NEW YORK – Researchers from the Johns Hopkins University Applied Physics Laboratory (JHUAPL) and New England Biolabs (NEB) have developed an optimized multiplex PCR method to improve nanopore sequencing of the influenza A virus, which could make it a useful tool for surveillance and outbreak response for the growing public health threat of avian influenza A/H5N1 (bird flu).
Peter Thielen, a senior scientist and molecular biologist at JHUAPL, discussed the new sequencing workflow, as well as other efforts from his lab and its partners to improve bird flu surveillance and epidemiology, at an NEB-sponsored workshop at the Association for Molecular Pathology annual meeting in Vancouver, British Columbia, last week.
New molecular surveillance tools are needed to help contain bird flu, Thielen said, an ongoing outbreak of which has plagued cattle in the US in recent months, with cases crossing species into wild birds, big cats, and even humans.
For influenza, one example of a widely used tool that has needed updating is multi-segment reverse-transcription PCR (MS-RT-PCR), which uses so-called "tailed primers" with added sequences to improve sequencing performance and quality. This is a single-step RT-PCR method that has seen multiple slight variations to optimize reliability and scalability but in general has proven to be inefficient, Thielen said.
With support from NEB, Thielen helped develop a new version of MS-RT-PCR that uses an approach called integrated indexing, or iiMS-PCR. The freely available protocol features universal influenza A primer pairs with indexing tails that allow for the generation of indexed genome segments covering the full flu A genome. The protocol incorporates ONT barcodes during targeted cDNA synthesis and amplification, allowing for the pooling of up to 48 samples per sequencing run.
"By incorporating sequencing indices during reverse transcription, the approach retains the ideal qualities of the original whole-genome amplification method while significantly reducing the reagents, hands-on steps, and overall time required to sequence a flu virus," Thielen said in a follow-up email. "These improvements enable same-working day analysis, which in turn allows for more agile local and global responses when time is of the essence."
"The difference between one and two working days is huge," he said during the workshop. "We can now generate data in about seven hours [resulting in] consensus sequences to upload to repositories."
A key element of the protocol is incorporation of NEB's LunaScript One-Step RT-PCR reagent, which incorporates the Q5 proofreading polymerase while additionally improving representation of long genome segments, Thielen said in a follow-up message. Additional workflow improvements maximize compatibility between iiMS-PCR products and Oxford Nanopore reagent sets, eliminating unnecessary cleanup steps and maximizing ligation efficiency, to increase overall data output from a sequencing run, he added.
Using this reagent, which is currently available only through an NEB early-access program, the researchers have seen improved library generation and QA/QC consistency on ONT sequencers.
In an email, Andrew Barry, asociate director of NGS portfolio management at NEB, noted that the company expects the LunaScript One-Step RT-PCR reagents to be fully commercially available in the "near future."
Optimizing nanopore sequencing
Thielen noted that his group "uses many different sequencing platforms with the goal of expediting decision-making from the underlying genetic information."
However, ONT sequencing has proven its mettle in viral sequencing applications because of its long read lengths and its ability to provide real-time whole-genome characterization and because the platform is highly portable. "If we're going to stay prepared for whatever the next thing is … the challenge we have is distributing these [tools] to places with lower resources in a cost-effective manner," Thielen said.
In a recently published study, Thielen's team and collaborators at the Institut Pasteur Cambodia compared ONT's R10.4.1 flow cells to the previous R9.4.1 flow cells in sequencing human and avian influenza isolates, concluding that the newer sequencing chemistry had "superior accuracy and data quality" and showed "improved resolution, including in the critical hemagglutinin multi-basic cleavage site," representing "a significant advancement in the field of influenza virus genomic surveillance."
Also, in a preprint posted to MedRxiv this month, researchers from the Institut Pasteur Cambodia, University of Hong Kong, US Centers for Disease Control and Prevention, and elsewhere, including Thielen, used the iiMS-PCR workflow and ONT sequencing to elucidate the timeline, transmission dynamics, and evolutionary patterns of 16 human H5N1 bird flu cases in Cambodia and Vietnam. They found that 14 of these cases involved a novel reassortant flu A/H5N1 virus with gene segments from clade 2.3.2.1c and clade 2.3.4.4b viruses, concluding that "the emergence of this novel genotype underscores the persistent and ongoing threat of avian influenza in Southeast Asia."
"These are reemerging viruses that are highly pathogenic, causing mild disease in humans … often in populations where they don't or can't stay home from work," Thielen said during his presentation. "This is a very important thing that's happening in the background [and] a powder keg waiting to explode."