Next Generation Sequencing: The Difference Between Short- and Long-Read Sequencing

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Researchers who apply next-generation sequencing (NGS) techniques must accurately characterize structural variants (SVs). These are regions of DNA that are key to various scientific fields, from clinical disease research to agricultural science. SVs contribute to numerous normal and aberrant phenotypes, and they can be difficult to detect using traditional short-read technologies because of their size, complexity, and position in a genome.

Before the development of long-read sequencing technologies, researchers had to sequence the variants in short sections and then reassemble them, as short reads can’t span many SVs. This kind of sequencing often led to incorrect or incomplete assemblies. However, modern long-read sequencing techniques make characterizing SVs much easier. In particular, the advent of Oxford Nanopore’s technology in 2005 saw researchers characterize SVs with unparalleled resolution. Now, many researchers and scientists utilize nanopore technology and long-read sequencing techniques, which offer several benefits over short-read sequencing alternatives.

As next-generation sequencing techniques and technologies progress, many research professionals use the peer-reviewed, open-access journal BioTechniques to keep up with developments in the field. BioTechniques publishes a high volume of information on nanopore technology and SV detection.

Here, we’ll examine the differences between short- and long-read sequencing, focusing on the benefits of Oxford Nanopore’s long-read technologies and the impact nanopore sequencing has had on Covid-19 diagnostics.

Table of Contents

Differences Between Short and Long-Read Sequencing

  • Read Length

Traditional short-read sequencing techniques generate reads of approximately 75-300 bases and work to a set run time with bulk data delivery. Therefore, wait times for results can be lengthy, and short-read sequencing isn’t ideal for time-critical applications. However, Oxford Nanopore’s technology doesn’t limit read length — the current record is over 4 Mb — which makes it easier for researchers to sequence fragments regardless of their length. Researchers can sequence SVs end to end in single reads.

  • Sample Batching

Short-read sequencing techniques offer limited flexibility. Sometimes, researchers need to batch samples, which can lead to delays in results until they have collected sufficient samples. On the other hand, nanopore technology makes it possible to run thousands of samples on a single device without any sample batching requirements.

  • Data Access

Unlike short-read sequencing, long-read sequencing allows researchers to stream data in real time. For example, nanopore technology enables researchers to access actionable data surrounding antimicrobial resistance, variant analysis, and pathogen identification instantly. They can stop sequencing when they have produced enough data. Then, they can wash and reuse the flow cell and use data analysis tools like EPI2ME.

  • Sample Preparation and Amplification

Researchers need to complete thorough sample preparation and amplification before performing short-read sequencing techniques. But this preparation and amplification increases the risk of sequencing bias. On the contrary, there is no need for PCR in long-read sequencing, which enables simple, accurate characterization of all variants, no matter how complex, in all genomic contexts. Researchers can perform sample preparation for nanopore sequencing in as few as 10 minutes. They can then assemble complete genomes from metagenomic samples, span and delineate challenging regions, resolve complete genomes and plasmids, and discriminate closely related species.

Oxford Nanopore Sequencing

Oxford Nanopore’s technology is one of the most commonly used long-read sequencing technologies. Oxford Nanopore sequencing devices use flow cells with nanopores (minute holes in an electro-resistant membrane) that correspond with electrodes, each of which connects to a channel and sensor chip. The chip measures the electric current that passes through the nanopores, disrupts the current, and produces a “squiggle”. The squiggle is specific to the base that has passed through the nanopore. Researchers decode squiggles using basecalling algorithms that identify RNA and DNA sequences in real-time.

Researchers can only perform short-read sequencing in a laboratory because of its high platform costs and complicated setup requirements. However, researchers can carry out nanopore sequencing in a variety of settings by using a portable MiniOn device, which comes complete with sequencing reagents. Where necessary, researchers can scale up with modular GridION and PromethION sequencers. These sequencers make ultra-high-throughput sequencing of pathogen and complex metagenomic samples possible. For example, researchers can sequence a whole human genome to high coverage on one PromethION flow cell, and the GridION sequencer provides the flexibility to scale up or down to meet experimental goals.

Understanding and Controlling Pathogen Outbreaks Like Covid-19

Since 2015, nanopore sequencing has proven essential to understanding and controlling pathogen outbreaks like Ebola, Zika, swine flu, tuberculosis, and yellow fever. Even more recently, scientists used nanopore sequencing during the Covid-19 crisis to track, identify, and control the virus in more than 100 countries.

By sequencing and sharing SARS-CoV-2 genomic data, researchers have been able to detect variants of Covid-19 and monitor their prevalence and distribution, which has been key to the development of vaccines and drug treatments. Nanopore sequencing has also enabled researchers to understand how strains of Covid-19 relate, identify and eliminate routes of transmission, locate and investigate clusters, and conceptualize strategies to minimize the spread of the virus.

Midnight and ARTIC Classic Nanopore Sequencing

Researchers choose between two methods — ARTIC Classic and Midnight — to perform whole genome nanopore sequencing of SARS-CoV-2. Both techniques use a PCR tiling approach that amplifies the viral genome in overlapping sections to maximize coverage across the whole genome.

During ARTIC Classic nanopore sequencing, researchers amplify the SARS-CoV-2 genome in ~400 base pair fragments. The shorter length can improve coverage for RNA samples that may be degraded, usually because of storage at temperatures above -80°C or freeze-thaw cycles. ARTIC Classic sequencing uses the ligation library preparation method and requires a normalization step. Although this type of sequencing requires more experience than Midnight sequencing and uses a third-party reagent, it does offer quicker turnaround times.

Midnight nanopore sequencing is a mostly automated process that amplifies the SARS-CoV-2 genome in ~1,200 base pair overlapping segments. This process is resilient to drop-outs caused by mutations in the viral genome. This quick, flexible method enables the on-demand sequencing of small numbers of samples and scales up to high-throughput sequencing requirements. Midnight sequencing uses the rapid library preparation method and doesn’t require a normalization step. It’s also more cost-effective than ARTIC Classic sequencing.

Discovering New Lab Techniques Through Research

BioTechniques is one of Future Science Group’s 34 industry-leading journals, which research professionals from all corners of the globe use to keep up with laboratory methodologies and technologies. These research professionals share interests in future-facing laboratory techniques like NGS, western blotting, polymerase chain reaction, CRISPR gene editing, and chromatography. Many of the journal’s readers specialize in fields such as the life sciences, computer science, plant and agricultural science, chemistry, and physics. Aside from reading the journal, they gain additional insights from BioTechniques’ multimedia website, which holds a trove of eBooks, articles, videos, podcasts, webinars, and interviews.

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