Comparing eDNA metabarcoding primers (12S and 18S) for assessing riparian biodiversity

Abstract

Biodiversity assays in both terrestrial and aquatic populations are being increasingly conducted using eDNA metabarcoding. The creation of a suitable primer and selection of one from hundreds of previously released primer packages will determine the efficacy and results of these efforts. However, only a few studies have compared the effectiveness of various metabarcoding primers in riparian settings. In this study, we assessed two widely used primer sets for rRNA barcoding gene amplification and compared the results with sediment samples taken from the highly biodiverse riparian zones of the Ciliwung River. Using the 1391f and Eukbr primers for the 18S target area and 12S-V5 primer sets for the 12S target region, we amplified the target region of the study. When classifying species at the species level using the 12S and 18S primer sets, we discovered 36 and 264 non-comparable species, respectively, based on a conservative 99% similarity threshold. Unfortunately, the phylum Chordata’s 12S-V5 primer set was limited to detecting the classes Actinopterygii, Aves, and Mammalia. The results were corroborated by the 1391f and Eukbr primers (target area 18S), which also identified the same class of microorganisms, including plankton and other species, for which the 12S-V5 primer combination could not be identified. These findings underscore the significance of utilizing several primer combinations and primers aimed at distinct genomic areas. Moreover, our results suggest that amplification of the target region of 18S using the 1391f and Eukbr primers would be the best choice for riparian biodiversity identification, as opposed to the 12S-V5 primer combination.