Ultrasensitive Sequencing ( <scp>SiMSen‐Seq</scp> ) for Environmental DNA Applications

ABSTRACT The use of Next‐Generation Sequencing (NGS) in environmental DNA (eDNA) research offers increased sensitivity in detecting rare species within vast marine ecosystems. However, conventional NGS methods often fail to accurately capture low‐frequency variants, posing risks of imprecise detections. Inspired by clinical research, innovations like SiMSen‐Seq (simple multiplexed PCR‐based barcoding of DNA for ultrasensitive mutation detection using sequencing) have emerged. SiMSen‐Seq utilizes molecular barcoding to track individual DNA templates, correcting polymerase‐induced errors and quantification biases. In this study, we adapted the SiMSen‐Seq protocol for eDNA analysis, targeting 18S rRNA metabarcoding in seawater samples to enhance marine biodiversity assessment, particularly for identifying rare non‐indigenous species (NIS). Comparative analysis against an Illumina MiSeq protocol (Illumina—DADA2) and the SiMSen‐Seq protocol with DADA2 bioinformatics (SiMSen‐Seq—DADA2) revealed that SiMSen‐Seq detected a higher number (1495) of molecular Operational Taxonomic Units (mOTUs) compared to the other two pipelines (Illumina—DADA2: 585 and SiMSen‐Seq—DADA2: 578). In terms of genera, SiMSen‐Seq detected the lowest number of genera, Illumina—DADA2 the highest number, and 45% of genera were detected across protocols. While all three protocols identified the predominant NIS, they differed in detecting rare taxa: SiMSen‐Seq uniquely detected Arenigobius bifrenatus and Pseudopolydora paucibranchiata but did not detect some others (e.g., Asterias amurensis / A. forbesi ). These findings suggest that for some species (especially Stylea plicata and Ciona savignyi ), SiMSen‐Seq could potentially be useful to detect intraspecific variability for population genetic studies, with uses in more precise ecological monitoring and effective marine conservation.