Identification and classification of eukaryotes increasingly depends on DNA sequences of standardized genetic markers, a concept known as DNA barcoding. Thus, molecular taxonomists continue to search for regions conserved enough to allow reliable priming but sufficiently variable to yield highly resolved and well-supported phylograms. The ideal characteristics include adequate species resolution, ease of amplification, absence of extreme length variation, the presence of only single copies and low intra-species variability.
Additional challenge that metabarcoding primers should overcome efficiently is the amplification of target DNA in samples rich in non-target DNA and environmental pollutants that may have been co-extracted with the DNA. What is more, a single primer set should cover a large proportion of the species that compose a community of interest expanding the general taxonomic to produce a reliable mapping of microbial diversity.
We evaluated the efficiency of a new set of ITS1-30F/ITS1-217R primers recently designed by Usyk et al. for the analysis of fungal cultures in three different human oral samples (A, B and C). We benchmarked their performance to the standard primers sets (ITS1-2 and ITS1-4) reported in the milestone paper on amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics by White et al. in 1990.
Fig 1. Domain taxonomy boxplot (%).
ITS1-2 amplification was least specific. It targeted more bacterial ‘contaminant’ DNA than the other two primer sets (Fig 1). In addition, it chronically generated more sequences that could not be classified (Fig 2).
Fig 2. Fungi genus taxonomy boxplot (%).
On the other hand, the performance of ITS1-30F/ITS1-217R and ITS1-4 primer pairs was comparable (Fig 2 and 3).
We observed 2 times higher amount of human ‘contaminant’ in the samples amplified with ITS1-30F/ITS1-217R primers in respect to those amplified with ITS1-4 (on average 16% vs. 8%). However, ITS1-30F/ITS1-217R primer set was more efficient in discriminating fungi’s OTUs and produced less ‘unknown’ reads (Fig 2).
Fig 3. Bray Curtis beta-diversity measures the distance or dissimilarity between pairs of samples.
Comparing the amplification efficiency and robustness of primers used in metabarcoding studies is important because differences in primer efficiency may result in strong biases in favor of more easily amplifiable sequences during PCR reactions, potentially influencing our view on fungal communities. Constant improvements and updates of fungal reference databases will inevitably result in design of new primer sets in order to ensure an adequate capture of the ever-growing fungal diversity.
Stay tuned, we will for sure continue to test and share the results. :-)