Comparative Study of Whole Genome Amplification and Next-Generation Sequencing Performance on Single Cancer Cells

BACKGROUND: Single cell genomics is a powerful tool, having many applications in cancer research and providing a next step towards individualized medicine. Single cell investigation became realizable due to progress in whole genome amplification (WGA) methods and development of next generation sequencing (NGS). METHODS: We analyzed performance of three different WGA techniques on single tumor cells obtained from EDTA- and CellSave-preserved blood, as well as, from formalin-fixed, paraffin-embedded material. In total, 120 single cells were amplified with PCR-based, multiple-displacement amplification (MDA) –based, and combined MDA-PCR technique. The efficacy of WGA was assessed by the ability of multiplex PCR producing sequential products of ≥200 bp. Subsequently, WGA DNA underwent whole exome next generation sequencing (NGS) with the Illumina HiSeq2000 and the Thermo Fisher IonProton platfornms in order to investigate SNPs, indel, and copy number alteration analysis. RESULTS: All tested WGA techniques were able to amplify single cells with average yield of 6.09, 3.74, and 34.04 µg per single cell for PCR-based, MDA-PCR combined, and MDA-based, respectively and quality control success rates of 93, 95, and 50% respectively. Illumina’s NGS platform outperformed IonProton allowing for more precise SNP, indel, and copy number alteration analysis. Combination of PCR-based WGA with sequencing on Illumina’s NGS platform demonstrated the best concordance with results obtained from unamplified DNA for SNP and indel calling (41.3 and 15.3% concordance respectively). Copy number alteration profile of single cell, amplified with combined MDA-PCR technique and sequenced on Illumina’s NGS platform, resembled unamplified DNA the most (Spearman correlation coefficient 0.81, p-value <0.001). MDA WGA technique produced the highest DNA yield, but not of sufficient quality for SNP, indel, and CNA analysis. CONCLUSION: Single cell genomics might provide valuable information, nevertheless WGA and NGS approaches has to be chosen carefully depending on the investigation aims.