Development of a Nucleic Acid-Based Life Detection Instrument Testbed

Future space instruments will explore increasingly complex questions about our universe, including the origin of life on Earth and the presence of life elsewhere. These instruments will likely integrate chemical and biological subsystems that will face unique challenges; existing protocols typically require non-stabilized components and manual handling. The Search for Extra-Terrestrial Genomes (SETG) instrument is being developed for in situ extraction and sequencing of nucleic acids as a biomarker of life on other planetary bodies. Such sequencing is being implemented using a nanopore-based device, the Oxford Nanopore Technologies (ONT) MinION; as such, it needs to integrate many benchtop-based protocols. Here we describe an automated testbed, designed and built to automate and rapidly prototype extraction, library preparation, and sequencing protocols that could be used in our instrument. The system is designed to be modular with respect to components, facilitating hardware and software modifications with minimal system impact, while also precise across multiple test runs, allowing for accurate evaluation of the impact of varying system inputs as well as exploration of system failure modes and potential solutions. We also present testing results from each of the three primary subsystems (extraction, library preparation, and sequencing) as well as a plan for and initial data on subsystem integration into an end-to-end system. The extraction subsystem is able to match or approach nucleic acid yields attained via manual testing for B. subtilis spores in water (~15 %) and spores in basalt (~ 12%). The library preparation subsystem can successfully prepare a library of E. coli DNA that can be identified after sequencing. The loading/sequencing subsystem has successfully automated sequencer loading, resulting in a sequencing run producing 1.4 billion bases after 1 day of sequencing from a pre-prepared sample. These testing results provide valuable data about the challenges of biological protocol automation, while directly informing future design decisions for SETG. In the process, the lessons learned from this milestone are relevant to the technological development of future planetary science instruments that take advantage of molecular biology techniques.