Unraveling the functional dark matter through global metagenomics
Georgios A. Pavlopoulos, Fotis A. Baltoumas, Sirui Liu, Oğuz Selvitopi, Antônio Pedro Camargo, Stephen Nayfach, Ariful Azad, Simon Roux, Lee Call, Natalia Ivanova, I. Min Chen, David Páez-Espino, Evangelos Karatzas, Silvia G. Acinas, Nathan A. Ahlgren, Graeme T. Attwood, Petr Baldrián, Timothy D. Berry, Jennifer Bhatnagar, Devaki Bhaya, Kay D. Bidle, Jeffrey L. Blanchard, Eric S. Boyd, Jennifer L. Bowen, Jeff S. Bowman, Susan H. Brawley, Eoin Brodie, Andreas Brune, Donald A. Bryant, Alison Buchan, Hinsby Cadillo‐Quiroz, Barbara J. Campbell, Ricardo Cavicchioli, Peter F. Chuckran, Maureen L. Coleman, Sean A. Crowe, Daniel R. Colman, Cameron R. Currie, Jeff Dangl, Nathalie Delherbe, Vincent J. Denef, Paul Dijkstra, Daniel D. Distel, Emiley A. Eloe‐Fadrosh, Kirsten M. Fisher, Christopher Francis, Aaron Garoutte, Amélie C. M. Gaudin, Lena Gerwick, Filipa Godoy‐Vitorino, Peter Guerra, Jiarong Guo, Mussie Y. Habteselassie, Steven Hallam, Roland Hatzenpichler, Ute Hentschel, Matthias Hess, Ann M. Hirsch, Laura Hug, Jenni Hultman, Dana E. Hunt, Marcel Huntemann, William P. Inskeep, Timothy Y. James, Janet Jansson, Eric R. Johnston, Marina Kalyuzhnaya, Charlene N. Kelly, Robert M. Kelly, Jonathan L. Klassen, Klaus Nüsslein, Joel E. Kostka, Steven E. Lindow, Erik A. Lilleskov, Mackenzie M. Lynes, Rachel Mackelprang, Francis Martin, Olivia U. Mason, R. Michael L. McKay, Katherine D. McMahon, David A. Mead, Mónica Medina, Laura K. Meredith, Thomas Möck, William W. Mohn, Mary Ann Moran, Alison E. Murray, Josh D. Neufeld, Rebecca B. Neumann, Jeanette M. Norton, Laila P. Partida‐Martínez, Nicole Pietrasiak, Dale A. Pelletier, T. B. K. Reddy, Brandi Kiel Reese, Nicholas J. Reichart, Rebecca A. Reiss, Mak A. Saito, Daniel P. Schachtman, R. Seshadri
Abstract
Abstract Metagenomes encode an enormous diversity of proteins, reflecting a multiplicity of functions and activities 1,2 . Exploration of this vast sequence space has been limited to a comparative analysis against reference microbial genomes and protein families derived from those genomes. Here, to examine the scale of yet untapped functional diversity beyond what is currently possible through the lens of reference genomes, we develop a computational approach to generate reference-free protein families from the sequence space in metagenomes. We analyse 26,931 metagenomes and identify 1.17 billion protein sequences longer than 35 amino acids with no similarity to any sequences from 102,491 reference genomes or the Pfam database 3 . Using massively parallel graph-based clustering, we group these proteins into 106,198 novel sequence clusters with more than 100 members, doubling the number of protein families obtained from the reference genomes clustered using the same approach. We annotate these families on the basis of their taxonomic, habitat, geographical and gene neighbourhood distributions and, where sufficient sequence diversity is available, predict protein three-dimensional models, revealing novel structures. Overall, our results uncover an enormously diverse functional space, highlighting the importance of further exploring the microbial functional dark matter.