Abstract
BackgroundSubunits of ribosomal RNA genes (rDNAs) characterized by PCR-based protocols have been the proxy for studies in microbial taxonomy, phylogenetics, evolution and ecology. However, relevant factors have shown to interfere in the experimental outputs in a variety of systems. In this work, a ‘theoretical’ to ‘actual’ delta approach was applied to data on culturable mock bacterial communities (MBCs) to study the levels of losses in operational taxonomic units (OTUs) detectability. Computational and lab-bench strategies based on 16S rDNA amplification by 799F and U1492R primers were employed, using a fingerprinting method with highly improved detectability of fragments as a case-study tool. MBCs were of two major types: in silico MBCs, assembled with database-retrieved sequences, and in vitro MBCs, with AluI digestions of PCR data generated from culturable endophytes isolated from cacao trees.ResultsInterfering factors for the 16 s rDNA amplifications, such as the type of template, direct and nested PCR, proportion of chloroplast DNA from a tropical plant source (Virola officinalis), and biased-amplification by the primers resulted in altered bacterial 16S rDNA amplification, both on MBCs and V. officinalis leaf-extracted DNA. For the theoretical data, the maximum number of fragments for in silico and in vitro cuts were not significantly different from each other. Primers’ preferences for certain sequences were detected, depending on the MBCs’ composition prior to PCR. The results indicated overall losses from 2.3 up to 8.2 times in the number of OTUs detected from actual AluI digestions of MBCs when compared to in silico and in vitro theoretical data.ConclusionsDue to all those effects, the final amplification profile of the bacterial community assembled was remarkably simplified when compared to the expected number of detectable fragments known to be present in the MBC. From these findings, the scope of hypotheses generation and conclusions from experiments based on PCR amplifications of bacterial communities was discussed.
Highlights
Subunits of ribosomal RNA genes characterized by Polymerase chain reaction (PCR)-based protocols have been the proxy for studies in microbial taxonomy, phylogenetics, evolution and ecology
Interference of chloroplast DNA and number of PCR amplifications The pattern of amplicons produced from the same samples were assessed by ‘Direct’ (1 PCR) and ‘nested’ (2 PCR) amplifications with the 799F and U1492R primers
For nested PCR, a strong interference effect of the chloroplast sequences was observed, whereas for direct PCR, a preferential amplification over bacterial DNA was noticed instead; a clearer amplification of the bacterial 16 s ribosomal RNA gene (rDNA) was only achieved in the samples lacking chloroplast DNA (cpDNA)
Summary
Subunits of ribosomal RNA genes (rDNAs) characterized by PCR-based protocols have been the proxy for studies in microbial taxonomy, phylogenetics, evolution and ecology. ‘Genetic fingerprinting’ is a first group of these techniques, including CAPS [17], ARDRA [18], T-RFLP [19], D/TGGE [20], A/RISA [7], and others [8, 15] These methods allow for sequencing of operational taxonomic units (OTUs), their major purpose is to address the effects of different treatments on structure/complexity of communities and on population dynamics [15, 21]. A second group of methods employs high throughput sequencing (HTS) of PCR-amplified rDNA subunits [22,23,24,25,26,27] These techniques have largely expanded OTU data generation by several orders of magnitude, allowing simultaneous analysis of richness, abundance and composition, and detection of much finer changes in the communities [8]
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