Biology subjects

Ruvkun, G.

Publications and source records attributed to Ruvkun, G..

17 recordsLinked to original sources

bioRxiv2026

Lysosome-related organelle genes are required for mitochondrial transformations during bacterial infections in Caenorhabditis elegans.

Detecting and responding to pathogenic bacteria is an essential function of eukaryotic cells. As bacteria-derived organelles, mitochondria carry lipids, proteins, and other molecules such as iron which are required for bacterial growth, and thus are subject to pathogenic bacterial attack. In fact, mitochondrial morphology and function are often altered during early bacterial infection. However, the mechanism by which bacterial pathogen attack triggers mitochondrial responses is unknown. Here, we demonstrate that infection by the pathogenic bacterium Staphylococcus aureus or Pseudomonas aeruginosa or hypoxia leads to remodeling of the mitochondrial network in the host Caenorhabditis elegans. Our analysis discovers that lysosome-related organelle (LRO) genes are required for this remodeling and indicates that LROs also precipitate downstream infection-response gene expression.

immunology↗

bioRxiv2025

The effects of intense light on Caenorhabditis elegans.

Caenorhabditis elegans utilize light receptors to modify foraging and locomotion in response to phototoxic blue and UV light. Our study investigated physiological and behavioral changes in C. elegans during and after exposure to high-intensity light-emitting diode (LED) light. Our findings corroborate previously reported blue-light-induced oxidative stress, mitochondrial damage, and avoidance behavior. Novel to our work is the identification of a protective role for lysosome-related organelles (LROs), the observation of a unique "shelter-seeking behavior" potentially mediated by light gradient sensing, and the seemingly dispensable role of canonical light receptors in this specific behavioral response.

developmental biology↗

bioRxiv2024

Caenorhabditis elegans SynMuv B gene activity is down-regulated during a viral infection to enhance RNA interference

Small RNA pathways regulate eukaryotic antiviral defense. Many of the Caenorhabditis elegans mutations that were identified based on their enhanced RNAi, the synMuv B genes, also emerged from unrelated genetic screens for increased growth factor signaling. The dozen synMuv B genes encode homologues of the mammalian dREAM complex found in nearly all animals and plants, which includes the lin-35/retinoblastoma oncogene. We show that a set of highly induced mRNAs in synMuv B mutants is congruent with mRNAs induced by Orsay RNA virus infection of C. elegans. In wild type animals, a combination of a synMuv A mutation and a synMuv B mutation are required for the Muv phenotype of increased growth factor signaling. But we show that Orsay virus infection of a single synMuv A mutant can induce a Muv phenotype, unlike the uninfected single synMuv A mutant. This suggests that decreased synMuv B activity, which activates the antiviral RNAi pathway, is a defense response to viral infection. Small RNA deep sequencing analysis of various dREAM complex mutants uncovers distinct siRNA profiles indicative of such an siRNA response. We conclude that the synMuv B mutants maintain an antiviral readiness state even in the absence of actual infection. The enhanced RNAi and conservation of the dREAM complex mutants suggests new therapeutic avenues to boost antiviral defenses.

genetics↗

bioRxiv2024

Mitochondrial fission surveillance is coupled to Caenorhabditis elegans DNA and chromosome segregation integrity

Mitochondrial fission and fusion are tightly regulated to specify mitochondrial abundance, localization, and arrangement during cell division as well as in the diverse differentiated cell types and physiological states. However, the regulatory pathways for such mitochondrial dynamics are less explored than the mitochondrial fission and fusion components. Here we report a large-scale screen for genes that regulate mitochondrial fission. Mitochondrial fission defects cause a characteristic asymmetric fluorescent pattern in embryos carrying mitochondrial stress reporter genes. Using this asymmetric activation, we performed RNAi screens that identified 3 kinase genes from a [~]500-kinase library and another 11 genes from 3,300 random genes that function in mitochondrial fission. Many of these identified genes play roles in chromosome segregation. We find that chromosome missegregation and genome instability lead to dysregulation of mitochondrial fission in a manner independent of Drp-1. ATL-1, the C. elegans ATR orthologue, plays a protective role in alleviating the mitochondrial fission defect caused by chromosome missegregation. This establishes a screening paradigm for identifying mitochondrial fission regulators which reveals the role of ATR in surveilling mitochondrial fission to mitigate dysregulation caused by improper chromosome segregation.

cell biology↗

bioRxiv2023

Perturbed sulfur homeostasis allows C. elegans to escape growth retardation on Actinobacteria from its natural microbiome

The rate at which organisms grow is influenced by their biotic environment. The nematode Caenorhabditis elegans grows slower in the presence of Actinobacteria, but it is unknown why. Here, we show how perturbed levels of hydrogen sulfide and cysteine modulate the growth rate of C. elegans on Actinobacteria. Using an unbiased forward genetic screen of C. elegans we discovered alleles of the conserved cystathionine gamma-lyase (cth-2/CTH) that improved animal growth rate on Actinobacteria. Conversely, null alleles of cth-2 cause developmental arrest of animals grown on Actinobacteria, which can be rescued by exogenous H2S. We also discovered a leucine rich repeat gene that regulates cysteine and H2S production, lrr-2/LRRC58. A wild isolate of C. elegans that naturally grows well on Actinobacteria has a mutant allele of lrr-2, suggesting this sulfur metabolism pathway is important for the regulation of animal growth rate in its natural ecological context. We propose a model in which wild-type worms use sulfurous compounds to promote growth of their favored bacterial food sources by inhibiting Actinobacteria growth. This strategy becomes a liability when Actinobacteria are the sole food source but can be bypassed by mutations in sulfur metabolism. This study reveals how the homeostasis of sulfurous compounds controls the growth rate of animals in an ecological context.

ecology↗

bioRxiv2023

Hypoxia-inducible factor promotes cysteine homeostasis in Caenorhabditis elegans

Dedicated genetic pathways regulate cysteine homeostasis. For example, high levels of cysteine activate cysteine dioxygenase, a key enzyme in cysteine catabolism in most animal and many fungal species. The mechanism by which cysteine dioxygenase is regulated is largely unknown. In an unbiased genetic screen for mutations that activate cysteine dioxygenase (cdo-1) in the nematode C. elegans, we isolated loss-of-function mutations in rhy-1 and egl-9, which encode proteins that negatively regulate the stability or activity of the oxygen-sensing hypoxia inducible transcription factor (hif-1). EGL-9 and HIF-1 are core members of the conserved eukaryotic hypoxia response. However, we demonstrate that the mechanism of HIF-1-mediated induction of cdo-1 is largely independent of EGL-9 prolyl hydroxylase activity and the von Hippel-Lindau E3 ubiquitin ligase, the classical hypoxia signaling pathway components. We demonstrate that C. elegans cdo-1 is transcriptionally activated by high levels of cysteine and hif-1. hif-1-dependent activation of cdo-1 occurs downstream of an H2S-sensing pathway that includes rhy-1, cysl-1, and egl-9. cdo-1 transcription is primarily activated in the hypodermis where it is also sufficient to drive sulfur amino acid metabolism. Thus, the regulation of cdo-1 by hif-1 reveals a negative feedback loop that maintains cysteine homeostasis. High levels of cysteine stimulate the production of an H2S signal. H2S then acts through the rhy-1/cysl-1/egl-9 signaling pathway to increase HIF-1-mediated transcription of cdo-1, promoting degradation of cysteine via CDO-1.

genetics↗

bioRxiv2021

Phylogenetic evidence for asparagine to aspartic acid protein editing of N-glycosylated SARS-CoV-2 viral proteins by NGLY1 deglycosylation/deamidation suggests an unusual vaccination strategy

Many viral proteins, including multiple SARS-CoV-2 proteins, are secreted via the endoplasmic reticulum, and viral particles are assembled and exported in ER-associated replication compartments. Viral coat proteins such as the SARS-CoV-2 Spike protein are N-glycosylated at NxS/T sites as they enter the ER. N-glycosylated sites in many eukaryotic proteins are deglycosylated by the NGLY1/PNG-1 deglycosylation enzyme which also deamidates the N-glycosylated asparagine to aspartic acid, thus editing the target protein sequence. Proteomic analysis of mammalian cell lines has revealed deamidation of many host N-glycosylated asparagines to aspartic acid by NGLY1/PNG-1 on peptides that are presented by mammalian HLA for immune surveillance. The key client protein for NGLY1/PNG-1 deglycosylation and N to D protein editing was revealed by genetic analysis of C. elegans proteasome regulation to be the intact endoplasmic reticulum-transiting SKN-1A transcription factor. Strikingly, an analysis of cancer cell genetic dependencies for growth revealed that the mammalian orthologue of SKN-1A, NRF1 (also called NFE2L1) is required by a highly correlated set of cell lines as NGLY1/PNG-1, supporting that NGLY1/PNG-1 and NRF1 act in the same pathway. NGLY1/PNG-1 edits N-glycosylated asparagines on the intact SKN-1 protein as it is retrieved by ERAD from the ER to in turn activate the transcription of target proteasomal genes. The normal requirement for NGLY1/PNG-1 editing of SKN-1A can be bypassed by a genomic substituion of N to D in four NxS/T N-glycosylation motifs of SKN-1A. Thus NGLY1/PNG-1-mediated N to D protein editing is more than a degradation step for the key client protein for proteasomal homeostasis in C. elegans or tumor growth in particular mammalian cell lines, SKN-1A/NRF1. In addition, such N to D substitutions in NxS/T N-glycosylation motifs occur in evolution: N to D substitutions are observed in phylogenetic comparisons of SKN-1A between nematode species that diverged hundreds of millions of years ago or of the vertebrate NRF1 between disparate vertebrates. Genomic N to D mutations bypass the many steps in N-glycosylation in the ER and deglycosylation-based editing of N to D, perhaps based on differences in the competency of divergent species for various N-glycosylation or deglycosylation steps. We surveyed the N-glycosylation sites in coronavirus proteins for such phylogenetic evidence for N to D protein editing in viral life cycles, and found evidence for preferential N to D residue substitutions in NxS/T N-glycosylation sites in comparisons of the genome sequences of hundreds of coronaviruses. This suggests that viruses use NGLY1/PNG-1 in some hosts, for example humans, to edit particular N-glycosylated residues to aspartic acid, but that in other hosts, often in bats, an N to D substitution mutation in the virus genome is selected. Single nucleotide mutations in Asp or Asn codons can produce viruses with N to D or D to N substitutions that might be selected in different animal hosts from the population of viral variants produced in any previous host. NGLY1/PNG-1 has been implicated in viral immunity in mammalian cell culture, favoring this hypothesis. Because of the phylogenetic evidence that the NGLY1/PNG-1 editing of protein sequences has functional importance for SKN-1A/NRF1 and viruses, and because most immunization protocols do not address the probable editing and functional importance of N-glycosylated aspargines to aspartic acid in normal viral infections, we suggest that immunization with viral proteins engineered to substitute D at genomically encoded NxS/T sites of N-glycosylated viral proteins that show a high frequency of N to D substitution in viral phylogeny may enhance immunological response to peptide antigens. Such genomically-edited peptides would not require ER-localization for N-glycosylation or other cell compartment localization for NGLY1/PNG-1 N to D protein editing. In addition, such N to D edited protein vaccines could be produced in bacteria since N-glycosylation and deglycosylation which do not occur in bacteria would no longer be required to immunize with a D-substituted peptide. Bacterially-expressed vaccines would be much lower cost and with fewer failure modes than attenuated viral vaccines or recombinant animal viruses produced in chicken eggs, mammalian tissue culture cells, or delivered by mRNA vectors to the patient directly. Because N to D edited peptides are clearly produced by NGLY1/PNG-1, and may be and presented by mammalian HLA, such peptides may more robustly activate T-cell killing or B-cell maturation to mediate more robust viral immunity.

genetics↗

bioRxiv2020

Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans

The molybdenum cofactor (Moco) is a 520 dalton prosthetic group that is synthesized in all domains of life. In animals, four oxidases (among them sulfite oxidase) use Moco as a prosthetic group. Moco is essential in animals; humans with mutations in genes that encode Moco-biosynthetic enzymes display lethal neurological and developmental defects. Moco supplementation seems a logical therapy, however the instability of Moco has precluded biochemical and cell biological studies of Moco transport and bioavailability. The nematode Caenorhabditis elegans can take up Moco from its bacterial diet and transport it to cells and tissues that express Moco-requiring enzymes, suggesting a system for Moco uptake and distribution. Here we show that protein-bound Moco is the stable, bioavailable species of Moco taken up by C. elegans from its diet and is an effective dietary supplement, rescuing a C. elegans model of Moco deficiency. We demonstrate that diverse Moco:protein complexes are stable and bioavailable, suggesting a new strategy for the production and delivery of therapeutically active Moco to treat human Moco deficiency.

developmental biology↗

bioRxiv2020

Two isoforms of the essential C. elegans Argonaute CSR-1 differentially regulate sperm and oocyte fertility through distinct small RNA classes

The C. elegans genome encodes nineteen functional Argonaute proteins that utilize 22G-RNAs, 26G-RNAs, miRNAs, or piRNAs to regulate their target transcripts. Only one Argonaute is essential under normal laboratory conditions: CSR-1. While CSR-1 has been studied widely, nearly all studies have overlooked the fact that the csr-1 locus encodes two isoforms. These isoforms differ by an additional 163 amino acids present in the N-terminus of CSR-1a. Using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG into the long (CSR-1a) and short (CSR-1b) isoforms, we found that CSR-1a is expressed during spermatogenesis and in several somatic tissues, including the intestine. CSR-1b is expressed constitutively in the germline. small RNA sequencing of CSR-1 complexes shows that they interact with partly overlapping sets of 22G-RNAs. Phenotypic analyses reveal that the essential functions of csr-1 described in the literature coincide with CSR-1b, while CSR-1a plays tissue specific functions. During spermatogenesis, CSR-1a integrates into an sRNA regulatory network including ALG-3, ALG-4, and WAGO-10 that is necessary for fertility at 25{degrees}C. In the intestine, CSR-1a silences immunity and pathogen-responsive genes, and its loss results in improved survival from the pathogen Pseudomonas aeruginosa. Our findings functionally distinguish the CSR-1 isoforms and highlight the importance of studying each AGO isoform independently.

molecular biology↗

bioRxiv2020

Induction of RNA interference by C. elegans mitochondrial dysfunction via the DRH-1/RIG-I homologue RNA helicase and the EOL-1/RNA decapping enzyme

RNA interference (RNAi) is an antiviral pathway common to many eukaryotes that detects and cleaves foreign nucleic acids. In mammals, mitochondrially localized proteins such as MAVS, RIG-I, and MDA5 mediate antiviral responses. Here, we report that mitochondrial dysfunction in Caenorhabditis elegans activates RNAi-directed silencing via induction of a pathway homologous to the mammalian RIG-I helicase viral response pathway. The induction of RNAi also requires the conserved RNA decapping enzyme EOL-1/DXO. The transcriptional induction of eol-1 requires DRH-1 as well as the mitochondrial unfolded protein response (UPRmt). Upon mitochondrial dysfunction, EOL-1 is concentrated into foci that depend on the transcription of mitochondrial RNAs that may form dsRNA, as has been observed in mammalian antiviral responses. The enhanced RNAi triggered by mitochondrial dysfunction contributes to the increase in longevity that is induced by mitochondrial dysfunction.

genetics↗

bioRxiv2020

Lysosomal activity regulates Caenorhabditis elegans mitochondrial dynamics through vitamin B12 metabolism

Mitochondrial fission and fusion are highly regulated by energy demand and physiological conditions to control the production, activity, and movement of these organelles. Mitochondria are arrayed in a periodic pattern in Caenorhabditis elegans muscle, but this pattern is disrupted by mutations in the mitochondrial fission component dynamin. Here we show that the dramatically disorganized mitochondria caused by a mitochondrial fission-defective dynamin mutation is strongly suppressed to a more periodic pattern by a second mutation in lysosomal biogenesis or acidification. Vitamin B12 is normally imported from the bacterial diet via lysosomal degradation of B12-binding proteins and transport of vitamin B12 to the mitochondrion and cytoplasm. We show that the lysosomal dysfunction induced by gene inactivations of lysosomal biogenesis or acidification factors causes vitamin B12 deficiency. Growth of the C. elegans dynamin mutant on an E. coli strain with low vitamin B12 also strongly suppressed the mitochondrial fission defect. Of the two C. elegans enzymes that require B12, gene inactivation of methionine synthase suppressed the mitochondrial fission defect of a dynamin mutation. We show that lysosomal dysfunction induced mitochondrial biogenesis which is mediated by vitamin B12 deficiency and methionine restriction. S-adenosylmethionine, the methyl donor of many methylation reactions, including histones, is synthesized from methionine by S-adenosylmethionine synthase; inactivation of the sams-1 S-adenosylmethionine synthase also suppresses the drp-1 fission defect, suggesting that vitamin B12 regulates mitochondrial biogenesis and then affects mitochondrial fission via chromatin pathways. SIGNIFICANCE STATEMENTThe balance of mitochondrial fission and fusion, two aspects of mitochondrial dynamics, is important for mitochondrial function. Here we show that Caenorhabditis elegans lysosomal activity regulates mitochondrial dynamics by affecting mitochondrial fission through interfering the metabolism of a micronutrient, vitamin B12. Vitamin B12 is exclusively obtained from diets in animals including C. elegans and humans, and its uptake is mediated by the lysosome. We show that lysosomal dysfunction causes vitamin B12 deficiency that leads to reduction of methionine and S-adenosylmethionine to in turn increase mitochondrial biogenesis and fission. Our study provides an insight on the interactions between mitochondrial function and micronutrient metabolism.

cell biology↗

bioRxiv2020

Nanopore Sequencing at Mars, Europa and Microgravity Conditions

Nanopore sequencing, as represented by Oxford Nanopore Technologies MinION, is a promising technology for in situ life detection and for microbial monitoring including in support of human space exploration, due to its small size, low mass ([~]100 g) and low power ([~]1W). Now ubiquitous on Earth and previously demonstrated on the International Space Station (ISS), nanopore sequencing involves translocation of DNA through a biological nanopore on timescales of milliseconds per base. Nanopore sequencing is now being done in both controlled lab settings as well as in diverse environments that include ground, air and space vehicles. Future space missions may also utilize nanopore sequencing in reduced gravity environments, such as in the search for life on Mars (Earth-relative gravito-inertial acceleration (GIA) g = 0.378), or at icy moons such as Europa (g = 0.134) or Enceladus (g = 0.012). We confirm the ability to sequence at Mars as well as near Europa or Lunar (g = 0.166) and lower g levels, demonstrate the functionality of updated chemistry and sequencing protocols under parabolic flight, and reveal consistent performance across g level, during dynamic accelerations, and despite vibrations with significant power at translocation-relevant frequencies. Our work strengthens the use case for nanopore sequencing in dynamic environments on Earth and in space, including as part of the search for nucleic-acid based life beyond Earth.

genomics↗

bioRxiv2020

Bacterial carotenoids suppress Caenorhabditis elegans surveillance and defense of translational dysfunction

Microbial toxins and virulence factors often target the eukaryotic translation machinery. Caenorhabditis elegans surveils for such microbial attacks by monitoring translational competence, and if a deficit is detected, particular drug detoxification and bacterial defense genes are induced. The bacteria Kocuria rhizophila has evolved countermeasures to animal translational surveillance and defense pathways. Here, we used comprehensive genetic analysis of Kocuria rhizophila to identify the bacterial genetic pathways that inhibit C. elegans translational toxin surveillance and defense. Kocuria rhizophila mutations that disrupt its ability to disable animal immunity and defense map to multiple steps in the biosynthesis of a 50-carbon bacterial carotenoid from 5 carbon precursors. Extracts of the C50 carotenoid from wild type K. rhizophila could restore this bacterial anti-immunity activity to K. rhizophila carotenoid biosynthetic mutant. Corynebacterium glutamicum, also inhibits the C. elegans translation detoxification response by producing the C50 carotenoid decaprenoxanthin, and C. glutamicum carotenoid mutants are defective in this suppression of C. elegans detoxification. Consistent with the salience of these bacterial countermeasures to animal drug responses, bacterial carotenoids sensitize C. elegans to drugs that target translation and inhibit food aversion behaviors normally induced by protein translation toxins or mutations. The surveillance and response to toxins is mediated by signaling pathways conserved across animal phylogeny, suggesting that these bacterial carotenoids may also suppress such human immune and toxin responses.

genomics↗

bioRxiv2019

Caenorhabditis elegans ADAR editing and the ERI-6/7/MOV10 RNAi pathway silence endogenous viral elements and LTR retrotransposons

Endogenous retroviruses and LTR retrotransposons are mobile genetic elements that are closely related to retroviruses. Desilenced endogenous retroviruses are associated with human autoimmune disorders and neurodegenerative diseases. C. elegans and related Caenorhabdites contain LTR retrotransposons and, as described here, numerous integrated viral genes including viral envelope genes that are part of LTR retrotransposons. We found that both LTR retrotransposons and endogenous viral elements are silenced by ADARs (adenosine deaminases acting on double-stranded RNA (dsRNA)) together with the endogenous RNAi factor ERI-6/7, a homolog of Mov10 helicase, a retrotransposon and retrovirus restriction factor in human. siRNAs corresponding to integrated viral genes and LTR retrotransposons, but not to DNA transposons, are dependent on the ADARs and ERI-6/7; on the contrary, siRNAs corresponding to palindromic repeats are increased in adar-eri mutants because of an antiviral RNAi response to dsRNA. Silencing of LTR retrotransposons is dependent on downstream RNAi factors and P granule components but is independent of the viral sensor DRH-1/RIG-I and the nuclear Argonaute NRDE-3. The activation of retrotransposons in the ADAR- and ERI-6/7/MOV10-defective mutant is associated with the induction of the Unfolded Protein Response (UPR), a common response to viral infection. The overlap between genes induced upon viral infection and infection with intracellular pathogens, and genes co-expressed with retrotransposons, suggests that there is a common response to different types of foreign elements that includes a response to proteotoxicity presumably caused by the burden of replicating pathogens and expressed retrotransposons.\n\nSIGNIFICANCESilencing of transposable elements and viruses is critical for the maintenance of genome integrity, cellular homeostasis and organismal health. Here we describe multiple factors that control different types of transposable elements, providing insight into how they are regulated. We also identify stress response pathways that are triggered upon mis-regulation of these transposable elements. The conservation of these factors and pathways in human suggests that our studies in C. elegans can provide general insight into the regulation of and response to transposable elements and viruses.

genetics↗

bioRxiv2019

Interkingdom competition for Fe(III) revealed by a synthetic lethal interaction between Caenorhabditis elegans mitochondrial electron transport and Escherichia coli siderophore iron release mutations

C. elegans consumes bacteria which can supply essential vitamins and cofactors especially for mitochondrial functions ancestrally related to bacteria. Therefore, we screened the Keio E. coli knockout library for mutations that induce a C. elegans mitochondrial damage response gene. We identified 45 E. coli mutations that induce a the C. elegans hsp-6::gfp response gene. Surprisingly, four of these E. coli mutations that disrupt the import or removal of iron from the bacterial siderophore enterobactin were lethal in combination with C. elegans mutations that disrupt particular iron-sulfur proteins of the electron transport chain. Bacterial mutations that fail to synthesize enterobactin are not synthetic lethal with these C. elegans mitochondrial mutants; it is the enterobactin-iron complex that is lethal in combination with the C. elegans mitochondrial mutations. Antioxidants suppress this inviability, suggesting that reactive oxygen species (ROS) are produced by the mutant mitochondria in combination with the bacterial enterobactin-iron complex.\n\nSignificance StatementThe animal mitochondrion has a bacterial origin and continues to have a dialogue with the bacterial metabolisms of their microbiome. We identified 45 E. coli gene disruptions that induce a C. elegans mitochondrial damage response gene. Four of these E. coli mutations that disrupt the import or retrieval of iron from the siderophore enterobactin were synthetic lethal with C. elegans mitochondrial mutants. Antioxidants strongly suppressed the inviability of C. elegans mitochondrial mutants grown on the E. coli enterobactin siderophore utilization or import mutants. We hypothesize that reactive oxygen species are produced by C. elegans mitochondrial mutations and that this non-lethal ROS triggers ferric-chelated enterobactin to induce dramatically increased ROS, which leads to lethality.

genetics↗

bioRxiv2018

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

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 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 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.

molecular biology↗

bioRxiv2017

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

MotivationLong-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.\n\nResultsHere 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 1 g Enterobacteria phage {lambda} 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). By treating reads as a Poisson arrival process, we implement a statistical test to reject data from channels dominated by HQNRs while retaining target reads.\n\nAvailabilityCarrierSeq is an open-source bash script with supporting python scripts which leverage a variety of bioinformatics software packages on macOS and Ubuntu. Supplemental documentation is available from Github - https://github.com/amojarro/carrierseq. In addition, we have compiled all required dependencies in a Docker image available from - https://hub.docker.com/r/mojarro/carrierseq.

bioinformatics↗