DNA sequencing at the picogram level to investigate life on Mars and Earth.

DNA is an incontrovertible biosignature whose sequencing aids in species identification, genome functionality, and evolutionary relationships. To study life within the rocks of Earth and Mars, we demonstrate, in an ISO5 clean room, a procedure based on nanopore technology that correctly identifies organisms at picogram levels of DNA without amplification. Our study with E. coli and S. cerevisiae DNA samples showed that MinION sequencer (Oxford Nanopore Technologies) can unequivocally detect and characterise microbes with as little as 2 pg of input with just 50 active nanopores. This result is an excellent advancement in sensitivity, immediately applicable to investigating low biomass samples. This value is also at the level of possible background contamination associated with the reagents and the environment. Cultivation of natural and heat-treated Martian analogue (MMS-2) regolith samples, exposed to atmospheric water vapour or in increasing water concentrations, led to the extraction of 600-1000 pg of DNA from 500 mg of soil. Applying the low detectability technology enabled through MinION sequencer for a natural low biomass setting, we characterised the dry MMS-2 and found few soil-related organisms and airborne contaminants. The picogram detection level and the procedure presented here, may be of interest for the future Mars sample Return program, and the life research and planetary protection studies that will be implemented through the sample safety assessment.

An Optimized Active Sampling Procedure for Aerobiological DNA Studies.

The Earth's atmosphere plays a critical role in transporting and dispersing biological aerosols. Nevertheless, the amount of microbial biomass in suspension in the air is so low that it is extremely difficult to monitor the changes over time in these communities. Real-time genomic studies can provide a sensitive and rapid method for monitoring changes in the composition of bioaerosols. However, the low abundance of deoxyribose nucleic acid (DNA) and proteins in the atmosphere, which is of the order of the contamination produced by operators and instruments, poses a challenge for the sampling process and the analyte extraction. In this study, we designed an optimized, portable, closed bioaerosol sampler based on membrane filters using commercial off-the-shelf components, demonstrating its end-to-end operation. This sampler can operate autonomously outdoors for a prolonged time, capturing ambient bioaerosols and avoiding user contamination. We first performed a comparative analysis in a controlled environment to select the optimal active membrane filter based on its ability to capture and extract DNA. We have designed a bioaerosol chamber for this purpose and tested three commercial DNA extraction kits. The bioaerosol sampler was tested outdoors in a representative environment and run for 24 h at 150 L/min. Our methodology suggests that a 0.22-µm polyether sulfone (PES) membrane filter can recover up to 4 ng of DNA in this period, sufficient for genomic applications. This system, along with the robust extraction protocol, can be automated for continuous environmental monitoring to gain insights into the time evolution of microbial communities within the air.

TIDE Analysis of Cryptosporidium Infections by gp60 Typing Reveals Obscured Mixed Infections.

BACKGROUND: Cryptosporidiosis is a parasitic disease associated with potentially fatal diarrhea. The most used method in Cryptosporidium subtyping is based on the glycoprotein gene gp60. Each infection can represent a parasite population, and it is important to investigate the influence on transmission and virulence, as well as any impact on public health investigations. However, an easy-to-use method for detection is lacking. METHODS: Here we report on the use of the bioinformatic program TIDE for deconvolution of gp60 chromatograms. A combination of single oocyst analysis and cloning successfully confirmed the within-sample parasite population diversity. Retrospective sample analysis was conducted on archived chromatograms. RESULTS: For Cryptosporidium parvum, 8.6% multistrain infections (13 of 152) obscured by currently used consensus base calling were detected. Importantly, we show that single oocysts can harbor a mixed population of sporozoites. We also identified a striking dominance of unappreciated polymerase stutter artefacts in all 218 chromatograms analyzed, challenging the uncritical use of gp60 typing. CONCLUSIONS: We demonstrate the value of a new, easy-to-use analytical procedure for critical characterization of C. parvum and Cryptosporidium hominis in epidemiological investigations, also applicable retrospectively. Our findings illuminate the hidden parasite diversity with important implications for tracing zoonotic and person-to-person transmissions.