The Impact of Primer Design on Amplicon-Based Metagenomic Profiling Accuracy: Detailed Insights into Bifidobacterial Community Structure

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.

Keywords: 16S rRNA profiling; Bifidobacterium; metagenomics; microbiota; primer pairs.

Figure 1

Amplification efficiency of primer pairs as calculated by web-tool TestPrime 1.0. A heat map showing primer pair amplification efficiency when targeting 54 bacterial taxa that are commonly found in the human gut microbiota. The PCR primer pair clusters were obtained by TM4 MeV software. The white cells indicate the inability of PCR primer pairs to amplify (members of) a bacterial genus.

Figure 2

16S rRNA gene-based microbial profiling analysis of human fecal samples. The heat map reports the deduced relative abundance of 54 bacterial taxa that are commonly found in the human gut microbiota. The black cell indicated the absence of the bacterial genus.

Figure 3

PCR primer pair amplification efficiency towards all so far known bifidobacterial species. A heat map illustrating PCR primer pair efficiency on all so far known bifidobacterial species. The bifidobacterial clusters were obtained by TM4 MeV software considering the primer pair efficiency. The inability of a given PCR primer pair to amplify a particular bifidobacterial species was highlighted by a white cell.