Michael D. J. Lynch

Generation of multimillion-sequence 16S rRNA gene libraries from complex microbial communities by assembling paired-end illumina reads.

ABSTRACT Microbial communities host unparalleled taxonomic diversity. Adequate characterization of environmental and host-associated samples remains a challenge for microbiologists, despite the advent of 16S rRNA gene sequencing. In order to increase the depth of sampling for diverse bacterial communities, we developed a method for sequencing and assembling millions of paired-end reads from the 16S rRNA gene (spanning the V3 region; ∼200 nucleotides) by using an Illumina genome analyzer. To confirm reproducibility and to identify a suitable computational pipeline for data analysis, sequence libraries were prepared in duplicate for both a defined mixture of DNAs from known cultured bacterial isolates (>1 million postassembly sequences) and an Arctic tundra soil sample (>6 million postassembly sequences). The Illumina 16S rRNA gene libraries represent a substantial increase in number of sequences over all extant next-generation sequencing approaches (e.g., 454 pyrosequencing), while the assembly of paired-end 125-base reads offers a methodological advantage by incorporating an initial quality control step for each 16S rRNA gene sequence. This method incorporates indexed primers to enable the characterization of multiple microbial communities in a single flow cell lane, may be modified readily to target other variable regions or genes, and demonstrates unprecedented and economical access to DNAs from organisms that exist at low relative abundances.

Ecology and exploration of the rare biosphere

The profound influence of microorganisms on human life and global biogeochemical cycles underlines the value of studying the biogeography of microorganisms, exploring microbial genomes and expanding our understanding of most microbial species on Earth: that is, those present at low relative abundance. The detection and subsequent analysis of low-abundance microbial populations — the rare biosphere — have demonstrated the persistence, population dynamics, dispersion and predation of these microbial species. We discuss the ecology of rare microbial populations, and highlight molecular and computational methods for targeting taxonomic blind spots within the rare biosphere of complex microbial communities.

Bacterial biogeography of the human digestive tract

We present bacterial biogeography as sampled from the human gastrointestinal tract of four healthy subjects. This study generated >32 million paired-end sequences of bacterial 16S rRNA genes (V3 region) representing >95,000 unique operational taxonomic units (OTUs; 97% similarity clusters), with >99% Goods coverage for all samples. The highest OTU richness and phylogenetic diversity was found in the mouth samples. The microbial communities of multiple biopsy sites within the colon were highly similar within individuals and largely distinct from those in stool. Within an individual, OTU overlap among broad site definitions (mouth, stomach/duodenum, colon and stool) ranged from 32–110 OTUs, 25 of which were common to all individuals and included OTUs affiliated with Faecalibacterium prasnitzii and the TM7 phylum. This first comprehensive characterization of the abundant and rare microflora found along the healthy human digestive tract represents essential groundwork to investigate further how the human microbiome relates to health and disease.

Diversity of basidiomycetes in Michigan agricultural soils

ABSTRACT We analyzed the communities of soil basidiomycetes in agroecosystems that differ in tillage history at the Kellogg Biological Station Long-Term Ecological Research site near Battle Creek, Michigan. The approach combined soil DNA extraction through a bead-beating method modified to increase recovery of fungal DNA, PCR amplification with basidiomycete-specific primers, cloning and restriction fragment length polymorphism screening of mixed PCR products, and sequencing of unique clones. Much greater diversity was detected than was anticipated in this habitat on the basis of culture-based methods or surveys of fruiting bodies. With “species” defined as organisms yielding PCR products with ≥99% identity in the 5′ 650 bases of the nuclear large-subunit ribosomal DNA, 241 “species” were detected among 409 unique basidiomycete sequences recovered. Almost all major clades of basidiomycetes from basidiomycetous yeasts and other heterobasidiomycetes through polypores and euagarics (gilled mushrooms and relatives) were represented, with a majority from the latter clade. Only 24 of 241 “species” had 99% or greater sequence similarity to named reference sequences in GenBank, and several clades with multiple “species” could not be identified at the genus level by phylogenetic comparisons with named sequences. The total estimated “species” richness for this 11.2-ha site was 367 “species” of basidiomycetes. Since >99% of the study area has not been sampled, the accuracy of our diversity estimate is uncertain. Replication in time and space is required to detect additional diversity and the underlying community structure.

Evaluating Bias of Illumina-Based Bacterial 16S rRNA Gene Profiles

ABSTRACT Massively parallel sequencing of 16S rRNA genes enables the comparison of terrestrial, aquatic, and host-associated microbial communities with sufficient sequencing depth for robust assessments of both alpha and beta diversity. Establishing standardized protocols for the analysis of microbial communities is dependent on increasing the reproducibility of PCR-based molecular surveys by minimizing sources of methodological bias. In this study, we tested the effects of template concentration, pooling of PCR amplicons, and sample preparation/interlane sequencing on the reproducibility associated with paired-end Illumina sequencing of bacterial 16S rRNA genes. Using DNA extracts from soil and fecal samples as templates, we sequenced pooled amplicons and individual reactions for both high (5- to 10-ng) and low (0.1-ng) template concentrations. In addition, all experimental manipulations were repeated on two separate days and sequenced on two different Illumina MiSeq lanes. Although within-sample sequence profiles were highly consistent, template concentration had a significant impact on sample profile variability for most samples. Pooling of multiple PCR amplicons, sample preparation, and interlane variability did not influence sample sequence data significantly. This systematic analysis underlines the importance of optimizing template concentration in order to minimize variability in microbial-community surveys and indicates that the practice of pooling multiple PCR amplicons prior to sequencing contributes proportionally less to reducing bias in 16S rRNA gene surveys with next-generation sequencing.

Aquatic metagenomes implicate Thaumarchaeota in global cobalamin production.

Cobalamin (vitamin B12) is a complex metabolite and essential cofactor required by many branches of life, including most eukaryotic phytoplankton. Algae and other cobalamin auxotrophs rely on environmental cobalamin supplied from a relatively small set of cobalamin-producing prokaryotic taxa. Although several Bacteria have been implicated in cobalamin biosynthesis and associated with algal symbiosis, the involvement of Archaea in cobalamin production is poorly understood, especially with respect to the Thaumarchaeota. Based on the detection of cobalamin synthesis genes in available thaumarchaeotal genomes, we hypothesized that Thaumarchaeota, which are ubiquitous and abundant in aquatic environments, have an important role in cobalamin biosynthesis within global aquatic ecosystems. To test this hypothesis, we examined cobalamin synthesis genes across sequenced thaumarchaeotal genomes and 430 metagenomes from a diverse range of marine, freshwater and hypersaline environments. Our analysis demonstrates that all available thaumarchaeotal genomes possess cobalamin synthesis genes, predominantly from the anaerobic pathway, suggesting widespread genetic capacity for cobalamin synthesis. Furthermore, although bacterial cobalamin genes dominated most surface marine metagenomes, thaumarchaeotal cobalamin genes dominated metagenomes from polar marine environments, increased with depth in marine water columns, and displayed seasonality, with increased winter abundance observed in time-series datasets (e.g., L4 surface water in the English Channel). Our results also suggest niche partitioning between thaumarchaeotal and cyanobacterial ribosomal and cobalamin synthesis genes across all metagenomic datasets analyzed. These results provide strong evidence for specific biogeographical distributions of thaumarchaeotal cobalamin genes, expanding our understanding of the global biogeochemical roles played by Thaumarchaeota in aquatic environments.

Prevalence of Anaerobic Ammonium-Oxidizing Bacteria in Contaminated Groundwater

Anaerobic ammonium-oxidizing (anammox) bacteria perform an important step in the global nitrogen cycle: anaerobic oxidation of ammonium and reduction of nitrite to form dinitrogen gas (N(2)). Anammox organisms appear to be widely distributed in natural and artificial environments. However, their roles in groundwater ammonium attenuation remain unclear and only limited biomarker-based data confirmed their presence prior to this study. We used complementary molecular and isotope-based methods to assess anammox diversity and activity occurring at three ammonium-contaminated groundwater sites: quantitative PCR, denaturing gradient gel electrophoresis, sequencing of 16S rRNA genes, and (15)N-tracer incubations. Here we show that anammox performing organisms were abundant bacterial community members. Although all sites were dominated by Candidatus Brocadia-like sequences, the community at one site was particularly diverse, possessing four of five known genera of anammox bacteria. Isotope data showed that anammox produced up to 18 and 36% of N(2) at these sites. By combining molecular and isotopic results we have demonstrated the diversity, abundance, and activity of these autotrophic bacteria. Our results provide strong evidence for their important biogeochemical role in attenuating groundwater ammonium contamination.

Targeted recovery of novel phylogenetic diversity from next-generation sequence data

Next-generation sequencing technologies have led to recognition of a so-called ‘rare biosphere’. These microbial operational taxonomic units (OTUs) are defined by low relative abundance and may be specifically adapted to maintaining low population sizes. We hypothesized that mining of low-abundance next-generation 16S ribosomal RNA (rRNA) gene data would lead to the discovery of novel phylogenetic diversity, reflecting microorganisms not yet discovered by previous sampling efforts. Here, we test this hypothesis by combining molecular and bioinformatic approaches for targeted retrieval of phylogenetic novelty within rare biosphere OTUs. We combined BLASTN network analysis, phylogenetics and targeted primer design to amplify 16S rRNA gene sequences from unique potential bacterial lineages, comprising part of the rare biosphere from a multi-million sequence data set from an Arctic tundra soil sample. Demonstrating the feasibility of the protocol developed here, three of seven recovered phylogenetic lineages represented extremely divergent taxonomic entities. These divergent target sequences correspond to (a) a previously unknown lineage within the BRC1 candidate phylum, (b) a sister group to the early diverging and currently recognized monospecific Cyanobacteria Gloeobacter, a genus containing multiple plesiomorphic traits and (c) a highly divergent lineage phylogenetically resolved within mitochondria. A comparison to twelve next-generation data sets from additional soils suggested persistent low-abundance distributions of these novel 16S rRNA genes. The results demonstrate this sequence analysis and retrieval pipeline as applicable for exploring underrepresented phylogenetic novelty and recovering taxa that may represent significant steps in bacterial evolution.

FUNGI AND EUKARYOTIC ALGAE

Publisher Summary This chapter provides an overview of fungi and eukaryotic algae as both these groups share a range of morphologies—that is, from filamentous to cellular. Fungi bind soil together by their filamentous form, exudates, and trophic interactions with all other groups of soil organisms. Fungi are enormously important in carbon and nitrogen cycling because of their ability to degrade complex substrates of plant origin, which represent up to 90% of net primary productivity in most terrestrial ecosystems. In addition, the usually mutualistic symbioses, known as “mycorrhizas,” between many fungi and plant roots as well as the parasitic interactions leading to many plant diseases have huge impacts in both ecological and economic terms. The temporarily or permanently wet margins of rivers, lakes, and ponds and the peaty substrates of bogs and fens are home to many freshwater algae that do not thrive far from water. The diverse soils of temperate and tropical agriculture, grasslands, forests, arctic and alpine tundra, and even deserts also harbor a surprisingly diverse and active flora of both eukaryotic algae and cyanobacteria. This chapter elaborates the classification and characteristics of fungi and eukaryotic algae along with the ecological roles of these groups in soil.

Tu1797 The Adoptive Transfer of Anxiety and Gut Dysfunction From IBS Patients to Axenic Mice Through Microbiota Transplantation

Studies of the effect of esophageal sensory stimulation on brain functional connectivity using magnetic resonance imaging (fc-MRI) requires insertion of perfusion or balloon catheters. The contribution of the presence of these catheters themselves on observed brain connectivity changes has not been systematically studied. Aim: To determine the effect of the presence of a catheter within the esophagus on a priori selected regional brain connectivity in healthy and patient volunteers. Methods: In 14 controls (7 female, age 27±4 years) and 12 nonerosive reflux disease (NERD) patients (6 female, age 46±5 years), fc-MRI data of amygdala connectivity to the rest of the brain were gathered during two 9-minute scans, one scan without transnasal esophageal intubation and one scan with esophageal intubation. Functional connectivity was quantified by the correlation coefficient (CC) between average fcMRI time series (seeds) in two spherical volumes centered in each of the right and left amygdala and time series of all fc-MRI volume elements (voxels) across the entire brain. The amygdala seeds were centered at the locations identified by the MNI atlas. Group analysis was performed using unpaired t-tests (with cluster size Monte Carlo simulation to correct for multiple comparison) of correlation coefficients to test amygdala-cortical connectivity differences across NERD patients and healthy subjects. Results: Amygdala connectivity was characterized by strong connections to the right and left superior temporal gyrus. The presence of esophageal catheter in the healthy controls resulted in significant differences in both left and right amygdala connectivity to the left superior temporal gyrus (Figure 1, p<0.001, corrected). No significant catheter effect was observed in the NERD patients. Presence of the catheter also effected the between group comparisons showing significant differences in amygdala connectivity. In the resting state without the catheter present, significant differences in both right and left amygdala connectivity to the right superior temporal gyrus were seen between the two groups (p<0,001, corrected). In the presence of catheter, significant differences were limited to the right amygdala connectivity to the right superior temporal gyrus (p<0,001, corrected). Conclusions: Pharyngo-esophageal stimulation by the presence of a catheter alters the functional connectivity of the amygdala in healthy controls. This response is absent in NERD patients which may represent a state of pre-existing maximum neural activity and associated BOLD signal alterations (ceiling effect). Furthermore, the presence of an esophageal catheter can affect the findings of comparative analyses between groups. This possibility needs to be taken into account when interpreting resting and non-resting state connectivity data involving esophageal stimulation.