Generation Amplicon Research Articles

Effect of body mass index on semen quality, sperm chromatin integrity and sperm DNA methylation

Obesity represents a growing problem due to its impacts on human health and reproduction. In this study, we analysed semen quality, sperm DNA integrity and gene-specific CpG methylation in 116 healthy men from normal population. The men were divided into three groups according to their body mass index (BMI), and their ejaculates were analysed using standard methods, sperm chromatin structure assay (SCSA), methylation next generation sequencing (NGS) and amplicon sequencing. The sperm methylation NGS revealed six significantly differentially methylated regions (DMRs). Using subsequent targeted amplicon sequencing in 116 men, two of the DMRs were proved as differentially methylated in sperm of men with normal BMI vs. BMI ≥ 25. The DMRs were located in the EPHA8 and ANKRD11 gene. Also, we detected a significant decline in the EPHA8, ANKRD11 and CFAP46 gene methylation in association with increasing BMI values. The genes EPHA8 and ANKRD11 are involved in the nervous system and brain development; the CFAP46 gene plays a role in a flagellar assembly and is associated with sperm motility. Significantly lower rates of motile and progressive motile sperm were observed in men with BMI ≥ 30. Our results show that excess body weight can modify CpG methylation of specific genes, affect sperm motility, and compromise sperm chromatin integrity. These factors can stand behind the observed reduced fertility in men with obesity. The methylation changes might be transmitted to their offspring through sperm, and become a basis for possible developmental and reproductive issues in the next generation.

Olivar: towards automated variant aware primer design for multiplex tiled amplicon sequencing of pathogens

Tiled amplicon sequencing has served as an essential tool for tracking the spread and evolution of pathogens. Over 15 million complete SARS-CoV-2 genomes are now publicly available, most sequenced and assembled via tiled amplicon sequencing. While computational tools for tiled amplicon design exist, they require downstream manual optimization both computationally and experimentally, which is slow and costly. Here we present Olivar, a first step towards a fully automated, variant-aware design of tiled amplicons for pathogen genomes. Olivar converts each nucleotide of the target genome into a numeric risk score, capturing undesired sequence features that should be avoided. In a direct comparison with PrimalScheme, we show that Olivar has fewer mismatches overlapping with primers and predicted PCR byproducts. We also compare Olivar head-to-head with ARTIC v4.1, the most widely used primer set for SARS-CoV-2 sequencing, and show Olivar yields similar read mapping rates (~90%) and better coverage to the manually designed ARTIC v4.1 amplicons. We also evaluate Olivar on real wastewater samples and found that Olivar has up to 3-fold higher mapping rates while retaining similar coverage. In summary, Olivar automates and accelerates the generation of tiled amplicons, even in situations of high mutation frequency and/or density. Olivar is available online as a web application at https://olivar.rice.edu and can be installed locally as a command line tool with Bioconda. Source code, installation guide, and usage are available at https://github.com/treangenlab/Olivar.

A cross-sectional survey of Blastocystis sp. and Dientamoeba fragilis in non-human primates and their caregivers in Czech zoos

Intestinal protists in the gut microbiome are increasingly studied, but their basic epidemiology is not well understood. We explored the prevalence, genetic diversity, and potential zoonotic transmission of two protists colonizing the large intestine - Blastocystis sp. and Dientamoeba fragilis - in 37 species of non-human primates (NHPs) and their caregivers in six zoos in the Czech Republic. We analyzed 179 fecal samples (159 from NHPs, 20 from humans) by qPCR. Blastocystis sp. was detected in 54.7% (98/179) of samples, in 24 NHP species and in 57.2% of NHP samples (prevalence ranged between 36 and 80%), and in 35% of human samples (prevalence ranged between 0 and 67%). Using next generation amplicon sequencing, nine Blastocystis subtypes (ST1-ST5, ST7, ST8, and two novel subtypes) were identified. The two new Blastocystis subtypes (named ST47 and ST48) were described using Nanopore sequencing to produce full-length reference sequences of the small subunit ribosomal RNA gene. Some subtypes were shared between NHPs and their caregivers, suggesting potential zoonotic transmission. Mixed subtype colonization was frequently observed, with 52% of sequenced samples containing two or more subtypes. Dientamoeba was found only in NHPs with a prevalence of 6%. This study emphasizes the critical role of molecular diagnostics in epidemiological and transmission studies of these protists and calls for further research to better understand their impact on public health.

Olivar: automated variant aware primer design for multiplex tiled amplicon sequencing of pathogens.

Tiled amplicon sequencing has served as an essential tool for tracking the spread and evolution of pathogens. Over 2 million complete SARS-CoV-2 genomes are now publicly available, most sequenced and assembled via tiled amplicon sequencing. While computational tools for tiled amplicon design exist, they require downstream manual optimization both computationally and experimentally, which is slow and costly. Here we present Olivar, a first step towards a fully automated, variant-aware design of tiled amplicons for pathogen genomes. Olivar converts each nucleotide of the target genome into a numeric risk score, capturing undesired sequence features that should be avoided. In a direct comparison with PrimalScheme, we show that Olivar has fewer SNPs overlapping with primers and predicted PCR byproducts. We also compared Olivar head-to-head with ARTIC v4.1, the most widely used primer set for SARS-CoV-2 sequencing, and show Olivar yields similar read mapping rates (~90%) and better coverage to the manually designed ARTIC v4.1 amplicons. We also evaluated Olivar on real wastewater samples and found that Olivar had up to 3-fold higher mapping rates while retaining similar coverage. In summary, Olivar automates and accelerates the generation of tiled amplicons, even in situations of high mutation frequency and/or density. Olivar is available as a web application at https://olivar.rice.edu. Olivar can also be installed locally as a command line tool with Bioconda. Source code, installation guide and usage are available at https://github.com/treangenlab/Olivar.

Planktonic and epilithic prokaryota community compositions in a large temperate river reflect climate change related seasonal shifts.

In freshwaters, microbial communities are of outstanding importance both from ecological and public health perspectives, however, they are threatened by the impact of global warming. To reveal how different prokaryotic communities in a large temperate river respond to environment conditions related to climate change, the present study provides the first detailed insight into the composition and spatial and year-round temporal variations of planktonic and epilithic prokaryotic community. Microbial diversity was studied using high-throughput next generation amplicon sequencing. Sampling was carried out monthly in the midstream and the littoral zone of the Danube, upstream and downstream from a large urban area. Result demonstrated that river habitats predominantly determine the taxonomic composition of the microbiota; diverse and well-differentiated microbial communities developed in water and epilithon, with higher variance in the latter. The composition of bacterioplankton clearly followed the prolongation of the summer resulting from climate change, while the epilithon community was less responsive. Rising water temperatures was associated with increased abundances of many taxa (such as phylum Actinobacteria, class Gammaproteobacteria and orders Synechococcales, Alteromonadales, Chitinophagales, Pseudomonadales, Rhizobiales and Xanthomonadales), and the composition of the microbiota also reflected changes of several further environmental factors (such as turbidity, TOC, electric conductivity, pH and the concentration of phosphate, sulphate, nitrate, total nitrogen and the dissolved oxygen). The results indicate that shift in microbial community responding to changing environment may be of crucial importance in the decomposition of organic compounds (including pollutants and xenobiotics), the transformation and accumulation of heavy metals and the occurrence of pathogens or antimicrobial resistant organisms.

Curated and harmonized gut microbiome 16S rRNA amplicon data from dietary fiber intervention studies in humans

Next generation amplicon sequencing has created a plethora of data from human microbiomes. The accessibility to this scientific data and its corresponding metadata is important for its reuse, to allow for new discoveries, verification of published results, and serving as path for reproducibility. Dietary fiber consumption has been associated with a variety of health benefits that are thought to be mediated by gut microbiota. To enable direct comparisons of the response of the gut microbiome to fiber, we obtained 16S rRNA sequencing data and its corresponding metadata from 11 fiber intervention studies for a total of 2,368 samples. We provide curated and pre-processed genetic data and common metadata for comparison across the different studies.

Proglacial slopes are protected against erosion by trait diverse and dense plant communities associated with specific microbial communities

Soil erosion is a severe threat for ecosystems and anthropogenic infrastructure. Glaciers are retreating rapidly due to global warming and the receding ice leaves unvegetated depositions of sediment, which are prone to mobilisation during precipitation events. Vegetation is known to serve as erosion protection, but how above- and belowground plant traits, vegetation cover and plant diversity on community level are jointly affecting erosion is unclear. Additionally, soil microbial communities may have effects on slope stability by promoting plant growth and function. We measured sediment transport on 30 plots of 2 × 3 m size on proglacial slopes of the Gepatschferner glacier (Kaunertal, Austria) over three years in a natural pristine high alpine environment. Vegetation cover, species abundances and relevant above- and belowground traits were measured for each occurring plant species on community level. Path model analysis revealed that dense and species-rich plant cover characterized by specific plant growth strategies and trait diversity was best suited to decrease erosion. Vegetation properties were also closely linked to the composition of soil microbial communities characterised by next generation amplicon sequencing, which may facilitate soil formation and further enhance the plants’ soil stabilising potential. Our study illustrates that erosion control may benefit from a high vegetation cover, which is facilitated by diverse plant communities with complementary morphological traits. These vegetation properties also affect soil microbiota, but their impact on slope protection in combination with the vegetation remains to be elucidated. Our findings may inform nature-based solutions against erosion such as revegetation of alpine slopes using specific seed mixtures.

Father and son are carriers of two different types of human inborn errors of immunity: a case report

IntroductionChronic granulomatous disease (CGD) is an inborn error of immunity (IEI) characterized by recurrent bacterial and fungal infections of skin, airways, liver, brain and bones with the estimated prevalence of 1 in 250,000 live births. CGD can inherited either in an X-linked (allelic variants in the CYBB gene) or autosomal recessive mode (allelic variants in the CYBA, NCF1, NCF2, NCF4, and CYBC1 genes). Wiskott-Aldrich syndrome is an X-linked disorder with an estimated incidence of 1 in 100 000 live male births caused by variants in the WAS gene, characterized by microthrombocytopenia, eczema, recurrent infections, increased risk for autoimmune disease and malignancy. Materials and MethodsIn this case report, we present our experience about two cases of blood relatives diagnosed with different types of human inborn errors of immunity. Genetic analysis of CYBB gene in 2007 was performed by Sanger sequencing and of WAS gene in 2022 by next generation amplicon sequencing. All subjects consented to the study. ResultsThe first case was a 15-year-old male (1992 y.o.b., proband) who was diagnosed as X-linked CGD patient in 2007 due to his clinical history and hemizygote allelic variant in the CYBB gene (NM_000397.4: c.484–1G>T, COSV101059810). The second case was a 2-month-old boy (biological son of proband) who was hospitalized in 2022 due to microthrombocytopenia, low platelet volume, cytomegalovirus infection, mild anemia. Genetic analysis revealed in the DNA sample of the second case the hemizygote allelic variant in the WAS gene (NM_000377.3: c.777 +3_777+6del GAGT). Genetic testing didn’t reveal such variant in the DNA sample of the proband's wife, that's why this variant was classified as “de novo”. ConclusionInborn errors of immunity (IEI) present clinically as increased susceptibility to infections, autoimmunity, autoinflammatory diseases, allergy, bone marrow failure, and/or malignancy. There are now a total of 485 IEI, which are segregating to 10 groups of disorders into overlapping phenotypes. Despite the fact that IEI are rare diseases, we have shown that even in one family two different types of disorders can occur. In our case the second IEI was occur due to spontaneously alterations during the process of DNA replication during cell division.

Diversity of Blastocystis Subtypes in Horses in Colombia and Identification of Two New Subtypes

Blastocystis is a common intestinal protist in humans and animals worldwide. Wild and domestic animals are thought to be reservoirs of Blastocystis subtypes that also infect humans. There are limited studies on the prevalence and subtype distribution of Blastocystis in horses. In this study, 185 fecal samples were collected from horses (1 month to 17 years of age) in four regions of Colombia (Sabana de Bogotá, Costa Atlántica, Llanos Orientales, and Bogotá D.C.). Blastocystis presence and subtypes were determined by PCR and next generation amplicon sequencing. Eighty-one (43.8%) horses were positive for Blastocystis, with positive horses in all four regions. Molecular characterization identified 12 Blastocystis subtypes, 10 known subtypes (ST1, ST3–ST6, ST10, ST14, ST25, ST26), and 2 novel subtypes (ST33 and ST34). The validity of the novel subtypes was confirmed via phylogenetic and pairwise distance analyses of the full-length SSU rRNA gene sequences. Mixed subtype infections were common (55.6% of Blastocystis-positive horses). ST10 was the most prevalent subtype, present in 82.8% of Blastocystis-positive horses. Potentially zoonotic subtypes were identified in 88.9% of the Blastocystis-positive horses. This constitutes the most comprehensive study of Blastocystis in horses. Our findings indicate that horses harbor potentially zoonotic subtypes and could contribute to the transmission of Blastocystis to humans.

Enhanced detection of Giardia duodenalis mixed assemblage infections in pre-weaned dairy calves using next generation sequencing

Giardia duodenalis is one of the most common parasitic causes of gastrointestinal illness in humans worldwide with widespread infections in mammalian hosts. It frequently infects cattle, producing a high number of cysts. Cattle can harbor both host-adapted assemblage E and human pathogenic assemblages A and B. Previous studies have demonstrated that conventional molecular methods lack the sensitivity required for detecting mixed infections and that the occurrence of mixed infections in cattle are likely underestimated. To investigate the presence of mixed assemblage infections in cattle, 2539 pre-weaned dairy calves from the United Stated were screened for the presence of G. duodenalis using molecular tools. Next generation amplicon sequencing (NGS) was then performed for a subset of around 30% of positive samples (n = 314) and the ability of NGS and Sanger sequencing to detect mixed assemblage infections was compared. The overall prevalence of G. duodenalis in pre-weaned dairy calves in the sample using PCR was high (1013/2539; 39.9%). Molecular genotyping identified only assemblage A and E, with assemblage E as the predominant assemblage. Out of the 314 samples examined by both Sanger and NGS, 9 samples (2.9%) were identified as mixed A/E infections by Sanger while NGS identified 56 samples (17.8%), which was six-times more mixed infections compared with Sanger sequencing. NGS demonstrated superior sensitivity to Sanger in detecting assemblages present in low abundances. The percentage of mixed A/E infections found in the sampled dairy calves was higher than was hypothesized using values from the literature. This underestimation could be present in the wider cattle population as well, though further exploration would be needed to verify that claim. These findings highlight the advantages of NGS application in molecular epidemiological studies of Giardia. To better understand Giardia epidemiology, establish routes of transmission, and assess the potential role of cattle and other animals as a source of environmental contamination with zoonotic assemblages it is necessary to uncover mixed assemblage infections.

Parasitic trophic mode of plant host affects the extent of colonization, but does not induce systematic shifts in the composition of foliar endophytic assemblages in temperate meadow ecosystems

Abstract Foliar endophytic bacteria and fungi are increasingly being recognized as important drivers of plant host phenotype—affecting a wide range of eco‐physiological processes. However, we are still lacking fundamental ecosystem‐level knowledge about the structure, function and inter‐species interactions in endophytic assemblages associated with plant hosts sharing a common life strategy or ecological specialization. In this study, we chose two groups of plants with contrasting physiology as model systems: parasites and their hosts. We assessed whether plant life‐history strategy, namely differences in nutrient acquisition and accumulation, plays a role in structuring above‐ground microbiomes under field conditions. We focused on the structure, colonization extent and potential function of foliar endophytic bacteria and fungi in three root hemiparasitic species (Orobanchaceae), one stem holoparasite (Convolvulaceae), and their potential host plants co‐occurring in species‐rich temperate grassland ecosystems. For this purpose, we combined next generation amplicon sequencing with quantitative real‐time PCR, chemical analyses of leaf tissue, and, in the case of bacteria, functional predictions using information deposited in available databases. We found the foliar endophytic assemblages to be diverse, dominated by generalist taxa, but highly similar across all studied species. Despite of the highly contrasting leaf tissue chemistry in the parasitic and non‐parasitic plant species, the parasitic trophic mode did not induce systematic shifts in the diversity, composition or predicted biogeochemical function of the endophytic microbiomes under field conditions. However, compared to their potential hosts, leaves of both hemiparasitic and holoparasitic species harboured significantly lower fungal counts, estimated as ß‐actin gene copies ng DNA−1, which suggests that parasitic plants may possess mechanisms to regulate the extent of colonization by endophytic fungi. A free Plain Language Summary can be found within the Supporting Information of this article.

A new diagnostic approach to fast-track and increase the accessibility of gastrointestinal nematode identification from faeces: FECPAKG2 egg nemabiome metabarcoding

Effective gastrointestinal nematode management in livestock industries is becoming increasingly difficult due to the rise of anthelmintic resistance and changes in the temporal and geographical distribution of major gastrointestinal nematodes. Underpinning the response to these challenges is the need for a fast-tracked diagnostic identification technique, making it easier for livestock producers to make informed gastrointestinal nematode management decisions. The traditional ‘gold-standard’ approach, larval culture followed by morphological differentiation, is laborious and potentially inaccurate. We developed a new diagnostic approach to identify gastrointestinal nematodes that integrates a remote-location digital faecal egg count platform, FECPAKG2, with internal transcribed spacer 2 (ITS2) nemabiome metabarcoding. The technique involves a quick and simple protocol to harvest concentrated strongyle eggs from the FECPAKG2 cassette utilising a repurposed pipette tip, followed by DNA isolation and Illumina next generation amplicon sequencing. The gastrointestinal nematode compositions and alpha diversity generated by our FECPAKG2 egg nemabiome metabarcoding approach was not significantly different to traditional morphological larval differentiation and nemabiome metabarcoding of larval and faecal samples. We demonstrated that storing FECPAKG2 harvested eggs in either DNA isolation lysis buffer or 80% ethanol (v/v) had no impact on gastrointestinal nematode identification outcomes for at least 60 days; enabling the transport of biological samples from their remote origins to a molecular diagnostic facility for nemabiome metabarcoding, in the absence of a cold chain. We discovered that sustained gastrointestinal nematode egg embryonation in the lysis buffer storage solution lead to higher yields of DNA compared with ethanol-stored gastrointestinal nematode eggs or faeces with gastrointestinal nematode eggs. Taking advantage of an already well-established platform such as FECPAKG2, and providing the livestock producers that use it with the option to identify gastrointestinal nematodesin their samples and contribute to large-scale gastrointestinal nematode distribution and/or anthelmintic resistance surveys, is an important future direction for the FECPAKG2 egg nemabiome metabarcoding approach.

The ratio of microbial-to-host nucleic acid impacts detection of microbial nucleic acid in placental tissue

The placental microbiome is characterized metagenomically as a low-biomass, sparse, reliably detected collection of genetic material that is vastly outnumbered by source host DNA. This complicates its molecular detection, leading some research teams to achieve detection while others attribute detection to contamination. Contributing to this misclassification is the failure to appreciate microbial:host nucleic acid stoichiometry in amplicon generation via polymerase chain reaction (PCR). We hypothesized that germ-free pregnant mice inoculated with specific microbes during gestation will have placentas with detectable microbial signatures via targeted quantitative PCR (qPCR) but not untargeted standard PCR methods.

DdPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samples

BackgroundOne limiting factor of short amplicon 16S rRNA gene sequencing approaches is the use of low DNA amounts in the amplicon generation step. Especially for low-biomass samples, insufficient or even commonly undetectable DNA amounts can limit or prohibit further analysis in standard protocols.ResultsUsing a newly established protocol, very low DNA input amounts were found sufficient for reliable detection of bacteria using 16S rRNA gene sequencing compared to standard protocols. The improved protocol includes an optimized amplification strategy by using a digital droplet PCR. We demonstrate how PCR products are generated even when using very low concentrated DNA, unable to be detected by using a Qubit. Importantly, the use of different 16S rRNA gene primers had a greater effect on the resulting taxonomical profiles compared to using high or very low initial DNA amounts.ConclusionOur improved protocol takes advantage of ddPCR and allows faithful amplification of very low amounts of template. With this, samples of low bacterial biomass become comparable to those with high amounts of bacteria, since the first and most biasing steps are the same. Besides, it is imperative to state DNA concentrations and volumes used and to include negative controls indicating possible shifts in taxonomical profiles. Despite this, results produced by using different primer pairs cannot be easily compared.

The role of genetic diversity and arbuscular mycorrhizal fungal diversity in population recovery of the semi-natural grassland plant species Succisa pratensis

BackgroundEcosystem restoration is as a critical tool to counteract the decline of biodiversity and recover vital ecosystem services. Restoration efforts, however, often fall short of meeting their goals. Although functionally important levels of biodiversity can significantly contribute to the outcome of ecosystem restoration, they are often overlooked. One such important facet of biodiversity is within-species genetic diversity, which is fundamental to population fitness and adaptation to environmental change. Also the diversity of arbuscular mycorrhizal fungi (AMF), obligate root symbionts that regulate nutrient and carbon cycles, potentially plays a vital role in mediating ecosystem restoration outcome. In this study, we investigated the relative contribution of intraspecific population genetic diversity, AMF diversity, and their interaction, to population recovery of Succisa pratensis, a key species of nutrient poor semi natural grasslands. We genotyped 180 individuals from 12 populations of S. pratensis and characterized AMF composition in their roots, using microsatellite markers and next generation amplicon sequencing, respectively. We also investigated whether the genetic makeup of the host plant species can structure the composition of root-inhabiting AMF communities.ResultsOur analysis revealed that population allelic richness was strongly positively correlated to relative population growth, whereas AMF richness and its interaction with population genetic diversity did not significantly contribute. The variation partitioning analysis showed that, after accounting for soil and spatial variables, the plant genetic makeup explained a small but significant part of the unique variation in AMF communities.ConclusionsOur results confirm that population genetic diversity can contribute to population recovery, highlighting the importance of within-species genetic diversity for the success of restoration. We could not find evidence, however, that population recovery benefits from the presence of more diverse AMF communities. Our analysis also showed that the genetic makeup of the host plant structured root-inhabiting AMF communities, suggesting that the plant genetic makeup may be linked to genes that control symbiosis development.

A DNA Nanoflower-Assisted Separation-Free Nucleic Acid Detection Platform with a Commercial Pregnancy Test Strip.

There is a constant drive for affordable point-of-care testing (POCT) technologies for the detection of infectious human diseases. Herein, we report a simple platform for DNA detection that takes advantage of four techniques: commercially available pregnancy test strips (PTS), amplicon generation via loop-mediated isothermal amplification (LAMP), toehold-mediated strand displacement, and noncovalent immobilization of DNA on paper surface with DNA nanoflowers. This simple, separation-free platform is highly specific, as demonstrated with the detection of rtL180M, a single-nucleotide polymorphism observed in hepatitis B virus (HBV) associated with antiviral drug resistance. It is very sensitive, capable of detecting the targeted mutation at 2 copies μL-1 . It is able to correctly identify the unmutated and rtL180M genome types of HBV in clinical samples. Given its wide adaptability, we expect this platform can be easily modified for the detection of genetic variations associated with various pathogens and human diseases.

A DNA Nanoflower‐Assisted Separation‐Free Nucleic Acid Detection Platform with a Commercial Pregnancy Test Strip

AbstractThere is a constant drive for affordable point‐of‐care testing (POCT) technologies for the detection of infectious human diseases. Herein, we report a simple platform for DNA detection that takes advantage of four techniques: commercially available pregnancy test strips (PTS), amplicon generation via loop‐mediated isothermal amplification (LAMP), toehold‐mediated strand displacement, and noncovalent immobilization of DNA on paper surface with DNA nanoflowers. This simple, separation‐free platform is highly specific, as demonstrated with the detection of rtL180M, a single‐nucleotide polymorphism observed in hepatitis B virus (HBV) associated with antiviral drug resistance. It is very sensitive, capable of detecting the targeted mutation at 2 copies μL−1. It is able to correctly identify the unmutated and rtL180M genome types of HBV in clinical samples. Given its wide adaptability, we expect this platform can be easily modified for the detection of genetic variations associated with various pathogens and human diseases.

Next generation sequencing based in-house HIV genotyping method: validation report

BackgroundHIV genotyping has had a significant impact on the care and treatment of HIV/AIDS. At a clinical level, the test guides physicians on the choice of treatment regimens. At the surveillance level, it informs policy on consolidated treatment guidelines and microbial resistance control strategies. Until recently, the conventional test has utilized the Sanger sequencing (SS) method. Unlike Next Generation Sequencing (NGS), SS is limited by low data throughput and the inability of detecting low abundant drug-resistant variants. NGS can improve sensitivity and quantitatively identify low-abundance variants; in addition, it has the potential to improve efficiency as well as lowering costs when samples are batched. Despite the NGS benefits, its utilization in clinical drug resistance profiling is faced with mixed reactions. These are largely based on a lack of a consensus regarding the quality control strategy. Nonetheless, transitional views suggest validating the method against the gold-standard SS. Therefore, we present a validation report of an NGS-based in-house HIV genotyping method against the SS method in Uganda.ResultsSince there were no established proficiency test panels for NGS-based HIV genotyping, 15 clinical plasma samples for routine care were utilized. The use of clinical samples allowed for accuracy and precision studies. The workflow involved four main steps; viral RNA extraction, targeted amplicon generation, amplicon sequencing and data analysis. Accuracy of 98% with an average percentage error of 3% was reported for the NGS based assay against the SS platform demonstrating similar performance. The coefficient of variation (CV) findings for both the inter-run and inter-personnel precision showed no variability (CV ≤ 0%) at the relative abundance of ≥ 20%. For both inter-run and inter-personnel, a variation that affected the precision was observed at 1% frequency. Overall, for all the frequencies, CV registered a small range of (0–2%).ConclusionThe NGS-based in-house HIV genotyping method fulfilled the minimum requirements that support its utilization for drug resistance profiling in a clinical setting of a low-income country. For more inclusive quality control studies, well-characterized wet panels need to be established.

Identifying optimal bioinformatics protocols for aerosol microbial community data.

Microbes are fundamental to Earth’s ecosystems, thus understanding ecosystem connectivity through microbial dispersal is key to predicting future ecosystem changes in a warming world. However, aerial microbial dispersal remains poorly understood. Few studies have been performed on bioaerosols (microorganisms and biological fragments suspended in the atmosphere), despite them harboring pathogens and allergens. Most environmental microbes grow poorly in culture, therefore molecular approaches are required to characterize aerial diversity. Bioinformatic tools are needed for processing the next generation sequencing (NGS) data generated from these molecular approaches; however, there are numerous options and choices in the process. These choices can markedly affect key aspects of the data output including relative abundances, diversity, and taxonomy. Bioaerosol samples have relatively little DNA, and often contain novel and proportionally high levels of contaminant organisms, that are difficult to identify. Therefore, bioinformatics choices are of crucial importance. A bioaerosol dataset for bacteria and fungi based on the 16S rRNA gene (16S) and internal transcribed spacer (ITS) DNA sequencing from parks in the metropolitan area of Auckland, Aotearoa New Zealand was used to develop a process for determining the bioinformatics pipeline that would maximize the data amount and quality generated. Two popular tools (Dada2 and USEARCH) were compared for amplicon sequence variant (ASV) inference and generation of an ASV table. A scorecard was created and used to assess multiple outputs and make systematic choices about the most suitable option. The read number and ASVs were assessed, alpha diversity was calculated (Hill numbers), beta diversity (Bray–Curtis distances), differential abundance by site and consistency of ASVs were considered. USEARCH was selected, due to higher consistency in ASVs identified and greater read counts. Taxonomic assignment is highly dependent on the taxonomic database used. Two popular taxonomy databases were compared in terms of number and confidence of assignments, and a combined approach developed that uses information in both databases to maximize the number and confidence of taxonomic assignments. This approach increased the assignment rate by 12–15%, depending on amplicon and the overall assignment was 77% for bacteria and 47% for fungi. Assessment of decontamination using “decontam” and “microDecon” was performed, based on review of ASVs identified as contaminants by each and consideration of the probability of them being legitimate members of the bioaerosol community. For this example, “microDecon’s” subtraction approach for removing background contamination was selected. This study demonstrates a systematic approach to determining the optimal bioinformatics pipeline using a multi-criteria scorecard for microbial bioaerosol data. Example code in the R environment for this data processing pipeline is provided.

Molecular Detection and Characterization of Blastocystis sp. and Enterocytozoon bieneusi in Cattle in Northern Spain.

Some enteric parasites causing zoonotic diseases in livestock have been poorly studied or even neglected. This is the case in stramenopile Blastocystis sp. and the microsporidia Enterocytozoon bieneusi in Spain. This transversal molecular epidemiological survey aims to estimate the prevalence and molecular diversity of Blastocystis sp. and E. bieneusi in cattle faecal samples (n = 336) in the province of Álava, Northern Spain. Initial detection of Blastocystis and E. bieneusi was carried out by polymerase chain reaction (PCR) and Sanger sequencing of the small subunit (ssu) rRNA gene and internal transcribed spacer (ITS) region, respectively. Intra-host Blastocystis subtype diversity was further investigated by next generation amplicon sequencing (NGS) of the ssu rRNA gene in those samples that tested positive by conventional PCR. Amplicons compatible with Blastocystis sp. and E. bieneusi were observed in 32.1% (108/336, 95% CI: 27.2–37.4%) and 0.6% (2/336, 95% CI: 0.0–1.4%) of the cattle faecal samples examined, respectively. Sanger sequencing produced ambiguous/unreadable sequence data for most of the Blastocystis isolates sequenced. NGS allowed the identification of 10 Blastocystis subtypes including ST1, ST3, ST5, ST10, ST14, ST21, ST23, ST24, ST25, and ST26. All Blastocystis-positive isolates involved mixed infections of 2–8 STs in a total of 31 different combinations. The two E. bieneusi sequences were confirmed as potentially zoonotic genotype BEB4. Our data demonstrate that Blastocystis mixed subtype infections are extremely frequent in cattle in the study area. NGS was particularly suited to discern underrepresented subtypes or mixed subtype infections that were undetectable or unreadable by Sanger sequencing. The presence of zoonotic Blastocystis ST1, ST3, and ST5, and E. bieneusi BEB4 suggest cross-species transmission and a potential risk of human infection/colonization.