[ODP-017] Little effect of unlinked rRNA genes on the rRNA operon-based metagenomic analysis in equine samples
Purpose: Metagenome analysis using the ribosomal RNA (rrn) operon is expected to improve the accuracy of taxonomic assignments than those by 16S rRNA analysis, but it has been pointed out that this analysis could underestimate the ratio of bacteria that harbor unlinked rRNA genes. The purpose of this study is to evaluate the impact of unlinked rRNA genes on equine microbiome analysis.
Materials and Methods: We performed nanopore-based shotgun metagenome sequencing on the three equine fecal samples, and checked the presence of unlinked rRNA genes. Besides, taxonomic assignments were performed on sequence reads obtained by amplicon sequencing (full-length 16S rRNA gene or rrn operon region), and cluster analysis based on dissimilarity index was performed.
Results and Discussion: Unlinked rRNA genes were detected in all samples and the reads were mainly assigned to genus Cellulosilyticum, of which percentage ranged from 0.7 to 1.5%. These bacteria could be underestimated by the rrn operon-based analysis. However, not the 16S rRNA gene, but amplicon sequencing with the rrn operon formed a cluster with shotgun sequencing which does not have PCR bias. These results show that the effects of the unlinked rRNA genes could be very small in the metagenome analysis using the rrn operon region for equine samples and its accurate bacterial identification was thought to be more beneficial.
Materials and Methods: We performed nanopore-based shotgun metagenome sequencing on the three equine fecal samples, and checked the presence of unlinked rRNA genes. Besides, taxonomic assignments were performed on sequence reads obtained by amplicon sequencing (full-length 16S rRNA gene or rrn operon region), and cluster analysis based on dissimilarity index was performed.
Results and Discussion: Unlinked rRNA genes were detected in all samples and the reads were mainly assigned to genus Cellulosilyticum, of which percentage ranged from 0.7 to 1.5%. These bacteria could be underestimated by the rrn operon-based analysis. However, not the 16S rRNA gene, but amplicon sequencing with the rrn operon formed a cluster with shotgun sequencing which does not have PCR bias. These results show that the effects of the unlinked rRNA genes could be very small in the metagenome analysis using the rrn operon region for equine samples and its accurate bacterial identification was thought to be more beneficial.