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16S analysis using real-time, long-read 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 unnecessary 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
  • Analysis of full-length 16S rRNA gene for enhanced microbe classification
  • Sample barcoding for cost-effective results
  • Stop sequencing as soon as result obtained, wash, and reuse flow cell
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Oxford Nanopore now offers an end-to-end analysis solution for researchers interested in 16S rRNA sequencing.

Prepare
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16S barcoding kits available
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Identification from less than 1 ng bacterial DNA
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No need to purify extracted DNA
Sequence
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MinION and Flongle: portable, cost-effective, real-time DNA / RNA sequencing
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Sequence samples on location — no transportation required
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Multiplex several samples in one flow cell
Analyse
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16S analysis workflow available in EPI2ME (watch video)
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Real-time identification
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Identification in under 40 mins

Prepare

The 16S Barcoding Kits 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.

16s workflow

Note: Sample preparation components not included in the kit.

In addition, the 16S Barcoding Kit and 16S Barcoding Kit 1-24 provides 12 and 24 barcodes respectively, for multiplexing up to 12 or 24 samples, enabling even more cost-effective results.  

16s barcoding

Sequence

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. Furthermore, analysis of the full-length 16S rDNA gene using long-read nanopore sequencing has been shown to enhance the resolution of microbial characterisation

Further resources
16S Workflow: see tutorial video in the Nanopore Community (login required)
Metagenomics application page, containing white papers, videos, and peer-reviewed publications

This is in contrast to the What's In My Pot (WIMP) application, which aligns all reads from a mixed metagenomic 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.

Analyse

Oxford Nanopore also provides a complete 16S analysis workflow, enabling users 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 over 20,000 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.

Analysis
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