RNA-seq analysis is easy as 1-2-3 with limma, Glimma and edgeR

Law, Charity W.; Alhamdoosh, Monther; Su, Shian; Smyth, Gordon K.; Ritchie, Matthew E.

doi:10.12688/f1000research.9005.2

Cited by 254 publications

(210 citation statements)

References 19 publications

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“…The number of mapped read-pairs were counted using cufflinks based on the GENCODE v23 annotation 48 . The raw read counts were transformed using voom 49, 50 , TMM-normalized 51 and QC-analysed using the arrayQualityMetrics package in Bioconductor 52 . Samples were scored based on 3 parameters: maplot, boxplot, heatmap.…”

Section: Methodsmentioning

confidence: 99%

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Reduced biological effect of e-cigarette aerosol compared to cigarette smoke evaluated in vitro using normalized nicotine dose and RNA-seq-based toxicogenomics

Haswell

Baxter

Banerjee

et al. 2017

Sci Rep

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Electronic cigarettes (e-cigarettes) use has increased globally and could potentially offer a lower risk alternative to cigarette smoking. Here, we assessed the transcriptional response of a primary 3D airway model acutely exposed to e-cigarette aerosol and cigarette (3R4F) smoke. Aerosols were generated with standard intense smoking regimens with careful consideration for dose by normalizing the exposures to nicotine. Two e-cigarette aerosol dilutions were tested for equivalent and higher nicotine delivery compared to 3R4F. RNA was extracted at 24 hrs and 48 hrs post exposure for RNA-seq. 873 and 205 RNAs were differentially expressed for 3R4F smoke at 24 hrs and 48 hrs using a pFDR < 0.01 and a [fold change] > 2 threshold. 113 RNAs were differentially expressed at the highest dose of e-cigarette aerosol using a looser threshold of pFDR < 0.05, 3 RNAs exceeded a fold change of 2. Geneset enrichment analysis revealed a clear response from lung cancer, inflammation, and fibrosis associated genes after 3R4F smoke exposure. Metabolic/biosynthetic processes, extracellular membrane, apoptosis, and hypoxia were identified for e-cigarette exposures, albeit with a lower confidence score. Based on equivalent or higher nicotine delivery, an acute exposure to e-cigarette aerosol had a reduced impact on gene expression compared to 3R4F smoke exposure in vitro.

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Section: Methodsmentioning

confidence: 99%

“…For each comparison, the null hypothesis was that there was no difference between the groups being compared. The Bioconductor package Limma was used 50 . The primary output from the statistical analysis is a set of fully annotated (when available) lists of genes differentially expressed in the comparison of interest.…”

Section: Methodsmentioning

confidence: 99%

Reduced biological effect of e-cigarette aerosol compared to cigarette smoke evaluated in vitro using normalized nicotine dose and RNA-seq-based toxicogenomics

Haswell

Baxter

Banerjee

et al. 2017

Sci Rep

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“…Genes have then been grouped into four classes: unexpressed , male - biased , female - biased and unbiased genes. Unexpressed genes have been determined as such if they displayed less than 1 count per million in at least three libraries as in [69]. Male - biased and female - biased genes have been determined as such using a FDR <0.05.…”

Section: Methodsmentioning

confidence: 99%

Dosage compensation and sex-specific epigenetic landscape of the X chromosome in the pea aphid

Richard

Legeai

Prunier-Leterme

et al. 2017

Epigenetics & Chromatin

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Background Heterogametic species display a differential number of sex chromosomes resulting in imbalanced transcription levels for these chromosomes between males and females. To correct this disequilibrium, dosage compensation mechanisms involving gene expression and chromatin accessibility regulations have emerged throughout evolution. In insects, these mechanisms have been extensively characterized only in Drosophila but not in insects of agronomical importance. Aphids are indeed major pests of a wide range of crops. Their remarkable ability to switch from asexual to sexual reproduction during their life cycle largely explains the economic losses they can cause. As heterogametic insects, male aphids are X0, while females (asexual and sexual) are XX.ResultsHere, we analyzed transcriptomic and open chromatin data obtained from whole male and female individuals to evaluate the putative existence of a dosage compensation mechanism involving differential chromatin accessibility of the pea aphid’s X chromosome. Transcriptomic analyses first showed X/AA and XX/AA expression ratios for expressed genes close to 1 in males and females, respectively, suggesting dosage compensation in the pea aphid. Analyses of open chromatin data obtained by Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE-seq) revealed a X chromosome chromatin accessibility globally and significantly higher in males than in females, while autosomes’ chromatin accessibility is similar between sexes. Moreover, chromatin environment of X-linked genes displaying similar expression levels in males and females—and thus likely to be compensated—is significantly more accessible in males.ConclusionsOur results suggest the existence of an underlying epigenetic mechanism enhancing the X chromosome chromatin accessibility in males to allow X-linked gene dose correction between sexes in the pea aphid, similar to Drosophila. Our study gives new evidence into the comprehension of dosage compensation in link with chromatin biology in insects and newly in a major crop pest, taking benefits from both transcriptomic and open chromatin data.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-017-0137-1) contains supplementary material, which is available to authorized users.

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“…Differential gene expression was then estimated using the lmFit function in limma::voom, a gene-wise linear model [105], and differentially expressed genes were defined as having an absolute fold-change > 2, with an FDR adjusted p-value < 0.05. Differentially expressed genes were first used "as is" for the gene ontology analysis as described above (previous section).…”

Section: Gene Expression and Ontology Analysis Of Root Transcriptomementioning

confidence: 99%

Salt Stress Induces Non-CG Methylation in Coding Regions of Barley Seedlings (Hordeum vulgare)

et al. 2018

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Salinity can negatively impact crop growth and yield. Changes in DNA methylation are known to occur when plants are challenged by stress and have been associated with the regulation of stress-response genes. However, the role of DNA-methylation in moderating gene expression in response to salt stress has been relatively poorly studied among crops such as barley. Here, we assessed the extent of salt-induced alterations of DNA methylation in barley and their putative role in perturbed gene expression. Using Next Generation Sequencing, we screened the leaf and root methylomes of five divergent barley varieties grown under control and three salt concentrations, to seek genotype independent salt-induced changes in DNA methylation. Salt stress caused increased methylation in leaves but diminished methylation in roots with a higher number of changes in leaves than in roots, indicating that salt induced changes to global methylation are organ specific. Differentially Methylated Markers (DMMs) were mostly located in close proximity to repeat elements, but also in 1094 genes, of which many possessed gene ontology (GO) terms associated with plant responses to stress. Identified markers have potential value as sentinels of salt stress and provide a starting point to allow understanding of the functional role of DNA methylation in facilitating barley's response to this stressor.

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RNA-seq analysis is easy as 1-2-3 with limma, Glimma and edgeR

Cited by 254 publications

References 19 publications

Reduced biological effect of e-cigarette aerosol compared to cigarette smoke evaluated in vitro using normalized nicotine dose and RNA-seq-based toxicogenomics

Reduced biological effect of e-cigarette aerosol compared to cigarette smoke evaluated in vitro using normalized nicotine dose and RNA-seq-based toxicogenomics

Dosage compensation and sex-specific epigenetic landscape of the X chromosome in the pea aphid

Salt Stress Induces Non-CG Methylation in Coding Regions of Barley Seedlings (Hordeum vulgare)

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