Browsing by Subject "Next Generation Sequencing"

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    Criteria for Clinical Reporting of Variants from a Broad Target Capture NGS Assay without Sanger Verification
    (JSM Biomarkers, 2015) Nelson, Andrew C.; Bower, Matthew; Baughn, Linda B.; Henzler, Christine; Onsongo, Getiria; Silverstein, Kevin A.T.; Schomaker, Matthew; Deshpande, Archana; Beckman, Kenneth B.; Yohe, Sophia; Thyagarajan, Bharat
    Increasing clinical interest and decreasing sequencing costs are driving the wider implementation of clinical next generation sequencing assays for the diagnosis of inherited disease, including among a growing number of small to medium sized clinical laboratories. Therefore, an optimal combination of cost-effectiveness and clinical specificity is required to continue this broad adoption of genomic technology for clinical diagnosis. Sanger confirmation of all NGS variants is a common practice that increases both cost and turnaround time for clinical reporting. We reviewed 300 cases of Sanger verified NGS results as well as 60 suspected (and subsequently confirmed) artifacts, and developed a set of multiple criteria to report NGS variants without Sanger verification with 100% accuracy. Using these criteria, we project greater than 80% of clinically reported variants could be confidently released without Sanger confirmation.
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    SVAtools for junction detection of genome-wide chromosomal rearrangements by mate-pair sequencing
    (2017-06) Johnson, Sarah
    Mate-pair sequencing (MPseq), using long-insert, paired-end genomic libraries, is a powerful next-generation sequencing-based approach for the detection of genomic structural variants. SVAtools is a set of algorithms to detect both chromosomal rearrangements and large (>10kb) copy number variants (CNVs) in genome-wide MPseq data. SVAtools can also predict gene disruptions, gene fusions, and characterize the genomic structure of complex rearrangements. To illustrate the power of SVAtools’ junction detection methods to provide comprehensive molecular karyotypes, MPseq data was compared against a set of samples previously characterized by traditional cytogenetic methods. Karyotype, fluorescence in situ hybridization (FISH) and chromosomal microarray (CMA), performed for 29 patients in a clinical laboratory setting, collectively revealed 285 breakpoints in 87 rearrangements. The junction detection methods of SVAtools detected 87% of these breakpoints compared to 48%, 42% and 57% for karyotype, FISH and CMA respectively. Breakpoint resolution was also reported to 1 kb or less and additional genomic rearrangement complexities not appreciable by standard cytogenetic techniques were revealed. For example, 63% of CNVs detected by CMA were shown by SVAtools’ junction detection to occur secondary to a rearrangement other than a simple deletion or tandem duplication. SVAtools with MPseq provides comprehensive and accurate whole-genome junction detection with improved breakpoint resolution, compared to karyotype, FISH, and CMA combined. This approach to molecular karyotyping offers considerable diagnostic potential for the simultaneous detection of both novel and recurrent genomic rearrangements in hereditary and neoplastic disorders.