Dataset Information

High content analysis enables high-throughput nematicide discovery screening for measurement of viability and movement behavior in response to natural product samples.

ABSTRACT: Historically, monitoring nematode movement and mortality in response to various potential nematicide treatments usually involved tedious manual microscopic analysis. High-content analysis instrumentation enables rapid and high-throughput collection of experimental data points on large numbers of individual worms simultaneously. The high-throughput platform outlined here should accelerate discovery of unique classes and types of promising lead molecules and sample types to control these plant pests. Also, the ability to automate the data analysis pipeline rather than relying on manual scoring reduces a potential source of data variance. Here we describe a high-throughput process based on high-content imaging. We demonstrate the use of time-lapse image acquisition to measure movement, and viability staining to confirm nematode mortality (versus paralysis) in targeted plant-pathogenic nematodes. We present screening results from a microbial-exudate library generated from approximately 2,300 microbial fermentations that demonstrate the robustness of this high-throughput process. The described methods should be applicable to other relevant nematode parasites with human, crop, or animal hosts.

SUBMITTER: Petitte JM

PROVIDER: S-EPMC6478374 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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High content analysis enables high-throughput nematicide discovery screening for measurement of viability and movement behavior in response to natural product samples.

Petitte Jennifer M JM Lewis Mary H MH Witsil Tucker K TK Huang Xiang X Rice John W JW

PloS one 20190423 4

Historically, monitoring nematode movement and mortality in response to various potential nematicide treatments usually involved tedious manual microscopic analysis. High-content analysis instrumentation enables rapid and high-throughput collection of experimental data points on large numbers of individual worms simultaneously. The high-throughput platform outlined here should accelerate discovery of unique classes and types of promising lead molecules and sample types to control these plant pes ...[more]

PMID: 31013269

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Project description:Microbiome analyses of low-biomass samples are challenging because of contamination and inefficiencies, leading many investigators to employ low-throughput methods with minimal controls. We developed a new automated protocol, KatharoSeq (from the Greek katharos [clean]), that outperforms single-tube extractions while processing at least five times as fast. KatharoSeq incorporates positive and negative controls to reveal the whole bacterial community from inputs of as few as 50 cells and correctly identifies 90.6% (standard error, 0.013%) of the reads from 500 cells. To demonstrate the broad utility of KatharoSeq, we performed 16S rRNA amplicon and shotgun metagenome analyses of the Jet Propulsion Laboratory spacecraft assembly facility (SAF; n = 192, 96), 52 rooms of a neonatal intensive care unit (NICU; n = 388, 337), and an endangered-abalone-rearing facility (n = 192, 123), obtaining spatially resolved, unique microbiomes reproducible across hundreds of samples. The SAF, our primary focus, contains 32 sOTUs (sub-OTUs, defined as exact sequence matches) and their inferred variants identified by the deblur algorithm, with four (Acinetobacter lwoffii, Paracoccus marcusii, Mycobacterium sp., and Novosphingobium) being present in >75% of the samples. According to microbial spatial topography, the most abundant cleanroom contaminant, A. lwoffii, is related to human foot traffic exposure. In the NICU, we have been able to discriminate environmental exposure related to patient infectious disease, and in the abalone facility, we show that microbial communities reflect the marine environment rather than human input. Consequently, we demonstrate the feasibility and utility of large-scale, low-biomass metagenomic analyses using the KatharoSeq protocol. IMPORTANCE Various indoor, outdoor, and host-associated environments contain small quantities of microbial biomass and represent a niche that is often understudied because of technical constraints. Many studies that attempt to evaluate these low-biomass microbiome samples are riddled with erroneous results that are typically false positive signals obtained during the sampling process. We have investigated various low-biomass kits and methods to determine the limit of detection of these pipelines. Here we present KatharoSeq, a high-throughput protocol combining laboratory and bioinformatic methods that can differentiate a true positive signal in samples with as few as 50 to 500 cells. We demonstrate the application of this method in three unique low-biomass environments, including a SAF, a hospital NICU, and an abalone-rearing facility.

Dataset Information

High content analysis enables high-throughput nematicide discovery screening for measurement of viability and movement behavior in response to natural product samples.

Publications

High content analysis enables high-throughput nematicide discovery screening for measurement of viability and movement behavior in response to natural product samples.

Similar Datasets

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