Search Results (173)

Fecal water syndrome (FWS) in horses is characterized by two-phase defecation, including both solid and liquid phases. While satisfactory explanations for FWS are unavailable, bacterial dysbiosis has been suggested as a contributing or causative factor. The objectives of this study were to determine whether fecal bacterial dysbiosis is associated with FWS in horses in the midwestern USA. Fecal samples were collected from horses with FWS and from unaffected horses at the same location. In total, 16S rRNA amplicon libraries produced from fecal bacterial DNA were sequenced using the Illumina sequencing platform. Significant differences in beta diversity were detected between affected and control horses (p = 7 × 10⁻⁴, F = 1.51), and differential abundance testing identified several features enriched in affected and control horses. These results agree with prior work regarding specific features in the bacterial microbiome associated with FWS, including Alloprevotella spp., and suggest fecal dysbiosis is associated with FWS. Full article

Treatment of Mycobacterium tuberculosis requires multi-drug regimens, and resistance to any individual antibiotic can compromise outcomes. For slow-growing organisms like M. tuberculosis, rapid detection of resistance-conferring mutations enables timely initiation of effective therapy. Conversely, confirming wild-type status in resistance-associated genes supports confidence in standard regimens. We developed an amplicon-based next generation sequencing (amplicon tNGS) assay on the Illumina platform targeting eight genes linked to resistance to isoniazid, rifampin, ethambutol, pyrazinamide, and fluoroquinolones. Sequencing results were analyzed using a custom bioinformatics pipeline. Forty-seven samples were used for assay development, and 37 additional samples underwent post-implementation clinical validation. Compared to whole genome sequencing (WGS), amplicon tNGS demonstrated 97.7% sensitivity, 98.9% specificity, and 98.7% overall accuracy for variant detection in targeted regions. Resistance prediction showed 79.3% concordance with WGS; discrepancies were primarily due to mutations outside of target regions. Among post-implementation samples, 27/37 passed quality metrics for all targets, with 95.7% concordance between amplicon tNGS results and final susceptibility results. This assay is now in use in our laboratory and offers significantly faster turnaround than both WGS and phenotypic methods on cultured isolates, enabling more rapid, informed treatment decisions for tuberculosis patients. Full article

Evolving technologies available to clinical laboratories and laboratory-related updates to clinical guidelines both drive the need for clinical laboratories to keep their test menu updated and in line with current technological and clinical developments. Our laboratory has developed a targeted Illumina-based amplicon next-generation sequencing (NGS) assay to interrogate the hsp65 and erm(41) genes of Mycobacterium spp. for the purposes of providing species-level ± subspecies-level identification of Mycobacterium spp. organisms in clinical samples and genotypic predictions for inducible macrolide resistance (in the case of M. abscessus complex members). The developed assay demonstrated 100% sensitivity and specificity for M. tuberculosis and M. abscessus complex cultured organisms, 98% ID overall concordance relative to the available reference identification, and a nearly 60% “rescue” rate for primary samples that could not be identified using our previous method. There was 94.6% concordance between genotypic and phenotypic results for inducible macrolide resistance. The developed assay was successfully implemented in our clinical laboratory and has been accredited for clinical use. Full article

Potato early dying disease complex (PED) leads to premature senescence and rapid decline in potato plants. Unlike potato wilt caused solely by Verticillium species, PED symptoms are more severe due to the synergistic effects of multiple pathogens, including root-lesion nematodes, fungi such as Colletotrichum and Fusarium, and soft-rot bacteria. To investigate the microbiome responsible for PED, soil and stem samples from healthy-looking and symptomatic plants were analyzed using amplicon-targeted next-generation sequencing (Illumina MiSeq and PacBio technologies). Samples were collected from four locations in New Brunswick, Canada from fields previously rotated with barley or oat. Comparative analysis of the bacterial, fungal, and eukaryotic diversity in soil samples showed minimal differences, with only bacterial alpha diversity influenced by the plant health status. Verticillium dahliae was abundant in all soil samples, and its abundance was significantly higher in the stems of diseased plants. Additional fungal species implicated in PED, including Plectosphaerella cucumerina, Colletotrichum coccodes, Botrytis sp., and Alternaria alternata, were also identified in the stems. This study highlights the complex, plant-associated microbial interactions underlying PED and provides a foundation for microbiome-informed disease management strategies. Full article

Androgenetic alopecia (AGA) is the most common form of patterned hair loss, exhibiting gender-specific clinical features. Recent studies highlight the importance of the skin microbiome in maintaining skin health, but the relationship between the hair follicle microbiome and hair loss, particularly AGA, remains understudied. Hair follicle layer samples were collected directly from the crown region of female pattern hair loss (FPHL), male pattern hair loss (MPHL), and healthy adult women (control) groups. Microbial DNA was extracted and analyzed using Illumina 16S rRNA V3–V4 gene amplicon sequencing. Alpha-diversity and beta-diversity analyses and taxonomic and functional profiling were conducted through relative abundance, LEfSe, and PICRUSt2 analyses. The alpha-diversity analysis showed a significant decrease in microbial richness in the hair loss groups. Unweighted UniFrac-based beta-diversity analysis revealed significant clustering between the control group and the FPHL group. Taxonomic profiling and LEfSe analysis identified differences in microbial composition and biomarkers. PICRUSt2 analysis further revealed altered pathways related to porphyrin metabolism, fatty acid biosynthesis, and steroid hormone metabolism. Additionally, differences in microbiome composition and potential functions were found between the FPHL and MPHL groups. This study provides comprehensive insights into the hair follicle microbiome, revealing unique microbial patterns and functional alterations associated with FPHL. Understanding these microbiome characteristics may contribute to targeted approaches for addressing AGA. Further research is warranted. Full article

DNA nanoball sequencing (DNBSEQ) is one of the most rapidly developing sequencing technologies and is widely applied in genomic and transcriptomic investigations. Recently, a new PE300 sequencing option primarily recommended for amplicon analysis was released for DNBSEQ-G99 and G400 devices. Given their unprecedentedly high data yield per flow cell, the new PE300 kits could be a great choice for various sequencing tasks, but we found that combining different types of DNA libraries in a single run could lead to undesired artifacts in the data. In this study, we investigate the occasional read cross-contamination that we first observed in our DNBSEQ PE300 run. The phenomenon, which we refer to as “software contamination”, is not actual contamination but primarily manifests as improper forward/reverse read pairing, improper demultiplexing, or as “digital chimeric” reads. Although rare, these artifacts were found in all runs we have analyzed, including several MGI demo datasets (both PE100 and PE150). In this study, we demonstrate that these artifacts arise primarily from the incorrect resolution of sequencing signals produced by neighboring DNA nanoballs, leading to mixing out forward and reverse reads or improper demultiplexing. The artifacts occur most frequently with read pairs where the length of insert sequence is shorter than the read length. Based on a few external NA12878 human exome sequencing data, we conclude that the total improper pairing rate in DNBSEQ data is comparable to Illumina ones. Overall, the problem only affects the analysis results when simultaneously sequenced libraries have markedly different insert size distribution or flow cell loading. Additionally, we demonstrate here that raw DNBSEQ data might contain ~2% optical duplicates, resulting from the same effect of close neighboring of DNB-sites in the flow cell. Full article

In this study, 172 Single-Nucleotide Polymorphisms (SNPs) (94 identity-informative SNPs, 56 ancestry-informative SNPs, and 22 phenotypic-informative SNPs) included in the ForenSeq™ DNA Signature Prep kit/DNA Primer Mix B (Verogen) were used for genotyping DNA samples from a population of twenty-one unrelated subjects, native to Northeast Italy. SNP sequencing was performed with the MiSeq FGx™ Forensic Genomics System (Illumina-Verogen), and data were analyzed using the Universal Analysis Software (UAS) v1.2. Raw data underwent further examination with STRait Razor v3 (SRv3) to compare the target SNPs’ genotype calls made with UAS and to identify the presence of microhaplotypes (MHs) due to SNPs associated with the same target SNP’s amplicon. The allele (haplotype) frequencies, Hardy–Weinberg equilibrium, linkage disequilibrium, number of effective alleles (A_e), and relevant forensic statistic parameters were calculated. Among the 172 SNPs evaluated, 45 unique microhaplotypes were found, comprising a novel sequence variant never previously described. The presence of MHs resulted in an 8.00% rise in the typologies of unique sequences, leading to changes in A_e. Notably, for 12 out of the 94 iiSNPs, the values of A_e exceeded 2.00, which is generally associated with a higher expected heterozygosity and increased power of discrimination. Full article

Improving the utilization of spent mushroom substrate and enhancing the digestibility of straw-based feed are critical for promoting environmental sustainability. However, the effects of replacing sawdust with straw in the cultivation of Pleurotus ostreatus—including changes in physicochemical properties, enzyme activities, and microbial community structure and function—remain unclear. In this study, corn straw was used as the substrate for P. ostreatus cultivation. Dynamic changes during the fermentation process were investigated through analyses of biological growth characteristics, physicochemical properties, enzyme activities, and amplicon sequencing. The results indicated a significant increase in mushroom yield, with the M80% treatment group achieving a yield of 156.09 ± 7.15 g. The nutritional value of the fermented feed was markedly improved; after 50 days of fermentation, crude protein (CP) and ether extract (EE) contents increased by 5.42% and 0.79%, respectively, while acid detergent fiber (ADF) and neutral detergent fiber (NDF) contents decreased by 18.5% and 22.3%, compared to day 0. Activities of cellulase, xylanase, and laccase were also elevated, contributing to more effective lignocellulose degradation. Furthermore, Illumina sequencing revealed shifts in bacterial and fungal metabolic pathways. The fungal community was dominated by Ascomycota and Basidiomycota, with Pleurotus as the prevailing genus, while the bacterial community was mainly composed of antagonistic genera such as Bacillus and Bacteroides. These findings provide a theoretical basis for understanding the role of microbial interactions during straw substrate fermentation in improving feed quality and increasing P. ostreatus yield. Full article

Camels survive in deserts through unique, adapted metabolic and immunological processes that are normally lethal to other species. Antioxidants and the gastrointestinal microbiota play major roles in redox homeostasis, yet they remain unexplored in camels to date. The objectives of this study were to characterize the dynamics of milk antioxidants, serum antioxidants, and the fecal microbiome of lactating Bactrian camels with different parities. In total, 30 lactating camels were selected and categorized into the following 3 groups: 10 were assigned to the first parity group (P_1), 10 were classified into the third parity group (P_3), and 10 belonged to the fifth parity group (P_5). The antioxidant parameters of the lactating camels were determined in milk and serum. The fecal microbial community of lactating camels was assessed using 16S rRNA amplicon sequencing, and the resulting library was sequenced on an Illumina NovaSeq platform. The amount of total antioxidant capacity (T-AOC) and antioxidant activity for polypepetides (DPPH) in the third parity was the highest among the groups both in the camel milk and its serum. In the case of hydroxyl radical (OH) and SOD, the amounts were 9.62 U/mL (SEM = 0.4950) and 13.64 U/mL (SEM = 0.5144), respectively, for P_1 in the serum, which were extremely significantly greater than those of the other groups. The Shannon index was significantly different between the P_1 group and either the P_3 or P_5 group. Additionally, Simpson’s diversity index significantly differed between the P_1 group and the P_5 group. Furthermore, the number of OH in camels is positively associated with the metabolic pathway of non-oxidative pentose phosphate pathway and ANAGLYCOLYSIS-PWY. In conclusion, this study revealed that different parities were associated with distinct levels of antioxidant parameters and fecal microbial ecologies in lactating Bactrian camels, where parity affects metabolic and microbial health. Understanding these dynamics in camels could optimize their nutritional management, enhancing their welfare in challenging environments. Full article

Despite providing highly accurate results, the short reads generated by second generation sequencing have major limitations in mapping complex genomic regions. Longer reads can resolve these issues and additionally phase distant variants. The third generation sequencing platform ONT currently achieves the longest sequencing reads but falls short in sequencing accuracy. Additionally, deriving phased haplotypes from amplicon-based NGS data remains a complex and time-consuming task that requires extensive bioinformatic expertise. We constructed an integrative, open-access modular data-analysis framework that allows for automated processing of high-throughput sequencing data from both second (Illumina) and third generation (ONT) sequencing platforms, combining the strengths of both technologies. Variant information is automatically evaluated and color-coded for discrepancies. Haplotypes are listed by frequency. All parts of the framework can be used independently. The framework’s performance was validated using synthetic and tested with real-life data by analyzing partly homologous FUT1/2/3 sequencing data from 400 blood donors. Full article

Understanding the interactions between the cervico-vaginal microbiome, immune responses, and sexually transmitted infections (STIs) is crucial for developing targeted diagnostic and therapeutic strategies. Although microbiome analyses are not yet standard practice, integrating them into routine diagnostics could enhance personalized medicine and therapies. We investigated the extent to which partial 16S short-read amplicon microbiome analyses could inform on the presence of six commonly encountered STI-causing pathogens in a patient cohort referred for colposcopy, and whether relevant taxonomic or diagnostic discrepancies occur when using vaginal rather than cervical swabs. The study cohort included cervical and vaginal samples collected from women referred for colposcopy at the University Hospital Bonn between November 2021 and February 2022, due to an abnormal PAP smear or positive hrHPV results. 16S rRNA gene sequencing libraries were prepared targeting the V1–V2 and V4 regions of the 16S RNA gene and sequenced on the Illumina MiSeq. PCR diagnostics for common STI-causing pathogens were conducted using the Allplex STI Essential Assay Kit (Seegene, Seoul, Republic of Korea). Concerning the bacterial microbiome, no significant differences were found between vaginal and cervical samples in terms of prevalence of taxa present or diversity. A total of 95 patients and 171 samples tested positive for at least one among Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Chlamydophila trachomatis or Neisseria gonorrhoeae. Sequencing the V1–V2 region enabled detection of one-third to half of the PCR-positive samples, with the detection likelihood increasing at lower cycle threshold (Ct) values. In contrast, sequencing the V4 region was less effective overall, yielding fewer species-level identifications and a higher proportion of undetermined taxa. We demonstrate that the vaginal microbiome closely mirrors the cervical microbiome, a relationship that has not been explored previously, but which broadens the possibilities for microbiome analysis and pathogen detection and establishes vaginal swabs as a reliable method for detecting the investigated pathogens, with sensitivities comparable with or superior to endocervical swabs. On the other hand, the sensitivity of partial 16S amplicon sequencing appears insufficient for effective STI diagnostics, as it fails to reliably identify or even detect pathogens at higher Ct values. Full article

Environmental DNA from bulk materials can be analyzed to gain an understanding of the bacterial, fungal, plant, and/or arthropod communities present. DNA metabarcoding is widely used to characterize these biological communities, by amplifying “barcode” regions and sequencing these amplicons via next-generation sequencing. The Earth Microbiome Project (EMP) adopted the use of indexed primers, PCR primers containing Illumina^® adapter sequences and a unique 12-nucleotide Golay barcode to simplify the identification of bacterial taxa via the 16S barcode. We sought to develop a wet laboratory workflow utilizing indexed primers that could cost-effectively reduce bench time while simultaneously targeting multiple DNA barcode regions to characterize bacterial (16S), fungal (ITS1), plant (ITS2, trnL p6 loop), and arthropod (COI) communities. The EMP primer constructs for 16S were modified to accommodate our DNA barcode regions of interest while also permitting successful demultiplexing following sequencing. A single indexed primer pair was designed for ITS1 and trnL p6 loop, and two primer pairs were developed for ITS2 and COI. To test the workflow, a total of 648 soil and 336 dust samples were processed, with key steps including DNA isolation, total DNA quantification, amplification with indexed primers, library purification and quantification, and Illumina MiSeq sequencing. Based on raw read counts and analysis of positive controls, the trnL p6 loop and ITS2 a primer pairs performed comparably to the originally designed 16S primers. Both COI primers pairs, ITS1 and ITS2 b primers, had lower raw reads compared to the other three primer pairs. The combination of the three plant targets successfully recovered all plant taxa in the positive controls except for Nephrolepis exaltata [Nephrolepidaceae] and the COI primers recovered all arthropod taxa except for the beetle. Notably, none of the taxa in the fungal positive control were recovered using ITS1. For environmental samples, sequencing was successful for all primers except COI c, and primer biases were observed for all three plant primers, in which a small number of families were uniquely amplified for each primer pair. This workflow can be applied to many disciplines that utilize DNA metabarcoding given its customizability and flexibility with Illumina sequencing chemistry. Full article

Colorectal cancer (CRC) is the third most commonly diagnosed cancer globally, and is a significant contributor to both morbidity and mortality rates. Emerging research has promptly highlighted the potential role of the gut microbiome in the development and progression of CRC. This study aims to investigate the differences in gut microbiota between CRC patients and healthy individuals in Iraq, using 16S rRNA metagenomic sequencing on Illumina NovaSeq (PE250-Seq). A total of 21 stool samples were analyzed: 12 from early-stage CRC patients and nine from healthy controls. Bacterial DNA was extracted, followed by 16S rRNA amplicon sequencing to profile the microbial communities. The results indicated significant differences between the fecal microbiome of the two groups. Remarkably, CRC patients exhibited a marked reduction in Bacteroidota and an increase in Verrucomicrobiota compared to healthy controls. At the genus level, Prevotella, Faecalibacterium, Roseburia, Barnesiella, Eubacterium Lachnospiraceae_UCG_004, and Lachnospira were significantly less abundant in CRC patients compared to the healthy individuals, while Actinomyces, Monoglobus, Desulfovibrio, Akkermansia, and Bacteroides were highly enriched. In addition, diversity analyses further indicated decreased α-diversity and distinct β-diversity patterns in the CRC patients, suggesting significant shifts in the gut microbial composition. These findings underscore the potential of microbiome-based diagnostics and therapeutic strategies, with microbial alterations serving as biomarkers for CRC diagnosis. Further research needs to focus on elucidating the causal relationships and therapeutic potential of these microbiome changes in CRC management. Full article

The spread of non-native species threatens biodiversity and exacerbates societal challenges like food security. To address this, effective conservation programs require detection methods that are easy to implement, accurate, and non-invasive. Over the past 15 years, environmental DNA (eDNA) techniques have gained popularity, surpassing traditional sampling methods. In this context, our study focused on tracking the invasive host–pathogen complex Pseudorasbora parva and Sphaerothecum destruens using eDNA metabarcoding. We collected water samples from freshwater canals over five months in the Camargue region, and once in Corsica Island, both in southern France. Total DNA was extracted from filtered water samples, and PCR-amplicons were sequenced using Illumina or Nanopore technologies. Our results revealed a high detection rate of P. parva in lentic ecosystems, aligning with habitat preferences of this small freshwater fish. Additionally, the detection rate in Camargue increased in May and June, likely due to the peak of the spawning season, which leads to more DNA being released into the environment (i.e., concentration and interaction of individuals). While eDNA successfully detected this invasive fish, we were unable to detect its cryptic fungal parasite, S. destruens, highlighting the challenges of identifying intracellular and cryptic fungal pathogens through eDNA methods. Full article

Sunflower Verticillium Wilt (SVW) caused by Verticillium dahliae is a significant threat to sunflower production in China. This soilborne disease is difficult to control. It has been observed that delayed sowing reduces the severity of SVW on different varieties and across various locations. Soil was collected from multiple locations with different sowing dates to understand the underlying biological mechanisms driving this phenomenon. The soil bacterial community was characterized through 16S rRNA gene amplicon sequencing performed on the Illumina MiSeq platform, followed by comprehensive bioinformatics analysis. Microsclerotia numbers in soil were detected using both NP-10 selective medium and quantitative polymerase chain reaction (qPCR). By delaying the sowing date, the number of microsclerotia in soil and the biomass of V. dahliae colonized inside sunflower roots were reduced during the early developmental stages (V2–V6) of sunflowers. Amplicon sequencing revealed an increased abundance of bacterial genera, such as Pseudomonas, Azoarcus, and Bacillus in soil samples collected from delayed sowing plots. Five bacterial strains isolated from the delayed sowing plot exhibited strong antagonistic effects against V. dahliae. The result of the pot experiments indicated that supplying two different synthetic communities (SynComs) in the pot did increase the control efficiencies on SVW by 19.08% and 37.82% separately. Additionally, soil temperature and humidity across different sowing dates were also monitored, and a significant correlation between disease severity and environmental factors was observed. In conclusion, delayed sowing appears to decrease microsclerotia levels by recruiting beneficial rhizosphere bacteria, thereby reducing the severity of SVW. Full article