Improving uniform coverage of targeted sequences for NGS

Learn about the challenges researchers face for obtaining uniform coverage of NGS data and how IDT xGen® Lockdown® Probes are uniquely positioned to facilitate uniform sequence coverage.

Successful targeted sequencing requires uniform coverage across those sequences. However, sequencing reads are often not distributed evenly over targeted areas. Truncated and/or hard-to-synthesize probes used for target capture often contribute to poor coverage uniformity. Target capture probes, therefore, benefit from quality control and normalization, which is generally not possible for the array-synthesized probes provided by most manufacturers. Thus, many probe pools and panels do not provide uniform coverage. IDT is uniquely positioned to prepare target capture probes—xGen® Lockdown® Probes—that can provide uniform coverage.

We use a proprietary IDT Ultramer® synthesis process, which has the highest coupling efficiency in the industry (99.5%; Figure 1).

DO-art21-CS-Target Capture Uniformity_Fig1

Figure 1. Full-length product synthesized under varying coupling efficiencies.

The high coupling rate allows us to synthesize long oligos (xGen Lockdown Probes are 60–120 bases long) with a high percentage of full-length product. Furthermore, because we synthesize each oligo individually, we can assess quality and normalize each oligo we manufacture, ensuring that each probe is represented in target capture pools. The presence of all probes and at full length within a pool provides consistent end-to-end tiling across the target(s) and improves coverage uniformity (Figure 2).


Figure 2. Excellent coverage uniformity obtained with xGen® Lockdown® AML Cancer Panel. Greater than 0.2X mean coverage is observed for >98% of targets. Libraries were prepared using the Illumina TruSeq® LT chemistry and sequenced on a MiSeq system using 250 x 250 paired-end reads. A cumulative of 31.8M reads was generated for all 4 samples.

Published Nov 3, 2014
Revised/updated Mar 16, 2017