SAN FRANCISCO (GenomeWeb) – Scientists at the nonprofit research organization MRIGlobal have designed a nanopore sequencing workflow to test environmental samples for pathogens in the field, using only lab equipment that fits in a backpack.
The researchers said that such portable molecular testing technology could enable better outbreak surveillance and point-of-care diagnostics in remote, underserved locations.
"One of the biggest challenges in infectious disease diagnostics is turnaround time," said Jonathan Jacobs, a senior advisor at MRIGlobal. "If you can bring the lab to the sample and do testing onsite, especially for outbreak response, that would deliver care faster and accelerate the entire public health response."
In a publication posted on the BioRxiv preprint server, the MRIGlobal team described performing qPCR on mosquitoes collected from forests in Southern Florida using a handheld device to identify samples positive for a pathogen, and then performing metatranscriptomic sequencing using Oxford Nanopore Technologies' MinIon to identify the pathogen as the Everglades virus. The equipment they used could all fit inside a 40-liter backpack.
The group is continuing to refine its protocols and hopes to have a prototype "lab-in-a-backpack" designed by next year, Jacobs said.
Joseph Russell, a postdoctoral research associate at MRIGlobal, said that the organization has been working on developing portable technologies for outbreak surveillance or disease diagnostics for some time, but it really came to a head when the organization went to West Africa during the recent Ebola outbreak. MRIGlobal had put together a makeshift lab in a shipping container that could be transported on the back of a semi truck, but once it was delivered, it was a permanent structure.
The semi-mobile labs that MRIGlobal operated out of West Africa were not quite mobile enough, Jacobs said. The problem was that many of the sick were still hundreds of miles away and blood samples would be delivered by motorbike. The vials would sometimes arrive cracked and damaged, they may not have been kept cold, and often there were no records of the patients, he said.
After that experience, "we wanted to see how far we could push the mobile concept," Russell said, and the researchers began figuring out what would be needed to develop a molecular biology lab-in-a-backpack.
The researchers had been collaborating with the University of Florida's Florida Medical Entomology Lab and accompanied researchers on fieldwork in southern Florida not far from Everglades National Park. The University of Florida team was studying a family of viruses known as Venezuelan Equine Encephalitis Virus (VEEV), an RNA virus that is transmitted by mosquitoes and is pathogenic in both humans and certain animals, where it can cause fever and other nonspecific flu-like symptoms.
The team captured around 500 mosquitoes and pooled them into groups of 20. The researchers first used the handheld Biomeme two3 qPCR machine to test each pool of mosquitos for VEEV. One pool tested positive and the researchers then performed metatranscriptomic sequencing on the MinIon, identifying a strain of VEEV called Everglades virus, which was later confirmed with sequencing on the Illumina MiSeq.
For the metatranscriptome nanopore sequencing, the researchers tested both Sigma's whole transcriptome amplification kit as well as Qiagen's REPLI-g transcriptome amplification kit and performed local basecalling using the MinKnow software. In addition, they tested several metagenomics taxonomy callers, as well as read-mapping and alignment tools.
Both the Sigma and REPLI-g kits worked for identifying the Everglades virus in the sample, although the Sigma kit performed slightly better with more reads aligning to the genome and lower rates of chimeric reads.
Other researchers have also been looking to develop portable solutions for field-based outbreak surveillance and diagnostics. For instance, Joshua Quick, a graduate student in Nick Loman's lab at the University of Birmingham in the UK took a lab-in-a-suitcase to Guinea during the Ebola outbreak in 2015 to perform nanopore sequencing. In that case, the researchers were sequencing samples that had tested positive for Ebola in order to generate more Ebola genomes that could be used to better understand the outbreak. Their results were published last year in Nature.
Loman said that the main challenges now are in the sample prep stage, including nucleic acid extraction and target enrichment. "The sequencing itself is trivially portable now," he said in an email, and even the library prep is becoming easier due to freeze-dried reagents and rapid kits.
Loman said that his lab plans to continue to work on developing portable sequencing solutions under a £3 million ($3.94 million) grant from the Wellcome Trust.
In the meantime, Oxford Nanopore is developing its own handheld sample prep device, VolTrax, which some early access users are currently operating. Jacobs said that MRIGlobal has not yet used the device but anticipated that it would first work on sample types such as blood or saliva and likely would not be immediately applicable to the types of environmental samples they analyze, like mosquitos.
The MRIGlobal researchers are continuing to refine their methodology. One critical aspect of the nanopore workflow for field deployment is the wash procedure, the researchers wrote in the study. Being able to reuse the flow cells is important in order to minimize the amount of consumables that have to be carried in the field. However, the researchers noted that when they reused the flow cells in this experiment, there was some cross-contamination that was not observed when a new flow cell was used.
There are a number of other challenges the researchers are also looking to address, namely refrigeration and having an adequate power supply. Jacobs said that instead of cooling, they are looking to use freeze-dried reagents when possible, and pre-cooled thermal packaging that is used to ship medical material. Such Credo Cubes can stay cold for two to three days, he said. For power, Jacobs said the team has a custom lithium ion battery array, which will power the MinIon and data analysis as well as the handheld qPCR. Biomeme's two3 qPCR system can actually run up to five times on just one iPhone charge, Jacbos noted. For computation, the researchers used the Intel NUC Skull Canyon.
Russell added that other challenges pop up as the group tests the technology in the field. Often, the technology itself has a small footprint, "but the operational footprint is not small," he said. For instance, bioinformatics tools often require significant amounts of computing power or run in the cloud, which cannot often be done in remote locations with limited or no internet service.
Another issue, he said, is biosafety. To address this, Russell said, the MRIGlobal team has designed a popup glove box that compresses down but unfolds and pops open when it is unpacked. The box, which is reusable and disposable, can be used either to protect the researchers from a potentially dangerous sample or to keep the samples isolated, Russell said.
In addition, the researchers are looking to collaborate with researchers at L'Institut Pasteur in Dakar, Senegal, to do real-time, field-based biosurveillance of arboviruses in mosquitoes, although Jacobs said that details have not yet been worked out.