with nanopore sequencing
Nanopore sequencing provides:
- Simple, rapid end-to-end workflows
- Library prep options taking as little as <10 minutes
- Analytical workflows, including WIMP (anchor) for real-time species identification, and ARMA (anchor) for real-time antimicrobial resistance characterisation
- Additional analysis workflows available from Oxford Nanopore users
- Insight into whether antimicrobial resistance genes are in plasmids or integrated into the chromosome, using genome assembly
- Long reads help the resolution of difficult regions, such as repetitive insertion sequences usually flanking horizontally acquired genes
- Portable analyses – the MinION weighs 100g and is powered by USB
- Scalable analyses – GridION X5 and PromethION are suitable for centralised, on-demand, high-throughput work, or large-scale studies
- Low cost to access the technology - $1,000 for a MinION starter pack
- Access to a supportive community of users
Antimicrobial resistance: the problem
At the root of the issue is the overuse, or unnecessary use of antibiotics, both in medicine and agriculture. This unnecessary use speeds up the development of drug resistance.
“Without policies to stop the worrying spread of AMR, today's already large 700,000 deaths every year would become an extremely disturbing 10 million every year [by 2050], more people than currently die from cancer.”
- The O’Neill report on antimicrobial resistance
What can be done about the rise of antimicrobial resistance?
The O’Neill report notes that to reduce the overuse of antibiotics, a step-change is needed in technologies that can identify pathogens. This would be used to aid decisions on whether to give antibiotics, and if so, which ones to choose.
“I call on the governments of the richest countries to mandate now that by 2020, all antibiotic prescriptions will need to be informed by up-to-date surveillance information and a rapid diagnostic test wherever one exists.”
Rapid characterisation of pathogens and their resistance profiles will allow rapid, precise treatment to improve outcomes and antibiotic stewardship.
Researching antimicrobial resistance?
Oxford Nanopore offers a simple, user-friendly solution for every step of your experiment.
Ligation and transposase-based library preparation kits (compatible with barcoding) for low and high genomic DNA input are available. Ligation kits are compatible with cDNA libraries for viral pathogens.
Whole genome prep: suitable for investigations of de novo AMR-conferring mutations or genome-wide screens for known resistance-associated mutations; start with a metagenomic or single pathogen-enriched culture sample.
Targeted enrichment: suitable for specific AMR-associated regions of interest, allowing greater coverage and faster time from sequencing initiation to result; start with a metagenomic or single pathogen-enriched culture sample.
Other: VolTRAX can make the sample library preparation even simpler and field-use friendly.
Real-time sequencing changes your workflow:
- The prepared library is loaded onto the flow cell on the device
- Sequencing begins with signal streaming to the MinKNOW software, where basecalling takes place in real time
- Basecalled data is uploaded into the ARMA EPI2ME workflow for real-time data analysis
- Sequencing can continue until sufficient data has been generated or the user decides to stop it early
- If more data is required the flow cell can be washed using the wash kit and new sample(s) can be loaded for further sequencing
- An option to use the ‘Read Until’ analysis method allows specific molecules to be selected to sequenced until the full fragment is completed, while all other molecules are rejected as undesirable
Depending on the scale of the investigation, different Oxford Nanopore sequencing devices are available.
For a comparative overview of sequencing platforms, click here
ARMA – the end-to-end workflow for antibiotic resistance
The Antimicrobial Resistance Mapping Application (ARMA) is an EPI2ME workflow used in conjunction with another EPI2ME workflow - What’s in my pot? (WIMP). While WIMP performs real-time taxonomic classification of microorganisms (bacteria, fungi, viruses, archaea) present in a sample, ARMA allows detection of antibiotic resistance genes also in real time.
- The ARMA workflow draws from resistance gene sequences and antibiotic-resistance ontology (ARO). It can be integrated with databases such as in the Comprehensive Antibiotic Resistance Database (CARD).
- Following local basecalling, ARMA uses lastal to align reads against the genes from the database in real time.
- The report that is produced highlights alignments indicating potential resistance to a given antibiotic.
ARMA is compatible with both a whole genome approach and a targeted approach which may be required when the experiment is focussed on a specific genomic region or when enrichment for a low abundance pathogen is performed.
The movie below shows an end-to-end bioinformatics workflow where WIMP is used for taxonomic classification and ARMA - for antibiotic resistance profiling.
Custom designed analysis
Data can also be taken into custom pipelines for further analysis where needed.
The long reads of nanopore sequencing can be used with a growing number of analysis tools so that new reference genomes can be produced in cases when:
- The pathogen under investigation does not already have a reference genome
- The available reference genome has poor resolution of repetitive regions associated with resistance-conferring genes
- The resistance profile is not well characterised
- Additional coverage is required for variant calling
- Assessment of whether the resistance is plasmid-based or integrated into the chromosome is required
- Working with fungi or viruses where a limited number of pathogen-specific resistance databases are available