The IDT xGen HS EGFR Pathway Amplicon Panel covers contiguous regions of the EGFR gene, and hotspot coverage of BRAF, KRAS, and NRAS with a panel of amplicon-generating primers. The final amplicons average only 136 bp making them compatible with DNA fragments from cell-free DNA (cfDNA) and/or FFPE DNA. The final target size of this panel is 1.5 kbp.
As seen in Figure 1, this workflow includes three PCR steps—one for the incorporation of unique molecular identifiers (UMIs) (PCR I), one for target amplification (PCR II), and one for the addition of combinatorial dual indexed adapters (PCR III), enabling multiplexing
of up to 96 unique libraries. Bead-based SPRI® cleanups (Beckman Coulter) are used to purify the sample by removing unused oligonucleotides and changing buffer composition between steps.