Next Generation Sequencing
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NGS Target Capture Custom Panels—Determining Probe Number and Cost

To determine the number (and cost) of probes you need when designing customized target capture panels, you should consider the following aspects of your experiment: 

CDS vs. Whole Gene vs. UTR

Most target capture strategies are designed against coding sequence (CDS), where the majority of mutations known to alter phenotype are located. Whole genes are usually not targeted because they often include long introns that do not affect protein coding. While mutations that occur in untranslated regions (UTRs) and introns have no known function, a few have been linked to mRNA expression [1,2]. A panel that is designed to capture the CDS and both UTRs of each gene would require 20–30% more probes, substantially increasing the cost of the panel. In addition, adding these additional target regions will decrease the amount of coverage per probe. Therefore, if you want to target UTR sequences, you should consider doing so only for specific genes, and design the rest of the probe pools to target only CDS. 

2X vs. 1X Tiling

Researchers sometimes require 2X tiling of their hybridization probes to get sufficient sequencing coverage (Figure 1). However, we have found that when using IDT xGen® Lockdown® target capture probes, 1X tiling provides excellent coverage results (Figure 2). Data obtained when using xGen Lockdown Probes suggest that 1X tiling is sufficient for most applications. 2X tiling requires more probes, and thus is more expensive; it is really most useful in special situations, such as trying to identify the site of a translocation or gene fusion event.


Figure 1. 2X vs. 1X Tiling.


Figure 2. Excellent Uniformity of Coverage Obtained With xGen® Pan-Cancer Panel. The uniformity of coverage of targeted regions is represented as the proportion of targets with >0.2 x mean coverage (blue), >0.5 x mean coverage (green) and >1.0 x mean coverage (grey). The data demonstrate that with a mean coverage depth of 100X, 99.9% of the targets captured using the xGen Pan-Cancer Panel will have >20X depth of coverage. Sequencing was performed on the MiSeq® System (Illumina) using 2 x 150 paired-end reads.

Number and Cost of Probes

When designing target capture experiments, the number of probes you will need to order and their total cost will vary with the set of genes being targeted. IDT provides a free, online xGen Lockdown Design Tool that provides an accurate assessment of the number and cost of probes required for capture. The tool will determine the probes required for optimal coverage depth and uniformity of your gene set, as well as provide a rudimentary design of the probes. The tool can accommodate up to 1000 genes or genomic regions, or up to 1 Mb of FASTA file sequence information. If you need probes for a larger set of sequences, please contact xgen@idtdna.com.

References

  1. Dvir S, Velten L, et al. (2013). Deciphering the rules by which 5′-UTR sequences affect protein expression in yeast. Proc Natl Acad Sci USA, 110(30):E2792–2801; doi: 10.1073/pnas.1222534110.
  2. Scheper GC, van der Knaap MS, Proud CG. (2007) Translation matters: protein synthesis defects in inherited diseases. Nat Rev Genet, 8:711–723.

Target capture reagents from IDT

xGen® Lockdown® Probes are individually synthesized, quality controlled, and normalized hybridization probes that offer:

  • Complete, uniform capture of the target space
  • 4-hour hybridization time
  • Sensitive detection of SNV, indels, CNV, LOH, and translocations
  • 21 CFR Part 820 manufacture for clinical and diagnostics research
  • Availability as stocked or custom panels
  • Quick delivery

Discover more about xGen Lockdown Probes.

xGen Lockdown Panels are preconfigured, validated, and stocked pools of xGen Lockdown Probes for targeted next generation sequencing of defined gene families:

  • xGen Exome Research Panel
  • xGen Acute Myeloid Leukemia Panel
  • xGen Pan-Cancer Panel
  • xGen Inherited Diseases Panel

Discover more about xGen Lockdown Panels.

xGen Blocking Oligos

xGen Universal Blocking Oligos for single- or dual-index adapters of common sequencing platforms improve on-target performance for multiplexed samples by reducing adapter participation in hybridization enrichment. Custom adapters can be manufactured for other barcodes or to meet the needs of customers who require specific modifications or services to improve performance in unique applications.

Discover more about xGen Blocking Oligos.


© 2015 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.


xGen® Blocking Oligos

Adapter blocking oligos increase the number of on-target reads by preventing non-specific binding during hybridization.

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