Comprehensive wet-bench and bioinformatics workflow for complex microbiota using Oxford Nanopore Technologies

We present a comprehensive analysis pipeline with sampling, storage, DNA extraction, library preparation, and bioinformatical evaluation for complex microbiomes sequenced with Oxford nanopore Technologies (ONT). Our findings from the swabs and DNA extraction experiments indicate that methods, which were approved for NGS microbiome analysis cannot be simply adapted to ONT. We recommend using swabs and DNA extractions protocols with appropriate medium or extended washing steps. Both 16S rRNA and metagenomic sequencing achieved reliable and reproducible results. Still, the relatively high error rate of ONT sequences remains a bioinformatical challenge. Our benchmarking experiments reveal thresholds for analysis parameters which achieved excellent precision, recall and AUPR values.