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All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

An Innovative High Throughput Genome Releaser for Rapid and Efficient PCR Screening

High-throughput PCR screening is vital in synthetic biology and metabolic engineering as it allows researchers to rapidly analyze and detect numerous targeted genetic mutation in the genome. Current challenges for high-throughput PCR screening in synthetic biology include efficiently preparing genomic DNA, optimizing protocols for diverse sample types, managing contamination risks, and effectively analyzing the large volumes of data generated while ensuring consistent and accurate results. In this study, we present the development of a High Throughput Genome Releaser (HTGR), an innovative device addressing common challenges in screening PCR. This genome DNA releaser is designed based on a squash method for rapid, cost-effective, and efficient DNA release, optimized for subsequent PCR reactions. After experimenting with various synthetic materials, we selected a plastic that closely replicates the smooth surface and compression properties of microscope slides, ensuring reliable performance. We engineered a device featuring a 96-Well Plate and a shear applicator, operable both manually and automatically, and compatible with standard liquid-handling robot platform. This compatibility enhances ease of use in high-throughput PCR workflows. Additionally, we developed software to support its automatic functions. Our results demonstrated that the specially engineered 96-Well Plate and HTGR can effectively squash fungal spores , which release enough genome DNA for PCR screening. The genome releaser facilitates the preparation of PCR-amplifiable genomic DNA substrate from 96 samples within minutes, eliminates the need for extraction buffers, and is adaptable to a wide range of microorganisms and cells, which could significantly advance biomanufacturing processes.

Yuan, Guoliang [BATTELLE (PACIFIC NW LAB)]↗
DOE OSTI2025

Efficient mutagenesis and genotyping of maize inbreds using biolistics, multiplex CRISPR/Cas9 editing, and Indel-Selective PCR

CRISPR/Cas9 based genome editing has advanced our understanding of a myriad of important biological phenomena. Important challenges to multiplex genome editing in maize include assembly of large complex DNA constructs, few genotypes with efficient transformation systems, and costly/labor-intensive genotyping methods. Here we present an approach for multiplex CRISPR/Cas9 genome editing system that delivers a single compact DNA construct via biolistics to Type I embryogenic calli, followed by a novel efficient genotyping assay to identify desirable editing outcomes. We first demonstrate the creation of heritable mutations at multiple target sites within the same gene. Next, we successfully created individual and stacked mutations for multiple members of a gene family. Genome sequencing found off-target mutations are rare. Multiplex genome editing was achieved for both the highly transformable inbred line H99 and Illinois Low Protein1 (ILP1), a genotype where transformation has not previously been reported. In addition to screening transformation events for deletion alleles by PCR, we also designed PCR assays that selectively amplify deletion or insertion of a single nucleotide, the most common outcome from DNA repair of CRISPR/Cas9 breaks by non-homologous end-joining. The Indel-Selective PCR (IS-PCR) method enabled rapid tracking of multiple edited alleles in progeny populations. The ‘end to end’ pipeline presented here for multiplexed CRISPR/Cas9 mutagenesis can be applied to accelerate maize functional genomics in a broader diversity of genetic backgrounds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Data for "Efficient Mutagenesis and Genotyping of Maize Inbreds Using Biolistics, Multiplex CRISPR/Cas9 Editing, and Indel-Selective PCR"

CRISPR/Cas9 based genome editing has advanced our understanding of a myriad of important biological phenomena. Important challenges to multiplex genome editing in maize include assembly of large complex DNA constructs, few genotypes with efficient transformation systems, and costly/labor-intensive genotyping methods. Here we present an approach for multiplex CRISPR/Cas9 genome editing system that delivers a single compact DNA construct via biolistics to Type I embryogenic calli, followed by a novel efficient genotyping assay to identify desirable editing outcomes. We first demonstrate the creation of heritable mutations at multiple target sites within the same gene. Next, we successfully created individual and stacked mutations for multiple members of a gene family. Genome sequencing found off-target mutations are rare. Multiplex genome editing was achieved for both the highly transformable inbred line H99 and Illinois Low Protein1 (ILP1), a genotype where transformation has not previously been reported. In addition to screening transformation events for deletion alleles by PCR, we also designed PCR assays that selectively amplify deletion or insertion of a single nucleotide, the most common outcome from DNA repair of CRISPR/Cas9 breaks by non-homologous end-joining. The Indel-Selective PCR (IS-PCR) method enabled rapid tracking of multiple edited alleles in progeny populations. The ‘end to end’ pipeline presented here for multiplexed CRISPR/Cas9 mutagenesis can be applied to accelerate maize functional genomics in a broader diversity of genetic backgrounds.

gene editing↗
DOE OSTI2025

Nanopore Activity Assays for Detection of Biomarker Protease Activity: Design and Testing of Substrates for Both Nanopore Sequencing and PCR-Based Detection Methods

The work performed in this project has demonstrated the ability to construct proteolytic enzyme substrates that are PCR and sequencing-readable reporter molecules. Specifically, the goal was to detect those reporter molecules via PCR and Oxford Nanopore Technologies MinION sequencing methods following exposure to the biomarker protease thrombin. The assay development focused on binding the constructed peptide-oligonucleotide chimera to immobilized streptavidin. The action of thrombin on the peptide portion of the molecule released the oligonucleotide for detection. Detection of protease activity was demonstrated in a concentration-dependent manner using MALDI-MS, RT-PCR and DNA sequencing. Additional steps to remove background release of reporter molecules during the assay was used to improve the difference in detected oligonucleotide reporter following protease activity. Additional steps in assay development will be to (1) test the assay in an appropriate matrix, (2) investigate detection using additional DNA sequencing platforms and (3) demonstrate multiplexed detection of multiple protease markers in a single reaction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Extraction of Pure Plastic Resins From PCR Plastic Waste by Solvent-Targeted Recovery and Precipitation (STRAP)

For this work, we have been developing a solvent‐based plastic recycling technology called STRAP. The technology is based on dissolving a targeted plastic resin in a specific solvent that does not dissolve other resins. We have demonstrated STRAP in thousands of bench scale experiments for a large variety of wastes. Recently we have demonstrated the technology for PCR, using mixed plastic wastes (MPWs), from a wet Material Recovery Facility (MRF). The process includes (1) infrared (IR) characterization to determine the plastic composition for accurate selection of the solvent to be used for the extraction of the pure resins. (2) Shredding to the right size and aspect ratio required for flowable and fast dissolvable process. (3) Mixing the MPW in the first solvent to dissolve the first resin. (4) Filtration of the solution plastic blend, to separate the nondissolved plastic from the solution. (5) Further filtration of the solution to remove micron‐sized particle of pigments and fibers. (6) Cooling for precipitation. (7) Filtration of pure resins. (8) Drying of a pure resin. (9) Extrusion of the resin to pellets. (10) Generating films or other products from the pure resin. Steps 1–10 can be considered as one‐cycle that extracted the first resin. (11) A second resin can be extracted with a respective solvent from the plastic that did not dissolve in the first cycle and following steps 1–10 described above. The process also includes characterization of interim and final products. The effort includes building a pilot system at 25 kg/h throughput. We will present specific results for various PCR.

IR characterization↗
DOE OSTI2024

Assessment of Different Application Grades of Post-Consumer Recycled (PCR) Polyolefins from Material Recovery Facilities (MRFs) in the United States

The recycling of plastics has been gaining traction in the following years as many companies, organizations, and governments are pushing toward a more circular economy. However, plastic recycling rates are still small compared to those of plastic production, especially for polyolefins, such as high-density polyethylene (HDPE) and polypropylene (PP). With the goal of finding applications for recycled plastic resin, recyclers can face some challenges associated with the heterogeneity in the polymer properties of plastic waste streams, which could hinder the full potential of recycling due to the lack of a consistent feedstock. Here, this study assessed how an additional sorting step of recycled HDPE and PP streams, separating them into two categories based on the previous application, impacts the recycled polymer properties, such as physical, molecular, and thermal. It was demonstrated that manually separating HDPE articles previously processed by extrusion blow molding (EBM) or injection molding (IM) and PP articles by thermoforming (TF) or IM could yield recycled resins with more consistent properties, considering the significant differences between them. This highlights that an additional sorting step would allow recyclers to target more premium or currently unattainable plastic processing applications with postconsumer recycled (PCR) resins, which could significantly promote an increase in plastic recycling rates. Nevertheless, the development of new technologies is needed to automate this extra sorting step, as with the increase in scale manual sortation could not be feasible and cost-effective, reducing the attractiveness of using PCR resins in comparison to virgin ones.

36 MATERIALS SCIENCE↗
DOE OSTI2025

Digital Droplet PCR and Mesocosm-Based Methods to Evaluate Biocontainment Strategies in a Native Soil Ecosystem

Genetically modified industrial production microbes and their associated bioproducts have emerged as an integral component of a sustainable bioeconomy. However, the rapid development of these innovative technologies raises biosecurity concerns, namely, the risk of environmental escape. Thus, the realization of a bioeconomy hinges not only on the development and deployment of microbial production hosts, but also on the development of secure biosystems and biocontainment designs. Current laboratory-based biocontainment testing systems do not accurately reflect the complexities found in natural environments, necessitating an environmentally relevant analysis pipeline that allows for the detection of rare escapees within a complex soil microbiome and differentiation between closely related strains. To this end, we have developed an approach that utilizes soil mesocosms and integrated digital droplet PCR (ddPCR) system to evaluate the efficacy of novel biocontainment strategies. We demonstrate the utility of this approach by modeling contamination with industrial microbial chasses versus their biocontained counterparts. Here we demonstrate the broad utility of this system by highlighting findings from strains of Saccharomyces cerevisiae that are contained with an inducible toxin anti-toxin system, strains of Synechocystis sp. PCC 6803 contained via gene knockout or toxin anti-toxin system, and strains of Escherichia coli that are contained via genomic recoding. We also show that ddPCR can be used to detect gene copies from E. coli equal to those counted by traditional spot plating assays. The resultant data demonstrates that this system has broad utility across diverse microbial chassis and biocontainment strategies and enables researchers to track the fate of our contaminating microbe with high sensitivity in the soil. The findings presented here support the use of this mesocosm-based approach to assess the environmental impact of industrial microbes and to validate biocontainment strategies.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

The Effects of Polyolefin Structure and Source on Pyrolysis-Derived Plastic Oil Composition

Seven types of plastics were pyrolyzed in a fluidized bed reactor: post-consumer recycled (PCR) high-density polyethylene (HDPE), PCR polypropylene (PP), virgin resins of varying molecular weights of HDPE, virgin resins of low-density polyethylene (LDPE), linear low-density polyethylene (LLDPE), and (PP). Pyrolysis produced non-condensable gases (C1-C3), liquid phase products (C4-C40), and solids (C40+ and chars), with alkane, alkene, alkadiene, aromatic, and multi-cycloaromatics as the predominant compounds. Polymer structure had the greatest impact on product distribution, with minimal influence from molecular weight. Branches in polyethylene (PE) acted as thermal defects initiating degradation. Higher branch density in PE led to increased concentrations of aromatics, branched alkanes, and internal alkenes. PP and PE exhibited distinct degradation mechanisms, with PP requiring less energy for decomposition and yielding more oil. Here, pyrolysis oil from PCR HDPE and PCR PP contained a higher proportion of branched compounds. Additives in PCR plastics may promote isomerization during pyrolysis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

High Throughput Genome Releaser

In this study, we present the development of a High Throughput Genome Releaser, an innovative device addressing common challenges in screening PCR. This genome DNA releaser is designed for rapid, cost-effective, and efficient DNA extraction, optimized for subsequent PCR reactions. Our experimentation with various synthetic materials led us to select a particular type of plastic that mirrors the properties of glass cover slides, providing a smooth surface and effective compression capabilities. We engineered a 96-well device equipped with a 96-well plate and a top rod, operable both manually and automatically, which is compatible with widely used liquid-handling robot decks. This compatibility enhances ease of use in high-throughput PCR setups. Additionally, we developed software to support its automatic functions. The genome releaser facilitates the extraction of PCR-amplifiable genomic DNA from 96 samples within minutes, eliminates the need for extraction buffers, and is adaptable to a wide range of microorganisms and cells. This versatility could significantly advance biomanufacturing processes.

42 ENGINEERING↗
DOE OSTI2026

Upcycling plastic waste into polyhydroxyalkanoates with high carbon conversion via CO 2 plasma-enabled deconstruction

Plastic deconstruction into fermentable intermediates is a key step for microbial bio-upcycling into value-added products. In this study, CO 2 plasma deconstruction was used as an electrified route to convert polyethylene into oxygenated intermediates and liquid (OIL). The resulting OIL was rich in fatty acids, fatty alcohols, and hydrocarbons, making it a suitable feedstock for medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida NRRL B-14688 and Pseudomonas resinovorans NRRL B-2649. Compared with batch fermentation and monocultures, fed-batch co-cultivation markedly improved biomass formation and PHA accumulation, likely due to complementary substrate utilization, particularly hydrocarbon conversion by P. resinovorans. Using virgin polyethylene-derived OIL (Vir-OIL), the co-culture achieved 40.11% mcl-PHA content and about 18% PHA yield based on total OIL fed. More importantly, OIL produced from post-consumer single-use plastic films (PCR-OIL) was directly fermented and well supported the cell growth and PHA accumulation, achieving 38.11% PHA content and about 14.7% PHA yield. Based on emulsified OIL fractions, PHA yields for Vir-OIL and PCR-OIL were comparable (∼28%). PHA granules were extracted from PCR-OIL-grown cells with high recovery (88.25%) and purity (94.8%). Five monomers were identified in the polymer, including 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), 3-hydroxydodecanoate (3HDD), and 3-hydroxytetradecanoate (3HTD), with 3HO (44.28%) and 3HD (40.60%) as the dominant units. The polymer exhibited moderate molecular weight and narrow dispersity (M n = 65.4 kDa, M w = 92.1 kDa, Đ = 1.40) and low crystallinity (T m ≈ 76.6 °C, X c ≈ 15.5%). These characteristics indicate elastomer-like behavior, making the material suitable for flexible applications such as films, coatings, adhesives, and blend modifiers. Overall, this study establishes a CO 2 plasma-assisted route for generating fermentable polyethylene-derived intermediates and demonstrates that fed-batch co-culture fermentation can effectively funnel plastic-derived carbon into mcl-PHA.

42 ENGINEERING↗
DOE OSTI2025

A 1-year study on SARS-CoV-2 variant shifts in wastewater using dPCR: comparison with clinical and GISAID data

Wastewater testing can be used to monitor SARS-CoV-2 infections in communities. Data from PCR-based wastewater testing are usually available to public health authorities within 5–7 days after excreta and other body fluids enter the sewer. While PCR-based methods can accurately detect and quantify SARS-CoV-2, sequencing-based methods are usually required to distinguish between variants, delaying the results and adding cost to the process. We developed and assessed a novel, customizable digital PCR (dPCR)-based genotyping method for SARS-CoV-2 variant detection in wastewater, which is more cost-effective, faster, and more accessible than sequencing. This approach was applied to more than 1,400 wastewater samples

Wilton, Rose↗
DOE OSTI2024

A cell-based Papain-like Protease (PLpro) activity assay for rapid detection of active SARS-CoV-2 infections and antivirals

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants are a continuous threat to human life. An urgent need remains for simple and fast tests that reliably detect active infections with SARS-CoV-2 and its variants in the early stage of infection. Here we introduce a simple and rapid activity-based diagnostic (ABDx) test that identifies SARS-CoV-2 infections by measuring the activity of a viral enzyme, Papain-Like protease (PLpro). The test system consists of a peptide that fluoresces when cleaved by SARS PLpro that is active in crude, unprocessed lysates from human tongue scrapes and saliva. Test results are obtained in 30 minutes or less using widely available fluorescence plate readers, or a battery-operated portable instrument for on-site testing. Proof-of-concept was obtained in a study on clinical specimens collected from patients with COVID-19 like symptoms who tested positive (n = 10) or negative (n = 10) with LIAT RT-PCR using nasal mid turbinate swabs. When saliva from these patients was tested with in-house endpoint RT-PCR, 17 were positive and only 5 specimens were negative, of which 2 became positive when tested 5 days later. PLpro activity correlated in 17 of these cases (3 out of 3 negatives and 14 out of 16 positives, with one invalid specimen). Despite the small number of samples, the agreement was significant (p value = 0.01). Two false negatives were detected, one from a sample with a late Ct value of 35 in diagnostic RT-PCR, indicating that an active infection was no longer present. The PLpro assay is easily scalable and expected to detect all viable SARS-CoV-2 variants, making it attractive as a screening and surveillance tool. Additionally, we show feasibility of the platform as a new homogeneous phenotypic assay for rapid screening of SARS-CoV-2 antiviral drugs and neutralizing antibodies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Surface Trap Dynamics and Gamma‐Ray Detection Enhancement in Passivated FAPbBr 3 Perovskite Crystals

Perovskite‐based direct radiation detectors offer a compelling platform for room‐temperature gamma spectroscopy due to their high sensitivity, tunable optoelectronic properties, and compatibility with solution processing. While prior studies on formamidinium lead tribromide (FAPbBr 3 ) have shown that surface passivation mitigates deep trap state impact on the charge collection efficiency (CCE), the influence of surface‐based shallow traps on time‐resolved detector response remains largely unexplored. In this article, a digital signal processing (DSP) method is applied to analyze waveform rising edge dynamics in FAPbBr 3 single‐crystal detectors, identifying prompt and delayed charge collection components. Using the second derivative of the collected charge signal, the onset of shallow trap reemission is isolated, occurring 1.1 µs after carrier generation. Surface passivation suppressed deep trap states, shifting carrier occupancy toward shallower traps and extending the temporal window of delayed charge collection. This delayed contribution correlates with measurable changes in the temporal and amplitude distributions of detector events. By leveraging time‐domain filtering informed by these dynamics, a selective enhancement in signal quality and gamma peak‐to‐Compton ratio (PCR) is achieved, while preserving over 90% of photoelectric events. An improvement in the PCR to 1.80, from the original 1.09, is enabled by focusing on high collection efficiency events corresponding to reemitted carriers in the 1.1 to 3 µs range after the generation event. These results demonstrate a pathway for leveraging shallow trap dynamics to enhance spectral fidelity in time‐resolved perovskite detectors.

36 MATERIALS SCIENCE↗
DOE OSTI2025

Upcycling of Waste Plastics into Carboxylic Acids for Biodegradable Surfactants

This work outlines a process for producing high‐purity (>95%) carboxylate surfactants from post‐consumer recycled high‐density polyethylene (PCR‐HDPE). The approach involves the thermal depolymerization of PCR‐HDPE via pyrolysis, followed by fractional distillation to isolate C9–C14 olefins. These olefins undergo hydroformylation using cobalt carbonyl catalysts to generate aldehydes, which are subsequently oxidized to carboxylic acids using Pinnick oxidation under mild aqueous‐phase conditions. Neutralization of the resulting carboxylic acids with sodium hydroxide produces plastic‐derived carboxylate surfactants (PDCs) in the form of sodium carboxylates. Subsequent purification steps ensure surfactant‐grade purity and enable accurate assessment of physicochemical properties. The resulting PDCs are evaluated for critical micelle concentration (CMC), foamability, surface tension reduction, and calcium ion tolerance, demonstrating competitive behavior with conventional anionic carboxylate surfactants. This route provides a sustainable alternative for surfactant production, reducing reliance on fossil‐derived feedstocks and valorizing plastic waste streams through chemical upcycling.

Biodegradable surfactants↗
DOE OSTI2025

Development of a high-throughput method for processing sponge-stick samples to detect viable Bacillus anthracis spores

Since the national validation of the sponge-stick based method for detection of Bacillus anthracis spores in environmental samples, there have not been focused efforts to address the low throughput nature of the method, which processes only one sample at one time. Sample processing remains a serious bottleneck for rapidly analyzing large numbers of samples expected from a biological warfare attack. Therefore, we developed a high-throughput method to simultaneously process multiple sponge-stick samples to be better prepared for rapid response and recovery after wide area anthrax incidents. In this method, sponges are placed in 50 mL tubes containing 25 mL extraction buffer and shaken to release spores, after which the suspension is recovered for analysis. Here, we determined that an additional extraction step, conducted in the same tubes with 10 mL buffer, further increased spore recovery from sponge-stick by approximately 10 %. We determined that orbital shaking and multi-tube vortexing were both more effective than reciprocating shaking for recovering spores. We conducted simultaneous processing of up to 12 sponge-stick samples and demonstrated comparable spore recovery efficiencies to the traditional low-throughput stomacher-based method (approximately 60 % recovery at 10 2 -spore level and 75 % recovery at 10 4 -spore level for both methods in three replicate experiments, P > 0.05 for two-tailed t-tests for each experiment and spore level). We also demonstrated that our high-throughput method could be integrated with Rapid Viability-Polymerase Chain Reaction (RV-PCR) analysis and could detect levels as low as 40 spores per sponge even when challenged by a PCR particulate contaminant.

Anthrax↗
DOE OSTI2025

Artemether-Lumefantrine Treatment Selects Plasmodium falciparum Multidrug Resistance 1 ( pfmdr1 ) Increased Copy Number Among African Malaria Infections

Abstract Background Decreased efficacy of artemether-lumefantrine, the globally most used antimalarial, has recently emerged in Africa. Methods An efficacy trial was carried out based on directly observed artemether-lumefantrine therapy at Bengo, Northern Angola. One-hundred Plasmodium falciparum uncomplicated malaria patients (2–10 years old) were enrolled, hospitalized for the treatment period, and followed up for 42 days. Polymerase chain reaction (PCR) correction was performed with pfmsp1/2 plus glurp, with analysis considering 2 or 3 coincident markers. Infections were tested by quantitative PCR (qPCR) for pfmdr1 copy number (pfmdr1×N), a potential P. falciparum marker of lumefantrine resistance previously identified in the region. In vitro clone mixtures were built and used to determine the relation between qPCR copy number scores and actual intrainfection quantitative fractions of pfmdr1×N. Results We observed a significant posttreatment selection of gene amplification, suggesting a role in the parasite in vivo response to this drug. pfmdr1×2 qPCR scores of 1.3, 1.4, and 1.5 were determined to correspond to 15%, 25%, and 35% intrainfection rates. Patients carrying infections with a score ≥1.4 at baseline were linked to decreased artemether-lumefantrine day 42 efficacy (79% vs 97% single-copy pfmdr1). All infections were pfmdr1 N86 carriers and no pfk13 mutations were found. Conclusions Our study suggests pfmdr1×N as a marker of P. falciparum in vivo response to lumefantrine in Africa, while indicating patients carrying infections with a pretreatment pfmdr1×N score ≥1.4 before treatment are a group experiencing decreased artemether-lumefantrine performance.

Fançony, Claudia (ORCID:0000000344211769)↗
DOE OSTI2024

Measurements of soil protist richness and community composition are influenced by primer pair, annealing temperature, and bioinformatics choices

ABSTRACT Protists are a diverse and understudied group of microbial eukaryotic organisms especially in terrestrial environments. Advances in molecular methods are increasing our understanding of the distribution and functions of these creatures; however, there is a vast array of choices researchers make including barcoding genes, primer pairs, PCR settings, and bioinformatic options that can impact the outcome of protist community surveys. Here, we tested four commonly used primer pairs targeting the V4 and V9 regions of the 18S rRNA gene using different PCR annealing temperatures and processed the sequences with different bioinformatic parameters in 10 diverse soils to evaluate how primer pair, amplification parameters, and bioinformatic choices influence the composition and richness of protist and non-protist taxa using Illumina sequencing. Our results showed that annealing temperature influenced sequencing depth and protist taxon richness for most primer pairs, and that merging forward and reverse sequencing reads for the V4 primer pairs dramatically reduced the number of sequences and taxon richness of protists. The data sets of primers that targeted the same 18S rRNA gene region (e.g., V4 or V9) had similar protist community compositions; however, data sets from primers targeting the V4 18S rRNA gene region detected a greater number of protist taxa compared to those prepared with primers targeting the V9 18S rRNA region. There was limited overlap of protist taxa between data sets targeting the two different gene regions (80/549 taxa). Together, we show that laboratory and bioinformatic choices can substantially affect the results and conclusions about protist diversity and community composition using metabarcoding. IMPORTANCE Ecosystem functioning is driven by the activity and interactions of the microbial community, in both aquatic and terrestrial environments. Protists are a group of highly diverse, mostly unicellular microbes whose identity and roles in terrestrial ecosystem ecology have been largely ignored until recently. This study highlights the importance of choices researchers make, such as primer pair, on the results and conclusions about protist diversity and community composition in soils. In order to better understand the roles protist taxa play in terrestrial ecosystems, biases in methodological and analytical choices should be understood and acknowledged.

Biotechnology & Applied Microbiology↗