One Library, Many Tests: The Evolution of Next Generation Sequencing Panel Testing

Abstract Number:

490 

First Author:

Devin Rhodenizer  
Emory Genetics Laboratory, Division of Medical Genetics, Department of Human Genetics, Emory Univers
Decatur, GA

Co-Author(s):

Cristina da Silva  
Emory Genetics Laboratory, Division of Medical Genetics, Department of Human Genetics, Emory Univers
Decatur, GA
Neil Skinner  
Emory Genetics Laboratory, Division of Medical Genetics, Department of Human Genetics, Emory Univers
Decatur, GA
Madhuri R Hegde, PhD, FACMG  
Emory Genetics Laboratory, Department of Human Genetics, Emory University
Atlanta, GA

Description:

Next Generation Sequencing for clinical diagnostics has been dominated by two assays: gene panels and Whole Exome Sequencing (WES). Traditional panel testing has the benefits of lower costs and higher coverage of the regions of interest with 95% of targets covered at >20X post experiment. However, logistical problems can arise as more libraries and panels are brought into use along with high sample volume leading to increased costs and slower sample processing. While WES has the benefit of capturing many of the regions of interest in one library, simplifying workflow, the library size limits multiplexing and carries a higher sequencing cost, which in turn can quickly raise costs and limit throughput. WES sequencing has the additional issue of incomplete coverage of known disease causing genes with 65% of exons in HGMD listed genes well covered post experiment with standard exome capture kits. This can lead to missed disease causing variants and incomplete coverage of exons targeted for a specific panel. These exons would need to be covered by another method for 100% coverage, creating additional costs and slowing result reporting. In order to combine the ease of WES with the high coverage and low cost for panels, we leveraged IDT Lockdown probes to create a library targeting all exons in HGMD listed genes. To test this methodology, we used 40 samples previously sequenced at Emory Genetics Laboratory using traditional panel testing. Each sample was indexed, hybridized in pools of 10 samples, and sequenced in groups of 2 pools, 20 samples total, on a single flow cell of a HiSeq2500 with 100bp paired end sequencing on Rapid Run Mode. Coverage and run statistics were recorded for each sample for all targets and only variants for the specified panel were returned, limiting incidental findings. HGMD exon coverage increased to 95% well covered post experiment, with each individual panel increased from 95% to 99% coverage with all known variants detected at an average coverage of 75X. The single library approach decreased cost over individual panels by 30% and decreased technologist hands on time by 20%. The creation of the one library allows for simplified workflow, lower costs, rapid sequencing, and the higher coverage necessary for clinical diagnostic Next Generation Sequencing.

Keywords:

Bioinformatics
Genetic Testing
Identification of Disease Genes
Methodology
Mutation Detection
NextGen Sequencing
Phenotype
Sequencing
Whole exome sequencing

Topic Focus:

Molecular Genetics

Secondary Topic Focus:

Clinical Genetics