Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the 16S-ITS-23S rRNA operon

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286–37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (∼1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full-length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost-effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput.     

Evaluating multiplexed next‐generation sequencing as a method in palynology for mixed pollen samples

The identification of pollen plays an important role in ecology, palaeo‐climatology, honey quality control and other areas. Currently, expert knowledge and reference collections are essential to identify pollen origin through light microscopy. Pollen identification through molecular sequencing and DNA barcoding has been proposed as an alternative approach, but the assessment of mixed pollen samples originating from multiple plant species is still a tedious and error‐prone task. Next‐generation sequencing has been proposed to avoid this hindrance. In this study we assessed mixed pollen probes through next‐generation sequencing of amplicons from the highly variable, species‐specific internal transcribed spacer 2 region of nuclear ribosomal DNA. Further, we developed a bioinformatic workflow to analyse these high‐throughput data with a newly created reference database. To evaluate the feasibility, we compared results from classical identification based on light microscopy from the same samples with our sequencing results. We assessed in total 16 mixed pollen samples, 14 originated from honeybee colonies and two from solitary bee nests. The sequencing technique resulted in higher taxon richness (deeper assignments and more identified taxa) compared to light microscopy. Abundance estimations from sequencing data were significantly correlated with counted abundances through light microscopy. Simulation analyses of taxon specificity and sensitivity indicate that 96% of taxa present in the database are correctly identifiable at the genus level and 70% at the species level. Next‐generation sequencing thus presents a useful and efficient workflow to identify pollen at the genus and species level without requiring specialised palynological expert knowledge.     

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

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.     

Unveiling Distribution Patterns of Freshwater Phytoplankton by a Next Generation Sequencing Based Approach

The recognition and discrimination of phytoplankton species is one of the foundations of freshwater biodiversity research and environmental monitoring. This step is frequently a bottleneck in the analytical chain from sampling to data analysis and subsequent environmental status evaluation. Here we present phytoplankton diversity data from 49 lakes including three seasonal surveys assessed by next generation sequencing (NGS) of 16S ribosomal RNA chloroplast and cyanobacterial gene amplicons and also compare part of these datasets with identification based on morphology. Direct comparison of NGS to microscopic data from three time-series showed that NGS was able to capture the seasonality in phytoplankton succession as observed by microscopy. Still, the PCR-based approach was only semi-quantitative, and detailed NGS and microscopy taxa lists had only low taxonomic correspondence. This is probably due to, both, methodological constraints and current discrepancies in taxonomic frameworks. Discrepancies included Euglenophyta and Heterokonta that were scarce in the NGS but frequently detected by microscopy and Cyanobacteria that were in general more abundant and classified with high resolution by NGS. A deep-branching taxonomically unclassified cluster was frequently detected by NGS but could not be linked to any group identified by microscopy. NGS derived phytoplankton composition differed significantly among lakes with different trophic status, showing that our approach can resolve phytoplankton communities at a level relevant for ecosystem management. The high reproducibility and potential for standardization and parallelization makes our NGS approach an excellent candidate for simultaneous monitoring of prokaryotic and eukaryotic phytoplankton in inland waters.     

Intracellular Diversity of the V4 and V9 Regions of the 18S rRNA in Marine Protists (Radiolarians) Assessed by High-Throughput Sequencing

Metabarcoding is a powerful tool for exploring microbial diversity in the environment, but its accurate interpretation is impeded by diverse technical (e.g. PCR and sequencing errors) and biological biases (e.g. intra-individual polymorphism) that remain poorly understood. To help interpret environmental metabarcoding datasets, we investigated the intracellular diversity of the V4 and V9 regions of the 18S rRNA gene from Acantharia and Nassellaria (radiolarians) using 454 pyrosequencing. Individual cells of radiolarians were isolated, and PCRs were performed with generalist primers to amplify the V4 and V9 regions. Different denoising procedures were employed to filter the pyrosequenced raw amplicons (Acacia, AmpliconNoise, Linkage method). For each of the six isolated cells, an average of 541 V4 and 562 V9 amplicons assigned to radiolarians were obtained, from which one numerically dominant sequence and several minor variants were found. At the 97% identity, a diversity metrics commonly used in environmental surveys, up to 5 distinct OTUs were detected in a single cell. However, most amplicons grouped within a single OTU whereas other OTUs contained very few amplicons. Different analytical methods provided evidence that most minor variants forming different OTUs correspond to PCR and sequencing artifacts. Duplicate PCR and sequencing from the same DNA extract of a single cell had only 9 to 16% of unique amplicons in common, and alignment visualization of V4 and V9 amplicons showed that most minor variants contained substitutions in highly-conserved regions. We conclude that intracellular variability of the 18S rRNA in radiolarians is very limited despite its multi-copy nature and the existence of multiple nuclei in these protists. Our study recommends some technical guidelines to conservatively discard artificial amplicons from metabarcoding datasets, and thus properly assess the diversity and richness of protists in the environment.     

An examination of the diversity of a novel Campylobacter reservoir

The diversity of eukaryotic populations, in particular protozoa, in the water supplies of intensively reared broilers has not been previously studied. This important food-rearing environment was screened for the molecular diversity of eukaryotes by the analysis of PCR-amplified 18S rRNA. DNA was extracted from filtered water samples that were collected from the poultry drinking water systems of five farms. The total genomic DNA was used to produce rRNA-PCR amplicons, which, with the application of TTGE, provided an overview of the eukaryotic population diversity. The rRNA-PCR amplicons were then used to generate 34 random clones that were subject to comparative sequence analysis. Twenty-five of the clones (73.5%) showed high similarity with yeasts and fungi (>92%) and 9 clones demonstrated similarity (>86%) with certain protozoan groups, including flagellates and alveolates. Further studies of the microbial diversity in the previously ignored niche of intensively reared poultry drinking water systems are required, along with subsequent in vitro co-culture assays of the detected protozoa and bacterial strains.     

Molecular detection of Campylobacter spp. in California gull (Larus californicus) excreta

We examined the prevalence, quantity, and diversity of Campylobacter species in the excreta of 159 California gull (Larus californicus) samples using culture-, PCR-, and quantitative PCR (qPCR)-based detection assays. Campylobacter prevalence and abundance were relatively high in the gull excreta examined; however, C. jejuni and C. lari were detected in fewer than 2% of the isolates and DNA extracts from the fecal samples that tested positive. Moreover, molecular and sequencing data indicated that most L. californicus campylobacters were novel (<97% 16S rRNA gene sequence identity to known Campylobacter species) and not closely related to species commonly associated with human illness. Campylobacter estimates were positively related with those of fecal indicators, including a gull fecal marker based on the Catellicoccus marimammalium 16S rRNA gene.     

Phylogenetic detection of novel Cryptomycota in an Iowa (United States) aquifer and from previously collected marine and freshwater targeted high‐throughput sequencing sets

Fungi are everywhere and interact with humans in countless ways, but a large group of fungi called ‘Cryptomycota’ has escaped detection until very recently. Still, the extent of diversity and ecological habits of this group remain largely unknown. We interrogated publically available 18S rRNA gene datasets, obtained via high‐throughput sequencing from marine and freshwater samples, for Cryptomycota sequences. Contrary to previous work, we found evidence of substantial Cryptomycota diversity in the marine upper water column. Additionally, we produced a sequencing set from a groundwater aquifer, an environment unrepresented among 18S rRNA gene pyrosequencing sets. The Cryptomycota community in this aquifer sample appears distinct from the community in both freshwater and marine environments with evidence of a unique aquifer clade. This study significantly expands the boundary of known Cryptomycota sequence diversity and characterizes the phylogenetic distribution of this diversity in aquatic environments. Furthermore, the approach utilized is generalizable to discovery of novel micro‐eukaryotic diversity from any lineage.     

Unveiling trophic functions of uncultured protist taxa by incubation experiments in the brackish baltic sea

Background

Our knowledge of the phylogeny and diversity of aquatic protists is rapidly increasing due to molecular surveys and next-generation sequencing approaches. This has led to a considerable discrepancy between the taxa known from cultures and those known from environmental 18S rRNA gene sequences. Hence, it is generally difficult to assign ecological functions to new taxa detected by culture-independent molecular approaches.

Methodology/Principal Findings

A combination of unamended dark incubations and 18S rRNA sequencing was chosen to link molecular diversity data of uncultured protists with heterotrophic, presumably bacterivorous, growth. The incubations, conducted with Baltic Sea brackish water, resulted in a consistent shift from a protistan community dominated by phototrophs to one in which heterotrophs predominated. This was determined on the basis of cell abundance and 18S rRNA sequences derived from fingerprint analysis and clone libraries. The bulk of enriched phylotypes after incubation were related to hitherto uncultured marine taxa within chrysophytes, ochrophytes, choanoflagellates, cercozoans, and picobiliphytes, mostly represented in recently established or here defined environmental clades. Their growth in the dark, together with coinciding results from studies with a similar objective, provides evidence that these uncultured taxa represent heterotrophic or mixotrophic species.

Conclusions/Significance

These findings shed some light into the trophic role of diverse uncultured protists especially within functionally heterogeneous groups (e.g., chrysophytes, ochrophytes) and groups that appear to be puzzling with regard to their nutrition (picobiliphytes). Additionally, our results indicate that the heterotrophic flagellate community in the southwestern Baltic Sea is dominated by species of marine origin. The combination of unamended incubations with molecular diversity analysis is thus confirmed as a promising approach to explore the trophic mode of environmentally relevant protist taxa for which only sequence data are currently available.     

Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4-V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8-V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of gamma-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.     

Diversity of Kenyan soda lake alkaliphiles assessed by molecular methods

DNA was extracted from water and sediment samples taken from soda lakes of the Kenyan-Tanzanian Rift Valley. DNA was also extracted from microbial enrichment cultures of sediment samples. 16S rRNA genes were amplified by the polymerase chain reaction and microbial diversity was studied using denaturing gradient gel electrophoresis (DGGE) of 16S rDNA amplicons. Cloning and sequencing of single DGGE bands showed that they usually contained mixed amplicons. Several of the amplicon sequences had high identities, up to 99%, with 16S rRNA genes of organisms previously isolated from soda lakes, while others were only distantly related, with identities as low as 82%. Phylogenetic analysis of the sequenced amplicons indicated that sequences were related to the haloarchaeal, Bacillus/Clostridium, Rhodobacterium/Thioalcalovibrio/ Methylobacter, and Cytophaga/Flavobacterium/Bacteroides (CFB) groups and the enterobacteria/Aeromonas/Vibrio part of the 3 subdivision of the Proteobacteria.Communicated by K. Horikoshi     

Identification and differentiation of Cryptosporidium species by capillary electrophoresis single-strand conformation polymorphism

Cryptosporidium species generally lack distinguishing morphological traits, and consequently, molecular methods are commonly used for parasite identification. Various methods for Cryptosporidium identification have been proposed, each with their advantages and disadvantages. In this study, we show that capillary electrophoresis coupled with single-strand conformation polymorphism (CE-SSCP) is a rapid, simple and cost-effective method for the identification of Cryptosporidium species and genotypes. Species could be readily differentiated based on the SSCP mobility of amplified 18S rRNA gene molecules. Clones that differed by single-nucleotide polymorphisms could be distinguished on CE-SSCP mobility. Profiles of species known to have heterogenic copies of 18S rRNA gene contained multiple peaks. Cloning and sequencing of Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium fayeri and Cryptosporidium possum genotype 18S rRNA gene amplicons confirmed that these multiple peaks represented type A and type B 18S rRNA gene copies. CE-SSCP provides a reliable and sensitive analysis for epidemiological studies, environmental detection and diversity screening.     

Analysis of the Cystic Fibrosis Lung Microbiota via Serial Illumina Sequencing of Bacterial 16S rRNA Hypervariable Regions

The characterization of bacterial communities using DNA sequencing has revolutionized our ability to study microbes in nature and discover the ways in which microbial communities affect ecosystem functioning and human health. Here we describe Serial Illumina Sequencing (SI-Seq): a method for deep sequencing of the bacterial 16S rRNA gene using next-generation sequencing technology. SI-Seq serially sequences portions of the V5, V6 and V7 hypervariable regions from barcoded 16S rRNA amplicons using an Illumina short-read genome analyzer. SI-Seq obtains taxonomic resolution similar to 454 pyrosequencing for a fraction of the cost, and can produce hundreds of thousands of reads per sample even with very high multiplexing. We validated SI-Seq using single species and mock community controls, and via a comparison to cystic fibrosis lung microbiota sequenced using 454 FLX Titanium. Our control runs show that SI-Seq has a dynamic range of at least five orders of magnitude, can classify >96% of sequences to the genus level, and performs just as well as 454 and paired-end Illumina methods in estimation of standard microbial ecology diversity measurements. We illustrate the utility of SI-Seq in a pilot sample of central airway secretion samples from cystic fibrosis patients.     

Formation of pseudo-terminal restriction fragments,a PCR-related bias affecting terminal restriction fragment length polymorphism analysis of microbial community structure

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called "pseudo-T-RFs" since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.     

Design and Experimental Application of a Novel Non-Degenerate Universal Primer Set that Amplifies Prokaryotic 16S rRNA Genes with a Low Possibility to Amplify Eukaryotic rRNA Genes

The deep sequencing of 16S rRNA genes amplified by universal primers has revolutionized our understanding of microbial communities by allowing the characterization of the diversity of the uncultured majority. However, some universal primers also amplify eukaryotic rRNA genes, leading to a decrease in the efficiency of sequencing of prokaryotic 16S rRNA genes with possible mischaracterization of the diversity in the microbial community. In this study, we compared 16S rRNA gene sequences from genome-sequenced strains and identified candidates for non-degenerate universal primers that could be used for the amplification of prokaryotic 16S rRNA genes. The 50 identified candidates were investigated to calculate their coverage for prokaryotic and eukaryotic rRNA genes, including those from uncultured taxa and eukaryotic organelles, and a novel universal primer set, 342F-806R, covering many prokaryotic, but not eukaryotic, rRNA genes was identified. This primer set was validated by the amplification of 16S rRNA genes from a soil metagenomic sample and subsequent pyrosequencing using the Roche 454 platform. The same sample was also used for pyrosequencing of the amplicons by employing a commonly used primer set, 338F-533R, and for shotgun metagenomic sequencing using the Illumina platform. Our comparison of the taxonomic compositions inferred by the three sequencing experiments indicated that the non-degenerate 342F-806R primer set can characterize the taxonomic composition of the microbial community without substantial bias, and is highly expected to be applicable to the analysis of a wide variety of microbial communities.     

Shotgun metagenomics and microscopy indicate diverse cyanophytes,other bacteria,and microeukaryotes in the epimicrobiota of a northern Chilean wetland Nostoc (Cyanobacteria)

Prokaryotic Nostoc, one of the world's most conspicuous and widespread algal genera (similar to eukaryotic algae, plants, and animals) is known to support a microbiome that influences host ecological roles. Past taxonomic characterizations of surface microbiota (epimicrobiota) of free‐living Nostoc sampled from freshwater systems employed 16S rRNA genes, typically amplicons. We compared taxa identified from 16S, 18S, 23S, and 28S rRNA gene sequences filtered from shotgun metagenomic sequence and used microscopy to illuminate epimicrobiota diversity for Nostoc sampled from a wetland in the northern Chilean Altiplano. Phylogenetic analysis and rRNA gene sequence abundance estimates indicated that the host was related to Nostoc punctiforme PCC 73102. Epimicrobiota were inferred to include 18 epicyanobacterial genera or uncultured taxa, six epieukaryotic algal genera, and 66 anoxygenic bacterial genera, all having average genomic coverage ≥90X. The epicyanobacteria Geitlerinemia, Oscillatoria, Phormidium, and an uncultured taxon were detected only by 16S rRNA gene; Gloeobacter and Pseudanabaena were detected using 16S and 23S; and Phormididesmis, Neosynechococcus, Symphothece, Aphanizomenon, Nodularia, Spirulina, Nodosilinea, Synechococcus, Cyanobium, and Anabaena (the latter corroborated by microscopy), plus two uncultured cyanobacterial taxa (JSC12, O77) were detected only by 23S rRNA gene sequences. Three chlamydomonad and two heterotrophic stramenopiles genera were inferred from 18S; the streptophyte green alga Chaetosphaeridium globosum was detected by microscopy and 28S rRNA genes, but not 18S rRNA genes. Overall, >60% of epimicrobial taxa were detected by markers other than 16S rRNA genes. Some algal taxa observed microscopically were not detected from sequence data. Results indicate that multiple taxonomic markers derived from metagenomic sequence data and microscopy increase epimicrobiota detection.