Next-generation mapping: a novel approach for detection of pathogenic structural variants with a potential utility in clinical diagnosis

Abstract

Background: Massively 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.

Methods: To 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.

Results: We 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.

Conclusions: We 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.

Keywords: Bionano; DMD; Duchenne muscular dystrophy; Nanochannel; Next-generation mapping; Optical mapping; Structural variants.

Conflict of interest statement

Ethics approval and consent to participate

Research involving human participants, human material, and human data have been performed in accordance with the principles of the Helsinki declaration and approved protocols by the UCLA Institutional Review Board: Genetic Modifiers of Duchenne and Becker Muscular Dystrophy (IRB#12-000769); Molecular Genetics of Degenerative Disease (IRB#11-001087). Informed consents were obtained during patients’ visits to the Pediatric Neuromuscular Clinic.

Consent for publication

Participants have consented by approved IRB protocols to share their de-identified information.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1

DNA labeling for NGM. The DNA labeling workflow is divided into four consecutive steps. First, the high molecular weight DNA is nicked with an endonuclease of choice that introduces single strand nicks throughout the genome. Second, Taq polymerase recognizes these sites and replaces several nucleotides with fluorescently tagged nucleotides added to the solution. Third, the two ends of the DNA are ligated together using DNA ligase. Fourth, the DNA backbone is stained with DNA Stain

Fig. 2

Irys/Saphyr chip nanochannel structure and DNA loading. The labeled dsDNA is loaded into two flowcells of either Irys or Saphyr chips. The applied voltage concentrates the coiled DNA at the lip (left). Later, DNA is pushed through pillars (middle) to uncoil/straighten, then into nanochannels (right). DNA is stopped and imaged in the nanochannels. Blue = staining of DNA backbone, green = fluorescently labeled nicked sites

Fig. 3

Visualization of the human genome coverage using NGM. Chromosome 1-22,X,Y are represented by G-banding patterns. The red shading represents centromere locations. Horizontal blue shading represents regions where long native-state DNA molecules have been aligned using the Bionano NGM platform

Fig. 4

Deletions identified in four DMD probands. For each case, the blue bar represents the reference X chromosome. The yellow bar represents the sample map generated based on long molecule assembly of the patient’s genome. The black vertical lines indicate Nt.BspQI endonuclease cut sites and corresponding matches between reference (blue) and sample (yellow) genomes. The lines between reference and assembled map show alignment of the two maps. The red area indicates the deletion where reference (blue) endonuclease sites are missing from the assembled map (yellow). The locations of the DMD exons are indicated at the top of the figure with vertical lines. Below each map, information such as size and type of the SV and deleted exons can be found

Fig. 5

NGM identified a hemizygous and heterozygous multi-exon deletion in a DMD patient and his biological mother, respectively. a Hemizygous deletion in the patient. Top: visual representation of the deletion (red) between the reference (blue) and patient (yellow) maps. Middle: representation of long molecules used to construct the sample maps. Bottom: Ref-seq locations on the X chromosome indicating possible size of the deletion based on MPLA and size identified using the NGM platform. b Heterozygous deletion in the biological mother. Top: The normal wild type allele (yellow) can be seen above reference (blue) where all nicking sites align to reference map. This is in contrary to the second allele (yellow) containing the deletion shown below the reference (blue) map. Maps were generated using Nt.BspQI nicking endonuclease

Fig. 6

NGM identified a hemizygous multi-exon deletion in a DMD patient that was not present in the biological mother. a, bTop: visual representation of the sample allele in yellow (a patient; b mother) compared to the reference (blue). The de novo deletion is shown in red. aMiddle: the lines below the patient’s contig represent the long molecules used to construct the sample map. Bottom: Ref-seq locations on the X chromosome indicating possible size of the deletion based on MPLA and size identified using the NGM platform. bBottom: location of Ref-Seq genes in the X chromosome within the shown region. Maps were generated using Nt.BspQI nicking endonuclease

Fig. 7

NGM identified a 13-Kbp insertion in a DMD patient and his biological mother. aTop: visual representation of the insertion (green) between the reference (blue) and patient (yellow) maps. Bottom: insertion size identified in the proband by chromosomal microarray and by NGM platform. bTop: the normal wild type allele of the mother (yellow) can be seen above reference (blue) where all nicking sites align to reference map. This is in contrary to the second allele of the mother (yellow) containing the insertion shown below the reference (blue) map. Maps were generated using Nt.BspQI nicking endonuclease

Fig. 8

NGM identified a 5.1-Mbp inversion disrupting DMD. Top: X chromosome and Ref-Seq genes (orange) present in the magnified region. Visual representation of the inversion where the middle section of the reference (blue) and patient (yellow) maps have inverted alignments. The sample maps were generated using Nb.BssSI (top) and Nt.BspQI (bottom) endonucleases. Nicked sites are represented by red (Nb.BssSI) or black (Nt.BspQI) vertical lines in the middle reference and top/bottom sample maps