Direct sequencing of 16S rRNA
The majority of microbiome studies to date have focused on the analysis of DNA to elucidate microbial composition and genome structure; however, advances in sequencing technology and workflows, have allowed researchers to investigate RNA, providing insights into gene expression in complex bacterial communities1. The analysis of epigenetic modifications adds a further level of characterisation to microbial communities and their environmental responses; however, such modifications are erased using traditional sequencing techniques2.
In a proof-of-principle study, Dr. Andrew Smith and colleagues at the University of California, Santa Cruz, utilised nanopore sequencing to directly analyse full-length (1.5 kb) 16S ribosomal RNA (rRNA) of Escherichia coli to characterise both nucleotide and epigenetic composition in a single sequencing run2. 16S rRNA is a core constituent of the small ribosomal subunit which is expressed in all living cells and plays an essential role in RNA translation. Numerous antibiotics target prokaryotic ribosomes which can acquire resistance via nucleotide substitutions, or by gain or loss of base modifications, making their analysis increasingly important2.
The team developed an enrichment strategy that allowed the analysis of just 5 pg of E. coli 16S rRNA in a background of 4.5 μg total human RNA. Furthermore, the team reported that analysis could be performed in a potentially clinically actionable timeframe of just 2 hours from sample to result. In fact, the real-time analysis provided by nanopore technology enabled 16S rRNA reads to be generated within 20 seconds of starting the sequencing run.
Examination of the data allowed the identification of 7-methyl guanosine (m7G) at known positions in the 16S rRNA and indicated the presence of additional epigenetic modifications, including pseudouridine.
In summary, the team suggested that:
‘This [nanopore] direct RNA sequencing technology has promise for rapid identification of microbes in the environment and in patient samples’2.*
* Nanopore devices are currently for research use only.
This case study is taken from the microbiome white paper.
1. Bashiardes, S. Zilberman-Schapira, G. & Elinav, E. Use of metatranscriptomics in microbiome research. Bioinform Biol Insights 10 (2016).
2. Smith, A.M. et al. Reading canonical and modified nucleotides in 16S ribosomal RNA using nanopore direct RNA sequencing. BioRxiv 132274 (2017).