Human genome consists of approximately three billion base pairs. The structure of the genome is very similar for all people, however, on closer examination it can be seen to contain plenty of individual variation. Human genome has many repeat regions and different structural variations. The majority of this variation is entirely harmless but, in some cases, a change in the genome can cause an illness or predispose a person to an illness. A new method has been developed for the study of large-scale variation at chromosome level: optical genome mapping.
Types of genomic structural variation
Genomic variation may consist of small point mutations, repeat regions or larger-scale structural variation. The latter, i.e. genomic structural variation, can be divided into six main types: deletion (loss of genetic material), duplication (gain of extra copies of genetic material), insertion (addition of new genetic material into DNA), translocation (rearrangement of genetic material, typically between two chromosomes), inversion (rearrangement of genetic material to reverse order) and aneuploidy (alteration in the number of entire chromosomes).
| Aneuploidy | An abnormal number of individual chromosomes in a set of chromosomes |
| Deletion | A part of the chromosome is lost |
| Duplication | A part of the chromosome is duplicated |
| Insertion | A part or parts of the same or another chromosome are attached to the chromosome |
| Inversion | The order of bases on a chromosome is inverted in a certain part of the chromosome |
| Translocation | Two different chromosomes switch parts |
Chromosome study produces information
The study of genomic structural variation provides vital information on genetic mechanisms. Because genetic alterations have an effect on the pathogenesis of diseases, new research data may also provide clinical benefits. Studying structural variation can help to diagnose hereditary diseases and predisposition to diseases, differentiate between different types of cancer or examine unknown diseases.
Methods: karyotyping, FISH, molecular karyotyping, NGS
A traditional way of studying genome structure is karyotyping, i.e. G-banding of chromosomes. In this technique, cells are cultured, and their chromosomes are stained and examined under microscope. Chromosomes are identified, and any chromosomal abnormalities are observed based on the typical size and G-banding pattern of each chromosome. This method is laborious, and the analysis requires a high level of professional skill and experience. Even though nowadays the analysis is partly automated, the results might vary between the persons performing the analysis.
Genomic structural variation can also be studied with fluorescence in situ hybridization (FISH), that uses targeted fluorescent probes designed for specific chromosome region. The analysis is carried out with a fluorescence microscope to see if the probes have attached to the chromosome. This method works well when looking for known changes in the genome.
Molecular karyotyping represents a more novel technology for chromosomal studies. In this technique, a sample DNA (e.g. DNA of cancerous cells) and reference DNA from normal tissue are labelled with fluorescent dyes of different colours and hybridized to a microchip. Based on the principle of competition, the sample and reference DNA attach to the DNA probes on the microchip. Microchips are scanned with a laser scanner, and image analysis software converts the data contained in the image to numerical format. The ratio of fluorescent labels represents the copy number of genes. This method is most commonly used to study copy number variation, for example, in cancer diagnostics.
In the last decade, next-generation sequencing (NGS) has provided the most new information about the genome. NGS is used to determine the sequence of nucleotides in the genome. In this approach, millions of short reads (less than 300 base pairs) are analysed simultaneously in a single run and aligned against a reference genome. Major strengths of NGS are it’s cost-effectiveness, speed and reliability. However, NGS cannot be used to study repeat regions or structural variation in the genome. The information contained in the short reads may not be sufficient enough to reliably align the sequenced part with the correct location on the genome, and this can lead to ambiguous results and errors. Due to the shortness of reads, identifying structural changes with NGS is usually based on indirect reasoning instead of direct observation.
A new study method: optical genome mapping
Optical genome mapping is a new method developed by Bionano to study the structural variation. Instead of amplifying DNA, this method uses high-resolution microscopy and automated image analysis. High-quality DNA is required for source material, and extremely long strands of DNA (more than 250,000 base pairs) are isolated. The isolated ultra-long DNA is labelled with a fluorescent dye and analysed on a microchip containing hundreds of thousands of adjacent nanochannels. One nanochannel allows only a single ultra-long strand of DNA to travel through, and the system images the fluorescent labels attached to the strand.
The longer the imaging period, the more precise the image of the DNA strand will be composed. During the analysis, the system creates an enormous stack of images, which the analysis software combines. The result is a genome map, which the software compares with a reference genome map and then lists all possible aberrations in the genome to the user.
This method does not require work-intensive cell culture, and only a very small amount of DNA sample is required. However, the studied DNA must be of high quality because the DNA strand needs to stay intact. For this reason very old or processed samples, such as formalin-fixed paraffin-embedded samples, are not suitable for analysis. The number of manual hands-on steps (isolation of DNA, fluorescence labelling and pipetting onto a microchip) is limited to minimise the time spent in a laboratory. The analysis itself is fully automated.
| Variant | Karyotyping | FISH | Molecular karyotyping | NGS | Optical genome mapping |
| Aneuploidy | + | Targeted | + | + | + |
| Deletion | > 5-10 Mbp | Targeted | + | Limited | + |
| Duplication | > 5-10 Mbp | Targeted | + | Limited | + |
| Insertion | > 5-10 Mbp | Targeted | - | Limited | + |
| Inversion | > 5-10 Mbp | Targeted | - | - | + |
| Translocation | > 5-10 Mbp | Targeted | - | Targeted | + |
Illustrative and unambiguous results
Because the comparison of genome maps is software-based, there will be no user-dependent variation. No bioinformaticians are required to interpret the results. Instead, the system performs the necessary data analysis and produces a graph that clearly shows all aberrations. However, optical genome mapping cannot be used to examine the centromeric region of a chromosome. DNA in the centromere is tightly packed, and it is not amplified in connection with normal cell division. As a result, the centromere region does not contain any binding sites for the fluorescent labels.
The reagents and microchips used in this method are supplied as a ready-to-use bundle. Since the number of hands-on steps is limited, optical genome mapping is less expensive than other new technologies. Optical genome mapping can be used for human, plant and animal applications and for both research and diagnostic purposes. The future trend is to combine NGS with optical genome mapping. NGS reveals the sequence of nucleotides in the genome, whereas optical genome mapping shows how the genetic material is arranged.
Optical genome mapping makes research easier
The study of structural variation in the genome increases the understanding of genetic mechanisms and their effect on the pathogenesis of hereditary diseases, for example. New optical genome mapping facilitates the study of structural variation.
