Amplicon Sequencing Data Research Articles

Addressing pandemic-wide systematic errors in the SARS-CoV-2 phylogeny.

The SARS-CoV-2 genome occupies a unique place in infection biology - it is the most highly sequenced genome on earth (making up over 20% of public sequencing datasets) with fine scale information on sampling date and geography, and has been subject to unprecedented intense analysis. As a result, these phylogenetic data are an incredibly valuable resource for science and public health. However, the vast majority of the data was sequenced by tiling amplicons across the full genome, with amplicon schemes that changed over the pandemic as mutations in the viral genome interacted with primer binding sites. In combination with the disparate set of genome assembly workflows and lack of consistent quality control (QC) processes, the current genomes have many systematic errors that have evolved with the virus and amplicon schemes. These errors have significant impacts on the phylogeny, and therefore over the last few years, many thousands of hours of researchers time has been spent in "eyeballing" trees, looking for artefacts, and then patching the tree. Given the huge value of this dataset, we therefore set out to reprocess the complete set of public raw sequence data in a rigorous amplicon-aware manner, and build a cleaner phylogeny. Here we provide a global tree of 4,471,579 samples, built from a consistently assembled set of high quality consensus sequences from all available public data as of June 2024, viewable at https://viridian.taxonium.org . Each genome was constructed using a novel assembly tool called Viridian ( https://github.com/iqbal-lab-org/viridian ), developed specifically to process amplicon sequence data, eliminating artefactual errors and mask the genome at low quality positions. We provide simulation and empirical validation of the methodology, and quantify the improvement in the phylogeny. We hope the tree, consensus sequences and Viridian will be a valuable resource for researchers.

Microbial community assembly across agricultural soil mineral mesocosms revealed by 16S rRNA gene amplicon sequencing data

Microbial community assembly across agricultural soil mineral mesocosms revealed by 16S rRNA gene amplicon sequencing data

Fecal Microbiome Composition Correlates with Pathologic Complete Response in Patients with Operable Esophageal Cancer Treated with Combined Chemoradiotherapy and Immunotherapy.

Background: Preclinical and clinical data indicate that chemoradiotherapy (CRT) in combination with checkpoint inhibitors may prime an anti-tumor immunological response in esophageal cancer. However, responses to neoadjuvant therapy can vary widely and the key biomarkers to determine response remain poorly understood. The fecal microbiome is a novel and potentially modifiable biomarker of immunotherapy response, and both fecal and tumor microbes have been found to associate with outcomes in esophageal cancer. Methods: Fecal and tumor samples were collected from patients with stage II-III resectable esophageal or gastroesophageal junction carcinoma treated with neoadjuvant immune checkpoint inhibitors (ICIs) plus CRT prior to surgical resection. Microbiome profiles were analyzed by 16S rRNA amplicon sequencing and taxonomic data were integrated with fecal metabolite analysis to assess microbial function. Results: The fecal microbiome of patients with pathological complete response (PCR) grouped in distinct clusters compared to patients with residual viable tumor (RVT) by Bray-Curtis diversity metric. Integrated taxonomic and metabolomic analysis of fecal samples identified a sphingolipid and primary bile acid as enriched in the PCR, the levels of which correlated with several bacterial species: Roseburis inulinivorans, Ruminococcus callidus, and Fusicantenibacter saccharivorans. Analysis of the tumor microbiome profiles identified several bacterial genera previously associated with esophageal tumors, including Streptococcus and Veillonella. Conclusions: These results further characterize the fecal and tumor microbiome of patients with operable esophageal cancer and identify specific microbes and metabolites that may help elucidate how microbes contribute to tumor response with neoadjuvant CRT combined with ICI.

The epidemic occurrence of decline disease in bayberry trees altered plant and soil related microbiome and metabolome

BackgroundIn China, decline disease with unknown etiology appeared as an epidemic among bayberry trees in the southern area of the Yangtze River. Furthermore, the use of beneficial microbes has been reported to be able to reduce the incidence of this disease, emphasizing the association of this disease with microorganisms. Therefore, it has become critical to uncover the microbiome’s function and related metabolites in remodeling the immunity of bayberry trees under biotic or abiotic stresses.ResultsThe amplicon sequencing data revealed that decline disease significantly altered bacterial and fungal communities, and their metabolites in the four distinct niches, especially in the rhizosphere soils and roots. Furthermore, the microbial communities in the four niches correlated with the metabolites of the corresponding niches of bayberry plants, and the fungal and bacterial networks of healthy trees were shown to be more complex than those of diseased trees. In addition, the role of microbiome in the resistance of bayberry trees to the occurrence of decline disease was justified by the isolation, identification, and characterization of important microorganisms such as significantly enriched Bacillus ASV804, Pseudomonas ASV815 in healthy plants, and significantly enriched Stenotrophomonas ASV719 in diseased plants.ConclusionOverall, our study revealed that the occurrence of decline disease altered the microbiome and its metabolites in four ecological niches in particular rhizosphere soils and roots of bayberry, which provides new insight into the control of bayberry decline disease.

Endemic, cosmopolitan, and generalist taxa and their habitat affinities within a coastal marine microbiome

The relative prevalence of endemic and cosmopolitan biogeographic ranges in marine microbes, and the factors that shape these patterns, are not well known. Using prokaryotic and eukaryotic amplicon sequence data spanning 445 near-surface samples in the Southern California Current region from 2014 to 2020, we quantified the proportion of taxa exhibiting endemic, cosmopolitan, and generalist distributions in this region. Using in-situ data on temperature, salinity, and nitrogen, we categorized oceanic habitats that were internally consistent but whose location varied over time. In this context, we defined cosmopolitan taxa as those that appeared in all regional habitats and endemics as taxa that only appeared in one habitat. Generalists were defined as taxa occupying more than one but not all habitats. We also quantified each taxon’s habitat affinity, defined as habitats where taxa were significantly more abundant than expected. Approximately 20% of taxa exhibited endemic ranges, while around 30% exhibited cosmopolitan ranges. Most microbial taxa (50.3%) were generalists. Many of these taxa had no habitat affinity (> 70%) and were relatively rare. Our results for this region show that, like terrestrial systems and for metazoans, cosmopolitan and endemic biogeographies are common, but with the addition of a large number of taxa that are rare and randomly distributed.

Management and rhizosphere microbial associations modulate genetic-driven nitrogen fate

The interplay between plant genotype and nutrient management affects rhizodeposition, which in turn modulates the rhizosphere-microbiome and microbe-mediated functions. Substituting mineral nitrogen (N) with an N-fixing inoculant reduces reliance on N fertilizer while supplying N to crops. We evaluated the effectiveness of integrating maize near-isogenic lines (NIL 1 and NIL 2) with the biological nitrification inhibition (BNI) trait into management practices aimed at optimizing N provisioning. Management strategies included mineral N inputs (0 and 67 kg ha⁻¹) with and without an N-fixing inoculant. Our approach synthesized insights from amplicon sequencing data and evaluated nitrification rates, rhizosphere N content, maize N uptake, and N use efficiency (NUE). Genotypes and management structured prokaryotic communities, while the developmental stages of genotypes further refined both fungal and prokaryotic communities. The N-fixing inoculant increased N availability, triggering the BNI capacity without increasing the nitrification rate. This was reflected in lower NO₃⁻ and higher NH₄⁺ levels in BNI-NIL leachate compared to B73, suggesting improved N retention. NIL2, characterized by distinct fungal biomarkers, exhibited higher N content (72.3 kg ha⁻¹) and superior NUE compared to NIL1 (65.0 kg ha⁻¹). NIL2’s enhanced N uptake was associated with a robust microbial network, featuring Archangium (prokaryote) and Trichoderma (eukaryote) as keystone taxa. Notably, Archangium was linked to rhizosphere N dynamics Synergizing BNI with diazotroph inoculants reduces N fertilizer reliance and increases maize N supply for sustainable agroecosystems.

Implementing high-throughput insect barcoding in microbiome studies: impact of non-destructive DNA extraction on microbiome reconstruction.

Symbiotic relationships with diverse microorganisms are crucial for many aspects of insect biology. However, while our understanding of insect taxonomic diversity and the distribution of insect species in natural communities is limited, we know much less about their microbiota. In the era of rapid biodiversity declines, as researchers increasingly turn towards DNA-based monitoring, developing and broadly implementing approaches for high-throughput and cost-effective characterization of both insect and insect-associated microbial diversity is essential. We need to verify whether approaches such as high-throughput barcoding, a powerful tool for identifying wild insects, would permit subsequent microbiota reconstruction in these specimens. High-throughput barcoding ("megabarcoding") methods often rely on non-destructive approaches for obtaining template DNA for PCR amplification by leaching DNA out of insect specimens using alkaline buffers such as HotSHOT. This study investigated the impact of HotSHOT on microbial abundance estimates and the reconstructed bacterial community profiles. We addressed this question by comparing quantitative 16S rRNA amplicon sequencing data for HotSHOT-treated or untreated specimens of 16 insect species representing six orders and selected based on the expectation of limited variation among individuals. We find that in 13 species, the treatment significantly reduced microbial abundance estimates, corresponding to an estimated 15-fold decrease in amplifiable 16S rRNA template on average. On the other hand, HotSHOT pre-treatment had a limited effect on microbial community composition. The reconstructed presence of abundant bacteria with known significant effects was not affected. On the other hand, we observed changes in the presence of low-abundance microbes, those close to the reliable detection threshold. Alpha and beta diversity analyses showed compositional differences in only a few species. Our results indicate that HotSHOT pre-treated specimens remain suitable for microbial community composition reconstruction, even if abundance may be hard to estimate. These results indicate that we can cost-effectively combine barcoding with the study of microbiota across wild insect communities. Thus, the voucher specimens obtained using megabarcoding studies targeted at characterizing insect communities can be used for microbiome characterizations. This can substantially aid in speeding up the accumulation of knowledge on the microbiomes of abundant and hyperdiverse insect species.

Improved species assignments across the entire Anopheles genus using targeted sequencing.

Accurate species identification of the mosquitoes in the genus Anopheles is of crucial importance to implement malaria control measures and monitor their effectiveness. We use a previously developed amplicon panel (ANOSPP) that retrieves sequence data from multiple short nuclear loci for any species in the genus. Species assignment is based on comparison of samples to a reference index using k-mer distance. Here, we provide a protocol to generate version controlled updates of the reference index and present its latest release, NNv2, which contains 91 species, compared to 56 species represented in its predecessor NNv1. With the updated reference index, we are able to assign samples to species level that previously could not be assigned. We discuss what happens if a species is not represented in the reference index and how this can be addressed in a future update. To demonstrate the increased power of NNv2, we showcase the assignments of 1789 wild-caught mosquitoes from Madagascar and demonstrate that we can detect within species population structure from the amplicon sequencing data.

Decay kinetics of human-associated pathogens in the marine microcosms reveals their new dynamics and potential indicators in the coastal waters of northern China

Pathogens in coastal waters cause infectious diseases and endanger public sanitation safety in humans and animals worldwide. To avoid these risks, timely detection of human-associated pathogens in waters is crucial. In this study, the decay kinetics of the molecular markers for human-associated pathogens, including enteric bacteria (Escherichia coli, Enterococcus, and Bacteroides), non-enteric bacteria (Staphylococcus aureus), crAssphage, and polyomavirus, were monitored over time at different temperatures and background microbes in seawater microcosms. The results indicated that temperature and native marine microbes were the main influential factors in attenuating bacterial pathogens. Remarkably, the effect of native microorganisms was more evidentially striking. Furthermore, Enterococcus was a more reliable and suitable fecal indicator bacterium than E. coli for the marine environment. The decay of crAssphage was like that of polyomavirus, indicating that it may be a good indicator of enterovirus in seawater. More importantly, the 16S amplicon sequencing data highlighted the decay kinetics of multiple bacterial pathogens in parallel with the dynamic changes of the whole bacterial communities. This study provides valuable information for public health risk management and a new approach to understanding the fate of bacteria in the coastal environment.

Sensitive and modular amplicon sequencing of Plasmodium falciparum diversity and resistance for research and public health.

Targeted amplicon sequencing is a powerful and efficient tool to interrogate the P. falciparum genome and generate actionable data from infections to complement traditional malaria epidemiology. For maximum impact, genomic tools should be multi-purpose, robust, sensitive and reproducible. We developed, characterized, and implemented MAD4HatTeR, an amplicon sequencing panel based on Multiplex Amplicons for Drug, Diagnostic, Diversity, and Differentiation Haplotypes using Targeted Resequencing, along with a bioinformatic pipeline for data analysis. MAD4HatTeR targets 165 highly diverse loci, focusing on multiallelic microhaplotypes; key markers for drug and diagnostic resistance, including duplications and deletions; and csp and potential vaccine targets. In addition, it can detect non-falciparum Plasmodium species. We used laboratory control and field sample data to demonstrate the high sensitivity and robustness of the panel. The successful implementation of this method in five laboratories, including three in malaria-endemic African countries, showcases its feasibility in generating reproducible data across laboratories. Finally, we introduce an analytical approach to detect gene duplications and deletions from amplicon sequencing data. MAD4HatTeR is thus a powerful research tool and a robust resource for malaria public health surveillance and control.

Clinical Investigation of Bacteriome in Primary Endodontic Infections With Apical Periodontitis Using High-Throughput Sequencing Analysis

IntroductionThis study characterized the bacteriome in primary endodontic infection (PEI) with apical periodontitis (AP), identified core and rare bacteriome species and community diversity metrics, and analyzed the relationship between the bacteriome composition, diversity and features, and patient variables. MethodsTwenty-seven patients with PEI and AP were sampled. The DNA was extracted and quantified using quantitative polymerase chain reaction. Raw V3-V4 amplicon sequencing data were processed with the DADA2 pipeline to generate amplicon sequence variants, and taxonomic assignment of the amplicon sequence variants up to the species level was done against the Human Oral Microbiome Database. Core bacteriome and differential abundance analyses were performed using ANCOM. Alpha diversity was determined using Chao1, Shannon, and Simpson indexes. LeFse analysis was used to identify abundant taxa. Sparse Estimation of Correlations among Microbiomes analysis estimated linear and nonlinear relationships among bacteria. ResultsOf 27, 24 root canal samples were analyzed, and 3 root canal sampling were filtered out with a low read count. The bacterial phyla with top mean relative abundance were Bacteroidetes, Firmicutes, Synergistetes, Fusobacteria, and Actinobacteria. A total of 113 genera and 215 species were identified. The samples were gathered into 3 clusters. LefSe analysis identified differences in abundant taxa between distinct age, gender, symptomatology, and lesion size groups. Sparse Estimation of Correlations among Microbiomes distance analysis indicated Slackia exigua as the node with the highest degree. ConclusionsThe bacteriome in PEI with AP among the patients in this study was complex and displayed high microbial heterogeneity. Moreover, age, gender, symptomatology, and lesion size were associated with differences in bacteriome features in PEI with AP.

Complete pipeline for Oxford Nanopore Technology amplicon sequencing (ONT-AmpSeq): from pre-processing to creating an operational taxonomic unit table.

Amplicon sequencing has long served as a robust method for characterising microbial communities, and despite inherent resolution limitations, it remains a preferred technique, offering cost- and time-effective insights into bacterial compositions. Here, we introduce ONT-AmpSeq, a user-friendly pipeline designed for processing amplicon sequencing data generated from Oxford Nanopore Technology (ONT) devices. Our pipeline enables efficient creation of taxonomically annotated operational taxonomic unit (OTU) tables from ONT sequencing data, with the flexibility to multiplex amplicons on the same barcode. The pipeline encompasses six main steps-statistics, quality filtering, alignment, clustering, polishing, and taxonomic classification-integrating various state-of-the-art software tools. We provide a detailed description of each step, along with performance tests and robustness evaluations using both test data and a ZymoBIOMICS® Microbial Community Standard mock community dataset. Our results demonstrate the ability of ONT-AmpSeq to effectively process ONT amplicon data, offering valuable insights into microbial community composition. Additionally, we discuss the influence of polishing tools on taxonomic insight and the impact of taxonomic annotation methods on the derived microbial composition. Overall, ONT-AmpSeq represents a comprehensive solution for analysing ONT amplicon sequencing data, facilitating streamlined and reliable microbial community analysis. The pipeline, along with test data, is freely available for public use.

Effects of humic acid application on bacterial diversity under maize cultivation

AbstractThe fundamental ecosystem processes in soil are regulated by microbial communities, and community diversity is implicated by soil environmental conditions. Humic acid (HA) improves soil quality and fertility, stimulating the microbial environment, but the detailed effects remain poorly understood. We investigated the effects of HA rates on soil bacterial diversity, particularly on species richness and community composition in the rhizosphere of corn (Zea mays). Inorganic fertilizer (T2), HA 0.5% (T3), HA 1.0% (T4), HA 1.5% (T5), and HA 2.0% (T6) were applied in soil. Initial soil (O1) and control after harvesting (T1) were included. A total of 3601 operational taxonomic units were captured from the overall sample, and analysis of 16S ribosomal ribonucleic acid amplicon sequencing data indicated that HA did not notably impact species richness. Intriguingly, HA induced changes in bacterial community composition, along with the relative prevalence of specific taxa. Certain associations between soil chemical properties and abundance distribution have been uncovered. Notably, exchangeable Mg2+, Ca2+, and available phosphorus were strongly related to the relative abundance of bacterial phyla. Furthermore, HA potentially shaped the specific bacterial taxa, as the application of HA at different rates had distinct effects on the member of bacterial abundance of each taxon. These findings enhance our understanding of communities potentially being increased or shifted by HA rate addition in short‐term corn cultivation.

Genetic surveillance reveals low, sustained malaria transmission with clonal replacement in Sao Tome and Principe.

Despite efforts to eliminate malaria in Sao Tome and Principe (STP), cases have recently increased. Understanding residual transmission structure is crucial for developing effective elimination strategies. This study collected surveillance data and generated amplicon sequencing data from 980 samples between 2010 and 2016 to examine the genetic structure of the parasite population. The mean multiplicity of infection (MOI) was 1.3, with 11% polyclonal infections, indicating low transmission intensity. Temporal trends of these genetic metrics did not align with incidence rates, suggesting that changes in genetic metrics may not straightforwardly reflect changes in transmission intensity, particularly in low transmission settings where genetic drift and importation have a substantial impact. While 88% of samples were genetically linked, continuous turnover in genetic clusters and changes in drug-resistance haplotypes were observed. Principal component analysis revealed some STP samples were genetically similar to those from Central and West Africa, indicating possible importation. These findings highlight the need to prioritize several interventions such as targeted interventions against transmission hotspots, reactive case detection, and strategies to reduce the introduction of new parasites into this island nation as it approaches elimination. This study also serves as a case study for implementing genetic surveillance in a low transmission setting.

Domestication and evolutionary histories of specialized gut symbionts across cephalotine ants.

The evolution of animals and their gut symbionts is a complex phenomenon, obscured by lability and diversity. In social organisms, transmission of symbionts among relatives may yield systems with more stable associations. Here, we study the history of a social insect symbiosis involving cephalotine ants and their extracellular gut bacteria, which come predominantly from host-specialized lineages. We perform multi-locus phylogenetics for symbionts from nine bacterial orders, and map prior amplicon sequence data to lineage-assigned symbiont genomes, studying distributions of rigorously defined symbionts across 20 host species. Based on monophyly and additional hypothesis testing, we estimate that these specialized gut bacteria belong to 18 distinct lineages, of which 15 have been successfully isolated and cultured. Several symbiont lineages showed evidence for domestication events that occurred later in cephalotine evolutionary history, and only one lineage was ubiquitously detected in all 20 host species and 48 colonies sampled with amplicon 16S rRNA sequencing. We found evidence for phylogenetically constrained distributions in four symbionts, suggesting historical or genetic impacts on community composition. Two lineages showed evidence for frequent intra-lineage co-infections, highlighting the potential for niche divergence after initial domestication. Nearly all symbionts showed evidence for occasional host switching, but four may, more often, co-diversify with their hosts. Through our further assessment of symbiont localization and genomic functional profiles, we demonstrate distinct niches for symbionts with shared evolutionary histories, prompting further questions on the forces underlying the evolution of hosts and their gut microbiomes.

Metagenomic insights into microbial community structure and metabolism in alpine permafrost on the Tibetan Plateau

Permafrost, characterized by its frozen soil, serves as a unique habitat for diverse microorganisms. Understanding these microbial communities is crucial for predicting the response of permafrost ecosystems to climate change. However, large-scale evidence regarding stratigraphic variations in microbial profiles remains limited. Here, we analyze microbial community structure and functional potential based on 16S rRNA gene amplicon sequencing and metagenomic data obtained from an ∼1000 km permafrost transect on the Tibetan Plateau. We find that microbial alpha diversity declines but beta diversity increases down the soil profile. Microbial assemblages are primarily governed by dispersal limitation and drift, with the importance of drift decreasing but that of dispersal limitation increasing with soil depth. Moreover, genes related to reduction reactions (e.g., ferric iron reduction, dissimilatory nitrate reduction, and denitrification) are enriched in the subsurface and permafrost layers. In addition, microbial groups involved in alternative electron accepting processes are more diverse and contribute highly to community-level metabolic profiles in the subsurface and permafrost layers, likely reflecting the lower redox potential and more complicated trophic strategies for microorganisms in deeper soils. Overall, these findings provide comprehensive insights into large-scale stratigraphic profiles of microbial community structure and functional potentials in permafrost regions.

Salinity and nutrient condition as key factors drive the assembly of sediment prokaryotic communities

Despite extensive research on the geographical patterns of microbial communities, our comprehension of the mechanisms underlying their spatial distribution is still limited. Natural ecosystems provide opportunities to investigate the structure, connectivity, and assembly processes of prokaryotic communities. Saline lakes, mangroves, ocean margins, cold seeps, and open oceans as five distinct natural ecosystems exhibit varied levels of salinity and nutrient condition (carbon sources, electron donors, and electron acceptors). Based on the analysis of 197 sets of published 16S rRNA gene amplicon sequencing data on sediment samples of the five habitats, differences in salinity and nutrient conditions were identified to play a critical role in governing the composition, connectivity, and assembly process of prokaryotic communities. Specifically, unique prokaryotic community patterns were observed in these habitats, e.g., mangrove sediment communities were shown to have the highest alpha diversity and the lowest community-level ribosomal RNA gene operon (rrn) copy numbers, compared to the open ocean sediment communities. Positive correlation predominated connections (>80% of total connections) of the prokaryotic microbial networks in the five habitats. Communities within nutrient-rich saline lake and cold seep sediments exhibit the strongest and closest connections. Using the dissimilarity-overlap curve and null model, differences in composition, connectivity, and assembly process were found to be predominantly governed by deterministic forces. These findings enhance our understanding of microbial ecology in typical saline environments and enable us to investigate intricate ecosystems.

Gardnerella diversity and ecology in pregnancy and preterm birth.

The vaginal microbiome has been linked to negative health outcomes including preterm birth. Specific taxa, including Gardnerella spp., have been identified as risk factors for these conditions. Historically, microbiome analysis methods have treated all Gardnerella spp. as one species, but the broad diversity of Gardnerella has become more apparent. We explore the diversity of Gardnerella clades and genomic species in the vaginal microbiome of pregnant women and their associations with microbiome composition and preterm birth. Relative abundance of Gardnerella clades and genomic species and other taxa was quantified in shotgun metagenomic sequencing data from three distinct cohorts of pregnant women. We also assessed the diversity and abundance of Gardnerella variants in 16S rRNA gene amplicon sequencing data from seven previously conducted studies in differing populations. Individual microbiomes often contained multiple Gardnerella variants, and the number of clades was associated with increased microbial load, or the ratio of non-human reads to human reads. Taxon co-occurrence patterns were largely consistent across Gardnerella clades and among cohorts. Some variants previously described as rare were prevalent in other cohorts, highlighting the importance of surveying a diverse set of populations to fully capture the diversity of Gardnerella. The diversity of Gardnerella both across populations and within individual vaginal microbiomes has long been unappreciated, as has been the intra-species diversity of many other members of the vaginal microbiome. The broad genomic diversity of Gardnerella has led to its reclassification as multiple species; here we demonstrate the diversity of Gardnerella found within and between vaginal microbiomes.IMPORTANCEThe present study shows that single microbiomes can contain all currently known species of Gardnerella and that multiple similar species can exist within the same environment. Furthermore, surveys of demographically distinct populations suggest that some species appear more commonly in certain populations. Further studies in broad and diverse populations will be necessary to fully understand the ecological roles of each Gardnerella sp., how they can co-exist, and their distinct impacts on microbial communities, preterm birth, and other health outcomes.

An amplicon panel for high-throughput and low-cost genotyping of Pacific oyster.

Maintaining genetic diversity in cultured shellfish can be challenging due to high variance in individual reproductive success, founder effects, and rapid genetic drift, but is important to retain adaptive potential and avoid inbreeding depression. To support broodstock management and selective breeding in cultured Pacific oysters (Crassostrea (Magallana) gigas), we developed an amplicon panel targeting 592 genomic regions and SNP variants with an average of 50 amplicons per chromosome. Target SNPs were selected based on elevated observed heterozygosity or differentiation in Pacific oyster populations in British Columbia, Canada. The use of the panel for parentage applications was evaluated using multiple generations of oysters from a breeding program on Vancouver Island, Canada (n = 181) and families selected for Ostreid herpesvirus-1 resistance from the Molluscan Broodstock Program in Oregon, USA (n = 136). Population characterization was evaluated using wild, naturalized, farmed, or hatchery oysters sampled throughout the Northern Hemisphere (n = 190). Technical replicates showed high genotype concordance (97.5%; n = 68 replicates). Parentage analysis found suspected pedigree and sample handling errors, demonstrating the panel's value for quality control in breeding programs. Suspected null alleles were identified and found to be largely population dependent, suggesting population-specific variation impacting target amplification. Null alleles were identified using existing data without the need for pedigree information, and once they were removed, assignment rates increased to 93.0% and 86.0% of possible assignments in the two breeding program datasets. A pipeline for analyzing the amplicon sequence data from sequencer output, amplitools, is also provided.

Absence of a pancreatic microbiome in intraductal papillary mucinous neoplasm

ObjectiveThis study aims to validate the existence of a microbiome within intraductal papillary mucinous neoplasm (IPMN) that can be differentiated from the taxonomically diverse DNA background of next-generation sequencing procedures.DesignWe...