Martina I. Lefterova, Peidong Shen, Justin I

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Presentation transcript:

Next-Generation Molecular Testing of Newborn Dried Blood Spots for Cystic Fibrosis Martina I. Lefterova, Peidong Shen, Justin I. Odegaard, Eula Fung, Tsoyu Chiang, Gang Peng, Ronald W. Davis, Wenyi Wang, Martin Kharrazi, Iris Schrijver, Curt Scharfe The Journal of Molecular Diagnostics Volume 18, Issue 2, Pages 267-282 (March 2016) DOI: 10.1016/j.jmoldx.2015.11.005 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Assay workflow for comprehensive CFTR sequencing using blood spots from newborns. Listed are the times for individual steps, when 96 specimens are multiplexed. Computation time for copy number variant (CNV) analysis can vary based on server cluster specifications, and therefore an estimated range is provided based on whether each sample is analyzed sequentially or in parallel (asterisk). DBS, dried blood spot; mPCR, multiplex PCR; MiSeq, MiSeq platform (Illumina, San Diego, CA); QC, quality control. The Journal of Molecular Diagnostics 2016 18, 267-282DOI: (10.1016/j.jmoldx.2015.11.005) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Quality-control (QC) algorithm for read coverage monitoring. A: Read coverage was examined on three different levels: individual samples, amplicons, and bp (red). The samples that failed to pass QC thresholds are indicated. B: Total reads per sample for all 193 samples that were sequenced. One sample (S33 from the 51-sample set) failed to amplify. C: Uniformity of amplicon coverage. Shown is the percentage of amplicons with read coverage >0.5-fold the mean amplicon coverage for each individual sample. Four samples did not pass the empirically established threshold of (mean – 2 × SD): S48, S60, and S92, all from the 96-sample set; in addition to S33 from the 51-sample set). D: Shown is the percentage of bp with coverage >100 reads per bp. Only three samples did not pass this threshold, indicating that the remaining 190 samples could be analyzed further. The asterisk indicates a 51-sample set; dagger, a 96-sample set. DBS, dried blood spot. The Journal of Molecular Diagnostics 2016 18, 267-282DOI: (10.1016/j.jmoldx.2015.11.005) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 CFTR copy number variant (CNV) detection in dried blood spot specimens. The results of three computational CNV prediction methods were integrated and plotted as the log2 ratio of observed versus expected read counts for each exon of each individual sample. Log2 ratios that significantly deviate from baseline are shaded in red if two consecutive exons show a difference in copy number, and in blue if a single exon/intron is affected. CNV results for the 51- and 48-sample sets are shown in A and B, respectively. No significant CNVs were detected in the 96-sample set. The Journal of Molecular Diagnostics 2016 18, 267-282DOI: (10.1016/j.jmoldx.2015.11.005) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions