Marine eDNA as an effective tool in the Climate Change Era: testing the analysis pipeline with mock communities

Climate change has caused major environmental shits, from habitat loss to the introduction of invasive species. The eDNA analysis is a promising tool for tracking climate change impacts on marine ecosystems. While innovative and cost-effective, eDNA analysis requires proper data interpretation. Mock communities (MCs) are useful for testing eDNA pipelines and identifying factors influencing results. DNA extracts from 13 fish species were used to create non-normalized (MC1) and normalized (MC2) mock communities. Primer sensitivity to mitochondrial DNA (mtDNA) was tested using tele02 primers targeting the 12s gene. Pre-PCR MCs were composed of pooled neat extracts, and post-PCR MCs from individually amplified extracts. The DNA libraries were sequenced on MiSeq Illumina, and the outputs were processed with Obitools and Vsearch. A strong positive Pearson correlation was found between DNA concentration and reads count when 12s gene was present in high copy number (post-PCR MC1). An even species ratio (Obitools: E>0.9; Vsearch: E>0.8) was observed only in post-PCR MCs, while no reads were assigned to Mullus barbatus in pre-PCR MCs regardless of concentration (except for 0.4% assigned with Obitools in pre-PCR MC2). Primers efficiency depended on mtDNA concentration, not on total DNA amount. The mtDNA concentration affects species detection in natural conditions, where different DNA traces are unevenly dispersed. These findings highlight the need to understand the chances of false negatives, especially when assessing climate change impacts on species distribution.