Comparison of a long-read amplicon sequencing approach to short-read amplicons for microbiome analysis

Most microbiome studies to date rely on sequencing short amplicons of the 16S rRNA gene on Illumina's platforms. Because of the short read length, sequences often can be identified reliably only to the family or genus levels. Long read sequencing with whole-length 16S rRNA sequencing can improve taxonomic resolution, but often only to the species level. StrainID is an alternative approach that amplifies a large segment of the ribosomal operon, including the entire 16S rRNA gene, internal transcribed spacer, and a portion of the 23S rRNA gene. This longer amplicon is designed to allow ribotype-level classification. Although studies have demonstrated the utility of StrainID for several sample types, it has not yet been validated for saliva. Here, we compared the performance of StrainID to short read amplicons with saliva samples as well as a synthetic mock DNA community and human and mouse fecal samples. Short reads were amplified with primer pairs appropriate for the corresponding sample type, and were classified with two different taxonomic databases. For both saliva and fecal samples, we found that StrainID performed similarly to short reads overall and demonstrated a key benefit with phylogenetic-based beta diversity tests and taxonomic classification. Our results further build on establishing StrainID as a valid method and specifically validate its use with saliva samples.