Sequence Variants Research Articles

Prediction and design of transcriptional repressor domains with large-scale mutational scans and deep learning.

Regulatory proteins have evolved diverse repressor domains (RDs) to enable precise context-specific repression of transcription. However, our understanding of how sequence variation impacts the functional activity of RDs is limited. To address this gap, we generated a high-throughput mutational scanning dataset measuring the repressor activity of 115,000 variant sequences spanning more than 50 RDs in human cells. We identified thousands of clinical variants with loss or gain of repressor function, including TWIST1 HLH variants associated with Saethre-Chotzen syndrome and MECP2 domain variants associated with Rett syndrome. We also leveraged these data to annotate short linear interacting motifs (SLiMs) that are critical for repression in disordered RDs. Then, we designed a deep learning model called TENet ( T ranscriptional E ffector Net work) that integrates sequence, structure and biochemical representations of sequence variants to accurately predict repressor activity. We systematically tested generalization within and across domains with varying homology using the mutational scanning dataset. Finally, we employed TENet within a directed evolution sequence editing framework to tune the activity of both structured and disordered RDs and experimentally test thousands of designs. Our work highlights critical considerations for future dataset design and model training strategies to improve functional variant prioritization and precision design of synthetic regulatory proteins.

Molecular diversity and seasonal dynamics of Ostreococcus (Mamiellophyceae, Chlorophyta) in typical mariculture bays based on metabarcoding analysis

Ostreococcus (Mamiellophyceae, Chlorophyta) is a cosmopolitan genus of marine pico-phytoplankton and the smallest free-living photosynthetic eukaryotes with cell size of 1–2 μm. To understand the diversity and spatio-temporal distribution of Ostreococcus in the Rongcheng coastal regions in northern China, metabarcoding analysis based on the 18S rDNA V4 molecular marker was applied to study the molecular diversity and seasonal dynamics of Ostreococcus in three typical mariculture bays (Rongcheng Bay, Lidao Bay and Sanggou Bay). A total of 103 amplicon sequence variants (ASVs) annotated as Ostreococcus were detected in these three typical mariculture bays throughout the year. The top five ASVs in terms of abundance were ASV4, ASV9, ASV14, ASV28 and ASV109, totally occupying 99.1% of Ostreococcus reads. Phylogenetic analysis showed that these five dominant ASVs represented two Ostreococcus ecotypes (OI and OII) and were grouped into four Ostreococcus clades including Ostreococcus lucimarinus (ASV9) and Ostreococcus tauri (ASV28 and ASV109) in OI, and Ostreococcus sp. RC1 (ASV4) and Ostreococcus sp. RC2 (ASV14) in OII, which provided direct evidence to support the co-existence of two ecotypes in the Rongcheng coastal regions. Five dominant ASVs in OI and OII exhibited two distinct seasonal distribution patterns. Three dominant ASVs (ASV9, ASV28 and ASV109) in OI could be detected in all four seasons of the year, exhibiting native distribution properties, while two ASVs (ASV4 and ASV14) in OII decreased sharply in winter and could not be detected in spring, exhibiting characteristics of alien inputs. The composition, succession and association of Ostreococcus community were mainly driven by water temperature in these mariculture bays. This study helps us systematically understand the molecular diversity and distribution patterns of Ostreococcus in typical mariculture bays in northern China, laying the foundation for understanding and revealing the ecological functions of pico-phytoplankton.

Cancer associated variant enrichment CAVE, a gene agnostic approach to identify low burden variants in chronic lymphocytic leukemia

Intratumoral heterogeneity is an important clinical challenge because low burden clones expressing specific genetic alterations drive therapeutic resistance mechanisms. We have developed CAVE (cancer-associated variant enrichment), a gene-agnostic computational tool to identify specific enrichment of low-burden cancer driver variants in next-generation sequencing (NGS) data. For this study, CAVE was applied to TP53 in chronic lymphocytic leukemia (CLL) as a cancer model. Indeed, as TP53 mutations are part of treatment decision-making algorithms and low-burden variants are frequent, there is a need to distinguish true variants from background noise. Recommendations have been published for reliable calling of low-VAF variants of TP53 in CLL and the assessment of the background noise for each platform is essential for the quality of the testing. CAVE is able to detect specific enrichment of low-burden variants starting at variant allele frequencies (VAFs) as low as 0.3%. In silico TP53 dependent and independent analyses confirmed the true driver nature of all these variants. Orthogonal validation using either ddPCR or NGS analyses of follow-up samples confirmed variant identification. CAVE can be easily deployed in any cancer-related NGS workflow to detect the enrichment of low-burden variants of clinical interest.

Conservation Agricultural Practices Increase Soil Fungal Diversity and Network Complexity in a 13-Year-Duration Conservation Agriculture System in the Loess Plateau of China

Agricultural practices can affect the diversity and community structure of soil fungi. This study investigates the impact of long-term agricultural practices on soil fungal diversity in the Loess Plateau of northwestern China. Different tillage practices have been implemented for 13 years, and their impact on soil fungi is assessed using high-throughput Illumina Sequencing. This study found a total of 2071 fungal Amplicon Sequence Variants (ASVs), and these were assigned to 25 different phyla, 372 families, and 496 genera. The fungal communities were dominated by Ascomycota (52.1%), followed by Zygomycota (14.3%) and Basidiomycota (9.0%). In general, the soil exhibited higher fungal community abundance, richness, and diversity in winter than in summer. Notably, no-tillage or stubble retention resulted in greater diversity than conventional tillage, with no-tillage combined with stubble retention resulting in the highest fungal richness, diversity, and network complexity in both summer and winter. These findings indicate that no-tillage with stubble retention is beneficial for biological soil components, which favors the establishment of abundant and diverse soil fungal communities in the Loess Plateau of China. The present study expands the knowledge of fungal communities in agro-ecosystems and the long-term ecosystem benefits of tillage practices.

Effect of Morchella esculenta polysaccharides on the rectal microbiota of mice challenged with lipopolysaccharides.

Intestinal dysfunction poses a severe problem by preventing the digestion and absorption of nutrients. The gut, being the most vital organ for these processes, plays a crucial role in ensuring our body receives the nutrients it needs. We explored the mitigating effect of Morchella esculenta polysaccharides (MEP) on intestinal injury induced by lipopolysaccharides (LPS) through the modulation of intestinal flora. For this purpose, Kunming mice (KM) were divided into three groups, namely, PC, PM, and PY. Group PY was treated with MEP, while groups PM and PY were induced with LPS. The results showed that weight loss in the PM group was significantly greater than that in the PY group (P < 0.05), and the organ indexes of the lung and spleen in the PM group were significantly higher than those in the PC (P < 0.01) and PY (P < 0.05) groups. LPS caused severe injuries in KM mice in the PM group, characterized by broken villi. However, MEP treatment could alleviate this damage in the PY group, resulting in relatively intact villi. The serum analysis showed that tumor necrosis factor alpha (TNF-ɑ) (P < 0.01), interleukin 6 (IL-6) (P < 0.01), and 3,4-methylenedioxyamphetamine (MDA) (P < 0.05) levels were significantly higher in the PM group, while IL-10 (P < 0.001), superoxide dismutase (SOD) (P < 0.01) and glutathione peroxidase (GSH-Px) (P < 0.01) were significantly lower in that group. Interestingly, supplementation with MEP could lower the levels of TNF-ɑ, IL-10, IL-6, MDA while increasing the levels of superoxide dismutase (SOD) (P < 0.01) and GSH-Px. The gut microbiota analysis yielded 630,323 raw reads and 554,062 clean reads, identifying 3,390 amplicon sequencing variants (ASVs). One phylum and five genera were notably different among animal groups, including Escherichia_Shigella, Limosilactobacillus, unclassified_Geminicoccaceae, unclassified_Rhodobacteraceae, and Parabacteroides (P. distasonis). In conclusion, we found that MEP could mitigate the intestinal damage caused by LPS by modulating the inflammatory response, oxidative resistance, and intestinal flora of KM mice. Our results may provide insights into novel treatment options for intestine-related diseases.

Molecular cytogenetic characterization of isolated recurrent 4q35.2 microduplication in Chinese population: a seven-year single-center retrospective study

BackgroundWith the extensive use of chromosomal microarray analysis (CMA), an increasing number of variants of uncertain significance (VOUS) have been detected. The objective of the present study was to elucidate the pathogenicity and clinical variability associated with isolated recurrent 4q35.2 microduplications within the Chinese population.MethodsThe present study involved 14 cases of isolated recurrent 4q35.2 microduplication (including 12 fetuses and 2 cases of pediatric patients) out of 5,188 subjects who sought genetic consultation at our hospital and received CMA detection. WES technology was subsequently utilized to identify additional sequence variants in a patient with multiple clinical anomalies.ResultsAll 14 cases exhibited isolated recurrent 4q35.2 microduplications spanning a 1.0-Mb region encompassing the ZFP42 gene. Among the 12 fetuses, 11 displayed normal clinical features, while one was born with renal duplication and hydronephrosis. Additionally, in the two pediatric patients, WES was performed for Case 1, who presented with congenital cataracts, severe intellectual disability, and seizures. This patient inherited the 4q35.2 microduplication from his phenotypically normal mother. WES identified a novel NM_000276:c.2042G > T (p.G681V) variant in the OCRL gene, which is associated with Lowe syndrome and may account for the observed phenotypic variability within this family.ConclusionA series of 14 cases with isolated recurrent 4q35.2 microduplications were investigated, highlighting a potential association with increased susceptibility to renal abnormalities. Further, the present findings may expand the mutation spectrum of the OCRL gene associated with Lowe syndrome and provide valuable insights for the genetic etiological diagnosis of patients with unexplained copy number variants.

Fine-mapping genomic loci refines bipolar disorder risk genes.

Bipolar disorder (BD) is a heritable mental illness with complex etiology. While the largest published genome-wide association study identified 64 BD risk loci, the causal SNPs and genes within these loci remain unknown. We applied a suite of statistical and functional fine-mapping methods to these loci, and prioritized 17 likely causal SNPs for BD. We mapped these SNPs to genes, and investigated their likely functional consequences by integrating variant annotations, brain cell-type epigenomic annotations, brain quantitative trait loci, and results from rare variant exome sequencing in BD. Convergent lines of evidence supported the roles of genes involved in neurotransmission and neurodevelopment including SCN2A, TRANK1, DCLK3, INSYN2B, SYNE1, THSD7A, CACNA1B, TUBBP5, PLCB3, PRDX5, KCNK4, CRTC3, AP001453.3, TRPT1, FKBP2, DNAJC4, RASGRP1, FURIN, FES, DPH1, GSDMB, MED24 and THRA in BD. These represent promising candidates for functional experiments to understand biological mechanisms and therapeutic potential. Additionally, we demonstrated that fine-mapping effect sizes can improve performance of BD polygenic risk scores across diverse populations, and present a high-throughput fine-mapping pipeline (https://github.com/mkoromina/SAFFARI).

Oral swabs as a proxy for direct ruminal microbiome sampling in Holstein dairy cows is correlated with sample color.

Using oral swabs to collect the remnants of stomach content regurgitation during rumination in dairy cows can replicate up to 70% of the ruminal bacterial community, offering potential for broad-scale population-based studies on the rumen microbiome. The swabs collected from dairy cows often vary widely with respect to sample quality, likely due to several factors such as time of sample collection and cow rumination behavior, which may limit the ability of a given swab to accurately represent the ruminal microbiome. One such factor is the color of the swab, which can vary significantly across different cows. Here, we hypothesize that darker-colored swabs contain more rumen contents, thereby better representing the ruminal bacterial community than lighter-colored swabs. To address this, we collected oral swabs from 402 dairy cows and rumen samples from 13 cannulated cows on a research farm in Wisconsin, United States and subjected them to 16S rRNA sequencing. In addition, given that little is known about the ability of oral swabs to recapitulate the ruminal fungal community, we also conducted ITS sequencing of these samples. To correlate swab color to the microbiota we developed and utilized a novel imaging approach to colorimetrically quantify each swab from a range of light to dark. We found that swabs with increasing darkness scores were significantly associated with increased bacterial alpha diversity (p < 0.05). Lighter swabs exhibited greater variation in their community structure, with many identified amplicon sequence variants (ASVs) categorized as belonging to known bovine oral and environmental taxa. Our analysis of the fungal microbiome found that swabs with increasing darkness scores were associated with decreased alpha diversity (p < 0.05) and were also significantly associated with the ruminal solids fungal community, but not with the ruminal liquid community. Our study refines the utility of oral swabs as a useful proxy for capturing the ruminal microbiome and demonstrates that swab color is an important factor to consider when using this approach for documenting both the bacterial and fungal communities.

The effect of supplemental arginine on the gut microbial homeostasis of broilers during sub-clinical necrotic enteritis challenge.

Necrotic enteritis (NE) is an enteric disease of poultry that alters the structure of the gut microbial community causing dysbiosis. This 28day experiment investigated the effects of 125% and 135% arginine diets on the gut microbial diversity and composition of broilers during a subclinical NE challenge. One hundred and twenty one-day-old chicks were randomly allocated to 4 treatments with six replicates each- Uninfected + Basal, NE + Basal, NE + Arg 125%, and NE + Arg 135% diet groups. NE was induced by inoculating 1 × 104E. maxima sporulated oocysts on day 14 and 1 × 108CFUC. perfringens on days 19, 20, and 21 of age. The NE challenge significantly decreased the number of observed amplicon sequence variants (p = 0.03), the abundance of the phylum Firmicutes (p < 0.01), and the species Mediterraneibacter cottocaccae (p = 0.01) in the ceca of birds on day 21. The NE challenge significantly increased the Bray-Curtis index (p < 0.01), and the abundance of the phylum Bacteroidota (p < 0.01), family Odoribacteraceae (p < 0.01), genus Odoribacter (p < 0.01), and species O. splanchnicus (p = 0.01) on day 21. During NE, the 125% arginine diet restored the abundance of the phylum Bacteroidota (p = 0.03), family Odoribacteraceae (p = 0.03) and Oscillospiraceae (p = 0.03), genus Odoribacter (p = 0.03), and species O. splanchnicus (p = 0.03) and M. cottocaccae (p < 0.01) on day 21. The 135% arginine diet effectively restored the loss in alpha diversity (p = 0.01) caused by NE, the abundance of the phylum Firmicutes (p = 0.01) and Bacteroidota (p < 0.01), family Oscillospiraceae (p = 0.03) and Odoribacteraceae (p < 0.01), genus Odoribacter (p < 0.01), and species O. splanchnicus (p < 0.01) and M. cottocaccae (p < 0.01) on day 21. On day 28, the treatments had a significant effect on the cecal propionate (p = 0.01), butyrate (p = 0.04), and total SCFA (p = 0.04) concentrations. In conclusion, the 125% and 135% arginine diets restored gut microbial composition during a subclinical NE challenge, but not the cecal SCFA profile. Hence, arginine in combination with other feed additives could be used in restoring gut microbial homeostasis during NE in poultry.

Sequence variants underlying severe combined immunodeficiency and leukocyte adhesion deficiency type 1 in six consanguineous families.

Inborn errors of immunity (IEI) are defined as genetic disorders affecting the immune system and resulting in diverse clinical signs and symptoms. Despite the lack of diagnosis and unavailability of IEI estimation in the Pakistani population, consanguinity is exacerbating its prevalence. The current study focuses on severe combined immunodeficiency (SCID) and leukocyte adhesion deficiency type 1 (LAD1). SCID is associated with the life-threatening symptoms developing at post-birth. LAD1 is clinically characterized by recurrent bacterial infections related to the skin, mouth, and respiratory tract owing to impaired leukocytes. Herein, in six consanguineous families, flow cytometry was used to evaluate the patient's immune status. Whole-exome sequencing (WES) was then conducted to search for the causative variations in immunodeficiency genes. Sanger sequencing was used to assess the segregation of the variants with the disorder within the families. Sequence analysis revealed five homozygous variants in four different causative genes. This included four novel nonsense variants in CD70 p.(Thr126Profs*33), CD3e p.(Trp151*), IL7R p.(Val138Ilefs*10), and ITGB2 p.(Ser627Valfs*61), and one previously reported in ITGB2 p.(Cys62*). In one of the families, two variants in two different genes, including DNAH6 p.(Tyr2653His) and NIPAL4 p.(Gly121Ser), were detected in an unclassified patient. All the identified variants were found in a homozygous state in the patient but in a heterozygous state in the available parents. The study will facilitate the diagnosis and management of IEI patients.

Yeasts of Pichia (Pichiaceae) dominate the mycobiome of Hermetia illucens (Diptera: Stratiomyidae) larvae during urban composting

Introduction: Hermetia illucens is a fly found worldwide in tropical and temperate regions that feeds on decaying organic matter in its larval stage, which makes them useful to accelerate composing processes. Bacterial component on the larval guts that helps to degrade organic matter is well studied, however fungal communities information is more scarse, specially in tropical regions. Objective: To determine fungal communities in the gut of H. illucens larvae naturally occurring during urban composting processes in a tropical region. Methods: ITS sequencing was employed to characterize fungal communities present in H. illucens larval gut. Results: The analysis of amplicon sequence variants (ASVs) unveiled a notable dominance of Saccharomycetales, being yeasts of the genus Pichia the most abundant. Other relatively abundant yeasts were Candida and Galactomyces and the genus Archaeospora from Archaeosporales. The last two groups not being reported in H. illucens before. Conclusions: Yeasts of the genus Pichia are the most abundant group, this result is in concordance with previous studies which suggest a stable insect-yeast relation. The description of previously unreported fungal groups highlights the importance of continuing to explore the mycobiome dynamics of this larva. This study offers insight into the mycobiome of naturally occurring H. illucens larvae in a tropical region.

Shifting microbial communities in acidified seawaters: insights from polychaetes living in the CO2 vent of Ischia, Italy.

Oceans' absorption of human-related CO2 emissions leads to a process called ocean acidification (OA), consisting of the decrease of the seawater pH with negative consequences for many marine organisms. In this study, we investigate the microbial community of two species of polychaetes found in naturally acidified CO2 vents: the nereid Platynereis massiliensis complex and the syllid Syllis prolifera. Animals were collected in the CO2 vents of Castello Aragonese (Gulf of Naples, Ischia, Italy) in three zones at decreasing pH. For the analysis of the microbiome, the V3-V4 hypervariable region of the 16S ribosomal RNA gene of 40 worm samples was sequenced on an Illumina MiSeq platform. No difference in the microbial alpha diversity of both species was highlighted. On the contrary, the microbial composition of worms collected in the site at normal pH was different from that of the individuals obtained from the sites at lower pH. This effect was evident also in samples from the site with a slight, but relevant, degree of acidification. Amplicon sequence variants showing a significant variation among the groups of samples collected from different pH zones were reported for both polychaetes, but no common trend of variation was observed. The present study deepens our knowledge about the composition of polychaete microbiome in marine naturally acidified sites. Our results stress the importance of future investigations about the connection between the variation of environmental and polychaete microbial communities induced by OA and about the effect of these variations on polychaete key biological and ecological traits.

Complex hydrothermal vent microbial mat communities used to assess primer selection for targeted amplicon surveys from Kama‘ehuakanaloa Seamount

The microbiota of hydrothermal vents has been widely implicated in the dynamics of oceanic biogeochemical cycling. Lithotrophic organisms utilize reduced chemicals in the vent effluent for energy, which fuels carbon fixation, and their metabolic byproducts can then support higher trophic levels and high-biomass ecosystems. However, despite the important role these microorganisms play in our oceans, they are difficult to study. Most are resistant to culturing in a lab setting, so culture-independent methods are necessary to examine community composition. Targeted amplicon surveying has become the standard practice for assessing the structure and diversity of hydrothermal vent microbial communities. Here, the performance of primer pairs targeting the V3V4 and V4V5 variable regions of the SSU rRNA gene was assessed for use on environmental samples from microbial mats surrounding Kama‘ehuakanaloa Seamount, an iron-dominated hydrothermal vent system. Using the amplicon sequence variant (ASV) approach to taxonomic identification, the structure and diversity of microbial communities were elucidated, and both primer pairs generated robust data and comparable alpha diversity profiles. However, several distinct differences in community composition were identified between primer sets, including differential relative abundances of both bacterial and archaeal phyla. The primer choice was determined to be a significant driver of variation among the taxonomic profiles generated. Based on the higher quality of the raw sequences generated and on the breadth of abundant taxa found using the V4V5 primer set, it is determined as the most efficacious primer pair for whole-community surveys of microbial mats at Kama‘ehuakanaloa Seamount.

Measuring X-Chromosome inactivation skew for X-linked diseases with adaptive nanopore sequencing.

X-linked genetic disorders typically affect females less severely than males owing to the presence of a second X Chromosome not carrying the deleterious variant. However, the phenotypic expression in females is highly variable, which may be explained by an allelic skew in X-Chromosome inactivation. Accurate measurement of X inactivation skew is crucial to understand and predict disease phenotype in carrier females, with prediction especially relevant for degenerative conditions. We propose a novel approach using nanopore sequencing to quantify skewed X inactivation accurately. By phasing sequence variants and methylation patterns, this single assay reveals the disease variant, X inactivation skew, and its directionality and is applicable to all patients and X-linked variants. Enrichment of X Chromosome reads through adaptive sampling enhances cost-efficiency. Our study includes a cohort of 16 X-linked variant carrier females affected by two X-linked inherited retinal diseases: choroideremia and RPGR-associated retinitis pigmentosa. As retinal DNA cannot be readily obtained, we instead determine the skew from peripheral samples (blood, saliva, and buccal mucosa) and correlate it to phenotypic outcomes. This reveals a strong correlation between X inactivation skew and disease presentation, confirming the value in performing this assay and its potential as a way to prioritize patients for early intervention, such as gene therapy currently in clinical trials for these conditions. Our method of assessing skewed X inactivation is applicable to all long-read genomic data sets, providing insights into disease risk and severity and aiding in the development of individualized strategies for X-linked variant carrier females.

Housing RCS rats under specific pathogen-free conditions mildly ameliorates retinal degeneration and alters intestine microbiota

Retinitis pigmentosa (RP) is a genetic blinding disease with over 80 causative genes. Disease progression varies between patients with similar genetic backgrounds. We assessed the association between environment, gut microbiota, and retinal degeneration in the RP rat model Royal College of Surgeons (RCS). The rats were born and raised for two generations under specific pathogen-free (SPF, n = 69) or non-SPF conditions (n = 48). At the age of four weeks, SPF rats had significantly shorter dark-adapted a-wave and dark and light-adapted b-wave implicit times by electroretinogram (p = 0.014, p = 9.5*10−6, p = 0.009, respectively). The SPF rats had significantly less photoreceptor apoptosis at ages four, eight, and twelve weeks (all p < 0.022), significantly thicker debris zone at age 14 weeks, and smaller hypofluorescent lesions in SPF rats at ages 10–16 weeks, especially in the inferior retina. The non-SPF rats had significantly higher microbiota alpha diversity (p = 0.037) and failed to present the age-related maturation of Proteobacteria, Spirochaetes, Actinobacteria, and Bacteroidetes seen in SPF conditions. Specific microbial amplicon sequence variants were reduced in rats with more severe retinal degeneration. Our data suggest an environmental effect on retinal deterioration in RCS rats. These findings may lead to the development of novel microbiome-related interventions for retinal degeneration.

PSV-8 Investigation of a haplotype and eQTL analysis of the LCORL-NCAPG locus associated with increased lean growth in beef cattle

Abstract Numerous studies have shown genetic variation at the LCORL-NCAPG locus is strongly associated with growth traits in beef cattle. However, a causative molecular variant has yet to be identified. To define all possible candidate variants, Charolais-sired calves (n = 34) were whole genome sequenced including 17 homozygous for a long-range haplotype associated with increased growth (QQ), and 17 homozygous for potential ancestral haplotypes for this region (qq). The Q haplotype was refined to an 814-kb region between chr6:37,199,897-38,014,080 and contained 218 variants not found in qq individuals. These variants include an insertion in an intron of NCAPG, a previously documented mutation in NCAPG (rs109570900), two coding sequence mutations in LCORL (rs109696064 and rs384548488), and 15 variants located within ATAC peaks that were predicted to affect transcription factor binding. Notably, rs384548488 is a frameshift variant likely resulting in loss of function for long isoforms of LCORL. To test the association of the coding sequence variants of LCORL with phenotype, 405 cattle from five populations were genotyped. The two variants were in complete linkage disequilibrium. Statistical analysis of the three populations that contained QQ animals revealed significant (P &amp;lt; 0.05) associations with genotype and birth weight, body weight, carcass weight, hip height, and average daily gain. Additionally, an RNAseq analysis was performed using 22 of the Charolais-sired calves. This, in conjunction with the WGS data, was used to run an eQTL analysis, which revealed 256 genes whose expression were impacted by eQTL in the region (FDR &amp;lt; 0.05). These findings affirm the link between this locus and growth in beef cattle and describe DNA variants that define the haplotype. However, further studies will be required to define the true causative mutation.

PSVI-5 Bacteriophage biocontrol of avian pathogenic Escherichia coli in laying hens does not produce collateral effects on the cecal microbiota

Abstract Bacteriophages have shown promise as an alternative to antibiotics for managing or preventing avian pathogenic Escherichia coli (APEC) in the egg industry. In a previous study, a phage cocktail comprising 3 virulent phages was shown to prevent APEC infections in laying hens. The aim of this study was to investigate whether orally and intramuscularly administered phages affect the intestinal barrier function of chickens, with a focus on cecal microbiome. In a 4-d trial, a total of 35 laying hens were randomly assigned to one of four treatment groups: 1) Medium control group (bacterial broth + phage buffer), 2) APEC only control (APEC + buffer); 3) IM group (Phage +APEC), or 4) DW group (phage in drinking water (4 d prior to APEC + APEC). Cecum samples were subjected to 16S rRNA gene sequencing targeting the V4 region to assess the effects of phages on cecal bacterial diversity. A 1-way ANOVA was used to evaluate alpha diversity with respect to treatment and time. Microbial community structure was analyzed using β-dispersion and permutational multivariate analysis of variance (PERMANOVA) to determine the effect of each treatment. Significant (P &amp;lt; 0.05) differentially abundant genera were identified with DESeq2 by fitting a negative binomial model to each treatment vs treatment comparison (“~Treatment”). Across the treatment and control groups, 13 phyla were identified. Firmicutes (53.81%), Bacteroidota (23.83%), Actinobacteriota (1.84%), and Proteobacteria (1.02%) were the most abundant phyla among the amplicon sequence variants (ASVs), with 18.39% being unclassified. Neither richness (P = 0.448) nor Shannon diversity (P = 0.687) exhibited significance in α- diversity metric between treatment and control groups. β-dispersion analysis showed no significant difference (P = 0.239) between treatment and control groups, indicating similar bacterial variability among groups. Moreover, the β-diversity representing microbiota structure was not affected by any phage treatment (P = 0.083). In each treatment group, 76 unique genera were identified, with Megamonas (15.80 %), Lactobacillus (8.76%), Faecalibacterium (7.70 %), Megasphaera (1.58%), Alloprevotella (1.54%), [Ruminococcus] torques group (1.29%), Prevotellaceae UCG-001 (1.14%), Bifidobacterium (1.04%), and Olsenella (0.48%) being the most abundant. Notably, Lactobacillus was more abundant (~ 10 to 20%) in medium control group compared with phage treated and APEC only treatment groups. Furthermore, gene expression profiles of various genera (pairwise) were analyzed using log2 fold change (log2FC), revealing differences (P &amp;lt; 0.05) between control and treatment groups for a total of 58 genera. It was concluded that bacteriophage selectively killed APEC population without comprising the population and structure of cecal microbiome in laying hens.

PSIX-10 Effects of Bacillus subtilis ATCC PTA-122264 on macronutrient digestibility and fecal characteristics, metabolites, and microbiota of healthy adult dogs subjected to an abrupt diet change

Abstract Probiotic use in dogs is increasing, with spore-forming Bacillus species gaining interest due to their viability during processing, storage, and in the gastrointestinal tract. B. subtilis is known for its adaptable metabolism that may be useful in pets subjected to abrupt dietary changes. The objective of this study was to determine dietary apparent total tract macronutrient digestibility (ATTD) and fecal characteristics, metabolites, and microbiota of healthy adult dogs supplemented with B. subtilis ATCC PTA-122264. All procedures were approved by the University of Illinois IACUC prior to experimentation. Dogs [n = 12; 6 ± 1 yr; 8.71 ± 0.91 kg body weight (BW)] were used in a replicated 3x3 Latin square design (n = 12/group) and fed to maintain BW. In each 42-d experimental period, dogs were allotted to one of three treatments: Control (kibble diet only), Low [kibble diet + 1×109 colony-forming units (CFU) of B. subtilis], and High (kibble diet + 5×109 CFU of B. subtilis). After a 22-d adaptation phase, a 5-d fecal collection phase was followed by a blood collection phase. On d 28, dogs were abruptly changed to a canned diet and fed for 14 d, with fecal samples collected on d 30 (2 d post change), 34 (6 d post change), 38 (10 d post change), and 42 (14 d post change). Fecal microbiota data were evaluated using QIIME2. All other data were analyzed using the Mixed Models procedure of SAS, with P &amp;lt; 0.05 being considered significant. B. subtilis supplementation of the kibble diet did not influence food or energy intake, fecal output, ATTD, most serum metabolites, hematology, fecal characteristics, fecal bacterial alpha and beta diversity indices, and dysbiosis index, demonstrating the safety of the strain in dogs. However, fecal Streptococcus and Blautia abundances were higher in High dogs than Low or Control dogs, while E. coli abundance was less in Low dogs than High and Control dogs. The abrupt change to a canned diet led to significant alterations in fecal characteristics, dysbiosis index, and bacterial abundances, which were not influenced by B. subtilis supplementation. Bacterial alpha diversity measures were affected, with observed ASV (amplicon sequence variants) and the Fisher Index reducing, and the Shannon Index increasing by dietary change. The abrupt dietary change caused a distinct shift in microbial communities, as evidenced by principal coordinates analysis plots. The relative abundances of various fecal bacterial taxa, including Bifidobacterium, Faecalibaculum, Turicibacter, and Lactobacillus were decreased, while Collinsella, Ruminococcus gnavus group, and Escherichia-Shigella were increased with the dietary change. Our data support the notion that daily supplementation of 1 – 5×109 CFU of B. subtilis ATCC PTA-122264 in healthy dogs is safe and does not affect markers of general health and fecal characteristics, warranting further exploration.

145 Utilization of 16s sequencing data and physiological measurements to produce a comparative analysis of the equine microbiome

Abstract Rapid advancements in microbiome research, particularly in the technologies associated with next-generation sequencing have enabled unprecedented investigations into the composition of the equine microbiome. The large datasets produced from these studies often present challenges to researchers seeking to understand the applicability of these data to equine physiology. To that end, a multi-year project was conducted with the goal of determining the core bacterial population of the equine hindgut through analysis of rectal swabs collected from thousands of horses and applying logic to the association between these ecologies and equine health. Samples were obtained from horses that varied across a diverse range of considerations including location, breed, age, and diet, amongst other metadata points including disease state, health history, vaccination and deworming status, body condition, and pregnancy status. These data were used in the development of a comparative score, that authors defined as the Microbiome Quotient (MQ score). Rectal swabs were collected, and DNA was extracted using the Quick-DNA Fecal/Soil Microbe Miniprep kit (Zymo Research, Irvine, CA) and the V3-V4 hypervariable region of the 16S rRNA gene was sequenced by following the Illumina 16S Protocol (San Diego, CA). Sequences were denoised using DADA2 within the QIIME2 pipeline to obtain amplicon sequence variant composition. These data were filtered again in R (4.2.2) to remove samples with missing data and low sequencing depth. Sample metadata was used to classify samples as either typical or atypical. The MQ score was calculated by computing the Euclidean distance difference between a sample and the centroids representing typical and atypical microbial ecologies, and then subtracting these distances to obtain the individual score. This score quantifies how similar the microbiome in a sample is to those of typical and atypical horses. These scores were then categorized into tertiles, representing the status of the individual horse’s microbiome. These statuses indicate differences in bacterial ecology among samples and their subsequent relevance to the health of the horse. While a large portion of the equine fecal microbiome is conserved through homeostatic and other mechanisms, there remains a high degree of variability in the lesser abundant genera that may be responsible for the different physiological interactions between the microbes and their hosts. Utilizing this MQ score, feeding and management recommendations can be made, providing veterinarians and horse owners with a tool to support the health and performance of horses through modulation of the bacterial population and its functionality. Further research is necessary to fully elucidate the impact of the various metadata components on the bacterial ecology. Additionally, the function of the metagenome is a critical component of microbiome sciences. Prediction of metagenomic function is likely to provide insight into the regulation of physiological function by the microbial population residing within the host.

161 Development, deployment, and calibration of genomic selection tools to improve performance traits for Canadian beef cattle

Abstract Genomic selection has the potential to accelerate the genetic improvement rate for difficult/expensive to measure traits such as feed efficiency and fertility traits in beef cattle. To develop genomic selection tools for Canadian beef cattle, we collected or sourced phenotypic data of feed efficiency along with other traits including carcass merit, female feed intake and fertility related traits, and their genotypes from both research and commercial herds. The phenotypic data were consolidated and recorded for contemporary groups, leading to the development of data sets including 11,292 beef cattle with residual feed intake, dry matter intake, average daily gain, metabolic body weight, 7,299 to 8,081 cattle with carcass merit traits, and 1,802 to 2,792 cows with feed intake and fertility traits after quality controls. Genotype data of the animals from various single nucleotide polymorphisms (SNP) panels were merged to the same allele format of 50K, and then imputed to 770K SNPs, and eventually to whole genome sequence variants. The refined Canadian beef cattle data sets have not only allowed us to investigate genetic architectures of the beef performance traits but also enabled the development of genomic selection tools including molecular breeding values and multiple trait selection indexes with a moderate to moderately high accuracy for the traits through a cross-validation of within reference data set. The genomic selection tools have been successfully deployed to over 10,415 breeding candidates submitted by Canadian beef producers through multiple demonstration projects. The average accuracy of the molecular breeding values and multiple trait selection index of the commercial beef cattle ranged from 0.38 to 0.49 for feed efficiency and carcass merit traits, and from 0.26 to 0.38 for female feed intake and fertility traits. Current research is focusing on calibrating the genomic selection tools with larger data sets and with more advanced genomic prediction methods.