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Simple Summary: We show how the relationship between the sub-network entropy of malignant up-regulated genes in twelve different types of cancer, spanning the entire spectrum of 5-year overall survival rates, can serve as a benchmark for optimizing RNA-seq workflows. Assessing the Shannon entropy of sub-networks formed by malignant up-regulated genes by several RNA-seq workflow approaches, such as DESeq2 and edgeR, but also by evaluating nine normalization methods on paired samples of TCGA RNA-seq, we found that the pipeline incorporating TPM normalization coupled with log2 fold change yielded the best correlation coefficient between cancer aggressiveness and tumor entropy. RNA-seq faces persistent challenges due to the ongoing, expanding array of data processing workflows, none of which have yet achieved standardization to date. It is imperative to determine which method most effectively preserves biological facts. Here, we used Shannon entropy as a tool for depicting the biological status of a system. Thus, we assessed the measurement of Shannon entropy by several RNA-seq workflow approaches, such as DESeq2 and edgeR, but also by combining nine normalization methods with log2 fold change on paired samples of TCGA RNA-seq representing datasets of 515 patients and spanning 12 different cancer types with 5-year overall survival rates ranging from 20% to 98%. Our analysis revealed that TPM, RLE, and TMM normalization, coupled with a threshold of log2 fold change ≥1, for identifying differentially expressed genes, yielded the best results. We propose that Shannon entropy can serve as an objective metric for refining the optimization of RNA-seq workflows and mRNA sequencing technologies. [ABSTRACT FROM AUTHOR]

Abstract Background Multiple studies have demonstrated a negative correlation between gene expression and positioning of genes at the nuclear envelope (NE) lined by nuclear lamina, but the exact relationship remains unclear, especially in light of the highly stochastic, transient nature of the gene association with the NE. Results In this paper, we ask whether there is a causal, systematic, genome-wide relationship between the expression levels of the groups of genes in topologically associating domains (TADs) of Drosophila nuclei and the probabilities of TADs to be found at the NE. To investigate the nature of this possible relationship, we combine a coarse-grained dynamic model of the entire Drosophila nucleus with genome-wide gene expression data; we analyze the TAD averaged transcription levels of genes against the probabilities of individual TADs to be in contact with the NE in the control and lamins-depleted nuclei. Our findings demonstrate that, within the statistical error margin, the stochastic positioning of Drosophila melanogaster TADs at the NE does not, by itself, systematically affect the mean level of gene expression in these TADs, while the expected negative correlation is confirmed. The correlation is weak and disappears completely for TADs not containing lamina-associated domains (LADs) or TADs containing LADs, considered separately. Verifiable hypotheses regarding the underlying mechanism for the presence of the correlation without causality are discussed. These include the possibility that the epigenetic marks and affinity to the NE of a TAD are determined by various non-mutually exclusive mechanisms and remain relatively stable during interphase. Conclusions At the level of TADs, the probability of chromatin being in contact with the nuclear envelope has no systematic, causal effect on the transcription level in Drosophila. The conclusion is reached by combining model-derived time-evolution of TAD locations within the nucleus with their experimental gene expression levels.

Background: Multiple studies have demonstrated a negative correlation between gene expression and positioning of genes at the nuclear envelope (NE) lined by nuclear lamina, but the exact relationship remains unclear, especially in light of the highly stochastic, transient nature of the gene association with the NE. Results: In this paper, we ask whether there is a causal, systematic, genome-wide relationship between the expression levels of the groups of genes in topologically associating domains (TADs) of Drosophila nuclei and the probabilities of TADs to be found at the NE. To investigate the nature of this possible relationship, we combine a coarse-grained dynamic model of the entire Drosophila nucleus with genome-wide gene expression data; we analyze the TAD averaged transcription levels of genes against the probabilities of individual TADs to be in contact with the NE in the control and lamins-depleted nuclei. Our findings demonstrate that, within the statistical error margin, the stochastic positioning of Drosophila melanogaster TADs at the NE does not, by itself, systematically affect the mean level of gene expression in these TADs, while the expected negative correlation is confirmed. The correlation is weak and disappears completely for TADs not containing lamina-associated domains (LADs) or TADs containing LADs, considered separately. Verifiable hypotheses regarding the underlying mechanism for the presence of the correlation without causality are discussed. These include the possibility that the epigenetic marks and affinity to the NE of a TAD are determined by various non-mutually exclusive mechanisms and remain relatively stable during interphase. Conclusions: At the level of TADs, the probability of chromatin being in contact with the nuclear envelope has no systematic, causal effect on the transcription level in Drosophila. The conclusion is reached by combining model-derived time-evolution of TAD locations within the nucleus with their experimental gene expression levels. [ABSTRACT FROM AUTHOR]

ObjectiveComplex local crosstalk amongst endocrine cells within the islet ensures tight coordination of their endocrine output. This is illustrated by the recent demonstration that the negative feedback control by delta cells within pancreatic islets determines the homeostatic set-point for plasma glucose during mouse postnatal development. However, the close association of islet endocrine cells that facilitates paracrine crosstalk also complicates the distinction between effects mediated directly on beta cells from indirect effects mediated via local intermediates, such as somatostatin from delta cells.MethodsTo resolve this problem, we generated reporter mice that allow collection of pure pancreatic delta cells along with alpha and beta cells from the same islets and generated comprehensive transcriptomes for each islet endocrine cell type. These transcriptomes afford an unparalleled view of the receptors expressed by delta, alpha and beta cells, and allow the prediction of which signal targets which endocrine cell type with great accuracy.ResultsFrom these transcriptomes, we discovered that the ghrelin receptor is expressed exclusively by delta cells within the islet, which was confirmed by fluorescent in situ hybridization and qPCR. Indeed, ghrelin increases intracellular calcium in delta cells in intact mouse islets, measured by GCaMP6 and robustly potentiates glucose-stimulated somatostatin secretion on mouse and human islets in both static and perfusion assays. In contrast, des-acyl-ghrelin at the same dose had no effect on somatostatin secretion and did not block the actions of ghrelin.ConclusionsThese results offer a straightforward explanation for the well-known insulinostatic actions of ghrelin. Rather than engaging beta cells directly, ghrelin engages delta cells to promote local inhibitory feedback that attenuates insulin release. These findings illustrate the power of our approach to resolve some of the long-standing conundrums with regard to the rich feedback that occurs within the islet that is integral to islet physiology and therefore highly relevant to diabetes.

Globodera pallida is a white potato cyst nematode present in the Andes, which causes huge losses to Peruvian farmers. An RNA-seq analysis allowed the identification of candidate genes that could mediate resistance against this pathogen. Two varieties, “María Huanca” (Solanum andigena) clone resistant (CIP 279142.12) and “Chimbina Colorada” (Solanum chaucha) (CIP 701013) clone susceptible to G. pallida, were used to identify differentially expressed genes. Total RNA from roots was extracted 72 hours post inoculation with second stage juveniles. Sequencing was done using the Illumina Hiseq 2500 platform. Reads were screened for quality issues and then mapped to the reference potato genome (clone DM1-3516 R44 v4.03). Here, we report 27717 and 27750 genes expressed in the resistant and susceptible variety respectively. The comparative analysis of expression identified 100 candidate genes. 91 genes were associated with resistance to G. pallida with Fold Change ≥ 2 (p

Abstract Background Comparative genomics has seen the development of many software performing the clustering, polymorphism and gene content analysis of genomes at different phylogenetic levels (isolates, species). These tools rely on de novo assembly and/or multiple alignments that can be computationally intensive for large datasets. With a large number of similar genomes in particular, e.g., in surveillance and outbreak detection, assembling each genome can become a redundant and expensive step in the identification of genes potentially involved in a given clinical feature. Results We have developed deltaRpkm, an R package that performs a rapid differential gene presence evaluation between two large groups of closely related genomes. Starting from a standard gene count table, deltaRpkm computes the RPKM per gene per sample, then the inter-group δRPKM values, the corresponding median δRPKM (m) for each gene and the global standard deviation value of m (s m ). Genes with m > = 2 ∗ s m (standard deviation s of all the m values) are considered as “differentially present” in the reference genome group. Our simple yet effective method of differential RPKM has been successfully applied in a recent study published by our group (N = 225 genomes of Listeria monocytogenes) (Aguilar-Bultet et al. Front Cell Infect Microbiol 8:20, 2018). Conclusions To our knowledge, deltaRpkm is the first tool to propose a straightforward inter-group differential gene presence analysis with large datasets of related genomes, including non-coding genes, and to output directly a list of genes potentially involved in a phenotype.

The emergence of new severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) has been a global concern. The B.1.1.7 variant of SARS CoV-2 is reported to cause higher transmission. The study investigates the replication cycle and transcriptional pattern of the B.1.1.7 to hypothesis the possible role of different genes in viral replication. It was observed that the B.1.1.7 variant required a longer maturation time. The transcriptional response demonstrated higher expression of ORF6 and ORF8 compared to nucleocapsid transcript till the eclipse period which might influence higher viral replication. The number of infectious viruses titer is higher in the B.1.1.7, despite a lesser copy number than B.1, indicating higher transmissibility. The experimental evidence published linked ORF6 and ORF8 to play important role in replication and we also observed their higher expression. This leads us to hypothesis the possible role of ORF6 and ORF8 in B.1.1.7 higher replication which causes higher transmission.

RNA-sequencing has been widely used to obtain high throughput transcriptome sequences in various species, but the assembly of a full set of complete transcripts is still a significant challenge. Judging by the number of expected transcripts and assembled unigenes in a transcriptome library, we believe that some unigenes could be reassembled. In this study, using the nitrate transporter (NRT) gene family and phosphate transporter (PHT) gene family in Salicornia europaea as examples, we introduced an approach to further assemble unigenes found in transcriptome libraries which had been previously generated by Trinity. To find the unigenes of a particular transcript that contained gaps, we respectively selected 16 NRT candidate unigene pairs and 12 PHT candidate unigene pairs for which the two unigenes had the same annotations, the same expression patterns among various RNA-seq samples, and different positions of the proteins coded as mapped to a reference protein. To fill a gap between the two unigenes, PCR was performed using primers that mapped to the two unigenes and the PCR products were sequenced, which demonstrated that 5 unigene pairs of NRT and 3 unigene pairs of PHT could be reassembled when the gaps were filled using the corresponding PCR product sequences. This fast and simple method will reduce the redundancy of targeted unigenes and allow acquisition of complete coding sequences (CDS).

Background: RNA-seq based on short reads generated by next generation sequencing technologies has become the main approach to study differential gene expression. Until now, the main applications of this technique have been to study the variation of gene expression in a whole organism, tissue or cell type under different conditions or at different developmental stages. However, RNA-seq also has a great potential to be used in evolutionary studies to investigate gene expression divergence in closely related species. Results: We show that the published genomes and annotations of the three closely related Drosophila species D. melanogaster, D. simulans and D. mauritiana have limitations for inter-specific gene expression studies. This is due to missing gene models in at least one of the genome annotations, unclear orthology assignments and significant gene length differences in the different species. A comprehensive evaluation of four statistical frameworks (DESeq2, DESeq2 with length correction, RPKM-limma and RPKM-voom-limma) shows that none of these methods sufficiently accounts for inter-specific gene length differences, which inevitably results in false positive candidate genes. We propose that published reference genomes should be re-annotated before using them as references for RNA-seq experiments to include as many genes as possible and to account for a potential length bias. We present a straight-forward reciprocal re-annotation pipeline that allows to reliably compare the expression for nearly all genes annotated in D. melanogaster. Conclusions: We conclude that our reciprocal re-annotation of previously published genomes facilitates the analysis of significantly more genes in an inter-specific differential gene expression study. We propose that the established pipeline can easily be applied to re-annotate other genomes of closely related animals and plants to improve comparative expression analyses. [ABSTRACT FROM AUTHOR]

Chlorophytum borivilianum is an important species of liliaceae family, owing to its vital medicinal properties. Plant roots are used for aphrodisiac, adaptogen, anti-aging, health-restorative and health-promoting purposes. Saponins, are considered to be the principal bioactive components responsible for the wide variety of pharmacological properties of this plant. In the present study, we have performed de novo root transcriptome sequencing of C. borivilianum using Illumina Hiseq 2000 platform, to gain molecular insight into saponins biosynthesis. A total of 33,963,356 high-quality reads were obtained after quality filtration. Sequences were assembled using various programs which generated 97,344 transcripts with a size range of 100-5,216 bp and N50 value of 342. Data was analyzed against non-redundant proteins, gene ontology (GO), and enzyme commission (EC) databases. All the genes involved in saponins biosynthesis along with five full-length genes namely farnesyl pyrophosphate synthase, cycloartenol synthase, β-amyrin synthase, cytochrome p450, and sterol-3-glucosyltransferase were identified. Read per exon kilobase per million (RPKM)-based comparative expression profiling was done to study the differential regulation of the genes. In silico expression analysis of seven selected genes of saponin biosynthetic pathway was validated by qRT-PCR. [ABSTRACT FROM AUTHOR]

The transcriptome ofKappaphycus alvareziiwas profiled using high-throughput Solexa paired-end sequencing technology. A total of 61 million sequencing reads was generated by filtering the low-quality reads, and 28 701 unigenes with a mean length of 901 bp were obtained based onde novoassembly. In similarity alignments against the NCBI non-redundant protein sequence (NR) database, Swissprot database, Cluster of Orthologous Groups (COG) database, Gene ontology (GO) database, and Kyoto Encyclopedia of Genes and Genomes (KEGG) database, 11 996 (41.79%) unigenes were identified with significant hits (E-value < 10−5) against existing genes. Functional annotation with the KEGG pathway database identified 8975 unigenes and mapped to 125 pathways. Through functional enrichment analysis of the genes with a higher expression value than the average RPKM (reads per kilobase per million reads), we found that some important pathways were highly expressed. The substantial number of transcript sequences ofK. alvareziiprovides a valuable resource for potential gene identification and comparative genomic studies. [ABSTRACT FROM PUBLISHER]

Measures of RNA abundance are important for many areas of biology and often obtained from high-throughput RNA sequencing methods such as Illumina sequence data. These measures need to be normalized to remove technical biases inherent in the sequencing approach, most notably the length of the RNA species and the sequencing depth of a sample. These biases are corrected in the widely used reads per kilobase per million reads (RPKM) measure. Here, we argue that the intended meaning of RPKM is a measure of relative molar RNA concentration (rmc) and show that for each set of transcripts the average rmc is a constant, namely the inverse of the number of transcripts mapped. Further, we show that RPKM does not respect this invariance property and thus cannot be an accurate measure of rmc. We propose a slight modification of RPKM that eliminates this inconsistency and call it TPM for transcripts per million. TPM respects the average invariance and eliminates statistical biases inherent in the RPKM measure. [ABSTRACT FROM AUTHOR]

The emergence of new severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) has been a global concern. The B.1.1.7 variant of SARS CoV-2 is reported to cause higher transmission. The study investigates the replication cycle and transcriptional pattern of the B.1.1.7 to hypothesis the possible role of different genes in viral replication. It was observed that the B.1.1.7 variant required a longer maturation time. The transcriptional response demonstrated higher expression of ORF6 and ORF8 compared to nucleocapsid transcript till the eclipse period which might influence higher viral replication. The number of infectious viruses titer is higher in the B.1.1.7, despite a lesser copy number than B.1, indicating higher transmissibility. The experimental evidence published linked ORF6 and ORF8 to play important role in replication and we also observed their higher expression. This leads us to hypothesis the possible role of ORF6 and ORF8 in B.1.1.7 higher replication which causes higher transmission. [ABSTRACT FROM AUTHOR]

In recent years, RNA-sequencing (RNA-seq) has emerged as a powerful technology for transcriptome profiling. For a given gene, the number of mapped reads is not only dependent on its expression level and gene length, but also the sequencing depth. To normalize these dependencies, RPKM (reads per kilobase of transcript per million reads mapped) and TPM (transcripts per million) are used to measure gene or transcript expression levels. A common misconception is that RPKM and TPM values are already normalized, and thus should be comparable across samples or RNA-seq projects. However, RPKM and TPM represent the relative abundance of a transcript among a population of sequenced transcripts, and therefore depend on the composition of the RNA population in a sample. Quite often, it is reasonable to assume that total RNA concentration and distributions are very close across compared samples. Nevertheless, the sequenced RNA repertoires may differ significantly under different experimental conditions and/or across sequencing protocols; thus, the proportion of gene expression is not directly comparable in such cases. In this review, we illustrate typical scenarios in which RPKM and TPM are misused, unintentionally, and hope to raise scientists' awareness of this issue when comparing them across samples or different sequencing protocols., (© 2020 Zhao et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Background: Comparative genomics has seen the development of many software performing the clustering, polymorphism and gene content analysis of genomes at different phylogenetic levels (isolates, species). These tools rely on de novo assembly and/or multiple alignments that can be computationally intensive for large datasets. With a large number of similar genomes in particular, e.g., in surveillance and outbreak detection, assembling each genome can become a redundant and expensive step in the identification of genes potentially involved in a given clinical feature. Results: We have developed deltaRpkm, an R package that performs a rapid differential gene presence evaluation between two large groups of closely related genomes. Starting from a standard gene count table, deltaRpkm computes the RPKM per gene per sample, then the inter-group δRPKM values, the corresponding median δRPKM (m) for each gene and the global standard deviation value of m (sm). Genes with m > = 2 ∗ sm (standard deviation s of all the m values) are considered as "differentially present" in the reference genome group. Our simple yet effective method of differential RPKM has been successfully applied in a recent study published by our group (N = 225 genomes of Listeria monocytogenes) (Aguilar-Bultet et al. Front Cell Infect Microbiol 8:20, 2018). Conclusions: To our knowledge, deltaRpkm is the first tool to propose a straightforward inter-group differential gene presence analysis with large datasets of related genomes, including non-coding genes, and to output directly a list of genes potentially involved in a phenotype. [ABSTRACT FROM AUTHOR]

The massive amount of information produced by ChIP-Seq, RNA-Seq, and other next-generation sequencing-based methods requires computational data analysis. However, biologists performing these experiments often lack training in bioinformatics. BioWardrobe aims to bridge this gap by providing a convenient user interface and by automating routine data-processing steps. This protocol details the use of BioWardrobe for identifying and visualizing ChIP-Seq peaks, calculating RPKMs, performing differential binding or gene expression analysis, and creating plots and heat maps. We specifically describe how to use BioWardrobe's quality control measures for troubleshooting NGS-based experiments.

Background & Aims Untreated necrotizing enterocolitis (NEC) can lead to massive inflammation resulting in intestinal necrosis with a high mortality rate in preterm infants. Limited access to human samples and relevant experimental models have hampered progress in NEC pathogenesis. Earlier evidence has suggested that bacterial colonization of an immature and developing intestine can lead to an abnormally high inflammatory response to bacterial bioproducts. The aim of our study was to use human fetal organoids to gain insights into NEC pathogenesis. Methods: RNA sequencing analysis was performed to compare patterns of gene expression in human fetal-derived enterospheres (FEnS) and adult-derived enterospheres (AEnS). Differentially expressed genes were analyzed using computational techniques for dimensional reduction, clustering, and gene set enrichment. Unsupervised cluster analysis, Gene Ontology, and gene pathway analysis were used to predict differences between gene expression of samples. Cell monolayers derived from FEnS and AEnS were evaluated for epithelium function and responsiveness to lipopolysaccharide and commensal bacteria. Results: Based on gene expression patterns, FEnS clustered according to their developmental age in 2 distinct groups: early and late FEnS, with the latter more closely resembling AEnS. Genes involved in maturation, gut barrier function, and innate immunity were responsible for these differences. FEnS-derived monolayers exposed to either lipopolysaccharide or commensal Escherichia coli showed that late FEnS activated gene expression of key inflammatory cytokines, whereas early FEnS monolayers did not, owing to decreased expression of nuclear factor-κB–associated machinery. Conclusions: Our results provide insights into processes underlying human intestinal development and support the use of FEnS as a relevant human preclinical model for NEC. Accession number of repository for expression data: GSE101531.

RNA-sequencing (RNA-seq) is a technique that employs next-generation DNA-sequencing technology to simultaneously sequence all of the RNA transcripts in a cell. It can provide valuable insights into transcript and operon structure, and is rapidly replacing expression microarrays as the technique of choice for determining global gene expression profiles in bacteria. Herein we outline the procedures involved in performing RNA-seq with samples of RNA from Staphylococcus aureus. We draw particular attention to key aspects of sample preparation, such as RNA integrity and removal of ribosomal RNA, and provide details of critical steps in downstream data analysis.

In this article, high-throughput de novo transcriptomic sequencing was performed in Alexandrium catenella, which provided the first view of the gene repertoire in this dinoflagellate based on next-generation sequencing (NGS) technologies. A total of 118,304 unigenes were identified with an average length of 673bp (base pair). Of these unigenes, 77,936 (65.9%) were annotated with known proteins based on sequence similarities, among which 24,149 and 22,956 unigenes were assigned to gene ontology categories (GO) and clusters of orthologous groups (COGs), respectively. Furthermore, 16,467 unigenes were mapped onto 322 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG). We also detected 1143 simple sequence repeats (SSRs), in which the tri-nucleotide repeat motif (69.3%) was the most abundant. The genetic facts and significance derived from the transcriptome dataset were suggested and discussed. All four core nucleosomal histones and linker histones were detected, in addition to the unigenes involved in histone modifications.190 unigenes were identified as being involved in the endocytosis pathway, and clathrin-dependent endocytosis was suggested to play a role in the heterotrophy of A. catenella. A conserved 22-nt spliced leader (SL) was identified in 21 unigenes which suggested the existence of trans-splicing processing of mRNA in A. catenella., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

The genomic architecture and expression of the Igf-1 gene are complex yielding multiple IGF-I transcript isoforms with putative functional contributions to growth and metabolism. Using RNA-seq on different tissues, physiological states, and species, the breadth of transcripts expressed was determined. Tissues from pre- and post-pubertal heifers and mature mice were collected and the transcript isoforms characterized. Three different IGF-I isoforms were detected in heifers with Class 1 transcripts most abundantly expressed. The pituitary reduced IGF-I expression post-pubertally whereas the uterus increased expression. Murine IGF-I transcript expression was more diverse utilizing multiple exons, start sites, and 3'UTRs. The RNA-seq methodology to characterize expression profiles permits assessment of the transcript isoforms yielding insight into functional roles of each transcript., (Copyright © 2013 Elsevier B.V. All rights reserved.)

To investigate the response of Plutella xylostella transcriptome in defending against a Bt toxin, high-throughput RNA-sequencing was carried out to examine Cry1Ac-susceptible and -resistant strains. The comparative analysis indentified over 2900 differentially expressed unigenes (DEUs) between these two strains. Gene Ontology analysis placed these unigenes primarily into cell, cell part, organelle, binding, catalytic, cellular process, metabolic process, and response to stimulus categories. Based on pathway analyses, DEUs were enriched in oxidoreductase activity and membrane lipid metabolic processes, and they were also significantly enriched in pathways related to the metabolic and biosynthesis of secondary metabolites. Most of the unigenes involved in the metabolic pathway were up-regulated in resistant strains. Within the ABC transporter pathway, majority of the down-regulated unigenes belong to ABCC2 and ABCC10, respectively, while up-regulated unigenes were mainly categorized as ABCG2. Furthermore, two aminopeptidases, and four cadherins encoding genes were significantly elevated as well. This study provides a transcriptome foundation for the identification and functional characterization of genes involved in the Bt resistance in an agriculturally important insect pest, P. xylostella., (© 2013 Elsevier B.V. All rights reserved.)

Plant researchers have focused much attention on leaf shape because of its importance in the identification. To evaluate the impact of intraspecies leaf-shape variation on the transcriptome, a series of Betula pendula 'Dalecarlica' and B. pendula saplings were generated through tissue culture. The leaf shapes and transcriptomes of B. pendula 'Dalecarlica' clones were compared with those of B. pendula clones. The leaf shape of B. pendula 'Dalecarlica' was incised and that of B. pendula was ovate. Transcriptome data revealed numerous changes in gene expression between B. pendula 'Dalecarlica' and B. pendula, including upregulation of 8767 unigenes and downregulation of 8379 unigenes in B. pendula 'Dalecarlica'. A pathway analysis revealed that the transport and signal transduction of auxin were altered in 'Dalecarlica', which may have contributed to its altered leaf shape. These results shed light on variation in birch leaf shape and help identify important genes for the genetic engineering of birch trees., (© 2013.)

Single Nucleotide Polymorphisms (SNPs) have become the marker of choice for genome wide association studies. In order to provide the best genome coverage for the analysis of disease, production and performance traits, a large number of relatively evenly distributed SNPs are needed. The main objective of present work was to identify large numbers of gene-associated SNPs using high-throughput sequencing in squamous cell carcinoma of horn. RNA-seq analysis was conducted on 2 tissues viz. Horn Cancer (HC) and Horn Normal (HN) in Kankrej breed of cattle. A total of 909,362 reads with average read length of 405 bp for HC and 583,491 reads with average read length of 411 bp for HN were obtained. We found 9532 and 7065 SNPs as well as 1771 and 1172 Indels in HC and HN, respectively, from which, 7889 SNPs and 1736 Indels were uniquely present in HC, 5886 SNPs and 1146 Indels were uniquely present in HN and reported first time in Bos indicus, whereas the rest are already reported in Bos taurus dbSNP database. The gene-associated SNPs and Indels were high in upregulated genes of HC as compared to HN. Analysis of differentially expressed genes was identified, these genes are involved in regulation of cell proliferation, apoptosis, gene transcription, cell survival and metabolism through various metabolic pathways. The result of transcriptome expression profiling was validated using Real Time quantitative PCR in nine randomly selected genes. We identified numbers aberrant signaling pathways responsible for carcinogenesis in HC which are also commonly altered in squamous cell carcinoma (SCC) of lung in human being. We conclude that a large number of altered genes and dysfunction of multiple pathways are involved in the development of Horn Cancer. The present findings contribute to theoretical information for further screening of genes and identification of markers for early diagnosis of HC as well as SNPs identified in this report provide a much needed resource for genetic studies in B. indicus and shall contribute to the development of a high density SNP array. Validation and testing of these SNPs using SNP arrays will form the material basis for gene associated SNPs in HC., (© 2013 Elsevier B.V. All rights reserved.)

'Purple Rain', a purple cultivar of Betula pendula, has dark purple leaves throughout the vegetative period. In this study, B. pendula 'Purple Rain' was found to have a higher anthocyanidin level compared with B. pendula, Transcriptome analysis revealed numerous changes in gene expression that could be attributed to color change, including the upregulation of 2467 unigenes and the downregulation of 2299 unigenes in 'Purple Rain'. Furthermore, anthocyanidin synthesis and transcriptional regulation were altered in 'Purple Rain', which may have contributed to phenotypic changes. These results provide unique molecular insights into the biochemical pathways and regulatory networks that function in a purple variety of B. pendula., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

Larval cuticle is degraded and replaced by the pupal counterpart during larval-pupal metamorphosis in the holometabolous insects. In addition to the extrinsic transformation, the epidermis goes through significant changes at molecular levels. To elucidate the intrinsic mechanism of epidermal metamorphosis, the dynamics of chitin content in the cuticle was examined in an important agricultural lepidopteran, the common cutworm, and the transcriptome was analyzed using Illumina sequencing technology. Gene expression profiles during the metamorphosis were further studied by both the digital gene expression (DGE) system and real-time quantitative PCR. The results showed that the chitin content decreased in prepupae and then increased in pupae. A total of 58 million sequencing reads were obtained and assembled into 70,346 unigenes. Over 9000 unigenes were identified to express differentially during the transformation process. As compared with the 6th instar feeding larvae, the most significant changes took place in the proteasome and metabolic pathways in prepupae and pupae, respectively. The cytochrome P450s, VHDLs, chitinase, serine protease and genes involved in sex pheromone biosynthesis changed their mRNA levels remarkably. Three chitinolytic enzymes (chitinase, β-N-acetylglucosaminidase and chitin deacetylase) showed distinct mRNA expression patterns, the former two enzymes revealed the highest expression in prepupae, however the latter one showed its climax mRNA level in pupae. The gene expression patterns suggest that chitinase and β-N-acetylglucosaminidase may be responsible for the degradation of larval cuticles, whereas chitin deacetylase may help to degrade the pupal counterparts. Gene expression dynamics also implied that the chitin of pupal cuticle might be formed by recycling of the degraded chitin of larval cuticle rather than through de novo synthesis. The 20E-induced nuclear receptors seem to be important factors regulating chitin metabolic enzymes during the cuticle remodeling. Our data provide a comprehensive resource for exploring the molecular mechanism of epidermal metamorphosis in insects., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Bumblebee males (Hymenoptera) produce species-specific labial gland secretions called marking pheromones (MPs). MPs generally consist of terpenoids and fatty-acid-derived aliphatic compounds with various chain lengths predominantly containing one or no double bonds. The unsaturated fatty-acid-derived MP components were hypothesized to be produced by fatty acid desaturases (FADs) that exhibit diverse substrate specificities. To address this hypothesis, we isolated and functionally characterized FADs from three bumblebee species: Bombus lucorum, Bombus terrestris, and Bombus lapidarius. By employing RNA sequencing of the male labial glands and fat bodies of B. lucorum and B. terrestris, we identified five paralogous FAD-like sequences but only two FAD lineages were abundant and differentially expressed in the labial glands. We found that abundant FAD lineages were also expressed in the labial gland and fat body of Bombus lapidarius. Functional characterization of FADs in a yeast expression system confirmed that Δ4-FADs exhibited a unique Δ4-desaturase activity exclusively on 14-carbon fatty acyls and Δ9-FADs displayed Δ9-desaturase activity on 14- to 18-carbon fatty acyls. These results indicate that Δ9-FADs are involved in the biosynthesis of major unsaturated components of MPs in B. lucorum and B. lapidarius despite the diverse MP composition of these bumblebee species. The contribution of lipases, acyltransferases, esterases, and fatty acid reductases to production of the species-specific MP composition is also discussed in light of the transcriptomic data obtained in this study., (Copyright © 2013 Elsevier Ltd. All rights reserved.)