Multicenter evaluation of the EasySeq full-length 16S sequencing kit for bacterial identification in clinical samples

Bacterial culture remains essential for pathogen detection and identification, but its sensitivity is limited in cases involving fastidious microorganisms, polymicrobial infections, or samples obtained after antibiotic treatment. Targeted next-generation sequencing (tNGS) offers a culture-independent alternative and is increasingly being implemented in clinical microbiology. This multicenter study evaluated the EasySeq Full-Length 16S Library Prep Kit for Oxford Nanopore Technologies (EasySeq; NimaGen) combined with the GenomeDetective pipeline (EasySeq-GD) against eight established 16S rRNA gene- and 16–23​S rRNA intergenic spacer (IS)-based workflows. Eight medical centers in the Netherlands and Belgium analyzed a mock community sample in triplicate and 20 clinical samples per center ( n = 160). Mock community analysis demonstrated consistent taxonomic profiles across centers with minimal intralaboratory variation. Of 160 clinical samples, 19 (11.9%) initially failed, of which nine (5.6%) remained unresolved. Among the remaining 151 samples, EasySeq-GD and comparator workflows shared 141 detected taxa. In addition, EasySeq-GD identified 92 unique bacterial taxa, whereas comparator workflows identified 32 unique taxa. These findings resulted in clinically relevant discrepancies in eight and three samples, respectively. Overall, EasySeq-GD demonstrated good interlaboratory reproducibility. Taxon-level results were concordant with comparator workflows in 107/151 samples (70.9%), while 140/151 samples (92.7%) showed no clinically relevant discrepancies.