Oxford Nanopore sequencing: new opportunities for plant genomics?

Abstract

DNA sequencing was dominated by Sanger’s chain termination method until the mid-2000s, when it was progressively supplanted by new sequencing technologies that can generate much larger quantities of data in a shorter time. At the forefront of these developments, long-read sequencing technologies (third-generation sequencing) can produce reads that are several kilobases in length. This greatly improves the accuracy of genome assemblies by spanning the highly repetitive segments that cause difficulty for second-generation short-read technologies. Third-generation sequencing is especially appealing for plant genomes, which can be extremely large with long stretches of highly repetitive DNA. Until recently, the low basecalling accuracy of third-generation technologies meant that accurate genome assembly required expensive, high-coverage sequencing followed by computational analysis to correct for errors. However, today’s long-read technologies are more accurate and less expensive, making them the method of choice for the assembly of complex genomes. Oxford Nanopore Technologies (ONT), a third-generation platform for the sequencing of native DNA strands, is particularly suitable for the generation of high-quality assemblies of highly repetitive plant genomes. Here we discuss the benefits of ONT, especially for the plant science community, and describe the issues that remain to be addressed when using ONT for plant genome sequencing.

Current Developments and Challenges in Plant Viral Diagnostics: A Systematic Review

“…Before the emergence of next-generation sequencing technologies, first-generation sequencing (Sanger sequencing) dominated the field of biological research. Even after the emergence of high-throughput sequencing technologies, these methods remained popular for analysis due to their higher throughput and relatively low cost [ 119 ]. NGS technologies have changed the approaches to both basic and applied research in plant virology.…”

“…For RNA viruses, MinION’s new direct RNA sequencing represents the next significant development; however, direct RNA sequencing studies are currently limited [ 169 ]. This technology is scalable from miniature devices to high-throughput installations like MinION, which was directly followed by GridION (designed to run five MinIONflowcells) and PromethION (designed to run 24 or 48 larger capacity flow cells), which use the same core technology as MinION but are designed for larger sequencing loads [ 119 ]. The pocket-sized MinION device from ONT is among the most popular devices for the real-time sequencing of DNA or RNA samples, as well as for whole genome sequencing [ 170 ].…”

“…ONT, therefore, developed as a flip–flop base-calling model that uses two overlapping windows to interpret the raw signal. Even though the output and error rates of the current generation of sequencers remain to be improved, new possibilities for evolutionary research using these novel techniques are expected to emerge [ 119 ].…”

SILEX: a fast and inexpensive high-quality DNA extraction method suitable for multiple sequencing platforms and recalcitrant plant species

“…The method was also used to extract high-molecularweight DNA from a recalcitrant wild species (Solanum elaeagnifolium). The DNA obtained was successfully used to construct long insert size Nanopore libraries for a de novo genome assembly, which can be difficult for recalcitrant species [40], thus proving its suitability for third-generation sequencing platforms.…”

SILEX: A fast and inexpensive high-quality DNA extraction method suitable for multiple sequencing platforms and recalcitrant plant species

“…The method was also used to extract high-molecular-weight DNA from a recalcitrant wild species (Solanum elaeagnifolium). The DNA obtained was successfully used to construct long insert size Nanopore libraries for a de novo genome assembly, which can be di cult for recalcitrant species [40], thus proving its suitability for third-generation sequencing platforms.…”