Using single-cell DNA sequencing as a dosimetric tool- An exploratory study
Abstract
Purpose: Radiation-associated tumours show mutation signatures in their genome, which distinguish them from radiation naïve tumours [1]. Our goal is to find if we can identify similar signatures, well in advance, before radiation-exposed cells transform into a tumour. We are also investigating if our technique can be used as a dosimetric tool to estimate the radiation quality and dose deposited in an individual during the event of an incident or an aerospace mission. We know that stochastic interactions of radiation introduce damage and mutations that are unique to each individual cell’s genome. But conventional genome sequencing [2] methods such as bulk cell sequencing cannot detect such unique mutations. Therefore, we are examining if single-cell DNA sequencing may be used to reveal the mutational effects of ionizing radiation in exposed cells. We expect to see different mutation patterns for high- and low-LET radiation and thus we hope to discern the radiation quality from genomic mutations in cells. Methods: In this work, four identical samples of a human B-lymphoblastoid cell line were irradiated in vitro using 6 MV X-rays from a medical linear accelerator. They were exposed to sham irradiation (control), 0.5 Gy, 1.5 Gy and 3 Gy respectively at a common dose rate. Irradiated samples were incubated for 24 hrs, and then DNA was extracted from approximately 500 cells per sample and subsequently subjected to single-cell whole-genome DNA sequencing technology. The well-characterized genome of our B-lymphoblastoid cell line was used to establish the baseline mutations in our control and to identify radiation-induced mutations in the three other samples. Copy number alterations (CNA) were identified and examined in individual sequence data from all four sample groups. Radiation-induced copy number (CN) gains and losses were counted. Results: We observed a dose-dependent increase in the number of CNA in our sample groups, where the number of CN losses increased significantly with radiation dose. We also observed a dose dependence for the size of the chromosomal aberrations. Conclusions: Our findings suggest that single-cell sequencing techniques may be used to directly examine the mutational effects of ionization radiation in human cells. We are presently working on reproducing these results with repeated experiments. If confirmed, we posit that our strategy of examining DNA anomalies will open up new avenues for radiation biodosimetry.
Felix Mathew
PhD Student
James Manalad
PhD Student, Former MSc Student
Luc Galarneau
Research Associate
Norma Ybarra
Assistant Professor, Collaborator on NICE radiobiology projects
