Nucleic acid extraction and sequencing from low-biomass synthetic Mars analog soils for in situ life detection

Mojarro, Angel; Hachey, Julie; Bailey, Ryan C.; Brown, Mark J.; Doebler, Robert; Ruvkun, Gary; Zuber, M. T.; Carr, Christopher E.

doi:10.1101/358218

Cited by 12 publications

(17 citation statements)

References 102 publications

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“…These protocols, developed using tough-to-lyse spores of Bacillus subtilis , allow us to achieve at least 5% extraction yield from a 50 mg sample containing 2 × 10 5 cells/g of soil without centrifugation [ 6 ]. Furthermore, in order to avoid possible amplification biases and additional points of contamination, we have experimented with utilizing a genomic carrier ( Enterobacteria phage λ ) to shuttle low-input amounts of target DNA ( B. subtilis ) through library preparation and sequencing with ideal stoichiometry [ 7 ]. This approach has allowed us to detect down to 0.2 ng of B. subtilis DNA prepared with 1000 ng of Lambda DNA using the Oxford Nanopore Technologies (ONT) MinION sequencer.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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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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Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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“…This revealed diverse microbial community developed in hydrated sand, which was also noted in other environments considered to be analog sites. Life detection methods based on sequencing nucleic acid are also suggested by some authors for application in extraterrestrial locations (e.g., Mojarro et al., 2017); however, this method is difficult to perform during robotic studies and is extremely sensitive to contamination with even trace amounts of terrestrial nucleic acids, which cause false positive results (e.g., Schulze‐Makuch et al., 2015).…”

Section: Discussionmentioning

confidence: 99%

Detection of Microorganisms and Metabolism in Dune Sand of a Low Organic Content

et al. 2021

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The planet Mars is a primary target in the search for extraterrestrial life. Metabolic experiments carried out during the Viking Mission (1976Mission ( -1982 designed to detect respiration and photosynthesis provided inconclusive results, and one type of experiment (Labeled Release) seemed to be positive (Klein, 1977;Levin & Straat, 2016;Schulze-Makuch et al., 2015). Precisely, the Labeled Release experiment demonstrated that addition of radioactively-labeled organic medium to Martian soil resulted in the release of radioactive gas, which suggested that organic compounds were metabolized. Moreover, the breakdown of organic matter was ceased by temperature treatment capable of killing terrestrial microorganisms (Klein, 1977;Levin & Straat, 2016). The positive result of Labeled Release experiment was not confirmed by other experiments carried out during the Viking Mission-the Pyrolytic Release and the Gas Exchange experiments (Klein, 1977). Moreover, during the Viking Mission, no organic matter was found in samples of the Martian soil (Klein, 1977). Results from the Viking Mission have been debated further in the context of geochemical data provided by subsequent missions, especially that of the Phoenix Mars Lander ( 2008) and the Curiosity Rover (since 2012), but the question about the presence of life on Mars remains unanswered (Goetz Abstract The main goal of astrobiological studies is the search for life beyond Earth. Developing life detection methods requires test locations that have similar environmental conditions to extraterrestrial sites or that simply have low organism abundances. In this study, we describe dune sand of a low organic matter content (0.11%) collected from a national park frequented by few people. It is located in temperate zone. We hypothesized that dune sand is characterized by the low abundance of microorganisms and metabolic rates that could be compared to analogs of extraterrestrial environments like the Antarctic McMurdo Dry Valleys or the Atacama Desert. Measurements of CO 2 efflux and ATP concentration demonstrated that hydrating dune sand with sterile distilled water initiated a short period of substantial microbial metabolic activity that lasted from 4 to 5 days. The maximum CO 2 efflux was 100 mgCO 2 m −2 d −1 , which was low compared to values reported for sandy dunes, deserts and poor soils, including McMurdo Dry Valleys. Microscopic observations demonstrated that the abundance of prokaryotic microorganisms in the dune sand was low at roughly one million per cm 3 of sand and was comparable to the abundance reported from the Atacama Desert. The microbial communities in the dune sand were studied based on 16S rRNA gene analyses. The most prominent bacterial genera were Massilia and Bacillus. Study demonstrated that dune sand sampled from a national park area was as useful for testing life detection methods as are other well-established analogs of extraterrestrial environments.Plain Language Summary One of the main goals of contemporary science is the search for life beyond Earth....

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“…Coupling these imaging and sequencing advances with novel microfluidics and nanotechnology would be the next important avenue to consider for OoL researchers. Further development of nanopore technology (used in the single molecule sequencing methods described above [152]) [169,170] would further allow analysis of a wider range of prebiological polymer systems, while additionally opening up the possibility of the incorporation of sequencing technologies onto in situ life detection missions [171]. While microfluidics technologies have already been incorporated into sequencing technologies [172], recent advances in coupling microfluidic systems with compartment formation and manipulation have been developed by Masahiro Takinoue's lab at Tokyo Institute of Technology [173] and Hiroyuki Noji's lab at University of Tokyo [174,175].…”

Section: The Future Of Ool Research: Incorporating Novel Biophysics Tmentioning

confidence: 99%

Recent Advances in Origins of Life Research by Biophysicists in Japan

Jia

Kuruma

2019

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Biophysics research tends to focus on utilizing multidisciplinary technologies and interdisciplinary collaborations to study biological phenomena through the lens of chemistry and physics. Although most current biophysics work is focused on studying extant biology, the fact remains that modern biological systems at some point were descended from a universal common ancestor. At the core of modern biology is the important question of how the earliest life on (or off) Earth emerged. Recent technological and methodological advances developed by biophysicists in Japan have allowed researchers to gain a new suite of knowledge related to the origins of life (OoL). Using these reports as inspiration, here, we highlight some of the significant OoL advances contributed by members of the biophysical research field in Japan with respect to the synthesis and assembly of biological (or pre-biological) components on early Earth, the co-assembly of primitive compartments with biopolymer systems, and the evolution of early genetic systems. We hope to provide inspiration to other biophysicists to not only use the always-advancing suite of available multidisciplinary technologies to continue their own line of work, but to also consider how their work or techniques can contribute to the ever-growing field of OoL research.

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Nucleic acid extraction and sequencing from low-biomass synthetic Mars analog soils for in situ life detection

Cited by 12 publications

References 102 publications

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

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

Detection of Microorganisms and Metabolism in Dune Sand of a Low Organic Content

Recent Advances in Origins of Life Research by Biophysicists in Japan

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