Recent studies regarding the origin of life and Mars-Earth meteorite transfer simulations suggest that biological informational polymers, such as nucleic acids (DNA and RNA), have the potential to provide unambiguous evidence of life on Mars. To this end, we are developing a metagenomics-based life-detection instrument which integrates nucleic acid extraction and nanopore sequencing: The Search for Extra-Terrestrial Genomes (SETG). Our goal is to isolate and sequence nucleic acids from extant or preserved life on Mars in order to determine if a particular genetic sequence (1) is distantly-related to life on Earth indicating a shared-ancestry due to lithological exchange, or (2) is unrelated to life on Earth suggesting a convergent origin of life on Mars. In this study, we validate prior work on nucleic acid extraction from cells deposited in Mars analog soils down to microbial concentrations observed in the driest and coldest regions on Earth. In addition, we report low-input nanopore sequencing results equivalent to 1 ppb life-detection sensitivity achieved by employing carrier sequencing, a method of sequencing sub-nanogram DNA in the background of a genomic carrier.

Due to meteoritic transfer between Earth and Mars, if life on Mars exists, it may be related to life on Earth and utilize nucleic acids as informational polymers. Thus, a Search for Extra-Terrestrial Genomes (SETG) could detect and sequence (deoxy) ribonucleic acids (DNA/RNA) utilized by any extant or recently dead life on Mars. The abiotic synthesis of common organic building blocks, such as nucleobases, sugars, and amino acids, in the solar nebula and potentially in diverse habitable environments could also bias a second genesis of life towards utilizing informational polymers similar to life as we know it. Here we build on prior work and describe the advancement of a SETG instrument to technology readiness level 4 through sample-to-sequence processing with limited manual handling. Another advance includes validation of nucleic acid extraction from Mars analogs at cell counts down to 10(4) per 50 mg sample, equivalent to a limit of detection of approximately 1 part per billion. In addition, we demonstrate that biological nanopore-based single molecule sequencing can be used to detect non-standard bases. Finally, we link sequence data to a statistical test to distinguish between any forward contamination and putative life beyond Earth. Nanopore-based sensing may ultimately enable characterization of nonstandard polymers and other molecules, highlighting the potential for nanopore-based life detection and sequencing on Mars or other words such as the icy moons Enceladus or Europa.