Quantitative analysis of dose dependent DNA fragmentation in dry pBR322 plasmid using long read sequencing and Monte Carlo simulations

Abstract

Exposure to ionizing radiation can induce genetic aberrations via unrepaired DNA strand breaks. To investigate quantitatively the dose-effect relationship at the molecular level, we irradiated dry pBR322 plasmid DNA with 3 MeV protons and assessed fragmentation yields at different radiation doses using long-read sequencing from Oxford Nanopore Technologies. This technology applied to a reference DNA model revealed dose-dependent fragmentation, as evidenced by read length distributions, showing no discernible radiation sensitivity in specific genetic sequences. In addition, we propose a method for directly measuring the single-strand break (SSB) yield. Furthermore, through a comparative study with a collection of previous works on dry DNA irradiation, we show that the irradiation protocol leads to biases in the definition of ionizing sources. We support this scenario by discussing the size distributions of nanopore sequencing reads in the light of Geant4 and Geant4-DNA simulation toolkit predictions. We show that integrating long-read sequencing technologies with advanced Monte Carlo simulations paves a promising path toward advancing our comprehension and prediction of radiation-induced DNA fragmentation.

Keywords: DNA damages; Ionizing radiation; Monte Carlo simulation; Nanopore sequencing.

Figure 1

Description of the Oxford Nanopore sequencing technologies. (a) Principle of Oxford Nanopore Sequencer. (b) Scheme of the different scenarios that can arise after DNA irradiation inducing single strand (SSB) and double strand breaks (DSB). (c–g) Read length probability distribution of reads from both sequencing runs for all experimental conditions at 0, 0.5, 1, 2 and 5 kGy. Note that y-scale is chosen to be the same as control. The lengths of reads mapping on the pBR322 genome were obtained from alignment files. Minor differences in the fragmentation rate were also observed between the two experimental runs (Exp1 and 2), particularly in the read size distribution. (h–l) Circular visualization of reads alignment coverage per irradiation dose ranging from 0, 0.5, 1, 2 and 5 kGy.

Figure 2

Quantitative analysis of radiation-induced plasmid DNA fragmentation. (a) Effect of 3 MeV proton irradiation on two sets of air-dried pBR322 plasmid DNA at 0, 0.5, 1, 2, and 5 kGy. Band sizes separated on 1% (w/v) agarose gel and visualized by ethidium bromide staining gels from Exp1 and Exp2. (b) Ratios of plasmid conformations observed on the agarose gel fitted on the McMahon statistical model. (c) Intact plasmid strand yields as a function of the proton dose on DNA. The average undamaged strand yields of both sequencing runs are calculated and corrected for the sequencer’s response function and are fitted according to the Poisson regression (Poisson 1). The expected intact strand yields are calculated from gel electrophoresis data fitted according to Poisson (Poisson 2) and McMahon’s models. (d) Comparison of SSB yields with published data. The SSB yields calculated using McMahon’s fitting model are compared with previously published data for irradiation of dry plasmid with hydrogen and proton beams. Experimental irradiation conditions: sealed symbols represent irradiation performed in air, while open symbols represent irradiation conducted in vacuum. Our AGE results (red) are in the expected range of damage according to the linear energy transfer of the proton beams (~ 16 keV µm⁻¹ in DNA).

Figure 3

From experience to Monte Carlo simulation. (a) Overview of the sample configuration. DNA drop was spotted on a 4 µm thick polypropylene foil (PP). The back-side of the cell dish is closed in air using a glass coverslip. (b) Geant4 simulation of the proton and secondary electron energy distributions produced in the experimental conditions. (c–f) Comparison of fragment length percentage yield between both sequencing runs and the Geant4-DNA simulations for irradiation doses of (c) 0.5, (d) 1, (e) 2 and (f) 5 kGy, respectively without the secondary electron contribution. (g–j) Comparison of fragment length percentage yield between both sequencing runs and the Geant4-DNA simulations for irradiation doses of (g) 0.5, (h) 1, (i) 2 and (j) 5 kGy, respectively with the secondary electron contribution. Experimental data from Exp1 and Exp2 are depicted in grey. Simulation results depicted in black were obtained with “molecularDNA” example with 17.5 eV energy threshold (t), and 5.5 Å hydration shell (r). A number of 10 simulations were run for each dose and error bars shown in red were calculated as standard deviation for simulation fragments. Distribution histograms have a bin width of 79 bp.