Biology subjects

Gohl, D. M.

Publications and source records attributed to Gohl, D. M..

9 recordsLinked to original sources

bioRxiv2026

Characterizing the Small Non-Coding RNA Pathways in the Invasive Zebra Mussel (Dreissena polymorpha)

The zebra mussel (Dreissena polymorpha) is an invasive species that causes extensive economic and ecological damage. Here, we identify and characterize the key components of the small RNA (sRNA) and RNA interference (RNAi) pathways in zebra mussels. Like other mollusks, zebra mussels have extensive microRNA (miRNA) and Piwi-interacting RNA (piRNA) machinery but lack or have modified canonical factors needed to produce small interfering RNA (siRNA). Specifically, the zebra mussel Dicer sequence displays substitutions in the conserved DEAD box motif that is required for substrate processivity, and this organism also lacks some attendant accessory factors such as R2D2. We sequenced the small RNA found in both isolated somatic tissue (adductor muscle) and whole animals (including germline), and identified both conserved and novel miRNA and diverse piRNA sequences, but few endogenous siRNAs. To determine whether their remaining sRNA machinery could still be co-opted to initiate gene silencing, we injected dsRNA targeting several genes into zebra mussel adductor muscle. The injected rpn8-targeting dsRNA reduced rpn8 mRNA levels and was processed into sRNA that resemble endogenous miRNAs and piRNAs. The levels of both sRNA types correlated with mRNA knockdown, suggesting that they may act together to initiate RNAi as seen elsewhere. dsRNA targeting other genes produced variable results suggesting that particular criteria may be needed to trigger an RNAi response in this assay. Our results characterize endogenous sRNA pathways in zebra mussels, establish that dsRNA can induce RNAi, and lay the groundwork for further optimizations to establish RNAi-based genetic manipulation tools for this damaging invasive species.

genomics↗

bioRxiv2025

Inhibitory columnar feedback neurons are required for motion processing in Drosophila

Visual motion information is essential to guiding the movements of many animals. The establishment of direction-selective signals, a hallmark of motion detection, is considered a core neural computation and has been characterized extensively in primates, mice and fruit flies. In flies, the circuits that produce direction-selective signals rely on feedforward visual pathways that connect peripheral visual inputs to the dendrites of the ON and OFF-direction selective cells. Here we describe a novel role for feedback inhibition in motion computation. Two GABAergic neurons, C2 and C3, connect to neurons upstream of the direction-selective T4 and T5 cells and blocking C2 and C3 affects direction selectivity in T4/T5. In the ON pathway, this is likely achieved by C2-mediated suppression of responses in the major T4 input neuron Mi1. Together, C2 and C3 suppress responses to non-preferred stimuli in both T4 and T5. At the behavioral level, feedback inhibition temporally sharpens responses to ON-moving stimuli, enhancing the flys ability to discriminate visual stimuli that occur in quick succession. GABAergic inhibitory feedback neurons thus constitute an essential component within the circuitry that computes visual motion.

neuroscience↗

bioRxiv2022

Rational probe design for efficient rRNA depletion and improved metatranscriptomic analysis of human microbiomes

BackgroundThe microbiota that colonize the human gut and other tissues are dynamic, varying both in composition and functional state between individuals and over time. In studying the function of the human microbiome and the mechanisms of microbiota-mediated phenotypes, gene expression measurements provide additional insights to DNA-based measurements of microbiome composition. However, efficient and unbiased removal of microbial ribosomal RNA (rRNA) presents a barrier to acquiring metatranscriptomic data, as rRNA typically accounts >90% of total RNA in microbial cells. ResultsHere we describe a probe set that achieves efficient enzymatic rRNA removal of complex human-associated microbial communities. We demonstrate that the custom probe set can be further refined through an iterative design process to efficiently deplete rRNA from a range of human microbiome samples, including adult and infant gut, as well as oral and vaginal communities. Using synthetic nucleic acid spike-ins, we show that the rRNA depletion process does not introduce substantial quantitative error in the resulting gene expression profiles. Successful rRNA depletion allows for efficient characterization of taxonomic and functional profiles, including during the development of the human gut microbiome. ConclusionsThe pan-human microbiome enzymatic rRNA depletion probes described here provide a powerful tool for studying the transcriptional dynamics and function of the human microbiome.

genomics↗

bioRxiv2021

Dissecting and Tuning Primer Editing by Proofreading Polymerases

Proofreading polymerases have 3 to 5 exonuclease activity that allows the excision and correction of mis-incorporated bases during DNA replication. In a previous study, we demonstrated that in addition to correcting substitution errors and lowering the error rate of DNA amplification, proofreading polymerases can also edit PCR primers to match template sequences. Primer editing is a feature that can be advantageous in certain experimental contexts, such as amplicon-based microbiome profiling. Here we develop a set of synthetic DNA standards to report on primer editing activity and use these standards to dissect this phenomenon. The primer editing standards allow next-generation sequencing-based enzymological measurements, reveal the extent of editing, and allow the comparison of different polymerases and cycling conditions. We demonstrate that proofreading polymerases edit PCR primers in a concentration-dependent manner, and we examine whether primer editing exhibits any sequence specificity. In addition, we use these standards to show that primer editing is tunable through the incorporation of phosphorothioate linkages. Finally, we demonstrate the ability of primer editing to robustly rescue the drop-out of taxa with 16S rRNA gene-targeting primer mismatches using mock communities and human skin microbiome samples.

molecular biology↗

bioRxiv2020

A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2

The global COVID-19 pandemic has led to an urgent need for scalable methods for clinical diagnostics and viral tracking. Next generation sequencing technologies have enabled large-scale genomic surveillance of SARS-CoV-2 as thousands of isolates are being sequenced around the world and deposited in public data repositories. A number of methods using both short- and long-read technologies are currently being applied for SARS-CoV-2 sequencing, including amplicon approaches, metagenomic methods, and sequence capture or enrichment methods. Given the small genome size, the ability to sequence SARS-CoV-2 at scale is limited by the cost and labor associated with making sequencing libraries. Here we describe a low-cost, streamlined, all amplicon-based method for sequencing SARS-CoV-2, which bypasses costly and time-consuming library preparation steps. We benchmark this tailed amplicon method against both the ARTIC amplicon protocol and sequence capture approaches and show that an optimized tailed amplicon approach achieves comparable amplicon balance, coverage metrics, and variant calls to the ARTIC v3 approach and represents a cost-effective and highly scalable method for SARS-CoV-2 sequencing.

genomics↗

bioRxiv2020

Analytical Validation of a COVID-19 qRT-PCR Detection Assay Using a 384-well Format and Three Extraction Methods

The COVID-19 global pandemic is an unprecedented health emergency. Insufficient access to testing has hampered effective public health interventions and patient care management in a number of countries. Furthermore, the availability of regulatory-cleared reagents has challenged widespread implementation of testing. We rapidly developed a qRT-PCR SARS-CoV-2 detection assay using a 384-well format and tested its analytic performance across multiple nucleic acid extraction kits. Our data shows robust analytic accuracy on residual clinical biospecimens. Limit of detection sensitivity and specificity was confirmed with currently available commercial reagents. Our methods and results provide valuable information for other high-complexity laboratories seeking to develop effective, local, laboratory-developed procedures with high-throughput capability to detect SARS-CoV-2.

pathology↗

bioRxiv2019

The Genome of the Zebra Mussel, Dreissena polymorpha: A Resource for Invasive Species Research

The zebra mussel, Dreissena polymorpha, continues to spread from its native range in Eurasia to Europe and North America, causing billions of dollars in damage and dramatically altering invaded aquatic ecosystems. Despite these impacts, there are few genomic resources for Dreissena or related bivalves, with nearly 450 million years of divergence between zebra mussels and its closest sequenced relative. Although the D. polymorpha genome is highly repetitive, we have used a combination of long-read sequencing and Hi-C-based scaffolding to generate the highest quality molluscan assembly to date. Through comparative analysis and transcriptomics experiments we have gained insights into processes that likely control the invasive success of zebra mussels, including shell formation, synthesis of byssal threads, and thermal tolerance. We identified multiple intact Steamer-Like Elements, a retrotransposon that has been linked to transmissible cancer in marine clams. We also found that D. polymorpha have an unusual 67 kb mitochondrial genome containing numerous tandem repeats, making it the largest observed in Eumetazoa. Together these findings create a rich resource for invasive species research and control efforts.

genomics↗

bioRxiv2019

Genome-wide assessment of Mycobacterium tuberculosis conditionally essential metabolic pathways

Better understanding of essential cellular functions in pathogenic bacteria is important for the development of more effective antimicrobial agents. We performed a comprehensive identification of essential genes in Mycobacterium tuberculosis, the major causative agent of tuberculosis, using a combination of transposon insertion sequencing (Tn-seq) and comparative genomic analysis. To identify conditional essential genes by Tn-seq, we used media with different nutrient composition. Although many conditional gene essentialities were affected by the presence of relevant nutrient sources, we also found that the essentiality of genes in a subset of metabolic pathways was unaffected by metabolites. Comparative genomic analysis revealed that not all essential genes identified by Tn-seq were fully conserved within the M. tuberculosis complex including some existing anti-tubercular drug target genes. In addition, we utilized an available M. tuberculosis genome-scale metabolic model, iSM810, to predict M. tuberculosis gene essentiality in silico. Comparing the sets of essential genes experimentally identified by Tn-seq to those predicted in silico reveals the capabilities and limitations of gene essentiality predictions highlighting the complexity of M. tuberculosis essential metabolic functions. This study provides a promising platform to study essential cellular functions in M. tuberculosis. Author SummaryMycobacterium tuberculosis causes 10 million cases of tuberculosis (TB) each year resulting in over one million deaths. TB therapy is challenging because it requires a minimum of six months of treatment with multiple drugs. Protracted treatment times and the emergent spread of drug resistant TB necessitate the identification of novel targets for drug discovery to curb this global health threat. Essential functions, defined as those indispensable for growth and/or survival, are potential targets for new antimicrobial drugs. In this study, we aimed to define gene essentialities of M. tuberculosis on a genome-wide scale to comprehensively identify potential targets for drug discovery. We utilized a combination of experimental (functional genomics) and in silico approaches (comparative genomics and flux balance analysis). Our functional genomics approach identified sets of genes whose essentiality was affected by nutrient availability. Comparative genomics revealed that not all essential genes were fully conserved within the M. tuberculosis complex. Comparing sets of essential genes identified by functional genomics to those predicted by flux balance analysis highlighted gaps in current knowledge regarding M. tuberculosis metabolic capabilities. Thus, our study identifies numerous potential anti-tubercular drug targets and provides a comprehensive picture of the complexity of M. tuberculosis essential cellular functions.

microbiology↗

bioRxiv2018

Measuring Illumina Size Bias Using REcount: A Novel Method for Highly Accurate Quantification of Engineered Genetic Constructs

Quantification of DNA sequence tags associated with engineered genetic constructs underlies many genomics measurements. Typically, such measurements are done using PCR to enrich sequence tags and add adapters, followed by next-generation sequencing (NGS). However, PCR amplification can introduce significant quantitative error into these measurements. Here we describe REcount, a novel PCR-free direct counting method for NGS-based quantification of engineered genetic constructs. By comparing measurements of defined plasmid pools to droplet digital PCR data, we demonstrate that this method is highly accurate and reproducible. We further demonstrate that the REcount approach is amenable to multiplexing through the use of orthogonal restriction enzymes. Finally, we use REcount to provide new insights into clustering biases due to molecule length across different Illumina sequencing platforms.

genomics↗