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Improving uniform coverage of targeted sequences for NGS

Individually synthesized and quality controlled xGen™ Lockdown™ Probes 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 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 synthesize target capture probes—xGen Lockdown Probes—that can provide uniform coverage.

We use a proprietary IDT Ultramer™ oligonucleotide synthesis process, which has the highest coupling efficiency in the industry (99.6%; Figure 1).

Long Ultramer oligos from high coupling efficiency
Figure 1. The better the coupling efficiency, the higher the yield of full-length oligos. The yield of different oligo lengths comparing IDT industry-leading coupling efficiency of 99.6% used to synthesize xGen Lockdown Probes to an industry-standard coupling efficiency of 98.5%. The highest coupling efficiency and proprietary synthesis technology provides you with high-quality individually synthesized probes for NGS. 

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 at full length within a pool provides consistent end-to-end tiling across the target(s) and improves coverage uniformity (Figure 2).

Uniform NGS coverage from Lockdown probes
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 (Illumina) using 250 x 250 paired-end reads. An average of 31.8M reads was generated for all 4 samples.

Published Nov 3, 2014
Revised/updated Sep 8, 2020