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Combining Highly Multiplexed PCR with Semiconductor-Based Sequencing for Rapid Cancer Genotyping
Carol Beadling, Tanaya L. Neff, Michael C. Heinrich, Katherine Rhodes, Michael Thornton, John Leamon, Mark Andersen, Christopher L. Corless The Journal of Molecular Diagnostics Volume 15, Issue 2, Pages (March 2013) DOI: /j.jmoldx Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions
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Figure 1 Protocol for multiplex PCR library preparation and semiconductor-based sequencing. The Journal of Molecular Diagnostics , DOI: ( /j.jmoldx ) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions
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Figure 2 Sequence read distribution across 190 amplicons generated from 45 FFPE specimens, normalized to 400,000 reads per sample. A: Average number of reads observed for each amplicon. B: Number of targets with a given read depth, sorted in bins of 100 reads. Data are expressed as means ± SD (A). The Journal of Molecular Diagnostics , DOI: ( /j.jmoldx ) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions
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Figure 3 Sensitivity and specificity of variant allele detection. A: Schematic summary of variant calls. B: The ROC curve shows sensitivity versus specificity of variant allele detection using variant allele cutoffs of 1%, 5%, 8%, 10%, 15%, 20%, and 50%. An 8% variant allele cutoff (vertical arrow) yields 100% sensitivity (95% CI = 97.3% to 100.0%) and 95.1% specificity (a 4.9% false-positive rate; 95% CI = 91.8% to 98.0%). C: Concordance of mutant allele ratios detected by a MassArray mass spectrometry–based platform and an Ion AmpliSeq kit. A total of 53 point mutations were evaluated in FFPE specimens. D: Concordance of mutant allele ratios detected in singleplex samples run on Ion 314 chips and fourplex samples run on Ion 316 chips. The Journal of Molecular Diagnostics , DOI: ( /j.jmoldx ) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions
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