Nuclease-Assisted, Multiplexed Minor-Allele Enrichment: Application in Liquid Biopsy of Cancer

The use of next-generation sequencing (NGS) to profile genomic variation of individual cancer species is revolutionizing the practice of clinical oncology. In liquid biopsy of cancer, sequencing of circulating-free DNA (cfDNA) is gradually applied to all stages of cancer diagnosis and treatment, serving as complement or replacement of tissue biopsies. However, analysis of cfDNA obtained from blood draws still faces technical obstacles due in part to an excess of wild-type DNA originating from normal tissues and hematopoietic cells. The resulting low-level mutation abundance often falls below routine NGS detection sensitivity and limits reliable mutation identification that meets clinical sensitivity and specificity standards. Despite sample preparation advances that reduce sequencing error rates via use of unique molecular identifiers (molecular barcodes) and error-suppression algorithms, excessive amounts of sequencing are still required to detect mutations at allelic frequency levels below 1%. This requirement reduces throughput and increases cost.In this chapter, we describe a sensitive multiplex mutation detection method that enriches mutation-containing DNA during sample preparation, prior to sequencing, thereby increasing signal-to-noise ratios and providing low-level mutation detection without excessive sequencing depth. We couple targeted next-generation sequencing with wild-type DNA removal using Nuclease-assisted Minor-allele Enrichment using Probe Overlap, NaME-PrO, a recently developed method to eliminate wild-type sequences from multiple targets simultaneously. A step by step guide to library preparation and data analysis are provided as well as some precautions during the sample handling.