title: Multiplexed Reference Materials as Controls for Diagnostic Next-Generation Sequencing: A Pilot Investigating Applications for Hypertrophic Cardiomyopathy.
abstract: Diagnostic next-generation sequencing (NGS)-based gene panels are increasingly used for prevalent disorders with genetic and clinical heterogeneity. Clinical development, validation, and quality management of these panels ideally includes reference samples containing prevalent pathogenic variants; however, clinical domain expertise to select appropriate variants may not be present, samples are often not publicly available, and their inclusion is associated with added cost. Expert-designed, multiplexed controls can remedy some of these challenges. One approach relies on spiking biosynthetic fragments carrying desired variants into human genomic DNA. We piloted the utility of this approach for hypertrophic cardiomyopathy. Data from >3000 previously sequenced probands were used to select 10 common pathogenic and/or technically challenging variants in the top hypertrophic cardiomyopathy genes. Multiplexed controls were constructed across a range of ideal and realistic allelic fractions for heterozygous germline variants. NGS was performed in quadruplicate, and results were compared with diagnostic NGS data for the source patient samples. Overall, results were indistinguishable from patient-derived data with variants being detected at or reasonably close to the targeted allelic fraction ratios. The exception was a common 25-bp deletion in MYBPC3, underscoring the importance of including such variants in test development. These controls may be an attractive addition to the repertoire of materials for development, validation, and quality monitoring of clinical NGS assays.
authors.affiliation: Laboratory for Molecular Medicine, Partners HealthCare Personalized Medicine, Cambridge, Massachusetts.
authors.forename: Emily M
authors.initials: EM
authors.lastname: Kudalkar
authors.affiliation: Laboratory for Molecular Medicine, Partners HealthCare Personalized Medicine, Cambridge, Massachusetts; Department of Pathology/MGH, Harvard Medical School, Boston, Massachusetts; Center for Genetics and Inherited Diseases, Taibah University, Almadinah Almunwarah, Kingdom of Saudi Arabia.
authors.forename: Naif A M
authors.initials: NA
authors.lastname: Almontashiri
authors.affiliation: SeraCare Life Sciences Inc., Milford, Massachusetts.
authors.forename: Catherine
authors.initials: C
authors.lastname: Huang
authors.affiliation: SeraCare Life Sciences Inc., Milford, Massachusetts.
authors.forename: Bharathi
authors.initials: B
authors.lastname: Anekella
authors.affiliation: Laboratory for Molecular Medicine, Partners HealthCare Personalized Medicine, Cambridge, Massachusetts.
authors.forename: Mark
authors.initials: M
authors.lastname: Bowser
authors.affiliation: Laboratory for Molecular Medicine, Partners HealthCare Personalized Medicine, Cambridge, Massachusetts.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Hynes
authors.affiliation: SeraCare Life Sciences Inc., Milford, Massachusetts.
authors.forename: Russell
authors.initials: R
authors.lastname: Garlick
authors.affiliation: Laboratory for Molecular Medicine, Partners HealthCare Personalized Medicine, Cambridge, Massachusetts; Department of Pathology/MGH, Harvard Medical School, Boston, Massachusetts. Electronic address: bfunke@partners.org.
authors.forename: Birgit H
authors.initials: BH
authors.lastname: Funke
mesh_terms: D000483:Alleles; D002312:Cardiomyopathy, Hypertrophic / Q000175:diagnosis* / Q000235:genetics*; D005787:Gene Frequency; D005819:Genetic Markers; D005820:Genetic Testing / Q000379:methods / Q000592:standards; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000592:standards; D006801:Humans; D009154:Mutation; D012015:Reference Standards
chemical_list: D005819:Genetic Markers