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. 2013;8(1):e53083.
doi: 10.1371/journal.pone.0053083. Epub 2013 Jan 11.

Comprehensive mutation analysis for congenital muscular dystrophy: a clinical PCR-based enrichment and next-generation sequencing panel

C Alexander Valencia  1 , Arunkanth Ankala, Devin Rhodenizer, Shruti Bhide, Martin Robert Littlejohn, Lisa Mari Keong, Anne Rutkowski, Susan Sparks, Carsten Bonnemann, Madhuri Hegde
Affiliations

Comprehensive mutation analysis for congenital muscular dystrophy: a clinical PCR-based enrichment and next-generation sequencing panel

C Alexander Valencia et al. PLoS One. 2013.

Abstract

The congenital muscular dystrophies (CMDs) comprise a heterogeneous group of heritable muscle disorders with often difficult to interpret muscle pathology, making them challenging to diagnose. Serial Sanger sequencing of suspected CMD genes, while the current molecular diagnostic method of choice, can be slow and expensive. A comprehensive panel test for simultaneous screening of mutations in all known CMD-associated genes would be a more effective diagnostic strategy. Thus, the CMDs are a model disorder group for development and validation of next-generation sequencing (NGS) strategies for diagnostic and clinical care applications. Using a highly multiplexed PCR-based target enrichment method (RainDance) in conjunction with NGS, we performed mutation detection in all CMD genes of 26 samples and compared the results with Sanger sequencing. The RainDance NGS panel showed great consistency in coverage depth, on-target efficiency, versatility of mutation detection, and genotype concordance with Sanger sequencing, demonstrating the test's appropriateness for clinical use. Compared to single tests, a higher diagnostic yield was observed by panel implementation. The panel's limitation is the amplification failure of select gene-specific exons which require Sanger sequencing for test completion. Successful validation and application of the CMD NGS panel to improve the diagnostic yield in a clinical laboratory was shown.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Variant detection by NGS (top) and Sanger sequencing confirmation (bottom).
(A) Example of an insertion mutation: IVS15-3-2insC in COL6A2 identified byNGS (to the left) and confirmed by Sanger sequencing (to the right). (B) Example of a 30-bp deletion: IVS16-8del30 in COL6A3 as represented by NGS analysis (top panel) and Sanger sequencing (bottom panel). The exact site or representation of the mutation is indicated by the red arrowheads.
Figure 2
Figure 2. Schematic diagram of the analysis workflow.
The flowchart demonstrates the criteria used to select variants that were Sanger sequencing confirmed. In essence, selected variants that had >20× coverage, a low allele frequency, and nonsynonymous changes were Sanger confirmed if they were listed on HGMD, frameshift, or nonsense changes. In addition, interesting variants with a coverage <20× were also confirmed (Table 7).
Figure 3
Figure 3. Congenital muscular dystrophy next-generation sequencing algorithm.
The diagram demonstrates the diagnostic work-up of congenital muscular dystrophies using RainDance enrichment in combination with next-generation sequencing.
See this image and copyright information in PMC

References

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