Evaluation of 16S rRNA Gene Primer Pairs for Monitoring Microbial Community Structures Showed High Reproducibility within and Low Comparability between Datasets Generated with Multiple Archaeal and Bacterial Primer Pairs.

Martin A Fischer,Sven C Neulinger,Ruth A Schmitz,Wolfgang R Streit,Simon Güllert

doi:10.3389/fmicb.2016.01297

Martin A Fischer, Sven C Neulinger + Show 3 more

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https://doi.org/10.3389/fmicb.2016.01297

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Abstract

The application of next-generation sequencing technology in microbial community analysis increased our knowledge and understanding of the complexity and diversity of a variety of ecosystems. In contrast to Bacteria, the archaeal domain was often not particularly addressed in the analysis of microbial communities. Consequently, established primers specifically amplifying the archaeal 16S ribosomal gene region are scarce compared to the variety of primers targeting bacterial sequences. In this study, we aimed to validate archaeal primers suitable for high throughput next generation sequencing. Three archaeal 16S primer pairs as well as two bacterial and one general microbial 16S primer pairs were comprehensively tested by in-silico evaluation and performing an experimental analysis of a complex microbial community of a biogas reactor. The results obtained clearly demonstrate that comparability of community profiles established using different primer pairs is difficult. 16S rRNA gene data derived from a shotgun metagenome of the same reactor sample added an additional perspective on the community structure. Furthermore, in-silico evaluation of primers, especially those for amplification of archaeal 16S rRNA gene regions, does not necessarily reflect the results obtained in experimental approaches. In the latter, archaeal primer pair ArchV34 showed the highest similarity to the archaeal community structure compared to observed by the metagenomic approach and thus appears to be the appropriate for analyzing archaeal communities in biogas reactors. However, a disadvantage of this primer pair was its low specificity for the archaeal domain in the experimental application leading to high amounts of bacterial sequences within the dataset. Overall our results indicate a rather limited comparability between community structures investigated and determined using different primer pairs as well as between metagenome and 16S rRNA gene amplicon based community structure analysis. This finding, previously shown for Bacteria, was as well observed for the archaeal domain.

Highlights

The investigation of the microbial community composition allows a detailed insight in diversity and potential ecosystem function and fosters understanding of complex microbial processes (Vanwonterghem et al, 2014)
In the sequences of the archaeal community, a clear abundances ranking of taxonomic orders (Methanosarcinales > Methanomicrobiales > Methanobacteriales ≥ Methanomassiliicoccales) was consistently conserved in all tested primer pairs as well as in the sequences obtained from the metagenome
The most promising combination to analyze the community of the sample would have been the combination of the BacV35 and ArchV46 primer pairs, the metagenomic sequences still show a different overall bacterial and archaeal community compared to that observed by those primer pairs (Figure 6). (B) From a technical point of view, efficiency strongly differed between the evaluated primer pairs, namely due to unspecific amplification by archaeal primer pairs

Summary

Introduction

The investigation of the microbial community composition allows a detailed insight in diversity and potential ecosystem function and fosters understanding of complex microbial processes (Vanwonterghem et al, 2014). Recent years have seen a strong increase in sequencing approaches targeting microbial communities via amplicon sequencing or metagenomic and metatranscriptomic approaches (Turnbaugh et al, 2007; Hamady et al, 2008; Raes and Bork, 2008; Caporaso et al, 2012; Grosskopf and Soyer, 2014; Ininbergs et al, 2015) These approaches play an important role in monitoring and comparing large numbers of samples in terms of their microbial composition (Caporaso et al, 2012; Kozich et al, 2013; Sundberg et al, 2013). Archaea are ubiquitously found under rather mesophilic conditions like in fresh and marine waters (DeLong, 1992; DeLong et al, 1994; Karner et al, 2001; Stahl and de la Torre, 2012), biogas reactors (Sundberg et al, 2013), and soil (Leininger et al, 2006), the intestinal tract of termites (Paul et al, 2012), ruminants (Jeyanathan et al, 2011; Kittelmann et al, 2013), and on the human skin (Probst et al, 2013; Oh et al, 2014), or in the intestine (recently reviewed in Bang and Schmitz, 2015), where they complete the microbiome together with their bacterial, eukaryotic and viral partners

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