A direct comparison of next generation sequencing enrichment methods using an aortopathy gene panel- clinical diagnostics perspective

Abstract

Background: Aortopathies are a group of disorders characterized by aneurysms, dilation, and tortuosity of the aorta. Because of the phenotypic overlap and genetic heterogeneity of diseases featuring aortopathy, molecular testing is often required for timely and correct diagnosis of affected individuals. In this setting next generation sequencing (NGS) offers several advantages over traditional molecular techniques.

Methods: The purpose of our study was to compare NGS enrichment methods for a clinical assay targeting the nine genes known to be associated with aortopathy. RainDance emulsion PCR and SureSelect RNA-bait hybridization capture enrichment methods were directly compared by enriching DNA from eight samples. Enriched samples were barcoded, pooled, and sequenced on the Illumina HiSeq2000 platform. Depth of coverage, consistency of coverage across samples, and the overlap of variants identified were assessed. This data was also compared to whole-exome sequencing data from ten individuals.

Results: Read depth was greater and less variable among samples that had been enriched using the RNA-bait hybridization capture enrichment method. In addition, samples enriched by hybridization capture had fewer exons with mean coverage less than 10, reducing the need for followup Sanger sequencing. Variants sets produced were 77% concordant, with both techniques yielding similar numbers of discordant variants.

Conclusions: When comparing the design flexibility, performance, and cost of the targeted enrichment methods to whole-exome sequencing, the RNA-bait hybridization capture enrichment gene panel offers the better solution for interrogating the aortopathy genes in a clinical laboratory setting.

Figure 1

Mean depth of coverage per aortopathies gene for the enrichment methods. In A, both custom-designed gene panels (SureSelect and RainDance) yielded significantly higher average coverage per gene compared to whole-exome sequencing (SureSelect 50 Mb All Exon Capture). Bars indicate one standard deviation. In B, the fraction of exons covered to the given depth for the enrichment methods is shown. The custom-designed SureSelect gene panel yielded greater average read depths across the 294 exons for all samples versus the RainDance or whole-exome enrichments.

Figure 2

Mean exon read depth as a function of reference sequence GC content for the custom-designed gene panels. High GC content resulted in decreased exon coverage in the SureSelect enriched samples. GC content had little to no effect on exon coverage of the RainDance enriched samples, which were more prone to sporadic amplicon failure.

Figure 3

Number of exons with mean read depth less than 20, across samples (x-axis) and genes (y-axis). The color of the block represents the number of exons with low coverage for a given sample and gene.

Figure 4

Variability in coverage: RainDance versus SureSelect. Standard deviation in coverage for each exon across enrichment samples was computed. The mean deviation across exons reflects the overall degree of coverage variability across samples 1–6, with zero indicating that each exon was covered by the same number of reads in every sample. Between sample variability was significantly higher for the RainDance enrichment.