Challenging current gold-standard infectious disease diagnostic methods with full-length 16S sequencing

Dr Anna Smielewska (Clinical Director of Infection and Immunity, Liverpool Clinical Laboratories, UK) and Prof. Alistair Darby (PI and Co-Director of the Centre for Genomic Research, University of Liverpool, UK) have carried out a successful pilot programme that enabled the rapid characterisation of bacterial pathogens from research samples. Effective pathogen identification is essential in improving patient outcomes and reducing antibiotic usage, and as a result, requires accessible and fast identification workflows to achieve these aims.

Antimicrobial resistance (AMR) is on the rise in healthcare settings, with bacterial AMR estimated to be associated with 4.95 million deaths in 2019¹. To combat resistance and save lives, rapid identification of microbes is critical to implement effective treatment options as fast as possible. Current gold-standard methods include time-consuming culture-based approaches, which can only identify organisms that are easy to culture or in high abundance, or use targeted PCR, which can only detect the specific organisms the assay is designed to target.

To tackle bacterial AMR, Anna and Alistair developed a nanopore workflow that sequences the entire 16S ribosomal RNA (rRNA) gene to accurately identify pathogens in research samples with a rapid turnaround time. Supplementary investigation tools using 16S sequencing with Sanger are widely accepted in clinical microbiology laboratories; however, the approach using nanopore sequencing analyses the entire 16S gene for genus-level bacterial identification from polymicrobial samples within 72 hours.

In this interview with Anna and Alistair, we discuss how they used nanopore technology in their pilot study, as discussed in their recent publication², to develop a rapid 16S sequencing workflow with the hopes to improve antibiotic stewardship and ultimately improve patient outcomes. They are currently working to validate this nanopore workflow for different sample types, including microbial keratitis and contaminated water², to use alongside routine diagnostic tests*.

What led you to research 16S sequencing as a supplementary investigation tool in microbiology labs?

Anna explained that 16S is not a new tool and has been previously used for microbial detection. Before the COVID-19 pandemic, her lab had used Sanger sequencing to investigate the 16S gene for identification purposes. However, ‘we were interested in introducing more sequencing’, and 16S assays have previously been used clinically, so ‘we knew there would be a demand for it, and it would be an easy assay to introduce’.

16S sequencing is a technique that Alistair has used for over 20 years and ‘it seems quite natural to me [to use], because we’re trying to look at samples where we’ve got fastidious organisms, and we don’t want to rely on culture’. In ecology and microbiome research, 16S sequencing has clearly demonstrated its power. However, Alistair noted that with nanopore sequencing there is more data generated from everything within a sample, including contaminants and variation in sequences, compared with gold-standard methods. By being able to analyse individual reads, they can ‘see the diversity that’s in the sample’, and they do not have to ‘make the assumption that the [microorganism] that is easiest to clone out … or the most abundant’ in a sample is the culprit. ‘This allows us to look at lots of different levels and to understand the sample in a lot more comprehensive ways than we would have done previously just working with a standard Sanger sequence’.

Why did you decide to use nanopore technology in this research?

Alistair turned to Oxford Nanopore Technologies because ‘nanopore provides something that is scalable and affordable, [so] there’s a low investment involved with actually bringing it into a lab’. He tested a nanopore workflow for full-length 16S sequencing, focusing on genus-level identification and ‘didn’t [encounter] any problem getting … the level of [identification] that we wanted from this data’. In fact, he found that ‘even if there were reads that were of lower quality, that wasn’t going to have an impact on our ability to assign taxonomy’.

Anna needed a tool ‘that was worthwhile to set up as a standalone test … something that was fast, that we could train people [to conduct], that was cost-effective and potentially scalable’. Anna found that the 16S nanopore workflow could be performed with a ‘comparatively small number of samples and relative ease, [and] we also liked the small footprint … so we could accommodate it in the lab’. Furthermore, Anna found that Sanger sequencing needed between a week and ten days to identify a fastidious organism. The faster turnaround time and matched sensitivity to targeted PCR assays cemented Anna’s decision to use nanopore technology.

How did you hear about Oxford Nanopore?

Anna’s background is in virology, and she briefly used a nanopore device at the beginning of the company’s journey. During the COVID-19 pandemic, she watched the technology improve ‘and being more and more used’. This 16S workflow project gave her the opportunity to use nanopore sequencing fully for the first time.

For Alistair, this was not his first experience with Oxford Nanopore Technologies: ‘we’ve used it a lot, both during COVID and before for viral, bacterial, and eukaryotic genomes’. During the pandemic, nanopore sequencing gave him an ‘extra level of confidence that sequencing amplicons on Oxford Nanopore was really reliable and robust’. This experience made him realise ‘that it could be relied upon in a [potentially] diagnostic situation’.

Do you have any highlights or successes that stand out in your research?

For Anna, the main success of this project has been using the rapid nanopore 16S workflow for a year where she and her team have ‘processed over 200 samples, which is quite a lot for something that is not automated’. She further explained that ‘this is very much a national first, and we’ve got very fast turnaround times; we process virgin samples within 24 hours’.

In microbiological labs, there is an arsenal of approaches to identify microbial infections and for Anna, full-length nanopore 16S sequencing ‘has found a niche within it for more complex samples [and] more complex cases’. One of her ‘favourite cases was when we [identified] a very, very fastidious, very difficult organism within 24 hours. This was a culture that would have taken two weeks and histology caught up with us in 72 hours’, demonstrating the power of the rapid nanopore workflow.

A highlight for Anna during this study was the opportunity to work with seven different hospitals across Cheshire and Merseyside, with requests to sequence research samples ‘from critical care units, paediatric hospitals, and cancer hospitals’. During this collaboration, she reviewed the potential impact this assay may have on the patient journey, with the hope that results will inform antibiotic treatment decisions, reduce patient stays, and stop unnecessary antimicrobial treatments — ultimately improving antibiotic stewardship.

The ability to use full-length nanopore 16S sequencing to look at complex samples and identify highly fastidious organisms is also one of Alistair’s highlights. He emphasised that these were not ‘200 standard, easy samples that’s being run through in the previous year’. These are samples that have undergone standard routine testing without a conclusive result — ‘these are the tricky ones’.

Alistair has been using 16S sequencing for over 20 years to solve biological problems and says, ‘there’s a tendency for the research community to dismiss 16S’. This project highlights that ‘in the right circumstances, with the right use, [it can give] very, very, clear answers and answers that can be easily translated’. He’s hoping that more researchers will see its benefits for analysing difficult samples and that more of them should consider this tool alongside shotgun metagenomics.

Do you have plans to use nanopore sequencing in your next project?

Anna is working on sequencing fungal samples and potentially viral samples as well, with the hopes to offer sequencing assays for these microorganisms. Currently, Anna sequences many research samples from a paediatric hospital because they need fast turnaround times as very sick children often ‘come in with very odd organisms compared [with] adults’. Alongside this, her main interest is evaluating research samples from immunosuppressed patients, ‘which have a very interesting cocktail of pathogens and a lot of the time, they are very hard to diagnose by conventional methods’, making nanopore sequencing an ideal option for these investigations.

Alongside Anna’s plans, Alistair is ‘keen to emphasise to people that really, they shouldn’t be doing any Sanger sequencing anymore in this sort of situation’, because longer reads from nanopore sequencing ‘gives you that key … to more information’.

What are you most excited for in your future research?

Alistair wants to investigate sequencing whole microbes and ‘using that as a universal test for pathogens and health diagnostics’. He hopes that it will happen in the next 5–10 years and will become routine because a universal test should be able to handle a large variety of samples. He notes that this is ‘a big piece of work, but I think it’s an exciting, exciting thing to explore and think about’, with Anna chipping in that ‘this is the ultimate goal’.

Anna explained that for research tools to be used as routine diagnostic tests, a lot of automation and minimal hands-on time is required. She thinks the full-length nanopore 16S sequencing workflow is a ‘very neat test, and it’s easy to explain’. She is also very interested to see what nanopore technology can do with ‘all the additional data that it brings, and the flexibility that it brings’; especially regarding the ‘discovery of new organisms’.

Since this interview took place, the Infection and Immunity Research, Development and Innovation team at Liverpool Clinical Laboratories in collaboration with the Centre of Genomic Research, University of Liverpool, have been awarded the RCPath Achievement Award 2024. This award celebrates excellence in pathology practice, as well as education, training, and research — a huge achievement for the team, highlighting the importance of this vital work. Additionally, they have also released their preprint publication², discussing the urgent need for enhancing antimicrobial stewardship.

To learn more about full-length nanopore 16S sequencing, see the targeted sequencing application page.

*Oxford Nanopore Technologies products are for research use only and are not approved to diagnose any disease or condition.

Here is the Infection and Immunity Research, Development and Innovation team behind the 16S nanopore sequencing project, with Dr Anna Smielewska in the centre front and Prof. Alistair Darby on the far left.