Genotyping by sequencing; a strategy for identification and mapping of induced mutation in newly developed wheat mutant lines.

Exposing genetic material with physical mutagens can create novel genetic resources capable of combating different stresses. High throughput GBS-DArTseq™ assay was deployed to estimate genetic diversity of 33 newly developed stable wheat mutants as compared to the wild type. The identified 1,57,608 PAVs markers were randomly distributed across wheat chromosomes and sub-genomes with the highest number detected on Chr-7D (2877) and Chr-7B (2711). The B sub-genome contained the most PAVs followed by D and A-sub genome. Among mutant lines, Pb-M-2061 and Pb-M-59 had the highest PAV count, while Pb-M-605 and Pb-M-196 had the lowest. A total of 7,910 PAVs were consistently present over all replicates, with 3,252 specifically present in mutants and absent in wild type. The maximum PAVs (1480) were found in Pb-M-1027 and Pb-M-1323 (656). Functional characterization revealed that out of 3,252, 1,238 were found in wheat transcriptome database that contained 152 characterized and 1,196 uncharacterized genes. COGs and GO-terms analysis linked many PAVs with pathways involving signaling, metabolism and defense. Maximum number of gene-containing PAVs were identified in Pb-M-1027, Pb-M-2302 and Pb-M-1323 which were involved in tolerance to diseases and abiotic stresses, improved photosynthetic efficiency, larger grain size, increased grain yield and harvest index pathways. This study provides valuable insights into the genetic diversity and potential agronomic benefits of PAVs in wheat mutant lines. These findings can help molecular geneticist and breeders for exploiting the induced genetic diversity for unravelling the genetic circuits as well as exploiting in wheat breeding for developing resilient cultivars.

Reprogramming Chromosome Ends by Functional Histone Acetylation.

Cancers harness embryonic programs to evade aging and promote survival. Normally, sequences at chromosome ends called telomeres shorten with cell division, serving as a countdown clock to limit cell replication. Therefore, a crucial aspect of cancerous transformation is avoiding replicative aging by activation of telomere repair programs. Mouse embryonic stem cells (mESCs) activate a transient expression of the gene Zscan4, which correlates with chromatin de-condensation and telomere extension. Head and neck squamous cell carcinoma (HNSCC) cancers reactivate ZSCAN4, which in turn regulates the phenotype of cancer stem cells (CSCs). Our study reveals a new role for human ZSCAN4 in facilitating functional histone H3 acetylation at telomere chromatin. Next-generation sequencing indicates ZSCAN4 enrichment at telomere chromatin. These changes correlate with ZSCAN4-induced histone H3 acetylation and telomere elongation, while CRISPR/Cas9 knockout of ZSCAN4 leads to reduced H3 acetylation and telomere shortening. Our study elucidates the intricate involvement of ZSCAN4 and its significant contribution to telomere chromatin remodeling. These findings suggest that ZSCAN4 induction serves as a novel link between 'stemness' and telomere maintenance. Targeting ZSCAN4 may offer new therapeutic approaches to effectively limit or enhance the replicative lifespan of stem cells and cancer cells.

Isolation and characterization of emerging Mpox virus from India.

Mpox (previously known as Monkeypox) has recently re-emerged, primarily through human-to-human transmission in non-endemic countries including India. Virus isolation is still considered as the gold standard for diagnosis of viral infections. Here, the qPCR positive skin lesion sample from a patient was inoculated in Vero E6 cell monolayer. Characteristic cytopathic effect exhibiting typical cell rounding and detachment was observed at passage-02. The virus isolation was confirmed by qPCR. The replication kinetics of the isolate was determined that revealed maximum viral titre of log 6.3 PFU/mL at 72 h postinfection. Further, whole genome analysis through next generation sequencing revealed that the Mpox virus (MPXV) isolate is characterized by several unique SNPs and INDELs. Phylogenetically, it belonged to A.2 lineage of clade IIb, forming a close group with all other Indian MPXV along with few from USA, UK, Portugal, Thailand and Nigeria. This study reports the first successful isolation and phenotypic and genotypic characterization of MPXV from India.

Identification of a novel mutation in thyroxine-binding globulin (TBG) gene associated with TBG-deficiency and its effect on the thyroid function.

PURPOSE: This study presents a case of familial transmission of thyroxine-binding globulin (TBG) deficiency. The SERPINA7-gene which codes for TBG is located on the X-chromosome (Xq21-22). More than 45 mutations have been reported to cause TBG- deficiency from various countries, but none from India so far. Genetic analysis of SERPINA7 gene was carried out to determine the cause of low TBG levels in one family. METHODS: DNA samples of the propositus and the family members were subjected to Polymerase Chain Reaction (PCR) followed by direct sequencing. Allele-specific PCR and Next-gen sequencing (NGS) were employed to confirm the site of the mutation. Thyroid function tests were estimated by Radioimmunoassay (RIA) and Immunoradiometric assay (IRMA) kits. X-chromosomal inactivation status was analyzed in the female members harboring the mutation. RESULTS: A mutational screening in this family revealed a novel frame-shift mutation S353Q, 354fs3X in the exon 4 of the SERPINA7 gene which will be referred to as TBG-complete deficiency-India (TBG-CD-Ind). One out of four female family members harboring the mutation showed selective X-chromosomal inactivation. The affected family members were clinically euthyroid initially, showed changes in the thyroid function when tested after a long time span. However, the changes in the thyroid function in the affected family members had an autoimmune etiology. CONCLUSION: This study presents the first report of TBG-CD from India wherein a novel frameshift mutation referred to as TBG-CD-Ind (S353Q, 354fs3X) in the SERPINA7 gene was detected. No apparent association was identified between thyroid function and the TBG-mutation in the affected subjects. A detailed biochemical and genomic testing to determine the exact cause of discordant TFT in the patients would certainly aid in the unequivocal diagnosis of the thyroid function and for the precise individualized treatment.

A computational framework for DNA sequencing microscopy.

We describe a method whereby microscale spatial information such as the relative positions of biomolecules on a surface can be transferred to a sequence-based format and reconstructed into images without conventional optics. Barcoded DNA "polymerase colony" (polony) amplification techniques enable one to distinguish specific locations of a surface by their sequence. Image formation is based on pairwise fusion of uniquely tagged and spatially adjacent polonies. The network of polonies connected by shared borders forms a graph whose topology can be reconstructed from pairs of barcodes fused during a polony cross-linking phase, the sequences of which are determined by recovery from the surface and next-generation (next-gen) sequencing. We developed a mathematical and computational framework for this principle called polony adjacency reconstruction for spatial inference and topology and show that Euclidean spatial data may be stored and transmitted in the form of graph topology. Images are formed by transferring molecular information from a surface of interest, which we demonstrated in silico by reconstructing images formed from stochastic transfer of hypothetical molecular markers. The theory developed here could serve as a basis for an automated, multiplexable, and potentially superresolution imaging method based purely on molecular information.

ContigExtender: a new approach to improving de novo sequence assembly for viral metagenomics data.

BACKGROUND: Metagenomics is the study of microbial genomes for pathogen detection and discovery in human clinical, animal, and environmental samples via Next-Generation Sequencing (NGS). Metagenome de novo sequence assembly is a crucial analytical step in which longer contigs, ideally whole chromosomes/genomes, are formed from shorter NGS reads. However, the contigs generated from the de novo assembly are often very fragmented and rarely longer than a few kilo base pairs (kb). Therefore, a time-consuming extension process is routinely performed on the de novo assembled contigs. RESULTS: To facilitate this process, we propose a new tool for metagenome contig extension after de novo assembly. ContigExtender employs a novel recursive extending strategy that explores multiple extending paths to achieve highly accurate longer contigs. We demonstrate that ContigExtender outperforms existing tools in synthetic, animal, and human metagenomics datasets. CONCLUSIONS: A novel software tool ContigExtender has been developed to assist and enhance the performance of metagenome de novo assembly. ContigExtender effectively extends contigs from a variety of sources and can be incorporated in most viral metagenomics analysis pipelines for a wide variety of applications, including pathogen detection and viral discovery.

MicroRNA expression in the heart of Xenopus laevis facilitates metabolic adaptation to dehydration.

Xenopus laevis survive severe dehydration during the summer months in their natural range. MicroRNA regulate translation of target mRNAs and have shown to be differentially expressed in response to dehydration in X. laevis. During dehydration, heart rate is elevated which appears to compensate for the reduced oxygen delivery capability due to increased hematocrit. We hypothesized that microRNAs would be differentially expressed in the heart to modulate gene expression levels in response to dehydration. The present study assessed changes in the microRNAome of X. laevis heart in response to severe dehydration (30% loss of body water) using microRNA-seq. We show that target genes are enriched for RNA, DNA, and transcription factor binding activities, cardiac muscle contraction, and glycolysis/gluconeogenesis. These results suggest that microRNAs contribute to gene expression reorganization in the heart in response to dehydration, putatively supporting the increased physiological demands and ATP production needs by the heart under these conditions.

Adaptive laboratory evolution induced novel mutations in Zymomonas mobilis ATCC ZW658: a potential platform for co-utilization of glucose and xylose.

A systematic adaptive laboratory evolution strategy was employed to develop a potential Zymomonas mobilis strain with the ability to co-utilize glucose and xylose. Z. mobilis ATCC ZW658, a recombinant xylose fermenting strain, was subjected to adaptive laboratory evolution over a period of 200 days under strict selection pressure of increasing concentration of xylose. The evolved strain exhibited 1.65 times increase in the overall specific xylose utilization rate when compared with the parent strain. Furthermore, the strain displayed significantly improved performance in terms of co-fermentation of xylose in the presence of glucose with specific glucose and xylose utilization rate of 1.24 g g-1 h-1 and 1.34 g g-1 h-1, respectively. Altered phenotypic response of the evolved strain, in terms of improved xylose utilization, co-utilization of mixed sugars, enhanced growth, ethanol production, and reduced xylitol production has been explained by novel mutations, identified using next-generation sequencing, in xylose assimilating, metabolizing, and crucial regulatory pathway genes and key enzyme activity assays.

Facing the challenge of genetic counselors' need for rapid continuing education about genomic technologies.

The last couple of decades have seen the rapid advancement of genomic technologies (GT) and their equally rapid adoption into clinical testing. Regardless of specialty, all genetic counselors are unified by the fundamental goal to aid in diagnosing patient's genetic disease underscoring the importance for genetic counselors to maintain an in-depth understanding of GT. The National Society of Genetic Counselors' (NSGC) GT Special Interest Group conducted an online survey of NSGC members to assess current genomic technologies knowledge gaps. A total of 171 individuals from a variety of primary work settings completed the survey sufficiently to be included in the analysis. The majority of respondents received their degree in genetic counseling in more recent years (2000-2015). On average across all technologies, >70% of respondents deemed knowledge of GTs as important for successful job performance, 55% responded that additional job training in GTs is needed to successfully perform job functions, and only 28% responded that graduate training in GTs was good. Overall, the data show that participating genetic counselors perceive that their knowledge of GTs is inadequate while it is a key component of their jobs. These results have implications both for training programs and for continuing education efforts. These data can be used as a starting point for additional research into GT educational needs of genetic counselors.

What lies beneath? Fungal diversity at the bottom of Lake Michigan and Lake Superior.

Fungi are phylogenetically diverse organisms found in nearly every environment as key contributors to the processes of nutrient cycling and decomposition. To date, most fungal diversity has been documented from terrestrial habitats leaving aquatic habitats underexplored. In particular, comparatively little is known about fungi inhabiting freshwater lakes, particularly the benthic zone, which may serve as an untapped resource for fungal biodiversity. Advances in technology allowing for direct sequencing of DNA from environmental samples provide a new opportunity to investigate freshwater benthic fungi. In this study, we employed both culture-dependent and culture-independent methods to evaluate the diversity of fungi in one of the largest freshwater systems on Earth, the North American Laurentian Great Lakes. This study presents the first comprehensive survey of fungi from sediment from Lake Michigan and Lake Superior, resulting in 465 fungal taxa with only 7% of sequence overlap between these two methods. Additionally, culture-independent analyses of the ITS1 and ITS2 regions revealed 49% and 72%, respectively, of the OTUs did not match a described fungal taxonomic group below kingdom Fungi. The low level of sequence overlap between methods and high percentage of fungal taxa that can only be classified at the kingdom level suggests an immense amount of fungal diversity remains to be studied in these aquatic fungal communities.

Reproducible simulations of realistic samples for next-generation sequencing studies using Variant Simulation Tools.

Computer simulations have been widely used to validate and evaluate the power of statistical methods for genetic epidemiological studies. Although a large number of simulation methods and software packages have been developed for genome-wide association studies, methodological and bioinformatics challenges have limited their applications in simulating datasets for whole-genome and whole-exome sequencing studies. With the development of more sophisticated statistical methods that make fuller use of available data and our knowledge of the human genome, there is a pressing need for genetic simulators that capture more features of empirical data (e.g., multiallele variants, indels, use of the Variant Call Format) and the human genome (e.g., functional annotations of genetic variants). This article introduces Variant Simulation Tools (VST), a module of Variant Tools for the simulation of genetic variants for sequencing-based genetic epidemiological studies. Although multiple simulation engines are provided, the core of VST is a novel forward-time simulation engine that simulates real nucleotide sequences of the human genome using DNA mutation models, fine-scale recombination maps, and a selection model based on amino acid changes of translated protein sequences. The design of VST allows users to easily create and distribute simulation methods and simulated datasets for a variety of applications and encourages fair comparison between statistical methods through the use of existing or reproduced simulated datasets.

Gene regulation and genetics in neurochemistry, past to future.

Ask any neuroscientist to name the most profound discoveries in the field in the past 60 years, and at or near the top of the list will be a phenomenon or technique related to genes and their expression. Indeed, our understanding of genetics and gene regulation has ushered in whole new systems of knowledge and new empirical approaches, many of which could not have even been imagined prior to the molecular biology boon of recent decades. Neurochemistry, in the classic sense, intersects with these concepts in the manifestation of neuropeptides, obviously dependent upon the central dogma (the established rules by which DNA sequence is eventually converted into protein primary structure) not only for their conformation but also for their levels and locales of expression. But, expanding these considerations to non-peptide neurotransmitters illustrates how gene regulatory events impact neurochemistry in a much broader sense, extending beyond the neurochemicals that translate electrical signals into chemical ones in the synapse, to also include every aspect of neural development, structure, function, and pathology. From the beginning, the mutability - yet relative stability - of genes and their expression patterns were recognized as potential substrates for some of the most intriguing phenomena in neurobiology - those instances of plasticity required for learning and memory. Near-heretical speculation was offered in the idea that perhaps the very sequence of the genome was altered to encode memories. A fascinating component of the intervening progress includes evidence that the central dogma is not nearly as rigid and consistent as we once thought. And this mutability extends to the potential to manipulate that code for both experimental and clinical purposes. Astonishing progress has been made in the molecular biology of neurochemistry during the 60 years since this journal debuted. Many of the gains in conceptual understanding have been driven by methodological progress, from automated high-throughput sequencing instruments to recombinant-DNA vectors that can convey color-coded genetic modifications in the chromosomes of live adult animals. This review covers the highlights of these advances, both theoretical and technological, along with a brief window into the promising science ahead. This article is part of the 60th Anniversary special issue.

Activation of cryptic biosynthetic gene clusters by fungal artificial chromosomes to produce novel secondary metabolites.

In 2017, we reported the discovery of Berkeleylactone A (BPLA), a novel, potent antibiotic produced exclusively in co-culture by two extremophilic fungi, Penicillium fuscum and P. camembertii/clavigerum, which were isolated from the Berkeley Pit, an acid mine waste lake, in Butte, Montana. Neither fungus synthesized BPLA when grown in axenic culture. Recent studies suggest that secondary metabolites (SMs) are often synthesized by enzymes encoded by co-localized genes that form "biosynthetic gene clusters" (BGCs), which might remain silent (inactive) under various fermentation conditions. Fungi may also harbor cryptic BGCs that are not associated with previously characterized molecules. We turned to the tools of Fungal Artificial Chromosomes (FAC)-Next-Gen-Sequencing (NGS) to understand how co-culture activated cryptic biosynthesis of BPLA and several related berkeleylactones and to further investigate the true biosynthetic potential of these two fungi. FAC-NGS enables the capture of BGCs as individual FACs for heterologous expression in a modified strain of Aspergillus nidulans (heterologous host, FAC-AnHH). With this methodology, we created ten BGC-FACs that yielded fourteen different SMs, including strobilurin, which was previously isolated exclusively from basidiomycetes. Eleven of these compounds were not detected in the extracts of the FAC-AnHH. Of this discrete set, only the novel compound citreohybriddional had been isolated from either Penicillium sp. before and only at very low yield. We propose that through heterologous expression, FACs activated these silent BGCs, resulting in the synthesis of new natural products (NPs) with yields as high as 50%-60% of the crude organic extracts.

miRNome-transcriptome analysis unveils the key regulatory pathways involved in the tumorigenesis of tongue squamous cell carcinoma.

Tongue squamous cell carcinoma (TSCC) is considered the most common malignant tumor among the oral squamous cell carcinomas with a poor prognosis. Understanding the underlying molecular mechanisms that underpin TSCC and its treatments is the focus of the research. Deregulated expression of microRNAs (miRNAs) has recently been implicated in various biological processes linked to cancer. Therefore, in this study, we attempted to investigate miRNAs and their targets expressed in TSCC, which could be involved in its oncogenesis. We performed next-generation sequencing of small RNAs and transcriptomes in H357 TSCC cell line and human oral keratinocytes as a control to find miRNAs and mRNAs that are differentially expressed (DE), which were then supplemented with additional expression datasets from databases, yielding 269 DE miRNAs and 2094 DE genes. The target prediction followed by pathway and disease function analysis revealed that the DE targets were significantly associated with the key processes and pathways, such as apoptosis, epithelial-mesenchymal transition, endocytosis and vascular endothelial growth factor signaling pathways. Furthermore, the top 12 DE targets were chosen based on their involvement in more than one cancer-related pathway, of which 6 genes are targeted by miR-128-3p. Real-time quantitative PCR validation of this miRNA and its targets in H357 and SCC9 TSCC cells confirmed their possible targeting from their reciprocal expression, with MAP2K7 being a critical target that might be involved in oncogenesis and progression of TSCC by acting as a tumor suppressor. Further research is underway to understand how miR-128-3p regulates oncogenesis in TSCC via MAP2K7 and associated pathways.

Challenges of genetic diagnosis of inborn errors of metabolism in a major tertiary care center in Lebanon.

Background: Inborn errors of metabolism are rare genetic disorders; however, these are prevalent in countries with high consanguinity rates, like Lebanon. Patients are suspected, based on a combination of clinical and biochemical features; however, the final confirmation relies on genetic testing. Using next generation sequencing, as a new genetic investigational tool, carries several challenges for the physician, the geneticist, and the families. Methods: In this retrospective study, we analyzed the clinical, biochemical, and genetic profile of inborn errors of metabolism suspected patients, seen at a major tertiary care center in Lebanon, between 2015 and 2018. Genetic testing was performed using next generation sequencing. Genotype-phenotype correlation and diagnostic yield of each testing modality were studied. Results: Out of 211 patients genetically tested, 126 were suspected to have an inborn error of metabolism. The diagnostic yield of next generation sequencing reached 64.3%. Single gene testing was requested in 53%, whole exome sequencing in 36% and gene panels in 10%. Aminoacid disorders were mostly diagnosed followed by storage disorders, organic acidemias and mitochondrial diseases. Targeted testing was performed in 77% of aminoacid and organic acid disorders and half of suspected storage disorders. Single gene sequencing was positive in 75%, whereas whole exome sequencing diagnostic yield for complex cases, like mitochondrial disorders, reached 49%. Good clinical and biochemical correlation allowed the interpretation of variants of unknown significance and negative mutations as well as therapeutic management of most patients. Conclusion: Tailoring the choice of test modality, by next generation sequencing, to the category of suspected inborn errors of metabolism may lead to rapid diagnosis, shortcutting the cost of repeated testing. Whole exome sequencing as a first-tier investigation may be considered mainly for suspected mitochondrial diseases, whereas targeted sequencing can be offered upon suspicion of a specific enzyme deficiency. Timing and modality of gene test remain challenging, in view of the cost incurred by families.

Tear miRNAs Identified in a Murine Model of Sjögren's Syndrome as Potential Diagnostic Biomarkers and Indicators of Disease Mechanism.

Objective: The tear miRNAome of the male NOD mouse, a model of ocular symptoms of Sjögren's syndrome (SS), was analyzed to identify unique miRNAs. Methods: Male NOD mice, aged 12-14 weeks, were used to identify tear miRNAs associated with development of autoimmune dacryoadenitis. Age- and sex-matched male BALB/c mice served as healthy controls while age-matched female NOD mice that do not develop the autoimmune dacryoadenitis characteristic of SS were used as additional controls. Total RNA was isolated from stimulated tears pooled from 5 mice per sample and tear miRNAs were sequenced and analyzed. Putative miRNA hits were validated in additional mouse cohorts as well as in tears of SS patients versus patients with another form of dry eye disease, meibomian gland disease (MGD) using qRT-PCR. The pathways influenced by the validated hits were identified using Ingenuity Pathway Analysis. Results: In comparison to tears from both healthy (male BALB/c) and additional control (female NOD) mice, initial analy1sis identified 7 upregulated and 7 downregulated miRNAs in male NOD mouse tears. Of these, 8 were validated by RT-qPCR in tears from additional mouse cohorts. miRNAs previously implicated in SS pathology included mmu-miR-146a/b-5p, which were significantly downregulated, as well as mmu-miR-150-5p and mmu-miR-181a-5p, which were upregulated in male NOD mouse tears. All other validated hits including the upregulated miR-181b-5p and mmu-miR-203-3p, as well as the downregulated mmu-miR-322-5p and mmu-miR-503-5p, represent novel putative indicators of autoimmune dacryoadenitis in SS. When compared to tears from patients with MGD, miRNAs hsa-miR-203a-3p, hsa-miR-181a-5p and hsa-miR-181b-5p were also significantly increased in tears of SS patients. Conclusions: A panel of differentially expressed miRNAs were identified in tears of male NOD mice, with some preliminary validation in SS patients, including some never previously linked to SS. These may have potential utility as indicators of ocular symptoms of SS; evaluation of the pathways influenced by these dysregulated miRNAs may also provide further insights into SS pathogenesis.

Germline mutations in RAD51C and RAD51D and hereditary predisposition to ovarian cancer.

Ovarian cancer is one of the most common gynecologic cancers with the highest mortality rate over a long period. Genetic predisposition to ovarian cancer is unusually high. In the Czech Republic, causal mutation in any ovarian cancer predisposition gene is identified in approximately 30% of the ovarian cancer patients. Therefore, according to the current guidelines, all ovarian cancer patients should be provided with genetic testing. The BRCA1 and BRCA2 are the two major ovarian cancer predisposition genes. Nevertheless, mutations in other predisposition genes, including RAD51C and RAD51D, are associated with high ovarian cancer risk. Mutations in RAD51C and RAD51D are found in 1% of ovarian cancer patients in each respective gene. Currently, identification of germline mutation in RAD51C and RAD51D is primarily of preventive importance but it potentially could make a prognostic difference. The aim of this review is to summarize the recent RAD51C and RAD51D knowledge, including the biological function, cancer risks associated with germline mutations, and recommendations for mutation carriers.

Metagenomic Insights in Activated Biomass Treating Industrial Wastewater at Different DO Levels.

Dissolved oxygen (DO) is an imperative parameter of the activated sludge process (ASP) for wastewater bioremediation. The effect of DO on microbial communities and corresponding metabolic functions in wastewater bioremediation was investigated using next-generation analysis techniques in this study. Illumina-based whole genome sequencing was applied to analyze the composition of the microbial community along with their functional diversity in activated sludge systems operating at three different DO levels. Activated biomass was collected from lab-scale reactors maintained at 1, 2, and 4 mg/L DO levels. Metagenomes were sequenced on an Illumina platform and analyzed using various tools. Results revealed that Proteobacteria phylum and Pseudomonas, Nitrobacter, Thauera, and Alicyclipilus genera were abundant in all reactor samples. Despite distinct DO levels, the microbial communities were conserved and consisted of a common population forming the core group governing the metabolic functions. However, higher diversity was observed at functional level indicating that microbes evolve and adapt to serve their role in a typical ASP. Metabolic pathway related to benzoate dominated at 1 mg/L DO level, while pathways for degradation of aromatic compounds like phenol, toluene, and biphenyl via central metabolic pathway were found dominating at 4 mg/L DO level. Pathways corresponding to homogentisate, naphthalene, cresol, and salicylate degradation enriched at 2 mg/L DO level.

Restricted epitope specificity determined by variable region germline segment pairing in rodent antibody repertoires.

Antibodies from B-cell clonal lineages share sequence and structural properties as well as epitope specificity. Clonally unrelated antibodies can similarly share sequence and specificity properties and are said to be convergent. Convergent antibody responses against several antigens have been described in humans and mice and include different classes of shared sequence features. In particular, some antigens and epitopes can induce convergent responses of clonally unrelated antibodies with restricted heavy (VH) and light (VL) chain variable region germline segment usage without similarity in the heavy chain third complementarity-determining region (CDR H3), a critical specificity determinant. Whether these V germline segment-restricted responses reflect a general epitope specificity restriction of antibodies with shared VH/VL pairing is not known. Here, we investigated this question by determining patterns of antigen binding competition between clonally unrelated antigen-specific rat antibodies from paired-chain deep sequencing datasets selected based solely on VH/VL pairing. We found that antibodies with shared VH/VL germline segment pairings but divergent CDR H3 sequences almost invariably have restricted epitope specificity indicated by shared binding competition patterns. This epitope restriction included 82 of 85 clonally unrelated antibodies with 13 different VH/VL pairings binding in 8 epitope groups in 2 antigens. The corollary that antibodies with shared VH/VL pairing and epitope-restricted binding can accommodate widely divergent CDR H3 sequences was confirmed by in vitro selection of variants of anti-human epidermal growth factor receptor 2 antibodies known to mediate critical antigen interactions through CDR H3. Our results show that restricted epitope specificity determined by VH/VL germline segment pairing is a general property of rodent antigen-specific antibodies.

Multi-Method Characterization of the Human Circulating Microbiome.

The term microbiome describes the genetic material encoding the various microbial populations that inhabit our body. Whilst colonization of various body niches (e.g., the gut) by dynamic communities of microorganisms is now universally accepted, the existence of microbial populations in other "classically sterile" locations, including the blood, is a relatively new concept. The presence of bacteria-specific DNA in the blood has been reported in the literature for some time, yet the true origin of this is still the subject of much deliberation. The aim of this study was to investigate the phenomenon of a "blood microbiome" by providing a comprehensive description of bacterially derived nucleic acids using a range of complementary molecular and classical microbiological techniques. For this purpose we utilized a set of plasma samples from healthy subjects (n = 5) and asthmatic subjects (n = 5). DNA-level analyses involved the amplification and sequencing of the 16S rRNA gene. RNA-level analyses were based upon the de novo assembly of unmapped mRNA reads and subsequent taxonomic identification. Molecular studies were complemented by viability data from classical aerobic and anaerobic microbial culture experiments. At the phylum level, the blood microbiome was predominated by Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. The key phyla detected were consistent irrespective of molecular method (DNA vs. RNA), and consistent with the results of other published studies. In silico comparison of our data with that of the Human Microbiome Project revealed that members of the blood microbiome were most likely to have originated from the oral or skin communities. To our surprise, aerobic and anaerobic cultures were positive in eight of out the ten donor samples investigated, and we reflect upon their source. Our data provide further evidence of a core blood microbiome, and provide insight into the potential source of the bacterial DNA/RNA detected in the blood. Further, data reveal the importance of robust experimental procedures, and identify areas for future consideration.