Clinical Next-Generation Sequencing Pipeline Outperforms a Combined Approach Using Sanger Sequencing and Multiplex Ligation-Dependent Probe Amplification.

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Presentation transcript:

Clinical Next-Generation Sequencing Pipeline Outperforms a Combined Approach Using Sanger Sequencing and Multiplex Ligation-Dependent Probe Amplification in Targeted Gene Panel Analysis Laila C. Schenkel, Jennifer Kerkhof, Alan Stuart, Jack Reilly, Barry Eng, Crystal Woodside, Alexander Levstik, Christopher J. Howlett, Anthony C. Rupar, Joan H.M. Knoll, Peter Ainsworth, John S. Waye, Bekim Sadikovic The Journal of Molecular Diagnostics Volume 18, Issue 5, Pages 657-667 (September 2016) DOI: 10.1016/j.jmoldx.2016.04.002 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 BRCA1/2 gene panel. A: Coverage plot for a 24-patient sample batch (each line represents one patient); y axis is sequence read depth; gene and exon locations are indicated. B: Sequence coverage depth for three representative BRCA1/2 screens; gene and exon locations are indicated; red lines indicate exon boundaries; x axis indicates nucleotide positions on corresponding genes and exons; y axis indicates depth of sequence coverage (approximate mean coverage for BRACA1/2 = 7000); y axis is on a linear scale so for a sample with a mean coverage of 2000, reduction in coverage to the half equates to approximately 1000 times coverage at that locus. C and D: Normalized copy number plot demonstrating deletion detection (arrows, C) and gene duplication (arrow, D). Gene and exon locations are indicated (x axis); blue lines indicate exon boundaries; y axis represents quantile normalized copy number data (for autosomal genes 0.5 = one copy, 1 = two copies, 1.5 = three copies). The Journal of Molecular Diagnostics 2016 18, 657-667DOI: (10.1016/j.jmoldx.2016.04.002) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 A: BRCA1 and BRCA2 variants identified by NGS in the retrospective cohort. SNPs were classified according to zygosity (homozygous/heterozygous) and location (intron or exon). B: Type of BRCA1 and BRCA2 variants identified by NGS in the prospective patient cohort. The number of deletions, insertions, and exon deletion/duplications identified are shown in each panel. n = 402 (A); n = 240 (B). del, deletion; dup, duplication; NGS, next-generation sequencing; SNP, single nucleotide polymorphism. The Journal of Molecular Diagnostics 2016 18, 657-667DOI: (10.1016/j.jmoldx.2016.04.002) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Sequence coverage depth for three representative patient screens for CMT panel (17 genes: MFN2, LMNA, MPZ, RAB7A, SH3TC2, FIG4, GARS, HSPB1, NEFL, GDAP1, EGR2, TRPV4, HSPB8, LITAF, PMP22, PRX, GJB1) (A); CANcer (25 genes: MUTYH, EPCAM, MSH2, MSH6, BARD1, MLH1, APC, PMS2, NBN, CDKN2A, BMPR1A, PTEN, ATM, CDK4, BRCA2, PALB2, CDH1, TP53, RAD51D, BRCA1, RAD51C, BRIP1, SMAD4, STK11, CHEK2) (B); and MtDNA panel (37 genes: ATP6, ATP8, COX1, COX2, COX3, CYTB, ND1, ND2, ND3, ND4, ND4L, ND5, ND6, TRNA, TRNC, TRND, TRNE, TRNF, TRNG, TRNH, TRNI, TRNK, TRNL1, TRNL2, TRNM, TRNN, TRNP, TRNQ, TRNR, TRNS1, TRNS2, TRNT, TRNV, TRNW, TRNY, RNR1, RNR2) (C). Red lines indicate exon boundaries. The x axis indicates nucleotide positions on corresponding genes and exons; y axis indicates depth of sequence coverage (approximate mean coverage for CMT, 2000; CANcer panel, 4000, and MtDNA, 5000 and 20,000 for blood and muscle, respectively). The y axis is on a linear scale so for a sample with a mean coverage of 2000, reduction in coverage to the half equates to approximately 1000 times coverage at that locus. CANcer, hereditary cancer; CMT, Charcot-Marie Tooth; MtDNA, mitochondrial genome sequencing. The Journal of Molecular Diagnostics 2016 18, 657-667DOI: (10.1016/j.jmoldx.2016.04.002) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 A: Normalized copy number plots for CMT; top image shows the comprehensive CMT panel; bottom image is a zoom in of PMP22 gene showing detection of one deletion and one duplication (indicated by arrows). Red lines indicate exon boundaries. B: CANcer panel; arrow indicates a patient with complex multi-exon deletion in BRCA2 (same patient as in Figure 1C). Red lines indicate exon boundaries. C: MtDNA panel; identification of patients with KSS, with approximately 10% heteroplasmy (in blue and pink) and approximately 50% heteroplasmy (in green). Asterisks indicate hypervariable D-loop regions (m.16,024 to m.576). A–C: The x axis indicates gene/exon positions; y axis represents quantile normalized copy number data (for autosomal genes 0.5 = one copy, 1 = two copies, 1.5 = three copies). CANcer, hereditary cancer; CMT, Charcot-Marie Tooth; KSS, Kearns-Sayre syndrome; MtDNA, mitochondrial genome sequencing. The Journal of Molecular Diagnostics 2016 18, 657-667DOI: (10.1016/j.jmoldx.2016.04.002) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions