Yan Wei Lim

Broad surveys of DNA viral diversity obtained through viral metagenomics of mosquitoes.

Viruses are the most abundant and diverse genetic entities on Earth; however, broad surveys of viral diversity are hindered by the lack of a universal assay for viruses and the inability to sample a sufficient number of individual hosts. This study utilized vector-enabled metagenomics (VEM) to provide a snapshot of the diversity of DNA viruses present in three mosquito samples from San Diego, California. The majority of the sequences were novel, suggesting that the viral community in mosquitoes, as well as the animal and plant hosts they feed on, is highly diverse and largely uncharacterized. Each mosquito sample contained a distinct viral community. The mosquito viromes contained sequences related to a broad range of animal, plant, insect and bacterial viruses. Animal viruses identified included anelloviruses, circoviruses, herpesviruses, poxviruses, and papillomaviruses, which mosquitoes may have obtained from vertebrate hosts during blood feeding. Notably, sequences related to human papillomaviruses were identified in one of the mosquito samples. Sequences similar to plant viruses were identified in all mosquito viromes, which were potentially acquired through feeding on plant nectar. Numerous bacteriophages and insect viruses were also detected, including a novel densovirus likely infecting Culex erythrothorax. Through sampling insect vectors, VEM enables broad survey of viral diversity and has significantly increased our knowledge of the DNA viruses present in mosquitoes.

TagCleaner: Identification and removal of tag sequences from genomic and metagenomic datasets

BackgroundSequencing metagenomes that were pre-amplified with primer-based methods requires the removal of the additional tag sequences from the datasets. The sequenced reads can contain deletions or insertions due to sequencing limitations, and the primer sequence may contain ambiguous bases. Furthermore, the tag sequence may be unavailable or incorrectly reported. Because of the potential for downstream inaccuracies introduced by unwanted sequence contaminations, it is important to use reliable tools for pre-processing sequence data.ResultsTagCleaner is a web application developed to automatically identify and remove known or unknown tag sequences allowing insertions and deletions in the dataset. TagCleaner is designed to filter the trimmed reads for duplicates, short reads, and reads with high rates of ambiguous sequences. An additional screening for and splitting of fragment-to-fragment concatenations that gave rise to artificial concatenated sequences can increase the quality of the dataset. Users may modify the different filter parameters according to their own preferences.ConclusionsTagCleaner is a publicly available web application that is able to automatically detect and efficiently remove tag sequences from metagenomic datasets. It is easily configurable and provides a user-friendly interface. The interactive web interface facilitates export functionality for subsequent data processing, and is available at http://edwards.sdsu.edu/tagcleaner.

Spatial distribution of microbial communities in the cystic fibrosis lung

Cystic fibrosis (CF) is a common fatal genetic disorder with mortality most often resulting from microbial infections of the lungs. Culture-independent studies of CF-associated microbial communities have indicated that microbial diversity in the CF airways is much higher than suggested by culturing alone. However, these studies have relied on indirect methods to sample the CF lung such as expectorated sputum and bronchoalveolar lavage (BAL). Here, we characterize the diversity of microbial communities in tissue sections from anatomically distinct regions of the CF lung using barcoded 16S amplicon pyrosequencing. Microbial communities differed significantly between different areas of the lungs, and few taxa were common to microbial communities in all anatomical regions surveyed. Our results indicate that CF lung infections are not only polymicrobial, but also spatially heterogeneous suggesting that treatment regimes tailored to dominant populations in sputum or BAL samples may be ineffective against infections in some areas of the lung.

Lytic to temperate switching of viral communities

Microbial viruses can control host abundances via density-dependent lytic predator–prey dynamics. Less clear is how temperate viruses, which coexist and replicate with their host, influence microbial communities. Here we show that virus-like particles are relatively less abundant at high host densities. This suggests suppressed lysis where established models predict lytic dynamics are favoured. Meta-analysis of published viral and microbial densities showed that this trend was widespread in diverse ecosystems ranging from soil to freshwater to human lungs. Experimental manipulations showed viral densities more consistent with temperate than lytic life cycles at increasing microbial abundance. An analysis of 24 coral reef viromes showed a relative increase in the abundance of hallmark genes encoded by temperate viruses with increased microbial abundance. Based on these four lines of evidence, we propose the Piggyback-the-Winner model wherein temperate dynamics become increasingly important in ecosystems with high microbial densities; thus ‘more microbes, fewer viruses’.

Identification and removal of ribosomal RNA sequences from metatranscriptomes

Summary: Here, we present riboPicker, a robust framework for the rapid, automated identification and removal of ribosomal RNA sequences from metatranscriptomic datasets. The results can be exported for subsequent analysis, and the databases used for the web-based version are updated on a regular basis. riboPicker categorizes rRNA-like sequences and provides graphical visualizations and tabular outputs of ribosomal coverage, alignment results and taxonomic classifications. Availability and implementation: This open-source application was implemented in Perl and can be used as stand-alone version or accessed online through a user-friendly web interface. The source code, user help and additional information is available at http://ribopicker.sourceforge.net/. Contact: rschmied@sciences.sdsu.edu; rschmied@sciences.sdsu.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Metagenomics and metatranscriptomics: Windows on CF-associated viral and microbial communities

BACKGROUND Samples collected from CF patient airways often contain large amounts of host-derived nucleic acids that interfere with recovery and purification of microbial and viral nucleic acids. This study describes metagenomic and metatranscriptomic methods that address these issues. METHODS Microbial and viral metagenomes, and microbial metatranscriptomes, were successfully prepared from sputum samples from five adult CF patients. RESULTS Contaminating host DNA was dramatically reduced in the metagenomes. Each CF patient presented a unique microbiome; in some Pseudomonas aeruginosa was replaced by other opportunistic bacteria. Even though the taxonomic composition of the microbiomes is very different, the metabolic potentials encoded by the community are very similar. The viral communities were dominated by phages that infect major CF pathogens. The metatranscriptomes reveal differential expression of encoded metabolic potential with changing health status. CONCLUSIONS Microbial and viral metagenomics combined with microbial transcriptomics characterize the dynamic polymicrobial communities found in CF airways, revealing both the taxa present and their current metabolic activities. These approaches can facilitate the development of individualized treatment plans and novel therapeutic approaches.

Clinical Insights from Metagenomic Analysis of Sputum Samples from Patients with Cystic Fibrosis

ABSTRACT As DNA sequencing becomes faster and cheaper, genomics-based approaches are being explored for their use in personalized diagnoses and treatments. Here, we provide a proof of principle for disease monitoring using personal metagenomic sequencing and traditional clinical microbiology by focusing on three adults with cystic fibrosis (CF). The CF lung is a dynamic environment that hosts a complex ecosystem composed of bacteria, viruses, and fungi that can vary in space and time. Not surprisingly, the microbiome data from the induced sputum samples we collected revealed a significant amount of species diversity not seen in routine clinical laboratory cultures. The relative abundances of several species changed as clinical treatment was altered, enabling the identification of the climax and attack communities that were proposed in an earlier work. All patient microbiomes encoded a diversity of mechanisms to resist antibiotics, consistent with the characteristics of multidrug-resistant microbial communities that are commonly observed in CF patients. The metabolic potentials of these communities differed by the health status and recovery route of each patient. Thus, this pilot study provides an example of how metagenomic data might be used with clinical assessments for the development of treatments tailored to individual patients.

Local genomic adaptation of coral reef-associated microbiomes to gradients of natural variability and anthropogenic stressors

Significance Microbial communities associated with coral reefs influence the health and sustenance of keystone benthic organisms (e.g., coral holobionts). The present study investigated the community structure and metabolic potential of microbes inhabiting coral reefs located across an extensive area in the central Pacific. We found that the taxa present correlated strongly with the percent coverage of corals and algae, while community metabolic potential correlated best with geographic location. These findings are inconsistent with prevailing biogeographic models of microbial diversity (e.g., distance decay) and metabolic potential (i.e., similar functional profiles regardless of phylogenetic variability). Based on these findings, we propose that the primary carbon sources determine community structure and that local biogeochemistry determines finer-scale metabolic function. Holobionts are species-specific associations between macro- and microorganisms. On coral reefs, the benthic coverage of coral and algal holobionts varies due to natural and anthropogenic forcings. Different benthic macroorganisms are predicted to have specific microbiomes. In contrast, local environmental factors are predicted to select for specific metabolic pathways in microbes. To reconcile these two predictions, we hypothesized that adaptation of microbiomes to local conditions is facilitated by the horizontal transfer of genes responsible for specific metabolic capabilities. To test this hypothesis, microbial metagenomes were sequenced from 22 coral reefs at 11 Line Islands in the central Pacific that together span a wide range of biogeochemical and anthropogenic influences. Consistent with our hypothesis, the percent cover of major benthic functional groups significantly correlated with particular microbial taxa. Reefs with higher coral cover had a coral microbiome with higher abundances of Alphaproteobacteria (such as Rhodobacterales and Sphingomonadales), whereas microbiomes of algae-dominated reefs had higher abundances of Gammaproteobacteria (such as Alteromonadales, Pseudomonadales, and Vibrionales), Betaproteobacteria, and Bacteriodetes. In contrast to taxa, geography was the strongest predictor of microbial community metabolism. Microbial communities on reefs with higher nutrient availability (e.g., equatorial upwelling zones) were enriched in genes involved in nutrient-related metabolisms (e.g., nitrate and nitrite ammonification, Ton/Tol transport, etc.). On reefs further from the equator, microbes had more genes encoding chlorophyll biosynthesis and photosystems I/II. These results support the hypothesis that core microbiomes are determined by holobiont macroorganisms, and that those core taxa adapt to local conditions by selecting for advantageous metabolic genes.

Case studies of the spatial heterogeneity of DNA viruses in the cystic fibrosis lung

Microbial communities in the lungs of patients with cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) have been shown to be spatially heterogeneous. Viral communities may also vary spatially, leading to localized viral populations and infections. Here, we characterized viral communities from multiple areas of the lungs of two patients with late-stage CF using metagenomics, that is, the explanted lungs from a transplant patient and lungs acquired postmortem. All regions harbored eukaryotic viruses that may infect the human host, notably herpesviruses, anelloviruses, and papillomaviruses. In the highly diseased apical lobes of explant lungs, viral diversity was extremely low, and only eukaryotic viruses were present. The absence of phage suggests that CF-associated microbial biofilms may escape top-down controls by phage predation. The phages present in other lobes of explant lungs and in all lobes of postmortem lungs comprised distinct communities, and encoded genes for clinically important microbial phenotypes, including small colony variants and antibiotic resistance. Based on the these observations, we postulate that viral communities in CF lungs are spatially distinct and contribute to CF pathology by augmenting the metabolic potential of resident microbes, as well as by directly damaging lung tissue via carcinomas and herpesviral outbreaks.

Breath gas metabolites and bacterial metagenomes from cystic fibrosis airways indicate active pH neutral 2,3-butanedione fermentation

The airways of cystic fibrosis (CF) patients are chronically colonized by patient-specific polymicrobial communities. The conditions and nutrients available in CF lungs affect the physiology and composition of the colonizing microbes. Recent work in bioreactors has shown that the fermentation product 2,3-butanediol mediates cross-feeding between some fermenting bacteria and Pseudomonas aeruginosa, and that this mechanism increases bacterial current production. To examine bacterial fermentation in the respiratory tract, breath gas metabolites were measured and several metagenomes were sequenced from CF and non-CF volunteers. 2,3-butanedione was produced in nearly all respiratory tracts. Elevated levels in one patient decreased during antibiotic treatment, and breath concentrations varied between CF patients at the same time point. Some patients had high enough levels of 2,3-butanedione to irreversibly damage lung tissue. Antibiotic therapy likely dictates the activities of 2,3-butanedione-producing microbes, which suggests a need for further study with larger sample size. Sputum microbiomes were dominated by P. aeruginosa, Streptococcus spp. and Rothia mucilaginosa, and revealed the potential for 2,3-butanedione biosynthesis. Genes encoding 2,3-butanedione biosynthesis were disproportionately abundant in Streptococcus spp, whereas genes for consumption of butanedione pathway products were encoded by P. aeruginosa and R. mucilaginosa. We propose a model where low oxygen conditions in CF lung lead to fermentation and a decrease in pH, triggering 2,3-butanedione fermentation to avoid lethal acidification. We hypothesize that this may also increase phenazine production by P. aeruginosa, increasing reactive oxygen species and providing additional electron acceptors to CF microbes.