Whole Genome Sequencing Research Articles

Genomic and transcriptomic landscape to decipher the genetic basis of hyperpigmentation in Lanping black-boned sheep (Ovis aries)

BackgroundLanping black-boned sheep (LPB) represent a distinctive mammalian species characterized by hyperpigmentation, resulting in black bone and muscle features, in contrast to their conventional counterparts exhibiting red muscle and white bone. The genetic basis underlying LPB hyperpigmentation has remained enigmatic.MethodsIn this study, we conducted whole-genome sequencing of 100 LPB and 50 Lanping normal sheep (LPN), and integrated this data with 421 sequenced datasets from wild and domestic sheep, shedding light on the genetic backdrop and genomic variations associated with LPB. Furthermore, we performed comparative RNA-Seq analysis using liver sample to pinpoint genes implicated in the pigmentation process. We generated a comprehensive dataset comprising 97,944,357 SNPs from 571 sheep, facilitating an in-depth exploration of genetic factors.ResultsPopulation genetic structure analysis revealed that the LPB breed traces its origin back to LPN, having evolved into a distinct breed. The integration of positively selected genes with differentially expressed genes identified two candidates, ERBB4 and ROR1, potentially linked to LPB hyperpigmentation. Comparative analysis of ERBB4 and ROR1 mRNA relative expression levels in liver, spleen, and kidney tissues of LPB, in comparison to Diqing sheep, revealed significant upregulation, except for ERBB4 in the liver. Gene expression heatmaps further underscored marked allelic frequency disparities in different populations.ConclusionOur findings establish the evolutionary lineage of the LPB breed from LPN and underscore the involvement of ERBB4 and ROR1 genes in melanin synthesis. These results enhance our comprehension of the molecular basis of hyperpigmentation and contribute to a more comprehensive depiction of sheep diversity.

Pan-genome analysis reveals novel chromosomal markers for multiplex PCR-based specific detection of Bacillus anthracis

BackgroundBacillus anthracis is a highly pathogenic bacterium that can cause lethal infection in animals and humans, making it a significant concern as a pathogen and biological agent. Consequently, accurate diagnosis of B. anthracis is critically important for public health. However, the identification of specific marker genes encoded in the B. anthracis chromosome is challenging due to the genetic similarity it shares with B. cereus and B. thuringiensis.MethodsThe complete genomes of B. anthracis, B. cereus, B. thuringiensis, and B. weihenstephanensis were de novo annotated with Prokka, and these annotations were used by Roary to produce the pan-genome. B. anthracis exclusive genes were identified by Perl script, and their specificity was examined by nucleotide BLAST search. A local BLAST alignment was performed to confirm the presence of the identified genes across various B. anthracis strains. Multiplex polymerase chain reactions (PCR) were established based on the identified genes.ResultThe distribution of genes among 151 whole-genome sequences exhibited three distinct major patterns, depending on the bacterial species and strains. Further comparative analysis between the three groups uncovered thirty chromosome-encoded genes exclusively present in B. anthracis strains. Of these, twenty were found in known lambda prophage regions, and ten were in previously undefined region of the chromosome. We established three distinct multiplex PCRs for the specific detection of B. anthracis by utilizing three of the identified genes, BA1698, BA5354, and BA5361.ConclusionThe study identified thirty chromosome-encoded genes specific to B.anthracis, encompassing previously described genes in known lambda prophage regions and nine newly discovered genes from an undefined gene region to the best of our knowledge. Three multiplex PCR assays offer an accurate and reliable alternative method for detecting B. anthracis. Furthermore, these genetic markers have value in anthrax vaccine development, and understanding the pathogenicity of B. anthracis.

Molecular epidemiology of SARS-CoV-2 genome sentinel surveillance in commercial COVID-19 testing sites targeting asymptomatic individuals during Japan’s seventh epidemic wave

Eight peaks of coronavirus disease 2019 (COVID-19) outbreak occurred in Japan, each associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern. The National Epidemiological Surveillance of Infectious Diseases (NESID) analyzed viral genome sequences from symptomatic patients and submitted the results to GISAID. Meanwhile, commercial testing services occasionally sequence samples from asymptomatic individuals. We compared a total of 1248 SARS-CoV-2 full-genome sequences obtained from the SB Coronavirus Inspection Center Corp. (SBCVIC) during Japan’s seventh wave, which was dominated by Omicron variants, with 1764 sequences obtained in Japan from GISAID during the same period using chronological phylogenies and molecular transmission networks. The number of SBCVIC sequences was consistent with the number of cases reported by NESID. The SBCVIC detected a shift in the PANGO lineage from BA.2 to BA.5 earlier than that of GISAID. BA.2 lineages from the SBCVIC were distributed at different locations in the transmission network dominated by GISAID entries, whereas BA.5 lineages from SBCVIC and GISAID often formed distinct subclusters. Test-based sentinel surveillance of asymptomatic individuals may be a more manageable approach compared to notifiable disease surveillance; however, it may not necessarily capture all infection populations throughout Japan.

Genome-wide analysis of DNA methylation and transcriptional changes associated with overwintering memory in Brassica rapa L. grown in the field

BackgroundWinter rapeseed, the sole overwintering oilseed crop in northern China, emphasizes winter resilience, yet epigenetic regulatory mechanisms governing overwintering memory remain poorly understood.ResultsIn this study, the root collar tissues from the robust cold-resistant variety Longyou-7 were sampled during the pre-winter period (S1), overwintering periods (S2–S5), and re-greening period (S6), to analyze overall genomic DNA methylation levels using high-performance liquid chromatography (HPLC). The result showed that DNA methylation level exceeded 80% in the S1 stage. Throughout the overwintering periods, methylation levels displayed a decreasing trend in S3, followed by an increase in S5, and a pronounced decrease in S6. Consequently, S1, S3, S5, and S6 periods were chosen for whole-genome bisulfite sequencing analyses to elucidate the overwintering memory mechanisms of Longyou-7. The result revealed that DNA methylation primarily occurs in the CG context in Longyou-7. However, methylation of mC sites is most prevalent in the CHH type, gradually decreasing during overwintering periods. Analysis of methylation patterns in specific genomic regions of Longyou-7 showed that the highest methylation levels in the intergenic region. Moreover, mC sites in repeats and transposon elements are distributed differently across the three contexts. Subsequently, differentially methylated regions and promoters of Longyou-7 were identified during various periods compared to the S1 stage, followed by joint analysis with transcriptome sequencing. Functional enrichment analysis highlighted the involvement of most overlapping genes in the MAPK signaling pathway, plant hormone signal transduction, and starch and sucrose metabolism pathways. Changes in candidate gene expression within these three pathways correlated closely with DNA methylation levels.ConclusionsOur findings underscored the critical role of DNA methylation in regulating the expression of overwintering memory genes in winter rapeseed. These results offer a comprehensive insights into the epigenetic regulatory mechanisms governing winter rapeseed's overwintering memory, while identified overwintering memory genes served as crucial genetic resources for multifaceted breeding of winter-resistant varieties.Graphical

Bacillus licheniformisYB06: A Rhizosphere–Genome-Wide Analysis and Plant Growth-Promoting Analysis of a Plant Growth-Promoting Rhizobacterium Isolated from Codonopsis pilosula

Codonopsis pilosula, commonly known as Dangshen, is a valuable medicinal plant, but its slow growth and susceptibility to environmental stress pose challenges for its cultivation. In pursuit of sustainable agricultural practices to enhance the yield and quality of Dangshen, the present study isolated a bacterial strain exhibiting plant growth-promoting potential from the rhizosphere of C. pilosula. This strain was subsequently identified as Bacillus licheniformisYB06. Assessment of its plant growth-promoting attributes revealed the potential of B. licheniformis YB06 as a biofertilizer. Whole-genome sequencing of B. licheniformis YB06 revealed a genome size of 4,226,888 bp with a GC content of 46.22%, harboring 4325 predicted protein-coding sequences. Genomic analysis of B. licheniformis YB06 revealed a diverse array of genes linked to induced systemic resistance (ISR) and plant growth-promoting (PGP) traits, encompassing phytohormone production, nitrogen assimilation and reduction, siderophore biosynthesis, phosphate solubilization, biofilm formation, synthesis of PGP-related amino acids, and flagellar motility. Seed germination assays demonstrated the positive effects of B. licheniformis YB06 on the germination and growth of C. pilosula seedlings. Furthermore, we explored various fertilization regimes, particularly the B. licheniformis YB06-based biofertilizer, were investigated for their impact on the structure and diversity of the C. pilosula rhizosphere soil bacterial community. Our findings revealed that fertilization significantly impacted soil bacterial composition and diversity, with the combined application of B. licheniformis YB06-based biofertilizer and organic fertilizer exhibiting a particularly pronounced enhancement of rhizosphere bacterial community structure and diversity. This study represents the first report on the beneficial effects of B. licheniformis YB06 on both the growth of C. pilosula and the composition of its rhizosphere soil microbial community. These findings provide a theoretical foundation and practical guidance for the development of novel bio-organic compound fertilizers, thereby contributing to the sustainable cultivation of C. pilosula.

First Record of the Complete Mitochondrial Genome for the Genus Borbo (Lepidoptera, Hesperiidae): Characterization and Comparative Genomic Analysis

Butterflies of the genus Borbo are mainly distributed in the Oriental and Australian regions and are considered pests of important crops. However, no mitochondrial genomes have been reported for this genus until now, leaving the evolutionary characteristics and differentiation patterns of their mitogenomes unclear. In this study, we present the first complete mitochondrial genome sequence of the rice swift, Borbo cinnara. The circular double-stranded mitogenome was 15,508 bp in length, comprising 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and 1 non-coding control region (CR). Among the mitogenomes of Hesperiinae, the ND3 gene was found to be the most variable PCG, while COX1 was the most conserved. Selection pressure analysis revealed that ND3 was under relaxed purifying selection, whereas COX1 was subjected to strong purifying selection. Phylogenetic trees reconstructed using both the Bayesian inference (BI) and maximum likelihood (ML) methods yielded robust and identical topologies, confirming the sister relationship between B. cinnara and Pelopidas mathias at the mitogenome level. Methodologically, this research enriches novel molecular markers for the species identification of butterflies and enhances our understanding of mitogenomic evolution in Lepidoptera.

Antimicrobial Resistance in the Terrestrial Environment of Agricultural Landscapes in Norway

The abundance and diversity of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs) in agricultural landscapes may be important for the spread of antimicrobial resistance (AMR) in the environment. The aim of this study was to apply screening methods for ARB and ARGs to investigate the impact of farming on the prevalence of AMR in a country with low antibiotic usage. We have analyzed samples (n = 644) from soil and wild terrestrial animals and plants (slugs, snails, mice, shrews, earthworms, and red clover) collected over two years in agricultural fields accompanied by nearby control areas with low human activity. All samples were investigated for the occurrence of 35 different ARGs using high-throughput quantitative PCR (HT-qPCR) on a newly developed DNA array. In addition, samples from the first year (n = 415) were investigated with a culture-based approach combined with whole-genome sequencing (WGS) to identify antimicrobial-resistant E. coli (AREC). ARGs were detected in 59.5% of all samples (2019 + 2020). AREC, which was only investigated in the 2019 samples, was identified in 1.9% of these. Samples collected in the autumn showed more ARGs and AREC than spring samples, and this was more pronounced for organic fields than for conventional fields. Control areas with low human activity showed lower levels of ARGs and a lack of AREC. The use of livestock manure was correlated with a higher level of ARG load than other farming practices. None of the soil samples contained antibiotics, and no association was found between AMR and the levels of metals or pesticides. High qualitative similarity between HT-qPCR and WGS, together with the positive controls to the validation of our 35 ARG assays, show that the microfluid DNA array may be an efficient screening tool on environmental samples. In conclusion, even in a country with a very low consumption of antimicrobials by production animals, our results support the hypothesis of these animals being a source of AREC and ARGs in agricultural environments, primarily through the use of manure.

High Diagnostic Yield and Clinical Utility of Next-Generation Sequencing in Children with Epilepsy and Neurodevelopmental Delays: A Retrospective Study.

Advances in genetics led to the identification of hundreds of epilepsy-related genes, some of which are treatable with etiology-specific interventions. However, the diagnostic yield of next-generation sequencing (NGS) in unexplained epilepsy is highly variable (10-50%). We sought to determine the diagnostic yield and clinical utility of NGS in children with unexplained epilepsy that is accompanied by neurodevelopmental delays and/or is medically intractable. A 5-year retrospective review was conducted at the American University of Beirut Medical Center to identify children who underwent whole exome sequencing (WES) or whole genome sequencing (WGS). Data on patient demographics, neurodevelopment, seizures, and treatments were collected. Forty-nine children underwent NGS with an overall diagnostic rate of 68.9% (27/38 for WES, and 4/7 for WGS). Most children (42) had neurodevelopmental delays with (18) or without (24) refractory epilepsy, and only three had refractory epilepsy without delays. The diagnostic yield was 77.8% in consanguineous families (18), and 61.5% in non-consanguineous families (26); consanguinity information was not available for one family. Genetic test results led to anti-seizure medication optimization or dietary therapies in six children, with subsequent improvements in seizure control and neurodevelopmental trajectories. Not only is the diagnostic rate of NGS high in children with unexplained epilepsy and neurodevelopmental delays, but also genetic testing in this population may often lead to potentially life-altering interventions.

Complete genome sequence analysis of Pestalotiopsis microspora, a fungal pathogen causing kiwifruit postharvest rots

BackgroundThe postharvest rot of kiwifruit is one of the most devastating diseases affecting kiwifruit quality worldwide. However, the genomic basis and pathogenicity mechanisms of kiwifruit rot pathogens are lacking. Here we report the first whole genome sequence of Pestalotiopsis microspora, one of the main pathogens causing postharvest kiwifruit rot in China. The genome of strain KFRD-2 was sequenced, de novo assembled, and analyzed.ResultsThe genome of KFRD-2 was estimated to be approximately 50.31 Mb in size, with an overall GC content of 50.25%. Among 14,711 predicted genes, 14,423 (98.04%) exhibited significant matches to genes in the NCBI nr database. A phylogenetic analysis of 26 known pathogenic fungi, including P. microspora KFRD-2, based on conserved orthologous genes, revealed that KFRD-2’s closest evolutionary relationships were to Neopestalotiopsis spp. Among KFRD-2’s coding genes, 870 putative CAZy genes spanned six classes of CAZys, which play roles in degrading plant cell walls. Out of the 25 other plant pathogenic fungi, P. microspora possessed a greater number of CAZy genes than 22 and was especially enriched in GH and AA genes. A total of 845 transcription factors and 86 secondary metabolism gene clusters were predicted, representing various types. Furthermore, 28 effectors and 109 virulence-enhanced factors were identified using the PHI (pathogen host-interacting) database.ConclusionThis complete genome sequence analysis of the kiwifruit postharvest rot pathogen P. microspora enriches our understanding its disease pathogenesis and virulence. This study establishes a theoretical foundation for future investigations into the pathogenic mechanisms of P. microspora and the development of enhanced strategies for the efficient management of kiwifruit postharvest rots.

Polymorphic microsatellite markers for genetic analysis of Berkeleyomyces rouxiae, a causal agent of black root rot

AbstractBerkeleyomyces rouxiae, a soil-borne fungal pathogen, causes black root rot on various plant species including carrot, okra, tobacco, lettuce, and other important crops. Here we analyzed the entire genome sequence of a Japanese isolate of B. rouxiae and discovered 25 microsatellite sequences. These sequences were amplified from the genomic DNA of several Japanese isolates by PCR and sequenced, revealing that the microsatellite sequences were polymorphic among isolates. These markers are useful for phylogenetic analysis, epidemiological population analysis, identification of specific isolates, and other genetic investigation of B. rouxiae.

Molecular epidemiology, microbiological features and infection control strategies for carbapenem-resistant Acinetobacter baumannii in a German burn and plastic surgery center (2020–2022)

BackgroundCarbapenem-resistant Acinetobacter baumannii (CRAB) frequently causes both healthcare-associated infections and nosocomial outbreaks in burn medicine/plastic surgery and beyond. Owing to the high antibiotic resistance, infections are difficult to treat, and patient outcomes are often compromised. The environmental persistence capability of CRAB favors its transmission in hospitals. A comprehensive analysis and understanding of CRAB epidemiology and microbiology are essential for guiding management.MethodsA three-year retrospective cohort study (2020–2022) was conducted in a German tertiary burn and plastic surgery center. In addition to epidemiological analyses, microbiological and molecular techniques, including whole-genome sequencing, were applied for the comprehensive examination of isolates from CRAB-positive patients.ResultsDuring the study period, eight CRAB cases were found, corresponding to an overall incidence of 0.2 CRAB cases per 100 cases and an incidence density of 0.35 CRAB cases per 1000 patient-days. Six cases (75%) were treated in the burn intensive care unit, and four cases (50%) acquired CRAB in the hospital. Molecular analyses comprising 74 isolates supported the epidemiologic assumption that hospital acquisitions occurred within two separate clusters. In one of these clusters, environmental CRAB contamination of anesthesia equipment may have enabled transmission. Furthermore, molecular diversity of CRAB isolates within patients was observed.ConclusionsCRAB can pose a challenge in terms of infection prevention and control, especially if cases are clustered in time and space on a ward. Our study demonstrates that high-resolution phylogenetic analysis of several bacterial isolates from single patients can greatly aid in understanding transmission chains and helps to take precision control measures.

Genome-Wide Association Studies and QTL Mapping Reveal a New Locus Associated with Resistance to Bacterial Pustule Caused by Xanthomonas citri pv. glycines in Soybean.

Bacterial pustule (BP), caused by Xanthomonas citri pv. glycines, is an important disease that, under favorable conditions, can drastically affect soybean production. We performed a genome-wide association study (GWAS) with a panel containing Brazilian and American cultivars, which were screened qualitatively and quantitatively against two Brazilian X. citri isolates (IBS 333 and IBS 327). The panel was genotyped using a genotyping by sequencing (GBS) approach, and we identified two main new regions in soybeans associated with X. citri resistance on chromosomes 6 (IBS 333) and 18 (IBS 327), different from the traditional rxp gene located on chromosome 17. The region on chromosome 6 was also detected by QTL mapping using a biparental cross between Williams 82 (R) and PI 416937 (S), showing that Williams 82 has another recessive resistance gene besides rxp, which was also detected in nine BP-resistant ancestors of the Brazilian cultivars (including CNS, S-100), based on haplotype analysis. Furthermore, we identified additional SNPs in strong LD (0.8) with peak SNPs by exploring variation available in WGS (whole genome sequencing) data among 31 soybean accessions. In these regions in strong LD, two candidate resistance genes were identified (Glyma.06g311000 and Glyma.18g025100) for chromosomes 6 and 18, respectively. Therefore, our results allowed the identification of new chromosomal regions in soybeans associated with BP disease, which could be useful for marker-assisted selection and will enable a reduction in time and cost for the development of resistant cultivars.

Exploring novel MYH7 gene variants using in silico analyses in Korean patients with cardiomyopathy

BackgroundPathogenic variants of MYH7, which encodes the beta-myosin heavy chain protein, are major causes of dilated and hypertrophic cardiomyopathy.MethodsIn this study, we used whole-genome sequencing data to identify MYH7 variants in 397 patients with various cardiomyopathy subtypes who were participating in the National Project of Bio Big Data pilot study in Korea. We also performed in silico analyses to predict the pathogenicity of the novel variants, comparing them to known pathogenic missense variants.ResultsWe identified 27 MYH7 variants in 41 unrelated patients with cardiomyopathy, consisting of 20 previously known pathogenic/likely pathogenic variants, 2 variants of uncertain significance, and 5 novel variants. Notably, the pathogenic variants predominantly clustered within the myosin motor domain of MYH7. We confirmed that the novel identified variants could be pathogenic, as indicated by high prediction scores in the in silico analyses, including SIFT, Mutation Assessor, PROVEAN, PolyPhen-2, CADD, REVEL, MetaLR, MetaRNN, and MetaSVM. Furthermore, we assessed their damaging effects on protein dynamics and stability using DynaMut2 and Missense3D tools.ConclusionsOverall, our study identified the distribution of MYH7 variants among patients with cardiomyopathy in Korea, offering new insights for improved diagnosis by enriching the data on the pathogenicity of novel variants using in silico tools and evaluating the function and structural stability of the MYH7 protein.

Large-scale analysis of whole genome sequencing data from formalin-fixed paraffin-embedded cancer specimens demonstrates preservation of clinical utility

Whole genome sequencing (WGS) provides comprehensive, individualised cancer genomic information. However, routine tumour biopsies are formalin-fixed and paraffin-embedded (FFPE), damaging DNA, historically limiting their use in WGS. Here we analyse FFPE cancer WGS datasets from England’s 100,000 Genomes Project, comparing 578 FFPE samples with 11,014 fresh frozen (FF) samples across multiple tumour types. We use an approach that characterises rather than discards artefacts. We identify three artefactual signatures, including one known (SBS57) and two previously uncharacterised (SBS FFPE, ID FFPE), and develop an “FFPEImpact” score that quantifies sample artefacts. Despite inferior sequencing quality, FFPE-derived data identifies clinically-actionable variants, mutational signatures and permits algorithmic stratification. Matched FF/FFPE validation cohorts shows good concordance while acknowledging SBS, ID and copy-number artefacts. While FF-derived WGS data remains the gold standard, FFPE-samples can be used for WGS if required, using analytical advancements developed here, potentially democratising whole cancer genomics to many.

Semi-supervised machine learning method for predicting homogeneous ancestry groups to assess Hardy-Weinberg equilibrium in diverse whole-genome sequencing studies.

Large-scale, multi-ethnic whole-genome sequencing (WGS) studies, such as the National Human Genome Research Institute Genome Sequencing Program's Centers for Common Disease Genomics (CCDG), play an important role in increasing diversity for genetic research. Before performing association analyses, assessing Hardy-Weinberg equilibrium (HWE) is a crucial step in quality control procedures to remove low quality variants and ensure valid downstream analyses. Diverse WGS studies contain ancestrally heterogeneous samples; however, commonly used HWE methods assume that the samples are homogeneous. Therefore, directly applying these to the whole dataset can yield statistically invalid results. To account for this heterogeneity, HWE can be tested on subsets of samples that have genetically homogeneous ancestries and the results aggregated at each variant. To facilitate valid HWE subset testing, we developed a semi-supervised learning approach that predicts homogeneous ancestries based on the genotype. This method provides a convenient tool for estimating HWE in the presence of population structure and missing self-reported race and ethnicities in diverse WGS studies. In addition, assessing HWE within the homogeneous ancestries provides reliable HWE estimates that will directly benefit downstream analyses, including association analyses in WGS studies. We applied our proposed method on the CCDG dataset, predicting homogeneous genetic ancestry groups for 60,545 multi-ethnic WGS samples to assess HWE within each group.

Complete genome sequences of Enterococcus casseliflavus strains ASE2 and ASE4 generated by PacBio sequencing.

Enterococcus casseliflavus strains ASE2 and ASE4 were isolated from the soil of a lettuce farm in Salinas, California, in 2020. Their complete genome sequences were generated by the PacBio Sequel II platform. They both consist of a single circular chromosome without plasmids.

Virulence factors, antibiotic susceptibility and sequence type distribution of hospital-associated Clostridioides difficile isolates in Israel, 2020–2022

Biofilm formation and toxin production are some of the virulence factors of Clostridioides difficile (C. difficile), which causes hospital-acquired C. difficile infection (HA-CDI). This work investigated the prevalence and distribution of different strains recovered from HA-CDI patients hospitalized in 4 medical centres across Israel, and characterized strains' virulence factors and antibiotic susceptibility. One-hundred and eighty-eight faecal samples were collected. C. difficile 's toxins were detected by the CerTest Clostridium difficile GDH + Toxin A + B combo card test kit. Toxin loci PaLoc and PaCdt were detected by whole-genome sequencing (WGS). Multi-locus sequence typing (MLST) was performed to classify strains. Biofilm production was assessed by crystal violet. Antibiotic susceptibility was determined using Etest. Fidaxomicin susceptibility was tested via agar dilution. Sequence type (ST) 42 was the most (13.8%) common strain. All strains harboured the 2 toxins genes; 6.9% had the binary toxin. Most isolates were susceptible to metronidazole (98.9%) and vancomycin (99.5%). Eleven (5.85%) isolates were fidaxomicin-resistant. Biofilm production capacity was associated with ST (p < 0.001). In conclusion, a broad variety of C. difficile strains circulate in Israel's medical centres. Further studies are needed to explore the differences and their contribution to HA-CDI epidemiology.

De novo Whole-Genome Assembly of the 10-Gigabase Fokienia Hodginsii Genome to Reveal Differential Epigenetic Events Between Callus and Xylem.

Fokienia hodginsii (F. hodginsii), belonging to the genus Fokienia of the Cupressaceae. F. hodginsii has significant application value due to its wood properties and great research value in evolutionary studies as a gymnosperm. However, the genome of F. hodginsii remains unknown due to the large size of gymnosperms genome. Pacific Bioscience sequencing, Hi-C mapping, whole-genome Bisulfite Sequencing (BS-Seq), long-read isoform sequencing (Iso-Seq), direct RNA sequencing (DRS), quantitative proteomics, and metabonomics analysis are employed to facilitate genome assembly, gene annotation, and investigation into epigenetic mechanisms. In this study, the 10G F. hodginsii genome is assembled into 11 chromosomes. Furthermore, 50521 protein-coding genes are annotated and determined that 65% of F. hodginsii genome comprises repetitive sequences. It is discovered that transposable element (TE)-including introns is associated with higher expression. The DNA methylome of F. hodginsii reveals that xylem has a higher DNA methylation level compared to callus. Moreover, DRS reveals the significant alterations in RNA full-length ratio, which potentially associated with poly(A) length (PAL) and alternative polyadenylation (APA). Finally, the morphology measurement and metabonomics analysis revealed the difference of 14 cultivars. In summary, the genomes and epigenetics datasets provide a molecular basis for callus formation in the gymnosperm family.

Molecular characterization to study the genetic diversity of begomoviruses occurring in the major chilli growing areas of Tamil Nadu state of India.

Chilli leaf curl disease (ChiLCD), which is a significant problem in chilli cultivation, is caused by begomoviruses that are transmitted by the whitefly Bemisia tabaci. This disease leads to severe impacts on crop yields. To determine the incidence of begomovirus in the chilli crop, infected chilli leaf samples exhibiting symptoms such as curling, yellowing, reduced leaf size, and overall stunted growth were collected from various districts of Tamil Nadu, namely, Coimbatore, Dharmapuri, Kancheepuram, Karur, Salem, Krishnagiri, Thoothukudi, Thiruvallur, Tiruchirappalli, Virudhunagar, Tiruvannamalai, Tenkasi, and Vellore, during the years 2018-2022. To determine the complete genome sequence of the begomoviruses, the rolling circle amplification (RCA) method was used to clone and sequence thebegomovirus genomes from the chilli samples collectedfrom various districts of Tamil Nadu. Here we characterized 17 DNA A genome sequences and 12 betasatellite sequences. BLAST results of the DNA A genome sequences revealed nucleotide identities ranging from 94.2 to 99.7% with five distinct begomovirus species of chilli, namely, chilli leaf curl Salem virus (HM007119), chilli leaf curl virus Bhavanisagar (NC_055130), chilli leaf curl Ahmedabad virus (MW795666), chilli leaf curl virus (NC_055131), and chilli leaf curl Sri Lanka virus (JN555600). BLAST results of the betasatellite sequences showed nucleotide identities of 96 to 98.8% with the tomato leaf curl Bangladesh betasatellite (MZ151286). In the present study, five distinct begomovirus species and one associated betasatellite were found to infect chilli crops in Tamil Nadu. This finding indicates a changing pattern of begomovirus occurrence in the different districts of Tamil Nadu. This study highlights the prevalence of chilli-infecting begomoviruses in the major chilli growing districts of Tamil Nadu, the identification of begomovirus species, and the significance of understanding and managing these viruses to safeguard chilli cultivation in the region.

Genomic profiling of pan-drug resistant proteus mirabilis Isolates reveals antimicrobial resistance and virulence gene landscape

Proteus mirabilis is a gram-negative pathogen that caused significant opportunistic infections. In this study we aimed to identify antimicrobial resistance (AMR) genes and virulence determinants in two pan-drug resistant isolate “Bacteria_11” and “Bacteria_27” using whole genome sequencing. Proteus mirabilis “Bacteria_11” and “Bacteria_27” were isolated from two different hospitalized patients in Egypt. Antimicrobial susceptibility determined using Vitek 2 system, then whole genome sequencing (WGS) using MinION nanopore sequencing was done. Antimicrobial resistant genes and virulence determinants were identified using ResFinder, CADR AMR database, Abricate tool and VF analyzer were used respectively. Multiple sequence alignment was performed using MAFFT and FastTree, respectively. All genes were present within bacterial chromosome and no plasmid was detected. “Bacteria_11” and “Bacteria_27” had sizes of approximately 4,128,657 bp and 4,120,646 bp respectively, with GC content of 39.15% and 39.09%. “Bacteria_11” and “Bacteria_27” harbored 43 and 42 antimicrobial resistance genes respectively with different resistance mechanisms, and up to 55 and 59 virulence genes respectively. Different resistance mechanisms were identified: antibiotic inactivation, antibiotic efflux, antibiotic target replacement, and antibiotic target change. We identified several genes associated with aminoglycoside resistance, sulfonamide resistance. trimethoprim resistance tetracycline resistance proteins. Also, those responsible for chloramphenicol resistance. For beta-lactam resistance, only blaVEB and blaCMY-2 genes were detected. Genome analysis revealed several virulence factors contribution in isolates pathogenicity and bacterial adaptation. As well as numerous typical secretion systems (TSSs) were present in the two isolates, including T6SS and T3SS. Whole genome sequencing of both isolates identify their genetic context of antimicrobial resistant genes and virulence determinants. This genomic analysis offers detailed representation of resistant mechanisms. Also, it clarifies P. mirabilis ability to acquire resistance and highlights the emergence of extensive drug resistant (XDR) and pan-drug resistant (PDR) strains. This may help in choosing the most appropriate antibiotic treatment and limiting broad spectrum antibiotic use.