Accurate detection of low-level mosaic mutations in pediatric acute lymphoblastic leukemia using single molecule tagging and deep-sequencing

Abstract

Pathogenic mutations in relapse-associated genes in pediatric acute lymphoblastic leukemia may improve risk stratification when detected at subclonal levels at primary diagnosis. However, to detect subclonal mutations upfront, a deep-sequencing approach with high specificity and sensitivity is required. Here, we performed a proof-of-principle study to detect low-level mosaic RAS pathway mutations by deep sequencing using random tagging-based single molecule Molecular Inversion Probes (smMIPs). The smMIP-based approach could sensitively detect variants with allele frequency as low as 0.4%, which could all be confirmed by other techniques. In comparison, with standard deep-sequencing techniques we reached a detection threshold of only 2.5%, which hampered detection of seven low-level mosaic mutations representing 24% of all detected mutations. We conclude that smMIP-based deep-sequencing outperforms standard deep-sequencing techniques by showing lower background noise and high specificity, and is the preferred technology for detecting mutations upfront, particularly in genes in which mutations show limited clustering in hotspots.

Keywords:

• Acute lymphoblastic leukemia
• low-level mosaic mutations
• deep sequencing
• molecular tagging
• single molecule Molecular Inversion Probes

Potential conflict of interest

Disclosure forms provided by the authors are available with the full text of this article online at https://doi.org/10.1080/10428194.2017.1390232.

Funding

This work was supported by grants from the Dutch Cancer Society (KWF; KUN2009-4298 to RPK, KUN2012-5366 to EW), Stichting Kinderen Kankervrij (KiKa 150 to RPK and AH), and the China Scholarship Council (CSC201304910347 to JY).