Agenda

Abstract: The NASA Research program funded-project seeks to develop and validate metagenomic methodologies using digital PCR (dPCR) and shotgun metagenomic sequencing to enhance and augment existing culture-based Planetary Protection (PP) protocols. Traditional NASA assays primarily detected a limited range of organisms, often missing PP-relevant microbes (e.g., anaerobic, psychrophilic, radiation-resistant species). The metagenomics/dPCR framework might overcame this limitation by providing comprehensive microbial detection across diverse domains of life, even in "ultra-low biomass" samples typical of spacecraft and associated environments. These samples posed unique challenges, as low microbial content and high contamination risks complicated analysis. To address these challenges, we employed mock microbial communities, nucleic acid spike-ins, and rigorous validation across all sample processing stages. This framework aimed to integrate metagenomics into NASA’s standard PP practices, supporting spacecraft cleanliness and reducing forward contamination risks. The outcomes supported more efficient microbial reduction strategies, enabling safer and more accurate space exploration.

Abstract: The European Space Agency (ESA) and Committee on Space Research (COSPAR) have established planetary protection requirements for Category III & IV missions to Europa and Enceladus. These requirements necessitate a probability assessment of inadvertent contamination by viable organisms transported by spacecraft to subsurface liquid oceans, with a threshold of 1x10-4. Currently, there are no standardized models for assessing this probability. Our objective was to leverage metagenomic analyses to estimate values for several poorly understood parameters, including: 1) Bioburden at launch, 2) Cruise survival, 3) Organism viability and proliferation before, during, and after subsurface transfer. We present preliminary results from the JUICE case study, which provide insights into these parameters and propose how metagenomic approaches can be integrated into mission-critical decision-making processes in the future.

Abstract: Starting in 2013, a large number of RNAseq data sets appeared in public data repositories for bacterial transcirptomes. This growth in these data sets demanded development of novel big data analytic approaches. We discovered that when ICA (independent component analysis) is applied to compendia of bacterial transcriptomes it leads to the definition of 'independently modulated sets of genes,' or iModulons. iModulons can correspond to known regulons, but often lead to discovery of new regulatory modalities. Surprisingly, most iModulons contain y-genes (ie genes of unknown function) leading to gene annotations to a cellular function. Once a y-gene is associated with an iModulon and a cellular function, focused hypotheses can be formulated about their molecular function. Specific cases will be presented.

Abstract: Vertebrates carry microorganisms concentrated on their mucosal surfaces.  The upper respiratory tract (URT) as the primary interface for sensing and responding to aerosolized microbes from our immediate environments. Culturing the aerobes from nasal swabs allows partial discovery of the diversity present compared to molecular profiling by DNA sequencing.  Not surprisingly, most DNA molecules do not match confidently into an existing taxon.  Thus, we utilized the StrainSelect reference database (PMID: 36814618) which provides durable provisional taxonomic categories where formal Latin nomenclature has not yet evolved. This approach enabled robust comparisons of microbial abundance across biospecimens, research sites, and timeframes.  The ability to scale and extend this system to be generalizable to categorize microbial dark matter based on arrays of protein domains encoded in metagenomic assemblies will be discussed.  From this type of large scale database, associations can be made between vertebrate respiratory diseases and recurring dark-matter entities.

Abstract: Cell counts in cleanrooms, like extreme environments, are low, making adequate recovery for nucleic-acid-based planetary protection (PP) monitoring challenging. We utilized low-input metagenomics (sub 1-ng libraries) to perform Illumina-based metagenomic sequencing from a transect in the hyper-arid region of the Atacama Desert. Nanopore sequencing offers accessible amplification-free sequencing not limited to short reads, and may also be used on future missions to detect forward contamination or ancestrally-related life. However, at present, its input requirements, typically up to 1 microgram, are challenging. In prior work, we demonstrated detection of a target microbe down to 2 pg of input DNA. We highlight prior and future strategies and opportunities to advance nanopore sequencing for PP applications, which will have important benefits in other contexts, including for science and crew health studies in space by astronauts, where current in situ sequencing is limited to culture-based workflows. We foresee metagenomics as a critical tool for enabling future robotic life detection missions and validating PP strategies on the moon in support of future human exploration of Mars. Finally, we propose ways in which metagenomics can inform integration of microbial growth potential into PP policies.

Abstract: Nanopore-based environmental metagenomics is often constrained by the requirement of high amounts of input DNA (1-3 µg). Here, we demonstrate the successful application of DNA quantities below these recommended levels (down to 1 ng) and explored its impact on sequencing quality, community composition, assembly accuracy, and the recovery of metagenome-assembled genomes (MAGs). We generated 27 Nanopore metagenomes using a microbial community standard, varying input DNA from 1000 ng down to 1 ng in nine input levels in triplicates. Our results show that read quality and mapping accuracy remains equally high across all input levels, with species' relative abundances remaining stable down to 50 ng. Remarkably, high-quality MAGs (> 95% completeness and < 5% contamination) are recoverable with as little as 35 ng of DNA. Even at the lowest levels (1 ng), Nanopore reads combined with Illumina data significantly enhance hybrid assemblies for recovery of large genome fragments. When applied to real environmental samples, our protocol enables low biomass Nanopore metagenomics from small sample quantities and reduce strain heterogeneity, thereby improving assembly and genome reconstruction even down to 7.x ng. These results demonstrate that Nanopore sequencing can successfully be performed with low amounts of environmental DNA, without compromising genome recovery and consequently offer new possibilities for advancing microbiome research, especially in low-biomass environments.

Abstract: The comprehensive genomic analysis of ultra-low biomass samples to monitor Spacecraft, Cleanroom, Satellites, Probes, and other samples related to planetary protection requires high efficiency methods and reagents.  This includes stringent controls, ultra-high efficiency procedures, and DNA-free sampling equipment and reagents to circumvent the noise caused by the “Kitome” and “Extractome”. While current methods to sample, extract, and analyze DNA are routine, the efficiency data of such procedures are lacking and not documented. Additionally, the ability to obtain complete microbial lysis along with methods to amplify DNA from sub-picogram amounts of DNA prior to downstream analysis is critical . This including Metapolyzyme for space applications, Exopolyzyme, as well as techniques to pre-amplify ultra- low biomass samples using multiple displacement amplification (MDA) and Primary Template-directed Amplification (PTA) prior to downstream sequencing and analysis techniques.

Abstract: I will discuss recent work using cultivation-independent methods to explore microbial diversity in ice-free soils of Antarctica as a case study to highlight some of the challenges encountered when working with low biomass samples. I will also emphasize selected ‘best practices’ that should be followed to minimize contamination when conducting metagenomic analyses of environmental samples.

Abstract: Planetary protection is crucial in space exploration, safeguarding both the integrity of extraterrestrial life investigations and Earth’s biosphere from potentially hazardous extraterrestrial material. Current protocols emphasize bioburden reduction and contamination control, requiring rigorous sterilization and detection in cleanroom environments. This talk will review these established standards, pinpointing gaps and exploring how advanced molecular techniques, such as metagenomics, can enhance planetary protection standards. Through examples from recent space missions, we will discuss practical applications and assess the potential of new biological tools in this field.

Abstract: SMCC is a repository for collections of space microbes originated by NASA scientists and research PIs and offers digital (genomic, phenotypic, and rich metadata) and physical formats (the isolates themselves) of space microbes, which can be requested for research purposes. SMCC could be a critical tool in enhancing NASA's planetary protection efforts. By systematically identifying and cataloging isolates, researchers can better understand the diversity of microbial life associated with spacecraft and mission environments. This understanding is vital for assessing contamination risks and developing strategies to mitigate the forward contamination of other planetary bodies and backward contamination of Earth. SMCC provides access to microbial isolates to enable researchers from various disciplines to explore new hypotheses, conduct comparative studies, and contribute to a more comprehensive understanding of life in extreme environments.

Abstract: EMBL-EBI is the home of big data in biology, empowering scientists to unravel complex information and drive discoveries that benefit humanity. MGnify, the microbiome data and analysis resource at EMBL-EBI, transforms microbiome-derived sequence data into robust, reproducible analysis products. We offer detailed taxonomic and functional profiles, biome-specific genome catalogues, and access to a staggering 2.4 billion non-redundant sequences in our protein database. These data products are ripe for biodiscovery mining. MGnify is dedicated to making microbiome research accessible with all public data, bioinformatic pipelines, and resources freely available, fostering an open research environment. In this talk, we will explore how these diverse data products are generated and how they can be harnessed in groundbreaking metagenomic research. We will also look to the future of MGnify and what the next release of our pipelines has in store.

Abstract: This presentation will highlight advancements in nanopore sequencing technology and its role in enabling metagenomics for microbial surveillance in diverse environments. We will discuss the latest methods, bioinformatics tools, and case studies that implement nanopore sequencing to detect and explore complex microbial communities, revealing insights into their composition and dynamics. These capabilities support our mission to enable the analysis of anything, by anyone, anywhere, particularly in the challenging contexts of space exploration.

Abstract: Microbes are ubiquitous and can be found even in very clean environments. Understanding which microbes persist after cleaning or are present during production of ‘clean’ items can be very important to understanding and anticipating potential effects from these residual microbes. Current metagenomics tools are ideal for investigating these questions, as they provide a universal and mostly unbiased view of the microbial content of a sample. However, when dealing with extremely low-biomass samples, trace amounts of contamination can skew the results and extreme care must be taken to ensure accurate testing. I will discuss our experience with these challenges, and describe some of the methods we have developed to address them.

Abstract: The Department of Energy (DOE) Joint Genome Institute (JGI) handles thousands of samples for metagenomes and metatranscriptomes on an annual basis across diverse research projects. We have developed methods to support low-biomass samples ranging from ice cores to rock surfaces, and include shotgun sequencing and targeted cell enrichments with amplification. In addition to short-read sequencing, the JGI increasingly supports long-read sequencing for samples with ultra-low input DNA quantities. We will also describe a new single-cell amplification technique, primary template-directed amplification (PTA), that has been shown to generate near-complete genomes.

Abstract: Kris Locken is a molecular microbiologist specializing in the development and application of microbiome tools, standards and controls at Zymo Research, with a focus on advancing reliable solutions for low-biomass metagenomic analysis. He holds an M.S. in Microbiology and a B.S. in Molecular Cell Biology from California State University, Long Beach. During his studies, Kris investigated Ustilago maydis, known for its edible galls on maize, providing insights into fungal pathology and food science. He later characterized multiple antibiotic-sensitive mutants in Saccharomyces cerevisiae, contributing to our understanding of TOR kinase regulation in cellular transport and stress response. Before joining Zymo’s Microbiome team in 2019, Kris served as Quality Control Manager in two medical device manufacturing settings. His passion lies in exploring the microbial world and fostering the development of robust microbiological tools and standards for diverse research.

Abstract: With the commencement of human occupation, the microbiome of the International Space Station (ISS) has been routinely monitored to assess risk to both spacecraft and crew. Historically, this monitoring has been achieved through onboard culture and ground-based analyses. While this approach has served to provide alerts to anomalies and overall confidence in the operational controls, the data are limited to the media type and growth conditions used; the bias toward the detection of culturable organisms has depicted an overall lack in biodiversity. As NASA moves to a focus on exploration beyond low-Earth orbit, it is critical to fully understand the microbiome of the ISS and its possible association with the noted positive influence on crew and vehicle health. The implementation of culture-independent, nanopore sequencing-based studies, both onboard the ISS and with returned ground samples, is revealing a more thorough depiction of the microbiome. Results from this study have implications for crew health, planetary protection, and controls used in future spacecraft systems. The ability to perform in situ profiling of the ISS microbiome is transforming how NASA assesses risk and is a critical tool towards monitoring the establishment of the environmental microbiome in exploration spacecraft.

Abstract: The objectives of this talk are to understand the link between the human and environmental microbiome as a reservoir for infections, and discuss further directions to detect and prevent infections.

Abstract: The objective of the talk is to discuss whether or not public health is a consideration for space exploration.