101. Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells.
- Author
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Babayan A, Alawi M, Gormley M, Müller V, Wikman H, McMullin RP, Smirnov DA, Li W, Geffken M, Pantel K, and Joosse SA
- Abstract
Background: Whole genome amplification (WGA) is required for single cell genotyping. Effectiveness of currently available WGA technologies in combination with next generation sequencing (NGS) and material preservation is still elusive., Results: In respect to the accuracy of SNP/mutation, indel, and copy number aberrations (CNA) calling, the HiSeq2000 platform outperformed IonProton in all aspects. Furthermore, more accurate SNP/mutation and indel calling was demonstrated using single tumor cells obtained from EDTA-collected blood in respect to CellSave-preserved blood, whereas CNA analysis in our study was not detectably affected by fixation. Although MDA-based WGA yielded the highest DNA amount, DNA quality was not adequate for downstream analysis. PCR-based WGA demonstrates superiority over MDA-PCR combining technique for SNP and indel analysis in single cells. However, SNP calling performance of MDA-PCR WGA improves with increasing amount of input DNA, whereas CNA analysis does not. The performance of PCR-based WGA did not significantly improve with increase of input material. CNA profiles of single cells, amplified with MDA-PCR technique and sequenced on both HiSeq2000 and IonProton platforms, resembled unamplified DNA the most., Materials and Methods: We analyzed the performance of PCR-based, multiple-displacement amplification (MDA)-based, and MDA-PCR combining WGA techniques (WGA kits Ampli1, REPLI-g, and PicoPlex, respectively) on single and pooled tumor cells obtained from EDTA- and CellSave-preserved blood and archival material. Amplified DNA underwent exome-Seq with the Illumina HiSeq2000 and ThermoFisher IonProton platforms., Conclusion: We demonstrate the feasibility of single cell genotyping of differently preserved material, nevertheless, WGA and NGS approaches have to be chosen carefully depending on the study aims., Competing Interests: CONFLICTS OF INTEREST Anna Babayan, Malik Alawi, Volkmar Müller, Harriet Wikman, Maria Geffken, and Simon A Joosse have no conflicts or disclosures to report. Michael Gormley: employed by Johnson and Johnson. Ryan P. McMullin: employed by LabConnect LLC through contract with Janssen R&D. No other conflicts or disclosures. Denis A. Smirnov: employed by Johnson and Johnson. No other conflicts or disclosures. Weimin Li: employed by Johnson and Johnson and is shareholder. Klaus Pantel: received a research grant from Janssen R&D.
- Published
- 2016
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