Short-read exome/genome sequencing (SRS) and chromosomal microarrays (CMA) have helped increase diagnostic rates across many genetic disorders. However, despite this success, about half of the cases remain undiagnosed. Due to the methodological limitations of both technologies (SRS, CMA), they fail to sensitively identify many structural variants and balanced rearrangements, respectively. Additionally, both technologies have limitations in assessment of epigenetic changes. For example, short-read based bisulfite sequencing or methylation arrays do not provide long-range haplotype specific methylation states, rather the detected signals are averaged for individual genomic positions. These limitations can be alleviated with a novel dual-label optical genome mapping (DL-OGM) technology for detection of both genetic and epigenetic variations in one assay over long stretches of single DNA molecules and phased haplotypes. The method relies on differential labeling of high molecular weight DNA. First, long DNA molecules are nicked with BspQI endonuclease and labeled with red fluorescent nucleotides. Second, the same DNA molecules undergo treatment with M.TaqI methyltransferase that attaches green fluorescent cofactor onto non-methylated CpGs in ATCG sequences throughout the genome. Third, the pattern of fluorescent labels is captured in nanochannel arrays for de novo genome assembly, variant calling and quantification of epigenetic marks. Here, we will show the ability of DL-OGM to detect large copy number variants and methylation levels for Facioscapulohumeral muscular dystrophy (FSHD) and Beckwith-Wiedemann syndrome (BWS). We successfully identified the molecular diagnosis (constriction of D4Z4 array and associated hypomethylation) in FSHD case/control samples in the sub-telomeric region of chromosome 4q35. Additionally, we tested the method for a case diagnosed with BWS, where DL-OGM identified a duplication in the paternally inherited allele carrying epigenetic states resulting in the syndrome. DL-OGM technology offers substantial advantages over the current clinical diagnostic practices for specific disorders tested here (FSHD, BWS) and can be broadly applied to other disorders with characteristic DNA methylation patterns such as CHARGE syndrome, disorders with skewed X-inactivation, and imprinting disorders.
Although the completion of the Human Genome Project resulted in a tremendous acceleration of the field of genetics and identification of thousands of pathogenic genomic mutations, the state of advancements of genomic technologies has seen slower development, where the genetics community primarily relies on array and short-read based sequencing methodologies for disease diagnosis. However, the repetitive nature of the majority of the human genome makes these ineffective in many regions known to be disease causing and they do not address epigenetic profiles in a single assay. To improve the clinical care and identification of causative genomic variants other than small variants, such as structural variants and epigenetic alterations, we propose a study that utilizes novel genome mapping technology to provide much higher sensitivity for structural variant identification as well as quantification of haplotype specific epigenetic changes by leveraging information from millions of single molecules ranging from 150kbp to 1Mb in size.
