Many types of banana (Musa spp.)—one of the most economically important horticultural crops—exist, and their ploidy (diploid, triploid, and tetraploid, usually) and genome types (containing A or/and B genome in most cases) differ. At present, observation and genome type detection are commonly used to identify banana germplasm resources. However, the former is tedious, while the latter cannot distinguish categories below genome types. It is therefore urgent that a simple and effective method for identifying banana germplasm resources is established. We sequenced and analyzed the ribosomal DNA internal transcribed spacer (ITS) sequences of 62 banana germplasm and found that the sequencing peaks, especially the 20 bp region near the 420 bp position (referred to as the “420 bp region”), exhibited relatively recognizable and repeatable polymorphism characteristics. Using the “420 bp region” as a marker, we were able to quickly distinguish bananas belonging to different genome type groups (comprising the AA, AB, AAA, ABB, AAB, ABBB, AAAB, and AAAA groups) or different subgroups in the same genome type group (for example, the Cavendish, Gros Michel, Red, Lakatan, and Ibota Bota subgroups in the AAA group; and the Pisang Raja, Plantain, Pome and Silk subgroups in the AAB group). Moreover, it appeared that Sanger sequencing of ITS could be used for identifying banana hybrid offspring. These results demonstrated that the banana ITS region contains rich genetic information that is useful for genotyping. In general, ITS sequencing simplifies the classification of banana germplasm resources and has potential application in several areas of Musa improvement.

DNA sequencing methods were first developed more than 20 years ago with the publication of two approaches to sequencing methodology that became known as Sanger sequencing (1), based on enzymatic synthesis from a single-stranded DNA template with chain termination using dideoxynucleotides (ddNTPs) and Maxim-Gilbert sequencing (2),which involved chemical degradation ofend-radio-labeled DNA fragments. Both methods relied on four-lane,high resolution polyacrylamide gel electrophoresis to separate the labeled fragment and allow the base sequence to be read in a staggered ladder-like fashion. Sanger sequencing was technically easier and faster, and thus became the main DNA sequencing method for the vast majority of applications.

Both SARS-CoV-2 and SARS-CoV initially appeared in China and spread to other parts of the world. SARS-CoV-2 has generated a COVID-19 pandemic causing more than 6 million human deaths worldwide while the SARS outbreak quickly ended in six months with a global total of 774 reported deaths. One of the factors contributing to this stunning difference in the outcome between these two outbreaks is the inaccuracy of the RT-qPCR tests for SARS-CoV-2, which generated a large number of false-negative and false-positive test results that have misled patient management and public health policy-makers. This article presented Sanger sequencing evidence to show that the RT-PCR diagnostic protocol established in 2003 for SARS-CoV can in fact detect SARS-CoV-2 accurately due to the well-known nonspecific PCR amplification of DNAs with similar sequences. Using nested RT-PCR followed by Sanger sequencing to retest 50 patient samples collected in January, 2022 and sold as RT-qPCR positive reference confirmed 21 (42%) were false-positive. Although the other 29 positive isolates were categorized as Omicron variant by partial sequencing of the N gene, and the RBD and the NTD of the S gene, 9 (31%) showed focal to complete sequencing failure in the S gene segments due to multi-allelic SNPs. During the course of the study, an Omicron variant isolate containing a BA.1 NTD and a BA.2 RBD in its S gene was also detected. Routine partial S gene sequencing of all PCR-positive samples can timely discover multi-allelic SNPs and viral recombination in the circulating variants for investigation of their impacts on vaccine efficacies, therapeutics and diagnostics.

As SARS-CoV-2 continues to spread among human populations, genetic changes occur and accumulate in the circulating virus. Some of these genetic changes have caused amino acid mutations, including deletions, which may have potential impact on critical SARS-CoV-2 countermeasures, including vaccines, therapeutics, and diagnostics. Considerable efforts have been made to categorize the amino acid mutations of the angiotensin-converting enzyme 2 (ACE2) receptor binding domain (RBD) of the spike (S) protein along with certain mutations in other regions within the S protein as specific variants in an attempt to study the relationship between these mutations and the biological behavior of the virus. However, the currently used whole genome sequencing surveillance technologies can test only a small fraction of the positive specimens with high viral loads and often generate uncertainties in nucleic acid sequencing that needs additional verification for precision determination of mutations. This article introduces a generic protocol to routinely sequence a 437-bp nested RT-PCR cDNA amplicon of the ACE2 RBD and a 490-bp nested RT-PCR cDNA amplicon of the N-terminal domain (NTD) of the S gene for detection of the amino acid mutations needed for accurate determination of all variants of concern and variants of interest according to the definitions published by the U.S. Centers for Disease Control and Prevention. This protocol was able to amplify both nucleic acid targets into cDNA amplicons to be used as templates for Sanger sequencing on all 16 clinical specimens that were positive for SARS-CoV-2.

Background: SARS-CoV-2 Omicron JN.1 has the highest number of amino acid mutations in its receptor binding domain (RBD) and has acquired a hallmark L455S mutation. The immune evasion capability of JN.1 is a subject of scientific investigation. The US CDC used RT-qPCR SGTF as the proxy indicator of JN.1 infections for evaluation of the effectiveness of updated monovalent XBB.1.5 COVID-19 vaccines against JN.1 and recommended that all persons aged ≥6 months should receive an updated COVID-19 vaccine dose. Objective: Recommend Sanger sequencing for diagnosis of JN.1 infections to generate the data based on which guidelines are made to direct vaccination policies. Methods: The RNA in nasopharyngeal swab specimens from patients with clinical respiratory infection was subjected to nested RT-PCR, targeting a 398-base segment of the N-gene and a 445-base segment of the RBD of SARS-CoV-2 for amplification. The DNA sequences were analyzed for amino acid mutations. Results: The N-gene sequence showed Q229K, usually associated with Omicron BA.2.86 (+JN.1). The RBD sequence showed 24 amino acid mutations, including the hallmark L455S mutation for JN.1 and the V483del for BA.2.86 lineage. Conclusions: The CDC should consider Sanger sequencing of the RBD to diagnose JN.1 infections for statistical analysis in making vaccination policies.

High resolution information on nucleotide arrangement in the genome of any or-ganism is critical to understand its genetic makeup, function, and evolutionary his-tory. For years, DNA sequencing has provided crucial breakthroughs in the field of genomics and molecular biology. With the advent of technology, Next Generation Sequencing has now transformed the whole genome sequencing prospects, thus unveiling the novel and interesting features of an organism genome that can behold. Next generation sequencing platforms offer realistic approach to identify and anal-yses whole genome with high throughput, accuracy, scalability in a very short time span. Information obtained can be further used in improving our existing healthcare systems, designing novel and personalized therapeutic modules, making the envi-ronment sustainable and proactively preparing ourselves for future pandemics and outbreaks. This article discusses the various NGS technologies and approaches, their advantages, drawbacks, and futuristic usage in different fields. Though still in nascent stages and marred with flaws, with ongoing advancement, NGS will indeed have promising impacts in the future.

The traditional practice of harvesting and processing raw date palm sap is not only culturally significant but also an essential nutritional source in South Asia. Yet, the potential for bacterial or viral contamination from animals and environmental sources during its collection remains a real and insufficiently studied risk. Implementing improved food safety measures and collection techniques could mitigate the risk of these infections. Additionally, the adoption of advanced food analytical methods has the potential to identify pathogens and uncover the natural bacterial diversity of these products. The advancement of NGS technologies, particularly nanopore sequencing, offers rapid and highly mobile capabilities. In this study, we employed nanopore sequencing for bacterial metabarcoding of a set of raw date palm sap samples collected without protective coverage against animals in Bangladesh in 2021. We identified several bacterial species with importance in the natural fermentation of the product and demonstrated the feasibility of this NGS method in the surveillance of raw palm sap products. We revealed two fermentation directions dominated by either Leuconostoc species or Lactococcus species in these products at the first 6 hours from harvest along with opportunistic human pathogens in the background, represented with low abundance. Plant pathogens, bacteria with the potential for opportunistic human infections and the sequences of the Exiguobacterium genus were also described and their potential role is discussed.

This study aimed to determine shifts in microbial populations regarding richness and diversity from the daily use of a popular over-the-counter nasal spray. In addition, the finding of nasal commensal bacterial species that overlap with the oral microbiome may prove to be potential probiotics for the “gateway microbiomes.” Nasal swab samples were obtained before and after using the most popular over-the-counter (OTC) nasal spray in 10 participants aged 18–48. All participants were healthy volunteers with no significant medical histories. The participants were randomly assigned a number by randomizing software and consisted of five men and five women. The sampling consisted of placing a nasal swab atraumatically into the nasal cavity. The samples were preserved and sent to Northwestern University Sequencing Center for whole-genome deep sequencing. After 21 days of OTC nasal spray use twice daily, the participants returned for further nasal microbiome sampling. The microbial analysis included all bacteria, archaea, viruses, molds, and yeasts via deep sequencing for species analysis. The Northwestern University Sequencing Center utilized artificial intelligence analysis to determine shifts in species and strains following nasal spray use that resulted in changes in diversity and richness.

Recently concluded, large-scale cancer genomics studies involving multiregion sequencing of primary tumors and paired metastases appear to indicate that many or most cancer patients have one or more “clonal" mutations in their tumors. Clonal mutations are those that are present in all of a patient’s cancer cells. Achilles Therapeutics is currently the only company specifically targeting clonal mutations. However, they are doing so with tumor-derived T cells. To address the potential limitations of immunotherapy, I have devised another approach for exploiting clonal mutations, which I call “Oncolytic Vector Efficient Replication Contingent on Omnipresent Mutation Engagement” (OVERCOME). The ideal version of OVERCOME would likely employ a bioengineered facultative intracellular bacterium. The bacterium would initially be attenuated, but (transiently) reverse its attenuation upon clonal mutation detection.

The ability to read and quantify nucleic acids such as DNA and RNA using sequencing technologies has revolutionized our understanding of life. With the emergence of synthetic biology, these tools are now being put to work in new ways - enabling de novo biological design. Here, we show how sequencing is supporting the creation of a new wave of biological parts and systems, as well as providing the vast data sets needed for the machine learning of design rules for predictive bioengineering. However, we believe this is only the tip of the iceberg and end by providing an outlook on recent advances that will likely broaden the role of sequencing in synthetic biology and its deployment in real-world environments.

During the last few years, advancement in the area of biochemistry, science of the material world, engineering and computer-aided testing has directed towards advancement of high-throughput tools for the sake of profiling information encoded in a gene. Single-cell RNA-sequencing (scRNA-seq) tools capable to examine the sequence data from each individual cells and it shows within population variety and permit exploring of cell conditions and transformation by extreme resolution, possibly give out cell sub-types or gene expression fluctuations that are shaded in mass sequencing processes, which shows population-averaged evaluations. Yet, the major disadvantage for this tool is the lack of success to pick out location related details of the RNA transcriptome, as this needs tissue detachment and cell isolation. Location based transcript determination is among the advancements in the area of medical biotechnology as this can find out the molecules like RNA dataset in their intact physical placement in tissue segment with spatial context at the scale of a single-cell, which is very advantageous as compare to single-cell sequencing techniques. These approaches give key observation in the areas of biomedical field sub-disciplines like neurology, embryology, carcinoma study, immune cell investigation and histological activities. Generally, this review mainly focused towards single-cell sequencing methods, technology development, its challenges observed, different expression data analysis mechanisms and their applications in different areas, such as in cancer research, microbes, central nervous system, sex organs and immune-biology, intensifying the essentiality of sequencing tools at single-cell level for the characterization of extremely dynamic individual cells.

Alfalfa is an important forage crop around the world. LncRNAs are considered to be a class of functional biomacromolecules, while little is known about lncRNAs in alfalfa. In this study, RNAs from different tissues of alfalfa were sequenced and anlyzed with full-length transcriptome sequencing technology, yielding the full-length transcripts dataset of alfalfa. Based on sequencing and public RNA-seq data, lncRNAs of alfalfa were predicted genome-wide by CPC2 and PLEK. The results showed most lncRNAs shared low sequence conservation with those in other plant species, part of which seems originate from plastid genome. We also identified 88563 lncRNAs, approximately 99.8% of total lncRNAs, with possibility of coding small ORFs using two prediction tools. Our research generated the biggest sequence set of alfalfa lncRNAs, and revealed some plastid originated lncRNAs with high sequence conservation.

There have been several developments in the field of nanopore biosensor development and sequencing applications that address previous limitations that restricted widespread nanopore use. These innovations, paired with the large-scale commercialization of biological nanopore sequencing by Oxford Nanopore Technologies, are making these platforms a mainstay in contemporary research labs. Equipped with the ability to provide long and short-read sequencing information with quick turn-around times and simple sample preparation, nanopore sequencers are quickly improving our understanding of unsolved genetic, transcriptomic, and epigenetic problems. However, there remain some key obstacles that have yet to be improved. In this review, we provide a general introduction to nanopore sequencing principles, discussing biological and solid-state nanopore developments, obstacles to single-base detection, and library preparation considerations. We conclude with examples of important clinical applications to give perspective on the potential future of nanopore sequencing in the field of molecular diagnostics.

Genetic disorders are preeminent determinants of infant mortality. The inherited pediatric-onset genetic disorders have consequential stress on child growth and development: several congenital, complex and rare disorders with indistinguishable clinical symptoms where diagnosis always remains a challenging task. Traditional diagnosis methods include biochemical tests followed by chromosomal microarray and sequencing of a single gene or panel of genes. These methods had several limitations, but with the advent of whole-exome sequencing (WES), genetic testing has become cost-effective and transformative. Exome sequencing has been known for its effectiveness, which appropriately elucidates and distinguishes the heterogeneous disorders to avoid misdiagnosis and decode the underlying genetic alterations. WES has led to discovering genes and genomic variants in a broad spectrum of diseases, including autism, epilepsy, congenital heart diseases, neurodevelopmental diseases, cancer, nephrotic disorders, neural tube defects and fetal structural anomalies. WES is significant in producing immense genomic biomarkers that can be made as appropriate pharmacogenomic targets for drug therapy. In this article, we analyze the recent exploration of WES technology to revolutionize not only the process of genetic variation and disease detection but also the convention of preventative and targeted drug discovery.

The world has now entered into a replacement era of genomics due to the continued advancements within the next generation high throughput sequencing technologies, which incorporates sequencing by synthesis-fluorescent in place sequencing (FISSEQ), pyrosequencing, sequencing by ligation using polony amplification, supported oligonucleotide detection (SOLiD), sequencing by hybridization alongside sequencing by ligation, and nanopore technology. Great impacts of those methods are often seen for solving the genome related problems of plant and Animalia which will open the door of a replacement era of genomics. This might ultimately overcome the Sanger sequencing that ruled for 30 years. NGS is predicted to advance and make the drug discovery process more rapid.

Multiple myeloma (MM) is an incurable, diverse cancer in which abnormal plasma cells produce and release monoclonal immunoglobulin (Ig), also known as monoclonal protein or M protein. MM is always preceded by monoclonal gammaglobulinemia of unknown significance (MGUS) and smoldering multiple myeloma (SMM). Diagnosis of MM is made from a bone marrow sample, which involves analysis of the appearance of plasma cells. Quantification of CD138+ plasma cells in core biopsies is performed using techniques such as immunohistochemistry, flow cytometry, fluorescence in situ hybridization (FISH) and conventional cytogenetics. Urinalysis includes total protein by immunofixation and 24-hour urine by serum protein electrophoresis. FISH probes should detect the presence of at least del 13, del 17p, t(4;14), t(11;14), t(14;16), and 1q21 amplifications. Indeed, next-generation sequencing (NGS) has become an increasingly important tool in the diagnosis and treatment of MM. It can be used to identify genetic mutations, copy number changes, and clonal structures to help determine disease prognosis and treatment strategies. NGS can identify subclonal populations of cancer cells that are resistant to therapy and may lead to disease relapse, and can be used to identify potential targets for personalized therapy. For example, NGS can identify mutations in genes involved in drug resistance, enabling the selection of drugs that may be more effective in treating a patient's cancer. This systematic review analyzes 182 papers to determine the position and role of NGS in the diagnosis of MM. We first analyze the platforms and sample types used in the selected studies, and then start discussing the results.

In recent years, the focus of disease resistance and susceptibility studies in cattle have been on determining patterns in the innate immune response of key proteins, such as Toll-like receptors (TLR). In the bovine genome, there are 10 TLR family members and, of these, TLR2, TLR4 and TLR6 are specialized in recognition of bacterial ligands. Indigenous cattle breeds of Anatolia have been reported to show fewer signs of clinical bacterial infections, such as bovine tuberculosis and mastitis, and it is hypothesized that this might be due to a less stringent genetic selection during breeding. In contrast, Holstein-Friesian cattle have been under strong selection for milk production, which may have resulted in greater susceptibility to diseases. To test this hypothesis, we have compared the TLR2, TLR4 and TLR6 genes of Anatolian Black (AB), East Anatolian Red (EAR), South Anatolian Red (SAR), Turkish Grey (TG), and Holstein (HOL) cattle using Next Generation Sequencing. The SAR breed had the most variations overall, followed by EAR, AB, TG and HOL. TG had the most variations for TLR2 whereas SAR had the most variations in TLR4 and TLR6. We compared these variants with those associated with disease and susceptibility traits. We used exon variants to construct haplotypes, investigated shared haplotypes within breeds and determined candidate haplotypes for disease resistance phenotype in Anatolian cattle breeds.

Single molecule de novo protein sequencing based on the 'superspecificity' of amino-acyl tRNA synthetases (aaRS) is proposed. An unfolded protein molecule is threaded through a nanopore in an electrolytic cell (e-cell) to expose the terminal residue in the e-cell's trans chamber. After the residue is cleaved with an exopeptidase, a set of tRNAs, their aaRSs, and ATP are added to trans. An aaRS charges a cognate tRNA molecule with the residue. The charged tRNA (along with the other reactants) is transferred to an extended e-cell with N (20 ≤ N ≤ 61) pores in N individual cis chambers and a single trans chamber. Each pore holds an RNA molecule ending in a unique codon that is exposed in trans. The charged tRNA's anticodon base-pairs with the terminal codon of an RNA. If tRNAs and residues are fluorescently tagged with two different colors, the residue can be identified from the observed position of the resulting color pair. As charging is 'superspecific' identification is unambiguous. The protein molecule in the first e-cell is advanced by one residue and the process repeated. In this approach there is no need for precise pore current or optical intensity measurements. Potential implementation issues are discussed. Other possibilities, including one in which the terminal residue is cleaved after charging, are also examined.

Background: The microbiome of the ocular surface has been characterised, but only limited in-formation is available on a possible silent intraocular microbial colonisation in normal eyes. Therefore, we performed next generation sequencing (NGS) of 16S rDNA genes in aqueous hu-mor. Methods: Aqueous humor was sampled from three patients during cataract surgery. Air swabs, patient’s and healthy donor conjunctival swab served as controls. Following DNA extraction, the V3 and V4 hypervariable regions of the 16S rDNA gene were amplified and sequenced fol-lowed by denoising. The resulting Amplicon Sequence Variants were matched to a subset of the Ribosomal Database Project 16S database. The deduced bacterial community was then statis-tically analysed. Results: DNA content in all samples was low (0-1.49 ng/µl) but sufficient for analysis. Main phyla in all samples were Acinetobacteria (48%), Proteobacteria (26%), Firmicutes (14%), Acidobac-teria (8%) and Bacteroidetes (2%). Patients conjunctival control samples and anterior chamber fluid showed similar patterns of bacterial species containing many waterborne species. Non-disinfected samples showed a different bacterial spectrum than the air swab sample. Conclusions: The data confirm the existence of an ocular surface microbiome. A distinct intraoc-ular microbiome was not discernable from the background, suggesting the absence of an intra-ocular microbiome in normal eyes.

Understanding microbial composition in upper respiratory infections (URIs) is critical for effective diagnosis and treatment. Precision Metagenomic next-generation sequencing p(mNGS) can provide a comprehensive yet clinically relevant profile of respiratory infection. The hybridization capture-based targeted sequencing generated a precision metagenomics profile of 29 patients with acute URIs. Nasopharyngeal samples were collected from the subjects suspected of respiratory infection, and p(mNGS) was performed using the Illumina®/IDbyDNA Respiratory Pathogen ID/AMR Panel (RPIP). The dataset obtained from the mNGS analysis contains a wealth of information on the composition of acute URI microbiota, including the relative abundance of known pathogens and potential clinical implications. The dataset represents a valuable resource for future research endeavors in respiratory medicine, infectious disease epidemiology, and therapeutic interventions. In addition, the dataset offers significant potential for reuse and integration with other omics datasets. The comprehensive nature of the mNGS data allows the exploration of associations between the respiratory microbiota and host factors such as clinical outcomes, immune responses, or genetic predisposition. Combining this dataset with other relevant datasets, such as transcriptomics or metabolomics, could provide a deeper understanding of the complex interactions between the microbiota and the host in acute URIs.

The quickest and easiest way to avoid greenhouse gas (GHG) emissions is to purchase renewable electricity and offset the remaining emissions. However, the industrial sector’s electricity needs already exceed renewable electricity generation. Moreover, electricity accounts for only one third of the industry’s energy needs. Simultaneously, the advance of sectoral coupling and the decarbonisation of industrial processes, as well as the desire to rapidly decrease dependence on fossil fuels, are creating significant additional demand for renewable energy. Neither existing nor planned generation and transmission infrastructure will suffice to meet the expected short-term demand. Based on survey data from the German Industry Energy Efficiency Index, this article therefore examines the share of GHG savings that companies intend to achieve on- and off-site. Understanding how much additional generation and transmission capacity is needed by the industry to decarbonise and by when is crucial to identify and address the extent of excess demand. On average, companies plan to avoid 22 % of their 2019 emissions by 2025 and 27 % by 2030, primarily through on-site measures. In combination with the extrapolation of the entire industry’s needs for off-site capacity, the data calls for a rapid expansion of planning authority and green generation capacities.

BACKGROUND: Glioblastoma (GBM) is driven by various genomic alterations. Next generation sequencing (NGS) could yield targetable alterations that may impact outcomes. The goal of this study was to describe how NGS can inform targeted therapy (TT) in this patient population. METHODS: The medical records of patients (pts) with a diagnosis of GBM from 2017-2019 were reviewed. Records of patients with recurrent GBM and genomic alterations were evaluated. Objective response rates and disease control rates were deter-mined. RESULTS: A total of 87 pts with GBM underwent NGS. Forty percent (n = 35) were considered to have actionable alterations. Of the 35, 40% (n=14) pts had their treatment changed due to an alteration. The objective response rate (ORR) of this population was 43%. The disease control rate (DCR) was 100%. The absolute mean decrease in contrast enhancing disease was 50.7% (95% CI 34.8 – 66.6). CONCLUSION: NGS for GBM, particularly in the recurrent setting, yields a high rate of actionable alterations. We observed a high ORR and DCR, reflecting the value of NGS in deciding on TT to match alterations that are likely to respond. In conclusion, patient selection and availability of NGS may impact outcomes in select pts with recurrent GBM.

Whole-genome DNA sequencing of Lactobacillus D75 and D76 strains (Vitaflor, Russia) was performed using the PacBio RS II platform, followed by de novo assembly with SMRT Portal 2.3.0. The average nucleotide identity (ANI) test showed that both strains belong to the Lactobacillus helveticus, but not the L. acidophilus as previously assumed. 31 exopolysaccharide (EPS) production genes (nine of which form a single genetic cluster), 13 adhesion genes, 38 milk protein and 11 milk sugar utilization genes, 13 genes for and against specific antagonistic activity, aight antibiotic resistance genes, and also three CRISPR blocks and eight Cas I-B system genes were identified in the genomes of the both strains. The expression of some genes was confirmed. In fact, the presence of identified genes suggests that L. helveticus D75 and D76 are able to form biofilms on the outer mucin layer, inhibit the growth of pathogens and pathobionts, utilize milk substrates with the formation of digestible milk sugars and bioactive peptides, resist bacteriophages and show some genome-determined resistance to antibiotics, stimulate the host’s immune system. Pathogenicity genes have not been identified. The study results confirm the safety and high probiotic potential of the strains.

Spontaneous tumours in higher plants can develop depending on plant genotype without any pathogen invasion. Spontaneous tumour formation on the taproots is consistently observed in certain inbred lines of radish (Raphanus sativus var. radicula Pers.). In this work, we have sequenced the genomes of two closely related radish inbred lines that differ in their ability to spontaneously form tumours. We have identified numerous single nucleotide variants (amino acid substitutions, insertions or deletions) that are likely to be associated with the spontaneous tumour formation. Furthermore, we performed the identification of CLE and WOX genes the genomic sequences of the radish inbred lines and, as a result, identified two unique radish CLE genes which probably encode proteins with multiple CLE domains. The results obtained provide a basis for investigating the mechanisms of plant tumour formation and also for future genetic and genomic studies of radish.

Background: The gut microbiome plays a major role in chronic diseases, of which several are characterized by an altered composition and diversity of bacterial communities. Large-scale sequencing projects allowed characterizing the perturbations of these communities. However, translating these discoveries into clinical applications remains a challenges. To facilitate routine implementation of microbiome profiling in clinical settings, portable, real-time, and low-cost sequencing technologies are needed. Results: Here, we propose a computational and experimental protocol for whole genome quantitative metagenomics studies of human gut microbiome with Oxford Nanopore sequencing technology (ONT) that could be applied to other microbial ecosystems. We developed a bioinformatic protocol to analyse ONT sequences taxonomically and functionally and optimized pre-analytic protocols including stool collection and DNA extraction methods to maximize read length. This is a critical parameter for the sequence alignment and classification. Our protocol was evaluated using simulations of metagenomic communities which reflect naturally occuring compositional variations. Next, we validated both protocols using stool samples from a bariatric surgery cohort, sequenced with ONT, Illumina and SOLiD technologies. Results revealed similar diversity and microbial composition profiles. Conclusion: This protocol can be implemented in the clinical or research setting, bringing rapid personalized whole genome profiling of target microbiome species.

Kluyveromyces marxianus (K. marxianus) is a newly emerging industrially relevant yeast. It is known to possess a highly efficient Non-Homologous End Joining (NHEJ) pathway that promotes random integration of non-homologous DNA fragments into its genome. The nature of the integration events was traditionally analyzed by Southern blot hybridization. However, the precise DNA sequence at the insertion sites were not fully explored. We transformed a PCR product of the Saccharomyces cerevisiae URA3 gene (ScURA3) into an uracil auxotroph K. marxianus wildtype strain and picked 24 stable Ura+ transformants for sequencing analysis. We took advantage of rapid advances in DNA sequencing technologies and developed a method using a combination of Illumina MiSeq and Oxford Nanopore sequencing. This approach enables us to uncover the Gross Chromosomal Rearrangements (GCRs) that are associated with the ScURA3 random integration. Moreover, it will shine a light on understanding DNA repair mechanisms in Eukaryotes, which could potentially provide insights for cancer research.

Species identification and phylogenetic relationships clarification are fundamental goals in species delimitation. Yet these tasks pose challenges when based on morphologies, geographic distribution, and genomic data. Previously, two new species of fern genus Bolbitis, B. × multipinna and B. longiaurita, were described based on morphological traits, are phylogenetically intertwined with B. sinensis and fail to form monophyletic groups. To address the unclear phylogenetic relationships within the B. sinensis species complex, RAD sequencing was performed on 65 individuals from five populations. Our integrated analysis of phylogenetic tree, neighbor net, and genetic structure, indicate that the B. sinensis species complex should not be considered as separate species. Moreover, our findings reveal differences in the degree of genetic differentiation among five populations, ranged from low to moderate, which might be influenced by geographical distance and gene flow. The Fst values also confirmed that genetic differentiation intensifies with increasing geographic distance. Collectively, this study clarifies the complex phylogenetic relationships within the B. sinensis species complex, elucidates the genetic diversity and differentiation across the studied populations, and offers valuable genetic insights that contribute to the broader study of evolutionary relationships and population genetics within the Bolbitis species.

A sufficient amount of tissue sample is required to perform next-generation sequencing (NGS) with a high success rate, but the majority of patients with advanced non-small-cell lung cancer (NSCLC) are diagnosed with small biopsy specimens. Biopsy samples were collected from 184 patients with bronchoscopically diagnosed NSCLC. The tissue surface area, tumor cell count, and tumor content rate of each biopsy sample were evaluated. The impact of the cut-off criteria for the tissue surface area (≥ 1 mm2) and tumor content rate (≥ 30%) on the success rate of Oncomine Dx Target Test (ODxTT) was evaluated. The mean tissue surface area of the transbronchial biopsies was 1.23 ± 0.85 mm2 when small endobronchial ultrasonography with a guide sheath (EBUS-GS) was used, 2.16 ± 1.49 mm2 with large EBUS-GS, and 1.81 ± 0.75 mm2 with endobronchial biopsy (EBB). The proportion of samples with a tissue surface area of ≥ 1 mm2 was 48.8% for small EBUS-GS, 79.2% for large EBUS-GS, and 78.6% for EBB. Sixty-nine patients underwent ODxTT. The success rate of DNA sequencing was 84.1% and that of RNA sequencing was 92.7% over all patients. The success rate of DNA (RNA) sequencing was 57.1% (71.4%) for small EBUS-GS (n = 14), 93.4% (96.9%) for large EBUS-GS (n = 32), 62.5% (100%) for EBB (n = 8), and 100% (100%) for endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) (n = 15). Regardless of the device used, a tissue surface area of ≥ 1 mm2 is adequate for samples to be tested with NGS.

In the last couple of years, the implementation of long-read sequencing (LRS) technologies for transcriptome profiling has uncovered an extreme complexity of viral gene expression. In this study, we carried out a systematic analysis on the pseudorabies virus transcriptome by combining our current data obtained by using Pacific Biosciences Sequel and Oxford Nanopore Technologies MinION sequencings with our earlier data generated by other LRS and short-read sequencing techniques. As a result, we identified a number of novel genes, transcripts, and transcript isoforms, including splice and length variants, and also confirmed earlier annotated RNA molecules. One of the major findings of this study is the discovery of a large number of 5’-truncated putative mRNAs embedded into larger host mRNAs. A large fraction of these RNA molecules contain in-frame ORFs, which may encode N-terminally truncated polypeptides. These study demonstrates that the PRV transcriptome is much more complex than previously appreciated.

Targeted next-generation sequencing (NGS) technology detects specific mutations that can provide treatment opportunities for colorectal cancer (CRC) patients. We included 145 CRC patients who underwent surgery. We analyzed the mutation frequencies of common actionable genes and their association with clinicopathological characteristics and oncologic outcomes using targeted NGS. Approximately 97.9% (142) of patients showed somatic mutations. Frequent mutations were observed in TP53 (70%), KRAS (49%), and APC (47%). TP53 mutations were significantly linked to higher overall stage (p=0.038) and lower disease-free survival (DFS) (p=0.039). ATM mutation was significantly associated with higher tumor stage (p=0.012) and shorter overall survival (OS) (p=0.041). Stage 3 and 4 patients with ATM mutations (p=0.023) had shorter OS, and FBXW7 mutation was significantly associated with shorter DFS (p=0.002). In multivariate Cox regression analysis, ATM mutation was an independent biomarker for poor prognosis of OS (p=0.022). TP53 and FBXW7 mutations are independent biomarkers for poor prognosis of DFS (p=0.042 and 0.030, respectively). A comprehensive analysis of the molecular markers for CRC can provide insights into the mechanisms underlying disease progression and help optimize a personalized therapy.

With the rise of long-read sequencers and long-range technologies, delivering high-quality plant genome assemblies is no longer reserved to large consortia. Indeed, sequencing techniques but also computer algorithms have reached a point where the reconstruction of assemblies at the chromosome-scale is now feasible at the laboratory scale. Current technologies, and especially long-range technologies, are numerous and selecting the most promising one for the genome of interest is crucial to obtain optimal results. In this study, we resequenced the genome of the yellow sarson, Brassica rapa cv. Z1, using the Oxford Nanopore PromethION sequencer and assembled the sequenced data using current assemblers. To reconstruct complete chromosomes, we used and compared three long-range techniques, optical mapping, Omni-C and Pore-C sequencing libraries commercialized by Bionano Genomics, Dovetail Genomics and Oxford Nanopore Technologies respectively, or a combination of the three, in order to evaluate the capability of each technology.

We consider the problem of scheduling $n$ jobs with identical processing times and given release as well as delivery times on $m$ uniform machines. The goal is to minimize the makespan, i.e., the maximum full completion time of any job. This problem is well-known to have an open complexity status even if the number of jobs is fixed. We present a polynomial-time algorithm for the problem which is based on the earlier introduced algorithmic framework blesscmore (``branch less and cut more''). We extend the analysis of the so-called behavior alternatives developed earlier for the version of the problem with identical parallel machines and show how the earlier used technique for identical machines can be extended to the uniform machine environment if a special condition on the job parameters is imposed. The time complexity of the proposed algorithm is $O(\gamma m^2 n\log n)$, where $\gamma$ can be either $n$ or the maximum job delivery time $q_{\max}$. This complexity can even be reduced further by using a smaller number $\kappa<n$ in the estimation describing the number of jobs of particular types. However, this number $\kappa$ becomes only known when the algorithm has terminated.

Study in black rice has gain prominence in recent times due to its high nutritive value, curative effect, and anti-oxidant properties. However, its poor agronomic traits, including low yield necessitates the incorporation of the colour-grain trait into elite varieties through plant breeding techniques. SSR markers play an important role in plant identification and breeding. Here, the generation of reference-based transcriptome, annotation of transcriptome datasets, and a large set of simple sequence repeat (SSR) markers derived from Black rice have been described. In all 28664 SSRs were predicted in 34978 (48.59%) expressed transcripts. However, 7068 (20.20%) transcripts were found to have more than one SSR. The identified SSRs were dominated by tri-nucleotide and tetra-nucleotide repeats representing about 54.11% and 33.31% respectively, of total SSRs. Validation of selected markers associated with anthocyanin trait performed across different black rice accessions established the reliability of the process used for mining SSR markers. The SSR markers identified in this study could be used to select varieties with desired traits, and to investigate the genetic mechanism underlying anthocyanin accumulation in the pericarps of black rice. Furthermore, the findings from this study may prove beneficial in future genetic diversity studies, primer development, and selective breeding programs.

Understanding ecological interactions is a key in managing phytopathology. Although entomologists rely mostly on both traditional molecular methods and morphological characteristics to identify pests, next-generation sequencing is becoming the go-to avenue for scientists studying fungal and oomycete phytopathogens. These organisms sometimes infect plants together with insects. There are many relationships yet to be discovered and much to learn about how these organisms interact with one another. Considering the growing number of exotic insect introductions in Canada, a high-throughput strategy for screening those insects is already implemented by the Canadian Food Inspection Agency (CFIA). However, no plan is deployed to investigate the phytopathogenic fungal and oomycete species interacting with insects. Metagenomics analysis was performed on the preservation fluids from CFIA’s insect traps across Canada. Using the Ion Torrent PGM technology and fusion primers for multiplexing and indexing, community profiling was conducted on the different semiochemicals used in the insect traps and the various areas where these traps were placed. Internal transcribed spacer 1 (fungi and oomycetes) and adenosine triphosphate synthase subunit 9-nicotinamide adenine dinucleotide dehydrogenase subunit 9 spacer amplicons were generated. Although direct links between organisms could not be established, moderately phytopathogenic fungi (e.g., Leptographium spp. and Meria laricis) and oomycetes (mainly Peronospora spp. and Pythium spp.) unique to every type of semiochemical were discovered. The entomopathogenic yeast Candida michaelii was also detected. This project demonstrated our ability to screen for unwanted species faster and at a higher scale and throughput than traditional pathogen diagnostic techniques. Additionally, minimal modifications to this approach would allow it to be used in other phytopathology fields.

Grain shape and chalkiness are important appearance quality traits of rice, which also affect the yield and milling quality. In this study, 190 F2 families derived from the cross between Hua 5178S and Hua 611 were sequenced to construct a genetic linkage map. Combined with F2 and F2:3 generations grain shape and chalkiness phenotype, a total of 15 QTL were detected. There are 8 grain shape QTL distributed on chromosomes 2, 5, 6, 8, and 10; 7 chalkiness QTL distributed on chromosomes 2, 3, and 6. A QTL cluster affecting multiple chalkiness traits was found on chromosome 6, qWBR6.1, qWCR6 and qCR6 were selected for genetic effect verification. It was found that qWBR6.1, qWCR6 and qCR6 explained 13% and 24%, and 29% of the phenotypic variations respectively, and qWCR6 and qCR6 were co-mapped. This study laid a foundation for further cloning of rice appearance quality genes.

The identification and molecular characterization of cellular hierarchies in complex tissues is key to understanding both normal cellular homeostasis and tumorigenesis. The mammary epithelium is a heterogeneous tissue consisting of two main cellular compartments, an outer basal layer containing myoepithelial cells and an inner luminal layer consisting of estrogen receptor-negative (ER⁻) ductal cells and secretory alveolar cells (in the fully functional differentiated tissue) and hormone-responsive estrogen receptor-positive (ER⁺) cells. Recent publications have used single-cell RNA-sequencing (scRNA-seq) analysis to decipher epithelial cell differentiation hierarchies in human and murine mammary glands, and reported the identification of new cell types and states based on the expression of the luminal progenitor cell marker KIT (c-Kit). These studies allow for comprehensive and unbiased analysis of the different cell types that constitute a heterogeneous tissue. Here we discuss scRNA-seq studies in the context of previous research in which mammary epithelial cell populations were molecularly and functionally characterized, and identified c-Kit⁺ progenitors and cell states analogous to those reported in the recent scRNA-seq studies.

To fully appreciate genetics, one must understand the link between genotype (DNA sequence) and phenotype (observable characteristics). Advances in high-throughput genomic sequencing technologies and applications, so-called “-omics”, have made genetic sequencing readily available across fields in biology from applications in non-traditional study organisms to precision medicine. Thus, understanding these tools is critical for any biologist, especially those early in their career. This comprehensive review discusses the chronological development of different sequencing methods, the bioinformatics steps to analyzing this data, and social and ethical issues raised by these techniques that must be discussed and evaluated.

Quebec is the third-largest wine grape producing province in Canada, and the industry is constantly expanding. Traditionally, 90% of the grapevine cultivars grown in Quebec were rustic or semi-rustic and largely dominated by winter hardy interspecific hybrid Vitis sp. cultivars. Over the years, the winter protection techniques adopted by growers and climate changes have offered an opportunity to establish V. vinifera L. cultivars (e.g. Pinot Noir). We characterized the virome of leafroll-infected interspecific hybrid cultivar and compared it to the virome of V. vinifera cultivar to support and facilitate the transition of the industry. A dsRNA sequencing method was used to sequence symptomatic and asymptomatic grapevine leaves of different cultivars. The results suggested a complex virome in terms of composition, abundance, richness, and phylogenetic diversity. Three viruses, grapevine Rupestris stem pitting-associated virus, grapevine leafroll-associated virus (GLR) 3 and 2 and hop stunt viroid (HSVd) largely dominated the virome. However, their presence and abundance varied among grapevine cultivars. The symptomless grapevine cultivar Vidal was frequently infected by multiple virus and viroid species and different strains of the same virus, including GLR virus 3 and 2. Our data shows that viruses and viroids associated with the highest number of grapevines expressing symptoms included HSVd, GLR3 and GLR2, in gradient order. However, co-occurrence analysis revealed that the presence of GLR species was randomly associated with the development of virus-like symptoms. These findings and their implication for grapevine leafroll disease management were discussed.

The rapid and accurate detection of viruses and the discovery of single nucleotide polymorphisms (SNPs) are crucial for disease management and understanding viral evolution. In this study, a pipeline for virus detection, validation, and SNP discovery from next-generation sequencing (NGS) data is presented. By integrating state-of-the-art bioinformatics tools with artificial intelligence, the pipeline processes raw sequencing data to identify viral sequences with high accuracy and sensitivity. Before aligning the reads to the reference genomes, quality control measures and adapter trimming are performed to ensure the integrity of the data. Unmapped reads are subjected to de novo assembly to reveal novel viral sequences and genetic elements. The effectiveness of the pipeline is demonstrated by the identification of virus sequences, illustrating its potential for the detection of known and emerging pathogens. SNP discovery is performed using a custom Python script that compares the entire population of sequenced viral reads to a reference genome. This approach provides a comprehensive overview of viral genetic diversity and identifies dominant variants and a spectrum of genetic variations. The robustness of the pipeline is confirmed by the recovery of complete viral sequences, which improves our understanding of viral genomics. This study highlights the synergy between traditional bioinformatics techniques and modern approaches, providing a robust tool for analyzing viral genomes and contributing to the broader field of viral genomics.

We investigated the potential genetic variants using whole exome sequencing (WES) and evalu-ated the disease course using T cell receptor (TCR) repertoire analysis in rheumatoid arthritis (RA). Fourteen patients with RA and five healthy controls (HCs) were enrolled. For patients with RA, only naïve patients were recruited before treatment, and data were collected at baseline and 6 and 12 months after the initiation of disease-modifying antirheumatic drug (DMARD) treatment. Laboratory data and disease parameters were also collected. Genetic variants were detected using WES, and the diversity of the TCR repertoire was assessed using Shannon-Weiner diversity. Some variants were detected by WES, but their clinical significance should be confirmed by further studies. The diversity of the TCR repertoire in patients with RA was lower than that in HCs; however, after DMARD treatment, it increased significantly. The diversity was negatively cor-related with laboratory findings and disease measures with statistical significance. Variants with a potential for RA pathogenesis were identified and the clinical significance of TCR repertoire was evaluated in Korean patients with RA. Further studies are required to confirm the findings of the present study.

The advent of immunotherapy has revolutionized the treatment of advanced non-small cell lung cancer (NSCLC), improving the prognosis of this disease and becoming a key part of the treatment, especially in the population without an oncogenic driver mutation. Two different groups of immune checkpoint inhibitors (ICIs) are used in lung cancer: Anti- Cytotoxic T-lymphocyte antigen-4 (Anti-CTLA 4) and Anti- T-cell receptor programmed cell death-1 or its ligand (Anti-PD-1 and Anti PD-L1). Here in, we review an uptodate on the use of immunotherapy for advanced NSCLC.

Recent changes in lung cancer care, including new approvals in first line and the introduction of high throughput molecular technologies in routine testing led us to question ourselves on how deeper molecular testing may be helpful for the optimal use of targeted drugs. In this article we review recent results in the scope of personalized medicine in lung cancer. We discuss biomarkers that have a therapeutic predictive value in lung cancer with a focus on recent changes and on the clinical value of large scale sequencing strategies. We review the use of second and third generation EGFR and ALK inhibitors with a focus on secondary resistance alterations. We discuss anti-BRAF and anti-MEK combo, emerging biomarkers as NRG1 and NTRKs fusions and immunotherapy. Finally we discuss the different technical issues of comprehensive molecular profiling and show how large screenings might refine the prediction value of individual markers. Based on a review of recent publications (2012–2018), we address promising approaches for the treatment of patients with lung cancers and the technical challenges associated to the identification of new predictive markers.

Alfalfa, an important legume forage, is an ideal crop for sustainable agriculture and a potential bioenergy plant. Drought, one of the most common environmental stresses, substantially affects plants’ growth, development and productivity. MicroRNAs (miRNAs) are newly discovered gene expression regulators that have been linked to several plant stress responses. To elucidate the role of miRNAs in drought stress regulation of alfalfa, a high-throughput sequencing approach was used to analyze 12 small RNA libraries comprising of 4 samples, each with 3 biological replicates. We identified 348 known miRNAs, belonging to 80 miRNA families, from the 12 libraries and 281 novel miRNAs using Mireap software. 18 known miRNAs in roots and 12 known miRNAs in leaves were screened out as drought-responsive miRNAs. Except for miR319d and miR157a which were upregulated under drought stress, the expression pattern of drought-responsive miRNAs were different between roots and leaves in alfalfa. This is the first study discovering miR157a, miR1507, miR3512, miR3630, miR5213, miR5294, miR5368 and miR6173 are drought-responsive miRNAs. Target transcripts of drought-responsive miRNAs were computationally predicted. All 447 target genes for the known miRNAs were predicted using an online tool. This study provides a significant insight on understanding drought-responsive mechanisms of alfalfa.

Advances in gene-specific therapeutics for patients with neuromuscular disorders (NMDs) have brought increased attention to the importance of genetic diagnosis. Genetic testing practices vary among adult neuromuscular clinics, with multi-gene panel testing currently being the most common approach; follow-up testing using broad-based methods, such as exome or genome sequencing, is less consistently offered. Here, we use five case examples to illustrate the unique ability of broad-based testing to improve diagnostic yield, resulting in identification of SORD-neuropathy, HADHB-related disease, ATXN2-ALS, MECP2 related progressive gait decline and spasticity, and DNMT1-related cerebellar ataxia, deafness, narcolepsy, and hereditary sensory neuropathy type 1E. We describe in each case the technological advantages that enabled identification of the causal gene, and the resultant clinical and personal implications for the patient, demonstrating the importance of offering exome or genome sequencing to adults with NMDs.

We explore three cases of pediatric neurological diseases, viz. Arthrogryposis, congenital bilateral cataract and Autism by analyzing clinical exomes. As genetic variation attributing to pathogenesis is a significant bottleneck, we attempted to understand and validate them using Sanger validation. We further employ our CONVEX pipeline to infer pathogenic variants and discern the candidate genes for phenotype correlation.

In the last decade, we have witnessed an upsurge in nuclei-based studies, particularly coupled with next-generation sequencing. Such studies aim at understanding the molecular states that exist in heterogeneous cell populations by applying increasingly more affordable sequencing approaches, in addition to optimized methodologies developed to isolate and select nuclei. Although these powerful new methods promise unprecedented insights, it is important to understand and critically consider the associated challenges. Here, we provide a comprehensive overview of the rise of nuclei-based studies and elaborate on their advantages and disadvantages. Improved designs and appropriate use of the various experimental strategies will result in acquiring biologically accurate and meaningful information.

Emerging infectious disease threats require rapid response tools to inform diagnostics, treatment, and outbreak control. RNA-based metagenomics offers this; however, most approaches are time-consuming and laborious. Here, we present a simple and fast protocol – the RAPIDprep assay – with the aim to provide cause agnostic laboratory diagnosis of infection within 24 hours of sample collection by sequencing ribosomal RNA-depleted total RNA. The method is based on the synthesis and amplification of double-stranded cDNA followed by short-read sequencing with minimal handling and clean-up steps to improve processing time. The approach was optimized and applied to a range of clinical respiratory samples to demonstrate diagnostic and quantitative performance. Our results showed robust depletion of both human and microbial rRNA, and library amplification across different sample types, qualities and extraction kits using a single protocol without input nucleic acid quantification or quality assessment. Furthermore, we demonstrate the genomic yield of both known and undiagnosed pathogens with complete genomes recovered in most cases to inform molecular epidemiological investigations and vaccine design. The RAPIDprep assay is a simple and effective tool, and representative of an important shift towards integration of modern genomic techniques to infectious disease investigations.

Human metapneumovirus (HMPV) is an important cause of upper and lower respiratory tract disease in individuals of all ages. It is estimated that most individuals will be infected by HMPV by the age of 5 years old. Despite this burden of disease, there remains caveats in our knowledge of virus global genetic diversity due to a lack of HMPV sequencing, particularly at whole genome scale. The purpose of this study was to create a simple and robust approach for HMPV whole genome sequencing to be used for genomic epidemiological studies. To design our assay, all available HMPV full length genome sequences were downloaded from the NCBI GenBank database and used to design four primer sets to amplify long, overlapping amplicons spanning the viral genome and, importantly, specific to all known HMPV subtypes. These amplicons were then pooled and sequenced on an Illumina iSeq; however the approach is suitable to other common NGS platforms. We demonstrate the utility of this method using a representative subset of clinical samples and examine these sequences using a phylogenetic approach. Here we present an amplicon-based method for the whole genome sequencing of HMPV from clinical extracts that can be used to better inform genomic studies of HMPV epidemiology and evolution.

Moonmilk are cave carbonate deposits that host a rich microbiome including antibiotic-producing Actinobacteria making these speleothems appealing for bioprospecting. Here we investigated the taxonomic profile of the actinobacterial community of three moonmilk deposits of the cave “Grotte des Collemboles” via high-throughput sequencing of 16S rRNA amplicons. Actinobacteria was the most common phylum after Proteobacteria, ranging from 9 to 23% of the total bacterial population. Next to actinobacterial OTUs attributed to uncultured organisms at the genus level (~44%), we identified 47 actinobacterial genera with Rhodoccocus (4 OTUs, 17%) and Pseudonocardia (9 OTUs, ~16%) as the most abundant in terms of absolute number of sequences. Streptomycetes presented the highest diversity (19 OTUs, 3%), with most of OTUs unlinked to the culturable Streptomyces strains previously isolated from the same deposits. 43% of OTUs were shared between the three studied collection points while 34% were exclusive to one deposit indicating that distinct speleothems host their own population despite their nearby localization. This important spatial diversity suggests that prospecting within different moonmilk deposits should result in the isolation of unique and novel Actinobacteria. These speleothems also host a wide range of non-streptomycetes antibiotic-producing genera, and should therefore be subjected to methodologies for isolating rare Actinobacteria.

Chimeric antigen receptor (CAR) T-cell therapy has greatly improved outcomes for patients with relapsed or refractory hematological malignancies. However, challenges such as treatment resistance, relapse, and severe toxicity still hinder its widespread clinical application. Traditional transcriptome analysis has provided limited insights into the complex transcriptional landscape of both leukemia cells and engineered CAR-T cells, as well as their interactions within the tumor microenvironment. However, with the advent of single-cell sequencing techniques, a paradigm shift has occurred, providing robust tools to unravel the complexities of these factors. These techniques enable unbiased analysis of cellular heterogeneity and molecular patterns. These insights are invaluable for precise receptor design, guiding gene-based T cell modification, and optimizing manufacturing conditions. Consequently, this review utilizes modern single-cell sequencing techniques to clarify the transcriptional intricacies of leukemia cells and CAR-Ts. The aim of this manuscript is to discuss the potential mechanisms that contribute to the clinical failures of CAR-T immunotherapy. We will examine the biological characteristics of CAR-Ts, the mechanisms that govern clinical responses, and the intricacies of adverse events. By exploring these aspects, we hope to gain a deeper understanding of CAR-T therapy, which will ultimately lead to improved clinical outcomes and broader therapeutic applications.

Crop output is directly impacted by plant infections (with fungi as the major pathogen), making accurate diagnosis of these threats crucial. Developing technology and multidisciplinary approaches are turning to genomic analyses in addition to traditional culture methods in diagnosing fungal plant diseases. The metagenomic next-generation sequencing (mNGS) method is preferred for genotyping identification of organisms, identification at the species level, illumination of metabolic pathways, and determination of microbiota. Moreover, the data obtained so far show that this new approach shows promise as an emerging new trend in fungal disease detection. Another approach covered by mNGS technologies, known as metabarcoding, enables use of specific markers specific to a genetic region and allows for genotypic identification by facilitating the sequencing of certain regions. Although the core concept of mNGS remains constant across applications, the specific sequencing methods and bioinformatics tools used to analyze the data do differ. In this review, we focus on how mNGS technology, including metabarcoding, is applied in fungal pathogenesis and its promising developments for the future.

Germline mutations in predisposition genes account for only 20% of all familial colorectal cancer (CRC) and the remaining genetic burden may be due to rare high-to-moderate-penetrance germline variants that are not explored. With the aim of identifying such potential cancer predisposing variants, we performed whole exome sequencing on three CRC cases and three unaffected members of a Polish family and identified two novel heterozygous variants; a coding variant in APC down-regulated 1 gene (APCDD1, p.R299H) and a non-coding variant in the 5’ untranslated region (UTR) of histone deacetylase 5 gene (HDAC5). Sanger sequencing confirmed the variants segregating with the disease and Taqman assays revealed 8 additional APCDD1 variants in a cohort of 1705 familial CRC patients and no further HDAC5 variants. Proliferation assays indicated an insignificant proliferative impact for the APCDD1 variant. Luciferase reporter assays using the HDAC5 variant resulted in an enhanced promoter activity. Targeting of transcription factor binding sites of SNAI-2 and TCF4 interrupted by HDAC5 variant showed a significant impact of TCF4 on promoter activity of mutated HDAC5. Our findings contribute not only to the identification of unrecognized genetic causes of familial CRC but also underline the importance of 5´UTR variants affecting transcriptional regulation and the pathogenesis of complex disorders.

Despite its remote discovery (1977) and numerous reports of its presence in vari-ous plant species in many countries, the available molecular information for the artichoke Italian latent virus (AILV) is still limited to a single complete genome sequence (RNA1 and 2) isolate from grapevine (AILV-V) and to a partial sequence portion from RNA2 of an isolate of unknow origin and host, both reported in the GenBank. Here we report the results of molecular analyses conducted on RNA2 of AILV isolates, sequenced for the first time in this study, together with the first-time finding of AILV in a new host plant species, i.e., chard (Beta vulgaris subsp. vulgaris). The different AILV isolates sequenced are from artichoke (AILV-C), gladiolus (AILV-G), sonchus (AILV-S) and chard (AILV-B). At the bio-logical level, the AILV-B was found to be involved with vein clearing and mott-ling symptoms on chard leaves. At the molecular level, sequencing results of RNA2 segments showed that AILV-C, AILV-G, AILV-S and AILV-B were 4,629 nt in length (excluding the 3’ terminal polyA tail), hence one nt shorter than that of AILV-V reported in the GenBank. The comparative sequence analysis between the CR of RNA2 of all isolates showed that AILV-V was the most variable isolate, with the lowest sequence identities of 83.2% and 84.7% at the nucleotides and amino acids levels, respectively. Putative intra-species sequence recombination sites were predicted among the AILV isolates, of which the AILV-V, AILV-C and AILV-B genomes were found to be mostly involved. This study adds further mo-lecular information on the previously unrecorded high genomic variability of the Nepoviruses of subgroup B, together with information on recombinations that may have conditioned the infection and transmission of AILV in nature; thus learning the way forward to design a new integrated pest management of chard plantation.

Plankton monitoring by microscopy offers long-term ecological perspective of plankton com-munities but different detection approaches are biased uniquely. Genetic identification of marine plankton has become standard but is still not used in routine monitoring. This study assessed the diversity of plankton taxa using 18S high throughput sequencing from 2011-2012 from small-volume (~200ml) samples from the Water and Microplankton Sampler (WaMS) deployed on the Continuous Plankton Recorder platform (CPR). The 18S-HTS survey revealed a bias towards heterotrophic taxa, and phototrophs under 10µm within the photosynthetic community. In comparison with phytoplankton microscopic counts from the CPR survey and Western Channel Observatory station L4, only 8-12 taxonomic families were common to all three surveys, with a bias towards larger diatoms and dinoflagellate taxa in microscopy surveys. The WaMS survey detected a contrasting but complementary taxa set to that of microscopic surveys. Additional Quantitative PCR was carried out on the picoeukaryotic pelagophyte, Aureococcus anophagefferens, and the nanoeukaryotic potential harmful algae, Pseudo-nitzschia delicatissima, from 2011-2013. This confirmed the persistence presence of A. anophagefferens in the Western Channel and an elevated abundance of both species in 2011. Species specific seasonality were distinct from those of aggregrate phytoplankton groups.

We presented here the first draft genome sequence of the trypanosomatid Herpetomonas muscarum ingenoplastis. This parasite was isolated repeatedly in the black blowfly, Phormia regina. This is the first draft genome of a flagellate from the phylogenetically distinct clade of Trypanosomatidae.

Cachexia is a complex metabolic syndrome characterized by loss of skeletal muscle, leading to a significant weight loss that impacts patient morbidity and mortality. Given the complexity of gene regulatory networks that control gene expression, our objective was to perform an integrative mRNA and miRNA profiling to identify genetic programs that capture essential mechanistic details that promote muscle atrophy in cancer cachexia. Here, we used RNA sequencing to analyze miRNAs and mRNAs expression profiles in tibialis anterior (TA) muscles of the Lewis lung carcinoma model of cancer cachexia. In addition, we compared these findings with RNA-Seq data from C2C12 myotubes treated in vitro with the cachectic factors tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). Extracellular matrix (ECM) alterations were validated by picrosirius staining, western blot, and fractal dimension analyses. We found 1,008 mRNAs and 18 miRNAs differentially expressed in cachectic mice. This set of genes was associated with the ECM, proteolysis, and inflammatory response. Enrichment analysis of transcriptional factor binding sites revealed activation of the atrophy-related transcriptional factors: NF-κB, Stat3, AP-1, and FoxO. Furthermore, the integration of mRNA and miRNA expression profiles identified post-transcriptional regulation by miRNAs of genes involved in ECM organization, cell migration, transcription factors binding, ion transport, and FoxO signaling pathway. C2C12 myotubes treated with TNF-α and IFN-γ similarly down-regulate subsets of ECM genes, including collagens. Our integrative analysis of miRNA-mRNA co-profiles comprehensive characterized regulatory relationships of molecular pathways and revealed miRNAs targeting ECM-associated genes in cancer cachexia. We also confirmed in C2C12 myotubes that changes in ECM-associated genes are dependent on inflammatory signaling of the cytokines TNF-α and IFN-γ.

Genotypic testing is often recommended to improve the management of patients infected with human immunodeficiency virus (HIV). To help combat this major pandemic, next-generation sequencing (NGS) techniques are widely used to analyse resistance to antiretroviral drugs. In this study, we used a Vela Sentosa kit, which is usually used for the Ion Torrent personal genome machine (PGM) platform, to sequence HIV using the Illumina Miseq platform. After RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR), minor modifications were applied to the Vela Sentosa kit to adapt it to Illumina Miseq platform. Analysis of the results showed the same mutations present in the samples using both sequencing platforms. The total number of reads varied from 185,069 to 752,343 and from 642,162 to 2,074,028 in the Ion Torrent PGM platform and the Illumina Miseq platform, respectively. The average depth was 21955 and 46856 for Ion Torrent PGM and Illumina Miseq platforms, respectively. The cost of sequencing a run of eight samples was closely similar between the two platforms (about 1 790 United States dollars [USD] for Illumina Miseq and about 1833 USD for Ion Torrent PGM platform). We have shown for the first time that it is possible to adapt and use the Vela Sentosa kit for the Illumina Miseq platform to obtain high quality results with a similar cost.

As exemplified by the global response to the SARS-CoV-2 pandemic, whole genome sequencing played an important role in monitoring the evolution of novel viral variants and provided guidance on potential antiviral treatments. The recent rapid and extensive introduction and spread of highly pathogenic avian influenza virus in Europe, North America and elsewhere raises the need for similarly rapid sequencing to aid in appropriate response and mitigation activities. To facilitate this objective, we investigated a next generation sequencing platform that uses a portable nanopore sequencing device to generate and present data in real time. This platform offers the potential to extend in-house sequencing capacities to laboratories that may otherwise lack resources to adopt sequencing technologies requiring large benchtop instruments. We evaluated this platform for routine use in a diagnostic laboratory. In this study we evaluated different primer sets for the whole genome amplification of influenza A virus and evaluated five different library preparation approaches for sequencing on the nanopore platform using the MinION flow-cell. A limited amplification procedure and a rapid procedure were found to be best among the approaches taken.

We attempted to understand the cervical cancer patient samples through Whole Exome Sequencing. We derived the variants from raw reads via our in-house benchmarked pipeline and validated the variants by IGV. This is the first cervical cancer exome data from the Indian cohort. Background: Cervical cancer (CC) is caused mainly by persistent infections of high-risk HPV, reduced parity, and factors like a decrease in average socioeconomic levels. We keep this because no cervical cancer exome data is available from the Indian cohort. Methods: The CC patient clinical samples were initially subjected to preparation using Qiagen DNA extraction, quantified, and the library preparation using the Agilent target enrichment system. Further, exome capture by Illumina platform (100X), quality check, alignment, and variant calling followed by the downstream analysis and finally visualized by IGV. Results: We observed a large number of SNVs or mutations from an Indian perspective, such as KMT2C, OR4M1, PDPR2P, EPHB1, FAS, OPCML, MGST1, C1QTNF9, HS6ST3, OR4K2, PRPSAP2, KCNJ12, FIGNL1, SFXN1, BAGE2, ARVCF, NAMPT variants of significance, unknown significance and possibly significant are reported. Conclusion: For the first time, KMT2C is observed as a novel potent mutation and pathogenic, showing the variant position at (7:152265091, T>A, SNV 62478356) from the Indian context in CC. Further, we visualized and validated the mutations using the Integrative Genomic Viewer (IGV) browser. Finally, we discuss the inherent challenges through KMT2C mutations.

In this study, a structural profiling of equid alphaherpesvirus 1 (EHV-1) transcriptome was carried out using next-generation (Illumina) and third-generation (Oxford Nanopore Technologies) sequencing platforms. We annotated the canonical mRNA molecules and their isoforms, including transcript start and end site isoforms, and splice variants. Additionally, a number of putative 5′-truncated mRNAs containing shorter in-frame ORFs were detected. We also demonstrated that EHV-1 produces a high number of non-coding transcripts, including antisense and intergenic RNAs. One of the most remarkable features of the EHV-1 is the generation of abundant fusion transcripts some of which encoding chimeric polypeptides. We observed a higher number of splicing and transcriptional overlaps than in related viruses. Additionally, we found that many upstream genes of tandem gene clusters have their own transcript end sites (TESs) besides the co-terminal TESs, which is rare in other alphaherpesviruses. We show here that the replication origins (OriS and OriL) of the virus are co-localized with promoter sequences and overlap with specific RNA molecules. Furthermore, we discovered a novel non-coding RNA (designated as NOIR) that overlaps the 5′-ends of the longer transcript variants encoded by the two main transactivator genes ORF64 and 65 bracketing the OriL. These all suggest the existence of a central regulatory system which controls the genome-wide transcription and the replication through a mechanism based on the interference between the machineries carrying out the synthesis of DNA and RNA.

In regions lacking genomic data, analysis of sequences from the early stages of an outbreak can provide important insights into the diversity of pathogens present. Following the detection of the first imported case of COVID-19 in the Northern sector of Ghana on 13^th March 2020, we have now molecularly characterized and phylogenetically analysed sequences including three (3) complete genomes of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) isolated from nine (9) patients observed in Ghana. Eight (8) of these patients reported with a recent history of foreign travel and one (1) with no history of foreign travel. We performed high throughput sequencing for 9 samples following the determination of high concentration of viral RNA. In addition, we estimated the potential impact that long distance transportation of samples to testing centres may have on sequencing outcomes. Here, two samples that were closest in terms of viral RNA concentration but transported from sites which are over 400km apart were assessed. All sequences were compared to previous sequences from Ghana and representative sequences from regions where our patients had previously travelled. Complete genomes were obtained for three (3) sequences and with another near complete genome with a coverage of 95.6%. Sequences with coverage in excess of 80% were found to belong to three lineages namely A, B.1 and B.2. Our sequences clustered in two different clades with the majority falling within a clade composed of sequences from sub-Saharan Africa. Less RNA fragmentation was seen in sample KATH23 which was collected 9km compared with sample TTH6 which was collected and transported over a distance of 400km to the testing site. The clustering of several sequences from sub-Saharan Africa suggests regional circulation of the viruses in the subregion. Importantly, there may be the need to decentralize testing sites and build more capacity across Africa to boost the sequencing output of the subregion.

After human genome sequencing and rapid changes in genome sequencing methods, we have entered in the era of rapidly accumulating genome-sequencing data. This has poses development of several types of methods for representing results of genome sequencing data. Circular genome visualizations tools are also critical in this area as they provide rapid interpretation and simple visualization of overall data. In the last 15 years, we have seen rapid changes in circular visualization tools after the development of the circos tool with 1–2 tools published per year. Herein we have summarized and revisited all these tools until the third quarter of 2018.

Chicory, a horticultural crop cultivated worldwide, presents many botanical varieties and local biotypes. Among these, Italian radicchio group cultivars of pure Cichorium intybus L. and interspecific hybrids with Cichorium endivia L., as the “Red of Chioggia” biotype which in turn includes several phenotypes. This study deals with a pipeline for the marker-assisted breeding of F1 hybrids: it presents the genotyping-by-sequencing results of four elite inbred lines using a RADseq approach and an original molecular assay based on CAPS markers for screening mutants with nuclear male-sterility in radicchio of Chioggia. Two thousand nine hundred fifty-three SNP-carrying RADtags were identified and used to compute the actual estimates of homozygosity, and overall genetic similarity and uniformity of the populations, as well as to determine their genetic distinctiveness and differentiation. Molecular data were further used to investigate the genomic distribution of the RADtags among the two Cichorium species, allowing their mapping in 1,131 and 1,071 coding sequences in chicory and endive, respectively. In parallel, an assay to screen the genotype at the male-sterility locus Cims-1 was developed to discriminate wild-type and mutant alleles of the causative gene myb80-like. Moreover, a RADtag mapped close to this genomic region proved the potential application of this method for future marker-assisted selection tools. Finally, combining the genotype information of the core collection, the best ten individuals from each inbred line were selected to compute the observed genetic similarity as a measure of uniformity as well as the expected homozygosity and heterozygosity estimates scorable by the putative progenies derived from selfing (pollen parent) and full-sibling (seed parent) or pair-wise crossing (F1 hybrids). This predictive approach was conducted as a pilot study for understanding the potential application of RADseq in improving molecular breeding strategies aimed at the development of inbred lines and F1 hybrids in leaf chicory.

Escherichia coli is an indicator microorganism in One Health antibiotic resistance surveillance programmes. The purpose of the study was to describe and compare E. coli isolates obtained from pigs and human contacts from a commercial farm in South Africa using conventional methods and whole genome sequencing (WGS). Porcine E. coli isolates were proportionally more resistant phenotypically and harboured a richer diversity of antibiotic resistance genes, as compared to human E. coli isolates. Different pathovars, namely ExPEC (12.43%, 21/169), ETEC (4.14%; 7/169), EPEC (2.96%, 5/169), EAEC (2.96%, 5/169) and STEC (1.18%, 2/169) were detected at low frequencies. Sequence type complex (STc) 10 were the most prevalent (85.51%, 59/169) among human and porcine isolates. Six STcs (STc10, STc86, STc168, STc206, STc278 and STc469) were shared at the human-livestock interface according to multilocus sequence typing (MLST). Core genome MLST and hierarchical clustering (HC) showed that human and porcine isolates were overall genetically diverse, but some clustering at HC2 to HC200 were observed. In conclusion, even though the isolates shared a spatio-temporal relationship, there were still differences in the virulence potential, antibiotic resistance profiles, and cgMLST and HC according to the source of isolation.

The present study aimed to determine the genetic diversity of isolates of Mycobacterium tuberculosis (Mtb) from presumed drug resistant tuberculosis patients from several states of Brazil. The isolates had been submitted to conventional drug susceptibility testing for first- and second-line drugs. Multidrug-resistant (MDR-TB) (54.8%) was the most frequent phenotypic resistance profile, in addition to an important high frequency of pre-extensively resistant (p-XDR-TB) (9.2%). Using whole-genome sequencing (WGS), we characterized 298 Mtb isolates from Brazil. Besides analysis of genotype distribution and possible correlations between molecular and clinical data, we determined the performance of an in-house WGS pipeline with other online pipelines for Mtb lineages and drug resistance profile definition. Sub-lineage 4.3 (52%) was the most frequent genotype and the genomic approach revealed a p-XDR-TB level of 22.5%. We detected 20 novel mutations in three resistance genes, and six of these were observed in eight phenotypically resistant isolates. Cluster analysis of 170 isolates showed that 43.5% of the TB patients belonged to 24 genomic clusters, suggesting considerable ongoing transmission of DR-TB, including two interstate transmissions. The in-house WGS pipeline showed the best overall performance in drug-resistance prediction, presenting the best accuracy values to five of nine drugs tested. Significant associations were observed between suffering from fatal disease and genotypic p-XDR-TB (p = 0.03) and either phenotypic (p = 0.006) and genotypic (p = 0.0007) ethambutol resistance. The use of WGS analysis improved our understanding of the population structure of MTBC in Brazil, genetic and clinical data correlations and demonstrating its utility for surveillance efforts for spread of DR-TB, hopefully helping to avoid the emergence of even more resistant strains and to reduce TB incidence and mortality rates.

Cerebellar ataxia, neuronopathy, and vestibular areflexia syndrome (CANVAS) is caused by biallelic pathogenic expansions, or compound heterozygosity with other pathogenic variants in the RFC1 gene. CANVAS is estimated to be underdiagnosed, both because of the lack of formal diagnostic criteria and molecular challenges that translate to lesser access and high cost of routine testing. Our aim was to address the need for making CANVAS genetic testing routine, by designing a streamlined two-step PCR consisting of a short-allele screening PCR and a confirmatory PCR with fragment capillary electrophoresis detection. Exome sequencing of RFC1 was additionally foreseen to resolve potential compound heterozygosity cases. Specificity of our approach was evaluated using ataxia patients with known non-CANVAS diagnoses, and optimized using Southern blot confirmed CANVAS patients. We evaluated our approach by testing patients consecutively referred for clinically suspected CANVAS using first the two-step PCR, followed by exome sequencing. Our approach was able to accurately identify negative and confirm positive cases in prospectively collected suspected CANVAS patients presenting with at least three typical clinical signs. The proposed testing approach provides an alternative method able to clearly distinguish between CANVAS negative and positive cases and can be easily incorporated into the genetic diagnostic laboratory workflow.

The study of the currently known >3,000 species of snakes can provide valuable insights into the evolution of their genomes. Deinagkistrodon acutus, also known as Sharp-nosed Pit Viper, one hundred-pacer viper or five-pacer viper, is a venomous snake with significant economic, medicinal and scientific importance. Widely distributed in southeastern China and South-East Asia, D. acutus has been primarily studied for its venom. Here, we employed next-generation sequencing to assemble and annotate a highly continuous genome of D. acutus. The genome size is 1.46 Gb; its scaffold N50 length is 6.21 Mb, the repeat content is 42.81%, and 24,402 functional genes were annotated. This study helps to further understand and utilize D. acutus and its venom at the genetic level.

Zika virus (ZIKV) is a widespread mosquito-borne pathogen. Phylogenetically, two lineages of the ZIKV are distinguished: African and Asian-American. The latter became the cause of the 2015-2016 pandemic with severe defeat to newborns. In West African countries the African lineage has been found, but there is evidence of the emergence of Asian-American lineage in Cape Verde and Angola. This highlights the need not only to monitor the ZIKV, but also to sequence the isolates. In this article, we present a case report of Zika fever in a pregnant woman from Guinea, identified in 2018. Viral RNA was detected by qRT-PCR in serum sample. In addition, seroconversion of anti-Zika IgM and IgG antibodies was detected in repeated blood samples. Subsequently, the virus was isolated in C6/36 cell line. The detected ZIKV belonged to the African lineage, the Nigerian sublineage. The strains with the closest sequences were isolated from mosquitoes in Senegal in 2011 and 2015. In addition, we conducted serological screening of 116 blood samples collected from patients presenting to the hospital of Faranah with fevers during the period 2018-2021. As a result, it was found that IgM-positive patients occurred each year, seroprevalence varied between 5.6% and 17.1%.

Rapeseed (Brassica napus L.) is one of the most oil crop, and its commercial value is contingent upon its agronomic characteristics and oil quality. In this study, 73,226 single nucleotide polymorphisms (SNPs) across 95 rapeseed mutant lines derived from gamma rays and their original cultivar (‘Tamra’) obtained from genotyping-by-sequencing (GBS) was investigated gene ontology (GO) analysis and genome-wide association study (GWAS). GWAS was conducted on agronomic (plant height, ear length, thousand seed weight and seed yield) and oil (fatty acid and crude fat) traits. GO analysis showed that many genes displaying SNPs were involved in cellular processes, intracellular anatomical structures and organic cyclic compound binding. A total of 149 significant SNPs were associated with the agronomic traits (76 SNPs) and oil traits (73 SNPs). Bna.A05p02350D (SFGH) and Bna.C02p22490D (MDN1) were selected as novel candid genes for thousand seed weight. In addition, Bna.C03p14350D (EXO70) and Bna.A09p05630D (PI4Kα1) were selected as novel candidate genes for the erucic acid and crude fat content, respectively. These findings could facilitate the identification of optimal genotypes for breeding new cultivars, and association studies represent an economically efficient tool for mutant screening and the selection of elite rapeseed-breeding lines.

Blueberries (Vaccinium section Cyanococcus) are perennial shrubs widely cultivated for their edible fruits. In this study, we used admixture and genetic relatedness analysis of northern highbush (NHB, V. corymbosum) and southern highbush (SHB, V. darrowii) blueberry genotypes and F2 progenies of the V. corymbosum × V. darrowii cross. Using genotyping-by-sequencing (GBS), we generated ~3.34 billion reads (75 bp). The GBS reads were aligned to the Vaccinium corymbosum cv. Draper v1.0 reference genome sequence, and ~2.8 million reads were successfully mapped. From the alignments, we identified 2,244,039 single nucleotide polymorphisms (SNPs), which were used for principal component, haplotype, and admixture analysis. PCA formed three main groups: 1) NHB cultivars, 2) SHB cultivars, and 3) BNJ16-5 progenies. The overall fixation index (FST) and nucleotide diversity for NHB and SHB, indicated wide genetic differentiation, and haplotype analysis revealed that SHB cultivars are more genetically diverse than NHB cultivars. The admixture analysis identified a mix of various lineages of parental genomic introgression. This study demonstrated the effectiveness of GBS-derived SNP markers in genetic and admixture analyses to reveal genetic relatedness and to examine parental lineages in blueberry, which may be useful for future breeding plans.

The accurate virus detection, strain discrimination, and source attribution of contaminated food items remains a persistent challenge because of the high mutation rates anticipated to occur in foodborne RNA viruses, such as Hepatitis A virus (HAV). This has led to predictions of the existence of more than one sequence variant between the hosts (inter-host) or within an individual host (intra-host). However, there have been no reports of intra-host variants from an infected single individual, and little is known about the accuracy of the single nucleotide variations (SNVs) calling with various methods. In this study, the presence and identity of viral SNVs, either between HAV clinical specimens or among a series of samples derived from HAV clone1-infected FRhK4 cells, were determined following analyses of nucleotide sequences generated using next-generation sequencing (NGS) and pyrosequencing methods. The results demonstrate the co-existence of inter- and intra-host variants both in the clinical specimens and the cultured samples. The discovery and confirmation of multi-viral RNAs in an infected individual is dependent on the strain discrimination at the SNV level, and critical for successful outbreak traceback and source attribution investigations. The detection of SNVs in a time series of HAV infected FRhK4 cells improved our understanding on the mutation dynamics determined probably by different selective pressures. Additionally, it demonstrated that NGS could potentially provide a valuable investigative approach toward SNV detection and identification for other RNA viruses.

Among all economically important plant species in the world, grapevine (Vitis vinifera L.) is the most cultivated fruit plant. It has a significant impact on the economies of many countries through wine and fresh and dried fruit production. In recent years, the grape and wine industry has been facing outbreaks of known and emerging viral diseases across the world. Although high-throughput sequencing (HTS) has been used extensively in grapevine virology, the application and potential of third-generation sequencing have not been explored in understanding grapevine viruses and their impact on the grapevine. Nanopore sequencing, a third-generation technology, can be used for direct sequencing of both RNA and DNA with minimal infrastructure. Compared to other HTS methods, the MinION nanopore platform is faster and more cost-effective and allows for long-read sequencing. Due to the size of the MinION device, it can be easily carried for field viral disease surveillance. This review article discusses grapevine viruses and their diagnostic methods, the principle of nanopore sequencing technology and its application in grapevine virus detection, virus–plant interactions, as well as the characterization of viral RNA modifications.

Illumina and nanopore sequencing technologies are powerful tools that can be used to determine the bacterial composition of complex microbial communities. In this study, we compared nasal microbiota results at genus level using both Illumina and nanopore 16S rRNA gene sequencing. We also monitored the progression of nanopore sequencing in the accurate identification of species, using pure, single species cultures, and evaluated the performance of the nanopore EPI2ME 16S data analysis pipeline. Fifty-nine nasal swabs were sequenced using Illumina MiSeq and Oxford Nanopore 16S rRNA gene sequencing technologies. In addition, five pure cultures of relevant bacterial species were sequenced with the nanopore sequencing technology. The Illumina MiSeq sequence data were processed using bioinformatics modules present in the Mothur software package. Albacore and Guppy base calling, a workflow in nanopore EPI2ME and an in house developed bioinformatics script were used to analyze the nanopore data. At genus level, similar bacterial diversity profiles were found, and five main and established genera were identified by both platforms. However, probably due to mismatching of the nanopore sequence primers, the nanopore sequencing platform identified Corynebacterium in much lower abundance compared to Illumina sequencing. Further, when using default settings in the EPI2ME workflow, almost all sequence reads that seem to belong to the bacterial genus Dolosigranulum and a considerable part to the genus Haemophilus were only identified at family level. Nanopore sequencing of single species cultures demonstrated at least 88% accurate identification of the species at genus and species level for 4/5 strains tested, including improvements in accurate sequence read identification when the basecaller Guppy and Albacore, and when flowcell versions R9.4 and R9.2 were compared.

Mallard (Anas platyrhynchos) are currently one of the most popular species in rare bird breeding in several southern provinces of China, but there are very few studies on the gut microbial communities of domestic and wild mallards. In this study, 16S rRNA gene high-throughput sequencing technology was used to compare the composition and diversity of gut microbial communities in domestic and wild mallards. Alpha diversity analysis revealed significant differences in the gut microbial communities of domestic and wild mallards. Beta diversity analysis showed that the two groups of stool samples were mostly separated on the principal coordinate analysis (PCOA) plot. In domestic mallards, Firmicutes (67.97%) was the most abundant bacterial phylum, followed by Proteobacteria (24.48%), Bacteroidetes (3.13%), Fusobacteria (2.21%), and Actinobacteria (1.10%). The dominant bacterial phyla in wild mallards were Firmicutes (79.02%), Proteobacteria (12.85%), Fusobacteria (3.37%), and Bacteroidetes (2.79%). At the genus level, a total of 10 dominant genera (Streptococcus, Enterococcus, Clostridium, Lactobacillus, Soilbacillus, Bacillus, Acinetobacter, Comamonas, Shigella, and Cetobacterium) with an average relative abundance greater than 1% were detected in the fecal samples of both groups. The average relative abundance of five potential pathogens (Streptococcus, Enterococcus, Acinetobacter, Comamonas, and Shigella) was higher in domestic mallards than in wild mallards. The enrichment of pathogenic bacteria in the intestinal tract of domestic mallards should be of sufficient concern.

Mallard (Anas platyrhynchos) are currently one of the most popular species in rare bird breeding in several southern provinces of China, but there are very few studies on the gut microbial communities of domestic and wild mallards. In this study, 16S rRNA gene high-throughput sequencing technology was used to compare the composition and diversity of gut microbial communities in domestic and wild mallards. Alpha diversity analysis revealed significant differences in the gut microbial communities of domestic and wild mallards. Beta diversity analysis showed that the two groups of stool samples were mostly separated on the principal coordinate analysis (PCOA) plot. In domestic mallards, Firmicutes (67.97%) was the most abundant bacterial phylum, followed by Proteobacteria (24.48%), Bacteroidetes (3.13%), Fusobacteria (2.21%), and Actinobacteria (1.10%). The dominant bacterial phyla in wild mallards were Firmicutes (79.02%), Proteobacteria (12.85%), Fusobacteria (3.37%), and Bacteroidetes (2.79%). At the genus level, a total of 10 dominant genera (Streptococcus, Enterococcus, Clostridium, Lactobacillus, Soilbacillus, Bacillus, Acinetobacter, Comamonas, Shigella, and Cetobacterium) with an average relative abundance greater than 1% were detected in the fecal samples of both groups. The average relative abundance of five potential pathogens (Streptococcus, Enterococcus, Acinetobacter, Comamonas, and Shigella) was higher in domestic mallards than in wild mallards. The enrichment of pathogenic bacteria in the intestinal tract of domestic mallards should be of sufficient concern.

Keywords: Cystic fibrosis, Cystic Fibrosis Transmembrane Conductance Regulator gene, CFTR, Next Generation Sequencing, Cystic fibrosis carriers

SARS-CoV-2 is a novel betacoronavirus and the aetiological agent of the current COVID-19 outbreak that originated in Hubei Province, China. While polymerase chain reaction is the front-line tool for SARS-CoV-2 surveillance, application of amplification-free and culture-free methods for isolation of SARS-CoV-2 RNA, partnered with next-generation sequencing, would provide a useful tool for both surveillance and research of SARS-CoV-2. We here release into the public domain a set of bait capture hybridization probe sequences for enrichment of SARS-CoV-2 RNA from complex biological samples. These probe sequences have been designed using rigorous bioinformatics methods to provide sensitivity, accuracy, and minimal off-target hybridization. Probe design was based on existing, validated approaches for detecting antimicrobial resistance genes in complex samples and it is our hope that this SARS-CoV-2 bait capture platform, once validated by those with samples in hand, will be of aid in combating the current outbreak.

The rapid expansion of transcriptomics from increased affordability of next-generation sequencing (NGS) technologies generates rocketing amounts of gene expression data across biology and medicine, and notably in cancer research. Concomitantly, many bioinformatics tools were developed to streamline gene expression analysis and quantification. We tested the concordance of NGS RNA sequencing (RNA-seq) analysis outcomes between the two predominant programs for reads alignment, HISAT2 and STAR, and the two most popular programs for quantifying gene expression in NGS experiments, edgeR and DESeq2, using RNA-seq data from a series of breast cancer progression specimens, which include histologically confirmed normal, early neoplasia, ductal carcinoma in situ and infiltrating ductal carcinoma samples microdissected from formalin fixed, paraffin embedded (FFPE) breast tissue blocks. We identified significant differences in aligners’ performance: HISAT2 was prone to misalign reads to retrogene genomic loci, STAR generated more precise alignments, especially for early neoplasia samples. edgeR and DESeq2 produced similar lists of differentially expressed genes in stage comparisons, with edgeR producing more conservative, though shorter, lists of genes. Albeit, Gene Ontology (GO) enrichment analysis revealed no skewness in significant GO categories identified among differentially expressed genes by edgeR vs DESeq2. As transcriptome analysis of archived FFPE samples becomes a vanguard of precision medicine, identification and fine-tuning of bioinformatics tools becomes critical for clinical research. Our results indicate that STAR and edgeR are well-suited tools for differential gene expression analysis from FFPE samples.

The grain filling rate (GFR) plays a crucial role in determining grain yield. However, the regulatory and molecular mechanism of grain filling rate (GFR) in foxtail millet remains unclear. In this study, we found the GFR of 'Changnong No.47' (CN47) was significantly higher at 14 days after flowering (DAF) and 21 DAF in comparison to 'Changsheng 13' (CS13). Furthermore, CN47 also exhibited higher thousand grain weight and yield than CS13. Therefore, we conducted transcriptomics and metabolomics to analyze the biological processes and functional genes associated with GFR during two stages of grain filling in both cultivars. A total of 765 differentially expressed genes (DEGs) and 246 differentially accumulated metabolites (DAMs) were identified at the 14 DAF stage, while at the 21 DAF stage, a total of 908 DEGs and 268 DAMs were identified. The integrated analysis of co-mapped DAMs and DEGs revealed enriched pathways, including flavonoid biosynthesis, plant hormone signal transduction, tyrosine metabolism, ATP-binding cassette (ABC) transporters, beta-alanine metabolism, as well as stilbenoid, diarylheptanoid and gingerol biosynthesis. In order to elucidate their potential functions in the context of GFR, we developed a gene-metabolite regulatory network for these metabolic pathways. Notably we found some genes associated with ABC transporters and plant hormone signal transduction pathway were implicated in auxin transport and signal transduction, highlighting the crucial role of auxin during grain filling. The results provide initial insights into the regulatory and molecular mechanisms underlying GFR in foxtail millet, as well as offer valuable genetic resources for further elucidation of GFR in future studies. The findings have established a theoretical basis for improving the efficiency of yield breeding in foxtail millet.

Trio-based exome sequencing and high-resolution HLA typing were employed to analyze three patients with autoimmune adrenal insufficiency (AAI), including those with autoimmune polyglandular syndrome (APS) type 2, as well as their parents. Benign or likely benign variants of the AIRE gene were identified in all participants of the study. These variants, coupled with clinical data and the results of antibody studies to type I interferons, helped to exclude APS-1. Patients with APS-2, in contrast to patient with AAI, inherited distinct variants of unknown significance in the CLEC16A gene, which is associated with autoimmune diseases including AAI. Various risk alleles in other genes associated with autoimmunity were identified in all patients. HLA typing of second class loci revealed alleles related to APS types 2, 3, and 4. Nevertheless, the frequencies of the haplotypes identified are substantial in the healthy Russian population, precluding from regarding these alleles as genetic determinants linked to APS development. Immunological examination can detect antibody carriers and predict the risk of autoimmune disease development. In the future, to identify genetic predictors of autoimmune endocrinopathies, it is recommended to analyze the whole genome of patients and their relatives, examining clinically relevant variants in non-coding regions.

Canine lymphoma (CL) is one of the most common malignant tumors in dogs. The cause of CL remains unclear. Genetic mutations that have been suggested as possible causes of CL are not fully understood. Whole-exome sequencing (WES) is a time- and cost-effective method for detecting genetic variants targeting only protein-coding regions (exons) that are part of the entire genome region. A total of eight patients with B-cell lymphomas were recruited, and WES analysis was performed on whole blood and lymph node aspirate samples from each patient. A total of 17 somatic variants (GOLIM4, ITM2B, STN1, UNC79, PLEKHG4, BRF1, ENSCAFG00845007156, SEMA6B, DSC1, TNFAIP1, MYLK3, WAPL, ADORA2B, LOXHD1, GP6, AZIN1, and NCSTN) with moderate to high impact were identified by WES analysis. Through a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of 17 genes with somatic mutations, a total of 16 pathways were identified. Overall, the somatic mutations identified in this study suggest novel candidate mutations for CL, and further studies are needed to confirm the role of these mutations.

Alfalfa primarily produces seeds through cross-pollination among different individuals. Self-pollination results in severe inbreeding depression, such as weak seedlings and termination of growth. Identifying the genetic loci associated with vigorous plant growth could enable the elimination of deleterious alleles and eventually develop inbred alfalfa lines for hybrid production. In this study, 215 alfalfa accessions were self-pollinated for three generations. Within accessions, pairs of weak and strong plants were sampled and bulked for exome sequencing. We extracted individual DNA from 534 plants that included parental clones, strong and weak pairs of plants, and plants selected based on the number of seeds produced. Among them, we formed 42 pools, including 16 with strong plants and 17 with weak plants, 3 top-seeded plants, 3 low-seeded plants, and 3 no-seeded plants. Along with 79 individuals, these pools were sequenced in the target regions covering 112,626 contigs across the entire alfalfa genome. From the 121 samples, (42+79) genotyped, 13.2 million SNPs including indels were generated. After filtering for MAF (>5%), depth (<20X), and no missing genotype and removing indels, a total of 588,136 SNPs were obtained which were used for final analysis. A genome-wide association study was performed and identified 11 genetic loci associated with alfalfa plant growth vigor. The GO analysis generated 12 significantly enriched GO terms. The associated SNPs were on, and near genes involved in stress response, defense responses against pathogens, and plant reproduction. These identified SNPs benefit the development of alfalfa inbred lines by purging the deleterious alleles and biomass improvement through marker-assisted selection.

Precision medicine seeks to use genomic data to help provide the right treatment to the right patient at the right time. Next-generation sequencing technology allows for the rapid and accurate sequencing of many genes at once. This technology is becoming more common in oncology, though the clinical benefit of incorporating it into precision medicine strategies remains under significant debate. In this manuscript, we discuss the early findings of the impact of next-generation sequencing on cancer patient outcomes. We investigate why not all patients with genomic variants linked to a specific therapy receive that therapy and describe current barriers. Finally, we explore the current state of health insurance coverage for individual genome sequencing and targeted therapies for cancer. Based on our analysis, we recommend increased transparency around the determination of “actionable mutations” and a heightened focus on investigating the variations in health insurance coverage across patients receiving sequencing-matched therapies.

Methicillin-resistant Staphylococcus aureus (MRSA) constitutes a serious public health concern with a considerable impact on patients' health and substantial healthcare costs. In this study, patients, and healthcare workers (HCWs)from six public hospitals in Benin were screened for MRSA. Strains were identified as MRSA using conventional microbiological methods in Benin and confirmed using Matrix-Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometry in Belgium. Whole genome sequencing (WGS) was used on confirmed MRSA isolates to characterize their genomic content and study their relatedness. In total,304 and 61 samples were collected from patients and HCWs, respectively. The mecA gene was detected in all isolates. The isolates were assigned to five sequence types (STs), with ST8 (55.6%, n=15), ST152 (18.52%, n=5), and ST121 (18.52%, n=5) being the most common. All isolates carried multiple virulence genes, including genes encoding the Panton-Valentine leukocidin toxin (48.15%, n=13) and the test gene (29.63%, n=8) associated with toxic shock syndrome. The presence of strains carrying numerous genomic features associated with antibiotic resistance and virulence in HCWs is a major public health concern. This study highlights the need to implement a multimodal strategy for reducing the risk of cross-transmission of MRSA in hospitals.

Evidence of familial inheritance in non-medullary thyroid cancer (NMTC) has accumulated over the last few decades. However, known variants account for a very small percentage of the genetic burden. Here, we focused on the identification of common pathways and networks enriched in NMTC families to better understand its pathogenesis with the final aim of identifying one novel high/moderate-penetrance germline predisposition variant segregating with the disease in each studied family. We performed whole genome sequencing on 23 affected and 3 unaffected family members from five NMTC-prone families and prioritized the identified variants using our Familial Cancer Variant Prioritization Pipeline (FCVPPv2). In total, 31 coding variants and 39 variants located in upstream, downstream, 5′ or 3′ untranslated regions passed FCVPPv2 filtering. Altogether, 210 genes affected by variants that passed the first three steps of the FCVPPv2 were analyzed using Ingenuity Pathway Analysis software. These genes were enriched in tumorigenic signaling pathways mediated by receptor tyrosine kinases and G-protein coupled receptors, implicating a central role of PI3K/AKT and MAPK/ERK signaling in familial NMTC. Our approach can facilitate the identification and functional validation of causal variants in each family as well as the screening and genetic counseling of other individuals at risk of developing NMTC.

Ostreococcus tauri is a simple unicellular green alga representing an ecologically important group of phytoplankton in oceans worldwide. Modern molecular techniques must be developed in order to understand the mechanisms that permit adaptation of microalgae to their environment. We present for the first time in O. tauri a detailed characterization of individual genomic integration events of foreign DNA of plasmid origin after PEG-mediated transformation. Vector integration appears to be random, occurring mainly at a single locus, and thus confirming the utility of this technique for insertional mutagenesis. While the mechanism of double-stranded DNA repair in the O. tauri model remains to be elucidated, we clearly demonstrate by genome resequencing that the integration of the vector leads to frequent structural variations (deletions/insertions and duplications) and some chromosomal rearrangements in the genome at the insertion loci, and often within the vector sequence itself. From these observations, we speculate that a non-homologous end joining-like mechanism is required during random insertion events, as described in plants and other freshwater algal models. PEG-mediated transformation is therefore a promising molecular biology tool, not only for functional genomic studies, but also for biotechnological research in ecologically important marine algae.

Distinctive strains of Pantoea are used as soil inoculants for their ability to promote plant growth. Pantoea agglomerans strain C1, previously isolated from the phyllosphere of lettuce, can produce indole-3-acetic acid (IAA), solubilize phosphate, and inhibit plant pathogens, such as Erwinia amylovora. In this paper, the complete genome sequence of strain C1 is reported. In addition, experimental evidence is provided on how the strain tolerates arseniate up to 100 mM, and on how secreted metabolites like IAA and siderophores act as biostimulants in tomato cuttings. The strain has a circular chromosome and two prophages for a total genome of 4,846,925-bp, with a GC content of 55.2%. Genes related to plant growth promotion and biocontrol activity, such as those associated with IAA and spermidine synthesis, solubilization of inorganic phosphate, acquisition of ferrous iron, and production of volatile organic compounds, siderophores and GABA, were found in the genome of strain C1. Genome analysis also provided better understanding of the mechanisms underlying strain resistance to multiple toxic heavy metals and transmission of these genes by horizontal gene transfer. Findings suggested that strain C1 exhibits high biotechnological potential as plant growth-promoting bacterium in heavy metal polluted soils.

The efficacy and cost-effectivness of Multigene Panel Next-Generation Sequencing (NGS) in directing patients towards genomically matched therapies remains uncertain. This study investigated metastatic colorectal cancer (mCRC) patients who underwent NGS analysis on formalin-fixed paraffin-embedded tumor samples. Data from 179 patients were analyzed, revealing no mutations in 39 patients (21.8%), one mutation in 83 patients (46.4%), and two or more mutations in 57 patients (31.8%). KRAS mutations were found in 87 patients (48.6%), including KRAS G12C mutations in 5 patients (2.8%), PIK3CA mutations in 40 patients (22.4%), BRAF mutations in 26 patients (14.5%). Less common mutations were identified: ERBB2 in 5 patients (2.8%) and SMO in 4 patients (2.2%). Additionally, MAP2K1, CTNNB1, and MYC were mutated in 3 patients (2.4%). Two mutations (1.1%) were observed in ERBB3, RAF1, MTOR, JAK1, and FGFR2. No significant survival differences were observed based on mutation numbers. 40% of patients had druggable molecular alterations, but only 1.1% received genomically guided treatment, suggesting limited application in standard practice. Despite this, expanded gene panel testing can identify actionable mutations, aiding personalized treatment strategies in metastatic CRC, although current eligibility for biomarker-guided trials remains limited.

African swine fever virus (ASFV) is the causative agent of African swine fever, an economically important disease of pigs, often with a high case fatality rate. ASFV has demonstrated low genetic diversity among isolates collected within Eurasia. To explore the influence of viral variants on clinical outcomes and infection-dynamics in pigs experimentally infected with ASFV, we have designed a deep sequencing strategy. Variant analysis revealed 3 distinct SNPs present in the virus inoculum (at 2.4%, 0.7%, and 13% frequency, respectively) that were maintained within all infected pigs (1-6 % frequency, and the latter at 16-21%). Several pigs displayed other unique SNPs at &lt;10% frequency. In addition, a deletion of 10487 bp (resulting in the complete loss of 21 genes) was present at nearly 100% frequency in the ASFV DNA from one pig at position 6362-16849. This deletion was also found to be present at low levels in the virus inoculum and in two other infected pigs. The current methodology can be used for the currently circulating Eurasian ASFVs and also adapted to other ASFV strains and genotypes. Comprehensive deep sequencing is critical for following ASFV molecular evolution, especially for identification of modifications that affect virus virulence.

Copepods are the most abundant metazoans on Earth, exhibit remarkable species diversity and global distribution. Accurate species identification of copepods presents a challenge due to their cryptic nature, necessitating meticulous examination and differentiation. To address this challenge, innovative approaches utilizing molecular markers have emerged, enabling more reliable and precise species identification. Mitochondrial genes, which are characterized by high variability, have proven invaluable in uncovering taxonomically significant variations among copepod species. In this study, we present the first complete mitochondrial genome analysis of the marine calanoid copepod Bestiolina similis (Sewell, 1914). Recent investigations employing whole mitochondrial genome-based phylogenetic analyses have generated considerable interest in exploring the deep branching patterns of arthropods. The complete mitochondrial genome of B. similis spans 23,704 base pairs and includes 13 protein-coding genes and 20 transfer RNA genes. The nucleotide composition of the mitochondrial genome is characterized by 43.42% adenine, 43.22% thymine, 6.48% guanine, and 6.88% cytosine. Maximum likelihood-based phylogenetic analysis of the mitochondrial genome were constructed of the available copepod species. Mitochondrial ge-nome-based phylogenies often reveal intriguing and distinctive topologies compared to traditional phylogenetic approaches. However, the sequencing of copepod mitochondrial genomes, particularly within the calanoid copepods remains limited. Therefore, this study aims to construct a phylogenetic tree based on the extracted mitochondrial genome, incorporating available data, to enhance our understanding of the evolutionary relationships among calanoid copepods.

Transposable elements (TEs) are mobile genetic elements capable of rapidly altering the genome through their movements. The importance of TE activity has been documented in many biological processes, such as introducing genetic instability, altering patterns of gene expression, and accelerating genome evolution. Increasing appreciation of TEs results in the growing number of bioinformatics software to identify insertion events. However, the application of existing TE finding tools is limited by either narrow-focused design of the package, too many dependencies on other tools, or prior knowledge required as input files that may not be readily available to all users. Here, we report a simple pipeline, TEfinder, developed for the detection of new TE insertions with minimal software dependencies using four inputs that can be easily generated with popular variant calling pipelines. The external software requirements are BEDTools, SAMtools, and Picard. Necessary inputs include TEs present in the reference genome, binary paired-end alignment, reference genome index, and a list of TE names. We tested TEfinder pipeline among several evolving populations of Fusarium oxysporum generated through a short-term adaptation study. Our results demonstrate that this easy-to-use tool can effectively detect new TE insertion events, making it accessible and practical for TE analysis.

Bread wheat (Triticum aestivum L.) is one of the most important cereal crops for food security. Of all the stresses that curtail wheat productivity, drought has the most detrimental effects. Especially terminal drought stress i.e. at the time of flowering imposes a big challenge to sustain grain production. In the current study, 339 pre-breeding lines derived from three-way crosses of exotics x elite lines were evaluated in the irrigated and drought stress environments at Obregon, Mexico for the year 2016 and 2018. Drought significantly reduced yield (Y), spike length (SL), number of grains per spikes (NGS) and thousand kernel weight (TKW) by 46.4, 19.2, 23.5 and 25.9%, respectively in comparison to irrigated conditions. Kernel abortion (KA), highly correlated with Y, increased significantly (11.6%) under drought stress environment. Population structure analysis in this panel revealed three sub-populations and a genome wide linkage disequilibrium (LD) decay was at 2.5 cM. Single marker and haplotypes-based genome wide association study (GWAS) revealed significant associations on three chromosomes; 4A (HB10.7), 2D (HB6.10) and 3B (HB8.12) with Y, SL and TKW, respectively. Likewise, associations on chromosomes 6B (HB17.1) and 3A (HB7.11) were identified for NGS and on 3A (HB7.12) for KA. Five traits i.e. normalized difference vegetation index (NDVI), canopy temperature depression (CTD) days to heading (DTH), NGS, KA were associated at chromosome 3A both under irrigated and drought conditions however, different haplotypes were estimated. Twenty-six SNPs were part of 10 haplotype blocks associated with Y, SL, TKW, NGS and KA. In silico analysis of the associated SNPs/haplotypes showed hits with candidate genes known to confer abiotic stress resistance in model species and crops. Potential candidate genes include those coding for sulfite exporter TauE/SafE family in Arabidopsis thaliana, TBC domain containing protein in Oryza sativa subsp. Japonica and heat shock proteins in Aegilops tauschii subsp. tauschii were revealed. The SNPs linked to the promising genes identified in the study can be used for marker-assisted selection.

The human gut microbiome (GM) plays an important role in human health and diseases. However, while substantial progress has been made in understanding the role of bacterial inhabitants of the gut, much less is known regarding the viral component of the GM. Bacteriophages (phages) are viruses attacking specific host bacteria and likely play important roles in shaping the GM. Although metagenomic approaches have led to the discoveries of many new viruses, they largely remain uncultured as their hosts have not been identified, which hampers our understanding of their biological roles. Existing protocols for isolation of viromes generally require relatively high input volumes and are generally more focused on extracting nucleic acids of good quality and purity for down-stream analysis and less on purification of still infective viruses. Here we report the development of an efficient protocol requiring low sample input yielding purified viromes containing still infective phages which also are of sufficient purity for genome sequencing. We validated the method through spiking of known phages followed by plaque assays, qPCR and metagenomic sequencing. The protocol should facilitate the culturing of novel viruses from the gut as well as large scale studies on gut viromes.

We present a case of Capnocytophaga canimorsus septic shock after a dog bite in an immunocompetent individual, where real-time nanopore metagenomic sequencing characterized the microbial agent within 19 hours, with subsequent confirmation using droplet digital PCR. Oral swabs from the dog demonstrated a nearly-identical C. canimorsus isolate by sequencing.

The phenolics concentrations of rapeseed are important because they are antinutritional com-pound or directly positive related to antioxidant actives. In this study, single nucleotide poly-morphisms (SNPs) identified from genotyping-by-sequencing (GBS), and genome-wide associa-tion study of phenolic contents were investigated in 95 rapeseed mutant lines derived from gamma rays and their original cultivar. A total of 3,196,318 SNPs were detected, identifying a set of 70,208 union SNPs used to perform association studies. Six phenolics and glucosinolate (pro-goitrin) were identified, sinapine was the major phenolic compound (91.2–99.2%) in all geno-types. The association study was conducted for six phenolic compounds, progoitrin, total sinap-ine content (TSC), total phenolic content (TPC) and seed yield. The association study identified 241 SNPs that were significantly associated with six phenolic compounds, progoitrin, TSC and TPC. Based on the detected SNP markers, BnaA06g31740D, a gene associated progoitrin, BnaC-nng39930D, a gene associated trans-sinapine 1, BnaA07g31720D, a gene associated trans-sinapine 2, BnaC03g31950D and BnaA06g09180D, a gene associated TSC, and BnaAnng27700D, a gene associ-ated TPC, were selected as candidate genes. The SNP makers associated major phenolic com-pounds identified and could be used as targets for breeding programs aimed at improving anti-oxidant contents and reducing antinutritional components.

[Objective] To investigate the expression of ALDH2 in hepatocellular carcinoma and its correlation with macrophage infiltration and with the activation of specific subtypes of macrophages. [Methods] Transcriptome and clinical data from patients with hepatocellular carcinoma were downloaded from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) databases and analyzed and collated using R and Perl software. In the TCGA hepatocellular carcinoma dataset, the Wilcoxon test was used to identify macrophage activation-related gene sets that show differential expression between hepatocellular carcinoma and normal tissues. Single-cell sequencing combined with bulk data was used to analyze macrophage infiltration of hepatocellular carcinoma immune cells and the activation and recruitment of different subtypes of macrophages. Single-factor Cox regression and minimum absolute convergence and selection operator (Lasso) were used to identify survival-related genes and construct prognostic models of HCC. The patients were divided into two groups based on their model scores. The Wilcoxon test was used to identify differentially expressed genes (DEGs) between the two groups, and gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses of the DEGs were performed. Single-sample gene set enrichment analysis (ssGSEA) was performed to explore the possible mechanism underlying the differences in prognosis. The reliability of the model was verified using the ICGC hepatocellular carcinoma dataset. A Kaplan‒Meier curve was constructed and used to analyze the influence of macrophage activation-related gene expression on survival and prognosis in patients with hepatocellular carcinoma. The log-rank test and the independent t test were used to analyze survival time data and to compare immune cell infiltration in the two groups. [Results] In a comparison of hepatocellular carcinoma and paracancerous tissues, the expression of six of 137 macrophage activation-related genes (ALDH2, CCNB2, CYP2C9, CYP3A4, F9 and KLKB1) was found to be significantly correlated with overall survival time (OS) in patients with hepatocellular carcinoma. Based on this finding, a prognosis assessment model for hepatocellular carcinoma was constructed. The OS of the patients in the high-scoring group was significantly shorter than the OS of the patients in the low-scoring group (P<0.05), and the model predicted the prognosis of patients with hepatocellular carcinoma independently of sex, age, tumor grade and stage. The area under the receiver operating characteristic (ROC) curve of the model used to predict 1-, 2-, and 3-year survival was greater than 0.7 in both the test set and the validation set. GO enrichment of DEGs between the high- and low-scoring groups of the model suggested that the DEGs are mainly related to immune function. GSEA suggested that the tumor-infiltrating immune cells in the high-scoring group were macrophages, Th2 cells and Treg cells and showed that the number of tumor-infiltrating immune cells, including NK cells, was lower in the high-scoring group than in the low-scoring group. With respect to immune function, the immune checkpoint of the high-scoring group was higher than that of the low-scoring group. Single-cell sequencing data indicated the recruitment of B cells, M0 cells, M1 cells and M2 cells to the hepatocellular carcinoma microenvironment and suggested that ALDH2 regulates related signaling pathways in a way that promotes the differentiation of macrophages into M2-type macrophages. [Conclusion] M2-type macrophages are the dominant type of macrophages associated with immune infiltration in hepatocellular carcinoma. The macrophage-associated activation gene ALDH2 activates the macrophage polarization signaling pathway and negatively regulates the differentiation of macrophages into M2-type macrophages.

The genetic aetiology of familial glioma is largely unknown. To gain further insight into the role of rare disruptive variants we performed whole exome sequencing (WES) of 17 glioma families, identifying two families where loss of function mutations in dynein axonemal heavy chain 11 (DNAH11) co-segregated with glioma (DNAH11 p.Ser1470ArgfsTer6 and p.Thr3900Lys) and single families implicating Telomere Replication Complex Component 1 (CTC1 p.Leu1058ProfsTer32) and filamin alpha (FLNA p.Ser1356Phe). We complemented linkage analysis by WES of 150 additional unrelated familial glioma cases and conducting gene burden tests leveraging WES data on 691 cases of glioma from TCGA. Our analysis does not provide support for a hitherto unidentified major cancer gene for glioma but highlights a series of novel candidate predisposition variants/genes, worthy of further investigation to shed light on glioma risk.

Forest health is dependent on the variability of microorganisms interacting with the host tree/holobiont. Symbiotic microbiota and pathogens engage in a permanent interplay, which influences the host. Thanks to the development of NGS technologies, a vast amount of genetic information on the virosphere of temperate forests has been gained the last seven years. To estimate the qualitative/quantitative impact of NGS in forest virology, we have summarized viruses affecting major tree/shrub species and their fungal associates, including fungal plant pathogens, mutualists and saprotrophs. The contribution of NGS methods is extremely significant for forest virology. Reviewed data about viral presence in holobionts, allowed us to address the role of the virome in the holobionts. Genetic variation is a crucial aspect in hologenome, significantly reinforced by horizontal gene transfer among all interacting actors. Through virus-virus interplays synergistic or antagonistic relations may evolve, which may drastically affect the health of the holobiont. Novel insights of these interplays may allow practical applications for forest plant protection based on endophytes and mycovirus biocontrol agents. The current analysis is conceived in light of the prospect that novel viruses may initiate an emergent infectious disease and that measures for avoidance of future outbreaks in forests should be considered.

Molecular identification via Internal Transcribed Spacer region (nrDNA-ITS) sequencing of Fusarium spp. isolates from wheat originated from Stara Zagora region, were performed for the first time in Bulgaria. А total of 60 wheat samples (Triticum aestivum) were morphologically identified at the genus level as Fusarium spp. in advance. The rDNA-ITS region of all isolates was successfully amplified and the PCR products obtained were directly sequenced. After a comparison of detected sequences with NCBI database, members of three different fungal genera (Fusarium, Chaetomium, and Alternaria) were identified. Among Fusarium isolates, the F. tricinctum was prevailing, followed by F. poae. A total of three isolate F. proliferatum, F. graminearum and F. equiseti were presented with a single probe. The lowest genetic distance (0.004) was detected between F. tricinctum isolates. On the base of genetic distances, fungal isolates were grouped in two main clusters – one comprising F. tricinctum isolates and F. proliferatum, and second including F. equiseti, F. graminearum and F. poae. It could be concluded that the rDNA-ITS genome region of the genus Fusarium may be used as a suitable marker of early detection, accurate and reliable identification of Fusarium spp. contamination of wheat. The timely and accurate information would assist in the selection of appropriate approaches for control of fusarium infections and possible mycotoxins contamination of agricultural production.

Autism spectrum disorder (ASD) is a common condition with lifelong implications and a strong hereditary component suggesting genetic underpinnings. The last decade has seen dramatic improvements in DNA sequencing and related bioinformatics and databases. We analyzed the raw DNA sequencing files on the Variantyx® bioinformatics platform for the last 50 ASD patients evaluated with trio whole genome sequencing (trio-WGS). “Qualified” variants were defined as coding, rare, and evolutionarily conserved. Primary Diagnostic Variants (PDV) additionally were in genes directly linked to ASD and matched clinical correlation. A PDV was identified in 34/50 (68%) of cases, including 25 (50%) cases with heterozygous de novo and 10 (20%) with inherited variants. De novo variants in genes directly associated with ASD were far more likely to be Qualifying than non-Qualifying versus a control group of genes not associated with ASD (P = 0.0002, odds ratio 29), validating that most are indeed disease related. Only 14/34 (41%) of PDV cases had the variant listed on the laboratory report, and reanalysis increased diagnostic yield from 28% to 68%. Variants that we assigned as PDVs yet not on the report were predominately de novo in genes not yet reported as ASD associated. Many subjects both with and without a PDV had inherited Qualifying variants in known ASD-associated genes, suggesting polygenic inheritance. Thirty-three subjects (66%) had treatment recommendation(s) based on DNA analyses. Our results demonstrate high yield of trio-WGS for revealing molecular diagnoses in ASD that is greatly enhanced by re-analyzing DNA sequencing files. In contrast to previous reports, de novo variants dominate the findings, mostly representing novel conditions. This has implications to the cause and rising prevalence of autism.