Genomic technologies such as 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 without a firm diagnosis. Due to the methodological limitations of both technologies (SRS, CMA) they fail to sensitively identify structural variants (SVs) or 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 changes 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 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 (i.e. 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 Beckwith-Wiedemann syndrome, where the paternally inherited allele carried a duplication and 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 applied to other types of disorders such as CHARGE syndrome.