DAVID: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update).

DAVID is a popular bioinformatics resource system including a web server and web service for functional annotation and enrichment analyses of gene lists. It consists of a comprehensive knowledgebase and a set of functional analysis tools. Here, we report all updates made in 2021. The DAVID Gene system was rebuilt to gain coverage of more organisms, which increased the taxonomy coverage from 17 399 to 55 464. All existing annotation types have been updated, if available, based on the new DAVID Gene system. Compared with the last version, the number of gene-term records for most annotation types within the updated Knowledgebase have significantly increased. Moreover, we have incorporated new annotations in the Knowledgebase including small molecule-gene interactions from PubChem, drug-gene interactions from DrugBank, tissue expression information from the Human Protein Atlas, disease information from DisGeNET, and pathways from WikiPathways and PathBank. Eight of ten subgroups split from Uniprot Keyword annotation were assigned to specific types. Finally, we added a species parameter for uploading a list of gene symbols to minimize the ambiguity between species, which increases the efficiency of the list upload and eliminates confusion for users. These current updates have significantly expanded the Knowledgebase and enhanced the discovery power of DAVID.

QuasiSeq: profiling viral quasispecies via self-tuning spectral clustering with PacBio long sequencing reads.

MOTIVATION: The existence of quasispecies in the viral population causes difficulties for disease prevention and treatment. High-throughput sequencing provides opportunity to determine rare quasispecies and long sequencing reads covering full genomes reduce quasispecies determination to a clustering problem. The challenge is high similarity of quasispecies and high error rate of long sequencing reads. RESULTS: We developed QuasiSeq using a novel signature-based self-tuning clustering method, SigClust, to profile viral mixtures with high accuracy and sensitivity. QuasiSeq can correctly identify quasispecies even using low-quality sequencing reads (accuracy <80%) and produce quasispecies sequences with high accuracy (≥99.55%). Using high-quality circular consensus sequencing reads, QuasiSeq can produce quasispecies sequences with 100% accuracy. QuasiSeq has higher sensitivity and specificity than similar published software. Moreover, the requirement of the computational resource can be controlled by the size of the signature, which makes it possible to handle big sequencing data for rare quasispecies discovery. Furthermore, parallel computation is implemented to process the clusters and further reduce the runtime. Finally, we developed a web interface for the QuasiSeq workflow with simple parameter settings based on the quality of sequencing data, making it easy to use for users without advanced data science skills. AVAILABILITY AND IMPLEMENTATION: QuasiSeq is open source and freely available at https://github.com/LHRI-Bioinformatics/QuasiSeq. The current release (v1.0.0) is archived and available at https://zenodo.org/badge/latestdoi/340494542. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Meta-Analysis of CTLA-4 +49 Gene Polymorphism and Susceptibility to Graves' Disease.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) plays an important role in the initiation and development of Graves' disease (GD), especially CTLA-4 +49A/G polymorphism and genetic susceptibility to GD. However, the current conclusions are consistent. Therefore, this study adopted meta-analysis method to assess the exact correlation between this polymorphism and GD risk. METHODS: Literature searches were performed in PubMed, Embase, Web of Science, China National Knowledge Infrastructure, China Wanfang, and other databases for data on the correlation between polymorphisms of CTLA-4 +49A/G and GD risk. The end date for publications was May 2020. Stata 15.0 software was used for data analysis. RESULTS: A total of 33 papers were included, covering 8,555 cases and 9,533 controls. The results showed that the CTLA-4 +49 allele G compared to allele A, odds ratio (OR) = 1.62, 95% CI [1.56, 2.09]. In the dominant, recessive, homozygous, and heterozygous genetic models, comparing the GD group and the control group, the combined OR was 1.81, 95% CI [1.56, 2.09]; 1.91, 95% CI [1.66, 2.21]; 2.75, 95% CI, [2.21, 3.41]; and 1.49, 95% CI [1.29, 1.71], respectively. When it was analyzed by dividing genetic models into subgroups according to ethnicity and published year, the data the of genetic model in each subgroup were also statistically significant. CONCLUSION: The CTLA-4 +49A/G polymorphism was strongly associated with genetic susceptibility to GD. Allele G, genotypes GG, GG + GA, and GA are correlated with an increase in the risk of GD.

Ontogenetic Expression of Lpin2 and Lpin3 Genes and Their Associations with Traits in Two Breeds of Chinese Fat-tailed Sheep.

Lipins play dual function in lipid metabolism by serving as phosphatidate phosphatase and transcriptional co-regulators of gene expression. Mammalian lipin proteins consist of lipin1, lipin2, and lipin3 and are encoded by their respective genes Lpin1, Lpin2, and Lpin3. To date, most studies are concerned with Lpin1, only a few have addressed Lpin2 and Lpin3. Ontogenetic expression of Lpin2 and Lpin3 and their associations with traits would help to explore their molecular and physiological functions in sheep. In this study, 48 animals with an equal number of males and females each for both breeds of fat-tailed sheep such as Guangling Large Tailed (GLT) and Small Tailed Han (STH) were chosen to evaluate the ontogenetic expression of Lpin2 and Lpin3 from eight different tissues and months of age by quantitative real-time polymerase chain reaction (PCR). Associations between gene expression and slaughter and tail traits were also analyzed. The results showed that Lpin2 mRNA was highly expressed in perirenal and tail fats, and was also substantially expressed in liver, kidney, reproductive organs (testis and ovary), with the lowest levels in small intestine and femoral biceps. Lpin3 mRNA was prominently expressed in liver and small intestine, and was also expressed at high levels in kidney, perirenal and tail fats as well as reproductive organs (testis and ovary), with the lowest level in femoral biceps. Global expression of Lpin2 and Lpin3 in GLT both were significantly higher than those in STH. Spatiotemporal expression showed that the highest levels of Lpin2 expression occurred at 10 months of age in two breeds of sheep, with the lowest expression at 2 months of age in STH and at 8 months of age in GLT. The greatest levels of Lpin3 expression occurred at 4 months of age in STH and at 10 months of age in GLT, with the lowest expression at 12 months of age in STH and at 8 months of age in GLT. Breed and age significantly influenced the tissue expression patterns of Lpin2 and Lpin3, respectively, and sex significantly influenced the spatiotemporal expression patterns of Lpin3. Meanwhile, Lpin2 and Lpin3 mRNA expression both showed significant correlations with slaughter and tail traits, and the associations appear to be related with the ontogenetic expression as well as the potential functions of lipin2 and lipin3 in sheep.

DAVID-WS: a stateful web service to facilitate gene/protein list analysis.

SUMMARY: The database for annotation, visualization and integrated discovery (DAVID), which can be freely accessed at http://david.abcc.ncifcrf.gov/, is a web-based online bioinformatics resource that aims to provide tools for the functional interpretation of large lists of genes/proteins. It has been used by researchers from more than 5000 institutes worldwide, with a daily submission rate of ∼1200 gene lists from ∼400 unique researchers, and has been cited by more than 6000 scientific publications. However, the current web interface does not support programmatic access to DAVID, and the uniform resource locator (URL)-based application programming interface (API) has a limit on URL size and is stateless in nature as it uses URL request and response messages to communicate with the server, without keeping any state-related details. DAVID-WS (web service) has been developed to automate user tasks by providing stateful web services to access DAVID programmatically without the need for human interactions. AVAILABILITY: The web service and sample clients (written in Java, Perl, Python and Matlab) are made freely available under the DAVID License at http://david.abcc.ncifcrf.gov/content.jsp?file=WS.html.

[Determination of trace element contents in Urtica laetevirens Maxim. reaped in different months by ICP-MS].

The contents of twenty kinds of trace elements, Al, Ba, Ca, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Mo, Na, Ni, P, Pb, S and Zn, were determined by ICP-MS with microwave digestion in Urtica laetevirens Maxim. The recovery (n=7) is 95.4%-101.2%, and the RSD (n=7) is 1.2%-5.3%. The contents of K, P, S, Ca and Na in the samples were abundant while Fe, Mg, Mn and Zn were less abundant. The contents of Al, Cr and Pb which were harmful to human were kept at low level. The contents of trace elements in Urtica laetevirens Maxim. also showed obvious seasonal dynamics. This result provides some theoretical basis for the study of internal relations between trace elements in Urtica laetevirens Maxim. and its efficacy. It's also useful for better development and utilization of the resource.

Complete Genome Sequence of Herpes Simplex Virus 2 Strain G.

Herpes simplex virus type 2 (HSV-2) is a common causative agent of genital tract infections. Moreover, HSV-2 and HIV infection can mutually increase the risk of acquiring another virus infection. Due to the high GC content and highly repetitive regions in HSV-2 genomes, only the genomes of four strains have been completely sequenced (HG52, 333, SD90e, and MS). Strain G is commonly used for HSV-2 research, but only a partial genome sequence has been assembled with Illumina sequencing reads. In the current study, we de novo assembled and annotated the complete genome of strain G using PacBio long sequencing reads, which can span the repetitive regions, analyzed the 'α' sequence, which plays key roles in HSV-2 genome circulation, replication, cleavage, and packaging of progeny viral DNA, identified the packaging signals homologous to HSV-1 within the 'α' sequence, and determined both termini of the linear genome and cleavage site for the process of concatemeric HSV-2 DNA produced via rolling-circle replication. In addition, using Oxford Nanopore Technology sequencing reads, we visualized four HSV-2 genome isomers at the nucleotide level for the first time. Furthermore, the coding sequences of HSV-2 strain G have been compared with those of HG52, 333, and MS. Moreover, phylogenetic analysis of strain G and other diverse HSV-2 strains has been conducted to determine their evolutionary relationship. The results will aid clinical research and treatment development of HSV-2.

Complete Genome Sequence of Herpes Simplex Virus 1 Strain McKrae.

Herpes simplex virus 1 (HSV-1) strain McKrae is highly virulent and relatively neuroinvasive in animal models compared with other wild-type HSV-1 strains. To identify the genetic determinants that lead to the unique phenotypes of the McKrae strain, we sequenced its genome with PacBio single-molecule real-time (SMRT) technology and resolved the complete sequence.

Complete Genome Sequence of Herpes Simplex Virus 1 Strain MacIntyre.

Herpes simplex virus type 1 (HSV-1) strain MacIntyre has a severe defect in the anterograde spread after replication in the nucleus. To better understand and identify the genetic determinants that lead to the unique phenotypes of the MacIntyre strain, we sequenced its genome with PacBio single-molecule real-time sequencing technology and resolved the complete sequence.

Gene and pathway identification with Lp penalized Bayesian logistic regression.

BACKGROUND: Identifying genes and pathways associated with diseases such as cancer has been a subject of considerable research in recent years in the area of bioinformatics and computational biology. It has been demonstrated that the magnitude of differential expression does not necessarily indicate biological significance. Even a very small change in the expression of particular gene may have dramatic physiological consequences if the protein encoded by this gene plays a catalytic role in a specific cell function. Moreover, highly correlated genes may function together on the same pathway biologically. Finally, in sparse logistic regression with Lp (p < 1) penalty, the degree of the sparsity obtained is determined by the value of the regularization parameter. Usually this parameter must be carefully tuned through cross-validation, which is time consuming. RESULTS: In this paper, we proposed a simple Bayesian approach to integrate the regularization parameter out analytically using a new prior. Therefore, there is no longer a need for parameter selection, as it is eliminated entirely from the model. The proposed algorithm (BLpLog) is typically two or three orders of magnitude faster than the original algorithm and free from bias in performance estimation. We also define a novel similarity measure and develop an integrated algorithm to hunt the regulatory genes with low expression changes but having high correlation with the selected genes. Pathways of those correlated genes were identified with DAVID http://david.abcc.ncifcrf.gov/. CONCLUSION: Experimental results with gene expression data demonstrate that the proposed methods can be utilized to identify important genes and pathways that are related to cancer and build a parsimonious model for future patient predictions.

STING is an essential mediator of the Ku70-mediated production of IFN-λ1 in response to exogenous DNA.

We previously identified Ku70, a subunit of a DNA repair protein complex, as a cytosolic DNA sensor that induces the production of interferon-λ1 (IFN-λ1) by human primary cells and cell lines. IFN-λ1 is a type III IFN and has similar antiviral activity to that of the type I IFNs (IFN-α and IFN-ß). We observed that human embryonic kidney (HEK) 293T cells, which are deficient in the innate immune adaptor protein STING (stimulator of IFN genes), did not produce IFN-λ1 in response to DNA unless they were reconstituted with STING. Conversely, parental HEK 293 cells produced IFN-λ1 after they were exposed to exogenous DNA; however, when STING was knocked out in the HEK 293 cells through the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system, they lost this response. Through confocal microscopy, we demonstrated that endogenous Ku70 was located in the nucleus and then translocated to the cytoplasm upon DNA exposure to form a complex with STING. Additionally, the DNA binding domain of Ku70 was essential for formation of the Ku70-STING complex. Knocking down STING in primary human macrophages inhibited their ability to produce IFN-λ1 in response to transfection with DNA or infection with the DNA virus HSV-2 (herpes simplex virus-2). Together, these data suggest that STING mediates the Ku70-mediated IFN-λ1 innate immune response to exogenous DNA or DNA virus infection.

Towards Better Precision Medicine: PacBio Single-Molecule Long Reads Resolve the Interpretation of HIV Drug Resistant Mutation Profiles at Explicit Quasispecies (Haplotype) Level.

Development of HIV-1 drug resistance mutations (HDRMs) is one of the major reasons for the clinical failure of antiretroviral therapy. Treatment success rates can be improved by applying personalized anti-HIV regimens based on a patient's HDRM profile. However, the sensitivity and specificity of the HDRM profile is limited by the methods used for detection. Sanger-based sequencing technology has traditionally been used for determining HDRM profiles at the single nucleotide variant (SNV) level, but with a sensitivity of only ≥ 20% in the HIV population of a patient. Next Generation Sequencing (NGS) technologies offer greater detection sensitivity (~ 1%) and larger scope (hundreds of samples per run). However, NGS technologies produce reads that are too short to enable the detection of the physical linkages of individual SNVs across the haplotype of each HIV strain present. In this article, we demonstrate that the single-molecule long reads generated using the Third Generation Sequencer (TGS), PacBio RS II, along with the appropriate bioinformatics analysis method, can resolve the HDRM profile at a more advanced quasispecies level. The case studies on patients' HIV samples showed that the quasispecies view produced using the PacBio method offered greater detection sensitivity and was more comprehensive for understanding HDRM situations, which is complement to both Sanger and NGS technologies. In conclusion, the PacBio method, providing a promising new quasispecies level of HDRM profiling, may effect an important change in the field of HIV drug resistance research.

Genome analysis of three Pneumocystis species reveals adaptation mechanisms to life exclusively in mammalian hosts.

Pneumocystis jirovecii is a major cause of life-threatening pneumonia in immunosuppressed patients including transplant recipients and those with HIV/AIDS, yet surprisingly little is known about the biology of this fungal pathogen. Here we report near complete genome assemblies for three Pneumocystis species that infect humans, rats and mice. Pneumocystis genomes are highly compact relative to other fungi, with substantial reductions of ribosomal RNA genes, transporters, transcription factors and many metabolic pathways, but contain expansions of surface proteins, especially a unique and complex surface glycoprotein superfamily, as well as proteases and RNA processing proteins. Unexpectedly, the key fungal cell wall components chitin and outer chain N-mannans are absent, based on genome content and experimental validation. Our findings suggest that Pneumocystis has developed unique mechanisms of adaptation to life exclusively in mammalian hosts, including dependence on the lungs for gas and nutrients and highly efficient strategies to escape both host innate and acquired immune defenses.

A Benchmark Study on Error Assessment and Quality Control of CCS Reads Derived from the PacBio RS.

PacBio RS, a newly emerging third-generation DNA sequencing platform, is based on a real-time, single-molecule, nano-nitch sequencing technology that can generate very long reads (up to 20-kb) in contrast to the shorter reads produced by the first and second generation sequencing technologies. As a new platform, it is important to assess the sequencing error rate, as well as the quality control (QC) parameters associated with the PacBio sequence data. In this study, a mixture of 10 prior known, closely related DNA amplicons were sequenced using the PacBio RS sequencing platform. After aligning Circular Consensus Sequence (CCS) reads derived from the above sequencing experiment to the known reference sequences, we found that the median error rate was 2.5% without read QC, and improved to 1.3% with an SVM based multi-parameter QC method. In addition, a De Novo assembly was used as a downstream application to evaluate the effects of different QC approaches. This benchmark study indicates that even though CCS reads are post error-corrected it is still necessary to perform appropriate QC on CCS reads in order to produce successful downstream bioinformatics analytical results.