Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells

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.

Next-generation Sequencing (NGS) Analysis on Single Circulating Tumor Cells (CTCs) with No Need of Whole-genome Amplification (WGA)

“…The WGA Ampli1™ system enables excellent qualitative and quantitative performance and is one of the preferred methods, due to its higher yield for CTCs isolated by the DEPArray™ cell separator according to our previous experience and recent data from the literature (12,23,24). This technique is based on the DNA cleavage of the TTAA MseI restriction-site motifs (http://www.siliconbiosystems.com/feefor-service) and, in addition to potential drawbacks mentioned above, there is the inconvenient of failure to generate amplicons suitable for subsequent sequencing in case of DNA tracts carrying this motif (19,24,29).…”

“…Moreover, the presence of some specific sequence variants in driver genes is currently under intensive investigation for cancer molecular diagnosis and pharmacogenomics including prediction of either susceptibility or resistance to the targeted therapy (5)(6)(7). In this context, introduction of innovative biotechnologies allows to identify, recover and analyze CTCs from the peripheral blood to perform the so-called "liquid biopsy" procedure (8)(9)(10)(11)(12)(13)(14)(15)(16).…”

New library construction method for single-cell genomes

“…DOP-PCR became the choice for the detection of CNVs, while MDA is preferred method for SNV [15]. We feel that there is need for a new single-cell library preparation method that suits both purposes.…”

Biology and clinical significance of circulating tumor cell subpopulations in lung cancer

“…However, the rapid technological development of reliable single cell assays have substantially increased assay sensitivity and currently enables analysis of mRNA and DNA of single cells (43)(44)(45). Accordingly, Liu et al, recently performed a meta-analysis on 170 patients from eight studies with EGFR mutation data on CTCs.…”