Preparation of multiplexed control materials for cancer mutation analysis by genome editing in GM12878 cells

Background Quality control materials are necessary for assay development, test validation, and proficiency testing in cancer mutation analysis. Most of the existing controls for somatic mutations only harbor a single variant and are derived from unstable cell lines. This study aimed to establish a method to create stable multianalyte controls in a defined background by genome editing in GM12878 cells, which also can be applied for the reference of next-generation sequencing. Methods GM12878 cells were electroporated with a donor plasmid containing a mutant DNA sequence and a Cas9/sgRNA expressing vector. The genome-edited GM12878 cell was validated with Sanger sequencing, amplification refractory mutation system (ARMS), and next-generation sequencing (NGS). Results We have successfully generated a mutant GM12878 cell line harboring the defined variants including single-nucleotide variants (SNVs), small insertions and deletions (indels), and structural variants (SVs). The introduction of intended mutations in GM12878 cell line was confirmed by both ARMS and sequencing methods. Conclusions We developed a method for the preparation of the multiplexed controls for reference mutations in cancer gene by genome editing in GM12878 cells. This methodology can be used to generate other stable cancer reference materials with an unlimited supply.