Development and performance of a comprehensive targeted sequencing assay for pan-ethnic screening of carrier status.

Identifying individuals as carriers of severe disease traits enables informed decision making about reproductive options. Although carrier screening has traditionally been based on ethnicity, the increasing ethnic admixture in the general population argues for the need for pan-ethnic carrier screening assays. Highly multiplexed mutation panels allow for rapid and efficient testing of hundreds of mutations concurrently. We report the development of the Pan-Ethnic Carrier Screening assay, a targeted sequencing assay for routine screening that simultaneously detects 461 common mutations in 91 different genes underlying severe, early-onset monogenic disorders. Mutation selection was aided by the use of an extensive mutation database from a clinical laboratory with expertise in newborn screening and lysosomal storage disease testing. The assay is based on the Affymetrix GeneChip microarray platform but generates genomic DNA sequence as the output. Analytical sensitivity and specificity, using genomic DNA from archived control cultures and from clinical specimens, was found to be >99% for all mutation types. This targeted sequencing assay has advantages over multiplex PCR and next-generation sequencing assays, including accuracy of mutation detection over a range of mutation types and ease of analysis and reporting of results.

Single assay for simultaneous detection and differential identification of human and avian influenza virus types, subtypes, and emergent variants.

For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based) or remarkably insensitive (antibody-based). Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR) have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu) is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A/HN subtype, virulence, host-range, and resistance to antiviral agents.