An introduction to the use of nanopore technology in next-generation sequencing. The term ‘structural variants’ (SVs) refers to a range of genetic alterations that are significant in many scientific fields, from clinical disease research to agricultural science.
Researchers must accurately characterize SVs as they contribute to many normal and aberrant phenotypes. For years, this proved difficult with short-read sequencing techniques, which restrict the detection of SVs. But, with the advent of Oxford Nanopore’s technology in 2005, researchers can now characterize SVs with unprecedented resolution.
Today, many researchers use nanopore technology in long-read sequencing techniques, a class of next-generation sequencing that is under active development and offers many benefits over short-read sequencing.
As next-generation sequencing techniques and applications evolve, many scientists keep up with the latest updates and practices by following the international journal of life science methods BioTechniques.
This journal curates numerous resources that spotlight developments in next-generation sequencing, nanopore technology, and its applications — particularly applications in COVID-19 research.
Next-Generation Sequencing: Nanopore Technology & Structural Variation
SVs are difficult to detect using traditional technologies because of their complexity, size, and position in a genome. As short reads cannot span many SVs, researchers used to sequence the variants in short sections and then reassemble these.
This sequencing often resulted in incorrect or incomplete assemblies. However, researchers have found a solution for these inaccurate assemblies: nanopore technology. With this technology, there is no limit to read length (the current record is over 4 Mb), making it easier to sequence both short and long fragments.
Nanopore technology makes it possible for researchers to sequence SVs end to end in single reads. There is no need for PCR in this long-read sequencing technique, which enables simple, accurate characterization of even the most complex variants in all genomic contexts.
As a result, researchers can resolve complete genomes and plasmids, span and delineate challenging regions, assemble complete genomes from metagenomic samples, and discriminate closely related species.
Differences Between Short-Read Sequencing & Long-Read Sequencing
Traditional short-read sequencing generates reads of approximately 75-300 bases and operates to a fixed run time with bulk data delivery. This can mean long wait times for results, so short-read sequencing isn’t usually ideal for time-critical applications. Meanwhile, long-read sequencing enables real-time data streaming.
For example, with nanopore technology, researchers can instantly access actionable data, including pathogen identification, antimicrobial resistance, and variant analysis. Researchers can stop sequencing when they have generated enough data, wash and reuse the flow cell, and use comprehensive data analysis tools like EPI2ME.
Short-read sequencing is also limited in terms of flexibility. Researchers may need to batch samples, which can cause delays in results until they have acquired sufficient samples. Meanwhile, nanopore technology allows researchers to run thousands of samples on a single device without any sample batching requirements.
Furthermore, short-read sequencing requires extensive sample preparation and amplification, which increases the risk of sequencing bias. Conversely, researchers can complete sample preparation for nanopore sequencing in as few as 10 minutes, and the process doesn’t require amplification.
How Nanopore Sequencing Works
Oxford Nanopore’s sequencing devices use flow cells that contain nanopores (tiny holes embedded in an electro-resistant membrane). Each nanopore corresponds with an electrode that connects to a channel and sensor chip. The chip measures the electric current that flows through the nanopore. When DNA passes through a nanopore, it disrupts the current and produces a ‘squiggle’.
This squiggle is specific to the base that has passed through the nanopore. Researchers can decode squiggles using basecalling algorithms that determine DNA and RNA sequences in real time. While short-read sequencing is only possible in a laboratory because of its complex setup requirements and high platform costs, researchers can perform nanopore sequencing in most settings with portable MinIOn devices, which come with sequencing reagents.
Researchers can scale up with modular GridION and PromethION sequencers, which enable ultra-high-throughput sequencing of pathogens and complex metagenomic samples. For example, researchers can sequence a full human genome to high coverage on a single PromethION flow cell. Meanwhile, the GridION sequencer offers the flexibility to scale up or down in line with experimental goals.
Nanopore Sequencing & COVID-19
Since 2015, scientists have adopted nanopore sequencing to understand and control pathogen outbreaks like Zika, Ebola, yellow fever, swine flu, and tuberculosis. More recently, scientific communities have adopted nanopore technology during the COVID-19 pandemic to track, identify, and control the virus. The technology’s easy access, simple workflow, and real-time nature have made it possible for researchers in over 100 countries to sequence and analyze the SARS-CoV-2 viral genome rapidly.
Sequencing and sharing SARS-CoV-2 genomic data have helped researchers detect variants and monitor their prevalence and distribution, informing the development of vaccines and drug treatments.
Nanopore sequencing has also helped researchers determine how strains of COVID-19 relate, allowing them to identify or rule out routes of transmission, detect and investigate clusters, and develop strategies to reduce the spread of the virus.
Researchers are using two methods to perform whole-genome nanopore sequencing of SARS-CoV-2: Midnight and ARTIC Classic. These techniques adopt a PCR tiling approach that amplifies the viral genome in overlapping sections. This maximizes the coverage across the full genome.
Midnight & ARTIC Classic Nanopore Sequencing
Midnight nanopore sequencing is a rapid, flexible method that allows the on-demand sequencing of small numbers of samples and scales up to high-throughput sequencing requirements. There is little hands-on practice: Instead, the automated process involves amplifying the SARS-CoV-2 genome in ~1,200 bp overlapping segments.
This process is resilient to drop-out caused by mutations in the viral genome. Meanwhile, ARTIC Classic nanopore sequencing involves the amplification of the SARS-CoV-2 genome in ~400 bp fragments. The shorter length can improve coverage for RNA samples that are likely to be degraded, often because of freeze-thaw cycles or storage at temperatures above -80°C.
While Midnight sequencing doesn’t involve a normalization step and uses the rapid library preparation method, ARTIC Classic sequencing does involve a normalization step and uses the ligation library preparation method.
ARTIC Classic sequencing also requires more experience than Midnight sequencing, uses a third-party reagent, and offers quicker turnaround times. However, Midnight sequencing is more cost-effective. Learn more about how nanopore sequencing enables COVID-19 research.
About BioTechniques: When BioTechniques published its first issue in 1983, the peer-reviewed, open-access journal became the first publication to review lab methodologies instead of treatments. To this day, BioTechniques spotlights the latest techniques and technologies in scientific and medical applications, including next-generation sequencing, western blotting, CRISPR gene editing, polymerase chain reaction (PCR), and chromatography. Print journal aside, BioTechniques also publishes a wide range of resources on its multimedia website, where scientists and lab workers of all levels develop their knowledge by accessing industry articles, interviews, eBooks, podcasts, webinars, and videos.
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