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

Abstract

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.

Microbial Identification Using rRNA Operon Region: Database and Tool for Metataxonomics with Long-Read Sequence

“…We provided MIrROR in a form that allows direct analysis by the most widely used universal primer sets. Therefore, the current release does not contain sequences amplified by the recently reported universal primer sets covering bacteria and archaea ( 38 , 39 ). We plan to provide a ready-to-analyze database for newly reported regions, including archaea.…”

“…Reported 16S-ITS-23S rRNA operon databases include rrn_DB ( 17 ), rrn_DBv2 (updated version of rrn_DB) ( 27 ), 16S-23S-rRNA encoding region database (16_mar) ( 28 ), ncbi_202006 database (DB) ( 39 ), and Athena database ( 55 ). Among those, publicly available databases rrn_DBv2 and 16_mar were used to evaluate the performance of MIrROR.…”

Short‐ and long‐read metabarcoding of the eukaryotic rRNA operon: Evaluation of primers and comparison to shotgun metagenomics sequencing

“…Furthermore, there is currently no database or collection of full eukaryotic rRNA operon sequences available, like it is attempted for bacteria (Benítez-Páez & Sanz, 2017;Kinoshita et al, 2021); such an initiative would greatly benefit the ease of application.…”

Microbial Community Analysis and Food Safety Practice Survey-Based Hazard Identification and Risk Assessment for Controlled Environment Hydroponic/Aquaponic Farming Systems

“…To improve the resolution and confidence of taxonomic classification, a novel amplicon sequencing approach was proposed, in which the full-length 16S rRNA, internal transcribed spacer (ITS), and partial 23S rRNA (16S-ITS-23S rRNA) was sequenced, resulting in a long read of ∼2,500 bp ( Martijn et al, 2019 ; Graf et al, 2021 ; Kinoshita et al, 2021 ). This sequencing method can be performed on PacBio systems at a cost similar to the full-length 16S rRNA sequencing.…”