Next Generation Sequencing
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Improving uniform coverage of targeted sequences for NGS

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).

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.

Author: Ellen Prediger, PhD, is a senior scientific writer at IDT.

Product focus: Target capture reagents from IDT

xGen® Lockdown® Probes

xGen Lockdown Probes are individually synthesized, quality controlled, and normalized hybridization probes for target enrichment by hybrid capture, specifically developed for next generation sequencing. They offer:

  • Sensitive detection of SNPs, indels, CNV, LOH, and translocations
  • Available for clinical and diagnostics research
  • Use to augment existing panels or create completely custom panels
  • Quick delivery

Discover more about xGen Lockdown Probes

xGen® Lockdown® Panels

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

  • xGen® Acute Myeloid Leukemia Panel
  • xGen® Pan-Cancer Panel
  • xGen® Inherited Diseases Panel

Discover more about xGen Lockdown Panels

xGen® Lockdown Reagents Hybridization and Wash Kit

Achieve uniform coverage with hybridization and wash buffers that are optimized for target enrichment using xGen Lockdown Probes and Panels. A short, 4-hour hybridization protocol generates results quickly.

Discover more about xGen Lockdown Reagents.

xGen® Blocking Oligos

xGen Universal Blocking Oligos for single- or dual-index adapters used with 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

Additional reading

For a further discussion on uniformity of coverage and coverage depth, read: How Important Are Those NGS Metrics?

Get the most out of your NGS samplesLearn how to create expandable NGS target capture panels that enrich for mutated genes implicated in tumors and genes associated with inherited diseases. Both panels return consistent results with high reproducibility and deep uniform coverage.

Gene panels vs. gene-by-gene analysis for assessing disease riskCitation summary: Authors compared an NGS-based gene panel and traditional testing data for diagnostic use and disease risk assessment in hereditary breast and ovarian cancer. Read how xGen® Lockdown® Probes (IDT) were able to rescue drop-out regions of SureSelect (Agilent) probe panels.

Generate consistent, reliable exome sequencing resultsSpanning 39 Mb of the human genome, the xGen® Exome Research Panel was designed to provide uniform and specific coverage of the coding regions for 19,396 genes. You can easily and cost-effectively expand this panel to include specific non-coding target regions through addition of xGen® Lockdown® Probes. 

Review other articles in the NGS application section of our DECODED Online newsletter.

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xGen® Blocking Oligos

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

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