Enrichment of genomic regions of interest is the most reliable and cost-effective way of performing pharmacogenomics and multi-gene disease targeted sequencing. One of the greatest challenges for clinical targeted Next-Generation Sequencing methods is obtaining complete and uniform NGS coverage for all target regions. Some popular methods for target enrichment rely on lengthy and inefficient hybrid capture or multiplexed PCR techniques, resulting in lower coverage and more off target reads. The need for improved NGS coverage is clear.
Now WaferGen has met the challenge to ensure more uniform NGS coverage with the development of the high-density SmartChip TE System. One SmartChip TE panel supports conducting hundreds to thousands of parallel, singleplex PCR reactions to efficiently enrich desired target regions in less than 3 hours. Since amplification of each target region occurs in an individual PCR reaction, the method is not susceptible to interference due to multiplexing. Following amplification, all PCR products from an individual sample are collected as amplicon pools in a single step and are ready for standard library preparation protocols. This format also enables quick and easy modifications to panel content during development and optimization.
The implications of the improvements in specificity, uniformity and NGS coverage are significant. While target enrichment with uniform NGS coverage approaching 100% has been difficult to achieve, until now, future implementations of routine Next-Gen Sequencing in diagnostic testing will require clinical labs to get as close to this goal as possible. Our proprietary approach to solving this challenge is based on massively-parallel, singleplex PCR reactions, in which amplification is cleaner and better controlled. WaferGen has set a new standard of possibility in the field of clinical Next-Generation Sequencing with superior coverage, specificity and uniformity in Target Enrichment.
Target enrichment methods providing complete coverage, uniform read depth across targets, and a simple and reproducible workflow are required for successful implementation in a clinical setting. Existing multiplex PCR-based methods and hybrid capture are often inadequate in these areas. Now, clinical investigators can utilize the SmartChip TE system to obtain the most complete and uniform coverage among target enrichment technologies. Unprecedented enrichment quality is achieved with flexibility of running up to 2500 unique singleplex reactions on a single SmartChip TE.
Figure 1. Performance data for a typical SmartChip TE Panel (140 kb target region) targeting all exonic regions in a 17 gene set. High design rates and % bases on target ensure specific amplifcation necessary for efficient enrichment. These panels result in high coverage rates up to 100x and very high uniformity of coverage (> 98%) at 10% of mean coverage.
Figure 2 Figure 2. Uniformity at 0.1x, 0.2x and 0.5x of the mean coverage is shown for a 140kb target region. Data is plotted for 20 samples on a single MiSeq run and is typical of high uniformity and repeatability of uniformity from sample to sample. For all samples, >95% of bases are covered at 0.1x and 0.2x of the mean coverage (green and orange bars, respectively) and >85% of bases are covered at 0.5x of the mean coverage (purple bars).