16S analysis using real-time nanopore sequencing
The 16S rRNA gene is present in all bacteria and archaea. The gene is ideal for sequence-based identification of these organisms, particularly in mixed samples, due to the presence of conserved and highly variable regions. By narrowing down to a specific region of interest, all the organisms present in the sample can be seen without sequencing unnecesary regions of the genome, making the analysis quicker and more economical.
16S analysis with nanopore technology offers:
- Rapid and easy workflow leading to genus-level bacterial identification
- Portability for in-field analyses minimising sample transportation
- Ability to stop the sequencing as soon as the report indicates sufficient confidence in the result
Oxford Nanopore now offers an end-to-end analysis solution for researchers interested in 16S.
The new 16S Rapid Sequencing Kit, SQK-RAS201, and EPI2ME 16S analysis workflow allows users to perform genus-level identification from single reads; with access to basecalled files for detailed investigations at the species and sub-species level.
Sample preparation components not included in the kit.
In addition, the 16S Rapid Amplicon Barcoding Kit provides 12 barcodes for multiplexing up to 12 samples. A rapid top-up kit contains extra Running Buffer and Rapid 1D Adapter, allowing using up of leftover barcodes in a cost-effective way.
The MinION has been used by many researchers for 16S analysis. By narrowing down to a specific region of interest, all the bacterial genera in the sample can be seen without sequencing unnecessary regions of the genome and avoiding problems of host contamination, making the test quicker and more economical.
|16S Workflow: see post in the Nanopore Community (login required)|
|Oxford Nanopore poster, December 2016. “Barcode of life: simple laboratory and analysis workflows for 16S and CO1 analysis”|
|Oxford Nanopore poster, December 2016: “Using long nanopore reads to assemble genomes from complex metagenomic samples”|
|Oxford Nanopore poster, December 2016: "DNA extraction and library preparation for rapid genus- and species-level identification, with or without PCR"|
|Publications focusing on pathogen or microbial analysis|
|Publications focusing on metagenomics analysis|
*This is in contrast to the What's In My Pot (WIMP) application, which uses Centrifuge to align all reads from a mixed sample to an NCBI database of bacteria, viruses, fungi and archaea. Between the WIMP and 16S workflows, nearly all scenarios for taxonomic assignment of small-genome species are covered.
Oxford Nanopore also provides a 16S analysis workflow. This enables users of its sequencing technologies to classify mixed samples and obtain accurate results for single reads at the genus level.
The workflow is designed to BLAST basecalled sequence against the NCBI 16S bacterial database, which contains 18,927 16S sequences from different organisms. Each read is classified based on % coverage and identity.
The 16S workflow will be useful to any researcher interested in identifying pathogens in a mixed sample or understanding the composition of a microbial community; both already thriving subjects of research within the nanopore community.