limma powers differential expression analyses for RNA-sequencing and microarray studies

Ritchie, Matthew E.; Phipson, Belinda; Wu, Di; Hu, Yifang; Law, Charity W.; Shi, Wei; Smyth, Gordon K.

doi:10.1093/nar/gkv007

Cited by 29,646 publications

(25,362 citation statements)

References 77 publications

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“…Cell line gene expression principal component analysis was performed using the R prcomp function with the 1000 genes with the largest 10–90% interpercentile range in signal intensities. Gene set analysis was performed using the camera function in the R Bioconductor package limma (Ritchie et al ., 2015; Wu and Smyth, 2012). Seventy gene sets were preselected based on relevance for CRC.…”

Section: Methodsmentioning

confidence: 99%

Prognostic, predictive, and pharmacogenomic assessments of CDX2 refine stratification of colorectal cancer

et al. 2018

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We aimed to refine the value of CDX2 as an independent prognostic and predictive biomarker in colorectal cancer (CRC) according to disease stage and chemotherapy sensitivity in preclinical models. CDX2 expression was evaluated in 1045 stage I–IV primary CRCs by gene expression (n = 403) or immunohistochemistry (n = 642) and in relation to 5‐year relapse‐free survival (RFS), overall survival (OS), and chemotherapy. Pharmacogenomic associations between CDX2 expression and 69 chemotherapeutics were assessed by drug screening of 35 CRC cell lines. CDX2 expression was lost in 11.6% of cases and showed independent poor prognostic value in multivariable models. For individual stages, CDX2 was prognostic only in stage IV, independent of chemotherapy. Among stage I–III patients not treated in an adjuvant setting, CDX2 loss was associated with a particularly poor survival in the BRAF‐mutated subgroup, but prognostic value was independent of microsatellite instability status and the consensus molecular subtypes. In stage III, the 5‐year RFS rate was higher among patients with loss of CDX2 who received adjuvant chemotherapy than among patients who did not. The CDX2‐negative cell lines were significantly more sensitive to chemotherapeutics than CDX2‐positive cells, and the multidrug resistance genes MDR1 and CFTR were significantly downregulated both in CDX2‐negative cells and in patient tumors. Loss of CDX2 in CRC is an adverse prognostic biomarker only in stage IV disease and appears to be associated with benefit from adjuvant chemotherapy in stage III. Early‐stage patients not qualifying for chemotherapy might be reconsidered for such treatment if their tumor has loss of CDX2 and mutated BRAF.

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Section: Methodsmentioning

confidence: 99%

Prognostic, predictive, and pharmacogenomic assessments of CDX2 refine stratification of colorectal cancer

et al. 2018

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“…Counts were converted to log2 counts per million, quantile normalized and precision weighted with the voom function of the limma package. 52,53 A linear model was fitted to each gene, and empirical Bayes moderated t-statistics were used to assess differences in expression. 54 A false discovery rate cut-off of 0.15 was applied for calling differentially expressed genes.…”

Section: Methodsmentioning

confidence: 99%

A point mutation in the Ncr1 signal peptide impairs the development of innate lymphoid cell subsets

et al. 2018

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NKp46 (CD335) is a surface receptor shared by both human and mouse natural killer (NK) cells and innate lymphoid cells (ILCs) that transduces activating signals necessary to eliminate virus-infected cells and tumors. Here, we describe a spontaneous point mutation of cysteine to arginine (C14R) in the signal peptide of the NKp46 protein in congenic Ly5.1 mice and the newly generated NCRB6C14R strain. Ly5.1C14R NK cells expressed similar levels of Ncr1 mRNA as C57BL/6, but showed impaired surface NKp46 and reduced ability to control melanoma tumors in vivo. Expression of the mutant NKp46C14R in 293T cells showed that NKp46 protein trafficking to the cell surface was compromised. Although Ly5.1C14R mice had normal number of NK cells, they showed an increased number of early maturation stage NK cells. CD49a+ILC1s were also increased but these cells lacked the expression of TRAIL. ILC3s that expressed NKp46 were not detectable and were not apparent when examined by T-bet expression. Thus, the C14R mutation reveals that NKp46 is important for NK cell and ILC differentiation, maturation and function.SignificanceInnate lymphoid cells (ILCs) play important roles in immune protection. Various subsets of ILCs express the activating receptor NKp46 which is capable of recognizing pathogen derived and tumor ligands and is necessary for immune protection. Here, we describe a spontaneous point mutation in the signal peptide of the NKp46 protein in congenic Ly5.1 mice which are widely used for tracking cells in vivo. This Ncr1 C14R mutation impairs NKp46 surface expression resulting in destabilization of Ncr1 and accumulation of NKp46 in the endoplasmic reticulum. Loss of stable NKp46 expression impaired the maturation of NKp46+ ILCs and altered the expression of TRAIL and T-bet in ILC1 and ILC3, respectively.

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“…Prediction of batch effects was carried out by singular value decomposition (SVD) using the champ bioconductor package (Morris et al ., 2014). Identification of differentially methylated CpG probes between sample groups was performed using a linear model fit of the M values with subsequent empirical Bayes reduction in standard errors from the estimated methylation differences, as described in the limma r package (Ritchie et al ., 2015). As the distribution of unadjusted P ‐values showed no influence of potential confounding factors (Fig.…”

Section: Methodsmentioning

confidence: 99%

Reduced DNA methylation patterning and transcriptional connectivity define human skin aging

et al. 2016

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SummaryEpigenetic changes represent an attractive mechanism for understanding the phenotypic changes associated with human aging. Age‐related changes in DNA methylation at the genome scale have been termed ‘epigenetic drift’, but the defining features of this phenomenon remain to be established. Human epidermis represents an excellent model for understanding age‐related epigenetic changes because of its substantial cell‐type homogeneity and its well‐known age‐related phenotype. We have now generated and analyzed the currently largest set of human epidermis methylomes (N = 108) using array‐based profiling of 450 000 methylation marks in various age groups. Data analysis confirmed that age‐related methylation differences are locally restricted and characterized by relatively small effect sizes. Nevertheless, methylation data could be used to predict the chronological age of sample donors with high accuracy. We also identified discontinuous methylation changes as a novel feature of the aging methylome. Finally, our analysis uncovered an age‐related erosion of DNA methylation patterns that is characterized by a reduced dynamic range and increased heterogeneity of global methylation patterns. These changes in methylation variability were accompanied by a reduced connectivity of transcriptional networks. Our findings thus define the loss of epigenetic regulatory fidelity as a key feature of the aging epigenome.

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limma powers differential expression analyses for RNA-sequencing and microarray studies

Cited by 29,646 publications

References 77 publications

Prognostic, predictive, and pharmacogenomic assessments of CDX2 refine stratification of colorectal cancer

Prognostic, predictive, and pharmacogenomic assessments of CDX2 refine stratification of colorectal cancer

A point mutation in the Ncr1 signal peptide impairs the development of innate lymphoid cell subsets

Reduced DNA methylation patterning and transcriptional connectivity define human skin aging

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