Using MinION ™ to characterize dog skin microbiota through full-length 16S rRNA gene sequencing approach

Abstract The most common strategy to assess microbiota is sequencing specific hypervariable regions of 16S rRNA gene using 2 nd generation platforms (such as MiSeq or Ion Torrent PGM). Despite obtaining high-quality reads, many sequences fail to be classified at the genus or species levels due to their short length. This pitfall can be overcome sequencing the full-length 16S rRNA gene (1,500bp) by 3 rd generation sequencers. We aimed to assess the performance of nanopore sequencing using MinION ™ on characterizing microbiota complex samples. First set-up step was performed using a staggered mock community (HM-783D). Then, we sequenced a pool of several dog skin microbiota samples previously sequenced by Ion Torrent PGM. Sequences obtained for full-length 16S rRNA with degenerated primers retrieved increased richness estimates at high taxonomic level (Bacteria and Archaea) that were missed with short-reads. Besides, we were able to obtain taxonomic assignments down to species level, although it was not always feasible due to: i) incomplete database; ii) primer set chosen; iii) low taxonomic resolution of 16S rRNA gene within some genera; and/or iv) sequencing errors. Nanopore sequencing of the full-length 16S rRNA gene using MinION ™ with 1D sequencing kit allowed us inferring microbiota composition of a complex microbial community to lower taxonomic levels than short-reads from 2 nd generation sequencers.