Introduction to genetic structural variation
It is an exciting time to be involved in genetics and its application to healthcare. It was only a little over two decades ago the first draft of the Human Genome Project was published, and last year the first telomere-to-telomere sequence of a single chromosome was achieved. And the impact of next-generation sequencing is seeing in increasingly valuable applications in the clinic: as a companion diagnostic for targeted cancer therapy, as a method for prenatal non-invasive testing for trisomy, and for rare disease diagnostics. Yet there are still so many big problems that remain unsolved.
One of the larger problems in genetics (and by association application and impact to healthcare) is the detection and characterization of structural variation. A single gene can be damaged via a multitude of mechanisms (such as non-homologous recombination), and is a different kind of variation compared to Single Nucleotide Polymorphisms (SNPs) which genotyping microarrays measure, or insertion / deletion mutations (called indels where a single base to several dozens of bases can be inserted or deleted) which sequencing can also detect.
The size of these insertions and deletions, however, can exceed the resolving power of next-generation sequencing, where readlengths can be limited to 150 to 300 bases. There can be insertions and deletions of kilobases or hundreds of kilobases long, which will be invisible to NGS analyses.
In a given individual’s whole-genome sequence, there will be some 4 to 5 million SNPs and indels detected. The structural rearrangements (above 50 bases of inserted or deleted nucleotides, to several million bases or even entire chromosome arms) go undetected. For clinical cases, a pathology cytogenetics laboratory routinely uses techniques such as Fluorescent In-Situ Hybridization (known by its acronym FISH), karyotyping and microarrays (typically aCGH or array Comparative Genomic Hybridization) to detect structural rearrangements and specific gene fusions for diagnosing and appropriately guiding the treatment of cancer.
Figure 1 below (kindly provided by Nabsys) compares conventional next-generation Sequencing by Synthesis (SBS) to genome mapping.
There are estimated over 20,000 structural variants in a single human genome, yet with current sequencing technology (including single molecule sequencing from manufacturers such as Pacific Biosciences or Oxford Nanopore Technologies) large swaths of genome sequence can be rearranged but go undetected.
For example, say there is a balanced structural variant, where a large multi-megabase region is inverted. It is called balanced because there is no gain or loss of DNA sequence, however there is a stretch of several megabases in the completely opposite orientation. Even with the technical advances of single-molecule sequencing to the tens or even hundreds of kilobases long, detecting all the different kinds of variation with a wide range of sizes and complexity remains a challenge.
Mapping versus long read sequencing
One definite trend over the past few years has been a consistent increase in throughput of short read sequencing, in addition to the similar throughput increases in long read sequencing as well. However on a cost-per-gigabase basis, long read sequencing remains 5-fold to 10-fold more expensive, severely limiting its applicability to clinical applications.
Genome mapping using an optical method has been on the market for several years from Bionano Genomics, and is accepted as a complement to whole genome or whole exome sequencing to understand the nature of structural variants and disease. Nabsys now offers better resolution of variants at lower cost, detecting SV’s as small as 300 base pairs with >100kb long segments of the genome electronically mapped.
Nabsys OhmX™ technology
For a Nabsys run, high-molecular weight genomic DNA (50 kb to 500 kb) is first nicked using sequence-specific nickase enzymes, that could be used alone or in combinations, then labeled and coated with a protein called RecA (the RecA protein serves to stiffen the DNA for analyses). The samples are injected into the instrument, and the data is collected.
Single DNA molecules are translocated through a silicon nanochannel, and the labeled locations are electronically detected to determine the distance between sequence-specific tags on individual molecules. While each electronic event is measured across the linear DNA molecule, there is a time-to-distance conversion and the entire genome has enough overlap to assemble what is effectively a restriction map of overlapping fragments (see figure 2).
This capability was showcased a few years ago for microbial genomes, and a few publications1, 2, 3 show the proof of the approach for analyzing DNA maps this way at single-molecule resolution in bacterial genomes.
With the recent commercial release of the Nabsys OhmX Analyzer system and OhmX-8 Detector consumables, a 10-fold increase in throughput has been achieved combined with 250 electronic detectors per channel. Nabsys uses a kit for efficient high molecular-weight DNA extraction and labeling in preparation for loading onto the system. (The sample input requirement is 5 ug of starting material, sufficient for several instrument runs if necessary; less input can be used if DNA quantities are limited.) In addition, as there are no optics (only fluidics and electronics) the Nabsys instrument is much more compact and less expensive than the equivalent optical instrument, as well as less expensive to run.
Applications for human disease: cancer and rare disease
Cancer has been correctly described as a ‘disease of the genome’, and as a research tool understanding the role structural variation has in cancer progression and treatment is an ongoing area of important work. Another important application of genomic mapping is for rare disease; currently it is estimated that about 70% of suspected Mendelian disorders go undiagnosed even with current short-read whole-genome sequencing4.
It remains to be seen whether better detection and characterization of structural variation can provide the needed insights into these two important research areas, currently limited by cost of existing technology.
Nabsys at ASHG 2023
At the upcoming American Society for Human Genetics conference in Washington DC November 2 – 5, 2023 Nabsys will be present in the Hitachi High-Tech America Booth 1423. Hitachi will present their Human Chromosome Explorer bioinformatics pipeline for a low-cost, scalable Structural Variation validation and discovery platform.
You can find out more about the Nabsys OhmX Analyzer here (a downloadable brochure is available on that page) and also more information about the overall approach to electronic genome mapping is here. A handy whitepaper about EGM can be found here (PDF).
- Passera A and Casati P et al. Characterization of Lysinibacillus fusiformis strain S4C11: In vitro, in planta, and in silico analyses reveal a plant-beneficial microbe. Microbiol Res. (2021) 244:126665. doi:10.1016/j.micres.2020.126665
- Weigand MR and Tondella ML et al. Screening and Genomic Characterization of Filamentous Hemagglutinin-Deficient Bordetella pertussis. Infect Immun. (2018) 86(4):e00869-17. doi:10.1128/IAI.00869-17
- Abrahams JS and Preston A et al. Towards comprehensive understanding of bacterial genetic diversity: large-scale amplifications in Bordetella pertussis and Mycobacterium tuberculosis. Microb Genom. (2022) 8(2):000761. doi:10.1099/mgen.0.000761
- Rehm HL. Evolving health care through personal genomics. Nat Rev Genet. (2017) 18(4):259-267. doi:10.1038/nrg.2016.162
