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Correcting for 16S rRNA gene copy numbers in microbiome surveys remains an unsolved problem.

ABSTRACT: The 16S ribosomal RNA gene is the most widely used marker gene in microbial ecology. Counts of 16S sequence variants, often in PCR amplicons, are used to estimate proportions of bacterial and archaeal taxa in microbial communities. Because different organisms contain different 16S gene copy numbers (GCNs), sequence variant counts are biased towards clades with greater GCNs. Several tools have recently been developed for predicting GCNs using phylogenetic methods and based on sequenced genomes, in order to correct for these biases. However, the accuracy of those predictions has not been independently assessed. Here, we systematically evaluate the predictability of 16S GCNs across bacterial and archaeal clades, based on ??6,800 public sequenced genomes and using several phylogenetic methods. Further, we assess the accuracy of GCNs predicted by three recently published tools (PICRUSt, CopyRighter, and PAPRICA) over a wide range of taxa and for 635 microbial communities from varied environments. We find that regardless of the phylogenetic method tested, 16S GCNs could only be accurately predicted for a limited fraction of taxa, namely taxa with closely to moderately related representatives (?15% divergence in the 16S rRNA gene). Consistent with this observation, we find that all considered tools exhibit low predictive accuracy when evaluated against completely sequenced genomes, in some cases explaining less than 10% of the variance. Substantial disagreement was also observed between tools (R2<0.5) for the majority of tested microbial communities. The nearest sequenced taxon index (NSTI) of microbial communities, i.e., the average distance to a sequenced genome, was a strong predictor for the agreement between GCN prediction tools on non-animal-associated samples, but only a moderate predictor for animal-associated samples. We recommend against correcting for 16S GCNs in microbiome surveys by default, unless OTUs are sufficiently closely related to sequenced genomes or unless a need for true OTU proportions warrants the additional noise introduced, so that community profiles remain interpretable and comparable between studies.

SUBMITTER: Louca S

PROVIDER: S-EPMC5828423 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Correcting for 16S rRNA gene copy numbers in microbiome surveys remains an unsolved problem.

Louca Stilianos S Doebeli Michael M Parfrey Laura Wegener LW

Microbiome 20180226 1

The 16S ribosomal RNA gene is the most widely used marker gene in microbial ecology. Counts of 16S sequence variants, often in PCR amplicons, are used to estimate proportions of bacterial and archaeal taxa in microbial communities. Because different organisms contain different 16S gene copy numbers (GCNs), sequence variant counts are biased towards clades with greater GCNs. Several tools have recently been developed for predicting GCNs using phylogenetic methods and based on sequenced genomes, i ...[more]

PMID: 29482646

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Project description:The human impact on natural habitats is increasing the complexity of human-wildlife interactions and leading to the emergence of infectious diseases worldwide. Highly successful synanthropic wildlife species, such as rodents, will undoubtedly play an increasingly important role in transmitting zoonotic diseases. We investigated the potential for recent developments in 16S rRNA amplicon sequencing to facilitate the multiplexing of the large numbers of samples needed to improve our understanding of the risk of zoonotic disease transmission posed by urban rodents in West Africa. In addition to listing pathogenic bacteria in wild populations, as in other high-throughput sequencing (HTS) studies, our approach can estimate essential parameters for studies of zoonotic risk, such as prevalence and patterns of coinfection within individual hosts. However, the estimation of these parameters requires cleaning of the raw data to mitigate the biases generated by HTS methods. We present here an extensive review of these biases and of their consequences, and we propose a comprehensive trimming strategy for managing these biases. We demonstrated the application of this strategy using 711 commensal rodents, including 208 Mus musculus domesticus, 189 Rattus rattus, 93 Mastomys natalensis, and 221 Mastomys erythroleucus, collected from 24 villages in Senegal. Seven major genera of pathogenic bacteria were detected in their spleens: Borrelia, Bartonella, Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia. Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia have never before been detected in West African rodents. Bacterial prevalence ranged from 0% to 90% of individuals per site, depending on the bacterial taxon, rodent species, and site considered, and 26% of rodents displayed coinfection. The 16S rRNA amplicon sequencing strategy presented here has the advantage over other molecular surveillance tools of dealing with a large spectrum of bacterial pathogens without requiring assumptions about their presence in the samples. This approach is therefore particularly suitable to continuous pathogen surveillance in the context of disease-monitoring programs. IMPORTANCE Several recent public health crises have shown that the surveillance of zoonotic agents in wildlife is important to prevent pandemic risks. High-throughput sequencing (HTS) technologies are potentially useful for this surveillance, but rigorous experimental processes are required for the use of these effective tools in such epidemiological contexts. In particular, HTS introduces biases into the raw data set that might lead to incorrect interpretations. We describe here a procedure for cleaning data before estimating reliable biological parameters, such as positivity, prevalence, and coinfection, using 16S rRNA amplicon sequencing on an Illumina MiSeq platform. This procedure, applied to 711 rodents collected in West Africa, detected several zoonotic bacterial species, including some at high prevalence, despite their never before having been reported for West Africa. In the future, this approach could be adapted for the monitoring of other microbes such as protists, fungi, and even viruses.

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Correcting for 16S rRNA gene copy numbers in microbiome surveys remains an unsolved problem.

Publications

Correcting for 16S rRNA gene copy numbers in microbiome surveys remains an unsolved problem.

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