Targeted resequencing of candidate genes using selector probes

Area of Science:

• Genomics
• Molecular Biology
• Cancer Research

Background:

• Next-generation sequencing (NGS) offers massive throughput but requires efficient methods for targeted genome enrichment.
• Existing protocols may have limitations in coverage, specificity, or compatibility with diverse sample types.
• The Selector technique provides a foundation for multiplex amplification of specific DNA regions.

Purpose of the Study:

• To present a simple, scalable, and updated protocol for multiplex amplification of target DNA regions using the Selector technique.
• To improve coverage and compatibility with NGS library construction for shotgun sequencing.
• To demonstrate the efficacy of the enhanced protocol in cancer gene enrichment.

Main Methods:

• Utilized an updated Selector technique for multiplex amplification of 501 exons from 28 cancer-associated genes.
• Applied the protocol to DNA from cell lines and both fresh frozen and formalin-fixed paraffin-embedded (FFPE) tumor biopsies.
• Performed shotgun sequencing on next-generation sequencing (NGS) platforms.

Main Results:

• Achieved 94% specificity and 98% coverage of targeted genomic regions.
• Demonstrated high reproducibility between replicates (R(2) = 0.98), enabling copy-number variation detection.
• The protocol is rapid, completable in under 24 hours, and requires no specialized instrumentation.

Conclusions:

• The enhanced Selector technique is a robust and efficient method for targeted genome enrichment.
• The protocol is suitable for various sample types, including challenging FFPE tissues.
• This technique facilitates deep sequencing of cancer-related genes and supports copy-number variation analysis in clinical and research settings.