Abstract
BackgroundMassively parallel DNA sequencing, such as exome sequencing, has become a routine clinical procedure to identify pathogenic variants responsible for a patient’s phenotype. Exome sequencing has the capability of reliably identifying inherited and de novo single-nucleotide variants, small insertions, and deletions. However, due to the use of 100–300-bp fragment reads, this platform is not well powered to sensitively identify moderate to large structural variants (SV), such as insertions, deletions, inversions, and translocations.MethodsTo overcome these limitations, we used next-generation mapping (NGM) to image high molecular weight double-stranded DNA molecules (megabase size) with fluorescent tags in nanochannel arrays for de novo genome assembly. We investigated the capacity of this NGM platform to identify pathogenic SV in a series of patients diagnosed with Duchenne muscular dystrophy (DMD), due to large deletions, insertion, and inversion involving the DMD gene.ResultsWe identified deletion, duplication, and inversion breakpoints within DMD. The sizes of deletions were in the range of 45–250 Kbp, whereas the one identified insertion was approximately 13 Kbp in size. This method refined the location of the break points within introns for cases with deletions compared to current polymerase chain reaction (PCR)-based clinical techniques. Heterozygous SV were detected in the known carrier mothers of the DMD patients, demonstrating the ability of the method to ascertain carrier status for large SV. The method was also able to identify a 5.1-Mbp inversion involving the DMD gene, previously identified by RNA sequencing.ConclusionsWe showed the ability of NGM technology to detect pathogenic structural variants otherwise missed by PCR-based techniques or chromosomal microarrays. NGM is poised to become a new tool in the clinical genetic diagnostic strategy and research due to its ability to sensitively identify large genomic variations.
Highlights
Parallel DNA sequencing, such as exome sequencing, has become a routine clinical procedure to identify pathogenic variants responsible for a patient’s phenotype
We performed next-generation mapping (NGM) on a cohort of eight affected Duchenne muscular dystrophy (DMD) individuals and three biological mothers, one of whom was a known carrier of a pathogenic deletion in DMD (Table 1)
Genetic diagnosis of DMD is most often achieved by polymerase chain reaction (PCR) and multiplex ligationdependent probe amplification (MLPA) of all 79 exons of DMD
Summary
Parallel DNA sequencing, such as exome sequencing, has become a routine clinical procedure to identify pathogenic variants responsible for a patient’s phenotype. Sanger sequencing is still widely used to sequence fragments of > 1 Kbp in length, massively parallel sequencing has emerged and dominates the global market for sequencing due to its comprehensiveness, supported by increasingly cheap price and fast turnaround times This is, in part, due to the improvements made in imaging, microengineering, and informatics techniques that enable acquisition of larger amounts of clean data from next-generation sequencing (NGS). Whole-genome sequencing (WGS) is capable of identifying single nucleotide variants (SNVs), insertions/deletions, and copy number variants in exons, and in non-coding regions of the genome This allows for identification of variants affecting gene regulation, which currently has limited clinical use due to inability to interpret most variants’ effect on the open reading frame of potential disease genes. Structural variation detection using WGS data presents challenges in highly repetitive genomic regions
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