Metagenomic sequencing to diagnose infectious diseases

An article published in Genome Medicine reports the ability to detect viruses in clinical samples using a nanopore sequencer. The senior author of the work tells more in this blog.

How can a nanopore sequencer be used to diagnose diseases?
How can a nanopore sequencer be used to diagnose diseases?
Flickr

I first heard about the Oxford Nanopore MinIONTM sequencer two years ago. It was being billed as the ‘future of sequencing’. My immediate thoughts were that a pocket-sized, USB-powered, real-time sequencer would be ideal for field-ready applications.

In particular, this technology should be perfectly suited for infectious disease outbreaks such as Ebola, where rapid diagnosis is needed both for managing the patient and containing spread of the disease.

However, we didn’t actually get our hands on a MinIONTM sequencer until September of 2014. Even worse, the first couple of flow cells that we analyzed yielded no usable data at all, as the platform was still at a very early stage. It was not until January of this year that we had a successful sequencing run and were off to the races.

Metagenomic sequencing

Our approach to diagnosing infectious diseases is called metagenomic sequencing. The general idea is that we sequence all of the nucleic acid – DNA and RNA – in clinical samples and analyze the data using computational software to find the proverbial ‘needle in the haystack’.

We search for the generally <0.1% of sequence reads that may correspond to a virus that may be causing the illness, whether it be Ebola or influenza. This is in contrast to other groups and commercial laboratories, which typically sequence using primers or probes that specifically target individual pathogens.

Our article published in Genome Medicine presents a ‘proof-of-concept’ study on leveraging MinION nanopore sequencing to metagenomically detect viruses in clinical samples.

Last year, we described the use of metagenomic sequencing to successfully diagnose a rare but treatable bacterial infection (neuroleptospirosis) in a critically ill 14-year old boy who had been sick without a diagnosis for over 4 months. For diseases such as acute tropical febrile illness that have a broad differential diagnosis (malaria, typhoid fever, dengue, Ebola, etc.), unbiased metagenomic sequencing is a very attractive diagnostic testing approach.

Our publication in Genome Medicine presents a ‘proof-of-concept’ study on leveraging MinION nanopore sequencing to metagenomically detect viruses in clinical samples. To demonstrate its utility for real-time detection of emerging viruses, we chose to focus on analyzing clinical samples collected from outbreak settings.

What did we do?

Through a bevy of international collaborators, we obtained blood samples from acute hemorrhagic fever patients during the 2014 Ebola outbreak in the Democratic Republic of the Congo, and from blood donors during the 2013-2014 chikungunya outbreak in the Caribbean.

We developed a computational pipeline, usable on the laptop, for real-time sequencing analysis of nanopore data, and ran it to demonstrate detection of Ebola and chikungunya virus from infected individuals within ten minutes of starting DNA sequencing and in under five hours turnaround time from sample-to-answer.

Screenshot of a laptop analyzing MinION nanopore data in real time for viral pathogen identification from clinical samples. The elapsed time is shown in the top right corner.
Screenshot of a laptop analyzing MinION nanopore data in real time for viral pathogen identification from clinical samples. The elapsed time is shown in the top right corner.
Charles Chiu

We then analyzed a blood sample from a patient infected with hepatitis C to show that the limit of detection with existing MinION technology is approximately 105 copies of virus per milliliter. Thus, there is still a way to go for the sensitivity of metagenomic detection to be comparable with ‘gold-standard’ PCR assays for viruses (102-103 copies per milliliter).

Looking to the future

I am excited about the power and potential of nanopore sequencing. As an infectious diseases physician who regularly sees critically ill patients, I am frustrated by our inability to diagnose a substantial proportion of acute infections in the hospital in a timely fashion. This problem is only magnified in field settings, where resources, equipment, and trained personnel are severely limited.

I envision a future role for nanopore sequencing as a universal test for infectious diseases, especially in point-of-care settings.

In the future, if we can overcome existing challenges – improve sensitivity, speeden up sample preparation time (the current rate-limiting step in the entire process), and enable local analysis on the laptop that does not require an internet connection – I envision a future role for nanopore sequencing as a universal test for infectious diseases, especially in point-of-care settings.

We are in the process of clinically validating a sequencing test for infectious diseases in acutely ill hospitalized patients in California (Illumina MiSeq).

In parallel, it would be wonderful to harness MinIONTM nanopore technology for infectious disease diagnostics in the developing world – to aid in the diagnosis and treatment of infected patients and ultimately, to prevent the spread of epidemics such as the 2014 West Africa Ebola outbreak in the future.

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