Abstract
Next Generation Sequencing (NGS) technologies have overcome the limitations of cultivation-dependent approaches and allowed detailed study of bacterial populations that inhabit the human body. The consortium of bacteria residing in the human intestinal tract, also known as the gut microbiota, impacts several physiological processes important for preservation of the health status of the host. The most widespread microbiota profiling method is based on amplification and sequencing of a variable portion of the 16S rRNA gene as a universal taxonomic marker among members of the Bacteria domain. Despite its popularity and obvious advantages, this 16S rRNA gene-based approach comes with some important limitations. In particular, the choice of the primer pair for amplification plays a major role in defining the accuracy of the reconstructed bacterial profiles. In the current study, we performed an in silico PCR using all currently described 16S rRNA gene-targeting primer pairs (PP) in order to assess their efficiency. Our results show that V3, V4, V5, and V6 were the optimal regions on which to design 16S rRNA metagenomic primers. In detail, PP39 (Probio_Uni/Probio_Rev), PP41 (341F/534R), and PP72 (970F/1050R) were the most suitable primer pairs with an amplification efficiency of >98.5%. Furthermore, the Bifidobacterium genus was examined as a test case for accurate evaluation of intra-genus performances at subspecies level. Intriguingly, the in silico analysis revealed that primer pair PP55 (527f/1406r) was unable to amplify the targeted region of any member of this bacterial genus, while several other primer pairs seem to rather inefficiently amplify the target region of the main bifidobacterial taxa. These results highlight that selection of a 16S rRNA gene-based PP should be done with utmost care in order to avoid biases in microbiota profiling results.
Highlights
During the last decade, a growing number of studies have described efforts to profile complex microbial communities, i.e., microbiota, hosted by various human body sites
In order to perform a comprehensive comparison of currently available primer pairs for 16S rRNA profiling, we performed an in-depth literature search for PCR primer pairs employed for 16S rRNA gene amplification and microbiota profiling (Figure S1 and Table S1)
The selection of specific PCR primer pairs is essential for assessing the microbiota composition
Summary
A growing number of studies have described efforts to profile complex microbial communities, i.e., microbiota, hosted by various human body sites. Scientific evidence has highlighted that the human microbiota plays a major role in several physiological host activities that are essential for health maintenance [1,2] Such in-depth studies of, in particular, the human gut microbiota has been facilitated by the development of high-throughput sequencing technologies, such as Roche 454, Ion Torrent, and Illumina which allow accurate profiling of bacterial populations without the need for their cultivation [1]. The 16S rRNA gene-based profiling method is currently the most commonly employed to catalogue components of the human microbiota This methodology has many advantages, such as reproducible and technically easy procedure, high efficiency in the identification of bacterial phylogeny and taxonomy, accessible bioinformatic pipelines, and low cost, which rapidly led to its wide spread use [3]. Several studies regarding the profiling of the infant gut microbiota, which used different metagenomics methodologies, have in some cases resulted in conflicting results, with certain discrepancies in the bifidobacterial relative abundance [4]
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