Radiation Tolerance of Nanopore Sequencing Technology for Life Detection on Mars and Europa

Abstract

The search for life beyond Earth is a key motivator in space exploration. Informational polymers, like DNA and RNA, are key biosignatures for life as we know it. The MinION is a miniature DNA sequencer based on versatile nanopore technology that could be implemented on future planetary missions. A critical unanswered question is whether the MinION and its protein-based nanopores can withstand increased radiation exposure outside Earth's shielding magnetic field. We evaluated the effects of ionizing radiation on the MinION platform - including flow cells, reagents, and hardware - and discovered limited performance loss when exposed to ionizing doses comparable to a mission to Mars. Targets with harsher radiation environments, like Europa, would require improved radiation resistance via additional shielding or design refinements.

Figure 1

Schematic representation of a DNA molecule translocating a protein nanopore. The double-stranded DNA (dsDNA) is split by a helicase enzyme, allowing only a single strand (ssDNA) to pass while slowing it enough to achieve sufficient resolution for sequencing.

Figure 2

The MinION sequencer. (a) A typical MinION flow cell. The flow cell contains an array with sufficient space for 2048 nanopores. (b) Two MinION devices sequencing DNA powered via USB laptop connection.

Figure 3

The experimental workflow. (A) Prior to the first irradiation, sequencing runs were attempted on both control and experimental MinION devices with lambda DNA libraries. Flow cells were removed from the MinION and stored at room temperature prior to the first irradiation. (B) Platform QCs were conducted on all flow cells prior to the first radiation dose. (C) Reagents (RAD and FRM) were aliquoted in 5 microliter volumes into 0.2 milliliter thin-walled PCR tubes and placed in the polystyrene cooler over dry ice. A total of 12 RAD aliquots (9 experimental and 3 control) and two FRM aliquots (1 experimental and 1 control) were made. (D) MinION hardware, flow cells, and reagents were irradiated simultaneously by a high energy gamma ray source. (E) The experimental MinION was removed from the chamber and short sequencing runs attempted on it and the control at cumulative doses of 250 (from the previous irradiation), 400, 600, 750, 1500, and 3000 gray. Flow cells were removed from both devices for irradiations. (F) One flow cell was dedicated to platform QCs at doses of 150, 300, 400, 500, 600, and 750 gray. This flow cell was placed back in the chamber after each QC. Additionally, platform QCs were conducted on three flow cells following doses of 50, 300, and 500 gray. Sequencing of prepared lambda DNA was then attempted on all flow cells. (G) One tube of RAD reagent was removed from the chamber and stored over dry ice in a control foam container at doses of 10, 50, 100, 150, 300, 400, 750, 1500, and 3000 gray. The tube of FRM was removed from the chamber at 400 gray. All RAD reagents were then barcoded and sequenced on a single flow cell. The two FRM reagents were sequenced on separate flow cells. (H) Raw sequencing data was basecalled by Oxford Nanopore’s Albacore software, aligned to the lambda genome with NanoOK, and further analyzed by custom Python code.

Figure 4

Sequencing alignment and quality analyses for flow cell experiments. No data is presented for flow cells at 500 or 750 gray as neither produced any reads during sequencing.

Figure 5

Sequencing alignment and quality analyses for RAD reagent experiments. Note that reads produced by all reagents showed nearly 100% of reads having alignments to the lambda genome. This was expected due to lower quality reads not being identified with a barcode by the basecalling software.

Figure 6

Sequencing alignment and quality analyses for FRM reagent experiments.

Figure 7

The radiation apparatus. (A) Schematic representation of the radiation apparatus. Reagents in PCR tubes were suspended over dry ice in a plastic tube rack enclosed by a polystyrene cooler. Flow cells in antistatic bags were placed in manufacturer boxes directly on top of the cooler. MinION hardware in an antistatic bag was placed on top of the flow cell boxes. (B) The radiation apparatus just prior to placement in the chamber. (C) The cooler opened to show the tube rack and dry ice.