Dataset Information

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.

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.

SUBMITTER: Fischer MA

PROVIDER: S-EPMC4994424 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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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.

Fischer Martin A MA Güllert Simon S Neulinger Sven C SC Streit Wolfgang R WR Schmitz Ruth A RA

Frontiers in microbiology 20160823

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 stu ...[more]

PMID: 27602022

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Project description:Short-amplicon 16S rRNA gene sequencing is currently the method of choice for studies investigating microbiomes. However, comparative studies on differences in procedures are scarce. We sequenced human stool samples and mock communities with increasing complexity using a variety of commonly used protocols. Short amplicons targeting different variable regions (V-regions) or ranges thereof (V1-V2, V1-V3, V3-V4, V4, V4-V5, V6-V8, and V7-V9) were investigated for differences in the composition outcome due to primer choices. Next, the influence of clustering (operational taxonomic units [OTUs], zero-radius OTUs [zOTUs], and amplicon sequence variants [ASVs]), different databases (GreenGenes, the Ribosomal Database Project, Silva, the genomic-based 16S rRNA Database, and The All-Species Living Tree), and bioinformatic settings on taxonomic assignment were also investigated. We present a systematic comparison across all typically used V-regions using well-established primers. While it is known that the primer choice has a significant influence on the resulting microbial composition, we show that microbial profiles generated using different primer pairs need independent validation of performance. Further, comparing data sets across V-regions using different databases might be misleading due to differences in nomenclature (e.g., Enterorhabdus versus Adlercreutzia) and varying precisions in classification down to genus level. Overall, specific but important taxa are not picked up by certain primer pairs (e.g., Bacteroidetes is missed using primers 515F-944R) or due to the database used (e.g., Acetatifactor in GreenGenes and the genomic-based 16S rRNA Database). We found that appropriate truncation of amplicons is essential and different truncated-length combinations should be tested for each study. Finally, specific mock communities of sufficient and adequate complexity are highly recommended.IMPORTANCE In 16S rRNA gene sequencing, certain bacterial genera were found to be underrepresented or even missing in taxonomic profiles when using unsuitable primer combinations, outdated reference databases, or inadequate pipeline settings. Concerning the last, quality thresholds as well as bioinformatic settings (i.e., clustering approach, analysis pipeline, and specific adjustments such as truncation) are responsible for a number of observed differences between studies. Conclusions drawn by comparing one data set to another (e.g., between publications) appear to be problematic and require independent cross-validation using matching V-regions and uniform data processing. Therefore, we highlight the importance of a thought-out study design including sufficiently complex mock standards and appropriate V-region choice for the sample of interest. The use of processing pipelines and parameters must be tested beforehand.

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Dataset Information

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.

Publications

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.

Similar Datasets

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