You can supplement an existing pool of array-synthesized baits, such as SureSelect® (Agilent) and SeqCap® (Nimblegen) target enrichment kits, with IDT xGen® Lockdown® Probes to improve capture of high GC content and gap regions. Just design xGen Lockdown Probes against target regions for which sequencing coverage is known or has been found to be poor; add 1 μL Lockdown Probes (1.6 μM) per 1 μL existing probe pool, and perform hybridization according to the manufacturer’s protocol*. Figure 1 demonstrates that as few as 3 xGen Lockdown Probes added to a 1.1 Mb array-based bait set enhanced coverage of a GC-rich exon. In an additional example, another research group used 4 Lockdown Probes to uniformly rescue gap regions in chromosome 7 of the HSPB1 gene (data not shown). As well as providing reduced GC bias, xGen Lockdown Probes can improve overall coverage uniformity of all target regions while maintaining the sensitivity and specificity of the enrichment. Learn more at www.idtdna.com/xgen.
* SureSelect reagents are not recommended for use with xGen® Lockdown® Probes when targeting genomic regions that have >70% A/T.
Figure 1. Rescue of Gap Regions Using xGen® Lockdown® Probes. 3 xGen Lockdown Probes were mixed with array-derived RNA baits and hybridized using standard conditions. Samples were sequenced on an Illumina HiSeq® 2000 platform using 49 x 49 paired-end reads. Supplementing with Lockdown Probes increased coverage from ~159X to ~495X. [Data courtesy of Foundation Medicine, Inc.]