Improved Library Prep Offers Higher Percentage of On-target Reads and Better Coverage for SureSelectXT2 Target Capture
Application Note Feb 13, 2015
Next-Generation Sequencing (NGS) has provided the scientific community with a powerful tool to sequence large and complex genomes in far less time than traditional Sanger sequencing methods. Even so, we have not yet reached a point in which sequencing and analyzing the 3.2 billion base human genome is either time or cost effective. Target capture strategies have been shown to provide a solution to this problem by reducing genome complexity to what is relevant to the question being asked. Target capture approaches require whole genome library preparation followed by systematic target selection through probe hybridization to genomic regions of interest followed by massively parallel sequencing. The size and complexity of the capture is user dependent but can range from large scale targeting of all protein coding regions (~50 Mb) to a subset of genes known to be involved in specific biological functions (<10 Mb). Pre-capture pooling of multiple samples prior to target capture is a cost effective method to maximize the amount of data obtained for multiple samples in a single sequencing run. Several target capture technologies are commercially available, all of which have their individual strengths and weaknesses. Agilent’s SureSelectXT2 system offers a wide range of probe sets including All Exon options for many different model organisms, as well as a flexible custom option allowing users to design their own capture panels, all of which can be purchased independent of upstream and downstream reagents required to construct sequence-ready capture libraries.
Target capture performance is dependent on several variables; however, one important factor is the barcode blocking strategy employed. Barcode blocking oligos bind to platform binding adapter sequences to reduce non-specific probe hybridization. A problematic class of unwanted molecules arise during in-solution hybridization target capture from the annealing of unrelated DNA sequences at complimentary terminal adapter sequences during probe hybridization. Identical adapter terminal sequences flank each template molecule in the hybridization pool. Thus, this molecule and its complement are in very high concentration relative to other molecules in solution. The joining of non-complementary template molecules at either terminal adapter sequence can be as little as two molecules, or a chain of many different unrelated sequences. Consequently, the presence of a bait binding site on one of the linked molecules will pull down the entire linked chain, resulting in a larger number of contaminating molecules and increasing the number of off-target reads in target capture sequencing results.
Bioo Scientific now offers all reagents required upstream and downstream of probe hybridization to prepare sequence-ready, multiplexed capture libraries compatible with all SureSelectXT2 bait sets. NEXTflex barcode blocker technology allows for maximum binding efficiency through index-specific blocking oligos, greatly reducing the number of off-target reads encountered in small and large target capture assays. Furthermore, utilization of NEXTflex reagents with SureSelectXT2 bait set improves target capture efficiency through robust library preparation workflow as well as important hybridization efficiency improvements.