The use of next generation sequencing (NGS) in discovery and diagnostic applications is rapidly expanding. Improvements in methods and reagents, as well as decreasing costs for sequencing, are the main drivers of this trend. High-throughput instruments sequence a remarkably large number of bases per run, but each instrument has an upper limit of bases that can be accurately read. For example, at its upper limit, the HiSeq® 2500 Instrument (Illumina) can sequence the human genome—approximately 3 billion base pairs—for about 10 individuals, at 20–30X coverage. While this is sufficient for investigating germline variations responsible for inherited traits or diseases, other applications may require larger sample sets or greater read depths.
By using a target enrichment strategy, researchers can focus the read capacity of their instrument to either run more samples, obtain more sequence reads in a subset of the genome, or both. The benefits of target enrichment are particularly important for sequencing rare variants, where hundreds of targeted reads are required to find key mutation(s) against a large genetic background. Areas where high read depth is indispensable include cancer research and studies of immune receptor diversity.
Of note, the target capture step can influence the quality of the final data, with panels and probe sets from some manufacturers performing poorly in GC-rich regions. This results has led some investigators to argue that it is better to just perform whole genome sequencing (WGS). However, we have demonstrated a method using individually synthesized, quality checked, DNA target capture probes (xGen® Lockdown® Probes) that eliminates the negative influence of poor captures. In her presentation titled, Analyzing the exome—focusing your NGS analysis with high performance target capture, Dr Kristina Giorda uses data from the xGen Exome Research Panel to show examples of how this target capture method performs across a variety of metrics and how it compares to other available solutions. Learn ways to improve your NGS target capture by watching the recorded presentation below.