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Nanopore Sequencing Helps Assemble Herpes Virus Genome, ID Resistance Mutations


NEW YORK (GenomeWeb) – A group of researchers from the University of Oxford and Public Health England have combined nanopore sequencing with short-read next-generation sequencing to de novo sequence and assemble the herpes virus genome.

The PHE researchers said that they will continue to use the Oxford Nanopore's MinIon to sequence herpes virus genomes in order to better understand factors that influence virulence and drug resistance.

Tamyo Mbisa, acting head of the Antiviral Unit and clinical scientist within the Virus Reference Laboratory at PHE, told GenomeWeb that the researchers were interested in using the MinIon to help assemble the herpes virus genome because it's very difficult to do so using shorter-read sequencing technology. Compared to other viral genomes, the herpes virus genome is big at 152 kilobase pairs; is GC-rich, which can pose challenges for NGS technology; and has repetitive regions of 100 to 200 tandem repeats.

"It's like looking at a jigsaw puzzle that has all the same colors," Mbisa said. "You don't know where to place your piece."

With the longer nanopore reads the entire genome can be captured in one to two single reads, he said. However, short-read sequencing technology is still needed since the longer nanopore reads contain more errors.

In the study, published in PLOS One last month, the PHE team collaborated with researchers at the University of Oxford. The PHE group used Roche's 454 GS Junior sequencer while the Oxford team conducted the nanopore sequencing portion. However, Mbisa said that now the PHE lab uses Illumina's MiSeq or HiSeq instruments and has retired the 454 instrument.

The researchers analyzed 18 herpes virus-1 genomes collected from 13 immunocompromised patients undergoing antiviral therapy. For seven isolates from four patients, they used both sequencing technologies to characterize the genome.

With the 454 sequencing technology, the team achieved average read lengths of about 379 bases, while with the MinIon technology, average read lengths were 930 bases.

In addition, a hybrid assembly protocol using the MinIon and MIRA assembler improved de novo assembly of the genomes, reducing the number of contigs and increasing the N50 contig for the samples, the authors wrote.

Having complete genomes enabled the team to compare variability between isolates, including variability in isolates collected at different time periods from the same patient undergoing therapy. They found that four genes accumulated the vast majority of mutations in the genome. Looking specifically at mutations that could cause antiviral resistance, the researchers identified both known and novel mutations in the TK gene and/or DNA polymerase genes of samples that showed drug susceptibility.

In addition, the researchers found that the de novo assembly, as opposed to a reference-based assembly, enabled the discovery of structural variants that would otherwise have been missed.

Mbisa said that his lab was particularly interested in understanding antiviral resistance development. He noted that there are two genes, UL23 and UL30, that tend to accumulate the majority of antiviral resistance mutations, and indeed in this study the UL23 gene in particular showed the highest level of genetic variability, at 32.5 percent, consistent with its role in drug resistance.

Typically, clinicians will target those two genes for sequencing to identify drug resistance, Mbisa said, "but we don't know what is going on in other parts of the genome." The ability to de novo assemble the entire genome will help enable the identification of other drug resistance mutations, initially in a research setting, he said, but that could eventually be translated to a clinical setting.

He said the group is also interested in continuing to use the MinIon for herpes virus assembly to do comparative genomics studies and study strain variation to identify genetic elements that cause some strains to be more virulent than others. One hypothesis is that the number of repetitive elements plays a role in the strain's virulence or pathogenicity, but that can only be tested using technology with long-enough read lengths to sequence through those regions, he added.

"We want to tease out the mechanisms of how that is occurring," he said.

Mbisa said that his lab plans to continue using the MinIon for full-genome sequencing and assembly for certain cases. "Our lab concentrates on resistance, so we'll continue to study the two genes [UL23 and UL30], but the nanopore technology will allow us to pick out a few interesting samples and look at those in more depth, sequence the whole genome, and see if we can get more information."

In the near future, he said that Public Health England did not plan to use the MinIon in routine clinical settings for herpes virus.

"You have to balance out what is the main public health issue you're facing and how much it will cost," he said. For routine herpes virus testing, it does not yet make sense.

However, he noted that the portability of the instrument would eventually make it "more amenable to clinical use." For instance, he noted that groups have already used it in outbreak settings, for example during the Ebola outbreak in West Africa.

In addition, he said that the device is continuing to improve. He noted that his group tested the first generation of the MinIon, and that since then, its accuracy, read lengths, and throughput have all improved and he anticipated that future versions would improve further.