Genome Shotgun Sequencing Research Articles

Abstract 6110: Multi-site evaluation of FFPE homologous recombination deficiency reference materials

Abstract Homologous recombination deficiency (HRD) arises when defects in DNA repair pathways occur, leading to genomic instability. HRD status is an emerging therapeutic biomarker; NGS assays that measure it can be used to stratify ovarian and breast cancer patients and determine eligibility for clinical trials, and PARP inhibitor- and platinum-based therapies. Seraseq FFPE HRD reference materials were developed to cover a range of genomic instability scores (GIS) to help NGS HRD assay validation and development. Here we discuss a multi-site evaluation across various platforms of our high-positive, low-positive, and negative HRD reference materials. Tumor cell lines were characterized by sequencing and evaluated in collaboration with several IVD partners. Three breast cancer cell lines (along with their SNP-matched normal cell lines) were selected based on their GIS. Tumor cells were blended with their SNP-matched normal cells to achieve ~65% tumor content. Biosynthetic DNA containing mutations in homologous recombination repair (HRR) genes (ATM, BRIP1, RAD51C, RAD51D) were added to the high-positive and negative reference materials targeted at >5% variant allele frequency (VAF) and measured by digital PCR. Formalin fixed paraffin embedded (FFPE) blocks were made and each block was tested for yield per curl using both the Qiagen QIAamp DNA FFPE Tissue Kit and the Maxwell RSC DNA FFPE Kit for extraction and Qubit dsDNA HS kit for concentration analysis. DNA quality was assessed using an Agilent gDNA ScreenTape Assay for the TapeStation. Whole genome shotgun sequencing was performed on extracted DNA using LGC NxSeq AmpFREE Low DNA Library Kit and Roche KAPA UDI adapters and Illumina NextSeq 2000 P1 flow cell. HRD status was evaluated by external collaborators with multiple NGS and microarray assays, including the Illumina TSO500 HRD RUO assay, the SOPHiA DDM HRD Solution and the OncoScan CNV Array A breast cancer cell line with a GIS of ~75 was selected as the HRD high-positive reference material, a second breast cancer cell line with a GIS of ~60 was selected as HRD low-positive, and a third breast cancer cell line with a GIS of ~30 was selected as HRD negative. Representative DNA yields per 10-micron section (determined using the HRD positive FFPE curls extracted by Qiagen QIAamp method) were 165 ± 28 ng/curl. Digital PCR confirmed the presence of the 8 HRR mutations (in 4 genes) at levels >5% VAF. GIS varied for each material across assays, but HRD status was consistent, confirming the wide applicability of the new reference materials We have developed the Seraseq HRD reference materials to meet the needs of laboratories looking to analyze HRD in cancer patient samples. These reference materials facilitate standardization and quality control in HRD testing by clinical labs for current and new PARP inhibitor treatment stratification in expanded patient populations that may include those with WT BRCA1/2 genes Citation Format: Dana Ruminski Lowe, Robert M. Whiting, Matthew G. Butler, Yves Konigshofer, Catherine Huang, Indira Chivukula, Krystyna Nahlik, Dianren Xia, Russell Garlick, Bharathi Anekella. Multi-site evaluation of FFPE homologous recombination deficiency reference materials. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6110.

Metagenomes from Coastal Sediments of Kuwait: Insights into the Microbiome, Metabolic Functions and Resistome.

Coastal sediments in the proximity of wastewater and emergency outfalls are often sinks of pharmaceutical compounds and other organic and inorganic contaminants that are likely to affect the microbial community. The metabolites of these contaminants affect microbial diversity and their metabolic processes, resulting in undesirable effects on ecosystem functioning, thus necessitating the need to understand their composition and functions. In the present investigation, we studied the metagenomes of 12 coastal surface sediments through whole genome shot-gun sequencing. Taxonomic binning of the genes predicted about 86% as bacteria, 1% as archaea, >0.001% as viruses and Eukaryota, and 12% as other communities. The dominant bacterial, archaeal, and fungal genera were Woeseia, Nitrosopumilus, and Rhizophagus, respectively. The most prevalent viral families were Myoviridae and Siphoviridae, and the T4 virus was the most dominant bacteriophage. The unigenes further aligned to 26 clusters of orthologous genes (COGs) and five carbohydrate-active enzymes (CAZy) classes. Glycoside hydrolases (GH) and glycoside transferase (GT) were the highest-recorded CAzymes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) level 3 functions were subjugated by purine metabolism > ABC transporters > oxidative phosphorylation > two-component system > pyrimidine metabolism > pyruvate metabolism > quorum sensing > carbon fixation pathways > ribosomes > and glyoxalate and dicarboxylate metabolism. Sequences allying with plasmids, integrons, insertion sequences and antibiotic-resistance genes were also observed. Both the taxonomies and functional abundances exhibited variation in relative abundances, with limited spatial variability (ANOVA p > 0.05; ANOSIM-0.05, p > 0.05). This study underlines the dominant microbial communities and functional genes in the marine sediments of Kuwait as a baseline for future biomonitoring programs.

Genomic plasticity and adaptive capacity of the quaternary alkyl-ammonium compound and copper tolerant Acinetobacter bohemicus strain QAC-21b isolated from pig manure

Here, we present the genomic characterization of an Acinetobacter bohemicus strain QAC-21b which was isolated in the presence of a quaternary alky-ammonium compound (QAAC) from manure of a conventional German pig farm. The genetic determinants for QAAC, heavy metal and antibiotic resistances are reported based of the whole genome shotgun sequence and physiological growth tests. A. bohemicus QAC-21b grew in a species typical manner well at environmental temperatures but not at 37 °C. The strain showed tolerance to QAACs and copper but was susceptible to antibiotics relevant for Acinetobacter treatments. The genome of QAC-21b contained several Acinetobacter typical QAAC and heavy metal transporting efflux pumps coding genes, but no key genes for acquired antimicrobial resistances. The high genomic content of transferable genetic elements indicates that this bacterium can be involved in the transmission of antimicrobial resistances, if it is released with manure as organic fertilizer on agricultural fields. The genetic content of the strain was compared to that of two other A. bohemicus strains, the type strain ANC 3994T, isolated from forest soil, and KCTC 42081, originally described as A. pakistanensis, a metal resistant strain isolated from a wastewater treatment pond. In contrast to the forest soil strain, both strains from anthropogenically impacted sources showed genetic features indicating their evolutionary adaptation to the anthropogenically impacted environments. Strain QAC-21b will be used as model strain to study the transmission of antimicrobial resistance to environmentally adapted Acinetobacter in agricultural environments receiving high content of pollutants with organic fertilizers from livestock husbandry.

Combining methods for non-invasive fecal DNA enables whole genome and metagenomic analyses in wildlife biology.

Non-invasive biological samples benefit studies that investigate rare, elusive, endangered, or dangerous species. Integrating genomic techniques that use non-invasive biological sampling with advances in computational approaches can benefit and inform wildlife conservation and management. Here, we used non-invasive fecal DNA samples to generate low- to medium-coverage genomes (e.g., >90% of the complete nuclear genome at six X-fold coverage) and metagenomic sequences, combining widely available and accessible DNA collection cards with commonly used DNA extraction and library building approaches. DNA preservation cards are easy to transport and can be stored non-refrigerated, avoiding cumbersome or costly sample methods. The genomic library construction and shotgun sequencing approach did not require enrichment or targeted DNA amplification. The utility and potential of the data generated was demonstrated through genome scale and metagenomic analyses of zoo and free-ranging African savanna elephants (Loxodonta africana). Fecal samples collected from free-ranging individuals contained an average of 12.41% (5.54-21.65%) endogenous elephant DNA. Clustering of these elephants with others from the same geographic region was demonstrated by a principal component analysis of genetic variation using nuclear genome-wide SNPs. Metagenomic analyses identified taxa that included Loxodonta, green plants, fungi, arthropods, bacteria, viruses and archaea, showcasing the utility of this approach for addressing complementary questions based on host-associated DNA, e.g., pathogen and parasite identification. The molecular and bioinformatic analyses presented here contributes towards the expansion and application of genomic techniques to conservation science and practice.

Limited population genetic variation but pronounced seascape genetic structuring in populations of the Mediterranean mussel (Mytilus galloprovincialis) from the eastern Adriatic Sea.

Population genetic analysis of variation at five neutral microsatellite loci for Mediterranean mussels (Mytilus galloprovincialis) from 18 sites along the eastern Adriatic Sea revealed little or no spatial variation. In contrast, seascape genetics analysis revealed a pronounced locus-specific gradient in allelic and genotypic frequencies across the study region. At a sixth locus, MGE7, the frequencies of two alleles, MGE7243 and MGE7249, were strongly associated, negatively and positively, respectively, with a single environmental variable - minimum salinity (minSAL). The frequency of the MGE7243/243 homozygous genotype was strongly negatively associated with minSAL, whereas the frequencies of the MGE7246/249 and the MGE7249/249 genotypes were strongly positively correlated with minSAL. Interpretation of these pronounced gradients is confounded by the fact that minSAL and another environmental variable, maximum sea surface temperature (maxSST), are highly correlated (R=-.911) and are therefore not necessarily acting independently. BLAST searches of the MGE7 locus against M. galloprovincialis whole genome shotgun sequence returned an alignment with contig mg10_S01094 (accession UYJE01010330.1) and 7 predicted M. galloprovincialis proteins VDI82194.1 - VDI82200.1. Conserved domain searches revealed a similar structure to the transcriptional regulator Msx2-interacting protein. The BLASTp search also returned significant alignments to Msx2-interacting proteins in Mytilus coruscus, Crassostrea virginica, and Haliotis rubra. The existence of the MGE7 gradient highlights the role that environmental variation may play in retarding gene flow among wild M. galloprovincialis populations, and also how the success of collection of young mussels (spat) from one site and their transfer to another site (the farm) may be influenced by a single factor such as minSAL or maxSST on a localized scale.

Phylogenetic Analysis of Wild Pomegranate (Punica granatum L.) Based on Its Complete Chloroplast Genome from Tibet, China

Pomegranates (Punica granatum L.) are one of the most fashionable fruits and can be cultivated in both tropical and subtropical regions of the world. It is a shiny and attractive pome due to its cultivation. It belongs to the Lythraceae family. In this study, we analyzed the chloroplast genome of wild pomegranate based on whole genome shotgun sequences. In wild pomegranate, we found 158,645 bp in size, 132 genes containing 84 protein genes, 37 tRNA genes, 8 rRNA genes, and 36.92% of GC content, one infA and two duplicated ycf15 pseudogenes. Moreover, 21 chloroplast genes contained intros that are detected in a large single copy (LSC), small single copy (SSC), and two inverted repeats (IRA and IRB) regions, 17 of which were involved in single introns, while four genes (ycf3, rps12, clpP and rsp12) located in LSC, IRA, and IRB region. In total, 26,272 codons are found in protein-coding genes (PCGs); relative synonymous codon usage (RSCU) analysis revealed that the most abundant amino acid is leucine containing 2773 codons (10.55%), less abundant is methionine amino acid containing 1 codon (0.0032) in the PCGs. Furthermore, a total of 233 cpSSRs were identified in the wild pomegranate cp genome, and their distribution was analyzed in three regions, namely IR, LSC, and SSC. However, 155 cpSSR were found in the LSC (66.5%), followed by 40 cpSSR in the SSC (17.2%) and 38 cpSSR in the IR (16.3%) regions. Phylogenetic validation revealed that wild pomegranate is close to the pemphis acidula species. We believe that the cp genome allocates significant information promising for breeding research of wild pomegranate to Lythraceae.

Gut microbial dysbiosis correlates with stroke severity markers in aged rats.

An imbalanced gut microbial community, or dysbiosis, has been shown to occur following stroke. It is possible that this dysbiosis negatively impacts stroke recovery and rehabilitation. Species level resolution measurements of the gut microbiome following stroke are needed to develop and test precision interventions such as probiotic or fecal microbiota transplant therapies that target the gut microbiome. Previous studies have used 16S rRNA amplicon sequencing in young male mice to obtain broad profiling of the gut microbiome at the genus level following stroke, but further investigations will be needed with whole genome shotgun sequencing in aged rats of both sexes to obtain species level resolution in a model which will better translate to the demographics of human stroke patients. Thirty-nine aged male and female rats underwent middle cerebral artery occlusion. Fecal samples were collected before stroke and 3 days post stroke to measure gut microbiome. Machine learning was used to identify the top ranked bacteria which were changed following stroke. MRI imaging was used to obtain infarct and edema size and cerebral blood flow (CBF). ELISA was used to obtain inflammatory markers. Dysbiosis was demonstrated by an increase in pathogenic bacteria such as Butyricimonas virosa (15.52 fold change, p < 0.0001), Bacteroides vulgatus (7.36 fold change, p < 0.0001), and Escherichia coli (47.67 fold change, p < 0.0001). These bacteria were positively associated with infarct and edema size and with the inflammatory markers Ccl19, Ccl24, IL17a, IL3, and complement C5; they were negatively correlated with CBF. Conversely, beneficial bacteria such as Ruminococcus flavefaciens (0.14 fold change, p < 0.0001), Akkermansia muciniphila (0.78 fold change, p < 0.0001), and Lactobacillus murinus (0.40 fold change, p < 0.0001) were decreased following stroke and associated with all the previous parameters in the opposite direction of the pathogenic species. There were not significant microbiome differences between the sexes. The species level resolution measurements found here can be used as a foundation to develop and test precision interventions targeting the gut microbiome following stroke. Probiotics that include Ruminococcus flavefaciens, Akkermansia muciniphila, and Lactobacillus murinus should be developed to target the deficit following stroke to measure the impact on stroke severity.

Genomic analysis reveals a new genus of Firetip skippers (Lepidoptera: Hesperiidae: Pyrrhopyginae).

We obtained whole genome shotgun sequence reads for a number of Firetip skippers (subfamily Pyrrhopyginae), including all known species from the genera Yanguna Watson, 1893 and Gunayan Mielke, 2002 and representative species of Pyrrhopyge Hübner, [1819]. Phylogenetic analysis of their protein-coding regions unexpectedly revealed that Yanguna tetricus Bell, 1931 was not monophyletic with the other species of Yanguna (type species Pyrrhopyga spatiosa Hewitson, 1870). Instead, Y. tetricus formed a phylogenetic lineage as ancient as other three genera in its clade (Pyrrhopyge, Yanguna and Gunayan) that rapidly diversified from their ancestor. Therefore a new genus, Guyanna Grishin, gen. n. (type species Yanguna tetricus), is proposed for this lineage. The specimen that we sequenced was the Y. tetricus holotype in the Natural History Museum, London, leaving no doubt that we are dealing with this species. Genomic sequencing and comparison of specimens from museum collections offers a powerful strategy to reveal unforeseen phylogenetic relationships, and sequencing of primary types ensures that the conclusions are accurate in terms of nomenclature.

Comparative Analysis of Potential Determinants of Resistance to Aminoglycosides in &lt;i&gt;Burkholderia pseudomallei&lt;/i&gt; Strains with Different Level of Sensitivity to Gentamicin

The aim of the study was to identify and compare potential determinants of aminoglycoside resistance in gentamicin susceptible Burkholderia pseudomallei strains.Materials and methods. A bioinformatics analysis of whole genome shotgun sequences of three B. pseudomallei strains having different levels of sensitivity to gentamicin was carried out.Results and discussion. B. pseudomallei is intrinsically resistant to aminoglycosides. Such strains, as a rule, are not taken into account in the classical scheme of isolation and identification. At the same time, there were no significant differences in the clinical manifestations of melioidosis during infection with gentamicin-resistant and sensitive strains. In B. pseudomallei strains of different sequence types (ST70, ST948, and ST1566), point missense mutations were found in the genes of three efflux pumps of the RND family: AmrAB-OprA, BpeAB-OprB, BpeEF-OprC, and one with unknown functions, as well as in the gene aminoglycoside-6’-N-acetyltransferase AAC(6’)-III. All three strains had amino acid substitutions in the AmrA periplasmic linker: ARG160SER, Arg116Gln and Gly237Arg, Thr317Lys, respectively. In moderately sensitive strains (ST948 and ST1566), an identical Val222Met substitution was found in the repressor of the AmrAB-OprA operon, AmrR. It is likely that the intermediate level of sensitivity to gentamicin in the studied strains is mediated by the constitutive expression of the AmrAB-OprA operon, which partially compensates for the structural defects. It is also possible that a dinucleotide deletion in the AAC (6’)-III aminoglycoside-6’-N-acetyltransferase gene, as well as detected mutations in the homologues of the periplasmic linker (BPSL2234) of an uncharacterized efflux operon of the RND family, are involved in the loss of resistance to gentamicin.

Functional recovery outcomes following acute stroke is associated with abundance of gut microbiota related to inflammation, butyrate and secondary bile acid.

Accumulating evidence suggests that gut microbes modulate brain plasticity via the bidirectional gut-brain axis and play a role in stroke rehabilitation. However, the microbial species alterations associated with stroke and their correlation with functional outcome measures following acute stroke remain unknown. Here we measure post-stroke gut dysbiosis and how it correlates with gut permeability and cognitive functions in 12 stroke participants, 18 controls with risk factors for stroke, and 12 controls without risk factors. Stool samples were used to measure the microbiome with whole genome shotgun sequencing and leaky gut markers. We genotyped APOE status and measured diet composition and motor, cognitive, and emotional status using NIH Toolbox. We used linear regression methods to identify gut microbial associations with cognitive and emotional assessments. We did not find significance differences between the two control groups. In contrast, the bacteria populations of the Stroke group were statistically dissimilar from the control groups. Relative abundance analysis revealed notable decreases in butyrate-producing microbial taxa, secondary bile acid-producing taxa, and equol-producing taxa. The Stroke group had higher levels of the leaky gut marker alpha-1-antitrypsin in the stool than either of the groups and several taxa including Roseburia species (a butyrate producer) were negatively correlated with alpha-1-antitrypsin. Stroke participants scored lower on memory testing than those in the two control groups. Stroke participants with more Roseburia performed better on the picture vocabulary task; more Bacteroides uniformis (a butyrate producer) and less Escherichia coli (a pro-inflammatory species) reported higher levels of self-efficacy. Intakes of fiber, fruit and vegetable were lower, but sweetened beverages were higher, in the Stroke group compared with controls. Vegetable consumption was correlated with many bacterial changes among the participants, but only the species Clostridium bolteae, a pro-inflammatory species, was significantly associated with stroke. Our findings indicate that stroke is associated with a higher abundance of proinflammatory species and a lower abundance of butyrate producers and secondary bile acid producers. These altered microbial communities are associated with poorer functional performances. Future studies targeting the gut microbiome should be developed to elucidate whether its manipulation could optimize rehabilitation and boost recovery.

Evolution of the facial skin microbiome during puberty in normal and acne skin.

The composition of the skin microbiome varies from infancy to adulthood and becomes most stable in adulthood. Adult acne patients harbour an 'acne microbiome' dominated by specific strains of Cutibacterium acnes. However, the precise timing of skin microbiome evolution, the development of the acne microbiome, and the shift to virulent C.acnes strain composition during puberty is unknown. We performed a cross-sectional pilot study in a paediatric population to understand how and when the skin microbiome composition transitions during puberty and whether a distinct 'acne microbiome' emerges in paediatric subjects. Forty-eight volunteers including males and females, ages 7-17 years, with and without acne were enrolled and evaluated for pubertal development using the Tanner staging criteria. Sebum levels were measured, and skin microbiota were collected by sterile swab on the subject's forehead. DNA was sequenced by whole genome shotgun sequencing. A significant shift in microbial diversity emerged between early (T1-T2) and late (T3-T5) stages of puberty, coinciding with increased sebum production on the face. The overall relative abundance of C.acnes in both normal and acne skin increased during puberty and individual C.acnes strains were uniquely affected by pubertal stage and the presence of acne. Further, an acne microbiome signature associated with unique C.acnes strain composition and metabolic activity emerges in late puberty in those with acne. This unique C.acnes strain composition is predicted to have increased porphyrin production, which may contribute to skin inflammation. Our data suggest that the stage of pubertal development influences skin microbiome composition. As children mature, a distinct acne microbiome composition emerges in those with acne. Understanding how both puberty and acne influence the microbiome may support novel therapeutic strategies to combat acne in the paediatric population.

The berberine-enriched gut commensal Blautia producta ameliorates high-fat diet (HFD)-induced hyperlipidemia and stimulates liver LDLR expression

Berberine (BBR) is an effective cholesterol-lowering drug. Although gut microbiota has been implicated in the pharmacological activities of BBR, little evidence exists on the specific species of gut microbiota involved in its therapeutic effects, nor on linking gut bacteria to its recognized hypercholesterolemia-alleviating mechanism–upregulation of the low-density lipoprotein receptor (LDLR) in the liver. The present study was performed to identify the specific species of gut microbiota involved in the anti-hyperlipdemic effect of BBR, and interpret its mechanism through linking the gut microbiota and LDLR. The BBR-enriched gut bacterial species were identified by whole genome shotgun sequencing. Pure cultured B. producta was orally administered to C57BL/6 mice to evaluate its anti-hyperlipdemic effect. The LDLR-upregulating effect of B. producta was evaluated both in vitro and in vivo. Orally administration of BBR (200 mg/kg) decreased serum and liver lipid levels in HFD-induced hyperlipidemic mice. Microbiome analysis indicated that Blautia was closely associated with BBR’s lipid-modulating activities. Further analysis revealed that BBR selectively promoted the growth of Blautia producta. Orally treatment of HFD mice with live B. producta reduced obesity and alleviated hyperlipidemia. Notably, the B. producta significantly increased LDLR expression in the liver, and its spent culture supernatant upregulated the LDLR level and promoted LDL uptake by HepG2 cells. Simultaneously, B. producta also linked butyrate-producing and bile salt hydrolase (BSH)-inhibiting effect of BBR. The gut microbiota, especially B. producta, may confers the hypercholesterolemia-alleviating effects of berberine. B. producta represents a novel probiotic that may be used for the treatment of dyslipidemia.

Differential co-expression networks of the gut microbiota are associated with depression and anxiety treatment resistance among psychiatric inpatients

BackgroundComorbid anxiety and depression are common and are associated with greater disease burden than either alone. Our recent efforts have identified an association between gut microbiota dysfunction and severity of anxiety and depression. In this follow-up, we applied Differential Co-Expression Analysis (DiffCoEx) to identify potential gut microbiota biomarker(s) candidates of treatment resistance among psychiatric inpatients. MethodsIn a sample of convenience, 100 psychiatric inpatients provided clinical data at admission and discharge; fecal samples were collected early during the hospitalization. Whole genome shotgun sequencing methods were used to process samples. DiffCoEx was used to identify clusters of microbial features significantly different based on treatment resistance status. Once overlapping features were identified, a knowledge-mining tool was used to review the literature using a list of microbial species/pathways and a select number of medical subject headlines (MeSH) terms relevant for depression, anxiety, and brain-gut-axis dysregulation. Network analysis used overlapping features to identify microbial interactions that could impact treatment resistance. ResultsDiffCoEx analyzed 10,403 bacterial features: 43/44 microbial features associated with depression treatment resistance overlapped with 43/114 microbial features associated with anxiety treatment resistance. Network analysis resulted in 8 biological interactions between 16 bacterial species. Clostridium perfringens evidenced the highest connection strength (0.95). Erysipelotrichaceae bacterium 6_1_45 has been most widely examined, is associated with inflammation and dysbiosis, but has not been associated with depression or anxiety. ConclusionDiffCoEx potentially identified gut bacteria biomarker candidates of depression and anxiety treatment-resistance. Future efforts in psychiatric microbiology should examine the mechanistic relationship of identified pro-inflammatory species, potentially contributing to a biomarker-based algorithm for treatment resistance.

Abundant antibiotic resistance genes in rhizobiome of the human edible Moringa oleifera medicinal plant.

Moringa oleifera (or the miracle tree) is a wild plant species widely grown for its seed pods and leaves, and is used in traditional herbal medicine. The metagenomic whole genome shotgun sequencing (mWGS) approach was used to characterize antibiotic resistance genes (ARGs) of the rhizobiomes of this wild plant and surrounding bulk soil microbiomes and to figure out the chance and consequences for highly abundant ARGs, e.g., mtrA, golS, soxR, oleC, novA, kdpE, vanRO, parY, and rbpA, to horizontally transfer to human gut pathogens via mobile genetic elements (MGEs). The results indicated that abundance of these ARGs, except for golS, was higher in rhizosphere of M. oleifera than that in bulk soil microbiome with no signs of emerging new soil ARGs in either soil type. The most highly abundant metabolic processes of the most abundant ARGs were previously detected in members of phyla Actinobacteria, Proteobacteria, Acidobacteria, Chloroflexi, and Firmicutes. These processes refer to three resistance mechanisms namely antibiotic efflux pump, antibiotic target alteration and antibiotic target protection. Antibiotic efflux mechanism included resistance-nodulation-cell division (RND), ATP-binding cassette (ABC), and major facilitator superfamily (MFS) antibiotics pumps as well as the two-component regulatory kdpDE system. Antibiotic target alteration included glycopeptide resistance gene cluster (vanRO), aminocoumarin resistance parY, and aminocoumarin self-resistance parY. While, antibiotic target protection mechanism included RbpA bacterial RNA polymerase (rpoB)-binding protein. The study supports the claim of the possible horizontal transfer of these ARGs to human gut and emergence of new multidrug resistant clinical isolates. Thus, careful agricultural practices are required especially for plants used in circles of human nutrition industry or in traditional medicine.

Measuring the frequency and distribution of meiotic crossovers in homozygous barley inbred lines

We report a novel approach for establishing the number and position of CO events in individual homozygous inbred plants by combining low level EMS mutagenesis, speed breeding, whole genome shotgun sequencing and sliding window analysis of the induced molecular variant data. We demonstrate the approach by exploring CO frequency and distribution in self-fertilised progeny of the inbred barley cultivar Bowman and compare these observations to similar data obtained from a Bowman nearly isogenic line (BW230 Hvmlh3) containing a mutation in the DNA mismatch repair gene HvMLH3. We have previously shown that Hvmlh3 decreases both plant fertility and recombination by ~50%. We compare our results to those from previously published traditional genetic analysis of F3 families derived from multiple F2 lines containing WT or mutant alleles of HvMLH3, revealing a high level of correspondence between analyses. We discuss possible applications of the approach in streamlining the assessment of recombination in plant meiosis research.

Metagenomic profile of the fecal microbiome of preterm infants consuming mother’s own milk with bovine milk–based fortifier or infant formula: a cross-sectional study

Preterm (PT) infants harbor a different gut microbiome than full-term infants. Multiple factors affect gut microbial colonization of PT infants, including low gestational age, high rates of Cesarean section, exposure to antibiotics, and diet. Human milk, whether it's mother's own milk (MOM) or donor human milk, is the preferred feeding mode for PT infants but needs to be fortified to achieve adequate nutrient content. Infant formulas are introduced at later stages if human milk is insufficient or unavailable. How these dietary exposures affect the gut microbiome of PT infants is poorly understood. The goal of this study was to evaluate the metagenomic potential of the fecal microbiome of PT infants consuming MOM with bovine milk-based fortifier compared with PT formula alone. Forty-two stool samples, from 27 infants consuming MOM or formula (21 samples in each group) were included. Twelve infants had repeated sampling (2-3 samples). Shotgun genomic DNA sequencing was performed and analyzed using MetaPhlAn and HUMAnN2. Multivariate regression analysis, adjusting by the repeated sampling, was used to identify the features that differed between PT infants consuming MOM compared with formula. The primary function of the fecal microbiome of PT infants was characterized by a high abundance of biosynthesis pathways. A set of core features was identified; these belonged to pathways for amino acid metabolism and vitamin K-2 biosynthesis. Five pathways significantly differed between the MOM and formula group. Pathways for fatty acid and carbohydrate degradation were significantly higher in the MOM group. Taxonomically, members of the phylum Actinobacteria and the genus Bifidobacterium were higher in PT infants exposed to MOM. This study provides insight into the influence of feeding MOM compared with infant formula on the structure and function of the fecal microbiome of PT infants.

Oral ingestion of the environmental toxicant trichloroethylene in rats induces alterations in the gut microbiome: Relevance to idiopathic Parkinson's disease

Microbial alterations within the gut microbiome appear to be a common feature of individuals with Parkinson's disease (PD), providing further evidence for the role of the gut-brain axis in PD development. As a major site of contact with the environment, questions have emerged surrounding the cause and effect of alterations to the gut microbiome by environmental contaminants associated with PD risk, such as pesticides, metals, and organic solvents. Recent data from our lab shows that ingestion of the industrial byproduct and environmental pollutant trichloroethylene (TCE) induces key Parkinsonian pathology within aged rats, including the degeneration of dopaminergic neurons, α-synuclein accumulation, neuroinflammation, and endolysosomal deficits. As TCE is the most common organic contaminant within drinking water, we postulated that ingestion of TCE associated with PD-related neurodegeneration may alter the gut microbiome to a similar extent as observed in persons with PD. To assess this, we collected fecal samples from adult rats treated with 200 mg/kg TCE over 6 weeks via oral gavage – the dose that produced nigrostriatal neurodegeneration – and analyzed the gut microbiome via whole genome shotgun sequencing. Our results showed changes in gut microorganisms reflective of the microbial signatures observed in individuals with idiopathic PD, such as decreased abundance of short-chain fatty acid producing Blautia and elevated lactic-acid producing Bifidobacteria, as well as genera who contain species previously reported as opportunistic pathogens such as Clostridium. From these experimental data, we postulate that TCE exposure within contaminated drinking water could induce alterations of the gut microbiome that contributes to chronic disease risk, including idiopathic PD.

Use of Metagenomic Whole Genome Shotgun Sequencing Data in Taxonomic Assignment of Dipterygium glaucum Rhizosphere and Surrounding Bulk Soil Microbiomes, and Their Response to Watering

The metagenomic whole genome shotgun sequencing (mWGS) approach was used to detect signatures of the rhizosphere microbiomes of Dipterygium glaucum and surrounding bulk soil microbiomes, and to detect differential microbial responses due to watering. Preliminary results reflect the reliability of the experiment and the rationality of grouping microbiomes. Based on the abundance of non-redundant genes, bacterial genomes showed the highest level, followed by Archaeal and Eukaryotic genomes, then, the least abundant viruses. Overall results indicate that most members of bacteria have a higher abundance/relative abundance (AB/RA) pattern in the rhizosphere towards plant growth promotion, while members of eukaryota have a higher pattern in bulk soil, most likely acting as pathogens. The results also indicate the contribution of mycorrhiza (genus Rhizophagus) in mediating complex mutualistic associations between soil microbes (either beneficial or harmful) and plant roots. Some of these symbiotic relationships involve microbes of different domains responding differentially to plant root exudates. Among these are included the bacterial genus Burkholderia and eukaryotic genus Trichoderma, which have antagonistic activities against the eukaryotic genus Fusarium. Another example involves Ochrobactrum phage POA1180, its bacterial host and plant roots. One of the major challenges in plant nutrition involves other microbes that manipulate nitrogen levels in the soil. Among these are the microbes that perform contraversal actions of nitrogen fixation (the methanogen Euryarchaeota) and ammonia oxidation (Crenarchaeota). The net nitrogen level in the soil is originally based on the AB/RA of these microbes and partially on the environmental condition. Watering seems to influence the AB/RA of a large number of soil microbes, where drought-sensitive microbes (members of phyla Acidobacteria and Gemmatimonadetes) showed an increased AB/RA pattern after watering, while others (Burkholderia and Trichoderma) seem to be among microbes assisting plants to withstand abiotic stresses. This study sheds light on the efficient use of mWGS in the taxonomic assignment of soil microbes and in their response to watering. It also provides new avenues for improving biotic and abiotic resistance in domestic plant germplasm via the manipulation of soil microbes.

Comparative Analysis of Metagenomics and Metataxonomics for the Characterization of Vermicompost Microbiomes.

The study of microbial communities or microbiotas in animals and environments is important because of their impact in a broad range of industrial applications, diseases and ecological roles. High throughput sequencing (HTS) is the best strategy to characterize microbial composition and function. Microbial profiles can be obtained either by shotgun sequencing of genomes, or through amplicon sequencing of target genes (e.g., 16S rRNA for bacteria and ITS for fungi). Here, we compared both HTS approaches at assessing taxonomic and functional diversity of bacterial and fungal communities during vermicomposting of white grape marc. We applied specific HTS workflows to the same 12 microcosms, with and without earthworms, sampled at two distinct phases of the vermicomposting process occurring at 21 and 63 days. Metataxonomic profiles were inferred in DADA2, with bacterial metabolic pathways predicted via PICRUSt2. Metagenomic taxonomic profiles were inferred in PathoScope, while bacterial functional profiles were inferred in Humann2. Microbial profiles inferred by metagenomics and metataxonomics showed similarities and differences in composition, structure, and metabolic function at different taxonomic levels. Microbial composition and abundance estimated by both HTS approaches agreed reasonably well at the phylum level, but larger discrepancies were observed at lower taxonomic ranks. Shotgun HTS identified ~1.8 times more bacterial genera than 16S rRNA HTS, while ITS HTS identified two times more fungal genera than shotgun HTS. This is mainly a consequence of the difference in resolution and reference richness between amplicon and genome sequencing approaches and databases, respectively. Our study also revealed great differences and even opposite trends in alpha- and beta-diversity between amplicon and shotgun HTS. Interestingly, amplicon PICRUSt2-imputed functional repertoires overlapped ~50% with shotgun Humann2 profiles. Finally, both approaches indicated that although bacteria and fungi are the main drivers of biochemical decomposition, earthworms also play a key role in plant vermicomposting. In summary, our study highlights the strengths and weaknesses of metagenomics and metataxonomics and provides new insights on the vermicomposting of white grape marc. Since both approaches may target different biological aspects of the communities, combining them will provide a better understanding of the microbiotas under study.

Chronic Exposure to Sublethal Concentrations of Aquatic Nitrite Change Nose and Gut Morphology and Associated Microbiomes in Channel Catfish

Nitrite is a common pollutant that can enter waterways via agricultural runoff or build up in aquaculture ponds when nitrogenous waste is converted to nitrite. Preliminary studies by the Huertas lab found that exposure to sublethal concentrations of aquatic nitrite alters the microbiome of goldfish and bioaccumulates in the olfactory epithelia, gills and brain. These studies also showed a negative impact on the olfactory tissue of exposed fish, which could lead to higher susceptibility to nasal pathogens. Our objective is to assess nitrite bioaccumulation in tissues and the alteration of the microbiome in the key aquaculture species channel catfish (Ictalurus punctatus). We hypothesize that the relative abundance and diversity of bacteria in the nose and gut microbiomes of channel catfish will be altered due to elevated nitrite exposure and that nitrite will bioaccumulate in tissues.Channel catfish were exposed to 0 mM (control), 0.2 mM, and 2 mM concentrations of nitrite. A total of six 500 liter tanks were used in a two‐phase experiment. For phase one, 16 fish per tank were placed into three 0 mM nitrite tanks and three 2 mM nitrite tanks. The 2 mM fish were kept for 12 hours before sampling. For phase two, 24 of the phase one control fish were placed into three 0.2 mM tanks and the remaining 24 control fish were left in the three control tanks for a total of 8 fish per tank. Phase two lasted 30 days with sub‐samplings at day five and 30. All fish were sampled for nose, gut, muscle, gill, brain, blood, liver, and kidney tissues for biochemical, microbial, or histological analysis. Upon dissection, fish from the 2 mM tanks exhibited marked methemoglobinemia due to nitrite‐induced oxidation of hemoglobin. The mortality rates were 0.04%, 0.08%, and 92% for the control, 0.2 mM, and 2 mM treatments respectively. The nose and gut microbiomes will be sequenced using whole genome shotgun sequencing to determine the microbial composition and measure changes in relative abundance. Histological analysis showed oxidative damage in all tissues from individuals exposed to 2 mM and partial damage in tissues of the 0.2 mM treatment.Because the microbiome is a powerful barrier against pathogens, disruption of the microbiome could lead to higher rates of disease infection and transmission in fish. Alteration of the nasal microbiome may increase mortality from the bacteria that cause septicemia in catfish, Edwardsiella ictaluri, whose common routes of infection are the nose or gut. Changes to the microbiome could be the missing link between nitrite and increased susceptibility to E. ictaluri infections in intensive farming. In the long term, this research aims to provide guidelines for water quality management in order to decrease the rate of bacterial infection and transmission in aquaculture systems.