-generation sequencing technology on genetics.» Trends Genet, 2008; Mar; 24(3): Petrosino, Highlander, Luna, Gibbs, y Versalovic. «Metagenomic Pyrosequencing and Microbial Identification.» Clin Chem, 2009: May 55(5): Qin et. al «A human gut microbial gene catalogue established by metagenomic sequencing.» Nature, 2010; 464, Rosselló-Mora, y Amann. «The species concept for prokaryotes.» FEMS Microbiology reviews 25, 2001: Segata, Boernigen, Tickle, Morgan, Garrett, y Hutternhower. «Computational meta'omics for microbial community studies.» Molecular Systems Biology, 2013: 9:666.

21 The Human Microbiome Consortium Jumpstart Reference Strains. «A catalog of reference genomes from the human microbiome.» Science, 2010; May 21; 328(5981): Tringe, y Rubin. «Metagenomics: DNA sequencing of environmental samples.» Nature Reviews Genetics, 2005: Nov 6(11): Turnbaugh, Ley, Hamady, Fraser-Liggett, y Knight. «The human microbiome project: exploring the microbial part of ourselves in a changing world.» Nature, 2007: Oct 449(7164): Venter, y otros. «Environmental Genome Shotgun Sequencing of the Sargasso Sea.» Science, 2004: April Vol. 304 no pp

Infectious Disease Omics

Infectious Disease Omics Metagenomics Ernest Diez Benavente LSHTM ernest.diezbenavente@lshtm.ac.uk Course outline What is metagenomics? In situ, culture-free genomic characterization of the taxonomic and