Comparative soft-tissue preservation in Holocene-age capelin concretions.

Determining how soft tissues are preserved and persist through geologic time are continuing challenge because decay begins immediately after senescence while diagenetic transformations generally progress over days to millions of years. However, in recent years, carbonate concretions containing partially-to-fully decayed macroorganisms have proven to be remarkable windows into the diagenetic continuum revealing insights into the fossilization process. This is because most concretions are the result of biologically induced mineral precipitation caused by the localized decay of organic matter, which oftentimes preserves a greater biological signal relative to their host sediment. Here we present a comparative lipid biomarker study investigating processes associated with soft-tissue preservation within Holocene-age carbonate concretions that have encapsulated modern capelin (Mallotus villosus). We focus on samples collected from two depositional settings that have produced highly contrasting preservation end-members: (1) Kangerlussuaq, Greenland: a marine environment, which, due to isostatic rebound, has exposed strata containing concretions exhibiting exceptional soft-tissue preservation (6-7 kya), and (2) Greens Creek, Ottawa, Canada: a paleo brackish-to-freshwater marine excursion containing concretions exhibiting skeletal remains (~11 kya). Lipid biomarker analysis reveals endogenous capelin tissues and productive waters at Kangerlussuaq that are in sharp contrast to Greens Creek concretions, which lack appreciable capelin and environmental signals. Comparable distributions of bacterial fatty acids and statistical analyses suggest soft-tissue preservation within concretions is agnostic to specific heterotrophic decay communities. We, therefore, interpret preservation within carbonate concretions may represent a race between microbially induced authigenic precipitation and decay. Namely, factors resulting in exceptional preservation within concretions likely include: (1) organic matter input, (2) rate of decay, (3) carbonate saturation, (4) porewater velocity, and (5) rate of authigenic (carbonate) precipitation resulting in arrested decay/bacterial respiration due to cementing pore spaces limiting the diffusion of electron acceptors into the decay foci.

In search of the RNA world on Mars.

Advances in origins of life research and prebiotic chemistry suggest that life as we know it may have emerged from an earlier RNA World. However, it has been difficult to reconcile the conditions used in laboratory experiments with real-world geochemical environments that may have existed on the early Earth and hosted the origin(s) of life. This challenge is due to geologic resurfacing and recycling that have erased the overwhelming majority of the Earth's prebiotic history. We therefore propose that Mars, a planet frozen in time, comprised of many surfaces that have remained relatively unchanged since their formation > 4 Gya, is the best alternative to search for environments consistent with geochemical requirements imposed by the RNA world. In this study, we synthesize in situ and orbital observations of Mars and modeling of its early atmosphere into solutions containing a range of pHs and concentrations of prebiotically relevant metals (Fe2+ , Mg2+ , and Mn2+ ) spanning various candidate aqueous environments. We then experimentally determine RNA degradation kinetics due to metal-catalyzed hydrolysis (cleavage) and evaluate whether early Mars could have been permissive toward the accumulation of long-lived RNA polymers. Our results indicate that a Mg2+ -rich basalt sourcing metals to a slightly acidic (pH 5.4) environment mediates the slowest rates of RNA cleavage, though geologic evidence and basalt weathering models suggest aquifers on Mars would be near neutral (pH ~ 7). Moreover, the early onset of oxidizing conditions on Mars has major consequences regarding the availability of oxygen-sensitive metals (i.e., Fe2+ and Mn2+ ) due to increased RNA degradation rates and precipitation. Overall, (a) low pH decreases RNA cleavage at high metal concentrations; (b) acidic to neutral pH environments with Fe2+ or Mn2+ cleave more RNA than Mg2+ ; and (c) alkaline environments with Mg2+ dramatically cleaves more RNA while precipitates were observed for Fe2+ and Mn2+ .

Nucleic Acid Extraction and Sequencing from Low-Biomass Synthetic Mars Analog Soils for In Situ Life Detection.

Recent studies regarding the origins 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 convergent origins 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 (i.e., 104 cells in 50 mg of soil) observed in the driest and coldest regions on Earth. In addition, we report low-input nanopore sequencing results from 2 pg of purified Bacillus subtilis spore DNA simulating ideal extraction yields equivalent to 1 ppb life-detection sensitivity. We achieve this by employing carrier sequencing, a method of sequencing sub-nanogram DNA in the background of a genomic carrier. After filtering of carrier, low-quality, and low-complexity reads we detected 5 B. subtilis reads, 18 contamination reads (including Homo sapiens), and 6 high-quality noise reads believed to be sequencing artifacts.

CarrierSeq: a sequence analysis workflow for low-input nanopore sequencing.

BACKGROUND: Long-read nanopore sequencing technology is of particular significance for taxonomic identification at or below the species level. For many environmental samples, the total extractable DNA is far below the current input requirements of nanopore sequencing, preventing "sample to sequence" metagenomics from low-biomass or recalcitrant samples. RESULTS: Here we address this problem by employing carrier sequencing, a method to sequence low-input DNA by preparing the target DNA with a genomic carrier to achieve ideal library preparation and sequencing stoichiometry without amplification. We then use CarrierSeq, a sequence analysis workflow to identify the low-input target reads from the genomic carrier. We tested CarrierSeq experimentally by sequencing from a combination of 0.2 ng Bacillus subtilis ATCC 6633 DNA in a background of 1000 ng Enterobacteria phage λ DNA. After filtering of carrier, low quality, and low complexity reads, we detected target reads (B. subtilis), contamination reads, and "high quality noise reads" (HQNRs) not mapping to the carrier, target or known lab contaminants. These reads appear to be artifacts of the nanopore sequencing process as they are associated with specific channels (pores). CONCLUSION: By treating sequencing as a Poisson arrival process, we implement a statistical test to reject data from channels dominated by HQNRs while retaining low-input target reads.

Nucleic Acid Extraction from Synthetic Mars Analog Soils for in situ Life Detection.

Biological informational polymers such as nucleic acids have the potential to provide unambiguous evidence of life beyond Earth. To this end, we are developing an automated in situ life-detection instrument that integrates nucleic acid extraction and nanopore sequencing: the Search for Extra-Terrestrial Genomes (SETG) instrument. Our goal is to isolate and determine the sequence of nucleic acids from extant or preserved life on Mars, if, for example, there is common ancestry to life on Mars and Earth. As is true of metagenomic analysis of terrestrial environmental samples, the SETG instrument must isolate nucleic acids from crude samples and then determine the DNA sequence of the unknown nucleic acids. Our initial DNA extraction experiments resulted in low to undetectable amounts of DNA due to soil chemistry-dependent soil-DNA interactions, namely adsorption to mineral surfaces, binding to divalent/trivalent cations, destruction by iron redox cycling, and acidic conditions. Subsequently, we developed soil-specific extraction protocols that increase DNA yields through a combination of desalting, utilization of competitive binders, and promotion of anaerobic conditions. Our results suggest that a combination of desalting and utilizing competitive binders may establish a "universal" nucleic acid extraction protocol suitable for analyzing samples from diverse soils on Mars. Key Words: Life-detection instruments-Nucleic acids-Mars-Panspermia. Astrobiology 17, 747-760.