Microbiota profiling with long amplicons using nanopore sequencing: full-length 16S rRNA gene and whole rrn operon
Background: Profiling the microbiome on low biomass samples is challenging for metagenomics since these samples are prone to present DNA from other sources, such as the host or the environment. The usual approach is sequencing specific hypervariable regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. Here, we aim to assess long-amplicon PCR-based approaches for assigning taxonomy at the genus and species level. We use Nanopore sequencing with two different markers: full-length 16S rRNA (~1,500 bp) and the whole rrn operon (16S rRNA gene - ITS - 23S rRNA gene; 4,500 bp).
Methods: We sequenced a clinical isolate of Staphylococcus pseudintermedius, two mock communities (HM-783D, Bei Resources; D6306, ZymoBIOMICS) and two pools of low-biomass samples (dog skin). Nanopore sequencing was performed on MinION (Oxford Nanopore Technologies) using the 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using the WIMP workflow on EPI2ME (ONT) or Minimap2 software with the rrn database.
Results: Full-length 16S rRNA and the rrn operon retrieved the microbiota composition from the bacterial isolate, the mock communities and the complex skin samples, even at the genus and species level. For the Staphylococcus pseudintermedius isolate, when using EPI2ME, the amplicons were assigned to the correct bacterial species in ~98% of the cases with rrn operon as the marker, and ~68% of the cases with 16S rRNA gene respectively. In both skin microbiota samples, we detected many species with an environmental origin. In chin, we found different Pseudomonas species in high abundance, whereas in the dorsal skin there were more taxa with lower abundance.
Conclusions: Both full-length 16S rRNA and the rrn operon retrieved the microbiota composition of simple and complex microbial communities, even from the low-biomass samples such as dog skin. For an increased resolution at the species level, rrn operon would be the best choice.