Biology subjects

Moore, R. M.

Publications and source records attributed to Moore, R. M..

10 recordsLinked to original sources

bioRxiv2026

A Sample to Results Workflow for Compositional Analysis of Multiplexed Amplicon Sequencing Experiments

Microbial communities play key roles in the transformation and cycling of elements ranging from required macronutrients to toxic metalloids. Next-generation sequencing has been applied across multiple ecosystems to probe the interplay of microbial community structure and functional potential with respect to elemental cycling. Shotgun metagenomics collects marker gene sequences without amplification and is costly for large numbers of samples and deep coverage. Conversely, amplicon sequencing of taxonomic marker genes, e.g. 16S and 18S rRNA, is cost-effective for large numbers of samples, but provides limited functional insight. A middle ground between the two approaches is needed to analyze community structure and functional potential within a sample while remaining cost-effective with high throughput. To address this need, we developed a standardized workflow for multiplexed amplicon sequencing from sample collection through data analysis for diverse sample types, including freshwater, sediments, and soils, that produces data and publication-ready figures for multiple taxonomic and functional genes for carbon, nitrogen, phosphorus, sulfur, and arsenic cycling for each sample analyzed. The workflows utility was shown by analyzing 11 taxonomic and functional gene amplicons sequenced from 25 samples with high technical replicate similarity. The workflow is named CAMASE for Compositional Analysis of Multiplex Amplicon Sequencing Experiments. This proof-of-concept shows that CAMASE economically produces standard amplicon sequencing outputs (ASV/OTU counts and taxonomy, PCA, and relative abundance plots) for hundreds of amplicon by sample combinations and provides specific recommendations for implementation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/741237v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@14c746corg.highwire.dtl.DTLVardef@6f9890org.highwire.dtl.DTLVardef@894038org.highwire.dtl.DTLVardef@d72b2a_HPS_FORMAT_FIGEXP M_FIG C_FIG Samples are collected in a preservative and material collected on filters prior to DNA extraction. Target gene amplicons are produced in parallel with internal barcodes enabling sequencing in a single run followed by compositional data analysis. All wet lab protocols, code markdowns, and templates for required metadata files are available at https://hansonlabgit.dbi.udel.edu/aprange/CAMASE. Created in BioRender. Bennett, A. (2026) https://BioRender.com/ymnojt0

microbiology↗

bioRxiv2026

Ribonucleotide reductases recapitulate biogeographic patterns within virioplankton according to ocean biogeochemistry

Many of the important biogeochemical implications of marine viruses focus on the activity of lytic viruses. It is generally thought that lytic viral activity contributes to maintaining bioavailable nutrient pools and controlling host community composition through specific infection and lysis processes. Among lytic viruses, genome replication is under stringent selection pressure as advantages in replication speed and fidelity can influence viral fitness. Deoxyribonucleotides (dNTPs) are the precursor substrate for DNA synthesis and ribonucleotide reductase (RNR) is the only known enzyme capable of reducing ribonucleotides into dNTPs. Thus, for a virus, encoding an RNR gene provides control over dNTP production. Overall, distributional patterns of the RNR-encoding virioplankton community mirrored that seen for total virioplankton throughout the global ocean. A majority of RNR-encoding virioplankton ([~]66%) carried the Class II enzyme and most of these carried the monomeric (NrdJm) gene. This was significant as NrdJm utilizes triphosphate ribonucleotides as opposed to the diphosphate ribonucleotides used by Class I and Class II dimeric RNRs. The distribution and frequency of Class I RNRs followed the concentration of required iron and manganese co-factors. Oceanic virioplankton encompassed a high diversity of Class I and II RNRs with the discovery of new phylogenetic clades not previously observed among viruses or within cellular life. Representing [~]10% of all virioplankton populations, RNR-encoding viral populations demonstrated surprising fidelity with the major biogeographical features defining oceanic ecosystems.

microbiology↗

bioRxiv2023

B12-dependent virioplankton demonstrate interseasonal dynamics and associate with a diversity of pelagic bacterioplankton

Through infection and lysis of their coexisting bacterial hosts, viruses impact the biogeochemical cycles sustaining globally significant pelagic oceanic ecosystems. Currently, little is known of the ecological interactions between lytic viruses and their bacterial hosts underlying these biogeochemical impacts at ecosystem scales. This study focused on populations of lytic viruses carrying the B12- dependent Class II monomeric ribonucleotide reductase (RNR) gene, ribonucleotide triphosphate reductase (RTPR), documenting seasonal changes in pelagic virioplankton and bacterioplankton using amplicon sequences of RTPR and the 16S rRNA gene, respectively. Amplicon sequence libraries were analyzed using compositional data analysis tools that account for the compositional nature of these data. Both virio- and bacterioplankton communities responded to environmental changes typically seen across seasonal cycles as well as shorter term upwelling-downwelling events. Defining RTPR-carrying viral populations according to major phylogenetic clades proved a more robust means of exploring virioplankton ecology than operational taxonomic units defined by percent sequence homology. Virioplankton RTPR populations showed positive associations with a broad phylogenetic diversity of bacterioplankton including dominant taxa within pelagic oceanic ecosystems such as Prochlorococcus and SAR11. Temporal changes in RTPR-virioplankton, occurring as both free viruses and within infected cells, indicated possible viral-host pairs undergoing sustained infection and lysis cycles throughout the seasonal study. Phylogenetic relationships inferred from RTPR sequences mirrored ecological patterns in virio- and bacterioplankton populations demonstrating possible genome to phenome associations for an essential viral replication gene.

ecology↗

bioRxiv2022

A Genome-wide CRISPR Screen Identifies WDFY3 as a Novel Regulator of Macrophage Efferocytosis

AbstractsPhagocytic clearance of dying cells, termed efferocytosis, must occur efficiently to maintain homeostasis and prevent disease. Yet, our understanding of this important biological process remains incomplete. To search for novel regulators of efferocytosis, we performed a FACS-based genome-wide CRISPR knockout screen in primary murine macrophages. We identified a novel role for WDFY3 in efferocytosis by macrophages. WDFY3 deficiency in macrophages specifically impaired uptake, not binding, of apoptotic cells due to defective actin depolymerization. We further revealed that WDFY3 directly interacts with GABARAP, thus facilitating LC3 lipidation and subsequent lysosomal acidification to permit the degradation of apoptotic cell components. Although the C-terminus of WDFY3 was sufficient to rescue impaired degradation, full-length WDFY3 is still required for regulating uptake. Finally, WDFY3 is required for efficient efferocytosis in vivo in mice and in primary human macrophages. The work expands our knowledge of the mechanisms of macrophage efferocytosis, and more broadly, provides a general strategy for genome-wide CRISPR screen to interrogate complex functional phenotypes in primary macrophages. HighlightsO_LIFunctional readout for pooled genome-wide CRISPR screen in primary macrophages. C_LIO_LIWDFY3 is discovered as a regulator of macrophage efferocytosis in vitro and in vivo. C_LIO_LIWDFY3 deficiency led to impaired uptake, as opposed to binding, of apoptotic cells due to defective actin depolymerization. C_LIO_LIWDFY3 directly interacts with GABARAP, facilitating LC3 lipidation and subsequent lysosomal acidification to permit the degradation of apoptotic cell components. C_LIO_LIC-terminal WDFY3 is sufficient to regulate the degradation of engulfed apoptotic cells while full-length WDFY is required for regulating uptake. C_LI

cell biology↗

bioRxiv2021

PASV: Automatic protein partitioning and validation using conserved residues

BackgroundIncreasingly, researchers use protein-coding genes from targeted PCR amplification or direct metagenomic sequencing in community and population ecology. Analysis of protein-coding genes presents different challenges from those encountered in traditional SSU rRNA studies. Most protein-coding sequences are annotated based on homology to other computationally-annotated sequences, which can lead to inaccurate annotations. Therefore, the results of sensitive homology searches must be validated to remove false-positives and assess functionality. Multiple lines of in silico evidence can be gathered by examining conserved domains and residues identified through biochemical investigations. However, manually validating sequences in this way can be time consuming and error prone, especially in large environmental studies. ResultsAn automated pipeline for protein active site validation (PASV) was developed to improve validation and partitioning accuracy for protein-coding sequences, combining multiple sequence alignment with expert domain knowledge. PASV was tested using commonly misannotated proteins: ribonucleotide reductase (RNR), alternative oxidase (AOX), and plastid terminal oxidase (PTOX). PASV partitioned 9,906 putative Class I alpha and Class II RNR sequences from bycatch in a global viral metagenomic investigation with >99% true positive and true negative rates. PASV predicted the class of 2,579 RNR sequences in >98% agreement with manual annotations. PASV correctly partitioned all 336 tested AOX and PTOX sequences. ConclusionsPASV provides an automated and accurate way to address post-homology search validation and partitioning of protein-coding marker genes. Source code is released under the MIT license and is found with documentation and usage examples on GitHub at https://github.com/mooreryan/pasv.

bioinformatics↗

bioRxiv2018

CRISPR spacers indicate preferential matching of specific virioplankton genes

Viral infection exerts selection pressure on marine microbes as viral-induced cell lysis causes 20 to 50% of cell mortality resulting in fluxes of biomass into oceanic dissolved organic matter. Archaeal and bacterial populations can defend against viral infection using the CRISPR-Cas system which relies on specific matching between a spacer sequence and a viral gene. If a CRISPR spacer match to any gene within a viral genome is equally effective in preventing lysis, then no viral genes should be preferentially matched by CRISPR spacers. However, if there are differences in effectiveness then certain viral genes may demonstrate a greater frequency of CRISPR spacer matches. Indeed, homology search analyses of bacterioplankton CRISPR spacer sequences against virioplankton sequences revealed preferential matching of replication proteins, nucleic acid binding proteins, and viral structural proteins. Positive selection pressure for effective viral defense is one parsimonious explanation for these observations. CRISPR spacers from virioplankton metagenomes preferentially matched methyltransferase and phage integrase genes within virioplankton sequences. These viriolankton CRISPR spacers may assist infected host cells in defending against competing phage. Analyses also revealed that half of the spacer-matched viral genes were unknown and that some genes matched several spacers and some spacers matched multiple genes, a many-to-many relationship. Thus, CRISPR spacer matching may be an evolutionary algorithm, agnostically identifying those genes under stringent selection pressure for sustaining viral infection and lysis. Investigating this subset of viral genes could reveal those genetic mechanisms essential to viral-host interactions and provide new technologies for optimizing CRISPR defense in beneficial microbes.

microbiology↗

bioRxiv2018

Reannotation of the ribonucleotide reductase in a cyanophage reveals life history strategies within the virioplankton

Ribonucleotide reductases (RNRs) are ancient enzymes that catalyze the reduction of ribonucleotides to deoxyribonucleotides. They are required for virtually all cellular life and are prominent within viral genomes. RNRs share a common ancestor and must generate a protein radical for direct ribonucleotide reduction. The mechanisms by which RNRs produce radicals are diverse and divide RNRs into three major classes and several subclasses. The diversity of radical generation methods means that cellular organisms and viruses typically contain the RNR best-suited to the environmental conditions surrounding DNA replication. However, such diversity has also fostered high rates of RNR misannotation within subject sequence databases. These misannotations have resulted in incorrect translative presumptions of RNR biochemistry and have diminished the utility of this marker gene for ecological studies of viruses. We discovered a misannotation of the RNR gene within the Prochlorococcus phage P-SSP7 genome, which caused a chain of misannotations within commonly observed RNR genes from marine virioplankton communities. These RNRs are found in marine cyanopodo- and cyanosiphoviruses and are currently misannotated as Class II RNRs, which are O2-independent and require cofactor B12. In fact, these cyanoviral RNRs are Class I enzymes that are O2-dependent and may require a di-metal cofactor made of Fe, Mn, or a combination of the two metals. The discovery of an overlooked Class I {beta} subunit in the P-SSP7 genome, together with phylogenetic analysis of the and {beta} subunits confirms that the RNR from P-SSP7 is a Class I RNR. Phylogenetic and conserved residue analyses also suggest that the P-SSP7 RNR may constitute a novel Class I subclass. The reannotation of the RNR clade represented by P-SSP7 means that most lytic cyanophage contain Class I RNRs, while their hosts, B12-producing Synechococcus and Prochlorococcus, contain Class II RNRs. By using a Class I RNR, cyanophage avoid a dependence on host-produced B12, a more effective strategy for a lytic virus. The discovery of a novel RNR {beta} subunit within cyanopodoviruses also implies that some unknown viral genes may be familiar cellular genes that are too divergent for homology-based annotation methods to identify.

bioinformatics↗

bioRxiv2018

Marine Fe-oxidizing Zetaproteobacteria: Historical, ecological, and genomic perspectives

The Zetaproteobacteria are a class of bacteria typically associated with marine Fe oxidizing environments. First discovered in the hydrothermal vents at Loihi Seamount, Hawaii, they have become model organisms for marine microbial Fe oxidation. In addition to deep sea and shallow hydrothermal vents, Zetaproteobacteria are found in coastal sediments, other marine subsurface environments, steel corrosion biofilms, as well as saline terrestrial aquifers and springs. Isolates from a range of environments all grow by Fe oxidation. Their success lies partly in their microaerophily, which enables them to compete with abiotic Fe oxidation at the low O2 concentrations common to Fe(II)-rich oxic/anoxic transition zones. Also, Zetaproteobacteria make a variety of biomineral morphologies as a repository for Fe(III) waste, and as attachment structures. To determine the known diversity of the Zetaproteobacteria, we have used 16S rRNA gene sequences to define 59 operational taxonomic units (OTUs), at 97% similarity. While some Zetaproteobacteria taxa appear to be cosmopolitan, various habitats enrich for different sets of Zetaproteobacteria. OTU networks show that certain Zetaproteobacteria co-exist, sharing compatible niches. These niches may correspond with adaptations to O2, H2, and nitrate availability, based on genomic analyses. Also, a putative Fe oxidation gene has been found in diverse Zetaproteobacteria taxa, suggesting that the Zetaproteobacteria evolved as specialists in Fe oxidation. In all, culture, genomic, and environmental studies suggest that Zetaproteobacteria are widespread, and therefore have a broad influence on marine and saline terrestrial Fe cycling.

microbiology↗

bioRxiv2018

ZetaHunter: a reproducible taxonomic classification tool for tracking the ecology of the Zetaproteobacteria and other poorly-resolved taxa

Like many taxa, the Zetaproteobacteria lack well-defined taxonomic divisions, 32 making it difficult to compare between studies. We designed ZetaHunter to reproducibly 33 assign 16S rRNA gene sequences to previously-described OTUs based on a curated 34 database. While ZetaHunter can use any given database, we include a curated 35 classification of publically-available Zetaproteobacteria.

bioinformatics↗

bioRxiv2017

Iroki: automatic customization for phylogenetic trees

Phylogenetic trees are an important analytical tool for evaluating community diversity and evolutionary history. In the case of microorganisms, the decreasing cost of sequencing has enabled researchers to generate ever-larger sequence datasets, which in turn have begun to fill gaps in the evolutionary history of microbial groups. However, phylogenetic analyses of these types of datasets create complex trees that can be challenging to interpret. Scientific inferences made by visual inspection of phylogenetic trees can be simplified and enhanced by customizing various parts of the tree. Yet, manual customization is time-consuming and error prone, and programs designed to assist in batch tree customization often require programming experience or complicated file formats for annotation. Iroki, a user-friendly web interface for tree visualization, addresses these issues by providing automatic customization of large trees based on metadata contained in tab-separated text files. Irokis utility for exploring biological and ecological trends in sequencing data was demonstrated through a variety of microbial ecology applications in which trees with hundreds to thousands of leaf nodes were customized according to extensive collections of metadata. The Iroki web application and documentation are available at https://www.iroki.net or through the VIROME portal (http://virome.dbi.udel.edu). Irokis source code is released under the MIT license and is available at https://github.com/mooreryan/iroki.

bioinformatics↗