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AbstractEnvironmental DNA ( eDNA ) metabarcoding from water samples has, in recent years, shown great promise for biodiversity monitoring. However, universal primers targeting the  cytochrome oxidase I  (  COI  ) marker gene popular in metazoan studies have displayed high levels of nontarget amplification. To date, enrichment methods bypassing amplification have not been able to match the detection levels of conventional metabarcoding. This study evaluated the use of universal metabarcoding primers as capture probes to either isolate target DNA or to remove nontarget DNA, prior to amplification, by using biotinylated versions of universal metazoan and bacterial barcoding primers, namely metazoan   COI   ( mlCOIintF ) and bacterial   16S   (515F). Additionally, each step of the protocol was assessed by amplifying for both metazoan   COI   ( mlCOIintF /jgHCO2198) and bacterial   16S   (515F/806R) to investigate the effect on the metazoan and bacterial communities. Bacterial read abundance increased significantly in response to the captures (  COI   library), while the quality of the captured DNA was also improved. The metazoan-oriented probe captured bacterial DNA in a range that was also amplifiable with the   16S   primers, demonstrating the ability of capture probes to isolate fragments of DNA spanning over a longer distance than perhaps expected, from  eDNA . Although the use of the tested   COI   probe cannot be recommended for metazoan enrichment, based on the experimental results, the concept of capturing these longer fragments could be applied to metazoan metabarcoding. By using a truly conserved site without a high-level taxonomic resolution as a target for capture, it may be possible to isolate DNA fragments large enough to span over a nearby barcoding region (e.g.,   COI  ), which can then be processed through a conventional metabarcoding-by-amplification protocol.

metabarcoding and metagenomics

﻿The bacterial hitchhiker’s guide to COI: Universal primer-based COI capture probes fail to exclude bacterial DNA, but 16S capture leaves metazoa behind

﻿The bacterial hitchhiker’s guide to COI: Universal primer-based COI capture probes fail to exclude bacterial DNA, but 16S capture leaves metazoa behind

The bacterial hitchhiker’s guide to COI: Universal primer-based COI capture probes fail to exclude bacterial DNA, but 16S capture leaves metazoa behind