Genome Shotgun Sequencing Research Articles

Inconsistencies in the published rabbit ribosomal rRNAs: a proposal for uniformity in sequence and site numbering.

Examination of all publicly available Oryctolagus cuniculus (rabbit) ribosome cryo-EM structures reveals numerous confusing inconsistencies. First, there are a plethora of single nucleotide differences among the various rabbit 28S and 18S rRNA structures. Second, two nucleotides are absent from the NCBI Reference Sequence for the 18S rRNA gene. Moving forward, we propose using the Broad Institute's rabbit whole genome shotgun sequence and numbering to reduce modeling ambiguity and improve consistency between ribosome models.

K-mer genome-wide association study for anthracnose and BCMV resistance in a Phaseolus vulgaris Andean Diversity Panel.

Access to broad genomic resources and closely linked marker-trait associations for common beans (Phaseolus vulgaris L.) can facilitate development of improved varieties with increased yield, improved market quality traits, and enhanced disease resistance. The emergence of virulent races of anthracnose (caused by Colletotrichum lindemuthianum) and bean common mosaic virus (BCMV) highlight the need for improved methods to identify and incorporate pan-genomic variation in breeding for disease resistance. We sequenced the P. vulgaris Andean Diversity Panel (ADP) and performed a genome-wide association study (GWAS) to identify associations for resistance to BCMV and eight races of anthracnose. Historical single nucleotide polymorphism (SNP)-chip and phenotypic data enabled a three-way comparison between SNP-chip, reference-based whole genome shotgun sequence (WGS)-SNP, and reference-free k-mer (short nucleotide subsequence) GWAS. Across all traits, there was excellent concordance between SNP-chip, WGS-SNP, and k-mer GWAS results-albeit at a much higher marker resolution for the WGS data sets. Significant k-mer haplotype variation revealed selection of the linked I-gene and Co-u traits in North American breeding lines and cultivars. Due to structural variation, only 9.1 to 47.3% of the significantly associated k-mers could be mapped to the reference genome. Thus, to determine the genetic context of cis-associated k-mers, we generated draft whole genome assemblies of four ADP accessions and identified an expanded local repertoire of disease resistance genes associated with resistance to anthracnose and BCMV. With access to variant data in the context of a pan-genome, high resolution mapping of agronomic traits for common bean is now feasible.

Museomics reveal origins of East African Pleophylla forest chafers and Miocene forest connectivity

Here we present a nearly complete species-level phylogeny including 23 of the 25 known species of the forest-dwelling herbivorous scarab chafer beetle genus Pleophylla (Coleoptera: Scarabaeidae: Sericinae), based on the analysis of 950 nuclear genes (metazoan-level universal single-copy orthologs; mzl-USCOs). DNA sequences were obtained from freshly collected, ethanol-preserved samples and from dried museum specimens by target enrichment or genome shotgun sequencing. Alignment completeness of mzl-USCOs newly obtained here by target DNA enrichment of ethanol samples were very heterogenous and lower (29–62 %) than in Dietz et al. (2023a), while that of sequences recovered from dried samples was even lower (∼19 %). Alignment completeness of the sequences obtained from low coverage shotgun sequencing was highest (∼92 %), although the average coverage was much lower than for the target enrichment samples. We used the resulting phylogeny to reconstruct the historical biogeography of the group. To estimate a time-calibrated tree, we combined the mzl-USCO data of Pleophylla with a nucleotide alignment from an available transcriptomic dataset of Scarabaeoidea and used two different sets of secondary calibration points. Despite the problems associated with the capture rate of mzl-USCO sequences from museum specimens, we were able to infer a well-resolved phylogeny of the genus Pleophylla that also provided reliable estimates of the phylogenetic position of species for which we had little sequence data. Our study clearly identified South Africa as the geographic origin of Pleophylla. Timing and biogeographic history confirm a persistent fragmentation of forests since the Eocene. The occurrence of only one long-distance dispersal event from southern Africa to the Eastern African Arc even during the Miocene highlights the limited dispersal possibilities for these forest-adapted chafers, which do not seem to have had important northerly range expansions along hypothetical forest corridors during the Pleistocene.

Constructing phylogenetic trees for microbiome data analysis: A mini-review

As next-generation sequencing technologies advance rapidly and the cost of metagenomic sequencing continues to decrease, researchers now face an unprecedented volume of microbiome data. This surge has stimulated the development of scalable microbiome data analysis methods and necessitated the incorporation of phylogenetic information into microbiome analysis for improved accuracy. Tools for constructing phylogenetic trees from 16S rRNA sequencing data are well-established, as the highly conserved regions of the 16S gene are limited, simplifying the identification of marker genes. In contrast, metagenomic and whole genome shotgun (WGS) sequencing involve sequencing from random fragments of the entire gene, making identification of consistent marker genes challenging owing to the vast diversity of genomic regions, resulting in a scarcity of robust tools for constructing phylogenetic trees. Although bacterial sequence tree construction tools exist for upstream bioinformatics, many downstream researchers—those integrating these trees into statistical models or machine learning—are either unaware of these tools or find them difficult to use due to the steep learning curve of processing raw sequences. This is compounded by the fact that public datasets often lack phylogenetic trees, providing only abundance tables and taxonomic classifications. To address this, we present a comprehensive review of phylogenetic tree construction techniques for microbiome data (16S rRNA or whole-genome shotgun sequencing). We outline the strengths and limitations of current methods, offering expert insights and step-by-step guidance to make these tools more accessible and widely applicable in quantitative microbiome data analysis.

A Multiomics Evaluation of the Countermeasure Influence of 4-Week Cranberry Beverage Supplementation on Exercise-Induced Changes in Innate Immunity.

This study examined the effect of a 4-week unsweetened cranberry beverage (CRAN) (317 mg polyphenols) versus placebo beverage (PLAC) ingestion (240 mL/day) on moderating exercise-induced changes in innate immunity. Participants included 25 male and female non-elite cyclists. A randomized, placebo-controlled, double-blind crossover design was used with two 4-week supplementation periods and a 2-week washout period. Supplementation periods were followed by an intensive 2.25 h cycling bout. Six blood samples were collected before and after supplementation (in an overnight fasted state) and at 0 h, 1.5 h, 3 h, and 24 h post-exercise. Stool and urine samples were collected pre- and post-supplementation. Outcome measures included serum creatine kinase, myoglobin, and cortisol, complete blood counts, plasma untargeted proteomics, plasma-targeted oxylipins, untargeted urine metabolomics, and stool microbiome composition via whole genome shotgun (WGS) sequencing. Urine CRAN-linked metabolites increased significantly after supplementation, but no trial differences in alpha or beta microbiota diversity were found in the stool samples. The 2.25 h cycling bout caused significant increases in plasma arachidonic acid (ARA) and 53 oxylipins (FDR q-value < 0.05). The patterns of increase for ARA, four oxylipins generated from ARA-cytochrome P-450 (CYP) (5,6-, 8,9-, 11,12-, and 14,15-diHETrEs), two oxylipins from linoleic acid (LA) and CYP (9,10-DiHOME, 12,13-DiHOME), and two oxylipins generated from LA and lipoxygenase (LOX) (9-HODE, 13-HODE) were slightly but significantly higher for the CRAN versus PLAC trial (all interaction effects, p < 0.05). The untargeted proteomics analysis showed that two protein clusters differed significantly between the CRAN and PLAC trials, with CRAN-related elevations in proteins related to innate immune activation and reduced levels of proteins related to the regulation of the complement cascade, platelet activation, and binding and uptake of ligands by scavenger receptors. No trial differences were found for cortisol and muscle damage biomarkers. CRAN versus PLAC juice resulted in a significant increase in CRAN-related metabolites but no differences in the gut microbiome. CRAN supplementation was associated with a transient and modest but significant post-exercise elevation in selected oxylipins and proteins associated with the innate immune system.

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.

Identification of Candidate Avirulence and Virulence Genes Corresponding to Stem Rust (Puccinia graminis f. sp. tritici) Resistance Genes in Wheat.

Stem rust, caused by the biotrophic fungal pathogen Puccinia graminis f. sp. tritici (Pgt), is an important disease of wheat. However, the majority of Pgt virulence/avirulence loci and underlying genes remain uncharacterized due to the constraints of developing bi-parental populations with this obligate biotroph. Genome-wide association studies (GWAS) using a sexual Pgt population mainly collected from the Pacific Northwestern United States were used to identify candidate virulence/avirulence effector genes corresponding to the six wheat Sr genes: Sr5, Sr21, Sr8a, Sr17, Sr9a, and Sr9d. The Pgt isolates were genotyped using whole-genome shotgun sequencing that identified approximately 1.2 million single nucleotide polymorphisms (SNPs) and were phenotyped at the seedling stage on six Sr gene differential lines. Association mapping analyses identified 17 Pgt loci associated with virulence or avirulence phenotypes on six Pgt resistance genes. Among these loci, 16 interacted with a specific Sr gene, indicating Sr-gene specific interactions. However, one avirulence locus interacted with two separate Sr genes (Sr9a and Sr17), suggesting two distinct Sr genes identifying a single avirulence effector. A total of 24 unique effector gene candidates were identified, and haplotype analysis suggests that within this population, AvrSr5, AvrSr21, AvrSr8a, AvrSr17, and AvrSr9a are dominant avirulence genes, while avrSr9d is a dominant virulence gene. The putative effector genes will be fundamental for future effector gene cloning efforts, allowing for further understanding of rust effector biology and the mechanisms underlying virulence evolution in Pgt with respect to race-specific R-genes. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Clinical Impact of Microbiome Characteristics in Treatment-Naïve Extranodal NK/T-Cell Lymphoma Patients.

Extranodal NK/T-cell lymphoma (ENKTL) predominantly manifests in East Asia and Latin America. Despite shared intrinsic factors, such as ethnic and genetic backgrounds, the progression of ENKTL can be influenced by extrinsic factors related to changing lifestyle patterns. This study collected stool samples from newly diagnosed (ND)-ENKTL patients (n=40) and conducted whole genome shotgun sequencing. ND-ENKTL revealed reduced alpha diversity in ND-ENKTL compared to healthy controls (HCs) (p=0.008), with Enterobacteriaceae abundance significantly contributing to the beta diversity difference between ENKTL and HCs (p<0.001). Functional analysis indicated upregulated aerobic metabolism and degradation of aromatic compounds in ND-ENKTL. Enterobacteriaceae were associated not only with clinical data explaining disease status (serum CRP, stage, prognosis index of natural killer cell lymphoma (PINK), and PINK-E) but also with clinical outcomes (early relapse and short progression-free survival). The relative abundance of Enterobacteriaceae at the family level was similar between ENKTL and diffuse large B-cell lymphoma (DLBCL) (p=0.140). However, the ENKTL exhibited a higher abundance of Escherichia, in contrast to the prevalence of Enterobacter and Citrobacter in DLBCL. Linear regression analysis demonstrated a significant association between Escherichia abundance and PD-L1 levels in tissue samples (p=0.025), whereas no correlation with PD-L1 was observed for Enterobacteriaceae at the family level (p=0.571). ND-ENKTL exhibited an abundance of Enterobacteriaceae and a dominant presence of Escherichia. These microbial characteristics correlated with disease status, treatment outcomes, and PD-L1 expression, suggesting the potential of the ENKTL microbiome as a biomarker and cause of lymphomagenesis, which warrants further exploration.

Novel Cascade Alpha Satellite HORs in Orangutan Chromosome 13 Assembly: Discovery of the 59mer HOR-The largest Unit in Primates-And the Missing Triplet 45/27/18 HOR in Human T2T-CHM13v2.0 Assembly.

From the recent genome assembly NHGRI_mPonAbe1-v2.0_NCBI (GCF_028885655.2) of orangutan chromosome 13, we computed the precise alpha satellite higher-order repeat (HOR) structure using the novel high-precision GRM2023 algorithm with Global Repeat Map (GRM) and Monomer Distance (MD) diagrams. This study rigorously identified alpha satellite HORs in the centromere of orangutan chromosome 13, discovering a novel 59mer HOR-the longest HOR unit identified in any primate to date. Additionally, it revealed the first intertwined sequence of three HORs, 18mer/27mer/45mer HORs, with a common aligned "backbone" across all HOR copies. The major 7mer HOR exhibits a Willard's-type canonical copy, although some segments of the array display significant irregularities. In contrast, the 14mer HOR forms a regular Willard's-type HOR array. Surprisingly, the GRM2023 high-precision analysis of chromosome 13 of human genome assembly T2T-CHM13v2.0 reveals the presence of only a 7mer HOR, despite both the orangutan and human genome assemblies being derived from whole genome shotgun sequences.

Genomic analysis of a spontaneous unifoliate mutant reveals gene candidates associated with compound leaf development in Vigna unguiculata [L] Walp

Molecular mechanisms which underpin compound leaf development in some legumes have been reported, but there is no previous study on the molecular genetic control of compound leaf formation in Vigna unguiculata (cowpea), an important dryland legume of African origin. In most studied species with compound leaves, class 1 KNOTTED-LIKE HOMEOBOX genes expressed in developing leaf primordia sustain morphogenetic activity, allowing leaf dissection and the development of leaflets. Other genes, such as, SINGLE LEAFLET1 in Medicago truncatula and Trifoliate in Solanum lycopersicum, are also implicated in regulating compound leaf patterning. To set the pace for an in-depth understanding of the genetics of compound leaf development in cowpea, we applied RNA-seq and whole genome shotgun sequence datasets of a spontaneous cowpea unifoliate mutant and its trifoliate wild-type cultivar to conduct comparative reference-based gene expression, de novo genome-wide isoform switch, and genome variant analyses between the two genotypes. Our results suggest that genomic variants upstream of LATE ELONGATED HYPOCOTYL and down-stream of REVEILLE4, BRASSINOSTERIOD INSENSITIVE1 and LATERAL ORGAN BOUNDARIES result in down-regulation of key components of cowpea circadian rhythm central oscillator and brassinosteroid signaling, resulting in unifoliate leaves and brassinosteroid-deficient-like phenotypes. We have stated hypotheses that will guide follow-up studies expected to provide more insights.

Whole-Genome Shotgun Metagenomic Sequencing Reveals Shifts in the Skin Microbiome and Bacteriophages of Psoriasis: An Extended Analysis of Published Data

Background Psoriasis is an immune-mediated cutaneous disease that may have shifts in the skin microbiome. Prior research on the skin microbiome in psoriasis has been limited to rRNA based approaches that lack resolution of taxonomic and functional level assessment. Objective To further illuminate strain and sub-strain level analysis of psoriatic lesions using the CosmosID-HUB Microbiome pipeline. Methods A previous study completed by Tett et al recruited patients with psoriasis who had skin microbiome samples taken from psoriatic plaques on the ear and the elbow as well as sites on the skin unaffected by psoriasis. They performed whole genome shotgun sequencing and made their dataset publicly available. We analyzed the dataset using the CosmosID-HUB Microbiome pipeline to evaluate the strain and sub-strain taxonomic analysis as well as functional gene profiling. Results When analyzed with the CosmosID pipeline, both ear and elbow sites in affected areas had decreased alpha diversity compared to unaffected areas. There was an increased relative abundance of Staphylococcus and Corynebacteria at affected sites. We identified distinguishing species and strains of the yeast Malassezia, including M. restricta. that were significantly enriched in healthy elbow samples. Vitamin B12 production genes were not present in psoriatic skin whereas it was present in healthy samples, supporting the notion of relative vitamin B12 deficiency in psoriatic plaques. Phage analysis revealed a greater diversity of Staphylococcus-related phages in unaffected elbow samples. Conclusion A greater diversity of microbial strains and their functional roles identified in this study may help to tailor treatment for psoriasis.

Validation of collection and anaerobic fermentation techniques for measuring prebiotic impact on gut microbiota

BackgroundDefining the ability of prebiotic dietary carbohydrates to influence the composition and metabolism of the gut microbiota is central to defining their health impact in diverse individuals. Many clinical trials are using indirect methods. This study aimed to validate collection and fermentation methods enabling their use in the context of clinical studies. Methods and ResultsParameters tested included stool sample acquisition, storage, and growth conditions. Stool from 3 infants and 3 adults was collected and stored under varying conditions. Samples were cultured anaerobically for two days in the presence of prebiotics, whereupon optical density and pH were measured across time. Whole genome shotgun sequencing and NMR metabolomics were performed. Neither the type of collection vial (standard vial and two different BD anaerobic collection vials) nor cryopreservation (-80 °C or 4 °C) significantly influenced either microbial composition at 16 h of anaerobic culture or the principal components of the metabolome at 8 or 16 h. Metagenomic differences were driven primarily by subject, while metabolomic differences were driven by fermentation sugar (2’-fucosyllactose or dextrose). ConclusionsThese data identified a feasible and valid approach for prebiotic fermentation analysis of individual samples in large clinical studies: collection of stool microbiota using standard vials; cryopreservation prior to testing; and collecting fermentation read-out at 8 and 16 hr. Thus, fermentation analysis can be a valid technique for testing the effects of prebiotics on human fecal microbiota.

Effect of a probiotic and an antibiotic on the mobilome of the porcine microbiota.

Introduction: To consider the growing health issues caused by antibiotic resistance from a "one health" perspective, the contribution of meat production needs to be addressed. While antibiotic resistance is naturally present in microbial communities, the treatment of farm animals with antibiotics causes an increase in antibiotic resistance genes (ARG) in the gut microbiome. Pigs are among the most prevalent animals in agriculture; therefore, reducing the prevalence of antibiotic-resistant bacteria in the pig gut microbiome could reduce the spread of antibiotic resistance. Probiotics are often studied as a way to modulate the microbiome and are, therefore, an interesting way to potentially decrease antibiotic resistance. Methods: To assess the efficacy of a probiotic to reduce the prevalence of ARGs in the pig microbiome, six pigs received either treatment with antibiotics (tylvalosin), probiotics (Pediococcus acidilactici MA18/5M; Biopower® PA), or a combination of both. Their faeces and ileal digesta were collected and DNA was extracted for whole genome shotgun sequencing. The reads were compared with taxonomy and ARG databases to identify the taxa and resistance genes in the samples. Results: The results showed that the ARG profiles in the faeces of the antibiotic and combination treatments were similar, and both were different from the profiles of the probiotic treatment (p < 0.05). The effects of the treatments were different in the digesta and faeces. Many macrolide resistance genes were detected in a higher proportion in the microbiome of the pigs treated with antibiotics or the combination of probiotics and antibiotics. Resistance-carrying conjugative plasmids and horizontal transfer genes were also amplified in faeces samples for the antibiotic and combined treatments. There was no effect of treatment on the short chain fatty acid content in the digesta or the faeces. Conclusion: There is no positive effect of adding probiotics to an antibiotic treatment when these treatments are administered simultaneously.

Abstract 6699: Delineating germline, tumor and extrinsic factors driving mucosal melanoma (MM) risk and response to immune checkpoint inhibitor (ICI) treatment

Abstract Introduction MM is a rare melanoma subtype with distinct biology, low immunogenicity and tumor mutational burden, and subsequently lower response rates to ICIs. The biology of MM is poorly understood. We sought to prospectively, uniformly, and comprehensively profile a cohort of MM patients to determine the interplay of molecular variation, germline predisposition, immunity, gut, mucosal and tumor microbiome and modifiable risk factors on ICI response and resistance in advanced MM. Methods Patients (pts) with MM treated with standard-of-care ICI as any treatment line presenting to MD Anderson are being prospectively enrolled for longitudinal collection for molecular, microbiome, and lifestyle factor profiling. Planned analyses include: whole genome sequencing of normal and tumor tissue, RNA sequencing, and T cell receptor sequencing from fresh tumor samples; germline sequencing and genotype analyses; NanoString Digital Spatial Profiling (DSP) from formalin-fixed paraffin embedded (FFPE) specimens; whole genome shotgun, microbiome profiling and 16S rRNA gene sequencing (16S) of fecal specimens and affected mucosal and tumor sites (swabs); participant lifestyle and patient reported outcomes (PROs) assessments. Fresh tissue is collected and used to develop PDX models and cell lines. Results 98 pts have been enrolled to date; 83 (85%) Caucasians, 8 (8%) Hispanic, 4 (4%) Asians. 67 (68%) females. Median age at MM diagnosis is 64 years (range 32-91 years). MM primary sites include 24 (24%) naso-oral, 29 (30%) urogenital, 26 (27%) anorectal, 7 (7%) conjunctival and 12 (12%) other. Clinical somatic mutation testing was performed in 76 (76%) pts and common mutations were KIT (n = 12, 16%), NRAS (n = 8, 11%) and BRAF V600 or non-V600 (n = 9, 12%). At data cut-off (November 2023), 18 (18%) pts deceased. Median follow-up from first diagnosis to last visit/death was 16 months (range 1-178 months). Sample collections at data cut-off include: blood samples (n = 95; 219 samples); fecal specimens (n = 50; 97 samples); mucosal swabs (tumor/tumor adjacent/oral cavity; n = 38; 101 samples); fresh frozen tumor/normal (n = 38; 55 samples), fresh tumor/normal (n = 28; 36 samples) and FFPE tumor (n = 21; 37 samples) tissue. PROs and dietary assessments have been collected from 47 pts. Fresh tissue for PDX model and cell line development has been collected from 11 pts. Conclusion This is an ongoing prospective study that is expected to drive insights into the tumor/microenvironment/host interactions and factors regulating immunogenicity to predict response and resistance to ICIs in a rare and understudied melanoma subtype. Interrogation of the role of the gut microbiome and its modifiable determinants will lead to the investigation of new therapeutic strategies to modulate the microbiome to improve treatment outcomes in MM. Data will be presented from initial cohort analyses. Citation Format: Florentia Dimitriou, Priyadharsini Nagarajan, Sabitha Prabhakaran, Neus Bota, Mark Knafl, Randy A. Chu, Ashish V. Damania, Pranoti V. Sahasrabhojane, Yasmine Hoballah, Jillian S. Losh, Nadim J. Ajami, Scott E. Woodman, Jennifer A. Wargo, Andrew Futreal, Jennifer L. McQuade. Delineating germline, tumor and extrinsic factors driving mucosal melanoma (MM) risk and response to immune checkpoint inhibitor (ICI) treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6699.

Abstract 3760: The importance of optimizing DNA extraction conditions for formalin fixed paraffin embedded tissues

Abstract The pre-analytic extraction of Formalin Fixed Paraffin Embedded Tissues (FFPETs) appears to have a profound effect on the ability to obtain meaningful data from the DNA therein. FFPETs represent important materials in Pathology that are used to diagnose and analyze cancer. As part of preparing FFPETs, the formalin fixation step preserves the structure of tissues through covalent modifications but can also damage nucleic acids through that same process. Over the last decade, the emergence of Molecular Pathology, which benefits from gentler fixation compared to Histopathology and especially Immunohistopathology, has led to changes in how FFPETs are prepared. However, the remnant FFPETs from over a decade ago may be far from representative of what is now prepared for Molecular Pathology by many laboratories. Here we discuss our optimization of FFPET extraction for gently fixed samples. When we prepared our Seraseq FFPE Homologous Recombination Deficiency (HRD) reference standards using fixation conditions that are recommended for Molecular Pathology and extracted the resulting FFPETs using generic conditions, our initial coverage data from Whole Genome Shotgun (WGS) sequencing was difficult to analyze for changes in copy number across the genome. A major contributor to nonuniform coverage was identified as the formalin reversal step, where deparaffinized proteinase K-digested tissue is heated in water to near boiling to attempt to detach formaldehyde from nucleic acids through hydrolysis. Given the gentler fixation conditions, this step was found to be mostly unnecessary, and omitting it improved the resulting data substantially. A reason for this appears to be that the gentler fixation conditions that are now in use may not protect the DNA from the effects of near-boiling water, where the lack of salt lowers the melting temperature of double stranded DNA fragments and causes them to denature unless they have a higher GC content. Different kits used for the extraction of nucleic acids from FFPETs may benefit from different optimizations to obtain improved sequencing data from today’s Molecular pathology samples, and reference standards that represent such samples provide a useful tool for such optimizations. Citation Format: Matthew G. Butler, Benedicta Forson, Maria Cowen, Jayanthi Ramprakash, Dana Ruminski Lowe, Yves Konigshofer. The importance of optimizing DNA extraction conditions for formalin fixed paraffin embedded tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3760.

Short-chain fatty acids in breast milk and their relationship with the infant gut microbiota.

The short-chain fatty acids (SCFAs) contained in breast milk play a key role in infant growth, affecting metabolism and enhancing intestinal immunity by regulating inflammation. In order to examine the associations between the microbiota and SCFA levels in breast milk, and explore the roles of SCFAs in regulating the infant gut microbiota, we enrolled 50 paired mothers and infants and collected both breast milk and infant fecal samples. Breast milk SCFA contents were determined by UPLC-MS, and whole genome shotgun sequencing was applied to determine the microbial composition of breast milk and infant feces. The SCFA levels in breast milk were grouped into tertiles as high, medium, or low, and the differences of intestinal microbiota and KEGG pathways were compared among groups. The results demonstrated that breast milk butyric acid (C4) is significantly associated with Clostridium leptum richness in breastmilk. Additionally, the specific Bifidobacterium may have an interactive symbiosis with the main species of C4-producing bacteria in human milk. Women with a low breast milk C4 tertile are associated with a high abundance of Salmonella and Salmonella enterica in their infants' feces. KEGG pathway analysis further showed that the content of C4 in breast milk is significantly correlated with the infants' metabolic pathways of lysine and arginine biosynthesis. This study suggests that interactive symbiosis of the microbiota exists in breast milk. Certain breast milk microbes could be beneficial by producing C4 and further influence the abundance of certain gut microbes in infants, playing an important role in early immune and metabolic development.

Metabolic analysis of the CAZy class glycosyltransferases in rhizospheric soil fungiome of the plant species Moringa oleifera

The target of the present work is to study the most abundant carbohydrate-active enzymes (CAZymes) of glycosyltransferase (GT) class, which are encoded by fungiome genes present in the rhizospheric soil of the plant species Moringa oleifera. The datasets of this CAZy class were recovered using metagenomic whole shotgun genome sequencing approach, and the resultant CAZymes were searched against the KEGG pathway database to identify function. High emphasis was given to the two GT families, GT4 and GT2, which were the highest within GT class in the number and abundance of gene queries in this soil compartment. These two GT families harbor CAZymes playing crucial roles in cell membrane and cell wall processes. These CAZymes are responsible for synthesizing essential structural components such as cellulose and chitin, which contribute to the integrity of cell walls in plants and fungi. The CAZyme beta-1,3-glucan synthase of GT2 family accumulates 1,3-β-glucan, which provides elasticity as well as tensile strength to the fungal cell wall. Other GT CAZymes contribute to the biosynthesis of several compounds crucial for cell membrane and wall integrity, including lipopolysaccharide, e.g., lipopolysaccharide N-acetylglucosaminyltransferase, cell wall teichoic acid, e.g., alpha-glucosyltransferase, and cellulose, e.g., cellulose synthase. These compounds also play pivotal roles in ion homeostasis, organic carbon mineralization, and osmoprotection against abiotic stresses in plants. This study emphasizes the major roles of these two CAZy GT families in connecting the structure and function of cell membranes and cell walls of fungal and plant cells. The study also sheds light on the potential occurrence of tripartite symbiotic relationships involving the plant, rhizospheric bacteriome, and fungiome via the action of CAZymes of GT4 and GT2 families. These findings provide valuable insights towards the generation of innovative agricultural practices to enhance the performance of crop plants in the future.

ECCO Grant Untangling the gut phagosome dynamics of the relapse/remission cycle in ulcerative colitis

Abstract Background and Aims The course of ulcerative colitis (UC) is usually characterized by intertwining periods of remission and relapse. Most microbiome UC studies focus on the gut bacteriome. However, recent studies on animal models suggest that phage-mediated bacterial cell lysis may play a role in sustaining gut inflammation, proving the phageome (collection of bacteriophages) should not be ignored in this disease. Therefore, this project aims to: 1) characterize the gut phageome of patients with UC who suffer from frequent relapses, 2) compare temporal dynamics of gut the phageome between patients and healthy controls, 3) To model interactions between bacteriome, mycobiome, phageome on one hand, and lifestyle factors (diet, stress levels, medication) on the other 4) To infer host status (relapse versus remission of the disease) based on phageome composition, and using interactions inferred in Objective 3. Methods The cohort comprises 24 patients with UC (14 of whom suffered from relapse) and 10 healthy controls, with stool samples collected monthly for a year. Phageome profiling will be achieved via shotgun sequencing of phage genomes from the same stool samples. This dataset will be interrogated together with bacterial (16S rRNA) and fungal (ITS) amplicon data, which have just been generated. Available metadata include disease symptoms, diet, stress levels, work productivity, medication, and faecal calprotectin. Dynamic time-wrapping algorithms will be used to align time series of microbial members with similar temporal dynamics. Dynamic Bayesian Network modelling will infer interactions between microbiome members and predict future relapse. Anticipated Impact The phageome is the often neglected (“dark matter”) part of the microbiome, but has defining interactions with bacteria. We will expand knowledge on inter-species dynamics during relapse and remission cycles of UC. This will potentially lead to new microbiome-related relapse predictors, which may contribute to new prognostic tools in UC, and potentially inform therapeutic avenues.

Human milk cream alters intestinal microbiome of preterm infants: a prospective cohort study.

In very low birth weight (VLBW) infants, human milk cream added to standard human milk fortification is used to improve growth. This study aimed to evaluate the impact of cream supplement on the intestinal microbiome of VLBW infants. Whole genome shotgun sequencing was performed on stool (n = 57) collected from a cohort of 23 infants weighing 500-1250 grams (control = 12, cream = 11). Both groups received an exclusive human milk diet (mother's own milk, donor human milk, and donor human milk-derived fortifier) with the cream group receiving an additional 2 kcal/oz cream at 100 mL/kg/day of fortified feeds and then 4 kcal/oz if poor growth. While there were no significant differences in alpha diversity, infants receiving cream significantly differed from infants in the control group in beta diversity. Cream group samples had significantly higher prevalence of Proteobacteria and significantly lower Firmicutes compared to control group. Klebsiella species dominated the microbiota of cream-exposed infants, along with bacterial pathways involved in lipid metabolism and metabolism of cofactors and amino acids. Cream supplementation significantly altered composition of the intestinal microbiome of VLBW infants to favor increased prevalence of Proteobacteria and functional gene content associated with these bacteria. We report changes to the intestinal microbiome associated with administration of human milk cream; a novel supplement used to improve growth rates of preterm very low birth weight infants. Since little is known about the impact of cream on intestinal microbiota composition of very low birth weight infants, our study provides valuable insight on the effects of diet on the microbiome of this population. Dietary supplements administered to preterm infants in neonatal intensive care units have the potential to influence the intestinal microbiome composition which may affect overall health status of the infant.

Genomic resources for a historical collection of cultivated two-row European spring barley genotypes

Barley genomic resources are increasing rapidly, with the publication of a barley pangenome as one of the latest developments. Two-row spring barley cultivars are intensely studied as they are the source of high-quality grain for malting and distilling. Here we provide data from a European two-row spring barley population containing 209 different genotypes registered for the UK market between 1830 to 2014. The dataset encompasses RNA-sequencing data from six different tissues across a range of barley developmental stages, phenotypic datasets from two consecutive years of field-grown trials in the United Kingdom, Germany and the USA; and whole genome shotgun sequencing from all cultivars, which was used to complement the RNA-sequencing data for variant calling. The outcomes are a filtered SNP marker file, a phenotypic database and a large gene expression dataset providing a comprehensive resource which allows for downstream analyses like genome wide association studies or expression associations.