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Metagenomes encode an enormous diversity of proteins, reflecting a multiplicity of functions and activities. 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 analyze 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. 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 neighborhood 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. 

Abstract Tropical cyclones play an increasingly important role in shaping ecosystems. Understanding and generalizing their responses is challenging because of meteorological variability among storms and its interaction with ecosystems. We present a research framework designed to compare tropical cyclone effects within and across ecosystems that: a) uses a disaggregating approach that measures the responses of individual ecosystem components, b) links the response of ecosystem components at fine temporal scales to meteorology and antecedent conditions, and c) examines responses of ecosystem using a resistance–resilience perspective by quantifying the magnitude of change and recovery time. We demonstrate the utility of the framework using three examples of ecosystem response: gross primary productivity, stream biogeochemical export, and organismal abundances. Finally, we present the case for a network of sentinel sites with consistent monitoring to measure and compare ecosystem responses to cyclones across the United States, which could help improve coastal ecosystem resilience.