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Alignment behaviors of short peptides provide a roadmap for functional profiling of metagenomic data.

ABSTRACT: Functional assignments for short-read metagenomic data pose a significant computational challenge due to perceived unpredictability of alignment behavior and the inability to infer useful functional information from translated protein-fragments/peptides. To address this problem, we have examined the predictability of short peptide alignments by systematically studying alignment behavior of large sets of short peptides generated from well-characterized proteins as well as hypothetical proteins in the KEGG database.Using test sets of peptides modeling the length and phylogenetic distributions of short-read metagenomic data, we observed that peptides from well-characterized proteins had indistinguishable alignments to proteins from the same orthologous family and proteins from different families. Nonetheless, the patterns contained remarkable phylogenetic and structural signals, with alignments of even very short peptides naturally restricted to their orthologous family and/or proteins having similar structural folds. In stark contrast, peptides from "hypothetical proteins" had only sparse hit patterns with low frequencies and much lower identities. By weighting the structure-driven alignments and filtering peptides with behaviors similar to those derived from "hypothetical proteins", we demonstrate that the accuracy of abundance predictions of protein families is dramatically improved.Evolutionary processes have dispersed protein folds across multiple protein families, precluding accurate functional assignment to short peptides, whose alignment behavior is non-random and driven by structure. Algorithms that filter sparse peptides and weight hit patterns of peptides from "known space" dramatically improve quantification of functions from diverse mixtures of peptides and should substantially improve applications of metagenomic analyses requiring accurate quantitative measures of functional families.

SUBMITTER: Sinha R

PROVIDER: S-EPMC4687345 | biostudies-other | 2015

REPOSITORIES: biostudies-other

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Alignment behaviors of short peptides provide a roadmap for functional profiling of metagenomic data.

Sinha Rohita R Clarke Jennifer J Benson Andrew K AK

BMC genomics 20151221

<h4>Background</h4>Functional assignments for short-read metagenomic data pose a significant computational challenge due to perceived unpredictability of alignment behavior and the inability to infer useful functional information from translated protein-fragments/peptides. To address this problem, we have examined the predictability of short peptide alignments by systematically studying alignment behavior of large sets of short peptides generated from well-characterized proteins as well as hypot ...[more]

PMID: 26691573

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Project description:Covering: up to 2021Metagenomics has yielded massive amounts of sequencing data offering a glimpse into the biosynthetic potential of the uncultivated microbial majority. While genome-resolved information about microbial communities from nearly every environment on earth is now available, the ability to accurately predict biocatalytic functions directly from sequencing data remains challenging. Compared to primary metabolic pathways, enzymes involved in secondary metabolism often catalyze specialized reactions with diverse substrates, making these pathways rich resources for the discovery of new enzymology. To date, functional insights gained from studies on environmental DNA (eDNA) have largely relied on PCR- or activity-based screening of eDNA fragments cloned in fosmid or cosmid libraries. As an alternative, shotgun metagenomics holds underexplored potential for the discovery of new enzymes directly from eDNA by avoiding common biases introduced through PCR- or activity-guided functional metagenomics workflows. However, inferring new enzyme functions directly from eDNA is similar to searching for a 'needle in a haystack' without direct links between genotype and phenotype. The goal of this review is to provide a roadmap to navigate shotgun metagenomic sequencing data and identify new candidate biosynthetic enzymes. We cover both computational and experimental strategies to mine metagenomes and explore protein sequence space with a spotlight on natural product biosynthesis. Specifically, we compare in silico methods for enzyme discovery including phylogenetics, sequence similarity networks, genomic context, 3D structure-based approaches, and machine learning techniques. We also discuss various experimental strategies to test computational predictions including heterologous expression and screening. Finally, we provide an outlook for future directions in the field with an emphasis on meta-omics, single-cell genomics, cell-free expression systems, and sequence-independent methods.

Project description:BackgroundThe microbial communities populating human and natural environments have been extensively characterized with shotgun metagenomics, which provides an in-depth representation of the microbial diversity within a sample. Microbes thriving in urban environments may be crucially important for human health, but have received less attention than those of other environments. Ongoing efforts started to target urban microbiomes at a large scale, but the most recent computational methods to profile these metagenomes have never been applied in this context. It is thus currently unclear whether such methods, that have proven successful at distinguishing even closely related strains in human microbiomes, are also effective in urban settings for tasks such as cultivation-free pathogen detection and microbial surveillance. Here, we aimed at a) testing the currently available metagenomic profiling tools on urban metagenomics; b) characterizing the organisms in urban environment at the resolution of single strain and c) discussing the biological insights that can be inferred from such methods.ResultsWe applied three complementary methods on the 1614 metagenomes of the CAMDA 2017 challenge. With MetaMLST we identified 121 known sequence-types from 15 species of clinical relevance. For instance, we identified several Acinetobacter strains that were close to the nosocomial opportunistic pathogen A. nosocomialis. With StrainPhlAn, a generalized version of the MetaMLST approach, we inferred the phylogenetic structure of Pseudomonas stutzeri strains and suggested that the strain-level heterogeneity in environmental samples is higher than in the human microbiome. Finally, we also probed the functional potential of the different strains with PanPhlAn. We further showed that SNV-based and pangenome-based profiling provide complementary information that can be combined to investigate the evolutionary trajectories of microbes and to identify specific genetic determinants of virulence and antibiotic resistances within closely related strains.ConclusionWe show that strain-level methods developed primarily for the analysis of human microbiomes can be effective for city-associated microbiomes. In fact, (opportunistic) pathogens can be tracked and monitored across many hundreds of urban metagenomes. However, while more effort is needed to profile strains of currently uncharacterized species, this work poses the basis for high-resolution analyses of microbiomes sampled in city and mass transportation environments.ReviewersThis article was reviewed by Alexandra Bettina Graf, Daniel Huson and Trevor Cickovski.

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Alignment behaviors of short peptides provide a roadmap for functional profiling of metagenomic data.

Publications

Alignment behaviors of short peptides provide a roadmap for functional profiling of metagenomic data.

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