High-throughput sequencing of 16S ribosomal RNA gene amplicons has facilitated understanding of complex microbial communities, but the inherent noise in PCR and DNA sequencing limits differentiation ...of closely related bacteria. Although many scientific questions can be addressed with broad taxonomic profiles, clinical, food safety, and some ecological applications require higher specificity. Here we introduce a novel sub-operational-taxonomic-unit (sOTU) approach, Deblur, that uses error profiles to obtain putative error-free sequences from Illumina MiSeq and HiSeq sequencing platforms. Deblur substantially reduces computational demands relative to similar sOTU methods and does so with similar or better sensitivity and specificity. Using simulations, mock mixtures, and real data sets, we detected closely related bacterial sequences with single nucleotide differences while removing false positives and maintaining stability in detection, suggesting that Deblur is limited only by read length and diversity within the amplicon sequences. Because Deblur operates on a per-sample level, it scales to modern data sets and meta-analyses. To highlight Deblur's ability to integrate data sets, we include an interactive exploration of its application to multiple distinct sequencing rounds of the American Gut Project. Deblur is open source under the Berkeley Software Distribution (BSD) license, easily installable, and downloadable from https://github.com/biocore/deblur.
Deblur provides a rapid and sensitive means to assess ecological patterns driven by differentiation of closely related taxa. This algorithm provides a solution to the problem of identifying real ecological differences between taxa whose amplicons differ by a single base pair, is applicable in an automated fashion to large-scale sequencing data sets, and can integrate sequencing runs collected over time.
Microbiota-based prediction of chronic infections is promising yet not well established. Early childhood caries (ECC) is the most common infection in children. Here we simultaneously tracked ...microbiota development at plaque and saliva in 50 4-year-old preschoolers for 2 years; children either stayed healthy, transitioned into cariogenesis, or experienced caries exacerbation. Caries onset delayed microbiota development, which is otherwise correlated with aging in healthy children. Both plaque and saliva microbiota are more correlated with changes in ECC severity (dmfs) during onset than progression. By distinguishing between aging- and disease-associated taxa and exploiting the distinct microbiota dynamics between onset and progression, we developed a model, Microbial Indicators of Caries, to diagnose ECC from healthy samples with 70% accuracy and predict, with 81% accuracy, future ECC onsets for samples clinically perceived as healthy. Thus, caries onset in apparently healthy teeth can be predicted using microbiota, when appropriately de-trended for age.
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•Oral microbiota in 50 four-year-old children were tracked for 2 years•Age-dependent microbiota development is perturbed by early childhood caries (ECC) onset•Shifts in microbiota precede manifestation of clinical symptoms of ECC•Microbial Indicators of Caries, when de-trended for age, can predict ECC onset
Teng et al. tracked plaque and saliva microbiota of 50 4-year-old children for 2 years. By distinguishing between aging- and disease-associated taxa and exploiting the distinct microbiota dynamics between disease onset and progression, a predictive model, Microbial Indicators of Caries, is proposed as a method to predict future caries onset.
•Toxicant-substitute based immunoassays for detecting mycotoxins were reviewed.•Toxicant substitute-based assays showed satisfying sensitivity and reliability.•Toxicant substitutes facilitate the ...standardization of immunoassays for mycotoxins.•Toxicant substitutes pose reduced harmfulness to operators and environment.•Toxicant substitutes contribute to develop eco-friendly immunoassays.
Recurring mycotoxins contamination has posedaseriousthreatto food safety worldwide. Competitive immunoassays are widely used techniques for high-throughput mycotoxins detection in agricultural products and foods. However, the inevitable introduction of mycotoxin conjugates produced by chemical conjugation usually results in complicated by-products, large batch errors and threats to operators and environment. Biologically derived surrogates of mycotoxin conjugates or mycotoxin standards are renewable immunoreagents. They can serve the same function as the responding counterparts in the immunoassays. The substitute-based immunoassays exhibit satisfactory sensitivity, pose less health threats to operators and environment, and contribute to the standardization of immunoassays for mycotoxins. This review focuses on the current applications of substitute-based immunoassays, clarifies their underlying mechanisms and provides a careful comparison. Challenges and future prospects are discussed.
The vitamin D receptor is highly expressed in the gastrointestinal tract where it transacts gene expression. With current limited understanding of the interactions between the gut microbiome and ...vitamin D, we conduct a cross-sectional analysis of 567 older men quantifying serum vitamin D metabolites using LC-MSMS and defining stool sub-Operational Taxonomic Units from16S ribosomal RNA gene sequencing data. Faith's Phylogenetic Diversity and non-redundant covariate analyses reveal that the serum 1,25(OH)
D level explains 5% of variance in α-diversity. In β-diversity analyses using unweighted UniFrac, 1,25(OH)
D is the strongest factor assessed, explaining 2% of variance. Random forest analyses identify 12 taxa, 11 in the phylum Firmicutes, eight of which are positively associated with either 1,25(OH)
D and/or the hormone-to-prohormone 1,25(OH)
D/25(OH)D "activation ratio." Men with higher levels of 1,25(OH)
D and higher activation ratios, but not 25(OH)D itself, are more likely to possess butyrate producing bacteria that are associated with better gut microbial health.
Our understanding of the interaction between the gut microbiota and host health has recently improved dramatically. However, the effects of toxic metal exposure on the gut microbiota remain poorly ...characterized. As this microbiota creates a critical interface between the external environment and the host's cells, it may play an important role in host outcomes during exposure. We therefore used 16S ribosomal RNA (rRNA) gene sequencing to track changes in the gut microbiota composition of rats exposed to heavy metals. Rats were exposed daily for five days to arsenic, cadmium, cobalt, chromium, nickel, or a vehicle control. Significant changes to microbiota composition were observed in response to high doses of chromium and cobalt, and significant dose-dependent changes were observed in response to arsenic, cadmium and nickel. Many of these perturbations were not uniform across metals. However, bacteria with higher numbers of iron-importing gene orthologs were overly represented after exposure to arsenic and nickel, suggesting some possibility of a shared response. These findings support the utility of the microbiota as a pre-clinical tool for identifying exposures to specific heavy metals. It is also clear that characterizing changes to the functional capabilities of microbiota is critical to understanding responses to metal exposure.
Recent studies suggest that gut microbiomes of urban-industrialized societies are different from those of traditional peoples. Here we examine the relationship between lifeways and gut microbiota ...through taxonomic and functional potential characterization of faecal samples from hunter-gatherer and traditional agriculturalist communities in Peru and an urban-industrialized community from the US. We find that in addition to taxonomic and metabolic differences between urban and traditional lifestyles, hunter-gatherers form a distinct sub-group among traditional peoples. As observed in previous studies, we find that Treponema are characteristic of traditional gut microbiomes. Moreover, through genome reconstruction (2.2-2.5 MB, coverage depth × 26-513) and functional potential characterization, we discover these Treponema are diverse, fall outside of pathogenic clades and are similar to Treponema succinifaciens, a known carbohydrate metabolizer in swine. Gut Treponema are found in non-human primates and all traditional peoples studied to date, suggesting they are symbionts lost in urban-industrialized societies.
Recent algorithmic advances in amplicon-based microbiome studies enable the inference of exact amplicon sequence fragments. These new methods enable the investigation of sub-operational taxonomic ...units (sOTU) by removing erroneous sequences. However, short (e.g., 150-nucleotide nt) DNA sequence fragments do not contain sufficient phylogenetic signal to reproduce a reasonable tree, introducing a barrier in the utilization of critical phylogenetically aware metrics such as Faith's PD or UniFrac. Although fragment insertion methods do exist, those methods have not been tested for sOTUs from high-throughput amplicon studies in insertions against a broad reference phylogeny. We benchmarked the SATé-enabled phylogenetic placement (SEPP) technique explicitly against 16S V4 sequence fragments and showed that it outperforms the conceptually problematic but often-used practice of reconstructing
phylogenies. In addition, we provide a BSD-licensed QIIME2 plugin (https://github.com/biocore/q2-fragment-insertion) for SEPP and integration into the microbial study management platform QIITA.
The move from OTU-based to sOTU-based analysis, while providing additional resolution, also introduces computational challenges. We demonstrate that one popular method of dealing with sOTUs (building a
tree from the short sequences) can provide incorrect results in human gut metagenomic studies and show that phylogenetic placement of the new sequences with SEPP resolves this problem while also yielding other benefits over existing methods.
The utility of cell-free nucleic acids in monitoring cancer has been recognized by both scientists and clinicians. In addition to human transcripts, a fraction of cell-free nucleic acids in human ...plasma were proven to be derived from microbes and reported to have relevance to cancer. To obtain a better understanding of plasma cell-free RNAs (cfRNAs) in cancer patients, we profiled cfRNAs in ~300 plasma samples of 5 cancer types (colorectal cancer, stomach cancer, liver cancer, lung cancer, and esophageal cancer) and healthy donors (HDs) with RNA-seq. Microbe-derived cfRNAs were consistently detected by different computational methods when potential contaminations were carefully filtered. Clinically relevant signals were identified from human and microbial reads, and enriched Kyoto Encyclopedia of Genes and Genomes pathways of downregulated human genes and higher prevalence torque teno viruses both suggest that a fraction of cancer patients were immunosuppressed. Our data support the diagnostic value of human and microbe-derived plasma cfRNAs for cancer detection, as an area under the ROC curve of approximately 0.9 for distinguishing cancer patients from HDs was achieved. Moreover, human and microbial cfRNAs both have cancer type specificity, and combining two types of features could distinguish tumors of five different primary locations with an average recall of 60.4%. Compared to using human features alone, adding microbial features improved the average recall by approximately 8%. In summary, this work provides evidence for the clinical relevance of human and microbe-derived plasma cfRNAs and their potential utilities in cancer detection as well as the determination of tumor sites.
Immediate freezing at -20°C or below has been considered the gold standard for microbiome preservation, yet this approach is not feasible for many field studies, ranging from anthropology to wildlife ...conservation. Here we tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including such types of variation as freeze-thaw cycles and the high temperature fluctuations often encountered under field conditions. We found that three of the methods-95% ethanol, FTA cards, and the OMNIgene Gut kit-can preserve samples sufficiently well at ambient temperatures such that differences at 8 weeks are comparable to differences among technical replicates. However, even the worst methods, including those with no fixative, were able to reveal microbiome differences between species at 8 weeks and between individuals after a week, allowing meta-analyses of samples collected using various methods when the effect of interest is expected to be larger than interindividual variation (although use of a single method within a study is strongly recommended to reduce batch effects). Encouragingly for FTA cards, the differences caused by this method are systematic and can be detrended. As in other studies, we strongly caution against the use of 70% ethanol. The results, spanning 15 individuals and over 1,200 samples, provide our most comprehensive view to date of storage effects on stool and provide a paradigm for the future studies of other sample types that will be required to provide a global view of microbial diversity and its interaction among humans, animals, and the environment.
Our study, spanning 15 individuals and over 1,200 samples, provides our most comprehensive view to date of storage and stabilization effects on stool. We tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including the types of variation often encountered under field conditions, such as freeze-thaw cycles and high temperature fluctuations. We show that several cost-effective methods provide excellent microbiome stability out to 8 weeks, opening up a range of field studies with humans and wildlife that would otherwise be cost-prohibitive.
Gut microbiota dysbiosis contributes to the onset and perpetuation of inflammatory bowel disease (IBD). Given that gut microbiotas vary across geography and ethnicity, it remains obscure whether any ...universal microbial signatures for IBD diagnosis and prognosis evaluation exist irrespective of populations. Here we profiled the fecal microbiota of a series of Chinese IBD patients and combined them with two Western IBD cohorts, PRISM and RISK, for meta-analyses. We found that the gut microbial alteration patterns in IBD are similar among Chinese and Westerners. Our prediction model based on gut microbiome for IBD diagnosis is robust across the cohorts, which showed 87.5% and 79.1% prediction accuracy in Crohn's disease (CD) and ulcerative colitis (UC) patients, respectively. A relative increase in the levels of
and
(
) and a relative decrease in the levels of
(
) were strongly correlated with IBD severity (
< 0.05). Additionally, restoration of gut microbiota diversity and a significant increase in
relative abundance were found in patients responding to infliximab (IFX Remicade) treatment compared to those in relapse. Moreover, certain microbes, mainly
, predicted the treatment effectiveness with 86.5% accuracy alone and 93.8% accuracy in combination with calprotectin levels and Crohn's disease activity index (CDAI). Taking the results together, we conclude that gut microbiota can offer a set of universal biomarkers for diagnosis, disease activity evaluation, and infliximab treatment response prediction in IBD.
In the present report, we show that the human fecal microbiota contains promising and universal biomarkers for the noninvasive evaluation of inflammatory bowel disease severity and IFX treatment efficacy, emphasizing the potential ability to mine the gut microbiota as a modality to stratify IBD patients and apply personalized therapy for optimal outcomes.
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