qPCR Primers Research Articles

Paenibacillus spp. may facilitate anaerobic soil disinfestation at low temperatures

We screened for bacteria that may facilitate the suppressive effect on Ralstonia pseudosolanacearum at temperatures < 30 °C after anaerobic soil disinfestation (ASD) with 1% v/v ethanol. Among 22 isolates grown anaerobically, we selected three that significantly suppressed R. pseudosolanacearum population after ASD at 25 °C. Based on the 16 S rRNA sequences, two isolates were inferred as Paenibacillus polymyxa and the other as Paenibacillus sp. Using qPCR primers designed for these strains, we found that all strains increased in the soil after ASD at 25 °C. After ASD with 1% v/v ethanol and with or without soil inoculation with these three strains in a concrete frame in the field at mean temperature of 26.3 °C, the R. pseudosolanacearum population was significantly lower in the inoculation treatment at a depth of 10 cm. These results suggest that these Paenibacillus strains have the potential to promote the ASD effect using 1% ethanol at low temperatures.

Quantitative real-time PCR assays for species-specific detection and quantification of Baltic Sea spring bloom dinoflagellates.

In the Baltic Sea, the dinoflagellates Apocalathium malmogiense, Biecheleria baltica, and Gymnodinium corollarium are important contributors to the spring bloom. However, their relative contribution to the bloom community cannot be unambiguously determined by conventional light microscopy due to a lack of resolution of distinctive morphological features of the three species. Here, we describe a molecular approach based on a quantitative real-time polymerase chain reaction (qPCR) primer and probe system, targeting the ITS1 and ITS2 regions of the rRNA gene for all three species and enabling their quantification. The specificity of the method was demonstrated using monocultures of A. malmogiense, B. baltica, G. corollarium as well as three other dinoflagellate species co-occurring in the Baltic Sea during spring and validated using field-collected phytoplankton samples.

QPrimerDB 2.0: an updated comprehensive gene-specific qPCR primer database for 1172 organisms.

High-quality primer design is essential for the success of all polymerase chain reaction (PCR)-based experiments. We previously developed a thermodynamics-based gene-specific quantitative PCR (qPCR) primer database for 147 organisms, which has been used extensively in gene expression studies. However, the number of organisms and the imperfection of function in the database limits its potential applications. Here, we improved the functionality of qPrimerDB to create a more comprehensive primer resource. Specifically, we (i) developed an improved primer design tool, qPrimer, building upon the previous qPrimerDB pipeline, to enhance the efficiency and simplicity of genome-scale qPCR primer design; (ii) pre-computed qPCR primer resources from 1 308 genomes of 1172 organismsand (iii) introduced a complete system for identifying, designing, checking, marking, and submitting qPCR primers. qPrimerDB 2.0 is freely available at https://qprimerdb.biodb.org. The qPrimer source code is available at https://github.com/swu1019lab/qPrimer.

ExonSurfer: a web-tool to design primers at exon–exon junctions

BackgroundReverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design.ResultsExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets.ConclusionExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.

An investigation of Inonotus obliquus in Estonia: Its distribution, abundance of conks, rot extent in infected trees, and peculiarities of basidiospore dispersal

Inonotus obliquus, a well-known forest fungal pathogen, started gaining attention for its potential medicinal uses. Known as Chaga, the fungus has become prominent due to the bioactive compounds present in its conks. Forest owners are cultivating it to produce conks as a source of non-timber profit. However, not much is known about the natural occurrence of this pathogen, its formation of conks, the extent of decay in the infected trees, and the distribution of its basidiospores. Across 80 forest stands in Estonia dominated by Betula pendula, B. pubescens, Alnus incana, and A. glutinosa, the conks of I. obliquus were documented in 17 stands. The conks were more common in continental Estonia compared to the western islands. Among 800 randomly chosen trees, I. obliquus conks were documented on ten trees. Additionally, an asymptomatic I. obliquus infection was detected in one tree. After two years of spore trapping, I. obliquus basidiospores were detected only on four occasions, between the end of July and the beginning of September, and only at night. The proportion of rot damage in the total volume of model tree wood ranged from 8.6% to 58.5%. For fast and reliable detection of I. obliquus, TaqMan species-specific qPCR primers were developed and tested.

Rapid detection and molecular epidemiology of β-lactamase producing Enterobacteriaceae isolated from food animals and in-contact humans in Nigeria.

The emergence and spread of β-lactamase-producing Enterobacteriaceae poses a significant threat to public health, necessitating the rapid detection and investigation of the molecular epidemiology of these pathogens. We modified a multiplex real-time (RT)-PCR to concurrently detect β-lactamase genes (blaCTX-M, blaTEM, and blaSHV) and Enterobacteriaceae 16S ribosomal RNA. qPCR probes and primers were validated using control isolates, and the sensitivity and specificity assessed. The optimised multiplex qPCR was used to screen 220 non-clinical Enterobacteriaceae from food animals and in-contact humans in Southeast Nigeria selected on cefotaxime-supplemented agar plates. Binary logistic regression was used to explore factors associated with the presence of the blaTEM and blaSHV genes in these isolates, and a subset of isolates from matched sampling sites and host species were whole genome sequenced, and their antimicrobial resistance (AMR) and plasmid profiles determined. The sensitivity and specificity of the qPCR assay was 100%. All isolates (220/220) were positive for Enterobacteriaceae ribosomal 16S rRNA and blaCTX-M, while 66.4% (146/220) and 9% (20/220) were positive for blaTEM and blaSHV, respectively. The prevalence of blaTEM and blaSHV varied across different sampling sites (farm, animal market and abattoirs). Isolates from Abia state were more likely to harbour blaTEM (OR = 2.3, p = 0.04) and blaSHV (OR = 5.12,p = 0.01) than isolates from Ebonyi state; blaTEM was more likely to be detected in isolates from food animals than humans (OR = 2.34, p = 0.03), whereas the reverse was seen for blaSHV (OR = 7.23, p = 0.02). Furthermore, Klebsiella and Enterobacter isolates harboured more AMR genes than Escherichia coli, even though they were isolated from the same sample. We also identified pan resistant Klebsiella harbouring resistance to ten classes of antimicrobials and disinfectant. Therefore, we recommend ESKAPE pathogens are included in AMR surveillance in future and suggest qPCRs be utilised for rapid screening of Enterobacteriaceae from human and animal sources.

An Optimized Genotyping Workflow for Identifying Highly SCRaMbLEd Synthetic Yeasts.

Synthetic Sc2.0 yeast strains contain hundreds to thousands of loxPsym recombination sites that allow restructuring of the Saccharomyces cerevisiae genome by SCRaMbLE. Thus, a highly diverse yeast population can arise from a single genotype. The selection of genetically diverse candidates with rearranged synthetic chromosomes for downstream analysis requires an efficient and straightforward workflow. Here we present loxTags, a set of qPCR primers for genotyping across loxPsym sites to detect not only deletions but also inversions and translocations after SCRaMbLE. To cope with the large number of amplicons, we generated qTagGer, a qPCR genotyping primer prediction tool. Using loxTag-based genotyping and long-read sequencing, we show that light-inducible Cre recombinase L-SCRaMbLE can efficiently generate diverse recombination events when applied to Sc2.0 strains containing a linear or a circular version of synthetic chromosome III.

Quantification of the dark fungal taxon Cryptomycota using qPCR.

Fungi are present in a wide variety of natural environments, and in the last years, various studies have shown that they are quite abundant in aquatic ecosystems. In addition, a whole new highly diverse phylum, the Cryptomycota, was discovered. Nevertheless, research on aquatic fungi and a detailed evaluation of their functions and distribution are still sparse. One of the main reasons is a limitation in reliable identification and quantification methods. To bridge part of the research gap, this study aims to implement a quantitative PCR method to detect and quantify the newly discovered phylum. We developed and validated a Cryptomycota-specific qPCR primer pair targeting the 5.8S region that detects the majority of Cryptomycota, but Microsporidia. The resulting amplicon is 102 bp long. We used different environmental samples to evaluate the primer pair, various fungal sequences as negative control and positive control sequences. Obtained amplicons were sequenced using Illumina, and the obtained ASVs were all classified as Cryptomycota. The qPCR method works reliably and specifically for the quantification of Cryptomycota in environmental samples.

Climate change can impair bacterial pathogen defences in sablefish via hypoxia-mediated effects on adaptive immunity

Low-oxygen levels (hypoxia) in aquatic habitats are becoming more common because of global warming and eutrophication. However, the effects on the health/disease status of fishes, the world's largest group of vertebrates, are unclear. Therefore, we assessed how long-term hypoxia affected the immune function of sablefish, an ecologically and economically important North Pacific species, including the response to a formalin-killed Aeromonas salmonicida bacterin. Sablefish were held at normoxia or hypoxia (100% or 40% air saturated seawater, respectively) for 6–16 weeks, while we measured a diverse array of immunological traits. Given that the sablefish is a non-model organism, this involved the development of a species-specific methodological toolbox comprised of qPCR primers for 16 key immune genes, assays for blood antibacterial defences, the assessment of blood immunoglobulin (IgM) levels with ELISA, and flow cytometry and confocal microscopy techniques. We show that innate immune parameters were typically elevated in response to the bacterial antigens, but were not substantially affected by hypoxia. In contrast, hypoxia completely prevented the ∼1.5-fold increase in blood IgM level that was observed under normoxic conditions following bacterin exposure, implying a serious impairment of adaptive immunity. Since the sablefish is naturally hypoxia tolerant, our results demonstrate that climate change-related deoxygenation may be a serious threat to the immune competency of fishes.

Detection and quantification of natural Wolbachia in Aedes aegypti in Metropolitan Manila, Philippines using locally designed primers.

The Philippines bears health and economic burden caused by high dengue cases annually. Presently, the Philippines still lack an effective and sustainable vector management. The use of Wolbachia, a maternally transmitted bacterium, that mitigate arbovirus transmission has been recommended. Cytoplasmic incompatibility and viral blocking, two characteristics that make Wolbachia suitable for vector control, depend on infection prevalence and density. There are no current Wolbachia release programs in the Philippines, and studies regarding the safety of this intervention. Here, we screened for Wolbachia in Aedes aegypti collected from Metropolitan Manila, Philippines. We designed location-specific primers for qPCR to test whether this improved Wolbachia detection in Ae. aegypti. We explored if host sex and Wolbachia strain could be potential factors affecting Wolbachia density. Ae. aegypti mosquitoes (n=429) were screened for natural Wolbachia by taqman qPCR using location-specific Wolbachia surface protein primers (wspAAML) and known 16S rRNA primers. Samples positive for wspAAML (n=267) were processed for Sanger sequencing. We constructed a phylogenetic tree using IQ-TREE 2 to further characterize Wolbachia present in the Philippine Ae. aegypti. We then compared Wolbachia densities between Wolbachia groups and host sex. Statistical analyses were done using GraphPad Prism 9.0. Wolbachia prevalence for 16S rRNA (40%) and wspAAML (62%) markers were high. Wolbachia relative densities for 16S rRNA ranged from -3.84 to 2.71 and wspAAML from -4.02 to 1.81. Densities were higher in male than female mosquitoes. Wolbachia strains detected in Ae. aegypti clustered into supergroup B. Some 54% (123/226) of these sequences clustered under a group referred to here as "wAegML," that belongs to the supergroup B, which had a significantly lower density than wAegB/wAlbB, and wAlbA strains. Location-specific primers improved detection of natural Wolbachia in Ae. aegypti and allowed for relative quantification. Wolbachia density is relatively low, and differed between host sexes and Wolbachia strains. An economical way of confirming sporadic or transient Wolbachia in Ae. aegypti is necessary while considering host sex and bacterial strain.

DNA from dives: Species detection of humpback whales (Megaptera novaeangliae) from flukeprint eDNA

AbstractNorthern British Columbia has been identified as an important habitat for several coastal cetacean species, including humpback whales (Megaptera novaeangliae). This species is listed as being of “Special Concern” under Canada's Species at Risk Act, partly due to data deficiencies concerning genetic population structure and demographics in British Columbia. Anthropogenic activities threaten North Coast humpback whale populations, with particular concern for the impact of vessel noise, entanglement, and ship strikes. Current methodology (i.e., biopsy sampling) for obtaining cetacean genetic data is invasive, challenging, and costly; therefore, there is an urgency to develop effective and minimally invasive methodologies for efficiently collecting this data. Environmental DNA (eDNA) has been identified as an ideal tool for monitoring the presence and distribution of numerous species within marine ecosystems; however, the feasibility for cetaceans is not yet well established. In this study, we opportunistically collected targeted 1 L seawater eDNA samples from flukeprints when individual humpback whales were observed diving between the years of 2020 and 2022. A total of 93 samples were collected from individual humpback whales identified using a photographic identification catalogue. We successfully detected humpback whale eDNA in 28 samples using novel species‐specific qPCR primers (~500 mL of sample), with relatively equal successful detection between immediate (0 days) and delayed (up to 10 days) sample filtration. Here, we have validated a qPCR assay for detecting humpback whale DNA from flukeprints and highlighted the future optimizations required to improve the potential application of flukeprint eDNA for conservation management.

Measuring the Distance and Effects of Weather Conditions on the Dispersal of Nothopassalora personata.

Nothopassalora personata is one of the most economically severe pathogens of peanut in the United States. The fungus primarily relies on wind and rain for dispersal, which has been documented up to 10 m from an inoculum source. Spore traps have been used in a wide variety of pathosystems to study epidemiology, document detection, develop alert systems, and guide management programs. The objective of this study was to use spore traps and N. personata-specific qPCR primers to quantitatively evaluate dispersal of N. personata conidia at distances up to 70 m from an infected peanut field and to examine relationships between quantities captured and weather variables. Impaction spore samplers were placed at 4, 10, 30, 50, and 70 m from peanut fields at the Edisto Research and Education Center (six fields) and commercial peanut fields in Barnwell and Bamberg counties (one field each) from 2020 to 2022. Following initial detection, samples were collected at a 48-, 48-, 72-h interval until harvest. N. personata conidia were detected at all locations and distances, documenting dispersal up to 70 m from an inoculum source. This result is a reminder that volunteer management is crucial when rotating peanut in nearby fields. A model for predicting log spore quantities was developed using temperature and humidity variables. Temperature variables associated with observed sampling periods had a negative correlation with N. personata quantities, whereas parameters of relative humidity and mean windspeed were positively correlated.

Occurrence of Rhodococcus sp. RR1 prmA and Rhodococcus jostii RHA1 prmA across microbial communities and their enumeration during 1,4-dioxane biodegradation

1,4-Dioxane, a likely human carcinogen, is a co-contaminant at many chlorinated solvent contaminated sites. Conventional treatment technologies, such as carbon sorption or air stripping, are largely ineffective, and so many researchers have explored bioremediation for site clean-up. An important step towards this involves examining the occurrence of the functional genes associated with 1,4-dioxane biodegradation. The current research explored potential biomarkers for 1,4-dioxane in three mixed microbial communities (wetland sediment, agricultural soil, impacted site sediment) using monooxygenase targeted amplicon sequencing, followed by quantitative PCR (qPCR). A BLAST analysis of the sequencing data detected only two of the genes previously associated with 1,4-dioxane metabolism or co-metabolism, namely propane monooxygenase (prmA) from Rhodococcus jostii RHA1 and Rhodococcus sp. RR1. To investigate this further, qPCR primers and probes were designed, and the assays were used to enumerate prmA gene copies in the three communities. Gene copies of Rhodococcus RR1 prmA were detected in all three, while gene copies of Rhodococcus jostii RHA1 prmA were detected in two of the three sample types (except impacted site sediment). Further, there was a statistically significant increase in RR1 prmA gene copies in the microcosms inoculated with impacted site sediment following 1,4-dioxane biodegradation compared to the control microcosms (no 1,4-dioxane) or to the initial copy numbers before incubation. Overall, the results indicate the importance of Rhodococcus associated prmA, compared to other 1,4-dioxane degrading associated biomarkers, in three different microbial communities. Also, the newly designed qPCR assays provide a platform for others to investigate 1,4-dioxane biodegradation potential in mixed communities and should be of particular interest to those considering bioremediation as a potential 1,4-dioxane remediation approach.

Selection of suitable reference genes for qPCR normalization in different developmental stages of Oenanthe javanica.

Gene expression analysis is widely used to unravel molecular regulatory mechanisms and identify key genes in plants. Appropriate reference gene is an important prerequisite to ensure the accuracy and reliability of qPCR analysis results. Water dropwort is a plant of the Oenanthe genus in the Apiaceae family, which has high economic benefits. However, the underlying molecular regulatory mechanisms in the growth and development of water dropwort have not been fully understood and the appropriate reference genes in different developmental stages of water dropwort not yet reported. In this study, 10 candidate reference genes (ACTIN, PP2A, SAND, EF-1α, GAPDH, UBQ, MIP, TBP, RPS-18, eIF-4α) were identified and cloned from Oenanthe javanica. The qPCR primers of candidate reference genes were designed and verified. Four statistical algorithms, geNorm, NormFinder, BestKeeper and RefFinder were used to evaluate the expression stability of 10 candidate reference genes in different developmental stages of water dropwort. The results showed that TBP and UBQ were the most stable genes in different developmental stages of water dropwort, while GAPDH was the most unstable gene. The normalization of EXP1 genes at different developmental stages further confirmed the reliability of internal reference genes. The results of this study provide a theoretical basis for selecting appropriate internal reference genes in different developmental stages of water dropwort. This study also provides technical support and reliable basis for the expression analysis of key genes in different developmental stages of water dropwort.

Bacterial genome-wide association study substantiates papGII of Escherichia coli as a major risk factor for urosepsis

BackgroundUrinary tract infections (UTIs) are among the most common bacterial infections worldwide, often caused by uropathogenic Escherichia coli. Multiple bacterial virulence factors or patient characteristics have been linked separately to progressive, more invasive infections. In this study, we aim to identify pathogen- and patient-specific factors that drive the progression to urosepsis by jointly analysing bacterial and host characteristics.MethodsWe analysed 1076 E. coli strains isolated from 825 clinical cases with UTI and/or bacteraemia by whole-genome sequencing (Illumina). Sequence types (STs) were determined via srst2 and capsule loci via fastKaptive. We compared the isolates from urine and blood to confirm clonality. Furthermore, we performed a bacterial genome-wide association study (bGWAS) (pyseer) using bacteraemia as the primary clinical outcome. Clinical data were collected by an electronic patient chart review. We concurrently analysed the association of the most significant bGWAS hit and important patient characteristics with the clinical endpoint bacteraemia using a generalised linear model (GLM). Finally, we designed qPCR primers and probes to detect papGII-positive E. coli strains and prospectively screened E. coli from urine samples (n = 1657) at two healthcare centres.ResultsOur patient cohort had a median age of 75.3 years (range: 18.00–103.1) and was predominantly female (574/825, 69.6%). The bacterial phylogroups B2 (60.6%; 500/825) and D (16.6%; 137/825), which are associated with extraintestinal infections, represent the majority of the strains in our collection, many of which encode a polysaccharide capsule (63.4%; 525/825). The most frequently observed STs were ST131 (12.7%; 105/825), ST69 (11.0%; 91/825), and ST73 (10.2%; 84/825). Of interest, in 12.3% (13/106) of cases, the E. coli pairs in urine and blood were only distantly related. In line with previous bGWAS studies, we identified the gene papGII (p-value < 0.001), which encodes the adhesin subunit of the E. coli P-pilus, to be associated with ‘bacteraemia’ in our bGWAS. In our GLM, correcting for patient characteristics, papGII remained highly significant (odds ratio = 5.27, 95% confidence interval = [3.48, 7.97], p-value < 0.001). An independent cohort of cases which we screened for papGII-carrying E. coli at two healthcare centres further confirmed the increased relative frequency of papGII-positive strains causing invasive infection, compared to papGII-negative strains (p-value = 0.033, chi-squared test).ConclusionsThis study builds on previous work linking papGII with invasive infection by showing that it is a major risk factor for progression from UTI to bacteraemia that has diagnostic potential.

IntI1 gene abundance from septic tanks in Thailand using validated intI1 primers.

Antimicrobial resistance is a global crisis, and wastewater treatment, including septic tanks, remains an important source of antimicrobial resistance (AMR) genes. The role of septic tanks in disseminating class 1 integron, and by extension AMR genes, in Thailand, where antibiotic use is unregulated remains understudied. We aimed to monitor gene abundance as a proxy to infer potential AMR from septic tanks in Thailand. We evaluated published intI1 primers due to the lack of consensus on optimal Q-PCR primers and the absence of standardization. Our findings confirmed septic tanks are a source of class 1 integron to the environment. We highlighted the significance of intI1 primer choice, in the context of interpretation of risk associated with AMR spread from septic tanks. We recommend the validated set (F3-R3) for optimal intI1 quantification toward the goal of achieving standardization across studies.

Detection and quantification of Cronartium pini from Scots pine bark and wood with Cronartium spp.‐specific quantitative PCR

AbstractThe forest disease, Scots pine blister rust, is caused by the rust fungus Cronartium pini. This pathogen was previously known as the host‐alternating type C. flaccidum and non‐host‐alternating type Peridermium pini. Recent epidemics of this disease in Northern European forests, especially young Scots pine forests in Sweden, caused significant economic and ecological losses. Cronartium pini can be identified based on the typical orange blister‐like aecia in Scots pine in summer, but any molecular identification and quantification method has not been available for Cronartium spp. This study developed qPCR primers that are specific to Cronartium spp. and evaluated DNA extraction protocols from pine bark and wood to enable robust qPCR assays. As little as three Cronartium ITS copies can be detected with the protocol. Since only C. pini is known to infect Scots pine in Northern Europe, the protocols were applied to detect C. pini from Scots pine samples without typical symptoms and investigate the C. pini colonization in Scots pine branches from the forest. These results will aid the detection and quantification of C. pini in asymptomatic or symptomatic samples and monitoring Scots pine blister rust in the forest in northern Europe.

The Fascinating Cross-Paths of Pathogenic Bacteria, Human and Animal Faecal Sources in Water-Stressed Communities of Vhembe District, South Africa.

Access to clean and safe drinking water still remains a major challenge in the developing world, causing public health risks in terms of waterborne infections, especially in rural areas of sub-Saharan Africa. This study aimed to track and detect enteric pathogens (Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, Shigella flexneri, and Campylobacter jejuni subsp. jejuni) in rural water sources. It also sought to establish a correlation between these pathogens and the sources of faecal pollution. Multiplex qPCR and specific primers and probes were used for detection and tracking. The study successfully correlated the occurrence of target pathogens with sources of human and animal faecal contamination using host-specific genetic markers (BacHum and HF183 for humans, BacCow for cows, Pig-2-Bac for pigs, Cytb for chickens, and BacCan for dogs). The study revealed that enteric pathogens were found in 47.69% and 32.80% of samples during the wet and dry seasons, respectively. These pathogens were associated with human or animal faecal contamination. Correlations between pathogens and contamination sources were significant (p ≤ 0.05), with varying strengths during the wet and dry seasons. The findings emphasize the importance of identifying faecal contamination sources to protect rural communities from waterborne infections.

New insights into the genome and transmission of the microsporidian pathogen Nosema muscidifuracis.

Nosema is a diverse genus of unicellular microsporidian parasites of insects and other arthropods. Nosema muscidifuracis infects parasitoid wasp species of Muscidifurax zaraptor and M. raptor (Hymenoptera: Pteromalidae), causing ~50% reduction in longevity and ~90% reduction in fecundity. Here, we report the first assembly of the N. muscidifuracis genome (14,397,169 bp in 28 contigs) of high continuity (contig N50 544.3 Kb) and completeness (BUSCO score 97.0%). A total of 2,782 protein-coding genes were annotated, with 66.2% of the genes having two copies and 24.0% of genes having three copies. These duplicated genes are highly similar, with a sequence identity of 99.3%. The complex pattern suggests extensive gene duplications and rearrangements across the genome. We annotated 57 rDNA loci, which are highly GC-rich (37%) in a GC-poor genome (25% genome average). Nosema-specific qPCR primer sets were designed based on 18S rDNA annotation as a diagnostic tool to determine its titer in host samples. We discovered high Nosema titers in Nosema-cured M. raptor and M. zaraptor using heat treatment in 2017 and 2019, suggesting that the remedy did not completely eliminate the Nosema infection. Cytogenetic analyses revealed heavy infections of N. muscidifuracis within the ovaries of M. raptor and M. zaraptor, consistent with the titer determined by qPCR and suggesting a heritable component of infection and per ovum vertical transmission. The parasitoids-Nosema system is laboratory tractable and, therefore, can serve as a model to inform future genome manipulations of Nosema-host system for investigations of Nosemosis.

Abstract 979: Development of a methylation-based classifier to identify pancreatic adenocarcinoma subtype

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) demonstrates variable prognosis and response to first-line FOLFIRINOX therapy depending on molecular subtype; patients with classical tumors showed a greater response compared to basal-like tumors. Identifying molecular subtype may inform treatment approaches, but current subtyping schemas rely on tumor biopsies which are invasive and can be technically challenging. Blood based epigenetic assays are commercially available, and identification of subtype-associated epigenetic modifications may provide a path toward blood-based PDAC molecular subtyping. We analyzed methylation levels and developed a subtype classifier using methylation data on PDAC tumors. Using this classifier, we then developed a methylation-specific qPCR assay to subtype PDAC tumors by analyzing methylation at particular sites in tumor DNA which may now be further adapted to more facile liquid biopsy approaches. Methods: Using methylation data (n=150) from TCGA PAAD, we applied a random forest (RF) model followed by a k-Top Scoring Pairs (k-TSP) algorithm to develop a methylation site-based classifier that allows prediction of subtype by comparing methylation levels between paired sites. The classifier was validated on two independent datasets, including ICGC (n=82) and patient-derived xenografts (PDXs) (n=21). We designed methylation-specific qPCR primers for these sites and validated their specificity with fully methylated or fully unmethylated DNA. Using genomic DNA extracted from 10 PDAC PDXs of known molecular subtype, we obtained methylation levels at sites identified by the classifier via a SYBR Green-based qPCR assay. We then used our classifier to predict subtype of the PDX tumors. Results: We validated our RF-kTSP methylation subtype classifier on two independent cohorts and predicted subtype using 20 methylation sites. In ICGC, we found balanced accuracy=0.94, AUROC=0.98, AU-PR=0.89. In PDX PDAC tumors, we found balanced accuracy=1, AUROC=1, AU-PR=1. We designed 14/20 primers for qPCR and confirmed specificity for methylated DNA. Using 14/20 methylation sites, we achieved 80% correct subtype prediction of 10 PDX tumors (5/5 basal-like, 3/5 classical). Conclusion: We have developed a methylation-based classifier that accurately and replicably predicts PDAC subtype. We continue to develop a qPCR assay for PDAC subtype that so far has 80% accuracy using 14/20 probes. We are completing validation of the remaining 6 primers and correlating methylation levels at each site with those obtained from EPIC array methylation analysis. The long-term goal is to apply our complete set of primers in a digital droplet PCR assay that will be amenable for subtyping using circulating tumor DNA (ctDNA). Citation Format: Zachary Schrank, Des Weighill, Hannah Thel, Hannah Trembath, Ashley Morrison, Jen Jen Yeh. Development of a methylation-based classifier to identify pancreatic adenocarcinoma subtype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 979.