Scientists must accurately characterize regions of DNA known as structural variants (SVs), which contribute to a variety of normal and aberrant phenotypes and are pivotal to research in several fields, such as agricultural science and clinical disease. But, until recently, scientists only had the option to employ short-read sequencing techniques when detecting SVs. Difficulties often arose from this process because of SVs' size, complexity, and position in a genome. As short reads can't span many SVs, scientists sequenced the variants in short sections and then reassembled these. This process often led to incomplete or incorrect assemblies.
However, next-generation sequencing techniques have advanced, and modern long-read sequencing makes it much simpler to characterize SVs today. One of the most commonly utilized long-read sequencing technologies is Oxford Nanopore's technology, which has seen scientists characterize SVs with unequaled resolution since 2005. Now, many scientists utilize long-read sequencing techniques and nanopore technology, which can offer many benefits over short-read sequencing alternatives.
Here, we'll explore the differences between short-read sequencing and long-read sequencing, highlight the benefits of Oxford Nanopore's long-read technologies, and delve into the ways that nanopore sequencing has enabled Covid-19 diagnostics.
Research professionals who would like to learn more about SV detection and nanopore technology will find a wealth of information in the life sciences journal BioTechniques.
Why Long-Read Sequencing Is Superseding Short-Read Sequencing
There are four main reasons why long-read sequencing is superseding short-read sequencing: Long-read sequencing doesn't come with batching requirements, doesn't limit read length, generates data in real time, and doesn't require PCR.
1. No Batching Requirements
Scientists often need to batch samples, and short-read sequencing techniques can limit flexibility, causing delays in results until scientists have collated enough samples. Meanwhile, nanopore technology enables scientists to run thousands of samples on one device without any sample batching requirements.
2. No Read Length Limit
Short-read sequencing techniques work to a set run time with bulk data delivery and generate reads of approximately 75-300 bases. As a result, wait times for results are often long, which isn't ideal when it comes to time-critical applications. Meanwhile, Oxford Nanopore's long-read technology doesn't limit read length and has a current record of over 4 Mb. This technology makes it easier for scientists to sequence fragments of any length, meaning they can sequence SVs in end-to-end single reads.
3. Real Time Data Generation
While short-read sequencing doesn't allow scientists to stream data in real time, scientists can employ nanopore technology to instantly access actionable data surrounding variant analysis, antimicrobial resistance, and pathogen identification. They can also stop sequencing when they have generated enough data. From here, they can wash and reuse the flow cell and use data analysis tools like EPI2ME.
4. No PCR Requirements
Scientists must complete sample preparation and amplification steps before they can perform short-read sequencing techniques. However, these steps increase the risk of sequencing bias. On the other hand, nanopore sequencing doesn't require amplification, therefore enabling simple, accurate characterization of all variants. This is true regardless of genomic context or how complex the variants are.
Scientists can complete sample preparation for nanopore sequencing in as few as 10 minutes. Then, they can assemble complete genomes from metagenomic samples, span and delineate challenging regions, resolve complete genomes and plasmids, and discriminate closely related species.
Long-Read Sequencing: Oxford Nanopore Sequencing
Oxford Nanopore's technology is one of the most-used long-read sequencing technologies. Oxford Nanopore designed its sequencing devices to use flow cells with nanopores, which are tiny holes in an electro-resistant membrane. The nanopores correspond with electrodes, which each connect to a channel and sensor chip. The chip measures the electric current that passes through the nanopores, disturbs the current, and produces a 'squiggle'. This squiggle is specific to the base that passes through the nanopore. Scientists decode squiggles using basecalling algorithms, which recognize DNA and RNA sequences in real time.
Because of short-read sequencing's complex setup requirements and high platform costs, scientists can only perform this type of sequencing in a laboratory. Meanwhile, scientists can perform nanopore sequencing in virtually any setting if they use a portable MiniOn device. These devices come with sequencing reagents.
Scientists can also scale up with modular GridION and PromethION sequencers, which enable ultra-high-throughput sequencing of pathogens and complex metagenomic samples. Scientists can sequence a whole human genome to high coverage using one PromethION flow cell, and GridION sequencers provide the flexibility scientists need to scale up or down to meet experimental goals.
Using Nanopore Sequencing to Control Pathogen Outbreaks Like Covid-19
Nanopore sequencing has become pivotal to scientists' understanding and controlling of pathogen outbreaks, including swine flu, Ebola, yellow fever, tuberculosis, and, more recently, Covid-19. In fact, nanopore sequencing made it possible for scientists to track, identify, and control Covid-19 in over 100 countries.
Scientists sequenced and shared SARS-CoV-2 genomic data to identify variants of the virus and monitor their prevalence and distribution, paving the way for the development of drug treatments and vaccines. Nanopore sequencing also made it possible for scientists to understand how Covid-19 strains relate, detect and eradicate routes of transmission, identify and investigate clusters, and come up with strategies to reduce the spread of the virus.
Midnight and ARTIC Classic Nanopore Sequencing
Scientists used two methods to sequence SARS-CoV-2: Midnight and ARTIC Classic. These techniques use a PCR approach that amplifies the viral genome in overlapping sections. This approach maximizes coverage across the genome.
Midnight nanopore sequencing is a mostly automated process that scientists used to amplify the SARS-CoV-2 genome in approximately 1,200 base pair overlapping segments. The method is resilient to drop-outs that mutations in the viral genome cause. The quick, flexible process made it possible to sequence small numbers of samples on demand and scale up to meet high-throughput sequencing requirements. Midnight sequencing doesn't require a normalization step, uses the rapid library preparation method, and is more cost-effective than ARTIC Classic sequencing.
On the other hand, scientists used ARTIC Classic nanopore sequencing to amplify the SARS-CoV-2 genome in approximately 400 base pair fragments. The shorter length can improve coverage for RNA samples that can degrade, often because of freeze-thaw cycles or storage temperatures that are above -80C. ARTIC Classic sequencing requires a normalization step and uses the ligation library preparation method. While this kind of sequencing uses a third-party reagent and requires more experience than Midnight sequencing, it does offer faster turnaround times.
Chronicling changing lab techniques
Scientists and research professionals from all over the world read BioTechniques (one of Future Science Group's 34 peer-reviewed, open-access journals) and access its multimedia website to stay on top of developments in laboratory methods and technologies. Many of these users are interested in lab techniques that are continuously evolving, such as next-generation sequencing, western blotting, CRISPR gene editing, chromatography, and polymerase chain reaction, and many specialize in fields like physics, chemistry, the life sciences, computer science, and plant and agricultural science.