Evaluation of Second-Generation Sequencing of 19 Dilated Cardiomyopathy Genes for Clinical Applications Sivakumar Gowrisankar, Jordan P. Lerner-Ellis,

Slides:

Advertisements

Similar presentations

Clinical Laboratory Analysis of Immunoglobulin Heavy Chain Variable Region Genes for Chronic Lymphocytic Leukemia Prognosis Philippe Szankasi, David.

Advertisements

Martina I. Lefterova, Peidong Shen, Justin I

Lawrence J. Jennings, Maria E

Detection of Exon 12 Mutations in the JAK2 Gene

Mutation Screening of EXT1 and EXT2 by Denaturing High-Performance Liquid Chromatography, Direct Sequencing Analysis, Fluorescence in Situ Hybridization,

Ane Y. Schmidt, Thomas v. O. Hansen, Lise B

The Use of COLD-PCR and High-Resolution Melting Analysis Improves the Limit of Detection of KRAS and BRAF Mutations in Colorectal Cancer Irene Mancini,

Implementation of a Reliable Next-Generation Sequencing Strategy for Molecular Diagnosis of Dystrophinopathies Melissa Alame, Delphine Lacourt, Reda.

Carol Beadling, Tanaya L. Neff, Michael C

Martina I. Lefterova, Peidong Shen, Justin I

Emily M. Kudalkar, Naif A. M

In Silico Proficiency Testing for Clinical Next-Generation Sequencing

A Method to Evaluate the Quality of Clinical Gene-Panel Sequencing Data for Single- Nucleotide Variant Detection Chung Lee, Joon S. Bae, Gyu H. Ryu, Nayoung.

Validation of a Next-Generation Sequencing Pipeline for the Molecular Diagnosis of Multiple Inherited Cancer Predisposing Syndromes Paula Paulo, Pedro.

Screening for Mutations in Kidney-Related Genes Using SURVEYOR Nuclease for Cleavage at Heteroduplex Mismatches Konstantinos Voskarides, Constantinos.

Performance of Common Analysis Methods for Detecting Low-Frequency Single Nucleotide Variants in Targeted Next-Generation Sequence Data David H. Spencer,

Targeted, High-Depth, Next-Generation Sequencing of Cancer Genes in Formalin- Fixed, Paraffin-Embedded and Fine-Needle Aspiration Tumor Specimens Andrew.

Robustness of Amplicon Deep Sequencing Underlines Its Utility in Clinical Applications Vera Grossmann, Andreas Roller, Hans-Ulrich Klein, Sandra Weissmann,

Improving Mutation Screening in Patients with Colorectal Cancer Predisposition Using Next-Generation Sequencing Jean-Marc Rey, Vincent Ducros, Pascal.

Yanggu Shi, Sharon F. Terry, Patrick F. Terry, Lionel G

Molecular Analysis of Circulating Cell-Free DNA from Lung Cancer Patients in Routine Laboratory Practice Stephan Bartels, Sascha Persing, Britta Hasemeier,

Application of Single-Molecule Amplification and Resequencing Technology for Broad Surveillance of Plasma Mutations in Patients with Advanced Lung Adenocarcinoma

Next Generation Sequencing for Clinical Diagnostics-Principles and Application to Targeted Resequencing for Hypertrophic Cardiomyopathy Karl V. Voelkerding,

B-Cell Clonality Determination Using an Immunoglobulin κ Light Chain Polymerase Chain Reaction Method Reetesh K. Pai, Artemis E. Chakerian, John M. Binder,

Application of Single-Molecule Amplification and Resequencing Technology for Broad Surveillance of Plasma Mutations in Patients with Advanced Lung Adenocarcinoma

Philippe Szankasi, Mohamed Jama, David W. Bahler

Association of Clinical Status of Follicular Lymphoma Patients after Autologous Stem Cell Transplant and Quantitative Assessment of Lymphoma in Blood.

Ming-Tseh Lin, Roy G. Rich, Royce F. Shipley, Michael J

Clinical Relevance of Sensitive and Quantitative STAT3 Mutation Analysis Using Next- Generation Sequencing in T-Cell Large Granular Lymphocytic Leukemia

Molecular Diagnosis of Autosomal Dominant Polycystic Kidney Disease Using Next- Generation Sequencing Adrian Y. Tan, Alber Michaeel, Genyan Liu, Olivier.

A Pyrosequencing-Based Assay for the Rapid Detection of IDH1 Mutations in Clinical Samples Prashanth Setty, Jennifer Hammes, Thomas Rothämel, Valentina.

Evaluation of the Cepheid GeneXpert BCR-ABL Assay

Influence of the Duplication of CFTR Exon 9 and Its Flanking Sequences on Diagnosis of Cystic Fibrosis Mutations Ayman El-Seedy, Tony Dudognon, Frédéric.

Single Monochrome Real-Time RT-PCR Assay for Identification, Quantification, and Breakpoint Cluster Region Determination of t(9;22) Transcripts Marina.

Detection of an Apparent Homozygous 3120G>A Cystic Fibrosis Mutation on a Routine Carrier Screen Denise LaMarche Heaney, Patrick Flume, Lauren Hamilton,

William L. Gerald, M.D., Ph.D, 1954–2008

Design and Validation of a Conformation Sensitive Capillary Electrophoresis-Based Mutation Scanning System and Automated Data Analysis of the More than.

SeqSharp The Journal of Molecular Diagnostics

Clinical Laboratory Analysis of Immunoglobulin Heavy Chain Variable Region Genes for Chronic Lymphocytic Leukemia Prognosis Philippe Szankasi, David.

High-Throughput and Sensitive Quantification of Circulating Tumor DNA by Microfluidic- Based Multiplex PCR and Next-Generation Sequencing Yinghui Guan,

Catherine E. Cottrell, Hussam Al-Kateb, Andrew J. Bredemeyer, Eric J

Detection of Exon 12 Mutations in the JAK2 Gene

Comprehensive Diagnostic Testing for Stereocilin

Hilmi Ozcelik, Xuejiang Shi, Martin C

Screening for Expanded Alleles of the FMR1 Gene in Blood Spots from Newborn Males in a Spanish Population Isabel Fernandez-Carvajal, Paulina Walichiewicz,

The c.1364C>A (p.A455E) Mutation in the CFTR Pseudogene Results in an Incorrectly Assigned Carrier Status by a Commonly Used Screening Platform Kristin.

Utility of NIST Whole-Genome Reference Materials for the Technical Validation of a Multigene Next-Generation Sequencing Test Bennett O.V. Shum, Ilya.

PIK3CA Hotspot Mutation Scanning by a Novel and Highly Sensitive High-Resolution Small Amplicon Melting Analysis Method Panagiotis A. Vorkas, Nikoleta.

Molecular Monitoring of Chronic Myelogenous Leukemia

Bassem A. Bejjani, Lisa G. Shaffer

Design and Evaluation of a Real-Time PCR Assay for Quantification of JAK2 V617F and Wild-Type JAK2 Transcript Levels in the Clinical Laboratory Jason.

Ye Bang-Ce, Chu Xiaohe, Fan Ye, Li Songyang, Yin Bincheng, Zuo Peng

Mutation Detection of Epidermal Growth Factor Receptor and KRAS Genes Using the Smart Amplification Process Version 2 from Formalin-Fixed, Paraffin-Embedded.

A Clinically Validated Diagnostic Second-Generation Sequencing Assay for Detection of Hereditary BRCA1 and BRCA2 Mutations Ian E. Bosdet, T. Roderick.

Performance and Clinical Evaluation of a Sensitive Multiplex Assay for the Rapid Detection of Common NPM1 Mutations Michael Hafez, Fei Ye, Keith Jackson,

Lawrence J. Jennings, Maria E

Avoiding Pitfalls in Molecular Genetic Testing

Amplification Refractory Mutation System, a Highly Sensitive and Simple Polymerase Chain Reaction Assay, for the Detection of JAK2 V617F Mutation in Chronic.

Feras M. Hantash, Arlene Rebuyon, Mei Peng, Joy B

Inherited Cardiomyopathies

Improved Detection of KIT Exon 11 Duplications in Formalin-Fixed, Paraffin-Embedded Gastrointestinal Stromal Tumors Jerzy Lasota, Bartosz Wasag, Sonja.

Statistical Treatment of Fluorescence in Situ Hybridization Validation Data to Generate Normal Reference Ranges Using Excel Functions Allison L. Ciolino,

Karen Snow-Bailey, Ph.D., 1961–2006

Custom Design of a GeXP Multiplexed Assay Used to Assess Expression Profiles of Inflammatory Gene Targets in Normal Colon, Polyp, and Tumor Tissue Janice.

Detection of BRCA1 and BRCA2 Ashkenazi Jewish Founder Mutations in Formalin- Fixed Paraffin-Embedded Tissues Using Conventional PCR and Heteroduplex/Amplicon.

Characterization of a Recurrent Novel Large Duplication in the Cystic Fibrosis Transmembrane Conductance Regulator Gene Feras M. Hantash, Joy B. Redman,

Monique A. Johnson, Marvin J. Yoshitomi, C. Sue Richards

Development of a Novel Next-Generation Sequencing Assay for Carrier Screening in Old Order Amish and Mennonite Populations of Pennsylvania Erin L. Crowgey,

Lawrence J. Jennings, Maria E

Quantification of bcl-2/JH Fusion Sequences and a Control Gene by Multiplex Real- Time PCR Coupled with Automated Amplicon Sizing by Capillary Electrophoresis

Presentation transcript:

Evaluation of Second-Generation Sequencing of 19 Dilated Cardiomyopathy Genes for Clinical Applications Sivakumar Gowrisankar, Jordan P. Lerner-Ellis, Stephanie Cox, Emily T. White, Megan Manion, Kevin LeVan, Jonathan Liu, Lisa M. Farwell, Oleg Iartchouk, Heidi L. Rehm, Birgit H. Funke The Journal of Molecular Diagnostics Volume 12, Issue 6, Pages 818-827 (November 2010) DOI: 10.2353/jmoldx.2010.100014 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Flow chart of experimental design and analytical pipeline. Pooled, positive controls were amplified by PCR as described in Materials and Methods. The number of mutations is indicated for each positive control sample. Each positive control sample was sequenced on one lane of one flow cell on the Illumina GAII instrument. The Illumina GAII pipeline uses three software packages for image analysis (Firecrest), base calling (Bustard), and analysis (Gerald). Variant detection was carried out using the NextGENe software package (SoftGenetics, LLC). The number of false positives (FP), false negatives (FN), and true positives (TP) identified by NextGENe are indicated for the given parameters and thresholds described in Materials and Methods. The Journal of Molecular Diagnostics 2010 12, 818-827DOI: (10.2353/jmoldx.2010.100014) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Illumina GAII sequence data. A: Average condensed coverage for each amplicon within the region of interest (ROI) for each sample. Amplicons are arranged by concatenation of the ROI in alphabetical order of gene names and by sequential order of exons (see Supplemental Table 2 at http://jmd.amjpathol.org). B: Distribution of the fraction of bases within the ROI with a given condensed coverage in intervals of 10 (see Supplemental Table 3 at http://jmd.amjpathol.org). C: Graphical representation of the distribution of condensed coverage within and surrounding the ROI. Dotted lines demarcate the boundaries of the ROI. The read depths of all bases within a given bin is averaged over all amplicons and plotted as a point in the graph. To do this, we divided each amplicon's region of interest into 10 bins and calculated the average read depth within each bin by taking the mean of the coverage of all of the bases. The flanking regions are obtained from a 200-bp window divided into four bins. The Journal of Molecular Diagnostics 2010 12, 818-827DOI: (10.2353/jmoldx.2010.100014) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Comparative analysis of array-based resequencing and Illumina GAII sequencing workflows with regard to cost and turnaround time. Laboratory workflows for the DCM array (left) and DCM SGS test (right) are shown. Steps that lead to increased test cost or turnaround time are highlighted in gray. The estimated relative impact of transitioning the DCM test from the array-based technology to SGS-based technology is shown on the right by arrows as well as the level of expected increase. The Journal of Molecular Diagnostics 2010 12, 818-827DOI: (10.2353/jmoldx.2010.100014) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions