Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells
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Anna Babayan1, Malik Alawi2,3, Michael Gormley4, Volkmar Müller5, Harriet Wikman1, Ryan P. McMullin6, Denis A. Smirnov4, Weimin Li4, Maria Geffken7, Klaus Pantel1 and Simon A. Joosse1
1Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
2Bioinformatics Core, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
3Heinrich-Pette-Institute, Leibniz-Institute for Experimental Virology (HPI), Hamburg, Germany
4Janssen Research and Development, Spring House, PA, USA
5Department of Gynecology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
6LabConnect LLC, Seattle, WA, USA
7Department of Transfusion Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Klaus Pantel, email: firstname.lastname@example.org
Simon A. Joosse, email: email@example.com
Keywords: NGS, WGA, SNP, allelic dropout, CellSave
Received: April 06, 2016 Accepted: June 09, 2016 Published: July 19, 2016
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.
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