Selecting competent reverse transcription strategies to maximize
biodiversity recovery with eRNA metabarcoding
Abstract
Both environmental DNA (eDNA) and environmental RNA (eRNA) have been
widely adopted for biodiversity assessment. While eDNA often persists
longer in environments, eRNA offers a more current view of biological
activities. In eRNA metabarcoding, extracted eRNA is reverse transcribed
into complementary DNA (cDNA) for metabarcoding. However, the efficacy
of various reverse transcription strategies has not been evaluated. Here
we compared the biodiversity recovery efficiency of three strategies:
random priming with hexamers, oligo(dT) priming, and taxa-specific
priming using Mifish-U for fish in both high- and low-biodiversity
regions. Our results demonstrate that reverse transcription strategies
significantly impact biodiversity recovery. Random priming consistently
detected the highest number of taxa in both low- and high-biodiversity
regions. In low-biodiversity areas, oligo(dT) performed comparably to
random hexamers; however, in high-biodiversity regions, random hexamers
outperformed oligo(dT), particularly in recovering rare taxa. While
taxa-specific priming was comparative to the other strategies for
high-abundance taxa, it was less effective for rare taxa, thus limiting
its utility for comprehensive biodiversity assessment. These differences
are largely due to the multiple binding sites for random hexamers
compared to the fewer or absent sites with oligo(dT) and taxa-specific
primers under high eRNA degradation. Combining random hexamers and
oligo(dT) significantly improved taxa recovery, especially for
low-abundance species, supporting its best practice in eukaryotes. For
prokaryotes or genes lacking polyadenylation, random priming is favored
over taxa- or gene-specific priming. Collectively, these findings
underscore the critical importance of selecting appropriate reverse
transcription strategies in eRNA metabarcoding, with significant
implications for effective biodiversity monitoring and conservation
efforts.