Development and validation of a targeted next generation DNA sequencing panel outperforming whole exome sequencing for the identification of clinically relevant genetic variants

Abstract

Next generation sequencing (NGS) technologies have revolutionized our approach to genomic research. The use of whole genome sequencing (WGS), whole exome sequencing (WES), transcriptome profiling, and targeted DNA sequencing has exponentially improved our understanding of the human genome and the genetic complexities underlying malignancy. Yet, WGS and WES clinical applications remain limited due to high costs and the large volume of data generated. When utilized to address biological questions in basic science studies, targeted sequencing panels have proven extremely valuable due to reduced costs and higher sequencing depth. However, the routine application of targeted sequencing to the clinical setting is limited to a few cancer subtypes. Some highly aggressive tumor types, like type 2 endometrial cancer (EC), could greatly benefit from routine genomic analysis using targeted sequencing. To explore the potential utility of a mid size panel (~150 genes) in the clinical setting, we developed and validated a custom panel against WGS, WES, and another commercially available targeted panel. Our results indicate that a mid size custom designed panel is as efficient as WGS and WES in mapping variants of biological and clinical relevance, rendering higher coverage, at a lower cost, with fewer variants of uncertain significance. Because of the much higher sequencing depth that could be achieved, our results demonstrate that targeted sequencing outperformed WGS and WES in the mapping of pathogenic variants in a breast cancer case, as well as a case of mixed serous and high-grade endometrioid EC, the most aggressive EC subtype.

The mutational landscape of recurrent versus nonrecurrent human papillomavirus–related oropharyngeal cancer

“…Our study implemented WES, in addition to the OncoPlex targeted sequencing assay, to interrogate a wide array of recurrence-promoting genetic alterations while testing the utility of a clinically implemented sequencing assay in identifying potentially actionable targets from the outset of treatment and at recurrence. We did find that the agreement between WES and OncoPlex was lower than described in other studies (39,40). However, this was the same general trend we would expect -higher depth of coverage sequencing of a targeted panel of cancer-related genes will yield a higher detection rate than WES, especially for variants that are present at lower variant allele frequencies.…”

Systematic characterization of germline variants from the DiscovEHR study endometrial carcinoma population

“…While there remain open questions related to this hypothesis, the investigation of loss of heterozygosity (sometimes referred to as “allelic loss”) of a tumor suppressors has provided support for this theory [17–20]. Furthermore, the analysis of germline variants has improved the detection of driver mutations when somatic variants have also been available [21, 22]. The Cancer Genome Atlas (TCGA) illustrated that cancer susceptibility genes could be identified across several cancer types using data produced from the germline, including but not limited to EMCA, by searching for enrichment of rare variants that resulted in truncations [23].…”

The High-Throughput Analyses Era: Are We Ready for the Data Struggle?

“…These techniques have impacted every field of molecular research, escalating previously used sequencing technologies [ 11 ], and opening the way to the -omic sciences foundation [ 1 , 2 ]. Indeed, NGS methods allow the sequencing of entire genomes [ 12 , 13 , 14 , 15 ], of exomes [ 16 , 17 , 18 ], of panels of genes related to a disease of interest [ 19 , 20 , 21 ], or of a single gene [ 22 , 23 , 24 , 25 , 26 ], but can also be used to explore the entire transcriptome [ 27 , 28 , 29 ], small RNAs [ 30 , 31 , 32 ], the epigenome [ 33 , 34 ], and the microbiome [ 35 , 36 , 37 , 38 ].…”

“…In this view, it is not surprising that NGS is also becoming a reference method for molecular diagnostics [ 10 ]. In particular, NGS allows not only the analysis, in more patients simultaneously, of disease-related genes in less time and at lower costs than traditional approaches, but also the sequencing of panels of genes up to the complete exome [ 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 ]. In this way, it is possible to increase the diagnostic sensitivity, to discover novel disease-related genes and also obtain data regarding other genes that were potentially acting as disease-phenotype modifiers [ 19 , 40 , 41 , 42 ].…”