Next-generation Amplicon Sequencing Research Articles

Abstract 978: Functional characterization and a real-world clinical laboratory pilot of the Foundation for the National Institutes of Health’s (FNIH) circulating tumor DNA (ctDNA) quality control materials (QCMs)

Abstract Background: Analysis of ctDNA now permits assessment of targetable mutations (e.g. EGFR L858R/Ex19del) using noninvasive blood tests. There are several FDA-approved tests using digital PCR (ddPCR) or next-generation sequencing (NGS) and many more laboratory developed tests in use. The FNIH Biomarkers Consortium identified the absence of validated standards to qualify the performance of ctDNA assays as a critical need and set out to aid in the characterization of QCMs. Here we report on a commutability study comparing the performance of three commercial QCMs to clinical samples at or above LoD (limit of detection) and update on a real-world assessment of these QCMs by 11 clinical laboratories spanning 4 continents and 3 assay modalities. Methods: QCMs were provided by Horizon Discovery (H), Thermo Fisher (T) and SeraCare (S). Clinical samples with known EGFR L858R and Ex19del mutations were prepared by diluting specimens at known concentrations into cell-free DNA (cfDNA) from healthy donors. The functional characterization compared detection of the mutations in QCM to clinical samples and consisted of 2 parts: (1) Evaluation of EGFR L858R/Ex19del (range 0.1% to 5% allele frequency [AF]) by ddPCR, amplicon and hybrid-capture NGS; (2) Evaluation near the LoD of the amplicon NGS assay at the assay LoD95 of 0.3% AF. For the clinical pilot, participants were sent QCM pools formulated at 0.5% and 1% AF by ddPCR. Some participants also tested clinical specimens with EGFR L858R and Ex19del at 0.5% and 1% AF by ddPCR. All participants were blinded to AF and identity of the manufacturer. Results: For Part 1, QCM results from hybrid-capture NGS of EGFR L858R were similar to ddPCR; for Ex19del, QCM AF trended lower compared to ddPCR. In contrast, L858R clinical samples showed a strong positive bias compared to ddPCR. For amplicon NGS, QCMs performed similarly to clinical samples, with amplicon NGS AF closest to ddPCR AF for T QCM. For Part 2, the observed hit rates mostly approximated expected values. For H QCM, observed LoD95 (10% for EGFR L858R; 50% for Ex19del) and subsequent dilution hit rates were lower than expected. For the clinical pilot, median Ex19del AF of amplicon NGS were higher than for hybrid-capture NGS for both the 1% QCM formulations and 0.5% QCM formulations. Median QCM L858R AF were similar for amplicon and hybrid-capture NGS, with greatest contributor to variance being inter-lab differences. Conclusions: This project, unprecedented in the number of participants and analytical rigor, revealed unexpected differences in performance of both assays and QCMs. These findings merit consideration by laboratories that rely on QCMs to develop and perform assays for ctDNA testing as diagnostics. Citation Format: Cloud P. Paweletz, Thomas D. Forbes, Grace A. Heavey, Laura M. Yee, Hua-Jun He, Zhiyong He, Dana E. Connors, Daniel Stetson, Susan Keating, Kenneth D. Cole, Gary Kelloff, Robert T. McCormack, Mickey P. Williams, Chris Karlovich. Functional characterization and a real-world clinical laboratory pilot of the Foundation for the National Institutes of Health’s (FNIH) circulating tumor DNA (ctDNA) quality control materials (QCMs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 978.

Abstract 2500: Technical feasibility of CRISPR-based functional profiling for solid tumor precision medicine

Abstract Introduction: Next generation sequencing (NGS) has revolutionized precision oncology by enabling identification of cancer patients harboring actionable mutations who may benefit from corresponding targeted therapies. However, sequencing-based biomarkers are not detected for most patients and imperfectly predict response to therapy even when present. To address this unmet need, we have developed a CRISPR-based diagnostics platform for ‘personalized functional genomics’, allowing identification of patient-specific therapeutic vulnerabilities irrespective of mutation status. We have previously applied this approach to primary hematologic malignancies, and here report application to solid tumor-derived samples. Methods: Patient-derived solid tumor samples from several tumor types including breast, ovarian, colon and lung were obtained from 3 sources: fresh tissue (obtained from patients via core biopsy, surgical resection, or malignant fluid sampling), primary Patient-Derived Cell Lines (PDCLs, generated from surgical material and adapted for ex vivo culture), and commercially obtained viably frozen dissociated tumor cells (DTCs). Samples were dissociated to single cell suspensions and transduced with a lentivirus encoding Cas9 and an sgRNA library covering a custom targeted oncology gene panel. The panel consists of genes encoding targets of FDA-approved oncology drugs, as well as reference genes. After a short culture period, transduced cells were harvested and relative sgRNA distribution was measured by amplicon NGS. Changes in sgRNA abundance were collapsed into gene-level log2-fold changes, and significantly depleted genes scored as functional dependencies. For select PDCLs, cells were treated with cognate small molecule inhibitors over the course of 4 days. Cell viability was read out using CTG assay and a calculated IC50 lower than 500nM inferred as inhibitor sensitive. Results: In a double-blind study using primary PDCLs, functional dependencies identified by CRISPR perfectly discriminated alpelisib sensitive (2/2) from resistant samples (6/6). Using fresh primary samples from varying cancer types and specimen sources, we achieve a technical success rate >70%. Among gene dependencies identified, targets of FDA-approved oncology drugs and those under late-stage clinical investigation were both observed including BRAF, CDK4/6, VEGFR1/2 and PARP1/2. Conclusion: We demonstrate technical proof of concept of the FX platform for CRISPR-based personalized functional genomics. This approach is feasible when applied to primary patient samples from a variety of tumor types and tissue sources, and results are validated by comparison with orthogonal methods. CRISPR-based functional profiling represents a powerful adjunct to traditional genomic profiling, for the first time allowing drug target dependency mapping in a patient-specific and mutation-agnostic manner. Citation Format: Karl Hodel, Andreas Varkaris, Nicholas Chevalier, Maci Meyers, Allison Kehlmann, Stephane Corbel, Anastasia Stavridi, Peter Kilfeather, Leonardo Hagmann, Kathleen Tran, Yali Li, Christian Schmedt, Ioannis Sanidas, Srinath Sampath, Srihari Sampath, Dejan Juric. Technical feasibility of CRISPR-based functional profiling for solid tumor precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2500.

Year-round dynamics of arbuscular mycorrhizal fungi communities in the roots and surrounding soils of Cryptomeria japonica

Arbuscular mycorrhizal fungi (AMF) live simultaneously inside and outside of host plant roots for a functional mycorrhizal symbiosis. Still, the year-round dynamics and relationships between soil properties and AMF communities of trees in forest ecosystems remain unclear. We collected paired root and soil samples of the same Cryptomeria japonica trees at two forest sites (five trees at each site) every 2 months over a year. Total DNA was extracted from roots and soil separately and soil physicochemical properties were measured. With Illumina’s next-generation amplicon sequencing targeting the small subunit of fungal ribosomal DNA, we clarified seasonal dynamics of soil properties and AMF communities. Soil pH and total phosphorus showed significant seasonality while total carbon, nitrogen, and C/N did not. Only pH was a good predictor of the composition and dynamics of the AMF community. The total AMF community (roots + soil) showed significant seasonality because of variation from May to September. Root and soil AMF communities were steady year-round, however, with similar species richness but contained significantly different AMF assemblages in any sampling month. Despite the weak seasonality in the communities, the top two dominant OTUs showed significant but different shifts between roots and soils across seasons with strong antagonistic relationships. In conclusion, few dominant AMF taxa are dynamically shifting between the roots and soils of C. japonica to respond to seasonal and phenological variations in their microhabitats. AMF inhabiting forest ecosystems may have high environmental plasticity to sustain a functional symbiosis regardless of seasonal variations that occur in the soil.

A targeted amplicon next-generation sequencing assay for tryptase genotyping to support personalized therapy in mast cell-related disorders.

Tryptase, the most abundant mast cell granule protein, is elevated in severe asthma patients independent of type 2 inflammation status. Higher active β tryptase allele counts are associated with higher levels of peripheral tryptase and lower clinical benefit from anti-IgE therapies. Tryptase is a therapeutic target of interest in severe asthma and chronic spontaneous urticaria. Active and inactive allele counts may enable stratification to assess response to therapies in asthmatic patient subpopulations. Tryptase gene loci TPSAB1 and TPSB2 have high levels of sequence identity, which makes genotyping a challenging task. Here, we report a targeted next-generation sequencing (NGS) assay and downstream bioinformatics analysis for determining polymorphisms at tryptase TPSAB1 and TPSB2 loci. Machine learning modeling using multiple polymorphisms in the tryptase loci was used to improve the accuracy of genotyping calls. The assay was tested and qualified on DNA extracted from whole blood of healthy donors and asthma patients, achieving accuracy of 96%, 96% and 94% for estimation of inactive α and βΙΙΙFS tryptase alleles and α duplication on TPSAB1, respectively. The reported NGS assay is a cost-effective method that is more efficient than Sanger sequencing and provides coverage to evaluate known as well as unreported tryptase polymorphisms.

Calcium carbonate precipitating extremophilic bacteria in an Alpine ice cave

Extensive research has provided a wealth of data on prokaryotes in caves and their role in biogeochemical cycles. Ice caves in carbonate rocks, however, remain enigmatic environments with limited knowledge of their microbial taxonomic composition. In this study, bacterial and archaeal communities of the Obstans Ice Cave (Carnic Alps, Southern Austria) were analyzed by next-generation amplicon sequencing and by cultivation of bacterial strains at 10 °C and studying their metabolism. The most abundant bacterial taxa were uncultured Burkholderiaceae and Brevundimonas spp. in the drip water, Flavobacterium, Alkanindiges and Polaromonas spp. in the ice, Pseudonocardia, Blastocatella spp., uncultured Pyrinomonadaceae and Sphingomonadaceae in carbonate precipitates, and uncultured Gemmatimonadaceae and Longimicrobiaceae in clastic cave sediments. These taxa are psychrotolerant/psychrophilic and chemoorganotrophic bacteria. On a medium with Mg2+/Ca2+ = 1 at 21 °C and 10 °C, 65% and 35% of the cultivated strains precipitated carbonates, respectively. The first ~ 200 µm-size crystals appeared 2 and 6 weeks after the start of the cultivation experiments at 21 °C and 10 °C, respectively. The crystal structure of these microbially induced carbonate precipitates and their Mg-content are strongly influenced by the Mg2+/Ca2+ ratio of the culture medium. These results suggest that the high diversity of prokaryotic communities detected in cryogenic subsurface environments actively contributes to carbonate precipitation, despite living at the physical limit of the presence of liquid water.

Molecular basis of DEL phenotype in the Indian population: Insights from next-generation sequencing analysis of two cases

The DEL phenotype represents an intriguing and challenging aspect of blood group serology. This condition is characterized by an extremely weak expression of the D antigen on red blood cells, to the extent that it often eludes detection through routine serological methods. Identifying the DEL phenotype necessitates more specialized techniques, such as adsorption and elution tests, to reveal the presence of the D antigen. This distinctive phenotype underscores the complexity and subtlety of blood group genetics and highlights the importance of using advanced methods to accurately classify individuals with this condition, as their ability to form anti-D antibodies can have clinical implications during transfusion and pregnancy scenarios. There is a paucity of data for the DEL phenotype in the Indian population, and the molecular basis has not been elucidated yet. Our investigation delves into the genetic underpinnings of two distinct DEL phenotype cases that pose challenges for resolution through conventional serological techniques. We employ next-generation amplicon sequencing to unravel the intricate genetic landscape underlying these cases. In the D-negative donor, the DEL phenotype was first identified serologically, which was subsequently confirmed by molecular analysis. In the second case, it was associated with an anti-D antibody in a D-positive patient. Initial data analysis unveiled a substantial reduction in coverage across the exonic segments of the RHD gene in both samples, suggesting the potential presence of RHD exon deletions. On both occasions, we identified a homozygous intronic RHD polymorphism that is well established to be linked to the RHD* 01EL.32/RHD*DEL32 variant.

Identification of Trichinella taxa by ITS-1 amplicon next-generation sequencing with an improved resolution for detecting underrepresented genotypes in mixed natural infections

BackgroundAmplicon-based next-generation sequencing (NGS) has rapidly gained popularity as a powerful method for delineating taxa in complex communities, including helminths. Here, we applied this approach to identify species and genotypes of zoonotic nematodes of the Trichinella genus. A known limitation of the current multiplex PCR (mPCR) assay recommended by the International Commission on Trichinellosis is that it does not differentiate Trichinella nativa from T. chanchalensis.MethodsThe new assay entails deep sequencing of an amplified variable fragment of the ribosomal cistron's (rDNA) internal transcribed spacer 1 using the Illumina platform. The assay was evaluated using first-stage larvae (L1) of select laboratory strains of various Trichinella taxa mixed in known proportions and then validated using archived L1 from 109 wildlife hosts. The species/genotypes of these L1 isolates from wildlife were previously determined using mPCR.ResultsNGS data analysis for Trichinella laboratory strains selected as representative of North American fauna revealed a sequence representation bias. Trichinella pseudospiralis, a non-encapsulated species, was the most underrepresented when mixed with T. spiralis, T. murrelli, T. nativa and Trichinella T6 in equal quantities. However, five L1 of T. pseudospiralis were readily revealed by NGS in a mix with 2000 L1 of T. nativa (1:400 ratio). From naturally infected wildlife, all Trichinella taxa revealed by mPCR were also identified by NGS in 103 of 107 (96.3%) samples amplified on both assays. NGS identified additional taxa in 11 (10.3%) samples, whereas additional taxa were revealed by mPCR in only four (3.7%) samples. Most isolates comprised single or mixed infections of T. nativa and Trichinella T6. On NGS, T. chanchalensis (T13) was detected in combination with Trichinella T6 in a wolverine (Gulo gulo) and in combination with T. nativa and Trichinella T6 in a marten (Martes americana) from the Northwest Territories, Canada.ConclusionsThis new NGS assay demonstrates strong potential as a single assay for identifying all recognised Trichinella taxa as well as improved sensitivity for detecting under-represented and novel genotypes in mixed infections. In addition, we report a new host record for T. chanchalensis in American marten.Graphical

Faecal microbiota and fatty acids in feline chronic enteropathy

BackgroundFeline chronic enteropathy is a set of disorders defined as the presence of clinical signs of gastrointestinal disease for at least three weeks. The most common final diagnoses are inflammatory bowel disease and alimentary small cell lymphoma. The etiopathogenesis of these diseases is incompletely understood; however, it is hypothesised that they involve a combination of factors, including altered composition and/or functionality of the intestinal microbiome. An important factor in the interplay of the microbiome and host is the production of short- and branched-chain fatty acids. The aim of this study was to evaluate the possible differences in faecal microbiota diversity, composition and fatty acid production between cats suffering from chronic enteropathy and healthy cats. Sixteen cats suffering from chronic enteropathy and fourteen healthy control cats were enrolled in the study. The microbiota compositions of faecal samples were analysed by using next-generation amplicon sequencing of the V3V4 fragment of the 16S rRNA gene. Fatty acids were evaluated by high-performance liquid chromatography.ResultsBoth the alpha and beta diversities were significantly lower in samples obtained from cats with chronic enteropathy. The relative abundance of the phylum Proteobacteria, orders Lactobacillales and Enterobacterales, family Enteriobacteriaceae and genus Escherichia Shigella were higher in diseased cats, whereas the abundance of the phylum Bacteroidota and order Peptococcales were higher in control cats. The faecal concentrations of short-chain fatty acids were higher in cats with chronic enteropathy, with lower propionate proportions and higher butyrate proportions.ConclusionThe study revealed alterations in microbiota compositions and short-chain fatty acid concentration in cats suffering from chronic enteropathy, which is an important finding both for research on the pathogenesis of the disease and for potential therapeutic interventions in the form of faecal microbiota transplantation and/or probiotic supplementation.

Performance of non-formalin fixed paraffin embedded samples in hybrid capture and amplicon next-generation sequencing panels.

Genomic profiling using next-generation sequencing (NGS) is fundamental for driving prognostic and therapy in cancer. Formalin-fixed paraffin embedded (FFPE) tissue is the widely used material, whereas non-FFPE may represent an alternative. However, studies comparing the NGS performance of non-FFPE materials to FFPE are still lacking in the literature. The objective of this study was to characterize in non-FFPE preparations the nucleic acid yield and NGS performance on both a capture-based and an amplicon-based NGS platform. NGS quality metrics obtained from non-FFPE preparations were compared to FFPE. We analyzed the cellularity and nucleic acid yield in 111 tumors from non-FFPE preparations. In addition, comprehensive hybrid capture panel sequencing metrics obtained from DNA and RNA libraries were compared between independent non-FFPE and FFPE samples. A paired comparison between non-FFPE and FFPE samples was performed to analyze concordance in mutant allele detection using an amplicon panel. The mean target coverage from DNA libraries was 2× higher in non-FFPE samples than in FFPE. The detection of exogenous DNA was 2.5× higher in non-FFPE than in FFPE. Conversely, a lower performance was observed in non-FFPE RNA libraries in comparison to FFPE DNA libraries with no impact in minimum standard cutoffs. The variant allele detection in non-FFPE was found to be comparable to that of FFPE tumor samples in matched samples. Non-FFPE was demonstrated to be a suitable material for DNA and RNA library preparations using a comprehensive NGS panel. This is the first study reporting library quality metrics according to the TSO500 analysis pipeline.

Comparative analysis of rhizobial and bacterial communities in experimental cotton fields: Impacts of conventional and conservation soil management in the Texas High Plains

Conservative agricultural management strategies pursue long-term ecological benefits through practices such as no-tillage, cover crop, and inherent soil properties management. Farmers, however, are often hesitant to adopt such practices due to lack of experience, initial expense, and concern for low crop productivity. Overcoming this barrier requires novel approaches, such as effectively managing the soil microbiome to attain high productivity at a low cost, especially in a semi-arid region. To study the potential of conservation agriculture, we investigated components of soil bacterial community and rhizobial diversity in long-term experimental cotton fields divided into conventional tillage monoculture systems with winter fallow (CT) and no-tillage with mixed cover crop (M-NT) system on the Texas High Plain (THP). We conducted next-generation amplicon sequencing targeting rpoB gene with collected soil samples from different soil managements and seasons. Our research revealed that although CT had significantly greater bacterial diversity and species richness than the M-NT management, rhizobial diversity and species richness were higher in M-NT than in CT management. Both bacterial and rhizobial diversity and richness were greater in summer than in fall. The abundance of the order Rhizobiales was consistently high in M-NT than in CT fields in both seasons. Soil management altered the dominant rhizobial genus associated with cotton production systems; Rhizobium and Pararhizobium dominated M-NT management, while Bradyrhizobium and Sinorhizobium were dominant under CT management. These outcomes suggest that incorporating legumes into a cover crop in this semi-arid cotton-growing region can initiate beneficial changes to the dynamics of the indigenous rhizobial assemblage. The high prediction accuracy of our machine learning model using bacterial community data classifying the managements as CT or M-NT validates the possibilities of a strong underlying relationship between soil management and the bacterial diversity in the soil.

Microbiome analysis of saliva from oral squamous cell carcinoma (OSCC) patients and tobacco abusers with potential biomarkers for oral cancer screening

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer and accounts for about 95% of all head and neck cancers with high mortality, usually at a late stage. Dysbiosis in the oral microbiome can lead to chronic inflammatory responses and may predispose to the development and progression of OSCC. Tobacco abuse plays an essential role in oral microbiome dysregulation and OSCC pathogenesis. We used 16S rRNA gene amplicon next-generation sequencing to examine microbial signatures unique to saliva from OSCC patients, tobacco abusers (TA) and controls (n = 10 for each group) to elucidate oral microbiome changes associated with tobacco abuse and OSCC. Overall, the oral microbiome compositions of class Betaproteobacteria and Epsilonproteobacteria, order Neisseriales, Burkholderiales and Campylobacterales, family Burkholderiaceae and Campylobacteraceae and genera Campylobacter and Leptotrichia revealed significant differences among OSCC patients, TA and control. Our preliminary pilot study not only serves as a basis for future studies with large sample size but also gives an indication of microbiome-based potential non-invasive biomarkers for early screening and monitoring of oral carcinogenesis transition due to tobacco abuse.

Comparing the microbial communities in and End-pit lake, active tailings ponds and freshwater bodies from the Athabasca oil sands region

The Athabasca oil sands region in northern Alberta is home to one of the largest bitumen deposits in the world. Oil sands are mostly recovered via surface mining and the oil extraction is achieved with hot caustic water and diluents, a process that produces liquid tailings waste (CEC 2020, Schramm et al. 2000). It is estimated that 1 m3 of bitumen produces around 4 m3 of tailings, which are contained in large tailings ponds (Mikula et al. 1996). End-pit lakes are a potential strategy to reclaim mining pits created by oil sands mining. They may be formed by filling a mined-out pit with tailings and then capping it with a layer of fresh water. With time, tailings undergo a dewatering process in which they become denser and release water to the cap water (Charette et al. 2012). Base Mine Lake (BML) is the first full-scale demonstration end-pit lake in the Canadian oil sands industry. This former tailings pond was initially capped in 2012 with a 5 m layer of freshwater to allow for consolidation of the tailings and the stimulation of aerobic microbial communities to biodegrade the organic pollutants. Since its establishment, BML has been extensively monitored to assess the improvement in water quality. The present research focused on determining how the eukaryotic and bacterial communities in BML compare to those in local freshwater bodies and active tailings ponds. Eleven reference sites, including freshwater reservoirs, natural lakes, and 9- to 14-year-old excavated pits filled with water, also known as borrow pits, were sampled along with 5 active tailings ponds and BML during the summer of 2022. Microbial communities were assessed via next-generation sequencing of PCR amplicons of the 16S rRNA gene for bacteria and the 18S rRNA gene for eukaryotes. Alpha-diversity analysis of the eukaryotic communities showed that BML has greater species richness and evenness than active tailings ponds, but lower than freshwater systems. The bacterial community in both BML and active tailings ponds is dominated by Proteobacteria, but the relative abundance of Actinobacteriota is similar between BML and freshwater. Beta-diversity analysis revealed that eukaryotic and bacterial communities in BML cluster distinctly from both the freshwater controls and active tailings ponds, however, the composition of the eukaryotic community shows some overlap with certain freshwater systems (Fig. 1). The results of this research suggest that, 10 years after its formation, the microbial communities in BML are intermediate between an active tailings pond and a freshwater lake.

PacBio next-generation sequencing uncovers Apicomplexa diversity in different habitats

The phylum Apicomplexa comprises a large group of intracellular protozoan parasites. These microorganisms are known to infect a variety of vertebrate and invertebrate hosts, leading to significant medical and veterinary conditions such as toxoplasmosis, cryptosporidiosis, theileriosis, and eimeriosis. Despite their importance, comprehensive data on their diversity and distribution, especially in riverine environments, remain scant. To bridge this knowledge gap, we utilized next-generation high-throughput 18S rRNA amplicon sequencing powered by PacBio technology to explore the diversity and composition of the Apicomplexa taxa. Principal component analysis (PCA) and principal coordinate analysis (PCoA) indicated the habitat heterogeneity for the physicochemical parameters and the Apicomplexa community. These results were supported by PERMANOVA (P < 0.001), ANOSIM (P < 0.001), Cluster analysis, and Venn diagram. Dominant genera of Apicomplexa in inlet samples included Gregarina (38.54%), Cryptosporidium (32.29%), and Leidyana (11.90%). In contrast, outlet samples were dominated by Babesia, Cryptosporidium, and Theileria. While surface water samples revealed 16% and 8.33% relative abundance of Toxoplasma and Cryptosporidium, respectively. To our knowledge, the next-generation high throughput sequencing covered a wide range of parasites in Egypt for the first time, which could be useful for legislation of the standards for drinking water and wastewater reuse.

TRiCit: A High-Throughput Approach to Detect Trichomonas vaginalis from ITS1 Amplicon Sequencing.

Trichomoniasis, caused by Trichomonas vaginalis (TV), is the most common non-viral sexually transmitted infection (STI) worldwide, affecting over 174 million people annually and is frequently associated with reproductive co-morbidities. However, its detection can be time-consuming, subjective, and expensive for large cohort studies. This case-control study, conducted at the Mount Sinai Adolescent Health Center in New York City, involved 36 women with prevalent TV infections and 36 controls. The objective was to examine Internal Transcribed Spacer region-1 (ITS1) amplicon-derived communities for the detection of prevalent TV infections with the same precision as clinical microscopy and the independent amplification of the TV-specific TVK3/7 gene. DNA was isolated from clinician-collected cervicovaginal samples and amplified using ITS1 primers in a research laboratory. Results were compared to microscopic wet-mount TV detection of concurrently collected cervicovaginal samples and confirmed against TV-specific TVK3/7 gene PCR. The area under the receiver operating characteristics curve (AUC) for diagnosing TV using ITS1 communities was 0.92. ITS1 amplicons displayed an intra-class correlation coefficient (ICC) of 0.96 (95% CI: 0.93-0.98) compared to TVK3/7 PCR fragment testing. TV cases showed an increased risk of bacterial vaginosis (BV) compared to the TV-negative controls (OR = 8.67, 95% CI: 2.24-48.54, p-value = 0.0011), with no significant differences regarding genital yeast or chlamydia infections. This study presents a bioinformatics approach to ITS1 amplicon next-generation sequencing that is capable of detecting prevalent TV infections. This approach enables high-throughput testing for TV in stored DNA from large-scale epidemiological studies.

Effect of long-term influx of tertiary treated wastewater on native bacterial communities in a dry valley topsoil: 16S rRNA gene-based metagenomic analysis of composition and functional profile.

Although dumping treated wastewater into soil might provide nutrients and organic matter, it can also expose the ecosystem to biological and chemical risks. A vital indication of soil health and quality is the soil microbial community. The current work used next-generation 16S rRNA gene amplicon sequencing to evaluate the effects of the long-term influx of tertiary treated wastewater (TWW) into Wadi Uranah, a dry valley in Makkah city, Saudi Arabia, on native topsoil bacterial community composition and predicted functions. The findings demonstrated that neither the compositions of microbial communities nor their predicted functions using PICRUSt2 differed significantly (p>0.05) between polluted valley soil (PolVS) and unpolluted valley soil (UPVS). Alpha and beta diversity, however, showed that the PolVS samples had a considerably higher level of diversity and variability. Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes were the most prevalent phyla in both groups. Noticeable relative variations existed in some metabolic pathways such as cofactor, prosthetic group, electron carrier degradation, aldehyde degradation, and Entner-Doudoroff (ED) pathways. Overall, our findings suggest that because both groups have very similar core microbiomes and functions, the long-term disposal of tertiary TWW into Wadi Uranah may have little to no influence on the composition and function of soil bacterial communities. In addition, the long-term discharge of tertiary TWW after partially treated wastewater's initial disposal may have helped the native soil microbial community recover.

Somatic EPAS1 Variants in Pheochromocytoma and Paraganglioma in Patients With Sickle Cell Disease.

Somatic EPAS1 variants account for 5% to 8% of all pheochromocytoma and paragangliomas (PPGL) but are detected in over 90% of PPGL in patients with congenital cyanotic heart disease, where hypoxemia may select for EPAS1 gain-of-function variants. Sickle cell disease (SCD) is an inherited hemoglobinopathy associated with chronic hypoxia and there are isolated reports of PPGL in patients with SCD, but a genetic link between the conditions has yet to be established. To determine the phenotype and EPAS1 variant status of patients with PPGL and SCD. Records of 128 patients with PPGL under follow-up at our center from January 2017 to December 2022 were screened for SCD diagnosis. For identified patients, clinical data and biological specimens were obtained, including tumor, adjacent non-tumor tissue and peripheral blood. Sanger sequencing of exons 9 and 12 of EPAS1, followed by amplicon next-generation sequencing of identified variants was performed on all samples. Four patients with both PPGL and SCD were identified. Median age at PPGL diagnosis was 28 years. Three tumors were abdominal paragangliomas and 1 was a pheochromocytoma. No germline pathogenic variants in PPGL-susceptibility genes were identified in the cohort. Genetic testing of tumor tissue detected unique EPAS1 variants in all 4 patients. Variants were not detected in the germline, and 1 variant was detected in lymph node tissue of a patient with metastatic disease. We propose that somatic EPAS1 variants may be acquired through exposure to chronic hypoxia in SCD and drive PPGL development. Future work is needed to further characterize this association.

A longitudinal study of Blastocystis in dairy calves from birth through 24 months demonstrates dynamic shifts in infection rates and subtype prevalence and diversity by age

Blastocystis is a common microeukaryotic intestinal parasite in humans and other animal hosts globally. However, no large-scale longitudinal study has ever been conducted for Blastocystis. To understand patterns of infection prevalence and subtype diversity and their relationship with host age, we have conducted the most comprehensive longitudinal study of Blastocystis infection ever performed. Dairy calves from a herd located in Maryland, USA, were followed from birth through 24 months of age, and 990 individual fecal samples from 30 calves were collected over the study period, representing three age groups (pre-weaned, post-weaned, and heifer). All samples were screened for Blastocystis via PCR, and subtype determination was performed using next-generation amplicon sequencing. Associations between age group and infection status were assessed using logistic regression analyses. Blastocystis infection prevalence increased with time, significant associations were observed between age groups and infection risk, and a cumulative prevalence of 100% was observed among the study population during the 24-month period. Thirteen previously reported subtypes (ST1-6, ST10, ST14, ST21, ST23–26) and one potentially novel subtype were observed. Diversity within ST10 supports the need for division of the subtype into new subtype designations. Associations between subtype and age group were explored, and relationships between subtypes and infection chronicity are described. While subtype diversity increased with age in the study population, distinct patterns of individual subtype prevalence and chronicity were observed, supporting the importance of subtype discrimination in studies of host infection and disease. The data from this study represent a significant advance in our understanding of Blastocystis infection dynamics within a single host population over time and can be used to inform future studies of Blastocystis epidemiology in both humans and other animal hosts.Graphical

Exploring Blastocystis genetic diversity in rural schoolchildren from Colombia using next-generation amplicon sequencing reveals significant associations between contact with animals and infection risk

Blastocystis is a common intestinal protist with a global distribution in humans and many other animals. Yet, the status of Blastocystis as a pathogen, the risk factors associated with its transmission, and its zoonotic potential remain ill-defined. Here, we explored subtype (ST) diversity and potential risk factors for Blastocystis infection in 98 children from Apulo, Colombia. Samples were screened for Blastocystis via PCR, and ST identification was performed through next-generation amplicon sequencing (NGS). Associations between the presence of Blastocystis and individual STs and sociodemographic variables were assessed via logistic regression analyses. Seventy-one samples (72.4%) were Blastocystis-positive, and NGS revealed the presence of five STs (ST1-ST5). ST1, ST2, and ST3 were common and observed in nearly equal proportions (~ 40%), while samples with ST4 (1.4%) and ST5 (5.6%) were comparatively rare. The presence of mixed STs in the same sample was also common (28.2%). Comparisons among children within the same household identified that shared ST profiles were common, but diversity within family units was also observed. Logistic regression analyses returned significant associations between the presence of Blastocystis, individual subtypes, or mixed subtypes for several variables. Intriguingly, the presence of animals was one of the most common significant associations. Taken together, these data represent an important step forward in understanding both the potential routes and risk factors that may influence Blastocystis transmission and will be useful in shaping future studies which seek to clarify the relationships between STs, pathogenicity, and zoonotic transmission.

Effects of sponge-to-sponge contact on the microbiomes of three spatially competing Caribbean coral reef species.

Sponges perform important ecosystem functions, host diverse microbial symbiont communities (microbiomes), and have been increasing in density on Caribbean coral reefs over the last decade. Sponges compete for space in coral reef communities through both morphological and allelopathic strategies, but no studies of microbiome impacts during these interactions have been conducted. Microbiome alterations mediate spatial competition in other coral reef invertebrates and may similarly impact competitive outcomes for sponges. In this study, we characterized the microbiomes of three common Caribbean sponges (Agelas tubulata, Iotrochota birotulata, and Xestospongia muta) observed to naturally interact spatially in Key Largo, Florida (USA). For each species, replicate samples were collected from sponges in contact with neighbors at the site of contact (contact) and distant from the site of contact (no contact), and from sponges spatially isolated from neighbors (control). Next-generation amplicon sequencing (V4 region of 16S rRNA) revealed significant differences in microbial community structure and diversity among sponge species, but no significant effects were observed within sponge species across all contact states and competitor pairings, indicating no large community shifts in response to direct contact. At a finer scale, particular symbiont taxa (operational taxonomic units at 97% sequence identity, OTUs) were shown to decrease significantly in some interaction pairings, suggesting localized effects for specific sponge competitors. Overall, these results revealed that direct contact during spatial competition does not significantly alter microbial community composition or structure of interacting sponges, suggesting that allelopathic interactions and competitive outcomes are not mediated by microbiome damage or destabilization.

Microbiome Profile of the Mediterranean Mussel (Mytilus galloprovincialis) from Northern Aegean Sea (Greece) Culture Areas, Based on a 16S rRNA Next Generation Sequencing Approach

Mediterranean mussels (Mytilus galloprovincialis), due to their nutritional mechanisms which involve filtering huge amounts of water, are affected by seawater pollution and can host microbial diversity of environmental origin, as well as pathogenic bacteria that must be constantly monitored. Herein, we applied a Next Generation Sequencing (NGS) metabarcoding approach in order to study the M. galloprovincialis microbiota. Collection of samples was conducted during winter and summer months from various mussel farm zones located in specific farm regions in the Thermaikos gulf, the northern Aegean Sea, Greece. A microbiological test was performed for the enumeration of Escherichia coli and the presence of Salmonella sp. DNA extraction and amplification of the whole bacterial 16S rRNA gene, followed by NGS amplicon sequencing and taxonomic classification, were carried out. Statistically significant differences (p &lt; 0.05) in the abundance of the most dominant bacterial phyla, families and genera between winter and summer time periods, regions, as well as zones within each region of sampling, were evaluated with z-score computation. According to the obtained results, the most prevalent taxa at the genus level were Mycoplasma (12.2%), Anaplasma (5.8%), Ruegeria (5.2%) and Mariniblastus (2.1%). Significant differences in the abundance of the most dominant genera were found at all levels of comparison (seasons, regions and zones within each region), highlighting the dynamic character of microorganisms, which might be affected by microenvironmental, temporal and spatial changes. The present research contributes to the characterization of M. galloprovincialis microbiome in areas that have not been studied previously, setting the baseline for future, more thorough investigations of the specific bivalve species and its bacterial profile in the above geographic regions.