Single-Tube, Switched Temperature Amplicon Barcoding for Multiplex Detection of Rare Mutations in Circulating Tumor DNA.

Detection and analysis of circulating tumor DNA (ctDNA) as a biomarker for cancer is a promising approach. Applications for ctDNA analysis include screening, diagnosis, treatment selection, treatment monitoring, minimal residual disease detection, and recurrence monitoring. Detection of ctDNA is challenging and requires highly sensitive methods. Approaches such as digital PCR are appropriate when only a small number of targets is being interrogated, whereas next-generation sequencing (NGS) is typically used when more targets are being analyzed. There are several NGS methods available, some of which are published and can be implemented in laboratories with the required expertise while other, commercial approaches are proprietary and are only available as a service. Of the published methods, most use some kind of unique molecular identifiers (or barcodes) to facilitate NGS error correction and detection of rare mutations at mutant allele frequencies of <0.1%. However, incorporation of barcodes and amplification of the resulting libraries are not trivial and typically require multiple steps and considerable hands-on time by an experienced molecular biologist. Herein, a novel approach for switched temperature amplicon barcoding was used, in which barcoding and library amplification were performed in the same tube using a two-stage PCR protocol with no additional manipulation. Total hands-on time was 10 to 15 minutes for reaction setup; the library was then cleaned and was ready for sequencing.