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Issue link: https://clinicalomics.epubxp.com/i/827649
www.clinicalomics.com May/June 2017 Clinical OMICs 11 NGS is in routine use for research and clinical appli- cations. While cost and data analysis complex- ity are still hurdles for whole genome sequencing, targeted approaches are cost-effective and generate less, but equally meaningful, data in a greatly reduced timescale. As NGS moves forward, there is a clear need for confident calling of all variants—without false negatives or positives. The choice of enrichment assay is important as, if poorly selected, can be one source of error. Recent advances in bait design and streamlined protocols make hybridization-based target enrichment an increas- ingly attractive option, offering enhanced perfor- mance at a speed and cost compa- rable to ampli- con (PCR)-based methods. Optimizing hybridization assays via advanced probe design and placement provides excellent uniformity of coverage, fewer false positives, and superior variant detection. The Importance of Bait Design Hybridization protocols use long oligonucleotide baits for target capture. Advanced bait design strategies and algorithms can be used to overcome common sequenc- ing challenges such as GC-rich regions and internal tan- dem repeats. In GC-rich regions, hybridization baits can be designed to capture efficiently giving a high level of uniform coverage. For example, the very high GC content (up to 90%) and number of repeat regions in the tumor suppressor gene, CEBPA, has hampered past sequencing attempts. Bait design expertise can overcome these diffi- culties, providing excellent depth of coverage and unifor- mity. Internal tandem duplications of up to 200bp can be sequenced efficiently by employing experience garnered from other probe hybridization technologies. Ironing Out the Kinks Duplicates are a common problem in target enrichment assays, leading to largely over-represented regions. Hybrid- ization assays employ random shearing of genomic DNA providing unique and overlapping captured fragments. Duplicates can be readily identified as those that align with each other, and be computationally removed—leaving high-quality data for analysis. The most common reason for false negatives is lack of cov- erage from non-uniform target enrichment. When enrich- ment is uniform, regions are represented more equally, ensuring any variants present are more likely to be iden- tified. This is particularly important when looking at het- erogenous samples. This increased confidence allows lower average sequencing depths to be used, enabling larger num- bers of samples to be multiplexed in a run, saving costs. In contrast, a common cause of false positives are artifacts introduced by PCR polymerases. As hybridization assays use very few PCR cycles, this risk is greatly reduced, as a consequence delivering higher detection sensitivity of low frequency variants—down to 1% variant allele frequency in some cases. Hybridization assays can be used for any target region size—from very small to entire exomes—and the higher accuracy of hybridization-based methods offers greater con- fidence when working with challenging samples, whether this be due to DNA quality or quantity, and can be opti- mized to perform well with FFPE samples and input quan- tities as little as 10ng. Streamlined Protocols Although traditionally more time consuming than ampl- icon-based approaches, work to streamline hybridization protocols means that users can now go from sample to sequencer in a single day. Protocol modifications such as the use of enzymatic fragmentation, optimizing and combining steps, and reducing the hybridization time down to as little as 30 minutes, have made this possible. Advances in hybridization-based assays are raising the bar in NGS target enrichment—providing new sequencing possibilities and increased confidence in data. Raising the Bar in Target Enrichment OP-ED Ed Cook Senior Product Manager, Cancer NGS Oxford Gene Technology CORRECTION: In last month's issue, on page 27, we referred to ddPCR as "digital droplet polymerase chain reaction" when the correct term is "droplet digital poly- merase chain reaction." We regret the error. iNueng/ Getty Images