Abstract
BACKGROUNDWhole 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.RESULTSIn 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 METHODSWe 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.CONCLUSIONWe 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.
Highlights
Introduction of single cell analysis led to paradigm shifts in almost all fields of biology and medical sciences as it allows for an accurate representation of the cell-tocell heterogeneity instead an average measure of an entire cell population [1]
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 copy number aberrations (CNA) analysis in our study was not detectably affected by fixation
SNP calling performance of multiple-displacement amplification (MDA)-PCR Whole genome amplification (WGA) improves with increasing amount of input DNA, whereas CNA analysis does not
Summary
Introduction of single cell analysis led to paradigm shifts in almost all fields of biology and medical sciences as it allows for an accurate representation of the cell-tocell heterogeneity instead an average measure of an entire cell population [1]. Single cell analysis empowers characterization of tumor heterogeneity, and most notably has potential for clinical impact through characterization of circulating tumor cells (CTCs). CTCs are tumor cells that have separated from primary tumor or current metastases and have infiltrated the systemic blood circulation [2]. Quantification and characterization of CTCs in blood of cancer patients was introduced as a concept of “liquid biopsy”. Enumeration of CTCs as a validated clinical biomarker has been utilized for disease prognosis, diagnosis of minimal residual disease, and monitoring of therapy effectiveness for breast, prostate, and colon cancer [3, 4]. Genomic characterization of CTCs provides insights into genetic heterogeneity of the cancer and metastases and might aid clinical management of cancer patients due to identification of therapy sensitive and resistant clones. Effectiveness of currently available WGA technologies in combination with generation sequencing (NGS) and material preservation is still elusive
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