The Creation and Implementation of a Clinical Exome and Exome Confirmation Pipeline Rhodenizer, Devin; Tanner, Alice; Skinner, Neil; daSilva, Cristina; Huang, Kuan-Hsiang; Hegde, Madhuri.

Department of Human Genetics, Emory University School of Medicine, Atlanta, GA

Abstract

Conclusions

Pipeline Results Clinical Exome and WorkFlow

Sample Exome Confirmation Plate Introduction Whole Exome Sequencing (WES) has rapidly moved from the research laboratory to the clinical laboratory and is increasing in both use and utility for clinical testing. The benefits of WES remains the same for clinical diagnostics as it does for the research setting, specifically sequencing large numbers of genes in a short time frame allowing for rapid evaluation of homogenous, ambiguous, or mild disorders and phenotypes. However, issues such as missing coverage and the high rate of false positives and false negatives associated with WES are amplified in the clinical setting. WES sequencing routinely identifies over 10,000 variants and typically 15 to 20% of the targeted exome is not covered well, with known disease causing genes at a higher rate of 30 to 40%. Exons with no or low coverage could be harboring a causative mutation and clinical labs must be able to verify that the mutations reported are a true positive. Methods Many methodologies have been implemented including using simultaneous WES of parental samples and family members, but the Sanger sequencing is still the gold standard. In order to facilitate better detection of variants in the 5000 genes associated with disease in the Human Gene Mutation Database (HGMD), we have created a Clinical Exome which includes higher coverage of these genes and created an Exome Confirmation pipeline to rapidly confirm chosen variants through Sanger Sequencing. The addition of probes in the genes of interest allows for greater interrogation of disease related genes and the Sanger pipeline allows for efficient confirmation of variants. The Exome Confirmation pipeline combines PrimerFinder, an automated primer design script, GLOBE, an internally developed LIMS system, robotics, and Bioneer Pyrostart lypholized Master Mix plates to rapidly and reliably amplify and sequence variants selected for confirmations. Here we show the comparison data between the Clinical Exome and commercially available exomes as well as the detection and failure rates of the Exome Confirmation pipeline developed and currently in use at Emory Genetics Laboratory. Results The coverage of HGMD listed genes was decreased from 36% average to 11% average in the Clinical Exome compared to commercially available products. The Exome Confirmation Pipeline boasts a 4% failure rate on the first PCR pass, with only 1% of the total needing manual redesign. The combination of the Clinical Exome and the Exome Confirmation pipeline allows for increased diagnostic utility of the test along with rapid validation of potentially disease causing variants.

WES Sequencing

Variant Report Generation

Variant Selection

Confirmation

Interpretation and Report

Sure Select Exome v5

Additional Probes

A representative gel Image depicting 3 Exome Confirmation plates with 3 different patient samples. Large Gaps are water blanks showing no contamination. Of these three plates, only a few amplicons failed to amplify.

Exome Coverage

Sanger Confirmation Rates

The coverage of HGMD listed genes was decreased from 36% average to 11% average in the Clinical Exome compared to commercially available products. The Exome Confirmation Pipeline boasts a 4% failure rate on the first PCR pass, with only 1% of the total needing manual redesign. 90% of all variants selected confirm by Sanger, and variant confirmation takes as little as 2 weeks before the final report is ready for reporting. The combination of the Clinical Exome and the Exome Confirmation pipeline allows for increased diagnostic utility of the test along with rapid validation of potentially disease causing variants. A collaborative effort between EGL, CHOP, and LMM has improved upon this design. Additional probes were added to the design for HGMD genes and some probes removed for unnecessary genes. This effort has increased coverage of HGMD listed genes to over 97% and strategies have been developed to increase this coverage to over 99%.

Coverage and Pipeline Statistics The Clinical Exome increased coverage of the Exome from 80% to 90%. HGMD listed genes were more fully covered, showing an overall increase of 40% over the standard exome. 90% of all variants selected are confirmed by the Confirmation Pipeline. PrimerFinder has a design failure rate of 5%, with designed primers failing the first pass at a rate of 4% and only a total of 1% needing manual redesign.