Short, long, or ultra-long: which read length is right for you?

In modern genomics research, selecting the appropriate sequencing technology is vital for generating accurate, comprehensive results. A key factor in this decision is read length — short, long, or ultra long — which can significantly impact your ability to resolve complex genomic regions and address specific research questions. So, what are the differences between these read lengths and their applications? And why does the ability of Oxford Nanopore technology to generate reads of any length provide unique advantages for diverse sequencing projects?

What are short, long, and ultra-long reads?

Short reads typically range from 50 to 300 base pairs (bp) in length. These relatively short fragments of DNA represent just a tiny portion of a genome and are usually generated by fragmenting nucleic acids during library preparation.

Long reads span from approximately 1–100 kilobases (kb) in length. Reads of 10–25 kb can span many common repeat elements and structural variants (SVs), providing valuable information for resolving complex genomic regions.

Ultra-long reads exceed 100 kb in length, with some reaching several megabases (Mb). These extraordinarily long reads can span the most challenging genomic regions, including large SVs, centromeres, and other highly repetitive sequences that shorter reads cannot resolve.

What read lengths do different sequencing technologies generate?

Illumina technology produces short reads through a sequencing-by-synthesis approach. DNA is fragmented into small pieces, amplified in clusters, and sequenced with fluorescently labelled nucleotides. While offering high throughput and base-level accuracy, the limited read length (typically 50–300 bp¹) creates inherent challenges for resolving complex genomic structures.

PacBio HiFi technology generates long reads through a circular consensus sequencing approach. This technology sequences the same DNA molecule multiple times to achieve high accuracy but limits read lengths to approximately 15–25 kb due to size selection of the circular template².

Oxford Nanopore technology can produce reads of any length, from short to ultra long (50 bp to >4 Mb). Nanopore sequencing directly analyses native DNA or RNA molecules without fragmentation, amplification, or dye labelling requirements, giving you an unbiased view of the nucleic acids in your sample.

Among these technologies, Oxford Nanopore stands out for its ability to generate ultra-long reads. This capability did not emerge overnight but evolved through continuous innovation and collaboration with the research community. Let's examine how these ultra-long reads developed.

A brief history of ultra-long reads from Oxford Nanopore

The evolution of long and ultra-long reads with nanopore sequencing began when the MinION Access Programme launched in 2014, where researchers were quickly achieving 10 kb reads during initial experiments. In 2016, Oxford Nanopore released the rapid library preparation kits with simplified chemistry that eliminated wet lab steps that were thought to limit read length. In 2017, Jain and colleagues used this new chemistry in the assembly of the first human centromere at UC Santa Cruz, USA³. At the same time, researchers achieved unprecedented 100 kb+ reads as part of the ‘nanopore human genome project’⁴, which led to the coining of the term ‘ultra-long’ for reads exceeding 100 kb, while reads longer than a megabase were dubbed ‘whales’. Since then, increasingly longer reads have been reported by various research groups, with the current record standing at over 4 Mb in a single continuous read.

Why read length matters for your research

Completing the human genome with ultra-long reads

For decades, approximately 8% of the human genome remained unresolved using short-read technologies due to highly repetitive regions, particularly in centromeres and segmental duplications. The groundbreaking paper by Nurk, Koren, Rhie, and Rautiainen et al. demonstrated that ultra-long reads generated by Oxford Nanopore sequencing were instrumental in completing the first truly gapless human genome assembly, filling in regions that had remained inaccessible to sequencing for two decades⁵.

Not only were ultra-long reads essential in finishing the human genome, but the long reads generated by nanopore technology have now been demonstrated as a solution for large-scale human genomics projects. Kolmogorov and Billingsley et al. achieved state-of-the-art small variant and SV calling using only Oxford Nanopore data produced by a single PromethION Flow Cell⁶. In addition, advances in bioinformatics have expanded the capabilities of Oxford Nanopore-only assemblies — Stanojević et al. developed a deep learning-based approach that used nanopore data to assemble telomere-to-telomere human genomes⁷.

While completing the human genome represents a significant achievement, the benefits of ultra-long reads extend across the biological spectrum — from organisms with big genomes to those with more compact ones. Below, we explore other examples where long read lengths matter.

Assembling the big and the small

Ultra-long reads are not only important for assembling human genomes, but also those that are much larger and much smaller. In terms of larger genomes, plant genomes are incredibly complex, with their high repeat content and polyploidy, and ultra-long nanopore reads were crucial for resolving extremely complex and large repetitive regions in the maize genome, including a massive 26.8 Mb array⁸ — something that would be impossible without ultra-long reads.

In microbiology, long and ultra-long reads mean that entire viral genomes, such as ocean phages⁹, and whole plasmids can be captured in single reads, dramatically simplifying assembly processes. This speeds up the time it takes for you to get results, which is key in pathogen surveillance and infectious disease outbreak situations.

Matching read length to clinical questions

Long and ultra-long reads are proving critical for resolving complex SVs associated with disease cases. Long reads detect five times more SVs in the human genome than short reads¹⁰ and 34% of disease variants are associated with SVs¹¹. This means that long reads can detect disease-causing variants that remain hidden to short-read approaches, offering the future potential to increase diagnostic yield for patients with previously undiagnosed conditions.

However, it is not just long Oxford Nanopore reads that are proving important in clinical research applications. Short reads generated by nanopore technology also offer advantages for specific clinical questions, particularly in cancer research and transcriptome analysis.

Harnessing short Oxford Nanopore reads

For cancer genomics, Baslan et al. showed that short reads generated by nanopore sequencing can be used for high-throughput copy number analysis. By targeting shorter DNA molecules at optimal concentrations, they achieved higher read counts from a single run, accurately detecting copy number alterations in leukaemia samples¹².

Oxford Nanopore technology is not limited to DNA sequencing — you can use the same platform for cDNA and RNA analysis. Research from Roman E. Reggiardo and their team demonstrates how Oxford Nanopore sequencing can detect a wide range of cell-free RNAs¹⁴. The team's COMPLETE-seq workflow revealed that cell-free RNAs in liquid biopsies can really vary in length — from ~150 bp to ~1,300 bp. This research revealed a high abundance of repetitive RNA sequences in blood, highlighting their potential as disease-specific diagnostic biomarkers.

Looking at even smaller RNA molecules, Koch and Reilly-O'Donnell et al. used a MinION device to successfully detect cardiovascular disease-associated microRNA directly from human serum without extraction or amplification¹⁵. Offering a one-hour turnaround time from sample to results, the team showcased how Oxford Nanopore technology is suitable to detect these molecules, which have an average length of 22 nucleotides.

Choosing the right library preparation for your read length needs

So, how can you get the best range of read lengths for your research? Oxford Nanopore offers various library preparation kits optimised for different read length requirements.

• Ligation Sequencing Kit: standard kit for routine sequencing where the read length matches the input fragment length
• Rapid Sequencing Kit: for quick sample-to-sequencer workflows — best for samples containing input fragments >30 kb
• Ultra-Long DNA Sequencing Kit: specialised for generating ultra-long reads exceeding 100 kb, ideal for resolving challenging genomic regions and achieving telomere-to-telomere assemblies
• PCR-based kits: for low input samples, typically yielding reads around 2 kb in length
• Direct RNA Sequencing Kit: to prepare native RNA where the read length is dependent on the input RNA molecule length, perfect for exploring attributes such as modified bases
• cDNA-PCR Sequencing Kit: optimised for identifying and quantifying full-length isoforms from low input amounts

Your choice of library preparation should align with your specific experimental goals. When targeting SVs or assembling complex genomes, ultra-long reads provide critical advantages. For variant detection, smaller genomes, or RNA analysis, standard read lengths may be sufficient and provide higher throughput.

Oxford Nanopore technology uniquely provides the full spectrum of read lengths on a single platform, allowing you to tailor your sequencing approach precisely to your research questions without being limited by technology constraints.

Explore the full range of flexible, scalable Oxford Nanopore sequencing devices