RepeatFS: a file system providing reproducibility through provenance and automation.

MOTIVATION: Reproducibility is of central importance to the scientific process. The difficulty of consistently replicating and verifying experimental results is magnified in the era of big data, in which bioinformatics analysis often involves complex multi-application pipelines operating on terabytes of data. These processes result in thousands of possible permutations of data preparation steps, software versions and command-line arguments. Existing reproducibility frameworks are cumbersome and involve redesigning computational methods. To address these issues, we developed RepeatFS, a file system that records, replicates and verifies informatics workflows with no alteration to the original methods. RepeatFS also provides several other features to help promote analytical transparency and reproducibility, including provenance visualization and task automation. RESULTS: We used RepeatFS to successfully visualize and replicate a variety of bioinformatics tasks consisting of over a million operations with no alteration to the original methods. RepeatFS correctly identified all software inconsistencies that resulted in replication differences. AVAILABILITYAND IMPLEMENTATION: RepeatFS is implemented in Python 3. Its source code and documentation are available at https://github.com/ToniWestbrook/repeatfs. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genetic drift, selection and the evolution of the mutation rate.

As one of the few cellular traits that can be quantified across the tree of life, DNA-replication fidelity provides an excellent platform for understanding fundamental evolutionary processes. Furthermore, because mutation is the ultimate source of all genetic variation, clarifying why mutation rates vary is crucial for understanding all areas of biology. A potentially revealing hypothesis for mutation-rate evolution is that natural selection primarily operates to improve replication fidelity, with the ultimate limits to what can be achieved set by the power of random genetic drift. This drift-barrier hypothesis is consistent with comparative measures of mutation rates, provides a simple explanation for the existence of error-prone polymerases and yields a formal counter-argument to the view that selection fine-tunes gene-specific mutation rates.

Variable Spontaneous Mutation and Loss of Heterozygosity among Heterozygous Genomes in Yeast.

Mutation and recombination are the primary sources of genetic variation. To better understand the evolution of genetic variation, it is crucial to comprehensively investigate the processes involving mutation accumulation and recombination. In this study, we performed mutation accumulation experiments on four heterozygous diploid yeast species in the Saccharomycodaceae family to determine spontaneous mutation rates, mutation spectra, and losses of heterozygosity (LOH). We observed substantial variation in mutation rates and mutation spectra. We also observed high LOH rates (1.65-11.07×10-6 events per heterozygous site per cell division). Biases in spontaneous mutation and LOH together with selection ultimately shape the variable genome-wide nucleotide landscape in yeast species.

Marker genes as predictors of shared genomic function.

BACKGROUND: Although high-throughput marker gene studies provide valuable insight into the diversity and relative abundance of taxa in microbial communities, they do not provide direct measures of their functional capacity. Recently, scientists have shown a general desire to predict functional profiles of microbial communities based on phylogenetic identification inferred from marker genes, and recent tools have been developed to link the two. However, to date, no large-scale examination has quantified the correlation between the marker gene based taxonomic identity and protein coding gene conservation. Here we utilize 4872 representative prokaryotic genomes from NCBI to investigate the relationship between marker gene identity and shared protein coding gene content. RESULTS: Even at 99-100% marker gene identity, genomes share on average less than 75% of their protein coding gene content. This occurs regardless of the marker gene(s) used: V4 region of the 16S rRNA, complete 16S rRNA, or single copy orthologs through a multi-locus sequence analysis. An important aspect related to this observation is the intra-organism variation of 16S copies from a single genome. Although the majority of 16S copies were found to have high sequence similarity (> 99%), several genomes contained copies that were highly diverged (< 97% identity). CONCLUSIONS: This is the largest comparison between marker gene similarity and shared protein coding gene content to date. The study highlights the limitations of inferring a microbial community's functions based on marker gene phylogeny. The data presented expands upon the results of previous studies that examined one or few bacterial species and supports the hypothesis that 16S rRNA and other marker genes cannot be directly used to fully predict the functional potential of a bacterial community.

PALADIN: protein alignment for functional profiling whole metagenome shotgun data.

MOTIVATION: Whole metagenome shotgun sequencing is a powerful approach for assaying the functional potential of microbial communities. We currently lack tools that efficiently and accurately align DNA reads against protein references, the technique necessary for constructing a functional profile. Here, we present PALADIN-a novel modification of the Burrows-Wheeler Aligner that provides accurate alignment, robust reporting capabilities and orders-of-magnitude improved efficiency by directly mapping in protein space. RESULTS: We compared the accuracy and efficiency of PALADIN against existing tools that employ nucleotide or protein alignment algorithms. Using simulated reads, PALADIN consistently outperformed the popular DNA read mappers BWA and NovoAlign in detected proteins, percentage of reads mapped and ontological similarity. We also compared PALADIN against four existing protein alignment tools: BLASTX, RAPSearch2, DIAMOND and Lambda, using empirically obtained reads. PALADIN yielded results seven times faster than the best performing alternative, DIAMOND and nearly 8000 times faster than BLASTX. PALADIN's accuracy was comparable to all tested solutions. AVAILABILITY AND IMPLEMENTATION: PALADIN was implemented in C, and its source code and documentation are available at https://github.com/twestbrookunh/paladin. CONTACT: anthonyw@wildcats.unh.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Large-scale detection of in vivo transcription errors.

Accurate transmission and expression of genetic information are crucial for the survival of all living organisms. Recently, the coupling of mutation accumulation experiments and next-generation sequencing has greatly expanded our knowledge of the genomic mutation rate in both prokaryotes and eukaryotes. However, because of their transient nature, transcription errors have proven extremely difficult to quantify, and current estimates of transcription fidelity are derived from artificial constructs applied to just a few organisms. Here we report a unique cDNA library preparation technique that allows error detection in natural transcripts at the transcriptome-wide level. Application of this method to the model organism Caenorhabditis elegans revealed a base misincorporation rate in mRNAs of ~4 × 10(-6) per site, with a very biased molecular spectrum. Because the proposed method is readily applicable to other organisms, this innovation provides unique opportunities for studying the incidence of transcription errors across the tree of life.

FlowClus: efficiently filtering and denoising pyrosequenced amplicons.

BACKGROUND: Reducing the effects of sequencing errors and PCR artifacts has emerged as an essential component in amplicon-based metagenomic studies. Denoising algorithms have been designed that can reduce error rates in mock community data, but they change the sequence data in a manner that can be inconsistent with the process of removing errors in studies of real communities. In addition, they are limited by the size of the dataset and the sequencing technology used. RESULTS: FlowClus uses a systematic approach to filter and denoise reads efficiently. When denoising real datasets, FlowClus provides feedback about the process that can be used as the basis to adjust the parameters of the algorithm to suit the particular dataset. When used to analyze a mock community dataset, FlowClus produced a lower error rate compared to other denoising algorithms, while retaining significantly more sequence information. Among its other attributes, FlowClus can analyze longer reads being generated from all stages of 454 sequencing technology, as well as from Ion Torrent. It has processed a large dataset of 2.2 million GS-FLX Titanium reads in twelve hours; using its more efficient (but less precise) trie analysis option, this time was further reduced, to seven minutes. CONCLUSIONS: Many of the amplicon-based metagenomics datasets generated over the last several years have been processed through a denoising pipeline that likely caused deleterious effects on the raw data. By using FlowClus, one can avoid such negative outcomes while maintaining control over the filtering and denoising processes. Because of its efficiency, FlowClus can be used to re-analyze multiple large datasets together, thereby leading to more standardized conclusions. FlowClus is freely available on GitHub (jsh58/FlowClus); it is written in C and supported on Linux.

Genome sequence and comparative analysis of a putative entomopathogenic Serratia isolated from Caenorhabditis briggsae.

BACKGROUND: Entomopathogenic associations between nematodes in the genera Steinernema and Heterorhabdus with their cognate bacteria from the bacterial genera Xenorhabdus and Photorhabdus, respectively, are extensively studied for their potential as biological control agents against invasive insect species. These two highly coevolved associations were results of convergent evolution. Given the natural abundance of bacteria, nematodes and insects, it is surprising that only these two associations with no intermediate forms are widely studied in the entomopathogenic context. Discovering analogous systems involving novel bacterial and nematode species would shed light on the evolutionary processes involved in the transition from free living organisms to obligatory partners in entomopathogenicity. RESULTS: We report the complete genome sequence of a new member of the enterobacterial genus Serratia that forms a putative entomopathogenic complex with Caenorhabditis briggsae. Analysis of the 5.04 MB chromosomal genome predicts 4599 protein coding genes, seven sets of ribosomal RNA genes, 84 tRNA genes and a 64.8 KB plasmid encoding 74 genes. Comparative genomic analysis with three of the previously sequenced Serratia species, S. marcescens DB11 and S. proteamaculans 568, and Serratia sp. AS12, revealed that these four representatives of the genus share a core set of ~3100 genes and extensive structural conservation. The newly identified species shares a more recent common ancestor with S. marcescens with 99% sequence identity in rDNA sequence and orthology across 85.6% of predicted genes. Of the 39 genes/operons implicated in the virulence, symbiosis, recolonization, immune evasion and bioconversion, 21 (53.8%) were present in Serratia while 33 (84.6%) and 35 (89%) were present in Xenorhabdus and Photorhabdus EPN bacteria respectively. CONCLUSION: The majority of unique sequences in Serratia sp. SCBI (South African Caenorhabditis briggsae Isolate) are found in ~29 genomic islands of 5 to 65 genes and are enriched in putative functions that are biologically relevant to an entomopathogenic lifestyle, including non-ribosomal peptide synthetases, bacteriocins, fimbrial biogenesis, ushering proteins, toxins, secondary metabolite secretion and multiple drug resistance/efflux systems. By revealing the early stages of adaptation to this lifestyle, the Serratia sp. SCBI genome underscores the fact that in EPN formation the composite end result - killing, bioconversion, cadaver protection and recolonization- can be achieved by dissimilar mechanisms. This genome sequence will enable further study of the evolution of entomopathogenic nematode-bacteria complexes.

Polybrominated Diphenyl Ether (PBDE)-Induced Suppression of Phosphoenolpyruvate Carboxykinase (PEPCK) Decreases Hepatic Glyceroneogenesis and Disrupts Hepatic Lipid Homeostasis.

Polybrominated diphenyl ethers (PBDE) are a class of flame-retardant chemicals that leach into the environment and enter the human body. PBDE have been shown to suppress activity of phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme in fatty acid esterification via hepatic glyceroneogenesis. The objective of this investigation was to assess hepatic glyceroneogenesis and lipid metabolism in PBDE-treated rats. Male, weanling Wistar rats were gavaged daily for 28 d with 14 mg/kg body weight of either DE-71, a commercial PBDE mixture (treated), or corn oil (control). After a 48-h fast, rats were euthanized, blood was obtained, and livers were excised. Suppression of hepatic PEPCK activity by 40% was noted. Serum ketone bodies were elevated by 27% in treated rats compared to controls, while hepatic glyceroneogenesis as measured by (14)C-pyruvate incorporation into triglycerides was 41% lower in explants from treated rats compared to controls. Liver lipid content was 29% lower in treated animals compared to controls. Taken together, these findings suggest that DE-71-induced inhibition of hepatic PEPCK activity alters lipid metabolism by redirecting fatty acids away from esterification and storage toward ketone synthesis.

Extraordinary genome stability in the ciliate Paramecium tetraurelia.

Mutation plays a central role in all evolutionary processes and is also the basis of genetic disorders. Established base-substitution mutation rates in eukaryotes range between ∼5 × 10(-10) and 5 × 10(-8) per site per generation, but here we report a genome-wide estimate for Paramecium tetraurelia that is more than an order of magnitude lower than any previous eukaryotic estimate. Nevertheless, when the mutation rate per cell division is extrapolated to the length of the sexual cycle for this protist, the measure obtained is comparable to that for multicellular species with similar genome sizes. Because Paramecium has a transcriptionally silent germ-line nucleus, these results are consistent with the hypothesis that natural selection operates on the cumulative germ-line replication fidelity per episode of somatic gene expression, with the germ-line mutation rate per cell division evolving downward to the lower barrier imposed by random genetic drift. We observe ciliate-specific modifications of widely conserved amino acid sites in DNA polymerases as one potential explanation for unusually high levels of replication fidelity.

An SNP-based second-generation genetic map of Daphnia magna and its application to QTL analysis of phenotypic traits.

BACKGROUND: Although Daphnia is increasingly recognized as a model for ecological genomics and biomedical research, there is, as of yet, no high-resolution genetic map for the genus. Such a map would provide an important tool for mapping phenotypes and assembling the genome. Here we estimate the genome size of Daphnia magna and describe the construction of an SNP array based linkage map. We then test the suitability of the map for life history and behavioural trait mapping. The two parent genotypes used to produce the map derived from D. magna populations with and without fish predation, respectively and are therefore expected to show divergent behaviour and life-histories. RESULTS: Using flow cytometry we estimated the genome size of D. magna to be about 238 mb. We developed an SNP array tailored to type SNPs in a D. magna F2 panel and used it to construct a D. magna linkage map, which included 1,324 informative markers. The map produced ten linkage groups ranging from 108.9 to 203.6 cM, with an average distance between markers of 1.13 cM and a total map length of 1,483.6 cM (Kosambi corrected). The physical length per cM is estimated to be 160 kb. Mapping infertility genes, life history traits and behavioural traits on this map revealed several significant QTL peaks and showed a complex pattern of underlying genetics, with different traits showing strongly different genetic architectures. CONCLUSIONS: The new linkage map of D. magna constructed here allowed us to characterize genetic differences among parent genotypes from populations with ecological differences. The QTL effect plots are partially consistent with our expectation of local adaptation under contrasting predation regimes. Furthermore, the new genetic map will be an important tool for the Daphnia research community and will contribute to the physical map of the D. magna genome project and the further mapping of phenotypic traits. The clones used to produce the linkage map are maintained in a stock collection and can be used for mapping QTLs of traits that show variance among the F2 clones.

The transcriptional consequences of mutation and natural selection in Caenorhabditis elegans.

The evolutionary importance of gene-expression divergence is unclear: some studies suggest that it is an important mechanism for evolution by natural selection, whereas others claim that most between-species regulatory changes are neutral or nearly neutral. We examined global transcriptional divergence patterns in a set of Caenorhabditis elegans mutation-accumulation lines and natural isolate lines to provide insights into the evolutionary importance of transcriptional variation and to discriminate between the forces of mutation and natural selection in shaping the evolution of gene expression. We detected the effects of selection on transcriptional divergence patterns and characterized them with respect to coexpressed gene sets, chromosomal clustering of expression changes and functional gene categories. We directly compared observed transcriptional variation patterns in the mutation-accumulation and natural isolate lines to a neutral model of transcriptome evolution to show that strong stabilizing selection dominates the evolution of transcriptional change for thousands of C. elegans expressed sequences.

Whole genome sequencing of Streptomyces antnestii sp. nov. with a potency to become an industrial strain.

Streptomyces Strain San01 is isolated from the soil of ant-nest found in the tea estate of Darjeeling, India. The morphology, biochemical, as well as the molecular characteristics, proved that San01 belonged to the genus Streptomyces. The average nucleotide identity (ANI) value between the genome sequence of the studied strain and its closest phylogenetic neighbors were very low and also could be distinguished from its closest neighbour with broad range of phenotypic data. The draft genome sequence of isolate San01 (NZ_RZYA00000000.1) was estimated to be 9.12 Mbp in size with 71.2% of GC content and it encompasses 39 biosynthetic gene clusters that emphasize the biotechnological potential of this isolate.Based on the phenotypic, genetic and genomic data, isolate San01 (=JCM 34633 = NCTC 14543) merits to be recognized as a type strain of a novel species and hereby propose the name Streptomyces antnestii sp. nov. Incidentally, this is the first report on Streptomyces genomes from Darjeeling, India.

Remarkably High Repeat Content in the Genomes of Sparrows: The Importance of Genome Assembly Completeness for Transposable Element Discovery.

Transposable elements (TE) play critical roles in shaping genome evolution. Highly repetitive TE sequences are also a major source of assembly gaps making it difficult to fully understand the impact of these elements on host genomes. The increased capacity of long-read sequencing technologies to span highly repetitive regions promises to provide new insights into patterns of TE activity across diverse taxa. Here we report the generation of highly contiguous reference genomes using PacBio long-read and Omni-C technologies for three species of Passerellidae sparrow. We compared these assemblies to three chromosome-level sparrow assemblies and nine other sparrow assemblies generated using a variety of short- and long-read technologies. All long-read based assemblies were longer (range: 1.12 to 1.41 Gb) than short-read assemblies (0.91 to 1.08 Gb) and assembly length was strongly correlated with the amount of repeat content. Repeat content for Bell's sparrow (31.2% of genome) was the highest level ever reported within the order Passeriformes, which comprises over half of avian diversity. The highest levels of repeat content (79.2% to 93.7%) were found on the W chromosome relative to other regions of the genome. Finally, we show that proliferation of different TE classes varied even among species with similar levels of repeat content. These patterns support a dynamic model of TE expansion and contraction even in a clade where TEs were once thought to be fairly depauperate and static. Our work highlights how the resolution of difficult-to-assemble regions of the genome with new sequencing technologies promises to transform our understanding of avian genome evolution.

Metagenomics for Pathogen Detection During a Mass Mortality Event in Songbirds.

Mass mortality events in wildlife can be indications of an emerging infectious disease. During the spring and summer of 2021, hundreds of dead passerines were reported across the eastern US. Birds exhibited a range of clinical signs including swollen conjunctiva, ocular discharge, ataxia, and nystagmus. As part of the diagnostic investigation, high-throughput metagenomic next-generation sequencing was performed across three molecular laboratories on samples from affected birds. Many potentially pathogenic microbes were detected, with bacteria forming the largest proportion; however, no singular agent was consistently identified, with many of the detected microbes also found in unaffected (control) birds and thus considered to be subclinical infections. Congruent results across laboratories have helped drive further investigation into alternative causes, including environmental contaminants and nutritional deficiencies. This work highlights the utility of metagenomic approaches in investigations of emerging diseases and provides a framework for future wildlife mortality events.

The genome of Romanomermis culicivorax: revealing fundamental changes in the core developmental genetic toolkit in Nematoda.

BACKGROUND: The genetics of development in the nematode Caenorhabditis elegans has been described in exquisite detail. The phylum Nematoda has two classes: Chromadorea (which includes C. elegans) and the Enoplea. While the development of many chromadorean species resembles closely that of C. elegans, enoplean nematodes show markedly different patterns of early cell division and cell fate assignment. Embryogenesis of the enoplean Romanomermis culicivorax has been studied in detail, but the genetic circuitry underpinning development in this species has not been explored. RESULTS: We generated a draft genome for R. culicivorax and compared its gene content with that of C. elegans, a second enoplean, the vertebrate parasite Trichinella spiralis, and a representative arthropod, Tribolium castaneum. This comparison revealed that R. culicivorax has retained components of the conserved ecdysozoan developmental gene toolkit lost in C. elegans. T. spiralis has independently lost even more of this toolkit than has C. elegans. However, the C. elegans toolkit is not simply depauperate, as many novel genes essential for embryogenesis in C. elegans are not found in, or have only extremely divergent homologues in R. culicivorax and T. spiralis. Our data imply fundamental differences in the genetic programmes not only for early cell specification but also others such as vulva formation and sex determination. CONCLUSIONS: Despite the apparent morphological conservatism, major differences in the molecular logic of development have evolved within the phylum Nematoda. R. culicivorax serves as a tractable system to contrast C. elegans and understand how divergent genomic and thus regulatory backgrounds nevertheless generate a conserved phenotype. The R. culicivorax draft genome will promote use of this species as a research model.

Nematodes from galls on Myrtaceae. IV. Fergusobia from flat leaf galls on Eucalyptus and Corymbia, with descriptions of two new species.

Two new species of Fergusobia are described. Both were collected from flat leaf galls from South Australia, one on Eucalyptus microcarpa and the other on E. porosa. Fergusobia microcarpae n. sp. Davies is characterised by the combination of a C-shaped parthenogenetic female with a short, broadly rounded conoid tail, an arcuate to open C-shaped infective female with an hemispherical tail tip, and arcuate to J-shaped males with angular spicules and short peloderan bursa. Fergusobia porosae n. sp. Davies is similar in having an arcuate to C-shaped parthenogenetic female with a small conoid tail, an almost straight to arcuate infective female with an hemispherical tail tip, and males that are almost straight to barely J-shaped with angular spicules and short peloderan bursa. They differ in that the bodies of parthenogenetic and infective females of F. microcarpae n. sp. are more curved than in F. porosae n. sp. Other known similar forms of Fergusobia/Fergusonina galls are outlined and the larval shield morphologies of their associated mutualistic Fergusonina fly species are discussed where known. An inventory of all known Fergusobia/Fergusonina associations from flat leaf galls from Corymbia spp. and Eucalyptus spp. is presented. Relationships of Fergusobia nematodes were inferred from analysis of sequences of 28S rDNA D2/D3 domains and a portion of mitochondrial DNA cytochrome oxidase subunit I (mtCOI). Nematodes from flat leaf galls appeared in two clades. 

Nematodes from galls on Myrtaceae. V. Fergusobia from large multilocular shoot bud galls from Angophora and Eucalyptus in Australia, with descriptions of six new species.

Six new species of Fergusobia, from large multilocular shoot bud galls on two species of Angophora and four species of Eucalyptus from both subgenera Eucalyptus and Symphyomyrtus, are described. Fergusobia cosmophyllae Davies n. sp. is characterized by the combination of a C-shaped parthenogenetic female with a short arcuate conoid tail, a broad (small a ratio) arcuate infective female with an hemispherical tail tip, and an arcuate to J-shaped male with broad, angular spicules and short bursa.  Fergusobia delegatensae Davies n. sp. has an open C-shaped parthenogenetic female with a broadly conoid tail, an infective female of variable shape with an hemispherical tail tip, and a male of open C-shape with a crenate bursa that arises 40-70% along the length of the body from the tail tip and terminates just anterior to the cloaca. Fergusobia diversifoliae Davies n. sp. has a C-shaped parthenogenetic female with a conoid tail, an arcuate infective female with a hemispherical tail tip, and an arcuate, C- or J-shaped male with angular spicule and a long peloderan bursa. Fergusobia floribundae Davies n. sp. has a C-shaped parthenogenetic female with a narrow, arcuate, conoid tail, an arcuate infective female with a hemispherical tail tip, and an arcuate or J-shaped male with an angular spicule and a short to mid-body length peloderan bursa. Fergusobia minimus Lisnawita n. sp. has a C-shaped parthenogenetic female with a conoid tail, an arcuate to open C-shaped infective female with a hemispherical tail tip, and an arcuate to open C-shaped male with an angular spicule and a peloderan bursa arising at about 10-30% of body length. Fergusobia pimpamensis Davies n. sp. has an open C to C-shaped parthenogenetic female with a narrow conoid tail, an arcuate to open C-shaped infective female with a hemispherical tail tip, and an arcuate to C-shaped male with an arcuate spicule and a long, crenate, peloderan bursa. An inventory of all known Fergusobia/Fergusonina associations from terminal shoot bud galls is presented. The larval shield morphology of the associated mutualistic Fergusonina species is discussed where known. Analyses of DNA sequences of D2/D3 and COI suggested that the six new species are distributed between three clades of Fergusobia.

Nematode mitochondrial metagenomics: A new tool for biodiversity analysis.

DNA barcoding approaches have greatly increased our understanding of biodiversity on the planet, and metabarcoding is widely used for classifying members of the phylum Nematoda. However, loci typically utilized in metabarcoding studies are often unable to resolve closely related species or are unable to recover all taxa present in a sample due to inadequate PCR primer binding. Mitochondrial metagenomics (mtMG) is an alternative approach utilizing shotgun sequencing of total DNA to recover the mitochondrial genomes of all species present in samples. However, this approach requires a comprehensive reference database for identification and currently available mitochondrial sequences for nematodes are highly dominated by sequences from the order Rhabditida, and excludes many clades entirely. Here, we analysed the efficacy of mtMG for the recovery of nematode taxa and the generation of mitochondrial genomes. We first developed a curated reference database of nematode mitochondrial sequences and expanded it with 40 newly sequenced taxa. We then tested the mito-metagenomics approach using a series of nematode mock communities consisting of morphologically identified nematode species representing various feeding traits, life stages, and phylogenetic relationships. We were able to identify all but two species through the de novo assembly of COX1 genes. We were also able to recover additional mitochondrial protein coding genes (PCGs) for 23 of the 24 detected species including a full array of 12 PCGs from five of the species. We conclude that mtMG offers a potential for the effective recovery of nematode biodiversity but remains limited by the breadth of the reference database.

Metagenomic Methods for Addressing NASA's Planetary Protection Policy Requirements on Future Missions: A Workshop Report.

Molecular biology methods and technologies have advanced substantially over the past decade. These new molecular methods should be incorporated among the standard tools of planetary protection (PP) and could be validated for incorporation by 2026. To address the feasibility of applying modern molecular techniques to such an application, NASA conducted a technology workshop with private industry partners, academics, and government agency stakeholders, along with NASA staff and contractors. The technical discussions and presentations of the Multi-Mission Metagenomics Technology Development Workshop focused on modernizing and supplementing the current PP assays. The goals of the workshop were to assess the state of metagenomics and other advanced molecular techniques in the context of providing a validated framework to supplement the bacterial endospore-based NASA Standard Assay and to identify knowledge and technology gaps. In particular, workshop participants were tasked with discussing metagenomics as a stand-alone technology to provide rapid and comprehensive analysis of total nucleic acids and viable microorganisms on spacecraft surfaces, thereby allowing for the development of tailored and cost-effective microbial reduction plans for each hardware item on a spacecraft. Workshop participants recommended metagenomics approaches as the only data source that can adequately feed into quantitative microbial risk assessment models for evaluating the risk of forward (exploring extraterrestrial planet) and back (Earth harmful biological) contamination. Participants were unanimous that a metagenomics workflow, in tandem with rapid targeted quantitative (digital) PCR, represents a revolutionary advance over existing methods for the assessment of microbial bioburden on spacecraft surfaces. The workshop highlighted low biomass sampling, reagent contamination, and inconsistent bioinformatics data analysis as key areas for technology development. Finally, it was concluded that implementing metagenomics as an additional workflow for addressing concerns of NASA's robotic mission will represent a dramatic improvement in technology advancement for PP and will benefit future missions where mission success is affected by backward and forward contamination.