Long read sequencing was recently named 2022’s method of the year by Nature Methods. Long read sequencing technologies, those that generate sequence reads with lengths of 10s of kilobases or longer have several advantages over short read sequencing technologies, which produce read lengths <300bp. A major limitation of short-read sequencing is that reconstruction and quantification of longer molecules (i.e., certain genomic regions, transcript isoforms) using fragments that are much shorter than the molecule being analyzed is difficult. Longer read lengths allow the more challenging regions of the genome (repetitive elements, centromeres) to be resolved and this has led to the first complete (telomere-to-telomere) assembly of a human genome. Long read lengths can also identify more complex structural variations in the genome (duplications, large insertions, and deletions), which are often missed by short-read technologies, thereby enhancing our understanding of genetic variation and disease mechanisms. In addition, long read sequencing can be applied to RNA molecules, enabling the identification of full-length transcript isoforms.
Oxford Nanopore Technology (ONT) is a company that manufactures long read sequencing platforms that can produce read lengths up to several megabases in length. ONT sequencing is fundamentally different from the sequencing-by-synthesis approach that other sequencing platforms are based on. Instead, the sequencing material (either DNA or RNA) is prepared by attaching a sequencing adapter containing a motor protein to the end of the molecule being sequenced. The motor protein then attaches to a synthetic membrane that contains thousands of nanopores and translocates the nucleic acid through the pore. As each individual nucleotide passes through the pore, it causes a characteristic disruption to an electric current flowing through the membrane. Real-time analysis of these changes in current allow for the determination of the sequence of the nucleotides in the molecule. The platform allows for direct sequencing of nucleic acid (i.e. both RNA and DNA), without additional manipulation or amplification steps that could introduce artifacts or biases. As a result, the platform allows for the detection of several nucleotide modifications, including 5-methylcytosine in DNA and N6-methyladenosine in RNA.
One of the purported challenges of long read technologies was the reported lower read accuracy (<85%) in the early days of their introduction. However, continual improvements in library preparation, flow cell technologies, and base calling algorithms, has led to the ONT platform producing read accuracies approaching those of short-read technologies. Currently, ONT reports accuracies of >99% with the soon-to-be-released version 10.4 flow cell in conjunction with the V14 sequencing kit.
ONT has several instrument types that differ with respect to their portability and throughput. These include the smaller flongle and MinION which are used for smaller genomes and transcriptomes, as well as targeted sequencing. The small size of these instruments make them ultra-portable and useful for field studies. ONT also offers larger benchtop sequencers including the GridION and PromethION platforms which allow for sequencing multi-sample, production-scale projects, involving whole genome and whole transcriptome analysis. Most recently, ONT has announced a partnership with 10X technologies, to provide workflows for sequencing full-length transcripts in single reads on the PromethION platform, allowing long-read sequencing to be applied at the single-cell and spatial level.
Two sequencing facilities at the NCI offer ONT sequencing. The CCR Genomics Core offers the MinION platform with plans to upgrade to the PromethION 2 Solo platform in the coming months. The Sequencing Facility in Frederick currently has the PromethION 2 Solo, GridION and MinION platforms. Interested researchers are encouraged to contact these facilities directly for more information.
— Desiree Tillo (GAU)