Long-read Amplicon Sequencing Research Articles

Molecular and Clinical Features of Congenital Hypothyroidism due to Multiple DUOX2 Variants.

Background: DUOX2 is one of the major causative genes of congenital hypothyroidism (CH). Still, the mutation spectrum and clinical outcomes of biallelic DUOX2 variants are not fully understood. This study aimed to elucidate the molecular features and long-term clinical manifestations of CH caused by multiple pathogenic DUOX2 variants. Methods: A total of 255 patients with CH were screened for rare variants of 11 known causative genes. DUOX2 variants were classified according to their protein structure and residual activity. In vitro assays were performed for several variants of unknown function. Clinical analyses were conducted for patients with multiple pathogenic variants of DUOX2 but not of other genes. Results: We identified 24 pathogenic variants of DUOX2, together with two benign variants and seven variants of uncertain significance, in 63 patients. The pathogenic variants included three missense substitutions and one frameshift variant that have not been linked to CH. Twenty-one patients carried multiple pathogenic DUOX2 variants without any other pathogenic gene variants. Three of the 21 patients harbored homozygous variants. Family analysis, long-read amplicon sequencing, and haplotype phasing confirmed compound heterozygosity of the DUOX2 variants in 14 patients, whereas the allelic positions of the variants in the remaining four patients could not be determined. Of the 21 patients, 19 were treated with levothyroxine; their ages at drug withdrawal ranged from 9 months to 21.4 years. Three patients required retreatment after drug-free intervals of 6 months, 8 months, and 10 years. There were no differences in the clinical severity among patients with DUOX2 amorphic/amorphic, amorphic/hypomorphic, and hypomorphic/hypomorphic variants. Conclusions: These results broaden the mutation spectrum of DUOX2. Furthermore, our data imply that patients with multiple pathogenic DUOX2 variants typically exhibit transient CH without significant genotype-phenotype correlations. Most importantly, this study demonstrated for the first time that these patients are at risk of developing recurrent hypothyroidism after a long drug-free interval.

EnsembleSeq: a workflow towards real-time, rapid, and simultaneous multi-kingdom-amplicon sequencing for holistic and resource-effective microbiome research at scale.

Human microbiome is a sum total of a variety of microbial genomes (including bacteria, fungi, protists, viruses, etc.) present in and on the human body. Yet, a majority of amplicon-based microbiome studies have largely remained skewed toward bacteriome as an assumed proxy of the total microbiome, primarily at a shallow genus level. Cost, time, effort, data quality/management, and importantly lack of guiding studies often limit progress in the direction of moving beyond bacteriome. Here, EnsembleSeq presents a proof-of-concept toward concomitantly capturing multiple-kingdoms of microorganisms (bacteriome and mycobiome) in a fully multiplexed (96-sample) single run of long-read amplicon sequencing. In addition, the workflow captures dynamic tracking of species-level saturation in a time- and resource-effective manner.

DuBA.flow─A Low-Cost, Long-Read Amplicon Sequencing Workflow for the Validation of Synthetic DNA Constructs.

Modern biological science, especially synthetic biology, relies heavily on the construction of DNA elements, often in the form of plasmids. Plasmids are used for a variety of applications, including the expression of proteins for subsequent purification, the expression of heterologous pathways for the production of valuable compounds, and the study of biological functions and mechanisms. For all applications, a critical step after the construction of a plasmid is its sequence validation. The traditional method for sequence determination is Sanger sequencing, which is limited to approximately 1000 bp per reaction. Here, we present a highly scalable in-house method for rapid validation of amplified DNA sequences using long-read Nanopore sequencing. We developed two-step amplicon and transposase strategies to provide maximum flexibility for dual barcode sequencing. We also provide an automated analysis pipeline to quickly and reliably analyze sequencing results and provide easy-to-interpret results for each sample. The user-friendly DuBA.flow start-to-finish pipeline is widely applicable. Furthermore, we show that construct validation using DuBA.flow can be performed by barcoded colony PCR amplicon sequencing, thus accelerating research.

Fungal Communities Associated with Corn in a Diverse Long-Term Crop Rotation in Ohio

Crop rotation is a cultural practice in disease management used to break the disease cycle, resulting in a reduction of inoculum. In Ohio, crop rotations have been reduced in diversity, with many farmers shifting to corn-soy rotations from more diverse rotations featuring wheat. We investigated the impact of this shift on soil fungal communities under corn by conducting synthetic long-read amplicon sequencing. DNA was extracted from soil sampled during the corn-growing season at two locations in Ohio with replicated long-term rotation plots in a corn–soybean (CS) and corn–soybean–winter wheat (CSW) rotation in 2018, 2019, and 2020. 18s-ITS amplicons were sequenced using Illumina paired with Loopgenomics technology. PERMANOVA analysis revealed that fungal communities were significantly impacted by location, rotation, and time. Shannon and Simpson diversity indices implied fungal species diversity was significantly higher in the CS rotation. Indicator species analysis revealed 20 species indicative of the CSW rotation, including Mortierella, Hymenoschyphus, Ascobolus, and Saitozyma species, and 36 species indicative of the CS rotation, including Fusarium, Neoaschocyta, Zalerion, and Trichoderma species, among others. Indicator species amplicon sequencing variant reads for the CSW rotation were not correlated with the decline in corn yield or soil nitrogen, carbon, or active carbon. However, they were positively correlated with soil organic matter. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Temporal dynamics of zooplankton community in an oyster farming area of the Yellow Sea in China via metabarcoding

IntroductionThe coastal ecosystem is a hub of both marine organisms and human activities. It plays a crucial role in human food production and affects facilities through biofouling. Long-read amplicon sequencing provides more accurate species identification and demonstrates numerous advantages in community diversity studies, making it an effective tool for ecological monitoring.MethodsTo investigate the zooplankton community characteristics in the oyster aquaculture area, the composition and temporal dynamics of zooplankton near Longwan Bay, Qingdao, China, were determined by the 18S rRNA gene long reads sequencing technique.ResultsA total of 89 zooplankton species were identified, among which copepods were the most abundant. Zooplankton composition and proportional abundances showed significant seasonal variations. The species richness in summer was the highest, while the species abundance in spring was the highest. Oyster farming showed weak influence on the zooplankton community variation. Paracalanus parvus abundance was higher in most assayed months. Chthamalus stellatus showed a strong temporal preference, with the highest percentage in May. Sea water temperature and species-species interactions were revealed to be the main contributors to the shifts in the community composition. DiscussionThe ubiquitous positive correlation between zooplankton suggests that species interactions are important in adaptation to the changing environment. The results reveal the seasonal occurrence of several major biofouling organisms and help improve biofouling management efficiency.

A genomic platform for surveillance and antigen discovery in Plasmodium spp. using long-read amplicon sequencing

Many vaccine candidate proteins in the malaria parasite Plasmodium falciparum are under strong immunological pressure and confer antigenic diversity. We present a sequencing and data analysis platform for the genomic surveillance of the insertion or deletion (indel)-rich antigens merozoite surface protein 1 (MSP1), MSP2, glutamate-rich protein (GLURP), and CSP from P.falciparum using long-read circular consensus sequencing (CCS) in multiclonal malaria isolates. Our platform uses 40 PCR primers per gene to asymmetrically barcode and identify multiclonal infections in pools of up to 384 samples. With msp2, we validated the method using 235 mock infections combining 10 synthetic variants at different concentrations and infection complexities. We applied this strategy to P.falciparum isolates from a longitudinal cohort in Tanzania. Finally, we constructed an analysis pipeline that streamlines the processing and interpretation of epidemiological and antigenic diversity data from demultiplexed FASTQ files. This platform can be easily adapted to other polymorphic antigens of interest in Plasmodium or any other human pathogen.

Host adaptation drives genetic diversity in a vector-borne disease system.

The range of hosts a pathogen can infect is a key trait, influencing human disease risk and reservoir host infection dynamics. Borrelia burgdorferi sensu stricto (Bb), an emerging zoonotic pathogen, causes Lyme disease and is widely considered a host generalist, commonly infecting mammals and birds. Yet the extent of intraspecific variation in Bb host breadth, its role in determining host competence, and potential implications for human infection remain unclear. We conducted a long-term study of Bb diversity, defined by the polymorphic ospC locus, across white-footed mice, passerine birds, and tick vectors, leveraging long-read amplicon sequencing. Our results reveal strong variation in host breadth across Bb genotypes, exposing a spectrum of genotype-specific host-adapted phenotypes. We found support for multiple niche polymorphism, maintaining Bb diversity in nature and little evidence of temporal shifts in genotype dominance, as would be expected under negative frequency-dependent selection. Passerine birds support the circulation of several human-invasive strains (HISs) in the local tick population and harbor greater Bb genotypic diversity compared with white-footed mice. Mouse-adapted Bb genotypes exhibited longer persistence in individual mice compared with nonadapted genotypes. Genotype communities infecting individual mice preferentially became dominated by mouse-adapted genotypes over time. We posit that intraspecific variation in Bb host breadth and adaptation helps maintain overall species fitness in response to transmission by a generalist vector.

Wood fibers are a crucial microhabitat for cellulose- and xylan- degrading bacteria in the hindgut of the wood-feeding beetle Odontotaenius disjunctus.

Wood digestion in insects relies on the maintenance of a mosaic of numerous microhabitats, each colonized by distinct microbiomes. Understanding the division of digestive labor between these microhabitats- is central to understanding the physiology and evolution of symbiotic wood digestion. A microhabitat that has emerged to be of direct relevance to the process of lignocellulose digestion is the surface of ingested plant material. Wood particles in the guts of some termites are colonized by a specialized bacterial fiber-digesting microbiome, but whether this represents a widespread strategy among insect lineages that have independently evolved wood-feeding remains an open question. In this study, we investigated the bacterial communities specifically associated with wood fibers in the gut of the passalid beetle Odontotaenius disjunctus. We developed a Percoll-based centrifugation method to isolate and enrich the wood particles from the anterior hindgut, allowing us to access the wood fibers and their associated microbiome. We then performed assays of enzyme activity and used short-read and long-read amplicon sequencing of the 16S rRNA gene to identify the composition of the fiber-associated microbiome. Our assays demonstrated that the anterior hindgut, which houses a majority of the bacterial load, is an important site for lignocellulose digestion. Wood particles enriched from the anterior hindgut contribute to a large proportion of the total enzyme activity. The sequencing revealed that O. disjunctus, like termites, harbors a distinct fiber-associated microbiome, but notably, its community is enriched in insect-specific groups of Lactococcus and Turicibacter. Our study underscores the importance of microhabitats in fostering the complex symbiotic relationships between wood-feeding insects and their microbiomes. The discovery of distinct fiber-digesting symbionts in O. disjunctus, compared to termites, highlights the diverse evolutionary paths insects have taken to adapt to a challenging diet.

SsDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid RPE1 and HCT116 cells

BackgroundRecent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, partly due to the complexity of long ssDNA preparation, it remains unclear whether ssDNA is the optimal type of HDR donors for insertion of long transgenes such as fluorescent reporters in human cells.ResultsIn this study, we established a nuclease-based simple method for the preparation of long ssDNA with high yield and purity, and comprehensively compared the performance of ssDNA and dsDNA donors with 90 bases of homology arms for endogenous gene tagging with long transgenes in human diploid RPE1 and HCT116 cells. Quantification using flow cytometry revealed lower efficiency of endogenous fluorescent tagging with ssDNA donors than with dsDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was lower with ssDNA donors than with dsDNA. Moreover, in off-target integration analyses using donors without homology arms, ssDNA and dsDNA were comparably prone to non-homologous integration.ConclusionsThese results indicate that ssDNA is not superior to dsDNA as long HDR donors with relatively short homology arms for gene knock-in in human RPE1 and HCT116 cells.

Deep haplotype analyses of target-site resistance locus ACCase in blackgrass enabled by pool-based amplicon sequencing.

Rapid adaptation of weeds to herbicide applications in agriculture through resistance development is a widespread phenomenon. In particular, the grass Alopecurus myosuroides is an extremely problematic weed in cereal crops with the potential to manifest resistance in only a few generations. Target-site resistances (TSRs), with their strong phenotypic response, play an important role in this rapid adaptive response. Recently, using PacBio's long-read amplicon sequencing technology in hundreds of individuals, we were able to decipher the genomic context in which TSR mutations occur. However, sequencing individual amplicons are costly and time-consuming, thus impractical to implement for other resistance loci or applications. Alternatively, pool-based approaches overcome these limitations and provide reliable allele frequencies, although at the expense of not preserving haplotype information. In this proof-of-concept study, we sequenced with PacBio High Fidelity (HiFi) reads long-range amplicons (13.2kb), encompassing the entire ACCase gene in pools of over 100 individuals, and resolved them into haplotypes using the clustering algorithm PacBio amplicon analysis (pbaa), a new application for pools in plants and other organisms. From these amplicon pools, we were able to recover most haplotypes from previously sequenced individuals of the same population. In addition, we analysed new pools from a Germany-wide collection of A. myosuroides populations and found that TSR mutations originating from soft sweeps of independent origin were common. Forward-in-time simulations indicate that TSR haplotypes will persist for decades even at relatively low frequencies and without selection, highlighting the importance of accurate measurement of TSR haplotype prevalence for weed management.

Metagenomics and metabarcoding experimental choices and their impact on microbial community characterization in freshwater recirculating aquaculture systems

BackgroundMicrobial communities in recirculating aquaculture systems (RAS) play a role in system success, nutrient cycling, and water quality. Considering the increasing socio-economic role of fish farming, e.g., regarding food security, an in-depth understanding of aquaculture microbial communities is also relevant from a management perspective, especially regarding the growth, development, and welfare of the farmed animal. However, the current data on the composition of microbial communities within RAS is patchy, which is partly attributable to diverging method choices that render comparative analyses challenging. Therefore, there is a need for accurate, standardized, and user-friendly methods to study microbial communities in aquaculture systems.ResultsWe compared sequencing approach performances (3 types of 16S short amplicon sequencing, PacBio long-read amplicon sequencing, and amplification-free shotgun metagenomics) in the characterization of microbial communities in two commercial RAS fish farms. Results showed that 16S primer choice and amplicon length affect some values (e.g., diversity measures, number of assigned taxa or distinguishing ASVs) but have no impact on spatio-temporal patterns between sample types, farms and time points. This implies that 16S rRNA approaches are adequate for community studies. The long-read amplicons underperformed regarding the quantitative resolution of spatio-temporal patterns but were suited to identify functional services, e.g., nitrification cycling and the detection of pathogens. Finally, shotgun metagenomics extended the picture to fungi, viruses, and bacteriophages, opening avenues for exploring inter-domain interactions. All sequencing datasets agreed on major prokaryotic players, such as Actinobacteriota, Bacteroidota, Nitrospirota, and Proteobacteria.ConclusionThe different sequencing approaches yielded overlapping and highly complementary results, with each contributing unique data not obtainable with the other approaches. We conclude that a tiered approach constitutes a strategy for obtaining the maximum amount of information on aquaculture microbial communities and can inform basic research on community evolution dynamics. For specific and/or applied questions, single-method approaches are more practical and cost-effective and could lead to better farm management practices.

Preclinical workup using long-read amplicon sequencing provides families with de novo pathogenic variants access to universal preimplantation genetic testing.

Can long-read amplicon sequencing be beneficial for preclinical preimplantation genetic testing (PGT) workup in couples with a de novo pathogenic variant in one of the prospective parents? Long-read amplicon sequencing represents a simple, rapid and cost-effective preclinical PGT workup strategy that provides couples with de novo pathogenic variants access to universal genome-wide haplotyping-based PGT programs. Universal PGT combines genome-wide haplotyping and copy number profiling to select embryos devoid of both familial pathogenic variants and aneuploidies. However, it cannot be directly applied in couples with a de novo pathogenic variant in one of the partners due to the absence of affected family members required for phasing the disease-associated haplotype. This is a prospective study, which includes 32 families that were enrolled in the universal PGT program at the University Hospital of Leuven between 2018 and 2022. We implemented long-read amplicon sequencing during the preclinical PGT workup to deduce the parental origin of the disease-associated allele in the affected partner, which can then be traced in embryos during clinical universal PGT cycles. To identify the parental origin of the disease-associated allele, genomic DNA from the carrier of the de novo pathogenic variant and his/her parent(s) was used for preclinical PGT workup. Primers flanking the de novo variant upstream and downstream were designed for each family. Following long-range PCR, amplicons that ranged 5-10 kb in size, were sequenced using Pacific Bioscience and/or Oxford Nanopore platforms. Next, targeted variant calling and haplotyping were performed to identify parental informative single-nucleotide variants (iSNVs) linked to the de novo mutation. Following the preclinical PGT workup, universal PGT via genome-wide haplotyping was performed for couples who proceeded with clinical PGT cycle. In parallel, 13 trophectoderm (TE) biopsies from three families that were analyzed by universal PGT, were also used for long-read amplicon sequencing to explore this approach for embryo direct mutation detection coupled with targeted long-read haplotyping. The parental origin of the mutant allele was identified in 24/32 affected individuals during the preclinical PGT workup stage, resulting in a 75% success rate. On average, 5.95 iSNVs (SD = 4.5) were detected per locus of interest, and the average distance of closest iSNV to the de novo variant was ∼1750 bp. In 75% of those cases (18/24), the de novo mutation occurred on the paternal allele. In the remaining eight families, the risk haplotype could not be established due to the absence of iSNVs linked to the mutation or inability to successfully target the region of interest. During the time of the study, 12/24 successfully analyzed couples entered the universal PGT program, and three disease-free children have been born. In parallel to universal PGT analysis, long-read amplicon sequencing of 13 TE biopsies was also performed, confirming the segregation of parental alleles in the embryo and the results of the universal PGT. The main limitation of this approach is that it remains targeted with the need to design locus-specific primers. Because of the restricted size of target amplicons, the region of interest may also remain non-informative in the absence of iSNVs. Targeted haplotyping via long-read amplicon sequencing, particularly using Oxford Nanopore Technologies, provides a valuable alternative for couples with de novo pathogenic variants that allows access to universal PGT. Moreover, the same approach can be used for direct mutation analysis in embryos, as a second line confirmation of the preclinical PGT result or as a potential alternative PGT procedure in couples, where additional family members are not available. This work was supported by KU Leuven funding (no. C1/018 to J.R.V.) and Fonds Wetenschappelijk Onderzoek (1241121N to O.T.). J.R.V. is co-inventor of a patent ZL910050-PCT/EP2011/060211-WO/2011/157846 'Methods for haplotyping single-cells' and ZL913096-PCT/EP2014/068315-WO/2015/028576 'Haplotyping and copy number typing using polymorphic variant allelic frequencies' licensed to Agilent Technologies. All other authors have no conflict of interest to declare. N/A.

Fungal communities associated with corn in a diverse long-term crop rotation in Ohio.

Abstract Crop rotation is a cultural practice in disease management used to break the disease cycle resulting in a reduction of inoculum. In Ohio, crop rotations have been reduced in diversity, with many farmers shifting to corn-soy rotations from more diverse rotations featuring wheat and forage crops. We investigated the impact of this shift on soil fungal communities under corn by conducting synthetic long-read amplicon sequencing. DNA was extracted from soil sampled during the corn growing season at two locations in Ohio with replicated long-term rotation plots in corn-soybean (CS) and a corn-soybean-winter wheat rotation (CSW) in 2018, 2019, and 2020.18s-ITS amplicons were sequenced using Illumina paired with Loopgenomics™ technology. PERMANOVA analysis revealed that fungal communities were significantly impacted by location, rotation, and time. Shannon and Simpson diversity indices implied fungal species diversity was significantly higher in the CS rotation. Indicator species analysis revealed 20 species indicative of the CSW rotation, including Mortierella , Hymenoschyphus , Ascobolus , and Saitozyma species, and 36 species indicative of the CS rotation, including Fusarium , Neoaschocyta , Zalerion , and Trichoderma species, among others. Indicator species ASV reads for the CSW rotation were not correlated with the decline in corn yield or soil N, C, or active carbon. However, they were positively correlated with soil organic matter.

MOCHI: a comprehensive cross-platform tool for amplicon-based microbiota analysis.

MotivationMicrobiota analyses have important implications for health and science. These analyses make use of 16S/18S rRNA gene sequencing to identify taxa and predict species diversity. However, most available tools for analyzing microbiota data require adept programming skills and in-depth statistical knowledge for proper implementation. While long-read amplicon sequencing can lead to more accurate taxa predictions and is quickly becoming more common, practitioners have no easily accessible tools with which to perform their analyses.ResultsWe present MOCHI, a GUI tool for microbiota amplicon sequencing analysis. MOCHI preprocesses sequences, assigns taxonomy, identifies different abundant species and predicts species diversity and function. It takes either taxonomic count table or FASTQ of partial 16S/18S rRNA or full-length 16S rRNA gene as input. It performs analyses in real time and visualizes data in both tabular and graphical formats.Availability and implementationMOCHI can be installed to run locally or accessed as a web tool at https://mochi.life.nctu.edu.tw.Supplementary information Supplementary data are available at Bioinformatics online.

P-561 Long-read amplicon guided haplotype imputation enabling comprehensive preimplantation genetic testing in families with de novo pathogenic variants

Abstract Study question Can we use comprehensive genome-wide haplotyping PGT for couples carrying de novo mutations, given the absence of phasing references to establish the disease-associated haplotype? Summary answer Targeted amplicon long-read sequencing represents a valuable approach for PGT workup that leverages comprehensive PGT application in families with de novo mutations. What is known already Preimplantation genetic testing (PGT) aims to select embryos devoid of inherited pathogenic variants. Current comprehensive genome-wide haplotyping PGT methods cannot be applied for couples where one of the partners carries a de novo mutation, as absence of affected close relatives restricts variant phasing to establish the disease-associated haplotype. For such families targeted approaches are currently used instead, thus missing genome-wide embryo analysis. Study design, size, duration To overcome the forementioned limitation, we developed a long-read amplicon guided haplotype imputation method. From January 2017 till June 2021 thirty-two couples were enrolled into the PGT program at the Centre for Human Genetics, UZ Leuven, in the context of a de novo variant causing a known monogenic disorder in one of the two partners. Participants/materials, setting, methods Genomic DNA from the partner carrying the variant (proband) and his/her parents was used for trio analysis via long-range PCR and long-read amplicon sequencing using the PacBio RSII and/or Oxford Nanopore platforms. Targeted haplotype phasing was then performed to impute the disease-associated allele. Main results and the role of chance The parental origin of the mutant allele was identified in 20 patients, resulting in the current 62.5% success rate. Most de novo mutations occurred on the paternal allele. In the remaining families, the region of interest either had no SNPs or had insufficient number of informative SNPs linked to mutation. From the successfully analyzed couples, eight have proceeded to PGT cycle and so far, three disease-free children have been born. Limitations, reasons for caution A costly family-specific work-up is required, allowing nevertheless the implementation of a generic genome-wide method at the embryo analysis stage. The sequencing cost itself is anticipated to decrease in time. To avoid non-informative results, the analysis of multiple amlpicons is recommended. Wider implications of the findings Current practices for the handling of couples with de novo variants are mostly targeted, require the combination of direct and indirect approaches and often require the multiple biopsies. Targeted amplicon long-read sequencing represents a valuable approach for PGT workup that leverages comprehensive PGT application in families with de novo mutations. Trial registration number not applicable

The Native Hymenoscyphus albidus and the Invasive Hymenoscyphus fraxineus Are Similar in Their Necrotrophic Growth Phase in Ash Leaves.

The populations of European ash and its harmless fungal associate Hymenoscyphus albidus are in decline owing to ash dieback caused by the invasive Hymenoscyphus fraxineus, a fungus that in its native range in Asia is a harmless leaf endophyte of local ash species. To clarify the behavior of H. albidus and its spatial and temporal niche overlap with the invasive relative, we used light microscopy, fungal species-specific qPCR assays, and PacBio long-read amplicon sequencing of the ITS1-5.8S-ITS2 region to examine fungal growth and species composition in attached leaves of European ash. The plant material was collected from a healthy stand in central Norway, where ash saplings in late autumn showed leaflet vein necrosis like that commonly related to H. fraxineus. For reference, leaflet samples were analyzed from stands with epidemic level of ash dieback in southeastern Norway and Estonia. While H. albidus was predominant in the necrotic veins in the healthy stand, H. fraxineus was predominant in the diseased stands. Otherwise, endophytes with pathogenic potential in the genera Venturia (anamorph Fusicladium), Mycosphaerella (anamorph Ramularia), and Phoma, and basidiomycetous yeasts formed the core leaflet mycobiome both in the healthy and diseased stands. In necrotic leaf areas with high levels of either H. albidus or H. fraxineus DNA, one common feature was the high colonization of sclerenchyma and phloem, a region from which the ascomata of both species arise. Our data suggest that H. albidus can induce necrosis in ash leaves, but that owing to low infection pressure, this first takes place in tissues weakened by autumn senescence, 1–2 months later in the season than what is characteristic of H. fraxineus at an epidemic phase of ash dieback. The most striking difference between these fungi would appear to be the high fecundity of H. fraxineus. The adaptation to a host that is phylogenetically closely related to European ash, a tree species with high occurrence frequency in Europe, and the presence of environmental conditions favorable to H. fraxineus life cycle completion in most years may enable the build-up of high infection pressure and challenge of leaf defense prior to autumn senescence.

Applying EDTA in Chelating Excess Metal Ions to Improve Downstream DNA Recovery from Mine Tailings for Long-Read Amplicon Sequencing of Acidophilic Fungi Communities.

The hostile environment of mine tailings contains unique microbial life capable of bioleaching. The metagenomic analysis of such an environment provides an in-depth understanding of the microbial life and its potential, especially in biomining operations. However, DNA recovery from samples collected in those environments is challenging due to the presence of metal ions that interfere with the DNA analysis. A varied concentration of EDTA (4–13 µg/µL) to chelate the metal ions of enriched tailing samples prior to DNA extraction was performed. The results show that 9 µg/µL of EDTA was effective in most samples. However, the increasing concentration of EDTA negatively affected the DNA recovery. The sequencing of the successfully extracted DNA revealed a diverse range of fungal genera, some of which have not been previously reported in tailing or bioleaching applications. The dominant genera include Fodinomyces, Penicillium, Recurvomuces, Trichoderma, and Xenoacremonium; their traits were determined using the FungalTraits database. This study demonstrates the need to include a preliminary metal-chelating step using EDTA before DNA extractions for samples collected from metal-rich environments. It further showed the need for optimization but provided a benchmark range, particularly for tailings. However, we caution that a further EDTA removal step from the extracted DNA should be included to avoid its interferences in downstream applications.

Long-read Amplicon Sequencing of the CYP21A2 in 48 Thai Patients With Steroid 21-Hydroxylase Deficiency

Congenital adrenal hyperplasia is most commonly caused by 21-hydroxylase deficiency (21-OHD), an autosomal recessive disorder resulting from biallelic pathogenic variants (PVs) in CYP21A2. With a highly homologous pseudogene and various types of single nucleotide and complex structural variants, identification of PVs in CYP21A2 has been challenging. To leverage long-read next-generation sequencing combined with locus-specific polymerase chain reaction (PCR) to detect PVs in CYP21A2 and to determine its diagnostic yield in patients with 21-OHD. Forty-eight Thai patients with 21-OHD comprising 38 sporadic cases and 5 pairs of siblings were enrolled. Two previously described locus-specific PCR methods were performed. Amplicons were subject to long-read sequencing. Ninety-six PVs in CYP21A2 in the 48 patients were successfully identified. The combined techniques were able to detect 26 structural chimeric variants (27%; 26/96) in 22 patients with 18 having monoallelic and 4 having biallelic chimeras. The remaining PVs were pseudogene-derived mutations (63%; 60/96), entire gene deletions (2%; 2/96), missense variants (3%; 3/96), a splice-site variant (2%; 2/96), frameshift variants (2%; 2/96), and a nonsense variant (1%; 1/96). Notably, a splice-site variant, IVS7 + 1G > T, which was identified in a pair of siblings, has not previously been reported. Our approach exploiting locus-specific PCR and long-read DNA sequencing has a 100% diagnostic yield for our cohort of 48 patients with 21-OHD.

Finding the right fit: evaluation of short-read and long-read sequencing approaches to maximize the utility of clinical microbiome data.

A long-standing challenge in human microbiome research is achieving the taxonomic and functional resolution needed to generate testable hypotheses about the gut microbiota’s impact on health and disease. With a growing number of live microbial interventions in clinical development, this challenge is renewed by a need to understand the pharmacokinetics and pharmacodynamics of therapeutic candidates. While short-read sequencing of the bacterial 16S rRNA gene has been the standard for microbiota profiling, recent improvements in the fidelity of long-read sequencing underscores the need for a re-evaluation of the value of distinct microbiome-sequencing approaches. We leveraged samples from participants enrolled in a phase 1b clinical trial of a novel live biotherapeutic product to perform a comparative analysis of short-read and long-read amplicon and metagenomic sequencing approaches to assess their utility for generating clinical microbiome data. Across all methods, overall community taxonomic profiles were comparable and relationships between samples were conserved. Comparison of ubiquitous short-read 16S rRNA amplicon profiling to long-read profiling of the 16S-ITS-23S rRNA amplicon showed that only the latter provided strain-level community resolution and insight into novel taxa. All methods identified an active ingredient strain in treated study participants, though detection confidence was higher for long-read methods. Read coverage from both metagenomic methods provided evidence of active-ingredient strain replication in some treated participants. Compared to short-read metagenomics, approximately twice the proportion of long reads were assigned functional annotations. Finally, compositionally similar bacterial metagenome-assembled genomes (MAGs) were recovered from short-read and long-read metagenomic methods, although a greater number and more complete MAGs were recovered from long reads. Despite higher costs, both amplicon and metagenomic long-read approaches yielded added microbiome data value in the form of higher confidence taxonomic and functional resolution and improved recovery of microbial genomes compared to traditional short-read methodologies.

EPCO-14. MULTIFACETED TRANSCRIPTOMIC AND PROTEOMIC ANALYSES IDENTIFIED PUTATIVE ALTERNATIVE SPLICING-DERIVED CELL SURFACE ANTIGENS IN GLIOMA

Abstract BACKGROUND To develop effective immunotherapy for gliomas, it is crucial to expand the repertoire of targetable antigens. Recent studies have suggested that alternative splicing (AS), or its deriving tumor-specific junctions (“neojunctions”), could generate cryptic amino acid sequences that can be a source of neoantigens. In this study, we investigated neojunctions based on multifaceted transcriptomic and proteomic analyses, seeking the potential cell surface antigens that may be targeted by CAR. METHODS For screening, we analyzed bulk RNA-sequencing data of TCGA-GBM/LGG with high tumor purity (n = 429) and GTEx normal tissues (n = 9,166). Cohorts of spatially mapped intratumoral samples and longitudinally collected tumors were used to determine clonality and stability of the candidate neojunctions. Nanopore long-read amplicon sequencing was deployed to confirm the full-length transcript sequence. Their protein-level expression was explored by analyzing the Clinical Proteomic Tumor Analysis Consortium (CPTAC)-GBM proteomics dataset. RESULTS In the screening analysis comparing TCGA and GTEx datasets, we identified 218 neojunctions with adequate expression, prevalence, and tumor-specificity. Of these, 12 were predicted to be cell-surface antigens. Eight of the 12, such as BCAN, DLL3, and PTPRZ1, were also observed in multiple cases of another validation dataset. In the analysis of tumors with spatially mapped intratumoral samples, 7 of the 12 were recurrently detected in no less than 50% of the samples in multiple cases. In addition, 5 of the 12 were found to be conserved in primary and recurrent pairs of tumors in multiple cases. Full-length transcript sequencing corroborated our predictions based on short reads, and also demonstrated more complex AS patterns. Finally, CPTAC-GBM proteomics analysis identified one cryptic peptide that substantiated the corresponding transcriptome-based prediction. CONCLUSION: We identified neojunctions with the potential to generate cell-surface antigens. These multifaceted transcriptomic and proteomic analyses provide the rationale to pursue the development of immunotherapy targeting neojunction-derived antigens.