Testing the Limits of 454 Pyrotag Sequencing: Reproducibility, Quantitative Assessment and Comparison to T-RFLP Fingerprinting of Aquifer Microbes

Uppsala University, Sweden
PLoS ONE (Impact Factor: 3.23). 07/2012; 7(7):e40467. DOI: 10.1371/journal.pone.0040467
Source: PubMed


The characterization of microbial community structure via 16S rRNA gene profiling has been greatly advanced in recent years by the introduction of amplicon pyrosequencing. The possibility of barcoding gives the opportunity to massively screen multiple samples from environmental or clinical sources for community details. However, an on-going debate questions the reproducibility and semi-quantitative rigour of pyrotag sequencing, similar to the early days of community fingerprinting. In this study we demonstrate the reproducibility of bacterial 454 pyrotag sequencing over biological and technical replicates of aquifer sediment bacterial communities. Moreover, we explore the potential of recovering specific template ratios via quantitatively defined template spiking to environmental DNA. We sequenced pyrotag libraries of triplicate sediment samples taken in annual sampling campaigns at a tar oil contaminated aquifer in Düsseldorf, Germany. The abundance of dominating lineages was highly reproducible with a maximal standard deviation of ~4% read abundance across biological, and ~2% across technical replicates. Our workflow also allows for the linking of read abundances within defined assembled pyrotag contigs to that of specific 'in vivo' fingerprinting signatures. Thus we demonstrate that both terminal restriction fragment length polymorphism (T-RFLP) analysis and pyrotag sequencing are capable of recovering highly comparable community structure. Overall diversity was roughly double in amplicon sequencing. Pyrotag libraries were also capable of linearly recovering increasing ratios (up to 20%) of 16S rRNA gene amendments from a pure culture of Aliivibrio fisheri spiked to sediment DNA. Our study demonstrates that 454 pyrotag sequencing is a robust and reproducible method, capable of reliably recovering template abundances and overall community structure within natural microbial communities.

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Available from: Michael S. Granitsiotis, Oct 07, 2015
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    • "In a recent study on sausage spoilage (Benson et al., 2014) a correlation coefficient of 0.805 was found among technical replicates using the 20 most abundant OTUs; this includes all steps extraction amplification and sequencing; although the authors claimed that most dispersion is found for relative abundances b0.001, significant dispersion is evident even for taxa appearing at higher relative abundances. On the other hand in a study on polluted aquifer communities (Pilloni et al., 2012) highly reproducible abundance estimates were obtained for dominant taxa and no significant bias for low abundance taxa. "
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    ABSTRACT: The microbiota of high-moisture Mozzarella cheese made from cow's milk and produced with different acidification methods was evaluated at the end of refrigerated storage by pyrosequencing of the 16S rRNA gene. The cheeses were clearly separated on the basis of the acidification methods. Cheeses produced with the addition of starters were dominated by Streptococcus thermophilus, but a variety of lactic acid bacteria and spoilage microorganisms appeared at low levels (0.01-1%). Cheeses produced by direct addition of citric acid were dominated by a diverse microbiota, including both lactic acid bacteria and psychrotrophic γ-proteobacteria. For five brands the acidification system was not declared on the label: the microbiota was dominated by thermophilic lactic acid bacteria (S. thermophilus, Lactobacillus delbrueckii, Lactobacillus helveticus) but a variety of other subdominant lactic acid bacteria, psychrotrophs and Enterobacteriaceae were present, with a diversity comparable or higher to cheeses produced by direct acid addition. This led to the conclusion that undefined starters were used for acidification. Both ordination methods and network analysis were used for the representation of beta-diversity: matrix cluster analysis, principal coordinate analysis and OTU networks uncovered different aspects of the microbial community structure. For three cheese brands both biological replicates (cheeses from different lots) and technical replicates (replicate cheeses from the same lot) were analyzed. Repeatability was acceptable for OTUs appearing at frequencies >1%, but was low otherwise. A linear mixed model showed that the starter system was responsible for most differences related to dairies, while difference due to psychrotrophic contaminants was more related to lot-to-lot variability.
    International journal of food microbiology 09/2015; 216:9-17. DOI:10.1016/j.ijfoodmicro.2015.09.002 · 3.08 Impact Factor
    • "[9] [11] [31] [47] [54]). Therefore, there was much higher information content [56], and identification was based on phylogenetic inference (OPUs) [26] [50] rather than only identity matches (OTUs) [4] [11] [31] [47] [54]. The procedure significantly reduced the diversity yields, but the results showed clearer trends than when only using clustering approaches (Fig. 1). "
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    ABSTRACT: Next generation sequencing approaches allow the retrieval of several orders of magnitude larger numbers of amplified single sequences in 16S rRNA diversity surveys than classical methods. However, the sequences are only partial and thus lack sufficient resolution for a reliable identification. The OPU approach used here, based on a tandem combination of high quality 454 sequences (mean >500nuc) applying strict OTU thresholds, and phylogenetic inference based on parsimony additions to preexisting trees, seemed to improve the identification yields at the species and genus levels. A total of thirteen biopsies of Crohn-diagnosed patients (CD) and seven healthy controls (HC) were studied. In most of the cases (73%), sequences were affiliated to known species or genera and distinct microbial patterns could be distinguished among the CD subjects, with a common depletion of Clostridia and either an increased presence of Bacteroidetes (CD1) or an anomalous overrepresentation of Proteobacteria (CD2). Faecalibacterium prausnitzii presence was undetectable in CD, whereas Bacteroides vulgatus-B. dorei characterized HC and some CD groups. Altogether, the results showed that a microbial composition with predominance of Clostridia followed by Bacteroidetes, with F. prausnitzii and B. vulgatus-B. dorei as major key bacteria, characterized what could be considered a balanced structure in HC. The depletion of Clostridia seemed to be a common trait in CD. Copyright © 2015 Elsevier GmbH. All rights reserved.
    Systematic and Applied Microbiology 07/2015; 38(6). DOI:10.1016/j.syapm.2015.06.008 · 3.28 Impact Factor
    • "Several techniques have been established to monitor the bacterial populations in WWTPs, such as plate counting (Guzman et al. 2007), qPCR (Yu et al. 2014), denaturing gradient gel electrophoresis (DGGE) (Boonnorat et al. 2014), terminal restriction fragment lengths polymorphism (T-RFLP) (Pervin et al. 2013b), fluorescence in situ hybridization (FISH) (Pervin et al. 2013a), and others. The characterization of microbial community structure via 16S ribosomal RNA (rRNA) gene amplicon sequencing is highly reproducible and has been greatly advanced in recent years by the introduction of the next generation sequencing (Pilloni et al. 2012). Therefore, bacterial community structure and phylogenetic diversity was assessed using 16S rRNA gene pyrotag analysis. "
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    ABSTRACT: Stabilized sewage sludge is applied to agricultural fields and farmland due to its high organic matter content. The aim of this study was to investigate the effects of two types of sludge stabilization, mesophilic anaerobic digestion (MAD) and thermophilic anaerobic digestion (TAD), on bacterial communities in sludge, including the presence of pathogenic microorganisms. Bacterial community structure and phylogenetic diversity were analyzed in four sewage sludge samples from the Czech Republic. Analysis of 16S ribosomal RNA (rRNA) genes showed that investigated sludge samples harbor diverse bacterial populations with only a few taxa present across all samples. Bacterial diversity was higher in sludge samples after MAD versus TAD treatment, and communities in MAD-treated sludge shared the highest genetic similarities. In all samples, the bacterial community was dominated by reads affiliated with Proteobacteria. The sludge after TAD treatment had considerably higher number of reads of thermotolerant/thermophilic taxa, such as the phyla Deinococcus-Thermus and Thermotogae or the genus Coprothermobacter. Only one operational taxonomic unit (OTU), which clustered with Rhodanobacter, was detected in all communities at a relative abundance >1 %. All of the communities were screened for the presence of 16S rRNA gene sequences of pathogenic bacteria using a database of 122 pathogenic species and ≥98 % identity threshold. The abundance of such sequences ranged between 0.23 and 1.57 % of the total community, with lower numbers present after the TAD treatment, indicating its higher hygienization efficiency. Sequences clustering with nontuberculous mycobacteria were present in all samples. Other detected sequences of pathogenic bacteria included Streptomyces somaliensis, Acinetobacter calcoaceticus, Alcaligenes faecalis, Gordonia spp., Legionella anisa, Bordetella bronchiseptica, Enterobacter aerogenes, Brucella melitensis, and Staphylococcus aureus.
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