Shotgun Sequencing Research Articles

PSVII-1 Widespread allele-specific expression across tissues and cells of the chicken genome

Abstract Chickens are a valuable livestock species, providing an affordable and nutritious food source worldwide through eggs and meat. Additionally, chickens are extensively used in scientific research as model organisms in virology, immunology, epigenetics, development, and conservation biology. Considering this, there are increasing efforts to deepen our understanding of their genome complexity, such as efforts by the Functional Annotation of Animal Genomes (FAANG) Consortium. Toward this goal, we are working to contribute to the annotation of the chicken transcriptome. Allele-specific expression (ASE) is an imbalance of allelic gene expression often driven by genetic and epigenetic changes in cis-regulatory regions. We, therefore, aimed to determine allelic imbalances in gene expression across 20 tissues and cells to shed light on mechanisms regulating gene expression. Using replicate paired-end (PE) RNA-seq samples, we identified around 7 million variants across 20 tissues and cells (e.g., reproductive tissues, intestine tissues, muscle, and immune tissues and cells). We applied quality control measures and filtered out monoallelic and homozygous variants. After that, we calculated read counts per allele to determine allelic expression. We found 365,894 significant ASE SNPs and 11,530 ASE genes in at least one tissue or cell type (FDR ≤ 0.05). We discovered that ASE SNPs are widely distributed throughout the chicken genome, and ASE SNPs and ASE genes showed a varied distribution across tissues and cells. Primary macrophage exhibited the most abundant, while the pectoralis major (breast muscle) had the lowest ASE genes and ASE SNPs, ranging from 7,592 to 32,505 ASE SNPs and 773 to 2,387 ASE genes. Our findings reveal several important pathways affected by allelic imbalance of expression. These pathways included fatty acid biosynthesis and regulation, cell proliferation and differentiation, cell metabolism, adipocytokine signaling, proteolysis and autophagy, and focal adhesion. In summary, our study provides insight into relevant biological processes critical to chickens and can contribute to improving genome annotation, helping to expand the transcriptomic reference source.

187 Exploring deep shotgun sequencing to understand the rumen microbial function in Angus bulls with divergent RFI EPD

Abstract We investigated the rumen microbiome of Angus bulls selected for Residual Feed Intake-Expected Progeny Difference (RFI-EPD) utilizing deep shotgun metagenomic sequencing. Negative RFI-EPD bulls (NegRFI: n = 10; RFI-EPD = -0.3883 kg/d) and Positive RFI-EPD bulls (PosRFI: n = 10; RFI-EPD = 0.2935 kg/d) were selected from a group of 59 Angus bulls [average body weight (BW) = 427.81 ± 18.77 kg] fed a high-forage total mixed ration after a 59-d testing period. At the end of the 59-d period, rumen fluid samples were collected for bacterial DNA extraction and subsequent shotgun metagenomic sequencing. Results of the metagenome analysis revealed greater gene richness in NegRFI bulls compared with PosRFI. Analysis of similarity revealed a significant difference (P = 0.05) in the rumen microbial community between the NegRFI and PosRFI. Linear Discriminant Analysis effect size (Lefse) was used to identify the differentially abundant taxa. The Lefse results showed that class Fibrobacteria (LDA = 5.1) and genus Fibrobacter (LDA = 4.8) were the most differentially enriched markers in NegRFI bulls, compared with PosRFI bulls. Relative abundance of the carbohydrate-active enzymes was also compared using lefse analysis. The results showed greater relative abundance of glycoside hydrolases and carbohydrate-binding modules such as GH5, CBM86, CBM35, GH43, and CBM6 (LDA > 3.0) in NegRFI bulls whereas GH13 and GT2 were greater in PosRFI. The distinct metabolic and microbial profiles observed in NegRFI, compared with PosRFI bulls, characterized by greater gene richness and specific taxa such as Fibrobacter, and variations in carbohydrate-active enzymes, underscore the potential genetic and functional differences in their rumen microbiome. These findings contribute to a deeper understanding of the interplay between host genetics, microbiota, and feed efficiency in Angus bulls, opening avenues for targeted interventions and advancements in livestock management practices.

COMPARISON OF COMPUTATIONAL METHODS FOR ANALYSING 16S rRNA SEQUENCING DATA PRODUCED WITH THE USE OF SHORT AND LONG READS

This research is driven by the importance of identifying species in microbial community for use in medical testing. Conventional methods including culturing, biochemical assays are often limited to the culturable organisms and do not provide information about relative abundance of different microbial taxa within a community. Comparative analysis of metagenomic programs method was used to identify the most appropriate option for long-read 16S rRNA data. Since the development of high-throughput sequencing technologies, novel methods of identifying species include taxonomic profiling of 16S rRNA. 16S rRNA gene contains variable regions (V1-V9) that are specific to different taxonomic groups. Illumina short-read sequencing technology provides read lengths 2*251 base pairs (bp) for paired-end sequencing, while fraction of 16S rRNA gene is around 1500 bp, thus reads can successfully cover 2 of 9 regions (300-400 bp), but not the whole gene. The Oxford Nanopore Technology (ONT) representing long-read sequencing or so-called 3rd generation sequencing is much more applicable for 16S rRNA profiling, as it can provide reads with length 1500 bp (exactly in line with the length of 16S rRNA gene). Extended read length provides high alignment quality making the downstream taxonomy classification and relative abundance calculation much easier and relevant. The samples from endometrium of uterus were studied in the first part of the research. Endometrial microbiome composition and balance can play an important role in the success of in vitro fertilization (IVF). Taxonomical classification was tested with NCBI, Silva databases. NCBI database consists of full genomes of Bacteria, Archaea and Eukaryote, Silva contains curated and annotated sequences of only small subunit ribosomal RNA of these domains. Among the enormous collection of metagenome classifiers there were selected three particular programs applying completely different algorithms: Emu, PathoScope and Kraken2. The comparative process was divided in three phases. In the first one, pipelines were run with three programs and 11 samples using Silva and NCBI databases, the results were compared both quantitively and visually. In the second phase, the results were compared to the short-read taxonomical classification, the discrepancies were analyzed and questions were put together for further investigation. During the third phase pipelines with best performed programs were run on sample with acknowledged microbial diversity and following metrics were assessed and visualized: True positive, False positive, Precision, Recall, F1-score, L1-norm, L2-norm. Results: After the first phase it was revealed that Kraken2, Emu, PathoScope produce very similar results, while MetaScope discrepancies exceeded substantially 10% threshold for 70% of the reads. Additionally, Kraken2 annotated significantly more genera upon the threshold than other programs indicating its low specificity. Consequently, these two programs were excluded from further analysis. The second phase brought to the conclusion that short-read sequencing reports reveal only one prevailing genus in the community, while long-read establish 3 of them, thus it was assumed that short-read classification is much less sensitive. In the third phase it was determined that Emu and PathoScope have least deviation from reference value of abundances (ground truth) and they successfully annotate all the species introduced in the sample. Therefore, the main outcome of the research is the recommendation to use Emu and PathoScope programs for the purpose of identifying species using long-read data.

GoEnrich: creating high quality genomic DNA resources from limited voucher specimen tissues or museum specimens of at-risk species for conservation-friendly use in the validation of environmental DNA assays

ObjectiveEnvironmental DNA (eDNA) methods are crucial for monitoring populations, particularly rare and cryptic species. For confident eDNA application, rigorous assay validation is required including specificity testing with genomic DNA (gDNA). However, this critical step is often difficult to achieve as obtaining fresh tissue samples from at-risk species can be difficult, highly limited, or impossible. Natural history museum collections could serve as a valuable and ethical voucher specimen resource for eDNA assay validation. The present study demonstrates the effectiveness of whole genome amplification (WGA) in providing enough gDNA to assemble high quality mitogenomes from which robust targeted eDNA assays can be designed.ResultsUsing fresh and historical museum tissue samples from six species spanning fish, birds, and mammals, we successfully developed a WGA method with an average yield of 380 to 1,268 ng gDNA per 20 µL reaction. This gDNA was used for whole genome shotgun sequencing and subsequent assembly of high quality mitogenomes using mtGrasp. These mitogenomes were then used to develop six new robust, targeted quantitative real time polymerase chain reaction-based eDNA assays and 200 ng WGA-enriched yielded satisfactory Cq values and near 100% detection frequencies for all assays tested. This approach offers a cost-effective and non-invasive alternative, streamlining eDNA research processes and aiding in conservation efforts.

A novel barcoded nanopore sequencing workflow of high-quality, full-length bacterial 16S amplicons for taxonomic annotation of bacterial isolates and complex microbial communities.

Due to recent improvements, Nanopore sequencing has become a promising method for experiments relying on amplicon sequencing. We describe a flexible workflow to generate and annotate high-quality, full-length 16S rDNA amplicons. We evaluated it for two applications, namely, (i) identification of bacterial isolates and (ii) species-level profiling of microbial communities. We assessed the identification of single bacterial isolates by sequencing, using a set of barcoded full-length 16S rRNA gene primer pairs (pair A), on 47 isolates encompassing multiple genera and compared those results with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based identification. Species-level community profiling was tested with two sets of barcoded full-length 16S primer pairs (A and B) and compared to the results obtained with shotgun Illumina sequencing using 27 stool samples. We developed a Nextflow pipeline to retain high-quality reads and taxonomically annotate them. We found high agreement between our workflow and MALDI-TOF data for isolate identification (positive predictive value = 0.90, Cramér's V = 0.857, and Theil's U = 0.316). For species-level community profiling, we found strong correlations (rs > 0.6) of alpha diversity indices between the two primer sets and Illumina sequencing. At the community level, we found significant but small differences when comparing sequencing techniques. Finally, we found a moderate to strong correlation when comparing the relative abundances of individual species (average rs = 0.6 and 0.533 for primers A and B). Despite identified shortcomings, the proposed workflow enabled accurate identification of single bacterial isolates and prominent features in microbial communities, making it a worthwhile alternative to MALDI-TOF MS and Illumina sequencing.IMPORTANCEA quick, robust, simple, and cost-effective method to identify bacterial isolates and communities in each sample is indispensable in the fields of microbiology and infection biology. Recent technological advances in Oxford Nanopore Technologies sequencing make this technique an attractive option considering the adaptability, portability, and cost-effectiveness of the platform, even with small sequencing batches. Here, we validated a flexible workflow to identify bacterial isolates and characterize bacterial communities using the Oxford Nanopore Technologies sequencing platform combined with the most recent v14 chemistry kits. For bacterial isolates, we compared our nanopore-based approach to matrix-assisted laser desorption ionization-time of flight mass spectrometry-based identification. For species-level profiling of complex bacterial communities, we compared our nanopore-based approach to Illumina shotgun sequencing. For reproducibility purposes, we wrapped the code used to process the sequencing data into a ready-to-use and self-contained Nextflow pipeline.

Investigating drug-gut microbiota interactions: reductive and hydrolytic metabolism of oral glucocorticoids by in vitro artificial gut microbiota

Elucidation of the role of gut microbiota in the metabolism of orally administered drugs may improve therapeutic effectiveness and contribute to the development of personalized medicine. In this study, ten different artificial gut microbiota (AGM), obtained by culturing fecal samples in a continuous fermentation system, were challenged for their metabolizing capacity on a panel of six glucocorticoids selected from either prodrugs or drugs. Data from metabolic stability assays highlighted that, while the hydrolysis-mediated conversion of prodrugs to drugs represented only a minor metabolic pathway, significant differences in the stability of parent compounds and in their conversion rates to multiple reductive metabolites were obtained for the selected drugs. In the latter case, a taxonomic composition-dependent ability to convert parent drugs to metabolites was observed. Indeed, the artificial microbial communities dominated by the genus Bacteroides showed the maximal conversion of parent glucocorticoids to several metabolites. Furthermore, the effect of drugs on AGM was also evaluated through shallow shotgun sequencing and flow cytometry-based total bacterial cell count highlighting that these drugs can affect both the taxonomic composition and growth performances of the human gut microbiota.

Characterization of larval gut microbiota of two endoparasitoid wasps associated with their common host, Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae).

Insect gut microbes play important roles in digestion, metabolism, development, and environmental adaptation. Parasitoid wasps are one of the most important biological control agents in pest control, while the gut microbial species compositions and the associated functions have been poorly investigated. Two endoparasitoid wasps, Cotesia vestalis and Diadromus collaris, parasitize the larval stage and pupal stage of the diamondback moth, Plutella xylostella, respectively. Using whole-genome shotgun metagenomic sequencing, we characterized the gut microbial composition, diversity, and potential functional roles associated with the two parasitoid wasp larvae. The results reveal that Proteobacteria and Firmicutes are the dominant phyla in the gut of C. vestalis and D. collaris larvae, with Rhizobium and Enterococcus being the dominant genera. The putative microbial functions associated with the two parasitoid wasps might play a virtual role in assisting in consuming the host's nutritional composition. The enriched CAZymes family genes are primarily involved in the degradation and synthesis of chitin. Despite the richness of microbial species and communities, the microbes species and the microbial community structure exhibit significant similarity between the two parasitoid wasps and between the parasitoid wasp and the host P. xylostella. Notably, the prevalence of the genus Enterococcus shared among them suggests a possible link of gut microbes between the host and their associated parasitoids. Our study offers insights into the gut microbe-based interactions between the host and parasitoid wasps for the first time, potentially paving the way for the development of an ecologically friendly biocontrol strategy against the pest P. xylostella.IMPORTANCEEndoparasitoid wasps spend the majority of their lifespan within their host and heavily rely on the host's nutrition for survival. There is limited understanding regarding the composition and physiological impacts of gut microbial communities in parasitoid wasps, particularly during the larval stage, which is directly linked to the host. Based on a thorough characterization of the gut microbe and comprehensive comparative analysis, we found the microbial species of the larval parasitoid wasp Cotesia vestalis and the pupal parasitoid wasp Diadromus collaris were similar, sharing 159 genera and 277 species, as were the microbial community structure. Certain of the dominant microbial strains of the two parasitoid wasps were similar to that of their host Plutella xylostella larvae, revealing host insect may affect the microbial community of the parasitoid wasps. The putative microbial functions associated with the parasitoid wasp larvae play an important role in dietary consumption.

Shotgun DNA sequencing for human identification: Dynamic SNP selection and likelihood ratio calculations accounting for errors

Shotgun sequencing is a DNA analysis method that potentially determines the nucleotide sequence of every DNA fragment in a sample, unlike PCR-based genotyping methods that is widely used in forensic genetics and targets predefined short tandem repeats (STRs) or predefined single nucleotide polymorphisms (SNPs). Shotgun DNA sequencing is particularly useful for highly degraded low-quality DNA samples, such as ancient samples or those from crime scenes. Here, we developed a statistical model for human identification using shotgun sequencing data and developed formulas for calculating the evidential weight as a likelihood ratio (LR). The model uses a dynamic set of binary SNP loci and takes the error rate from shotgun sequencing into consideration in a probabilistic manner. To our knowledge, the method is the first to make this possible. Results from replicated shotgun sequencing of buccal swabs (high-quality samples) and hair samples (low-quality samples) were arranged in a genotype-call confusion matrix to estimate the calling error probability by maximum likelihood and Bayesian inference. Different genotype quality filters may be applied to account for genotyping errors. An error probability of zero resulted in the commonly used LR formula for the weight of evidence. Error probabilities above zero reduced the LR contribution of matching genotypes and increased the LR in the case of a mismatch between the genotypes of the trace and the person of interest. In the latter scenario, the LR increased from zero (occurring when the error probability was zero) to low positive values, which allow for the possibility that the mismatch may be due to genotyping errors. We developed an open-source R package, wgsLR, which implements the method, including estimation of the calling error probability and calculation of LR values. The R package includes all formulas used in this paper and the functionalities to generate the formulas.

The Comparative Full-Length Genome Characterization of African Swine Fever Virus Detected in Thailand.

African swine fever virus (ASFV) has been responsible for the globally devastating epidemics in wild and domesticated pigs. Of the 24 identified ASFV genotypes, genotype II is the primary cause for the pandemic occurring in Europe and Asia since its emergence in Georgia in 2007. The current study aimed to characterize the full-length genomic pattern of the ASFV strain from Thailand, TH1_22/CR (Accession No. PP915735), which was then compared with genomic diversity across other Asian isolates using Georgia 2007/1 (Accession No. FR682468) as the reference. Viral DNA was isolated from the pig spleen sample following library preparation and paired-end sequencing using the MiSeq Illumina platform. The sequenced TH1_22/CR isolate spanned 189,395 nucleotides encoding 193 open reading frames (ORFs), exhibiting maximum nucleotide similarity (99.99%) with Georgian (Georgia 2007/1) and Chinese (Wuhan 2019-1 and China HLJ) isolates. Based on phylogenetic analysis, the TH1_22/CR isolate (Accession No. PP915735) was characterized as genotype II, serogroup 8, and IGR-II due to the presence of three tandem repeat sequences (TRSs). Genetic variations including SNPs and single and polynucleotide indels were identified in TH1_22/CR in agreement with other Asian isolates. For comprehensive analysis, the genome was divided into four regions (I-IV) based on gene location. Overall, the TH1_22/CR isolate demonstrated eight SNPs and indels in its genome. Two unique SNPs were reported in the coding regions of the TH1_22/CR isolate, out of which, a C-591-T substitution was seen in MGF 360-4L and a C-297-T was found in A238L, and four unique SNPs were reported in non-coding regions (NCRs). Furthermore, a 29 bp deletion was observed in the IGR between MGF 110-13La and MGF 110-13Lb, as well as 52 bp deletion in the ASFV G ACD 00350 gene. This comparative analysis establishes the foundational information for future studies on the diversity and phylogeography of this regionally significant genetic sub-group of ASFV.

The gut microbiota modifies antibody durability and booster responses after SARS-CoV-2 vaccination

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are pivotal in combating coronavirus disease 2019 (COVID-19); however, the declining antibody titers postvaccination pose challenges for sustained protection and herd immunity. Although gut microbiome is reported to affect the early antibody response after vaccination, its impact on the longevity of vaccine-induced antibodies remains unexplored.MethodsA prospective cohort study was conducted involving 44 healthy adults who received two doses of either the BNT162b2 or ChAdOx1 vaccine, followed by a BNT162b2 booster at six months. The gut microbiome was serially analyzed using 16S rRNA and shotgun sequencing, while humoral immune response was assessed using a SARS-CoV-2 spike protein immunoassay.ResultsFaecalibacterium prausnitzii was associated with robust and persistent antibody responses post-BNT162b2 vaccination. In comparison, Escherichia coli was associated with a slower antibody decay following ChAdOx1 vaccination. The booster immune response was correlated with metabolic pathways involving cellular functions and aromatic amino acid synthesis.ConclusionsThe findings of this study underscored the potential interaction between the gut microbiome and the longevity/boosting effect of antibodies following vaccination against SARS-CoV-2. The identification of specific microbial associations suggests the prospect of microbiome-based strategies for enhancing vaccine efficacy.

Dysbiosis of the gut microbiota is associated with in-hospital mortality in patients with antibiotic-associated diarrhoea: A metagenomic analysis

BackgroundThe increasing incidence of antibiotic-associated diarrhoea (AAD) is a serious health care problem. Dysbiosis of the gut microbiota is suspected to play a role in the pathogenesis of AAD, but its impact on the clinical outcomes of patients remains unclear. MethodsBetween May and October 2022, 210 patients with AAD admitted to a university hospital and 100 healthy controls were recruited. DNA extraction from stool specimens and shotgun sequencing were performed. Machine learning was conducted to assess profiling at different taxonomic levels and to select variables for multivariable analyses. ResultsPatients were classified into two groups: Clostridioides difficile infection (CDI, n = 39) and non-CDI AAD (n = 171). The in-hospital mortality rate for the patients was 20.0%, but the presence of C. difficile in the gut microbiota was not associated with mortality. Machine learning showed that taxonomic profiling at the genus level best reflected patient prognosis. The in-hospital mortality of patients was associated with the relative abundance of specific gut microbial genera rather than alpha-diversity: each of the five genera correlated either positively (Enterococcus, Klebsiella, Corynebacterium, Pseudomonas, and Anaerofustis) or negatively (Bifidobacterium, Bacteroides, Streptococcus, Faecalibacterium, and Dorea). Genes for vancomycin resistance were significantly associated with in-hospital mortality in patients with AAD (adjusted hazard ratios, 2.45; 95% CI, 1.20–4.99). ConclusionThis study demonstrates the potential utility of metagenomic studies of the gut microbial community as a biomarker for prognosis prediction in AAD patients.

Comparative assessment of microbiome and resistome of influent and effluent of sewage treatment plant and common effluent treatment plant located in Delhi, India using shotgun approach

Antimicrobial resistance (AMR) is a significant threat that demands surveillance to identify and analyze trends of the emerging antibiotic resistance genes (ARGs) and potential microbial carriers. The influent of the wastewater treatment plants (WWTPs) reflects the microbes derived from the population and effluent being the source of dissemination of potential pathogenic microbes and AMR. The present study aimed to monitor microbial communities and antibiotic resistance genes in WWTPs employing a whole metagenome shotgun sequencing approach. The samples were collected from a sewage treatment plant (STP) and a common effluent treatment plant (CETP) in Delhi, India. The results showed the influent of STP to be rich in Bifidobacterium, Bacteroides, Escherichia, Arcobacter, and Pseudomonas residents of gut microbiota and known to cause diseases in humans and animals; whereas the CETP sample was abundant in Aeromonas, Escherichia, and Shewanella known to be involved in the degradation of different compounds. Interestingly, the effluent samples from both STPs and CETP were rich in microbial diversity, comprising organic and xenobiotic compound degrading and disease-causing bacteria, indicating the effluent being the source of dissemination of concerning bacteria to the environment. The functional profile at both sites displayed similarity with an abundance of housekeeping function genes as analyzed by Clusters of Orthologous Genes (COG), KEGG Orthology (KO), and subsystem databases. Resistome profiling by MEGARes showed the dominance of ARGs corresponding to beta-lactams having relative abundance ranging from 16% to 34% in all the metagenome datasets, followed by tetracycline (8%–16%), aminoglycosides (7%–9%), multi-drug (5%–9%), and rifampin (3%–9%). Also, AMR genes oxa, ant3-DPRIME, and rpoB, which are of clinical importance were predominantly and most prevalently present in all the samples. The presence of AMR in effluents from both types of treatment plants indicates that wastewater from both sources contributes to the spread of pathogenic bacteria and resistance genes, increasing the environmental AMR burden and therefore requires tertiary treatment before discharge. This work will facilitate further research towards the identification of suitable biomarkers for monitoring antibiotic resistance.

The Microleaks study: 16S community profile and metagenomic shotgun sequencing signatures associated with anastomotic leak

Anastomotic leaks (AL) are the most severe complications of colorectal surgery. The cause of AL is unclear, but recent studies have implicated the intestinal microbiota in its development. We aimed to determine whether there is an identifiable microbial pattern in the mucosal microbiota associated with AL. A pragmatic series of 162 patients undergoing colorectal resection with anastomosis had swabs taken from the proximal and distal mucosa of the bowel resection immediately after the tissue was excised. DNA was extracted for 16S rRNA amplicon gene sequencing and a subset for metagenomic shotgun sequencing (MGS). The AL rate in the cohort was approximately 15% (25/162). The alpha diversity measures from the intraoperative swabs were all significantly increased for AL, and there were significant differences in the beta diversity measures for AL from both the 16S and MGS datasets. The predictive power of AL was more sensitive when both proximal and distal communities were considered, and the species-level classifier AUC-ROC was stronger for the MGS dataset than for the 16S data (AUC = 0.92 and 0.76, respectively). We also report, for the first time, the functional changes in intraoperative AL microbes and noted an increase in the relative abundance of pathways with fermentation end products. This result was also found in our murine model of anastomoses (n = 20). At the time of surgery, the mucosal microbiota of the anastomotic extremities exhibits subtle differences at the species level and altered fermentation capacity, which may be associated with AL outcomes. A greater understanding of these insights could improve AL prognosis and preoperative management to reduce the occurrence of this life-threatening condition.

Comparative transcriptomics of broad-spectrum and synthetic cannabidiol treated C2C12 skeletal myotubes.

Cannabidiol (CBD) is widely used in sports for recovery, pain management, and sleep improvement, yet its effects on muscle are not well understood. This study aimed to determine the transcriptional response of murine skeletal muscle myotubes to broad-spectrum CBD and synthetic CBD (sCBD). Differentiated C2C12 myotubes were treated with 10 μM CBD, sCBD, or vehicle control (DMSO) for 24 h before RNA extraction. Poly-A tail-enriched mRNA libraries were constructed and sequenced using 2 × 50 bp paired-end sequencing. CBD and sCBD treatment induced 4489 and 1979 differentially expressed genes (DEGs; p < 0.001, FDR step-up <0.05), respectively, with common upregulation of 857 genes and common downregulation of 648 genes. Common upregulated DEGs were associated with "response to unfolded protein," "cell redox homeostasis," "endoplasmic reticulum stress," "oxidative stress," and "cellular response to hypoxia." Common downregulated DEGs were linked to "sarcomere organization," "skeletal muscle tissue development," "regulation of muscle contraction," and "muscle contraction." CBD treatment induced unique DEGs compared to sCBD. The data indicate CBD may induce mild cellular stress, activating pathways associated with altered redox balance, unfolded protein response, and endoplasmic reticulum stress. We hypothesize that CBD interacts with muscle and may elicit a "mitohormetic" effect that warrants further investigation.

Abstract P381: Sex differences in association between gut microbiome and 24-hour blood pressure variability

Background: Blood pressure (BP) variability is an independent risk factor for cardiovascular disease (CVD). BP variability has been associated with cardiovascular organ damage and is a potential therapeutic target for the prevention of CVD. Recently, the gut microbiome (GM) has been shown to have a pathological role in hypertension, however, the association between GM and BP variability is poorly understood. Methods: 241 community-dwelling individuals free of symptomatic CVD from Hong Kong (113 males and 128 females, mean age 54±6 years) underwent ambulatory BP monitoring and stool microbiota shotgun sequencing. BP variability was determined as systolic/diastolic BP (SBP/DBP) coefficient of variation (CoV), nighttime dipping, and morning BP surge (MBPS). Sleep latency was estimated using 7-day Actigraphy data. Associations of BP variability with GM, and plasma and stool short-chain fatty acids (SCFAs) were analyzed under statistical models adjusting for age, sex, serum glucose and lipids, sodium intake assessed by urine analysis, menopause status, smoking, fatty liver, BP and sleep latency. Results: Women had a significantly higher 24-hour SBP ( p =0.0005) and DBP ( p =0.002) CoV than men. 24-hour SBP CoV had a negative association with GM α-diversity (Shannon and Simpson’s index: all P &lt;0.05) and a positive association with Firmicutes/Bacteroidetes ratio ( P &lt;0.05), suggesting gut dysbiosis in participants with higher systolic BP variability. Several GM species and SCFAs were significantly associated with the indices of BP variability in both men and women. Notably, Parabacteroides merdae had a negative association with both systolic and diastolic BP CoV (all P &lt;0.05-0.01). Bacteroidetes dorei and Bacteroides intestinalis were reduced in women with higher SBP CoV ( P &lt;0.05) and nondipping status ( P&lt; 0.01), respectively. Prewaking and sleepthrough MBPS had a positive association with Faecalibacterium prausnitzii in women (all P &lt;0.001) and plasma acetic acid levels in men ( P &lt;0.05-0.001). Further analysis indicated that Bacteroidetes dorei may mediate SBP CoV via plasma iso-butyric acid in women (bootstrapping 95% CI: -3.6 to -0.19; P &lt;0.05). Conclusions: This cross-sectional study suggests sex-specific associations between GM and BP variability. Most noteworthy, higher systolic BP variability was associated with a significant reduction in potentially beneficial bacterial species, primarily in women which may be explored for therapeutic potential.

MBCN: A novel reference database for Effcient Metagenomic analysis of human gut microbiome

Metagenomic shotgun sequencing data can identify microbes and their proportions. But metagenomic shotgun data profiling results obtained from multiple projects using different reference databases are difficult to compare and apply meta-analysis. Our work aims to create a novel collection of human gut prokaryotic genomes, named Microbiome Collection Navigator (MBCN). 2379 human gut metagenomic samples are screened, and 16,785 metagenome-assembled genomes (MAGs) are assembled using a standardized pipeline. In addition, MAGs are combined with the representative genomes from public prokaryotic genomes collections to cluster, and pan-genomes for each cluster's genomes are constructed to build Kraken2 and Bracken databases. The databases built by MBCN are more comprehensive and accurate for profiling metagenomic reads comparing with other collections on simulated reads and virtual bio-projects. We profile 1082 human gut metagenomic samples with MBCN database and organize profiles and metadata on the web program. Meanwhile, using MBCN as a reference database, we also develop a unified, standardized, and systematic metagenomic analysis pipeline and platform, named MicrobiotaCN (http://www.microbiota.cn) and common statistical and visualization tools for microbiome research are integrated into the web program. Taken together, MBCN and MicrobiotaCN can be a valuable resource and a powerful tool that allows researchers to perform metagenomic analysis by a unified pipeline efficiently.

Randomized Clinical Trial to Evaluate the Effect of Probiotic Intake on Androgenic Alopecia.

This study aimed to assess the impact of a combination of probiotic strains of Lactiplantibacillus on the treatment of androgenic alopecia (AGA). To this end, 136 individuals with AGA (62 men and 74 women) aged 18-65 years were enrolled in a double-blind, parallel-group clinical trial. A total of 115 individuals (57 in the probiotic group and 58 in the placebo group) completed this study within a 16-week intervention period. Capillary density, thickness, and length of hair were analyzed before and after the intervention using FotoFinder Trichoscale Pro. In addition, the gut microbiota was assessed by paired-end sequencing on the Illumina MiSeq platform (2 × 300 bp). At the conclusion of the treatment period, a notable decline (p < 0.05) in the number of telogen hairs was evident in the probiotic group while hair thickness decreased in the placebo group (p < 0.05). However, the remaining variables did not exhibit any statistically significant changes. In the probiotic-treated group, individuals aged less than 37.5 years exhibited a reduction in the number and density of telogen hair (p = 0.0693 and p = 0.0669, respectively) and an increase in hair length (p = 0.0871). Furthermore, a notable decline in the number and density of vellus hair (p < 0.05) was observed, and this was accompanied by no change in the hair thickness. The probiotic-treated group exhibited a significantly higher abundance of Lactobacillus (p-adjusted < 0.05, DEseq2 test) and demonstrated a notable reduction in the number and density of telogen hair, and this was accompanied by an increase in the percentage of anagen hair. The probiotic mixture was well tolerated by the participants, with a treatment adherence rate of 90%. In light of this study's limitations, it can be concluded that a mixture of three strains of Lactiplantibacillus promotes the presence of terminal follicles, preventing their gradual miniaturization, which is a characteristic of AGA.

Shotgun Sequencing of 512-mer Copolyester Allows Random Access to Stored Information.

Digital information encoded in polymers has been exclusively decoded by mass spectrometry. However, the size limit of analytes in mass spectrometry restricts the storage capacity per chain. In addition, sequential decoding hinders random access to the bits of interest without full-chain sequencing. Here we report the shotgun sequencing of a 512-mer sequence-defined polymer whose molecular weight (57.3 kDa) far exceeds the analytical limit of mass spectrometry. A 4-bit fragmentation code was implemented at aperiodic positions during the synthetic encoding of 512-bit information without affecting storage capacity per chain. Upon activating the fragmentation code, the polymer chain splits into 18 oligomers, which could be individually decoded by tandem-mass sequencing. These sequences were computationally reconstructed into a full sequence using an error-detection method. The proposed sequencing method eliminates the storage limit of a single polymer chain and allows random access to the bits of interest without full-chain sequencing.

The role of halophyte-induced saline fertile islands in soil microbial biogeochemical cycling across arid ecosystems

Halophyte shrubs, prevalent in arid regions globally, create saline fertile islands under their canopy. This study investigates the soil microbial communities and their energy utilization strategies associated with tamarisk shrubs in arid ecosystems. Shotgun sequencing revealed that high salinity in tamarisk islands reduces functional gene alpha-diversity and relative abundance compared to bare soils. However, organic matter accumulation within islands fosters key halophilic archaea taxa such as Halalkalicoccus, Halogeometricum, and Natronorubrum, linked to processes like organic carbon oxidation, nitrous oxide reduction, and sulfur oxidation, potentially strengthening the coupling of nutrient cycles. In contrast, bare soils harbor salt-tolerant microbes with genes for autotrophic energy acquisition, including carbon fixation, H2 or CH4 consumption, and anammox. Additionally, isotope analysis shows higher microbial carbon use efficiency, N mineralization, and denitrification activity in tamarisk islands. Our findings demonstrate that halophyte shrubs serve as hotspots for halophilic microbes, enhancing microbial nutrient transformation in saline soils.

The chromosome level genome assembly of the Asian green mussel, Perna viridis

The Asian green mussel, Perna viridis is an important aquaculture species in the family Mytilidae contributing substantially to molluscan aquaculture. We generated a high-quality chromosome level assembly of this species by combining PacBio single molecule sequencing technique (SMRT), Illumina paired-end sequencing, high-throughput chromosome conformation capture technique (Hi-C) and Bionano mapping. The final assembly resulted in a genome of 723.49 Mb in size with a scaffold N50 of 49.74 Mb with 99% anchored into 15 chromosomes. A total of 49654 protein-coding genes were predicted from the genome. The presence of 634 genes associated with the cancer pathway and 408 genes associated with viral carcinogenesis indicates the potential of this species to be used as a model for cancer studies. The chromosome-level assembly of this species is also a valuable resource for further genomic selection and selective breeding for improving economically important aquaculture traits and augmenting aquaculture productivity.