Assessment of target enrichment platforms using massively parallel sequencing for the mutation detection for congenital muscular dystrophy

C. Alexander Valencia, Devin Rhodenizer, Shruti Bhide, Ephrem Chin, Martin Robert Littlejohn, Lisa Mari Keong, Anne Rutkowski, Carsten Bonnemann, Madhuri Hegde

Research output: Contribution to journalArticlepeer-review

45 Scopus citations


Sequencing individual genes by Sanger sequencing is a time-consuming and costly approach to resolve clinically heterogeneous genetic disorders. Panel testing offers the ability to efficiently and cost-effectively screen all of the genes for a particular genetic disorder. We assessed the analytical sensitivity and specificity of two different enrichment technologies, solution-based hybridization and microdroplet-based PCR target enrichment, in conjunction with next-generation sequencing (NGS), to identify mutations in 321 exons representing 12 different genes involved with congenital muscular dystrophies. Congenital muscular dystrophies present diagnostic challenges due to phenotypic variability, lack of standard access to and inherent difficulties with muscle immunohistochemical stains, and a general lack of clinician awareness. NGS results were analyzed across several parameters, including sequencing metrics and genotype concordance with Sanger sequencing. Genotyping data showed that both enrichment technologies produced suitable calls for use in clinical laboratories. However, microdroplet-based PCR target enrichment is more appropriate for a clinical laboratory, due to excellent sequence specificity and uniformity, reproducibility, high coverage of the target exons, and the ability to distinguish the active gene versus known pseudogenes. Regardless of the method, exons with highly repetitive and high GC regions are not well enriched and require Sanger sequencing for completeness. Our study demonstrates the successful application of targeted sequencing in conjunction with NGS to screen for mutations in hundreds of exons in a genetically heterogeneous human disorder.

Original languageEnglish (US)
Pages (from-to)233-246
Number of pages14
JournalJournal of Molecular Diagnostics
Issue number3
StatePublished - May 2012
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Medicine
  • Pathology and Forensic Medicine


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