Multiplexed In Vivo Screening Using Barcoded Aptamer Technology to Identify Oligonucleotide-Based Targeting Reagents.

Recent FDA approvals of mRNA vaccines, short-interfering RNAs, and antisense oligonucleotides highlight the success of oligonucleotides as therapeutics. Aptamers are excellent affinity reagents that can selectively label protein biomarkers, but their clinical application has lagged. When formulating a given aptamer for in vivo use, molecular design details can determine biostability and biodistribution; therefore, extensive postselection manipulation is often required for each new design to identify clinically useful reagents harboring improved pharmacokinetic properties. Few methods are available to comprehensively screen such aptamers, especially in vivo, constituting a significant bottleneck in the field. In this study, we introduce barcoded aptamer technology (BApT) for multiplexed screening of predefined aptamer formulations in vitro and in vivo. We demonstrate this technology by simultaneously investigating 20 aptamer formulations, each harboring different molecular designs, for targeting Non-Small Cell Lung Cancer cells and tumors. Screening in vitro identified a 45 kDa bispecific formulation as the best cancer cell targeting reagent, whereas screening in vivo identified a 30 kDa monomeric formulation as the best tumor-specific targeting reagent. The multiplexed analysis pipeline also identified biodistribution phenotypes shared among formulations with similar molecular architectures. The BApT approach we describe here has the potential for broad application to fields where oligonucleotide-based targeting reagents are desired.

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

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

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

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

Impact of TP53 gene variants on prognosis and survival of childhood acute lymphoblastic leukemia.

The tumor suppressor protein 53 (TP53) gene is one of the most studied genes in cancer. Although TP53 variants are rare events in acute leukemia, recent observations showed that relapse samples might harbor TP53 variants. Here, we aimed to determine TP53 variants (hotspot region, exon 4-11) in childhood acute lymphoblastic leukemia (B and T-ALL) patients (n = 94) including diagnostic-relapse pairs (n = 15) by amplicon sequencing and evaluate the clinical impact of these variants. In total, nine different (E298*, R283C, R273H, L252F, C229F, I195T, E286G, c.955_956insC, and c.920-1G > C) variants were identified in 17 (18%) childhood ALL patients. c.(920-1G> C) splice site variant and c.(955_956insC) insertion were reported for the first time. In diagnose-relapse pair samples, we identified acquired and/or loss of TP53 variants in the samples at the time of relapse. TP53 variants were found to be more common in T-ALL (37%) than in B-ALL patients (9%). Pathogenic TP53 variants were associated with a shorter overall survival time (p = 0.001).TP53 variants were found to be associated with inferior outcomes in our cohort and can be an independent risk factor for poor prognosis in childhood acute leukemia patients. Identification of low-frequent variants with next-generation sequencing approaches enables better knowledge of the clonal dynamics of ALL.

Identification of Multiple Blastocystis Subtypes in Domestic Animals From Colombia Using Amplicon-Based Next Generation Sequencing.

Blastocystis is frequently reported in fecal samples from animals and humans worldwide, and a variety of subtypes (STs) have been observed in wild and domestic animals. In Colombia, few studies have focused on the transmission dynamics and epidemiological importance of Blastocystis in animals. In this study, we characterized the frequency and subtypes of Blastocystis in fecal samples of domestic animals including pigs, minipigs, cows, dogs, horses, goats, sheep, and llama from three departments of Colombia. Of the 118 fecal samples included in this study 81.4% (n = 96) were positive for Blastocystis using a PCR that amplifies a fragment of the small subunit ribosomal RNA (SSU rRNA) gene. PCR positive samples were sequenced by next generation amplicon sequencing (NGS) to determine subtypes. Eleven subtypes were detected, ten previously reported, ST5 (50.7%), ST10 (47.8%), ST25 (34.3%), ST26 (29.8%), ST21 (22.4%), ST23 (22.4%), ST1 (17.9%), ST14 (16.4%), ST24 (14.9%), ST3 (7.5%), and a novel subtype, named ST32 (3.0%). Mixed infection and/or intra -subtype variations were identified in most of the samples. Novel ST32 was observed in two samples from a goat and a cow. To support novel subtype designation, a MinION based sequencing strategy was used to generate the full-length of the SSU rRNA gene. Comparison of full-length nucleotide sequences with those from current valid subtypes supported the designation of ST32. This is the first study in Colombia using NGS to molecularly characterize subtypes of Blastocystis in farm animals. A great diversity of subtypes was observed in domestic animals including subtypes previously identified in humans. Additionally, subtype overlap between the different hosts examined in this study were observed. These findings highlight the presence of Blastocystis subtypes with zoonotic potential in farm animals indicating that farm animals could play a role in transmission to humans.

An Illumina MiSeq-Based Amplicon Sequencing Method for the Detection of Mixed Parasite Infections Using the Blastocystis SSU rRNA Gene as an Example.

Parasite mixed infections remain a relatively unexplored field in part due to the difficulties of unraveling complex mixtures of parasite DNA using classical methods of sequencing. Next-generation amplicon sequencing (NGS) is a powerful tool for exploring mixed infections of multiple genetic variants of the same parasite in clinical, environmental (water or soil), or food samples. Here, we provide a method for NGS-based detection of mixed parasite infections which uses the Blastocystis SSU rRNA gene as an example and includes steps for parasite concentration, DNA extraction, sequencing library preparation, and bioinformatic analysis.

Diversity and Abundance of Microbial Communities in UASB Reactors during Methane Production from Hydrolyzed Wheat Straw and Lucerne.

The use of straw for biofuel production is encouraged by the European Union. A previous study showed the feasibility of producing biomethane in upflow anaerobic sludge blanket (UASB) reactors using hydrolyzed, steam-pretreated wheat straw, before and after dark fermentation with Caldicellulosiruptor saccharolyticus, and lucerne. This study provides information on overall microbial community development in those UASB processes and changes related to acidification. The bacterial and archaeal community in granular samples was analyzed using high-throughput amplicon sequencing. Anaerobic digestion model no. 1 (ADM1) was used to predict the abundance of microbial functional groups. The sequencing results showed decreased richness and diversity in the microbial community, and decreased relative abundance of bacteria in relation to archaea, after process acidification. Canonical correspondence analysis showed significant negative correlations between the concentration of organic acids and three phyla, and positive correlations with seven phyla. Organic loading rate and total COD fed also showed significant correlations with microbial community structure, which changed over time. ADM1 predicted a decrease in acetate degraders after a decrease to pH ≤ 6.5. Acidification had a sustained effect on the microbial community and process performance.

Soil microbial communities associated with giant sequoia: How does the world's largest tree affect some of the world's smallest organisms?

Giant sequoia (Sequoiadendron giganteum) is an iconic conifer that lives in relict populations on the western slopes of the California Sierra Nevada. In these settings, it is unusual among the dominant trees in that it associates with arbuscular mycorrhizal fungi rather than ectomycorrhizal fungi. However, it is unclear whether differences in microbial associations extend more broadly to nonmycorrhizal components of the soil microbial community. To address this question, we used next-generation amplicon sequencing to characterize bacterial/archaeal and fungal microbiomes in bulk soil (0-5 cm) beneath giant sequoia and co-occurring sugar pine (Pinus lambertiana) individuals. We did this across two groves with distinct parent material in Yosemite National Park, USA. We found tree-associated differences were apparent despite a strong grove effect. Bacterial/archaeal richness was greater beneath giant sequoia than sugar pine, with a core community double the size. The tree species also harbored compositionally distinct fungal communities. This pattern depended on grove but was associated with a consistently elevated relative abundance of Hygrocybe species beneath giant sequoia. Compositional differences between host trees correlated with soil pH and soil moisture. We conclude that the effects of giant sequoia extend beyond mycorrhizal mutualists to include the broader community and that some but not all host tree differences are grove-dependent.

Blastocystis subtype distribution in domestic and captive wild bird species from Brazil using next generation amplicon sequencing.

Blastocystis is a food and water borne intestinal parasite commonly identified in humans and many other animals worldwide. Of the nine potentially zoonotic subtypes of Blastocystis, seven have been reported in bird species. However molecular studies of Blastocystis subtype diversity in birds are limited. In this study, fecal samples from 109 domestic and captive wild birds from Minas Gerais, Brazil were tested for the presence of Blastocystis subtypes using PCR and next generation amplicon sequencing of a fragment of the small subunit ribosomal RNA (SSU rRNA) gene. Birds from 11 orders and 38 species from both local markets and bird conservation facilities were sampled. Blastocystis was present in 14.7% of samples, and eight subtypes, six previously reported (ST5, ST6, ST7, ST10, ST14, ST24) and two novel subtypes (named ST27 and ST28), were identified. The most commonly identified subtypes were ST7 and ST6 identified in 10 (62.5%) and 6 (37.5%) of 16 Blastocystis positive samples. At least one of the three zoonotic subtypes identified (ST5, ST6, and ST7) was found in 81.3% of Blastocystis positive samples. Infection with multiple Blastocystis subtypes was common and identified in 62.5% of positive samples. This study is the first to use next generation amplicon sequencing to characterize Blastocystis subtype diversity in birds. The findings presented here confirm that birds may serve as reservoirs of zoonotic subtypes of Blastocystis and that the role of birds in transmission of Blastocystis to humans requires further study.

Microbial Succession of Anaerobic Chitin Degradation in Freshwater Sediments.

Chitin is massively produced by freshwater plankton species as a structural element of their exoskeleton or cell wall. At the same time, chitin does not accumulate in the predominantly anoxic sediments, underlining its importance as carbon and nitrogen sources for sedimentary microorganisms. We studied chitin degradation in littoral sediment of Lake Constance, Central Europe's third largest lake. Turnover of the chitin analog methyl-umbelliferyl-N,N-diacetylchitobioside (MUF-DC) was highest in the upper oxic sediment layer, with 5.4 nmol MUF-DC h-1 (g sediment [dry weight])-1 In the underlying anoxic sediment layers, chitin hydrolysis decreased with depth from 1.1 to 0.08 nmol MUF-DC h-1 (g sediment [dry weight])-1 Bacteria involved in chitin degradation were identified by 16S rRNA (gene) amplicon sequencing of anoxic microcosms incubated in the presence of chitin compared to microcosms amended either with N-acetylglucosamine as the monomer of chitin or no substrate. Chitin degradation was driven by a succession of bacteria responding specifically to chitin only. The early phase (0 to 9 days) was dominated by Chitinivibrio spp. (Fibrobacteres). The intermediate phase (9 to 21 days) was characterized by a higher diversity of chitin responders, including, besides Chitinivibrio spp., also members of the phyla Bacteroidetes, Proteobacteria, Spirochaetes, and Chloroflexi In the late phase (21 to 43 days), the Chitinivibrio populations broke down with a parallel strong increase of Ruminiclostridium spp. (formerly Clostridium cluster III, Firmicutes), which became the dominating chitin responders. Our study provides quantitative insights into anaerobic chitin degradation in lake sediments and linked this to a model of microbial succession associated with this activity.IMPORTANCE Chitin is the most abundant biopolymer in aquatic environments, with a direct impact on the carbon and nitrogen cycles. Despite its massive production as a structural element of crustaceans, insects, or algae, it does not accumulate in sediments. Little is known about its turnover in predominantly anoxic freshwater sediments and the responsible microorganisms. We proved that chitin is readily degraded under anoxic conditions and linked this to a succession of the members of the responsible microbial community over a 43-day period. While Fibrobacteres and Firmicutes members were driving the early and late phases of chitin degradation, respectively, a more diverse community was involved in chitin degradation in the intermediate phase. Entirely different microorganisms responded toward the chitin monomer N-acetylglucosamine, which underscores that soluble monomers are poor and misleading substrates to study polymer-utilizing microorganisms. Our study provides quantitative insights into the microbial ecology driving anaerobic chitin degradation in freshwater sediments.

The Active Sulfate-Reducing Microbial Community in Littoral Sediment of Oligotrophic Lake Constance.

Active sulfate-reducing microorganisms (SRM) in freshwater sediments are under-examined, despite the well-documented cryptic sulfur cycle occurring in these low-sulfate habitats. In Lake Constance sediment, sulfate reduction rates of up to 1,800 nmol cm-3 day-1 were previously measured. To characterize its SRM community, we used a tripartite amplicon sequencing approach based on 16S rRNA genes, 16S rRNA, and dsrB transcripts (encoding the beta subunit of dissimilatory sulfite reductase). We followed the respective amplicon dynamics in four anoxic microcosm setups supplemented either with (i) chitin and sulfate, (ii) sulfate only, (iii) chitin only, or (iv) no amendment. Chitin was used as a general substrate for the whole carbon degradation chain. Sulfate turnover in sulfate-supplemented microcosms ranged from 38 to 955 nmol day-1 (g sediment f. wt.)-1 and was paralleled by a decrease of 90-100% in methanogenesis as compared to the respective methanogenic controls. In the initial sediment, relative abundances of recognized SRM lineages accounted for 3.1 and 4.4% of all bacterial 16S rRNA gene and 16S rRNA sequences, respectively. When normalized against the 1.4 × 108 total prokaryotic 16S rRNA gene copies as determined by qPCR and taking multiple rrn operons per genome into account, this resulted in approximately 105-106 SRM cells (g sediment f. wt.)-1. The three amplicon approaches jointly identified Desulfobacteraceae and Syntrophobacteraceae as the numerically dominant and transcriptionally most active SRM in the initial sediment. This was corroborated in the time course analyses of sulfate-consuming sediment microcosms irrespective of chitin amendment. Uncultured dsrAB family-level lineages constituted in sum only 1.9% of all dsrB transcripts, with uncultured lineage 5 and 6 being transcriptionally most active. Our study is the first holistic molecular approach to quantify and characterize active SRM including uncultured dsrAB lineages not only in Lake Constance but for lake sediments in general.

Substrate-Induced Response in Biogas Process Performance and Microbial Community Relates Back to Inoculum Source.

This study investigated whether biogas reactor performance, including microbial community development, in response to a change in substrate composition is influenced by initial inoculum source. For the study, reactors previously operated with the same grass⁻manure mixture for more than 120 days and started with two different inocula were used. These reactors initially showed great differences depending on inoculum source, but eventually showed similar performance and overall microbial community structure. At the start of the present experiment, the substrate was complemented with milled feed wheat, added all at once or divided into two portions. The starting hypothesis was that process performance depends on initial inoculum source and microbial diversity, and thus that reactor performance is influenced by the feeding regime. In response to the substrate change, all reactors showed increases and decreases in volumetric and specific methane production, respectively. However, specific methane yield and development of the microbial community showed differences related to the initial inoculum source, confirming the hypothesis. However, the different feeding regimes had only minor effects on process performance and overall community structure, but still induced differences in the cellulose-degrading community and in cellulose degradation.

Comprehensive Study on Enteric Viruses and Indicators in Surface Water in Kyoto, Japan, During 2014-2015 Season.

Certain enteric viruses that are present in the water environment are potential risk factors of waterborne infections. To better understand the impact of viruses in water, both enteric viruses and their potential indicators should be comparatively investigated. In this study, occurrences of GI- and GII-noroviruses (NoVs), sapovirus (SaV), rotavirus (RoV), Aichi virus 1 (AiV-1), enterovirus (EV), and pepper mild mottle virus (PMMoV) were quantitatively determined in surface water samples in Japan. Additionally, the genotype distribution of GI- and GII-NoVs was determined using a next-generation amplicon sequencing. PMMoV was the most abundant virus regardless of season and location, indicating its usefulness as an indicator for the viral contamination of water. Other potential indicators, AiV and EV, were less abundant than GII-NoV. Viruses other than PMMoV showed seasonality, i.e., EV and other viruses (NoVs, SaV, RoV, and AiV-1) became prevalent during summer and winter, respectively. SaV showed a relatively high abundance at a location that was affected by untreated wastewater. Regarding NoV genotypes, GI.1, GI.2, GI.4, GI.5, GI.6, GII.3, GII.4, GII.6, and GII.17 were found from the surface water samples. GII.4 and GII.17 seemed to have contributed to the high abundance of GII-NoV in the samples. Interestingly, GII.17 strains became prevalent in the water samples before becoming prevalent among gastroenteritis patients in Japan. These findings provide further insights into the properties of viruses as contaminants in the water environment.

Making and Sequencing Heavily Multiplexed, High-Throughput 16S Ribosomal RNA Gene Amplicon Libraries Using a Flexible, Two-Stage PCR Protocol.

Deep sequencing of polymerase chain reaction (PCR)-amplified small subunit (16S or 18S) ribosomal RNA (rRNA) genes fragments is commonly employed to characterize the composition and structure of microbial communities. Preparing genomic DNA for sequencing of such gene fragments on Illumina sequencers can be performed in a straightforward, two-stage PCR method, described herein. The protocol described allows for up to 384 samples to be sequenced simultaneously, and provides great flexibility in choice of primers.

Co-infections with multiple genotypes of Anaplasma marginale in cattle indicate pathogen diversity.

BACKGROUND: Only a few studies have examined the presence of Anaplasma marginale and Anaplasma centrale in South Africa, and no studies have comprehensively examined these species across the whole country. To undertake this country-wide study we adapted a duplex quantitative real-time PCR (qPCR) assay for use in South Africa but found that one of the genes on which the assay was based was variable. Therefore, we sequenced a variety of field samples and tested the assay on the variants detected. We used the assay to screen 517 cattle samples sourced from all nine provinces of South Africa, and subsequently examined A. marginale positive samples for msp1α genotype to gauge strain diversity. RESULTS: Although the A. marginale msp1ß gene is variable, the qPCR functions at an acceptable efficiency. The A. centrale groEL gene was not variable within the qPCR assay region. Of the cattle samples screened using the assay, 57% and 17% were found to be positive for A. marginale and A. centrale, respectively. Approximately 15% of the cattle were co-infected. Msp1α genotyping revealed 36 novel repeat sequences. Together with data from previous studies, we analysed the Msp1a repeats from South Africa where a total of 99 repeats have been described that can be attributed to 190 msp1α genotypes. While 22% of these repeats are also found in other countries, only two South African genotypes are also found in other countries; otherwise, the genotypes are unique to South Africa. CONCLUSIONS: Anaplasma marginale was prevalent in the Western Cape, KwaZulu-Natal and Mpumalanga and absent in the Northern Cape. Anaplasma centrale was prevalent in the Western Cape and KwaZulu-Natal and absent in the Northern Cape and Eastern Cape. None of the cattle in the study were known to be vaccinated with A. centrale, so finding positive cattle indicates that this organism appears to be naturally circulating in cattle. A diverse population of A. marginale strains are found in South Africa, with some msp1α genotypes widely distributed across the country, and others appearing only once in one province. This diversity should be taken into account in future vaccine development studies.

Importance of inoculum source and initial community structure for biogas production from agricultural substrates.

This study evaluated the importance of inoculum source for start-up and operation of biogas processes. Three different inocula with different community structure were used to initiate six laboratory continuous stirred tank reactor (CSTR) processes operated with a grass manure mixture as substrate. The processes were evaluated by chemical and microbiological analysis, by targeting the overall bacterial community and potential cellulose-degrading bacteria. As expected, the results showed a large difference in community structure in the inocula and in process performance during the first hydraulic retention time (HRT). However, the performance and overall microbial community structure became similar in the reactors over time. An inoculum from a high-ammonia process, characterized by low diversity and low degradation efficiency, took the longest time to reach stability and final methane yield. The overall bacterial community was mainly shaped by the operating conditions but, interestingly, potential cellulose-degrading bacteria seemed mainly to originate from the substrate.

A Small Number of Low-abundance Bacteria Dominate Plant Species-specific Responses during Rhizosphere Colonization.

Plant growth can be affected by soil bacteria. In turn, plants are known to influence soil bacteria through rhizodeposits and changes in abiotic conditions. We aimed to quantify the phylotype richness and relative abundance of rhizosphere bacteria that are actually influenced in a plant species-specific manner and to determine the role of the disproportionately large diversity of low-abundance bacteria belonging to the rare biosphere (<0.1 relative abundance) in this process. In addition, we aimed to determine whether plant phylogeny has an influence on the plant species-specific rhizosphere bacterial community. For this purpose, 19 herbaceous plant species from five different plant orders were grown in a common soil substrate. Bacterial communities in the initial soil substrate and the established rhizosphere soils were compared by 16S rRNA gene amplicon sequencing. Only a small number of bacterial operational taxonomic units (OTUs, 97% sequence identity) responded either positively (ca. 1%) or negatively (ca. 1%) to a specific plant species. On average, 91% of plant-specific positive response OTUs comprised bacteria belonging to the rare biosphere, highlighting that low-abundance populations are metabolically active in the rhizosphere. In addition, low-abundance OTUs were in terms of their summed relative abundance major drivers of the bacterial phyla composition across the rhizosphere of all tested plant species. However, no effect of plant phylogeny could be observed on the established rhizosphere bacterial communities, neither when considering differences in the overall established rhizosphere communities nor when considering plant species-specific responders only. Our study provides a quantitative assessment of the effect of plants on their rhizosphere bacteria across multiple plant orders. Plant species-specific effects on soil bacterial communities involved only 18-111 bacterial OTUs out of several 1000s; this minority may potentially impact plant growth in plant-bacteria interactions.

The microbial community structure in industrial biogas plants influences the degradation rate of straw and cellulose in batch tests.

BACKGROUND: Materials rich in lignocellulose, such as straw, are abundant, cheap and highly interesting for biogas production. However, the complex structure of lignocellulose is difficult for microbial cellulolytic enzymes to access, limiting degradation. The rate of degradation depends on the activity of members of the microbial community, but the knowledge of this community in the biogas process is rather limited. This study, therefore, investigated the degradation rate of cellulose and straw in batch cultivation test initiated with inoculums from four co-digestion biogas plants (CD) and six wastewater treatment plants (WWTP). The results were correlated to the bacterial community by 454-pyrosequencing targeting 16S rRNA gene and by T-RFLP analysis targeting genes of glycoside hydrolase families 5 (cel5) and 48 (cel48), combined with construction of clone libraries. RESULTS: UniFrac principal coordinate analysis of 16S rRNA gene amplicons revealed a clustering of WWTPs, while the CDs were more separated from each other. Bacteroidetes and Firmicutes dominated the community with a comparably higher abundance of the latter in the processes operating at high ammonia levels. Sequences obtained from the cel5 and cel 48 clone libraries were also mainly related to the phyla Firmicutes and Bacteroidetes and here ammonia was a parameter with a strong impact on the cel5 community. The results from the batch cultivation showed similar degradation pattern for eight of the biogas plants, while two characterised by high ammonia level and low bacterial diversity, showed a clear lower degradation rate. Interestingly, two T-RFs from the cel5 community were positively correlated to high degradation rates of both straw and cellulose. One of the respective partial cel5 sequences shared 100 % identity to Clostridium cellulolyticum. CONCLUSION: The degradation rate of cellulose and straw varied in the batch tests dependent on the origin of the inoculum and was negatively correlated with the ammonia level. The cellulose-degrading community, targeted by analysis of the glycoside hydrolase families 5 (cel5) and 48 (cel48), showed a dominance of bacteria belonging the Firmicutes and Bacteriodetes, and a positive correlation was found between the cellulose degradation rate of wheat straw with the level of C. cellulolyticum.

Cross-Comparison of Exome Analysis, Next-Generation Sequencing of Amplicons, and the iPLEX(®) ADME PGx Panel for Pharmacogenomic Profiling.

Whole-exome sequencing (WES) has been widely used for analysis of human genetic diseases, but its value for the pharmacogenomic profiling of individuals is not well studied. Initially, we performed an in-depth evaluation of the accuracy of WES variant calling in the pharmacogenes CYP2D6 and CYP2C19 by comparison with MiSeq(®) amplicon sequencing data (n = 36). This analysis revealed that the concordance rate between WES and MiSeq(®) was high, achieving 99.60% for variants that were called without exceeding the truth-sensitivity threshold (99%), defined during variant quality score recalibration (VQSR). Beyond this threshold, the proportion of discordant calls increased markedly. Subsequently, we expanded our findings beyond CYP2D6 and CYP2C19 to include more genes genotyped by the iPLEX(®) ADME PGx Panel in the subset of twelve samples. WES performed well, agreeing with the genotyping panel in approximately 99% of the selected pass-filter variant calls. Overall, our results have demonstrated WES to be a promising approach for pharmacogenomic profiling, with an estimated error rate of lower than 1%. Quality filters, particularly VQSR, are important for reducing the number of false variants. Future studies may benefit from examining the role of WES in the clinical setting for guiding drug therapy.