It is difficult to untangle the mixed influences of high-and low-latitude climate forcing in the eastern equatorial Pacific (EEP). Here we test the hypothesis that the Southern Ocean drove change in the EEP via subsurface intermediate waters during the last deglaciation. We use the δ 18 O signature of benthic foraminifera to reconstruct water density changes during the last 25 kyr at three intermediate water depths (370 m, 600 m, and 1000 m) in the EEP. Carbonate δ 18 O records a combined signature of temperature and salinity and is therefore more closely related to density than temperature or salinity alone. We find that benthic foraminiferal δ 18 O values decreased first in the subsurface, simultaneously with rising temperatures over Antarctica, and propagated up to the surface within~3 kyr. The early subsurface response initiated a rapid decrease in density stratification over the upper water column as indicated by reduced δ 18 O gradients between surface and intermediate depths. Stratification of the upper water column remained low through the termination, with stratification minima reached during Heinrich Stadial 1 and the Younger Dryas (YD), synchronous with the two-part deglacial rise in atmospheric CO 2 . Centennial-scale shifts toward heavier δ 18 O signatures at 370 and 600 m during the YD indicate short-lived shifts in the Subantarctic Mode Water/Antarctic Intermediate Water boundary to shallower intermediate depths. We suggest that decreased density gradients during the deglaciation accelerated vertical mixing across the EEP, and potentially the entire South Pacific subtropical gyre, which enhanced CO 2 delivery from depth to the surface ocean and atmosphere.

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.

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BackgroundLong-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.ResultsHere 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).ConclusionBy 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.

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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 metagenomicsbased 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.

The origins of life can best be understood as a series of plausible steps culminating in the emergence of a "self-sustaining chemical system capable of Darwinian evolution" (NASA Astrobiology). However, life as we know it is a highly complex collection of molecular machinery and genetic information. The central dogma of molecular biology stipulates that deoxyribonucleic acid (DNA) makes ribonucleic acid (RNA) via transcription, RNA makes protein via translation, and information cannot be transferred backwards from proteins to nucleic acids

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 a 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., 10 4 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.