Accurate identification of single-nucleotide variants in whole-genome-amplified single cells

The authors present a novel methodology to address the artifacts associated with cell lysis and whole genome amplification during genome-wide DNA mutation analysis, termed Single-Cell Multiple Displacement Amplification (SCMDA). SCMDA and SCcaller were validated by direct comparison of SNVs from amplified single cells and unamplified clones derived from cells in the same population of early passage human primary fibroblasts. The CellRaft® Technology was utilized for multiple steps in the validation workflow, including isolation of single cells and generation of single cell clones. Single cells were plated on CytoSort™ Arrays, isolated, and subjected to downstream SCMDA, library preparation, and sequencing. Empty CellRafts® were also isolated to serve as negative controls in WGA. To generate single cell clones, cells were plated on CytoSort™ Arrays and isolated once the clones reached confluency on the CellRaft®, approximately 10-16 cells per raft, for further expansion in 96-well plates. Lastly, to generate kindred single cells and clones, small clones were transferred from 96-well and seeded on a fresh CytoSort Array to isolate single cells. This method, and the corresponding single-cell variant caller SCcaller, provide a foundation for standardizing somatic mutation analysis in single-cell genomics.