What’s the best strategy for finding low-frequency genetic variants?

targeted DNA sequencing

Whole-exome sequencing (WES) is sequencing the entire protein-coding region in a genome. By contrast, targeted panel sequencing is the sequencing of a small subset of genes or genomic regions. Lower cost per sample, reduced data analysis volume and faster turn-around time are some of the practical reasons why targeted sequencing is preferred over WES or whole genome sequencing (WGS). But is there a research situation where these benefits are not limited to workflow enhancements, but are also the better scientific choice?

Sequencing the whole exome or whole genome is the preferred approach for pan-genomic approaches looking to uncover novel variants in inherited disease. Typically, both WGS and WES offer moderate levels of sequencing between 30X and 100X, but targeted panel sequencing offers the ability to increase coverage by at least a factor of 10 with coverages starting at 1000X. These samples are for the most part homogeneous, as most non-cancerous tissues do not display significantly different clonal populations. Heterogeneous tissues, on the other hand, like tumor samples, can be trickier. These clonal populations may present mutations with disease significance, but only make up varying percentages of the mass of malignant tissue. Because sequencing fidelity is based on many factors including sequencing depth, library efficiency and isolation, targeted panels offer increased sensitivity and the ability to detect these low percentage allelic variants. By sequencing to a higher on-target depth, the ability to detect the presence of low-frequency genetic variants increases as they become less likely to be sequencing artifacts. This deep sequencing approach enables clearer analysis of clonal diversity which may not be clear at low depth. With the deeper coverage offered by panel approaches, you can more confidently identify low allele frequency variants in heterogeneous samples for further analysis and interpretation.

In a recent study, Cornella et al. showed how WES followed by targeted panel sequencing enabled them to identify new low-frequency mutations in fibrolamellar hepatocellular carcinoma (FLC). In this study, the authors analyzed 78 FLC samples by multiple techniques including whole-genome expression profiling, SNP array, WES, TDS and fusion transcript analysis and identified a unique genome profile that differentiates FLC from other common liver cancers such as hepatocellular carcinoma (HCC) or intrahepatic cholangiocarcinoma (ICC). Using whole-genome expression profiling, they showed that there are 3 distinct molecular classes of FLC, two of which are associated with proliferative or inflammatory molecular traits of liver cancers, and the third showing an overall enrichment of neuro-endocrine markers and EGFR protein expression.

The team also evaluated the mutational landscape of FLC by sequencing the entire exome and identified 276 somatic variants. Of these, 68 were nonsynonymous mutations and 23 of these were damaging mutations such as BRCA2-Y2789C, CSMD2-G1055E, ARMCX1 and COL6A6. As BRCA2 is a known tumor suppressor gene, they verified the data by sequencing the entire BRCA2 gene. They used GeneRead DNAseq Targeted Panels V2 to enrich the BRCA2 genes from 47 FLC samples along with 14 paired non-tumoral samples. Using the targeted panel sequencing strategy, they not only confirmed the WES data but also identified a new mutation in BRCA2: P2612S. This clearly demonstrates that the TDS strategy enables discovery of rare, low-frequency mutations with potential for future use as biomarkers.

Do you still have questions about the scope of targeted panel sequencing? Let me know!

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Reference

Cornella, H. et al. (2015) Unique genome profile of fibrolamellar hepatocellular carcinoma. Gastroenterology 148, 806.

Krishnan Allampallam, Ph.D.

Senior Global Marketing Manager, Translational Sciences

Dr. Krishnan Allampallam has spent over 10 years in the biotechnology industry. Before beginning his career in biotech, Dr. Allampallam worked on pathophysiology of myelodysplastic syndrome and authored and co-authored multiple publications, abstracts and book chapters. He received his Ph.D. from University of Toledo Medical Center in 1996 and did post-doctoral research at The Cleveland Clinic and Rush University Medical Center. He has been at QIAGEN since 2008.

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