A computational strategy for rapid on-site 16S metabarcoding with Oxford Nanopore sequencing

Abstract

The investigation of microbial communities through nucleotide sequencing has become an essential asset in environmental science, not only for research oriented activities but also for on-site monitoring; one technology, in particular, holds great promises for its application directly in the field: the Oxford Nanopore Technologies (ONT) MinION sequencer is a portable and affordable device, that produces long reads, with a remarkable sequencing output (in terms of bases/hour). One of the most common approaches in microbiological investigations through sequencing is the analysis of the 16S rRNA gene, known as 16S metabarcoding. Only recently the application of MinION has extended to 16S metabarcoding; to date, a limitation is still represented by the available computational protocols: due to the intrinsic, unique features of the technology ONT long reads cannot be adequately analyzed with tools developed for previous technologies (e.g. for Illumina). In this work a computational pipeline, specifically tailored to the usage of ONT reads in 16S metabarcoding, is developed, tested and discussed. This study is particularly addressed to on site evaluations, for environmental investigations or monitoring, where running time, costs and overall efficient usage of resources are particularly important.