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Superoxide dismutases (SODs), as a family of metalloenzymes related to the removal of reactive oxygen species (ROS), have not previously been investigated at genome-wide level in tea plant. In this study, 10 CsSOD genes were identified in tea plant genome, including 7 Cu/Zn-SODs (CSDs), 2 Fe-SODs (FSDs) and one Mn-SOD (MSD), and phylogenetically classified in three subgroups, respectively. Physico-chemical characteristic, conserved motifs and potential protein interaction analyses about CsSOD proteins were carried out. Exon-intron structures and codon usage bias about CsSOD genes were also examined. Exon-intron structures analysis revealed that different CsSOD genes contained various number of introns. On the basis of the prediction of regulatory miRNAs of CsSODs, a modification 5' RNA ligase-mediated (RLM)-RACE was performed and validated that csn-miR398a-3p-1 directly cleaves CsCSD4. By prediction of cis-acting elements, the expression patterns of 10 CsSOD genes and their regulatory miRNAs were detected under cold, drought, exogenous methyl jasmonate (MeJA) and gibberellin (GA3) treatments. The results showed that most of CsSODs except for CsFSD2 were induced under cold stress and CsCSDs may play primary roles under drought stress; exogenous GA3 and MeJA could also stimulated/inhibited distinct CsSODs at different stages. In addition, we found that csn-miR398a-3p-1 negatively regulated the expression of CsCSD4 may be a crucial regulatory mechanism under cold stress. This study provides a certain basis for the studies about stress resistance in tea plants, even provide insight into comprehending the classification, evolution, diverse functions and influencing factors of expression patterns for CsSOD genes. [ABSTRACT FROM AUTHOR]

MicroRNAs are conserved, endogenous small RNAs with critical post-transcriptional regulatory functions throughout eukaryota, including prominent roles in development and disease. Despite much effort, microRNA annotations still contain errors and are incomplete due especially to challenges related to identifying valid miRs that have small numbers of reads, to properly locating hairpin precursors and to balancing precision and recall. Here, we present miRWoods, which solves these challenges using a duplex-focused precursor detection method and stacked random forests with specialized layers to detect mature and precursor microRNAs, and has been tuned to optimize the harmonic mean of precision and recall. We trained and tuned our discovery pipeline on data sets from the well-annotated human genome, and evaluated its performance on data from mouse. Compared to existing approaches, miRWoods better identifies precursor spans, and can balance sensitivity and specificity for an overall greater prediction accuracy, recalling an average of 10% more annotated microRNAs, and correctly predicts substantially more microRNAs with only one read. We apply this method to the under-annotated genomes of Felis catus (domestic cat) and Bos taurus (cow). We identified hundreds of novel microRNAs in small RNA sequencing data sets from muscle and skin from cat, from 10 tissues from cow and also from human and mouse cells. Our novel predictions include a microRNA in an intron of tyrosine kinase 2 (TYK2) that is present in both cat and cow, as well as a family of mirtrons with two instances in the human genome. Our predictions support a more expanded miR-2284 family in the bovine genome, a larger mir-548 family in the human genome, and a larger let-7 family in the feline genome. [ABSTRACT FROM AUTHOR]

Cultivated watermelon (Citrullus lanatus) is one of the most important food crops in the Cucurbitaceae family. Diversification after domestication has led cultivated watermelons to exhibit diverse fruit flesh colors, including red, yellow, and orange. Recently, there has been increased interest in red-fleshed watermelons because they contain the antioxidant cis-isomeric lycopene. We performed whole genome resequencing (WGRS) of 24 watermelons with different flesh colors to identify single-nucleotide polymorphisms (SNPs) related to high lycopene content. The resequencing data revealed 203,894–279,412 SNPs from read mapping between inbred lines and the 97103 reference genome. In total, 295,065 filtered SNPs were identified, which had an average polymorphism information content of 0.297. Most of these SNPs were intergenic (90.1%) and possessed a transversion (Tv) rate of 31.64%. Overall, 2,369 SNPs were chosen at 0.5 Mb physical intervals to analyze genetic diversity across the 24 inbred lines. A neighbor-joining dendrogram and principal coordinate analysis (PCA) based on the 2,369 SNPs revealed that the 24 inbred lines could be grouped into high and low lycopene-type watermelons. In addition, we analyzed SNPs that could discriminate high lycopene content, red-fleshed watermelon from low lycopene, yellow or orange watermelon inbred lines. For validation, 19 SNPs (designated as WMHL1–19) were chosen randomly, and cleavage amplified polymorphic sequence (CAPS) markers were designed. Genotyping of the above 24 lines and 12 additional commercial cultivars using WMHL1–19 CAPS markers resulted in match rates of over 0.92 for most validated markers in correlation with the flesh color phenotypes. Our results provide valuable genomic information regarding the high lycopene content phenotype of red-fleshed cultivated watermelons, and the identified SNPs will be useful for the development of molecular markers in the marker-assisted breeding of watermelons with high lycopene content. [ABSTRACT FROM AUTHOR]

Candida auris is an invasive and multidrug-resistant ascomycetous yeast that is under global surveillance. All clinical cases of C. auris infection diagnosed from 1997 to 2019 in Japan were non-invasive and sporadic otitis media cases. In the present study, we performed whole-genome sequencing of seven C. auris strains isolated from patients with otitis media in Japan, all of which belonged to clade II. Comparative genome analysis using the high-quality draft genome sequences JCM 15448T revealed that single nucleotide variations (SNVs), clade-specific accessory genes, and copy number variations (CNVs) were identified in each C. auris clade. A total of 61 genes involved in cell wall and stress response-related functions was absent in clade II, and the pattern of conserved CNVs in each clade was more stable in clade II than in other clades. Our data suggest that the genomic structural diversity is stable in C. auris isolated from each biogeographic location, and Japanese strains isolated from patients with otitis media might belong to an ancestral type of C. auris. One Japanese strain, TWCC 58362, with reduced susceptibility to fluconazole, exhibited no mutation in ergosterol biosynthesis-related genes (ERG). However, TWCC 58362-specific variations, including SNVs, indels, and CNVs were detected, suggesting that gene duplication events in C. auris might contribute to antifungal drug resistance. Taken together, we demonstrated that genomic structural variations in C. auris could correlate to geographical dissemination, epidemiology, lesions in the host, and antifungal resistance. [ABSTRACT FROM AUTHOR]

Two novel and related pathogenic variants of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene were structurally and functionally characterized. These alterations have not been previously described in literature. Two patients with diagnosis of Cystic Fibrosis (CF) based on the presence of one mutated allele, p.Phe508del, pathological sweat test and clinical symptoms were studied. To complete the genotypes of both patients, an extensive genetic and functional analysis of the CFTR gene was performed. Extensive genetic characterization confirmed the presence of p.Phe508del pathogenic variant and revealed, in both patients, the presence of an insertion of part of intron 10 in intron 9 of the CFTR gene, within the (TG)m repeat, with a variable poly-T stretch. The molecular lesions resulted to be very similar in both patients, with only a difference in the number of T in the poly-T stretch. The functional characterization at RNA level revealed a complete anomalous splicing, without exon 10, from the allele with the insertion of both patients. Consequently, the alleles with the insertions are expected not to contribute to the formation of a functional CFTR protein. Molecular and functional features of these alterations are compatible with the definition of novel CF-causing variants of the CFTR gene. This also allowed the completion of the genetic characterization of both patients. [ABSTRACT FROM AUTHOR]

Drug-induced interstitial lung disease (DIILD) is a serious side effect of chemotherapy in cancer patients with an extremely high mortality rate. In this study, to identify genetic variants with greater risk of DIILD, we carried out whole genome sequencing (WGS) of germline DNA samples from 26 patients who developed DIILD, and conducted a case-control association study between these 26 cases and general Japanese population controls registered in the integrative Japanese Genome Variation Database (iJGVD) as a screening study. The associations of 42 single nucleotide variants (SNVs) showing P < 0.0001 were further validated using an independent cohort of 18 DIILD cases as a replication study. A further combined analysis of the screening and replication studies showed a possible association of two SNVs, rs35198919 in intron 1 of the chromosome 22 open reading frame 34 (C22orf34) and rs12625311 in intron 1 of the teashirt zinc finger homeobox 2 (TSHZ2), with DIILD (Pcombined = 1.87 × 10−5 and 5.16 × 10−5, respectively). Furthermore, in a subgroup analysis of epidermal growth factor receptor (EGFR)–tyrosine kinase inhibitor (TKI)-induced interstitial lung disease (ILD), we observed seven candidate SNVs that were possibly associated with ILD (P < 0.00001). This is the first study to identify genetic markers for the risk of DIILD using WGS. Collectively, our novel findings indicate that these SNVs may be applicable for predicting the risk of DIILD in patients receiving chemotherapy. [ABSTRACT FROM AUTHOR]

Alternative splicing diversifies mRNA transcripts in human cells. While the spliceosome pairs exons with a high degree of accuracy, the rates of rare aberrant and non-canonical pre-mRNA splicing have not been evaluated at the nucleotide level to determine the quantity and identity of these events across splice junctions. Using ultra-deep sequencing the frequency of aberrant and non-canonical splicing events for three splice junctions flanking exon 7 of SMN1 were determined at single nucleotide resolution. After correction for background noise introduced by PCR amplification and sequencing steps, pre-mRNA splicing was shown to maintain a low overall rate of aberrant and non-canonically spliced events. Several previously unannotated splicing events across 3 exon|intron junctions in SMN1 were identified. Mutations within SMN exon 7 were shown to affect splicing fidelity by modulating RNA secondary structures, by altering the binding site of regulatory proteins and by changing the 5’ splice site strength. Mutations also create a truncated SMN1 exon 7 through the introduction of a de novo non-canonical 5’ splice site. The results from the ultra-deep sequencing approach highlight the impressive fidelity of pre-mRNA splicing and demonstrate that the immediate sequence context around splice sites is the main driving force behind non-canonical splice site pairing. [ABSTRACT FROM AUTHOR]

In mammals, dosage compensation of sex chromosomal genes between females (XX) and males (XY) is achieved through X-chromosome inactivation (XCI). The X-linked X-inactive-specific transcript (Xist) long noncoding RNA is indispensable for XCI and initiates the process early during development by spreading in cis across the X chromosome from which it is transcribed. During XCI, Xist RNA triggers gene silencing, recruits a plethora of chromatin modifying factors, and drives a major structural reorganization of the X chromosome. Here, we review our knowledge of the multitude of epigenetic events orchestrated by Xist RNA to allow female mammals to survive through embryonic development by establishing and maintaining proper dosage compensation. In particular, we focus on recent studies characterizing the interaction partners of Xist RNA, and we discuss how they have affected the field by addressing long-standing controversies or by giving rise to new research perspectives that are currently being explored. This review is dedicated to the memory of Denise Barlow, pioneer of genomic imprinting and functional long noncoding RNAs (lncRNAs), whose work has revolutionized the epigenetics field and continues to inspire generations of scientists. [ABSTRACT FROM AUTHOR]

Members of the plant Heme Activator Protein (HAP) or NUCLEAR FACTOR Y (NF-Y) are trimeric transcription factor complexes composed of the NF-YA, NF-YB and NF-YC subfamilies. They bind to the CCAAT box in the promoter regions of the target genes and regulate gene expressions. Plant NF-Ys were reported to be involved in adaptation to several abiotic stresses as well as in development. In silico analysis of Sorghum bicolor genome resulted in the identification of a total of 42 NF-Y genes, among which 8 code for the SbNF-YA, 19 for SbNF-YB and 15 for the SbNF-YC subunits. Analysis was also performed to characterize gene structures, chromosomal distribution, duplication status, protein subcellular localizations, conserved motifs, ancestral protein sequences, miRNAs and phylogenetic tree construction. Phylogenetic relationships and ortholog predictions displayed that sorghum has additional NF-YB genes with unknown functions in comparison with Arabidopsis. Analysis of promoters revealed that they harbour many stress-related cis-elements like ABRE and HSE, but surprisingly, DRE and MYB elements were not detected in any of the subfamilies. SbNF-YA1, 2, and 6 were found upregulated under 200 mM salt and 200 mM mannitol stresses. While NF-YA7 appeared associated with high temperature (40°C) stress, NF-YA8 was triggered by both cold (4°C) and high temperature stresses. Among NF-YB genes, 7, 12, 15, and 16 were induced under multiple stress conditions such as salt, mannitol, ABA, cold and high temperatures. Likewise, NF-YC 6, 11, 12, 14, and 15 were enhanced significantly in a tissue specific manner under multiple abiotic stress conditions. Majority of the mannitol (drought)-inducible genes were also induced by salt, high temperature stresses and ABA. Few of the high temperature stress-induced genes are also induced by cold stress (NF-YA2, 4, 6, 8, NF-YB2, 7, 10, 11, 12, 14, 16, 17, NF-YC4, 6, 12, and 13) thus suggesting a cross talk among them. This work paves the way for investigating the roles of diverse sorghum NF-Y proteins during abiotic stress responses and provides an insight into the evolution of diverse NF-Y members. [ABSTRACT FROM AUTHOR]

Chloroplasts significantly influence species phylogenies because of their maternal inheritance and the moderate evolutionary rate of their genomes. Avocado, which is a member of the family Lauraceae, has received considerable attention from botanists, likely because of its position as a basal angiosperm. However, there is relatively little avocado genomic information currently available. In this study, six complete avocado chloroplast genomes from three ecological races were assembled to examine the sequence diversity among the three avocado ecological races. A comparative genomic analysis revealed that 515 simple sequence repeat loci and 176 repeats belonging to four other types were polymorphic across the six chloroplast genomes. Three highly variable regions (trnC-GCA-petN, petN-psbM, and petA-psbJ) were identified as highly informative markers. A phylogenetic analysis based on 79 common protein-coding genes indicated that the six examined avocado accessions from three ecological races form a monophyletic clade. The other three genera belonging to the Persea group clustered to form a sister clade with a high bootstrap value. These chloroplast genomes provide important genetic information for future attempts at identifying avocado races and for the related biological research. [ABSTRACT FROM AUTHOR]

Plasmodium vivax invasion of human erythrocytes depends on the Duffy Binding Protein (PvDBP) which interacts with the Duffy antigen. PvDBP copy number has been recently shown to vary between P. vivax isolates in Sub-Saharan Africa. However, the extent of PvDBP copy number variation, the type of PvDBP multiplications, as well as its significance across broad samples are still unclear. We determined the prevalence and type of PvDBP duplications, as well as PvDBP copy number variation among 178 Ethiopian P. vivax isolates using a PCR-based diagnostic method, a novel quantitative real-time PCR assay and whole genome sequencing. For the 145 symptomatic samples, PvDBP duplications were detected in 95 isolates, of which 81 had the Cambodian and 14 Malagasy-type PvDBP duplications. PvDBP varied from 1 to >4 copies. Isolates with multiple PvDBP copies were found to be higher in symptomatic than asymptomatic infections. For the 33 asymptomatic samples, PvDBP was detected with two copies in two of the isolates, and both were the Cambodian-type PvDBP duplication. PvDBP copy number in Duffy-negative heterozygotes was not significantly different from that in Duffy-positives, providing no support for the hypothesis that increased copy number is a specific association with Duffy-negativity, although the number of Duffy-negatives was small and further sampling is required to test this association thoroughly. [ABSTRACT FROM AUTHOR]

Terpenes are responsible for most or all of the odor and flavor properties of Cannabis sativa, and may also impact effects users experience either directly or indirectly. We report the diversity of terpene profiles across samples bound for the Washington dispensary market. The remarkable degree of variation in terpene profiles ultimately results from action of a family of terpene synthase genes, only some of which have been described. Using a recently available genome assembly we describe 55 terpene synthases with genomic context, and tissue specific expression. The family is quite diverse from a protein similarity perspective, and subsets of the family are expressed in all tissues in the plant, including a set of root specific monoterpene synthases that could well have agronomic importance. Ultimately understanding and breeding for specific terpene profiles will require a good understanding of the gene family that underlies it. We intend for this work to serve as a foundation for that. [ABSTRACT FROM AUTHOR]

Background: Tyrosine kinase inhibitors targeted to anaplastic lymphoma kinase (ALK) have been demonstrated to be effective for lung cancer patients with an ALK fusion gene. Application of liquid biopsy, i.e., detection and quantitation of the fusion product in plasma cell-free DNA (cfDNA), could improve clinical practice. To detect ALK fusions, because fusion breakpoints occur somewhere in intron 19 of the ALK gene, sequencing of the entire intron is required to locate breakpoints. Results: We constructed a target sequencing system using an adapter and a set of primers that cover the entire ALK intron 19. This system can amplify fragments, including breakpoints, regardless of fusion partners. The data analysis pipeline firstly detected fusions by alignment to selected target sequences, and then quantitated the fusion alleles aligning to the identified breakpoint sequences. Performance was validated using 20 cfDNA samples from ALK-positive non-small cell lung cancer patients and samples from 10 healthy volunteers. Sensitivity and specificity were 50 and 100%, respectively. Conclusions: We demonstrated that PCR-based target sequencing using a tiling primer set and two-step mapping/alignment quantitatively detected ALK fusions in cfDNA from lung cancer patients. The system offers an alternative to existing approaches based on hybridization capture. [ABSTRACT FROM AUTHOR]

SWItch/Sucrose NonFermentable (SWI/SNF) is a set of multi-subunits chromatin remodeling complexes, playing important roles in a variety of biological processes. Loss-of-function mutations in the genes encoding SWI/SNF subunits have been reported in more than 20% of human cancers. Thus, it was widely considered as a tumor suppressor in the past decade. However, recent studies reported that some genes encoding subunits of SWI/SNF complexes were amplified and play oncogenic roles in human cancers. In present study, we summarized the genetic alteration spectrum of SWI/SNF complexes, and firstly systematically estimated both the copy number variations and point mutations of all 30 genes encoding the subunits in this complex. Additionally, the bioinformatics analyses were performed for two significantly amplified genes, ACTL6A and BRD9, to investigate their oncogenic roles in human cancers. Our findings may lay a foundation for the discovery of potential treatment targets in SWI/SNF complexes of cancers. [ABSTRACT FROM AUTHOR]

Plant mitochondrial genomes are usually assembled and displayed as circular maps based on the widely-held view across the broad community of life scientists that circular genome-sized molecules are the primary form of plant mitochondrial DNA, despite the understanding by plant mitochondrial researchers that this is an inaccurate and outdated concept. Many plant mitochondrial genomes have one or more pairs of large repeats that can act as sites for inter- or intramolecular recombination, leading to multiple alternative arrangements (isoforms). Most mitochondrial genomes have been assembled using methods unable to capture the complete spectrum of isoforms within a species, leading to an incomplete inference of their structure and recombinational activity. To document and investigate underlying reasons for structural diversity in plant mitochondrial DNA, we used long-read (PacBio) and short-read (Illumina) sequencing data to assemble and compare mitochondrial genomes of domesticated (Lactuca sativa) and wild (L. saligna and L. serriola) lettuce species. We characterized a comprehensive, complex set of isoforms within each species and compared genome structures between species. Physical analysis of L. sativa mtDNA molecules by fluorescence microscopy revealed a variety of linear, branched, and circular structures. The mitochondrial genomes for L. sativa and L. serriola were identical in sequence and arrangement and differed substantially from L. saligna, indicating that the mitochondrial genome structure did not change during domestication. From the isoforms in our data, we infer that recombination occurs at repeats of all sizes at variable frequencies. The differences in genome structure between L. saligna and the two other Lactuca species can be largely explained by rare recombination events that rearranged the structure. Our data demonstrate that representations of plant mitochondrial genomes as simple, circular molecules are not accurate descriptions of their true nature and that in reality plant mitochondrial DNA is a complex, dynamic mixture of forms. [ABSTRACT FROM AUTHOR]

Introduction: Genetic polymorphisms and social factors (alcohol consumption, betel quid (BQ) usage, and cigarette consumption), both separately or jointly, play a crucial role in the occurrence of oral malignant disorders such as oral and pharyngeal cancers and oral potentially malignant disorders (OPMD). Material and methods: Simultaneous analyses of multiple single nucleotide polymorphisms (SNPs) and environmental effects on oral malignant disorders are essential to examine, albeit challenging. Thus, we conducted a case-control study (N = 576) to analyze the risk of occurrence of oral malignant disorders by using binary particle swarm optimization (BPSO) with an odds ratio (OR)-based method. Results: We demonstrated that a combination of SNPs (CYP26B1 rs887844 and CYP26C1 rs12256889) and socio-demographic factors (age, ethnicity, and BQ chewing), referred to as the combined effects of SNP-environment, correlated with maximal risk diversity of occurrence observed between the oral malignant disorder group and the control group. The risks were more prominent in the oral and pharyngeal cancers group (OR = 10.30; 95% confidence interval (CI) = 4.58–23.15) than in the OPMD group (OR = 5.42; 95% CI = 1.94–15.12). Conclusions: Simulation-based “SNP-environment barcodes” may be used to predict the risk of occurrence of oral malignant disorders. Applying simulation-based “SNP-environment barcodes” may provide insight into the importance of screening tests in preventing oral and pharyngeal cancers and OPMD. [ABSTRACT FROM AUTHOR]

Splicing expands, reshapes, and regulates the transcriptome of eukaryotic organisms. Despite its importance, key questions remain unanswered, including the following: Can splicing evolve when organisms adapt to new challenges? How does evolution optimize inefficiency of introns’ splicing and of the splicing machinery? To explore these questions, we evolved yeast cells that were engineered to contain an inefficiently spliced intron inside a gene whose protein product was under selection for an increased expression level. We identified a combination of mutations in Cis (within the gene of interest) and in Trans (in mRNA-maturation machinery). Surprisingly, the mutations in Cis resided outside of known intronic functional sites and improved the intron’s splicing efficiency potentially by easing tight mRNA structures. One of these mutations hampered a protein’s domain that was not under selection, demonstrating the evolutionary flexibility of multi-domain proteins as one domain functionality was improved at the expense of the other domain. The Trans adaptations resided in two proteins, Npl3 and Gbp2, that bind pre-mRNAs and are central to their maturation. Interestingly, these mutations either increased or decreased the affinity of these proteins to mRNA, presumably allowing faster spliceosome recruitment or increased time before degradation of the pre-mRNAs, respectively. Altogether, our work reveals various mechanistic pathways toward optimizations of intron splicing to ultimately adapt gene expression patterns to novel demands. [ABSTRACT FROM AUTHOR]

Introns are a prevalent feature of eukaryotic genomes, yet their origins and contributions to genome function and evolution remain mysterious. In budding yeast, repression of the highly transcribed intron-containing ribosomal protein genes (RPGs) globally increases splicing of non-RPG transcripts through reduced competition for the spliceosome. We show that under these “hungry spliceosome” conditions, splicing occurs at more than 150 previously unannotated locations we call protointrons that do not overlap known introns. Protointrons use a less constrained set of splice sites and branchpoints than standard introns, including in one case AT-AC in place of GT-AG. Protointrons are not conserved in all closely related species, suggesting that most are not under positive selection and are fated to disappear. Some are found in non-coding RNAs (e. g. CUTs and SUTs), where they may contribute to the creation of new genes. Others are found across boundaries between noncoding and coding sequences, or within coding sequences, where they offer pathways to the creation of new protein variants, or new regulatory controls for existing genes. We define protointrons as (1) nonconserved intron-like sequences that are (2) infrequently spliced, and importantly (3) are not currently understood to contribute to gene expression or regulation in the way that standard introns function. A very few protointrons in S. cerevisiae challenge this classification by their increased splicing frequency and potential function, consistent with the proposed evolutionary process of “intronization”, whereby new standard introns are created. This snapshot of intron evolution highlights the important role of the spliceosome in the expansion of transcribed genomic sequence space, providing a pathway for the rare events that may lead to the birth of new eukaryotic genes and the refinement of existing gene function. [ABSTRACT FROM AUTHOR]

In this study, we report the plastome of Eriocaulon decemflorum (Eriocaulaceae) and make an effort to understand the genome evolution, structural rearrangements and gene content of the order Poales by comparing it with other available plastomes. The size of complete E. decemflorum plastome is 151,671 bp with an LSC (81,477bp), SSC (17,180bp) and a pair of IRs (26,507 bp). The plastome exhibits GC content of 35.8% and 134 protein-coding genes with 19 genes duplicated in the IR region. The Eriocaulaceae plastome is characterized by the presence of accD, ycf1 and ycf2 genes and presence of introns in clpP and rpoC1 genes which have been lost in the Graminid plastomes. Phylogenomic analysis based on 81 protein-coding genes placed Eriocaulaceae sister to Mayacaceae. The present study enhances our understanding of the evolution of Poales by analyzing the plastome data from the order. [ABSTRACT FROM AUTHOR]

There is a need to investigate and better understand the inherited risk of cancer to ensure that clinical applications provide more accurate assessments and management strategies. Developing research-based next-generation sequencing gene panels that not only target (present-day) clinically actionable susceptibility genes but also genes that currently lack sufficient evidence for risk as well as candidate genes, such as those in DNA repair pathways, can help aid this effort. Therefore, gene panel B.O.P. (reast, varian, and rostate) was developed to evaluate the genetic risk of breast, ovarian and/or prostate cancer, and this manuscript serves as an introduction to B.O.P. and highlights its initial analytical validity assessment. B.O.P targets 87 genes that have been suggested, predicted, or clinically proven to be associated with breast, ovarian, and/or prostate cancer risk using Agilent Technologies Haloplex probes. The probes were designed for 100 base pair reads on an Illumina platform and target both coding and non-coding exons as well as 10 intronic base pairs flanking the intron-exon boundaries. The initial B.O.P screening involved 43 individuals from the Alabama Hereditary Cancer Cohort, and an average sequencing depth of 809X was obtained. Upon variant filtering and validation, true positives had an average sequencing depth of 659X and allele balance of 0.51. The average false positive sequencing depth was 34X and allele balance was 0.33. Although low sequencing depth was not always indicative of a false positive, high sequencing depths (>100X) signified a true positive. Furthermore, sensitivity and specificity of BRCA1/2 were calculated to be 100% and 92.3%, respectively. Overall, this screening enabled the establishment of criteria to alleviate future validation efforts and strongly supports the use of B.O.P. to further elucidate hereditary cancer susceptibility. Ultimately, continued B.O.P. screening will provide insights toward the genetic risk of and overlap between breast, ovarian, and/or prostate cancer. [ABSTRACT FROM AUTHOR]

Vesicoureteral reflux (VUR) is a complex, heritable disorder. Genome-wide linkage analyses of families affected by VUR have revealed multiple genomic loci linked to VUR. These loci normally harbor a number of genes whose biologically functional variant is yet to be identified. DNA copy number variations (CNVs) have not been extensively studied at high resolution in VUR patients. In this study, we performed array comparative genomic hybridization (aCGH) on a cohort of patients with a history of both VUR and urinary tract infection (UTI) with the objective of identifying genetic variations responsible for VUR and/or UTI susceptibility. UTI/VUR-associated CNVs were identified by aCGH results from the 192 Randomized Intervention for Children With Vesicoureteral Reflux (RIVUR) patients compared to 683 controls. Rare, large CNVs that are likely pathogenic and lead to VUR development were identified using stringent analysis criteria. Because UTI is a common affliction with multiple risk factors, we utilized standard analysis to identify potential disease-modifying CNVs that can contribute to UTI risk. Gene ontology analysis identified that CNVs in innate immunity and development genes were enriched in RIVUR patients. CNVs affecting innate immune genes may contribute to UTI susceptibility in VUR patients and may provide the first step in assisting clinical medicine in determining adverse outcome risk in children with VUR. [ABSTRACT FROM AUTHOR]

Essential tremor (ET) is one of the most common movement disorders. The etiology of ET remains largely unexplained. Whole genome sequencing (WGS) is likely to be of value in understanding a large proportion of ET with Mendelian and complex disease inheritance patterns. In ET families with Mendelian inheritance patterns, WGS may lead to gene identification where WES analysis failed to identify the causative single nucleotide variant (SNV) or indel due to incomplete coverage of the entire coding region of the genome, in addition to accurate detection of larger structural variants (SVs) and copy number variants (CNVs). Alternatively, in ET families with complex disease inheritance patterns with gene x gene and gene x environment interactions enrichment of functional rare coding and non-coding variants may explain the heritability of ET. We performed WGS in eight ET families (n = 40 individuals) enrolled in the Family Study of Essential Tremor. The analysis included filtering WGS data based on allele frequency in population databases, rare SNV and indel classification and association testing using the Mixed-Model Kernel Based Adaptive Cluster (MM-KBAC) test. A separate analysis of rare SV and CNVs segregating within ET families was also performed. Prioritization of candidate genes identified within families was performed using phenolyzer. WGS analysis identified candidate genes for ET in 5/8 (62.5%) of the families analyzed. WES analysis in a subset of these families in our previously published study failed to identify candidate genes. In one family, we identified a deleterious and damaging variant (c.1367G>A, p.(Arg456Gln)) in the candidate gene, CACNA1G, which encodes the pore forming subunit of T-type Ca(2+) channels, CaV3.1, and is expressed in various motor pathways and has been previously implicated in neuronal autorhythmicity and ET. Other candidate genes identified include SLIT3 which encodes an axon guidance molecule and in three families, phenolyzer prioritized genes that are associated with hereditary neuropathies (family A, KARS, family B, KIF5A and family F, NTRK1). Functional studies of CACNA1G and SLIT3 suggest a role for these genes in ET disease pathogenesis. [ABSTRACT FROM AUTHOR]

Adeno-associated viruses (AAV) are Dependoparvoviruses that have shown promise as recombinant vectors for gene therapy. While infectious pathways of AAV are well studied, gaps remain in our understanding of host factors affecting vector genome expression. Here, we map the role of ring finger protein 121 (RNF121), an E3 ubiquitin ligase, as a key regulator of AAV genome transcription. CRISPR-mediated knockout of RNF121 (RNF121 KO) in different cells markedly decreased AAV transduction regardless of capsid serotype or vector dose. Recombinant AAV transduction is partially rescued by overexpressing RNF121, but not by co-infection with helper Adenovirus. Major steps in the AAV infectious pathway including cell surface binding, cellular uptake, nuclear entry, capsid uncoating and second strand synthesis are unaffected. While gene expression from transfected plasmids or AAV genomes is unaffected, mRNA synthesis from AAV capsid-associated genomes is markedly decreased in RNF121 KO cells. These observations were attributed to transcriptional arrest as corroborated by RNAPol-ChIP and mRNA half-life measurements. Although AAV capsid proteins do not appear to be direct substrates of RNF121, the catalytic domain of the E3 ligase appears essential. Inhibition of ubiquitin-proteasome pathways revealed that blocking Valosin Containing Protein (VCP/p97), which targets substrates to the proteasome, can selectively and completely restore AAV-mediated transgene expression in RNF121 KO cells. Expanding on this finding, transcriptomic and proteomic analysis revealed that the catalytic subunit of DNA PK (DNAPK-Cs), a known activator of VCP, is upregulated in RNF121 KO cells and that the DNA damage machinery is enriched at sites of stalled AAV genome transcription. We postulate that a network of RNF121, VCP and DNA damage response elements function together to regulate transcriptional silencing and/or activation of AAV vector genomes. [ABSTRACT FROM AUTHOR]

Spinal Muscular Atrophy (SMA) is a disorder characterized by the degeneration of motor neurons in the spinal cord, leading to muscular atrophy. In the majority of cases, SMA is caused by the homozygous absence of the SMN1 gene. The disease severity of SMA is strongly influenced by the copy number of the closely related SMN2 gene. In addition, an SMN variant lacking exons 7 and 8 has been reported in 8% and 23% of healthy Swedish and Spanish individuals respectively. We tested 1255 samples from the 1000 Genomes Project using a new version of the multiplex ligation-dependent probe amplification (MLPA) P021 probemix that covers each SMN exon. The SMN variant lacking exons 7 and 8 was present in up to 20% of individuals in several Caucasian populations, while being almost completely absent in various Asian and African populations. This SMN1/2Δ7–8 variant appears to be derived from an ancient deletion event as the deletion size is identical in 99% of samples tested. The average total copy number of SMN1, SMN2 and the SMN1/2Δ7–8 variant combined was remarkably comparable in all populations tested, ranging from 3.64 in Asian to 3.75 in African samples. [ABSTRACT FROM AUTHOR]

WRKY proteins are a large group of plant transcription factors that are involved in various biological processes, including biotic and abiotic stress responses, hormone response, plant development, and metabolism. WRKY proteins have been identified in several plants, but only a few have been identified in Capsicum annuum. Here, we identified a total of 62 WRKY genes in the latest pepper genome. These genes were classified into three groups (Groups 1–3) based on the structural features of their proteins. The structures of the encoded proteins, evolution, and expression under normal growth conditions were analyzed and 35 putative miRNA target sites were predicted in 20 CaWRKY genes. Moreover, the response to cold or CMV treatments of selected WRKY genes were examined to validate the roles under stresses. And alternative splicing (AS) events of some CaWRKYs were also identified under CMV infection. Promoter analysis confirmed that CaWRKY genes are involved in growth, development, and biotic or abiotic stress responses in hot pepper. The comprehensive analysis provides fundamental information for better understanding of the signaling pathways involved in the WRKY-mediated regulation of developmental processes, as well as biotic and abiotic stress responses. [ABSTRACT FROM AUTHOR]

Microvascular proliferation (MVP), an aberrant vascular structure containing multilayered mitotically active endothelial- and smooth-muscle cells/pericytes, is a histopathological hallmark of glioblastoma multiforme (GBM). Although MVP tends to be associated with high-grade glioma, it has also been detected in WHO grade I pilocytic astrocytoma (PA). However, little is known about the mechanism underlying its formation. Using TP53 point mutations as a marker for tumor-derived cells, we earlier reported that MVP was partially converted from tumor cells via mesenchymal transition. In the current study we used the KIAA1549-BRAF fusion gene as a marker to assess whether MVPs in PA contained tumor-derived cells and/or phenotypically distinct tumor cells expressing vascular markers. cDNA synthesized from frozen tissue of six PA patients operated at our institute was analyzed to detect the KIAA1549-BRAF fusion gene by reverse transcription polymerase chain reaction (RT-PCR) assay. The breakpoint in the fusion gene was identified by long and accurate PCR (LA-PCR) and Sanger sequencing of genomic DNA. Distinct tumor cells and cellular components of MVP were obtained by laser microdissection. For the qualitative and quantitative detection of the KIAA1549-BRAF fusion gene we performed genomic and digital PCR assays. Fluorescence in situ hybridization (FISH) was used to assess gene fusion in cellular components of MVP. Samples from three PA patients harbored the KIAA1549 exon 15, BRAF exon 9 fusion gene. In two patient samples with abundant MVP, RT-PCR assay detected strong bands arising from the KIAA1549-BRAF fusion gene in both tumor cells and cellular components of MVP. Digital PCR showed that vis-à-vis tumor tissue, its relative expression in cellular components of MVP was 42% in one- and 76% in another sample. FISH revealed amplified signals in both tumor cells and cellular components of MVP indicative of tandem duplication. Our findings suggest that in patients with PA, some cellular components of MVP contained tumor derived cell and/or phenotypically distinct tumor cells expressing vascular markers. [ABSTRACT FROM AUTHOR]

X-box binding protein 1 (XBP1) mRNA processing plays a crucial role in the unfolded protein response (UPR), which is activated in response to endoplasmic reticulum (ER) stress. Upon accumulation of the UPR-converted XBP1 mRNA splicing from an unspliced (u) XBP1 (inactive) isoform to the spliced (s) XBP1 (active) isoform, inositol-requiring enzyme 1 α (IRE1α) removes a 26-nucleotide intron from uXBP1 mRNA. Recent studies have reported the assessment of ER stress by examining the ratio of sXBP1 to uXBP1 mRNA (s/uXBP1 ratio) via densitometric analysis of PCR bands relative to increased levels of sXBP1 to uXBP1 using a housekeeping gene for normalization. However, this measurement is visualized by gel electrophoresis, making it very difficult to quantify differences between the two XBP1 bands and complicating data interpretation. Moreover, most commonly used housekeeping genes display an unacceptably high variable expression pattern of the s/uXBP1 ratio under different experimental conditions, such as various phases of development and different cell types, limiting their use as internal controls. For a more quantitative determination of XBP1 splicing activity, we measured the expression levels of total XBP1 (tXBP1: common region of s/uXBP1) and sXBP1 via real-time PCR using specific primer sets. We also designed universal real-time PCR primer sets capable of amplifying a portion of each u/s/tXBP1 mRNA that is highly conserved in eukaryotes, including humans, monkeys, cows, pigs, and mice. Therefore, we provide a more convenient and easily approachable quantitative real-time PCR method that can be used in various research fields to assess ER stress. [ABSTRACT FROM AUTHOR]

Sugarcane (Saccharum spp.) has a complex genome with variable ploidy and frequent aneuploidy, which hampers the understanding of phenotype and genotype relations. Despite this complexity, genome-wide association studies (GWAS) may be used to identify favorable alleles for target traits in core collections and then assist breeders in better managing crosses and selecting superior genotypes in breeding populations. Therefore, in the present study, we used a diversity panel of sugarcane, called the Brazilian Panel of Sugarcane Genotypes (BPSG), with the following objectives: (i) estimate, through a mixed model, the adjusted means and genetic parameters of the five yield traits evaluated over two harvest years; (ii) detect population structure, linkage disequilibrium (LD) and genetic diversity using simple sequence repeat (SSR) markers; (iii) perform GWAS analysis to identify marker-trait associations (MTAs); and iv) annotate the sequences giving rise to SSR markers that had fragments associated with target traits to search for putative candidate genes. The phenotypic data analysis showed that the broad-sense heritability values were above 0.48 and 0.49 for the first and second harvests, respectively. The set of 100 SSR markers produced 1,483 fragments, of which 99.5% were polymorphic. These SSR fragments were useful to estimate the most likely number of subpopulations, found to be four, and the LD in BPSG, which was stronger in the first 15 cM and present to a large extension (65 cM). Genetic diversity analysis showed that, in general, the clustering of accessions within the subpopulations was in accordance with the pedigree information. GWAS performed through a multilocus mixed model revealed 23 MTAs, six, three, seven, four and three for soluble solid content, stalk height, stalk number, stalk weight and cane yield traits, respectively. These MTAs may be validated in other populations to support sugarcane breeding programs with introgression of favorable alleles and marker-assisted selection. [ABSTRACT FROM AUTHOR]

Development of eye tissue is initiated by a conserved set of transcription factors termed retinal determination network (RDN). In the fruit fly Drosophila melanogaster, the zinc-finger transcription factor Glass acts directly downstream of the RDN to control identity of photoreceptor as well as non-photoreceptor cells. Tight control of spatial and temporal gene expression is a critical feature during development, cell-fate determination as well as maintenance of differentiated tissues. The molecular mechanisms that control expression of glass, however, remain largely unknown. We here identify complex regulatory mechanisms controlling expression of the glass locus. All information to recapitulate glass expression are contained in a compact 5.2 kb cis-acting genomic element by combining different cell-type specific and general enhancers with repressor elements. Moreover, the immature RNA of the locus contains an alternative small open reading frame (smORF) upstream of the actual glass translation start, resulting in a small peptide instead of the three possible Glass protein isoforms. CRISPR/Cas9-based mutagenesis shows that the smORF is not required for the formation of functioning photoreceptors, but is able to attenuate effects of glass misexpression. Furthermore, editing the genome to generate glass loci eliminating either one or two isoforms shows that only one of the three proteins is critical for formation of functioning photoreceptors, while removing the two other isoforms did not cause defects in developmental or photoreceptor function. Our results show that eye development and function is largely unaffected by targeted manipulations of critical features of the glass transcript, suggesting a strong selection pressure to allow the formation of a functioning eye. [ABSTRACT FROM AUTHOR]

Bioinformatics research is frequently performed using complex workflows with multiple steps, fans, merges, and conditionals. This complexity makes management of the workflow difficult on a computer cluster, especially when running in parallel on large batches of data: hundreds or thousands of samples at a time. Scientific workflow management systems could help with that. Many are now being proposed, but is there yet the “best” workflow management system for bioinformatics? Such a system would need to satisfy numerous, sometimes conflicting requirements: from ease of use, to seamless deployment at peta- and exa-scale, and portability to the cloud. We evaluated Swift/T as a candidate for such role by implementing a primary genomic variant calling workflow in the Swift/T language, focusing on workflow management, performance and scalability issues that arise from production-grade big data genomic analyses. In the process we introduced novel features into the language, which are now part of its open repository. Additionally, we formalized a set of design criteria for quality, robust, maintainable workflows that must function at-scale in a production setting, such as a large genomic sequencing facility or a major hospital system. The use of Swift/T conveys two key advantages. (1) It operates transparently in multiple cluster scheduling environments (PBS Torque, SLURM, Cray aprun environment, etc.), thus a single workflow is trivially portable across numerous clusters. (2) The leaf functions of Swift/T permit developers to easily swap executables in and out of the workflow, which makes it easy to maintain and to request resources optimal for each stage of the pipeline. While Swift/T’s data-level parallelism eliminates the need to code parallel analysis of multiple samples, it does make debugging more difficult, as is common for implicitly parallel code. Nonetheless, the language gives users a powerful and portable way to scale up analyses in many computing architectures. The code for our implementation of a variant calling workflow using Swift/T can be found on GitHub at , with full documentation provided at . [ABSTRACT FROM AUTHOR]

Recent plastid genome (plastome) studies of legumes (family Fabaceae) have shown that this family has undergone multiple atypical plastome evolutions from each of the major clades. The tribe Desmodieae belongs to the Phaseoloids, an important but systematically puzzling clade within Fabaceae. In this study, we investigated the plastome evolution of Desmodieae and analyzed its phylogenetic signaling. We sequenced six complete plastomes from representative members of Desmodieae and from its putative sister Phaseoloid genus Mucuna. Those genomes contain 128 genes and range in size from 148,450 to 153,826 bp. Analyses of gene and intron content revealed similar characters among the members of Desmodieae and Mucuna. However, there were also several distinct characters identified. The loss of the rpl2 intron was a feature shared between Desmodieae and Mucuna, whereas the loss of the rps12 intron was specific to Desmodieae. Likewise, gene loss of rps16 was observed in Mucuna but not in Desmodieae. Substantial sequence variation of ycf4 was detected from all the sequenced plastomes, but pseudogenization was restricted to the genus Desmodium. Comparative analysis of gene order revealed a distinct plastome conformation of Desmodieae compared with other Phaseoloid legumes, i.e., an inversion of an approximately 1.5-kb gene cluster (trnD-GUC, trnY-GUA, and trnE-UUC). The inversion breakpoint suggests that this event was mediated by the recombination of an 11-bp repeat motif. A phylogenetic analysis based on the plastome-scale data set found the tribe Desmodieae is a highly supported monophyletic group nested within the paraphyletic Phaseoleae, as has been found in previous phylogenetic studies. Two subtribes (Desmodiinae and Lespedezinae) of Desmodieae were also supported as monophyletic groups. Within the subtribe Lespedezinae, Lespedeza is closer to Kummerowia than Campylotropis. [ABSTRACT FROM AUTHOR]

Testicular or ovotesticular disorders of sex development (DSD) in individuals with female karyotype (XX) lacking the SRY gene has been observed in several mammalian species, including dogs. A genetic background for this abnormality has been extensively sought, and the region harboring the SOX9 gene has often been considered key in canine DSD. Three types of polymorphism have been studied in this region to date: a) copy number variation (CNV) in a region about 400 kb upstream of SOX9, named CNVR1; b) duplication of SOX9; and c) insertion of a single G-nucleotide (rs852549625) approximately 2.2 Mb upstream of SOX9. The aim of this study was thus to comprehensively analyze these polymorphisms in a large multibreed case-control cohort containing 45 XX DSD dogs, representing 23 breeds. The control set contained 57 fertile females. Droplet digital PCR (ddPCR) was used to study CNVR1 and the duplication of SOX9. Fluorescent in situ hybridization (FISH) was used to visualize copy numbers on a cellular level. The Sanger sequencing approach was performed to analyze the region harboring the G-insertion. We confirmed that CNVR1 is highly polymorphic and that copy numbers varied between 0 and 7 in the case and control cohorts. Interestingly, the number of copies was significantly higher (P = 0.038) in XX DSD dogs (mean = 2.7) than in the control females (mean = 2.0) but not in all studied breeds. Duplication of the SOX9 gene was noted only in a single XX DSD dog (an American Bully), which had three copies of SOX9. Distribution of the G-nucleotide insertion was similar in the XX DSD (frequency 0.20) and control (frequency 0.14) cohorts. Concluding, our study showed that CNVR1, located upstream of SOX9, is associated with the XX DSD phenotype, though in a breed-specific manner. Duplication of the SOX9 gene is a rare cause of this disorder in dogs. Moreover, we did not observe any association of G-insertion with the DSD phenotype. We assume that the genetic background of XX DSD can be different in certain breeds. [ABSTRACT FROM AUTHOR]

Failures in control of tan spot of pyrethrum, caused by Didymella tanaceti, has been associated with decreased sensitivity within the pathogen population to the succinate dehydrogenase inhibitor (SDHI) fungicide boscalid. Sequencing the SdhB, SdhC, and SdhD subunits of isolates with resistant and sensitive phenotypes identified 15 mutations, resulting in three amino acid substitutions in the SdhB (H277Y/R, I279V), six in the SdhC (S73P, G79R, H134R, H134Q, S135R and combined H134Q/S135R), and two in the SdhD (D112E, H122R). In vitro testing of their boscalid response and estimation of resistance factors (RF) identified isolates with wild-type (WT) Sdh genotypes were sensitive to boscalid. Isolates with SdhB-I279V, SdhC-H134Q and SdhD-D112E exhibited moderate resistance phenotypes (10 ≥ RF < 100) and isolates with SdhC-H134R exhibited very high resistance phenotypes (RF ≥ 1000). All other substitutions were associated with high resistance phenotypes (100 ≥ RF < 1000). High-resolution melt assays were designed and used to estimate the frequencies of substitutions in four field populations (n = 774) collected in August (pre-boscalid application) and November (post-boscalid application) 2012. The SdhB-H277Y, SdhC-H134R and SdhB-H277R genotypes were most frequently observed across populations at 56.7, 19.0, and 10.3%, respectively. In August 92.9% of D. tanaceti contained a substitution associated with decreased sensitivity. Following boscalid application, this increased to 98.9%, with no WT isolates detected in three fields. Overlaying previously obtained microsatellite and mating-type data revealed that all ten recurrent substitutions were associated with multiple genotypes. Thus, boscalid insensitivity in D. tanaceti appears widespread and not associated with clonal spread of a limited pool of individuals. [ABSTRACT FROM AUTHOR]

Sequencing data have been instrumental in identifying oncogenic drivers in prostatic carcinoma and highlighting biomarkers that define aggressive disease. A review of a series of 30 primary and metastatic prostatic carcinomas clinically sequenced at our cancer genomics laboratory utilizing a targeted gene panel identified recurrent structural variants in the TP53 gene. These structural variants were found in 27% of all sequenced cases and represented 36% of the cases with metastatic disease. TP53 structural rearrangements have been previously reported in a significant subset of osteosarcomas, where they result in loss of p53 protein expression by immunohistochemistry. Similarly, in our prostate cases with TP53 structural rearrangements for which tissue was available for testing, we find loss of p53 protein expression by immunohistochemistry. In the eight TP53-rearranged cases, concurrent PTEN loss was identified in 4 cases, TMPRSS2-ERG fusion in 5 cases, and AR and FOXA1 amplification in 1 case each. Our results from this small case series suggest that TP53 rearrangements with loss of expression represent a frequent alternative mechanism of inactivation of this key tumor suppressor gene with potential utility as a marker of aggressive disease. Recognition of this TP53 rearrangement pathway is essential to accurately identify prostatic carcinomas with loss of TP53 function. [ABSTRACT FROM AUTHOR]

In contrast to human cells, very few HSV-1 genes are known to be spliced, although the same pre-mRNA processing machinery is shared. Here, through global analysis of splice junctions in cells infected with HSV-1 and an HSV-1 mutant virus with deletion of infectious cell culture protein 27 (ICP27), one of two viral immediate early (IE) genes essential for viral replication, we identify hundreds of novel alternative splice junctions mapping to both previously known HSV-1 spliced genes and previously unknown spliced genes, the majority of which alter the coding potential of viral genes. Quantitative and qualitative splicing efficiency analysis of these novel alternatively spliced genes based on RNA-Seq and RT-PCR reveals that splicing at these novel splice sites is efficient only when ICP27 is absent; while in wildtype HSV-1 infected cells, the splicing of these novel splice junctions is largely silenced in a gene/sequence specific manner, suggesting that ICP27 not only promotes accumulation of ICP27 targeted transcripts but also ensures correctness of the functional coding sequences through inhibition of alternative splicing. Furthermore, ICP27 toggles expression of ICP34.5, the major viral neurovirulence factor, through inhibition of splicing and activation of a proximal polyadenylation signal (PAS) in the newly identified intron, revealing a novel regulatory mechanism for expression of a viral gene. Thus, through the viral IE protein ICP27, HSV-1 co-opts both splicing and polyadenylation machinery to achieve optimal viral gene expression during lytic infection. On the other hand, during latent infection when ICP27 is absent, HSV-1 likely takes advantages of host splicing machinery to restrict expression of randomly activated antigenic viral genes to achieve immune evasion. [ABSTRACT FROM AUTHOR]

The nosocomial pathogen Acinetobacter baumannii acquired clinical significance due to the rapid development of its multi-drug resistant (MDR) phenotype. A. baumannii strains have the ability to colonize several ecological niches including soil, water, and animals, including humans. They also survive under extremely harsh environmental conditions thriving on rare and recalcitrant carbon compounds. However, the molecular basis behind such extreme adaptability of A. baumannii is unknown. We have therefore determined the complete genome sequence of A. baumannii DS002, which was isolated from agricultural soils, and compared it with 78 complete genome sequences of A. baumannii strains having complete information on the source of their isolation. Interestingly, the genome of A. baumannii DS002 showed high similarity to the genome of A. baumannii SDF isolated from the body louse. The environmental and clinical strains, which do not share a monophyletic origin, showed the existence of a strain-specific unique gene pool that supports niche-specific survival. The strains isolated from infected samples contained a genetic repertoire with a unique gene pool coding for iron acquisition machinery, particularly those required for the biosynthesis of acinetobactin. Interestingly, these strains also contained genes required for biofilm formation. However, such gene sets were either partially or completely missing in the environmental isolates, which instead harbored genes required for alternate carbon catabolism and a TonB-dependent transport system involved in the acquisition of iron via siderophores or xenosiderophores. [ABSTRACT FROM AUTHOR]

In this study, we perform a full genome-wide association study (GWAS) to identify statistically significantly associated single nucleotide polymorphisms (SNPs) with three red blood cell (RBC) components and follow it with two independent PheWASs to examine associations between phenotypic data (case-control status of diagnoses or disease), significant SNPs, and RBC component levels. We first identified associations between the three RBC components: mean platelet volume (MPV), mean corpuscular volume (MCV), and platelet counts (PC), and the genotypes of approximately 500,000 SNPs on the Illumina Infimum DNA Human OmniExpress-24 BeadChip using a single cohort of 4,673 Northern Nevadans. Twenty-one SNPs in five major genomic regions were found to be statistically significantly associated with MPV, two regions with MCV, and one region with PC, with p<5x10-8. Twenty-nine SNPs and nine chromosomal regions were identified in 30 previous GWASs, with effect sizes of similar magnitude and direction as found in our cohort. The two strongest associations were SNP rs1354034 with MPV (p = 2.4x10-13) and rs855791 with MCV (p = 5.2x10-12). We then examined possible associations between these significant SNPs and incidence of 1,488 phenotype groups mapped from International Classification of Disease version 9 and 10 (ICD9 and ICD10) codes collected in the extensive electronic health record (EHR) database associated with Healthy Nevada Project consented participants. Further leveraging data collected in the EHR, we performed an additional PheWAS to identify associations between continuous red blood cell (RBC) component measures and incidence of specific diagnoses. The first PheWAS illuminated whether SNPs associated with RBC components in our cohort were linked with other hematologic phenotypic diagnoses or diagnoses of other nature. Although no SNPs from our GWAS were identified as strongly associated to other phenotypic components, a number of associations were identified with p-values ranging between 1x10-3 and 1x10-4 with traits such as respiratory failure, sleep disorders, hypoglycemia, hyperglyceridemia, GERD and IBS. The second PheWAS examined possible phenotypic predictors of abnormal RBC component measures: a number of hematologic phenotypes such as thrombocytopenia, anemias, hemoglobinopathies and pancytopenia were found to be strongly associated to RBC component measures; additional phenotypes such as (morbid) obesity, malaise and fatigue, alcoholism, and cirrhosis were also identified to be possible predictors of RBC component measures. [ABSTRACT FROM AUTHOR]

Paralogues pairs are more frequently observed in eels (Anguilla sp.) than in other teleosts. The paralogues often show low phylogenetic distances; however, they have been assigned to the third round of whole genome duplication (WGD), shared by all teleosts (3R), due to their conserved synteny. The apparent contradiction of low phylogenetic difference and 3R conserved synteny led us to study the duplicated gene complement of the freshwater eels. With this aim, we assembled de novo transcriptomes of two highly relevant freshwater eel species: The European (Anguilla anguilla) and the Japanese eel (Anguilla japonica). The duplicated gene complement was analysed in these transcriptomes, and in the genomes and transcriptomes of other Actinopterygii species. The study included an assessment of neutral genetic divergence (4dTv), synteny, and the phylogenetic origins and relationships of the duplicated gene complements. The analyses indicated a high accumulation of duplications (1217 paralogue pairs) among freshwater eel genes, which may have originated in a WGD event after the Elopomorpha lineage diverged from the remaining teleosts, and thus not at the 3R. However, very similar results were observed in the basal Osteoglossomorpha and Clupeocephala branches, indicating that the specific genomic regions of these paralogues may still have been under tetrasomic inheritance at the split of the teleost lineages. Therefore, two potential hypotheses may explain the results: i) The freshwater eel lineage experienced an additional WGD to 3R, and ii) Some duplicated genomic regions experienced lineage specific rediploidization after 3R in the ancestor to freshwater eels. The supporting/opposing evidence for both hypotheses is discussed. [ABSTRACT FROM AUTHOR]

The human dopamine transporter gene SLC6A3 is involved in substance use disorders (SUDs) among many other common neuropsychiatric illnesses but allelic association results including those with its classic genetic markers 3’VNTR or Int8VNTR remain mixed and unexplainable. To better understand the genetics for reproducible association signals, we report the presence of recombination hotspots based on sequencing of the entire 5’ promoter regions in two small SUDs cohorts, 30 African Americans (AAs) and 30 European Americans (EAs). Recombination rate was the highest near the transcription start site (TSS) in both cohorts. In addition, each cohort carried 57 different promoter haplotypes out of 60 and no haplotypes were shared between the two ethnicities. A quarter of the haplotypes evolved in an ethnicity-specific manner. Finally, analysis of five hundred subjects of European ancestry, from the 1000 Genome Project, confirmed the promoter recombination hotspots and also revealed several additional ones in non-coding regions only. These findings provide an explanation for the mixed results as well as guidance for selection of effective markers to be used in next generation association validation (NGAV), facilitating the delineation of pathogenic variation in this critical neuropsychiatric gene. [ABSTRACT FROM AUTHOR]

Background: Inflammatory bowel disease (IBD) is an idiopathic, chronic disorder of unclear etiology with an underlying genetic predisposition. Recent genome-wide association studies have identified more than 200 IBD susceptibility loci, but the causes of IBD remain poorly defined. We hypothesized that rare (<0.1% population frequency) gene copy number variations (CNVs) could play an important mechanism for risk of IBD. We aimed to examine changes in DNA copy number in a population-based cohort of patients with IBD and search for novel genetic risk factors for IBD. Methods: DNA samples from 243 individuals with IBD from the Manitoba IBD Cohort Study and 2988 healthy controls were analyzed using genome-wide SNP microarray technology. Three CNV calling algorithms were applied to maximize sensitivity and specificity of CNV detection. We identified IBD-associated genes affected by rare CNV from comparing the number of overlapping CNVs in IBD samples with the number of overlapping CNVs in controls for each gene. Results: 4,402 CNVs detected by two or three algorithms intersected 7,061 genes, in at least one analyzed sample. Four genes (e.g. DUSP22 and IP6K3) intersected by rare deletions and fourteen genes (e.g. SLC25A10, PSPN, GTF2F1) intersected by rare duplications demonstrated significant association with IBD (FDR-adjusted p-value < 0.01). Of these, ten genes were functionally related to immune response and intracellular signalling pathways. Some of these genes were also identified in other IBD related genome-wide association studies. These suggested that the identified genes may play a role in the risk of IBD. Conclusion: Our results revealed new genomic loci associated with IBD, which suggested the role of rare CNVs in IBD risk. [ABSTRACT FROM AUTHOR]

The Hirschsprung disease (HSCR) is an inherited disease that is controlled by multiple genes and has a complicated genetic mechanism. HSCR patients suffer from various extents of constipation due to dysplasia of the enteric nervous system (ENS), which can be so severe as to cause complete intestinal obstruction. Many genes have been identified as playing causative roles in ENS dysplasia and HSCR, among them the endothelin receptor type B gene (Ednrb) has been identified to play an important role. Mutation of Ednrb causes a series of symptoms that include deafness, pigmentary abnormalities, and aganglionosis. In our previous studies of three rat models carrying the same spotting lethal (sl) mutation on Ednrb, the haplotype of a region on chromosome (Chr) 2 was found to be responsible for the differing severities of the HSCR-like symptoms. To confirm that the haplotype of the responsible region on Chr 2 modifies the severity of aganglionosis caused by Ednrb mutation and to recreate a rat model with severe symptoms, we selected the GK inbred strain, whose haplotype in the responsible region on Chr 2 resembles that of the rat strain in which severe symptoms accompany the Ednrbsl mutation. An Ednrb mutation was introduced into the GK rat by crossing with F344-Ednrbsl and by genome editing. The null mutation of Ednrb was found to cause embryonic death in F2 progeny possessing the GK haplotype in the responsible region on Chr 2. The results of this study are unexpected, and they provide new clues and animal models that promise to contribute to studies on the genetic regulatory network in the development of ENS and on embryogenesis. [ABSTRACT FROM AUTHOR]

Genomic imprinting results in monoallelic expression of genes in mammals and flowering plants. Understanding the function of imprinted genes improves our knowledge of the regulatory processes in the genome. In this study, we have employed classification and clustering algorithms with attribute weighting to specify the unique attributes of both imprinted (monoallelic) and biallelic expressed genes. We have obtained characteristics of 22 known monoallelically expressed (imprinted) and 8 biallelic expressed genes that have been experimentally validated alongside 208 randomly selected genes in bovine (Bos taurus). Attribute weighting methods and various supervised and unsupervised algorithms in machine learning were applied. Unique characteristics were discovered and used to distinguish mono and biallelic expressed genes from each other in bovine. To obtain the accuracy of classification, 10-fold cross-validation with concerning each combination of attribute weighting (feature selection) and machine learning algorithms, was used. Our approach was able to accurately predict mono and biallelic genes using the genomics and proteomics attributes. [ABSTRACT FROM AUTHOR]

Hepatic steatosis is the most common chronic liver disease in Western countries. Both genetic and environmental factors are known as causes of the disease although their underlying mechanisms have not been fully understood. This study investigated the association of DNA methylation with oleic acid-induced hepatic steatosis. It also examined effects of food components on DNA methylation in hepatic steatosis. Genome-wide DNA methylation of oleic acid (OA)-induced lipid accumulation in vitro cell model was investigated using reduced representation bisulfite sequencing. Changes of DNA methylation were also analyzed after treatment with food components decreasing OA-induced lipid accumulation in the model. We identified total 81 regions that were hypermethylated by OA but hypomethylated by food components or vice versa. We determined the expression of seven genes proximally located at the selected differentially methylated regions. Expression levels of WDR27, GNAS, DOK7, MCF2L, PRKG1, and CMYA5 were significantly different between control vs OA and OA vs treatment with food components. We demonstrated that DNA methylation was associated with expression of genes in the model of hepatic steatosis. We also found that food components reversely changed DNA methylation induced by OA and alleviated lipid accumulation. These results suggest that DNA methylation is one of the mechanisms causing the hepatic steatosis and its regulation by food components provides insights that may prevent or alleviate lipid accumulation. [ABSTRACT FROM AUTHOR]

The computational reconstruction of genome sequences from shotgun sequencing data has been greatly simplified by the advent of sequencing technologies that generate long reads. In the case of relatively small genomes (e.g., bacterial or viral), complete genome sequences can frequently be reconstructed computationally without the need for further experiments. However, large and complex genomes, such as those of most animals and plants, continue to pose significant challenges. In such genomes, assembly software produces incomplete and fragmented reconstructions that require additional experimentally derived information and manual intervention in order to reconstruct individual chromosome arms. Recent technologies originally designed to capture chromatin structure have been shown to effectively complement sequencing data, leading to much more contiguous reconstructions of genomes than previously possible. Here, we survey these technologies and the algorithms used to assemble and analyze large eukaryotic genomes, placed within the historical context of genome scaffolding technologies that have been in existence since the dawn of the genomic era. [ABSTRACT FROM AUTHOR]

A morphospecies is defined as a taxonomic species based wholly on morphology, but often morphospecies consist of clusters of cryptic species that can be identified genetically or molecularly. The nature of the evolutionary novelty that accompanies speciation in a morphospecies is an intriguing question. Morphospecies are particularly common among ciliates, a group of unicellular eukaryotes that separates 2 kinds of nuclei—the silenced germline nucleus (micronucleus [MIC]) and the actively expressed somatic nucleus (macronucleus [MAC])—within a common cytoplasm. Because of their very similar morphologies, members of the Tetrahymena genus are considered a morphospecies. We explored the hidden genomic evolution within this genus by performing a comprehensive comparative analysis of the somatic genomes of 10 species and the germline genomes of 2 species of Tetrahymena. These species show high genetic divergence; phylogenomic analysis suggests that the genus originated about 300 million years ago (Mya). Seven universal protein domains are preferentially included among the species-specific (i.e., the youngest) Tetrahymena genes. In particular, leucine-rich repeat (LRR) genes make the largest contribution to the high level of genome divergence of the 10 species. LRR genes can be sorted into 3 different age groups. Parallel evolutionary trajectories have independently occurred among LRR genes in the different Tetrahymena species. Thousands of young LRR genes contain tandem arrays of exactly 90-bp exons. The introns separating these exons show a unique, extreme phase 2 bias, suggesting a clonal origin and successive expansions of 90-bp–exon LRR genes. Identifying LRR gene age groups allowed us to document a Tetrahymena intron length cycle. The youngest 90-bp exon LRR genes in T. thermophila are concentrated in pericentromeric and subtelomeric regions of the 5 micronuclear chromosomes, suggesting that these regions act as genome innovation centers. Copies of a Tetrahymena Long interspersed element (LINE)-like retrotransposon are very frequently found physically adjacent to 90-bp exon/intron repeat units of the youngest LRR genes. We propose that Tetrahymena species have used a massive exon-shuffling mechanism, involving unequal crossing over possibly in concert with retrotransposition, to create the unique 90-bp exon array LRR genes. [ABSTRACT FROM AUTHOR]

The invasion of the Western Hemisphere native fall armyworm (Spodoptera frugiperda; J. E. Smith) (Lepidoptera: Noctuidae) into the Eastern Hemisphere has been notable for the rapidity and geographical breadth of new detections. In the year following the first discovery in western sub-Saharan Africa in 2016, infestations have been documented in most sub-Saharan maize growing regions and has now expanded beyond Africa with populations recently reported in India. These observations could indicate a remarkable capacity for rapid establishment and long-distance dissemination. However, while fall armyworm does exhibit extended migration in North America where it annually traverses thousands of kilometers, this behavior is known to be dependent on highly favorable wind patterns and so can’t be assumed to occur in all locations. An alternative possibility is that the species has long been present in Africa, and perhaps the rest of the hemisphere, but was undetected until the enhanced monitoring that resulted after its initial discovery. Determining whether the fall armyworm in the Eastern Hemisphere is newly arrived or long pre-existing is important for assessing the risks of significant economic impacts, as the former indicates a change in pest composition while the latter does not. This study examined this issue by comparing collections from two geographically distant locations, South Africa and India. Sequence comparisons were used to quantify differences between the South Africa and India collections, assess the likelihood of their sharing a common source population, and their possible relationship with previously characterized fall armyworm from other regions of Africa. The results indicate genetic homogeneity between the South African and Indian fall armyworm populations tested and substantial similarities between these and collections from eastern Africa. The implications of these findings on fall armyworm population behavior and composition are discussed. [ABSTRACT FROM AUTHOR]

Motivation: Recently, copy number variation (CNV) has gained considerable interest as a type of genomic variation that plays an important role in complex phenotypes and disease susceptibility. Since a number of CNV detection methods have recently been developed, it is necessary to help investigators choose suitable methods for CNV detection depending on their objectives. For this reason, this study compared ten commonly used CNV detection applications, including CNVnator, ReadDepth, RDXplorer, LUMPY and Control-FREEC, benchmarking the applications by sensitivity, specificity and computational demands. Taking the DGV gold standard variants as a standard dataset, we evaluated the ten applications with real sequencing data at sequencing depths from 5X to 50X. Among the ten methods benchmarked, LUMPY performs the best for both high sensitivity and specificity at each sequencing depth. For the purpose of high specificity, Canvas is also a good choice. If high sensitivity is preferred, CNVnator and RDXplorer are better choices. Additionally, CNVnator and GROM-RD perform well for low-depth sequencing data. Our results provide a comprehensive performance evaluation for these selected CNV detection methods and facilitate future development and improvement in CNV prediction methods. [ABSTRACT FROM AUTHOR]

Drug screening studies typically involve assaying the sensitivity of a range of cancer cell lines across an array of anti-cancer therapeutics. Alongside these sensitivity measurements high dimensional molecular characterizations of the cell lines are typically available, including gene expression, copy number variation and genomic mutations. We propose a sparse multitask regression model which learns discriminative latent characteristics that predict drug sensitivity and are associated with specific molecular features. We use ideas from Bayesian nonparametrics to automatically infer the appropriate number of these latent characteristics. The resulting analysis couples high predictive performance with interpretability since each latent characteristic involves a typically small set of drugs, cell lines and genomic features. Our model uncovers a number of drug-gene sensitivity associations missed by single gene analyses. We functionally validate one such novel association: that increased expression of the cell-cycle regulator C/EBPδ decreases sensitivity to the histone deacetylase (HDAC) inhibitor panobinostat. [ABSTRACT FROM AUTHOR]

Proteins of the Fanconi Anemia (FA) complementation group are required for crosslink (CL) repair in humans and their loss leads to severe pathological phenotypes. Here we characterize a homolog of the Fe-S cluster helicase FANCJ in the model plant Arabidopsis, AtFANCJB, and show that it is involved in interstrand CL repair. It acts at a presumably early step in concert with the nuclease FAN1 but independently of the nuclease AtMUS81, and is epistatic to both error-prone and error-free post-replicative repair in Arabidopsis. The simultaneous knock out of FANCJB and the Fe-S cluster helicase RTEL1 leads to induced cell death in root meristems, indicating an important role of the enzymes in replicative DNA repair. Surprisingly, we found that AtFANCJB is involved in safeguarding rDNA stability in plants. In the absence of AtRTEL1 and AtFANCJB, we detected a synergetic reduction to about one third of the original number of 45S rDNA copies. It is tempting to speculate that the detected rDNA instability might be due to deficiencies in G-quadruplex structure resolution and might thus contribute to pathological phenotypes of certain human genetic diseases. [ABSTRACT FROM AUTHOR]

The Pol32 protein is one of the universal subunits of DNA polymerase δ (Pol δ), which is responsible for genome replication in eukaryotic cells. Although the role of Pol32 in DNA repair has been well-characterized, its exact function in genome replication remains obscure as studies in single cell systems have not established an essential role for Pol32 in the process. Here we characterize Pol32 in the context of Drosophila melanogaster development. In the rapidly dividing embryonic cells, loss of Pol32 halts genome replication as it specifically disrupts Pol δ localization to the nucleus. This function of Pol32 in facilitating the nuclear import of Pol δ would be similar to that of accessory subunits of DNA polymerases from mammalian Herpes viruses. In post-embryonic cells, loss of Pol32 reveals mitotic fragile sites in the Drosophila genome, a defect more consistent with Pol32’s role as a polymerase processivity factor. Interestingly, these fragile sites do not favor repetitive sequences in heterochromatin, with the rDNA locus being a striking exception. Our study uncovers a possibly universal function for DNA polymerase ancillary factors and establishes a powerful system for the study of chromosomal fragile sites in a non-mammalian organism. [ABSTRACT FROM AUTHOR]