“There are several ways to overcome this, one of which we are working on at UCSC,” he says.īenedict Paten, also at UCSC, anticipates that this technology will be able to read out the sequence of bases and their individual methylation statuses, too, which could potentially help to discern haplotypes. The one systematic bias he has found relates to homopolymers longer than five bases, such as the sequence TTTTTT. In his work, accuracy with the MinION now reaches 92% for a single-molecule 2D read. “All other sequencing devices make copies, which masks modifications and doesn't work at all for proteins,” says Mark Akeson of the University of California at Santa Cruz (UCSC), who codeveloped the technology and is an ONT consultant. Nanopore sequencing directly analyzes a sample. For instance, it can help to quickly authenticate a cell line before the start of an experiment. The MinION has applications in the lab too, he says.
#Minion backpack software
Software such as poretools can help determine variants relative to a viral reference sequence generated early in an outbreak. During an outbreak a virus will also mutate as a result of selection pressure, which can cause diagnostic PCR primers to fail and lead to false negative results.
A virus might be transmitted from parent to child or across a village rapid sequencing and analysis can reveal chains of transmission. Rapid sequence analysis helps map the relatedness of Ebola cases, says Loman. There is currently a flurry of software development for analyzing nanopore sequence data ( Box 1). Together with Aaron Quinlan at the University of Virginia, he has developed software called poretools that converts MinION data into FASTA or FASTQ files for further analysis with traditional sequence-alignment or genome-assembly software. “It takes longer to make the libraries than it does to do the sequencing,” he says. Sequencing the Ebola virus with the MinION takes around half an hour, says Loman. Despite a drop in case numbers, a few stubborn chains of viral transmission still need sorting out. Quick transmitted nanopore sequence data to Loman in Birmingham, who analyzed them and returned results both to Quick and to the World Health Organization to guide treatment. In a different project, Joshua Quick, a researcher in Loman's lab, took three nanopore sequencers to Guinea to sequence the genomes of Ebola patients and to help with efforts to monitor the outbreak using PCR-based diagnostics. They later confirmed results with sequencing performed on the Illumina MiSeq instrument. In a few hours, Loman and colleagues used the MinION to identify the species of Salmonella responsible for an outbreak that had closed eight wards in a local hospital. “That's why we are excited about it,” he says.
#Minion backpack portable
The portable nanopore sequencer also opens up a slew of potential applications in the field, such as rapid surveillance of the flu, malaria, tuberculosis or Ebola, says Nicholas Loman of the University of Birmingham. Longer reads help researchers build complete or near-complete assemblies more quickly. These long reads are unlike the output of most high-throughput sequencers, which produce many shorter snippets that must then be computationally glued together. As Clive Brown, ONT's chief technology officer, said in a recent company presentation, a nanopore read can be as long as the DNA fragment researchers prepare. These sequencers can process DNA fragments that are 10,000–50,000 bases-and potentially 100,000 bases-in length. Plenty of challenges remain, including the need for higher accuracy, increased throughput and more data-analysis software, but the technology is rapidly evolving. Small sequencers could become ubiquitous sensors that test the environment for pathogens, for example. Nanopore sequencing conjures futuristic scenarios. The technology can also be used to analyze RNA and proteins. As the molecule zips through, it causes telltale fluctuations in the current that are specific to different DNA sequences. Voltage is applied across the membrane, creating an ionic current and an electrophoretic force that pulls the DNA through the opening. That pore might be a protein such as α-hemolysin that is embedded in a polymer membrane or a hole formed in a solid material such as silicon nitride (SiN) 7. The technology identifies a nucleic acid sequence by threading a molecule through a pore a few nanometers in diameter. Nanopore sequencing technology has been emerging in fits and starts 4, 5, 6. UCSC graduate student Miten Jain loads the MinION, a nanopore sequencer.
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