S. Sho1, C. M. Court1, P. Winograd1, J. S. Tomlinson1 1University Of California – Los Angeles,General Surgery,Los Angeles, CA, USA
Introduction:
Obtaining circulating tumor cells from a peripheral blood draw opens the door to the possibility of applying precision oncology strategies from a “liquid biopsy.” Realization of this potential requires whole genome amplification (WGA) for genomic analysis of single cells. However, WGA remains prone to non-linear and uneven amplifications, resulting in inaccurate representation of the starting genome. WGA reactions are also known to be stoichastic, resulting in highly varied amplified DNA quality between samples. Thus, appropriate selection of the high-quality WGA product is needed to ensure the accuracy and reliability of downstream analysis. Currently, quality analysis methods for WGA products is not well defined, and no clear workflow exists for obtaining and selecting optimal WGA products for downstream molecular assay.
Methods:
Laser microdissection (LMD) was used isolate pancreatic cancer cell line (HPAF-II) cells. Nine single-cells, six 5-cell groups and six 10-cell groups were obtained for WGA using multiple displacement amplification (MDA) method. Quality of WGA products was assessed by performing real-time quantitative (RT-qPCR) PCR for 8 cancer related genes. Quality score (QS) of 0-8 was assigned based on the ability of RT-qPCR to detect these cancer genes in the WGA DNA product (0: absence, 1: presence). Single-cell WGA with perfect QS of 8 and low QS of 1 were subjected to array comparative genomic hybridization (aCGH) and next generation sequencing (NGS), in order to assess if QS could predict the success of downstream analysis. WGA products of 5-cells and 10-cells with QS of 8 were also subjected to aCGH and NGS, in order to determine the impact of increasing number of starting cells on the quality of downstream analysis.
Results:
Successful DNA amplification was noted in all samples undergoing WGA. However, only 22% (2 of 9) single-cell WGAs had the perfect QS of 8, with the rest of ranging between 1-3. For both 5- and 10-cell WGA, 50% (3 of 6) of the WGA products had QS of 8. When applied to aCGH and NGS, the single-cell WGA product with low QS of 1 resulted in poor quality data (Table 1). On the other hand, the single-cell WGA with the perfect QS of 8 resulted in a significantly higher quality aCGH and NGS data. When the WGA products of 5-/10-cells with perfect QS of 8 were subjected to aCGH and NGS, results were even better, approximating that of nonamplified DNA.
Conclusion:
WGA quality control by RT-qPCR offers a simple, inexpensive way of selecting optimal WGA products for downstream analysis. Furthermore, aCGH/NGS results approximating nonamplified DNA can be obtained by using high quality WGA product (QS of 8) obtained from using 5-10 cells as starting template.
