Establishment and assessment of an amplicon sequencing method targeting the 16S-ITS-23S rRNA operon for analysis of the equine gut microbiome

Microbial communities are commonly studied by using amplicon sequencing of part of the 16S rRNA gene. Sequencing of the full-length 16S rRNA gene can provide higher taxonomic resolution and accuracy. To obtain even higher taxonomic resolution, with as few false-positives as possible, we assessed a method using long amplicon sequencing targeting the rRNA operon combined with a CCMetagen pipeline. Taxonomic assignment had > 90% accuracy at the species level in a mock sample and at the family level in equine fecal samples, generating similar taxonomic composition as shotgun sequencing. The rRNA operon amplicon sequencing of equine fecal samples underestimated compositional percentages of bacterial strains containing unlinked rRNA genes by a fourth to a third, but unlinked rRNA genes had a limited effect on the overall results. The rRNA operon amplicon sequencing with the A519F + U2428R primer set was able to detect some kind of archaeal genomes such as Methanobacteriales and Methanomicrobiales, whereas full-length 16S rRNA with 27F + 1492R could not. Therefore, we conclude that amplicon sequencing targeting the rRNA operon captures more detailed variations of equine microbiota.

Automated summary

A method using long amplicon sequencing targeting the rRNA operon provides higher taxonomic resolution for microbial communities, with over 90% accuracy at species level and performance similar to shotgun sequencing in equine fecal samples. However, it may underestimate the compositional percentages of certain bacterial strains, although the overall results are minimally affected.