Agilent’s SureSelect Target Enrichment System significantly improves the cost- and process-efficiency of next-generation sequencing. Based on an extremely efficient hybrid selection technique, the SureSelect Target Enrichment System can be automated and easily scaled to meet the needs of larger sequencing projects. This is unlike any other commercially available target enrichment method. With sample input requirements at or below 3 µg of gDNA, researchers can perform highly targeted next-generation sequencing on the most precious of samples.
The SureSelect Target Enrichment Platform is available through eArray, our online custom microarray design tool. For more information, visit the Agilent’s eArray.
As shown in Figure 1, the SureSelect Target Enrichment System workflow is solution-phase based and is performed in microcentrifuge tubes or microtiter plates. It has been designed to work with the Illumina Genome Analyzer system in order to optimize end sequencing. Protocol optimization for the Life Technologies SOLiD system is also well underway. Utilizing Agilent’s eArray, an intuitive web-based design tool with wizards, researchers can create customized target enrichment kits. Each target enrichment kit comes with the mixture of custom SureSelect RNA oligonucleotides, or baits, that are biotinylated for easy capture onto streptavidin-labeled magnetic beads, as well as buffers and blocking agents necessary for performing the capture process.
Zoom
Figure 1. SureSelect Target Enrichment System Workflow.
Because each new custom kit is composed of a unique set of baits, validation of kits may be of concern. To test this, a diverse set of enrichment designs were created and tested in parallel against the same sample. To measure performance, we analyzed the sequencing reads for percent in the targeted regions because this is one of the most direct measures of enrichment efficiency. The percent on target for eight designs is within approximately 40-80%, demonstrating efficiency in focusing on only regions of interest across several diverse kit designs. This translates into enrichments of 300-7400 fold, which is largely dependent upon the size of the targeted region and the number of reads per sequencing run – small capture designs typically yield a higher level of enrichment due to the kit’s efficiency in focusing sequencing reads on a smaller subset of the targeted genome.
Zoom
Figure 2. SureSelect Target Enrichment Robustness Across a Range of Custom Designs
No matter how specific the capture methodology, because of random shearing, both the targeted region and nearby DNA targets will be captured. Agilent has optimized the design algorithms and protocol to limit the capture of near target sequences. To this end, the specificity of the SureSelect System was measured by analyzing sequencing reads exactly on target, within 100 bp of the target, and within 200 bp of the target. Figure 2 shows the off-target capture rate is not substantially increased by including sequence reads within up to 200 bp of the target.
Zoom
Figure 3. SureSelect Target Enrichment Sequence Coverage
Another metric of great importance to DNA sequencing researchers is read distribution, as it affects the ability to adequately cover genomes to identify sequence variation. There are two ways of looking at this metric: 1) plot the distribution of actual percent of bases with a certain number of reads; and 2) plot the cumulative number of reads with at least a certain depth. Figure 3 is a plot of a representative sample showing that roughly 80% of all sequences have at least a 20X read depth, indicating the suitability of this method for identifying SNPs with targeted re-sequencing.
The design robustness, specificity and comprehensive sequence coverage distinguish the SureSelect Target Enrichment System from other commercially available target enrichment products.