Genes Genomics

SM-RCNV: a statistical method to detect recurrent copy number variations in sequenced samples Abstract Background Copy number variation (CNV) is an important form of genomic structural variation and is linked to dozens of human diseases. Using next-generation sequencing (NGS) data and developing computational methods to characterize such structural variants is significant for understanding the mechanisms of diseases. Objective The objective of this study is to develop a new statistical method of detection recurrent CNVs across multiple samples from genomic sequences. Methods A statistical method is carried out to detect recurrent CNVs, referred to as SM-RCNV. This method uses a statistic associated with each location by combining the frequency of variation at one location across whole samples and the correlation among consecutive locations. The weights of the frequency and correlation are trained using real datasets with known CNVs. P-value is assessed for each location on the genome by permutation testing. Results Compared with six peer methods, SM-RCNV outperforms the peer methods under receiver operating characteristic curves. SM-RCNV successfully identifies many consistent recurrent CNVs, most of which are known to be of biological significance and associated with diseased genes. The validation rate of SM-RCNV in the CEU call set and YRI call set with Database of Genomic Variants are 258/328 (79%) and (157/309) 51%, respectively. Conclusion SM-RCNV is a well-grounded statistical framework for detecting recurrent CNVs from multiple genomic sequences, providing valuable information to study genomes in human diseases. The source code is freely available at https://sourceforge.net/projects/sm-rcnv/.

Enzymatic construction of shRNA library from oligonucleotide library Abstract Background Short hairpin RNAs (shRNAs) expressed from vectors have been used as an effective means of exploiting the RNA interference (RNAi) pathway in mammalian cells. Genome-scale screening with shRNA libraries has been used to investigate the relationship between genotypes and phenotypes on a large scale. Although several methods have been developed to construct shRNA libraries, their broad application has been limited by the high cost of constructing these libraries. Objective We develop a new method that efficiently constructs a shRNA library at low cost, using treatments with several enzymes and an oligonucleotide library. Methods The library of shRNA expression cassettes, which were cloned into a lentiviral plasmid, was produced through several enzymatic reactions, starting from a library of 20,000 different short oligonucleotides produced by microarray-based oligonucleotide synthesis. Results The NGS sequence analysis of the library shows that 99.8% of them (19,956 from 20,000 sequences) were contained in the library: 63.2% of them represent the correct sequences and the rest showed one or two base pair differences from the expected sequences. Conclusion Considering the ease of our method, shRNA libraries of new genomes and of specific populations of genes can be prepared in a short period of time for genome-scale RNAi library screening.

Comparative transcriptome analyses provide insights into the adaptation mechanisms to acute salt stresses in juvenile Sinonovacula constricta Abstract Background Sinonovacula constricta is an economically important bivalve species in China, Korea and Japan that widely resides in estuarine and coastal areas where salinity fluctuates rapidly. However, little is known about its adaptation mechanisms to acute salt stresses. Objective To reveal the underlying molecular mechanisms involved in acute salt stresses in juvenile S. constricta. Methods Nine cDNA libraries (triplicate each trial) were established from juvenile S. constricta, which were subjected to low salinity (5 psu), optimal salinity (15 psu) and high salinity (25 psu) for 6 h, respectively. Results Illumina sequencing generated 478,587,310 clean reads totally, which were assembled into 427,057 transcripts of 246,672 unigenes. Compared with the control, 1259 and 2163 differentially expressed genes (DEGs) were identified under acute low and high salt stresses, respectively. GO and KEGG enrichment analyses of DEGs revealed that several key metabolic modulations were mainly responsible for the acute salt stresses. According to the significantly highlighted KEGG pathways, some key DEGs were identified and discussed in details. Notably, based on which, some potential osmolytes were further speculated. Conclusion Here, we carried out a unique report of comparative transcriptome analyses in juvenile S. constricta in response to acute salt stresses. The identified DEGs and their significantly enriched GO terms and KEGG pathways were critical for understanding and further investigating the underlying the physical and biochemical performances, and ultimately facilitated S. constricta breeding. Besides, the transcriptome data greatly enriched the genetic information of S. constricta, which were valuable for promoting its molecular biology researches.

Genetic risk score combining six genetic variants associated with the cellular NRF2 expression levels correlates with Type 2 diabetes in the human population Abstract Background Type 2 diabetes (T2D) is known as an inflammatory disease. NRF2 (Nuclear Factor Erythroid 2 Like2) encodes a transcription factor that binds to antioxidant response elements (AREs) and regulates the expression of genes involved in many antioxidant responses. Objective This study aimed to gain insight into individual anti-inflammatory activity to prevent T2D development in humans. Methods We performed a genome-wide association study (GWAS) to identify genetic variants influencing NRF2 expression in LCLs (lymphoblastoid cell lines) generated from 74 different individuals. Association analyses between T2D or its related traits and genetic risk score (GRS) calculated by combining genetic variants detected from GWAS for cellular NRF2 expression were performed using data from 8715 subjects. The T2D prediction model using GRS was evaluated by measuring the area under the curve (AUC) of the receiver operating characteristics (ROC) curve. Results Our GWAS identified six genetic variants (SNP) showing suggestive evidence of associations with cellular NRF2 expression (P < 10− 6). Logistic regression analysis demonstrated that GRS was associated with an increased risk of T2D (P value = 0.003, OR = 1.13). In addition, linear regression analyses showed positive associations between GRS and fasting glucose (P value = 0.028, β = 0.62), 2-h glucose (P value = 0.0004, β = 1.13) and HbA1C (P value = 0.033, β = 0.03). In the T2D prediction model using GRS, the AUC of the ROC curve was 0.69. Conclusion This study highlights genetic variants associated with cellular NRF2 expression and suggests that the GRS of NRF2 expression-associated variants is likely to be a useful indicator of T2D development in the human population.

Markov chain Monte Carlo simulation of a Bayesian mixture model for gene network inference Abstract Background Simultaneous measurement of gene expression level for thousands of genes contains the rich information about many different aspects of biological mechanisms. A major computational challenge is to find methods to extract new biological insights from this wealth of data. Complex biological processes are often regulated under the various conditions or circumstances and associated gene interactions are dynamically changed depending on different biological contexts. Thus, inference of such dynamic relationships between genes with consideration of biological conditions is very challenging. Method In this study, we propose a comprehensive and integrated approach to infer the dynamic relationships between genes and evaluate this approach on three distinct gene networks. Results This study demonstrates the advantage of integrating Markov chain Monte Carlo (MCMC) simulation into a Bayesian mixture model to overcome the high-dimension, low sample size (HDLSS) problem as well as to identify context-specific biological modules. Such biological modules were identified through the summarization of sampled network structures obtained from MCMC simulation. Conclusion This novel approach gives a comprehensive understanding of the dynamically regulated biological modules.

Identification of a novel plant RNA virus species of the genus Amalgavirus in the family Amalgaviridae from chia ( Salvia hispanica ) Abstract Background Chia (Salvia hispanica) is a flowering plant in the family Lamiaceae, which produces seeds that are a rich source of various nutritional compounds. Objective To identify a novel RNA virus potentially associated with chia. Methods Transcriptome data obtained from developing chia seeds were assembled into contigs. Sequence contigs containing an open reading frame (ORF) that showed amino acid identities with a viral RNA-dependent RNA polymerase (RdRp) were identified and analyzed. Results A genomic sequence of a novel plant RNA virus named Salvia hispanica RNA virus 1 (ShRV1) was identified in a chia seed transcriptome dataset. The ShRV1 genome sequence has two ORFs that showed high sequence identities with ORFs of known members of the genus Amalgavirus in the family Amalgaviridae. Amalgaviridae is a family of positive-sense double-stranded non-segmented RNA viruses that infect plants, fungi, and animals. The ShRV1 genome encodes two proteins: a putative replication factory matrix-like protein from ORF1 and an RdRp from the fused ORF of ORF1 and ORF2 by a + 1 programmed ribosomal frameshifting (PRF) mechanism. A conserved + 1 PRF motif sequence UUU_CGU was found at the ORF1/ORF2 boundary. A comparison of 31 amalgavirus ORF1 + 2 fusion proteins revealed that only three positions were repeatedly used as a + 1 PRF site during amalgavirus evolution. Conclusion ShRV1 is a novel virus found to be associated with chia and may be useful for studying the molecular features of amalgaviruses.

Inhibition of TNFα-induced interleukin-6 gene expression by barley ( Hordeum vulgare ) ethanol extract in BV-2 microglia Abstract Background Inflammation in the central nervous system is closely associated with pathological neurodegenerative diseases as well as psychiatric disorders. Prolonged activation of microglia can produce many inflammatory mediators, which may result in pathological neurotoxic side effects. Interleukin (IL)-6 serves as a hallmark of the injured brain. Objective Whole grains are known to contain many bioactive components. However, little information is available about anti-neuroinflammatory effects of grains in the CNS. This study aims to investigate the effect of Hordeum vulgareethanol extract (HVE) on the suppression of IL-6 expression in BV2 microglia. Methods Inhibitory effects of HVE on IL-6 expression were analyzed by immunoblot anaysis, immunofluoresce microscopic analysis, reverse transcription-polymerase chain reaction, and luciferase promoter reporter assay. Results HVE inhibited TNFα-induced phosphorylation of IKKα/β, IκB, and p65/RelA NF-κB. TNFα-induced IL-6 mRNA expression and promoter activity were reduced by HVE. Point mutation of NF-κB-binding site within the IL-6 gene promoter abolished TNFα-induced reporter activity, whereas exogenous expression of p65 NF-κB enhanced IL-6 promoter activity. Conclusion NF-κB-binding site within the IL-6 promoter region is a HVE target element involved in the inhibition of TNFα-induced IL-6 gene transcription. HVE inhibits TNFα-induced IL-6 expression via suppression of NF-κB signaling in BV2 microglial cells.

Molt-dependent transcriptome analysis of claw muscles in Chinese mitten crab Eriocheir sinensis Abstract Background Molting is a critical developmental process for crustaceans, during which the claw muscles undergo periodic atrophy and restoration. But the mechanism underlying this special muscle reshuffle around ecdysis is not yet thoroughly understood. Objective To investigate the molecular mechanism underlying the muscle's reshuffle over the molting cycle of Chinese mitten crab Eriocheir sinensis. Methods The Illumina high-throughput sequencing technique were used to sequence the transcriptome of the whole claw muscles from Chinese mitten crab Eriocheir sinensis in three molting stages (inter-molt C stage, pre-molt D3−4 and post-molt A–B stage); the de novo assembly, annotation and functional evaluation of the contigs were performed with bioinformatics tools. Results Totally 129,149 unigenes, 128,190 CDS, 33,770 SSRs and a large number of SNP sites were obtained, and 3700 and 12,771 differentially expressed genes (DEGs) were identified respectively in A–B and D3−4 stage compared with that in C stage. The identified DEGs were enriched to 746 and 1 408 GO terms respectively in A–B and D3–4 stage compared with C stage (p ≤ 0.05). KEGG pathway analysis showed that the DEGs were significantly enriched in 14 and 11 pathways in A–B vs C comparison and D3−4 vs C comparison (p ≤ 0.05), respectively. These DEGs are involved in several biological processes critical for the animal's growth and development, such as gene expression, protein synthesis, muscle development, new cuticle reconstruction, oxidation–reduction process and glycolytic process. Conclusion The data generated in this study is the first transcriptomic resource from the muscles of Chinese mitten crab, which would facilitate to characterize key molecular processes underlying crab muscle's growth and development during the molting cycles.

Pattern recognition receptors and their interactions with bacterial type III effectors in plants Abstract Innate immune signaling of plants is initiated by pattern recognition receptors (PRRs) at the plasma membrane. Upon pathogen attack, PRRs recognize pathogen-associated molecular patterns (PAMPs) via ectodomain and lead to signaling cascade via cytoplasmic kinase domain. PAMP-triggered immunity (PTI) activates basal defense responses sufficient to confer broad-spectrum disease resistance by inhibiting pathogen entry and growth. On the other hand, one of the major virulence factors in plant-pathogenic bacteria is type III secretion system, which can deliver effector proteins into the host cell and modulate host cellular processes. Most type III effectors are implicated in PTI suppression, and PRRs have been identified as targets of multiple type III effectors. Mutants defective in T3SS lack pathogenicity in many bacterial species, revealing that T3SS-mediated PTI suppression is critical for host colonization and subsequent disease development. This review summarizes molecular basis of bacterial pathogen perception by plant PRRs and also interaction between PRRs and type III effectors during early stages of plant-pathogen interaction.

De novo transcriptomic analysis of gonad of Strongylocentrotus nudus and gene discovery for biosynthesis of polyunsaturated fatty acids Abstract Background Strongylocentrotus nudus is an important cultured sea urchin species in north China, because its gonad is rich in unsaturated fatty acids, particularly long polyunsaturated fatty acids (LC-PUFAs). These PUFAs play pleiotropic and crucial roles in a wide range of biological process. Objective However, the genes contributing to biosynthesis PUFAs have not been elucidated yet, and the molecular mechanism relative to the difference in PUFA composition between male and female gonad as been revealed but the corresponding has not been understood. Methods In this paper, solexa sequencing based transcriptomic approach was used to identify and characterize the key genes relative to PUFA synthesis and further conducted different expressed genes between male and female gonad. Results A total of 130,124 transcripts and 189330 unigenes were de novo assembled from 64.32 Gb data. Next, these unigenes were subjected to functional annotation by mapping to six public databases, and this process revealed a lot of genes involving in lipid metabolism. In addition, three types of fatty acids front-end desaturase and three species of very long fatty acids elongase were identified and the pathway for PUFA biosynthesis was hypothesized. Last, comparative analysis revealed the higher expression level of Δ5 desaturase, Δ6 desaturase, ELOVL-4, -6 and -7 in male gonad compared with female. Conclusion This results could plausible explain the differ in composition of PUFAs between male and female gonad of sea urchin.