Effect of low-expression gene filtering on detection of differentially expressed genes in RNA-seq data

Sha, Ying; Phan, John H.; Wang, May D.

doi:10.1109/embc.2015.7319872

Cited by 68 publications

(43 citation statements)

References 19 publications

(28 reference statements)

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“…Samples were analyzed statistically using the tool Empirical analysis of DGE ( Differential Gene Expression ) in CLC Genomics Workbench as described in Materials and Methods, with original total exon counts from each sample used as input. For each gene, if the average exon counts from all biological replicates of a sample were identified as <10, the gene was defined as not expressed in that sample (see Soneson and Delorenzi, 2013 ; Sha et al, 2015 ). Based on this condition, 14,491, 14,157 and 14,259 genes from Arabidopsis were identified as not expressed in each of the three experimental comparisons (i), (ii) and (iii), respectively, and these were consequently excluded from further analyses (Table 1 ).…”

Section: Resultsmentioning

confidence: 99%

Transcript Profiling Identifies NAC-Domain Genes Involved in Regulating Wall Ingrowth Deposition in Phloem Parenchyma Transfer Cells of Arabidopsis thaliana

Hou

et al. 2018

Front. Plant Sci.

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Transfer cells (TCs) play important roles in facilitating enhanced rates of nutrient transport at key apoplasmic/symplasmic junctions along the nutrient acquisition and transport pathways in plants. TCs achieve this capacity by developing elaborate wall ingrowth networks which serve to increase plasma membrane surface area thus increasing the cell's surface area-to-volume ratio to achieve increased flux of nutrients across the plasma membrane. Phloem parenchyma (PP) cells of Arabidopsis leaf veins trans-differentiate to become PP TCs which likely function in a two-step phloem loading mechanism by facilitating unloading of photoassimilates into the apoplasm for subsequent energy-dependent uptake into the sieve element/companion cell (SE/CC) complex. We are using PP TCs in Arabidopsis as a genetic model to identify transcription factors involved in coordinating deposition of the wall ingrowth network. Confocal imaging of pseudo-Schiff propidium iodide-stained tissue revealed different profiles of temporal development of wall ingrowth deposition across maturing cotyledons and juvenile leaves, and a basipetal gradient of deposition across mature adult leaves. RNA-Seq analysis was undertaken to identify differentially expressed genes common to these three different profiles of wall ingrowth deposition. This analysis identified 68 transcription factors up-regulated two-fold or more in at least two of the three experimental comparisons, with six of these transcription factors belonging to Clade III of the NAC-domain family. Phenotypic analysis of these NAC genes using insertional mutants revealed significant reductions in levels of wall ingrowth deposition, particularly in a double mutant of NAC056 and NAC018, as well as compromised sucrose-dependent root growth, indicating impaired capacity for phloem loading. Collectively, these results support the proposition that Clade III members of the NAC-domain family in Arabidopsis play important roles in regulating wall ingrowth deposition in PP TCs.

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Section: Resultsmentioning

confidence: 99%

Transcript Profiling Identifies NAC-Domain Genes Involved in Regulating Wall Ingrowth Deposition in Phloem Parenchyma Transfer Cells of Arabidopsis thaliana

Hou

et al. 2018

Front. Plant Sci.

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“…For reliable differential expression analysis, a further improvement of eFDR levels is thus needed. Additional filtering steps have been successfully used to that effect [1–3, 24]. For RNA-seq, unlike for microarrays, beyond filters for small effect size (fold change) also filters for small expression levels are necessary.…”

Section: Resultsmentioning

confidence: 99%

“…Especially for RNA-seq a plethora of new tools is being developed, and the selection of an effective pipeline is not trivial [24]. Going beyond the comparisons of the original SEQC study [2, 3], we here present comprehensive benchmark results covering all known genes and a range of effect sizes typically observed in experiments.…”

Section: Discussionmentioning

confidence: 99%

Sensitivity, specificity, and reproducibility of RNA-Seq differential expression calls

Łabaj

Kreil²

2016

Biol Direct

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BackgroundThe MAQC/SEQC consortium has recently compiled a key benchmark that can serve for testing the latest developments in analysis tools for microarray and RNA-seq expression profiling. Such objective benchmarks are required for basic and applied research, and can be critical for clinical and regulatory outcomes. Going beyond the first comparisons presented in the original SEQC study, we here present extended benchmarks including effect strengths typical of common experiments.ResultsWith artefacts removed by factor analysis and additional filters, for genome scale surveys, the reproducibility of differential expression calls typically exceed 80% for all tool combinations examined. This directly reflects the robustness of results and reproducibility across different studies. Similar improvements are observed for the top ranked candidates with the strongest relative expression change, although here some tools clearly perform better than others, with typical reproducibility ranging from 60 to 93%.ConclusionsIn our benchmark of alternative tools for RNA-seq data analysis we demonstrated the benefits that can be gained by analysing results in the context of other experiments employing a reference standard sample. This allowed the computational identification and removal of hidden confounders, for instance, by factor analysis. In itself, this already substantially improved the empirical False Discovery Rate (eFDR) without changing the overall landscape of sensitivity. Further filtering of false positives, however, is required to obtain acceptable eFDR levels. Appropriate filters noticeably improved agreement of differentially expressed genes both across sites and between alternative differential expression analysis pipelines.ReviewersAn extended abstract of this research paper was selected for the Camda Satellite Meeting to Ismb 2015 by the Camda Programme Committee. The full research paper then underwent one round of Open Peer Review under a responsible Camda Programme Committee member, Lan Hu, PhD (Bio-Rad Laboratories, Digital Biology Center-Cambridge). Open Peer Review was provided by Charlotte Soneson, PhD (University of Zürich) and Michał Okoniewski, PhD (ETH Zürich). The Reviewer Comments section shows the full reviews and author responses.Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-016-0169-7) contains supplementary material, which is available to authorized users.

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“…Similarly, the correlation between the lncRNA expression and the closest coding gene was performed for the coding genes that were expressed (RPKM >0.5) in at least 3 samples among the 8 studied. This criteria has been defined in order to remove low counts in the libraries to improve the sensitivity and the precision of the differential genes expression ( 20 ). Moreover, this threshold was selected because, for GRO-Seq, reads are counted throughout the gene body, which represents more total reads per genes than RNA-seq ( 12 , 15 , 21 , 22 ).…”

Section: Methodsmentioning

confidence: 99%

Transcriptional Profiling of Hypoxia-Regulated Non-coding RNAs in Human Primary Endothelial Cells

Moreau

Örd

Downes

et al. 2018

Front. Cardiovasc. Med.

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Hypoxia occurs in human atherosclerotic lesions and has multiple adverse effects on endothelial cell metabolism. Recently, key roles of long non-coding RNAs (lncRNAs) in the development of atherosclerosis have begun to emerge. In this study, we investigate the lncRNA profiles of human umbilical vein endothelial cells subjected to hypoxia using global run-on sequencing (GRO-Seq). We demonstrate that hypoxia regulates the nascent transcription of ~1800 lncRNAs. Interestingly, we uncover evidence that promoter-associated lncRNAs are more likely to be induced by hypoxia compared to enhancer-associated lncRNAs, which exhibit an equal distribution of up- and downregulated transcripts. We also demonstrate that hypoxia leads to a significant induction in the activity of super-enhancers next to transcription factors and other genes implicated in angiogenesis, cell survival and adhesion, whereas super-enhancers near several negative regulators of angiogenesis were repressed. Despite the majority of lncRNAs exhibiting low detection in RNA-Seq, a subset of lncRNAs were expressed at comparable levels to mRNAs. Among these, MALAT1, HYMAI, LOC730101, KIAA1656, and LOC339803 were found differentially expressed in human atherosclerotic lesions, compared to normal vascular tissue, and may thus serve as potential biomarkers for lesion hypoxia.

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Effect of low-expression gene filtering on detection of differentially expressed genes in RNA-seq data

Cited by 68 publications

References 19 publications

Transcript Profiling Identifies NAC-Domain Genes Involved in Regulating Wall Ingrowth Deposition in Phloem Parenchyma Transfer Cells of Arabidopsis thaliana

Transcript Profiling Identifies NAC-Domain Genes Involved in Regulating Wall Ingrowth Deposition in Phloem Parenchyma Transfer Cells of Arabidopsis thaliana

Sensitivity, specificity, and reproducibility of RNA-Seq differential expression calls

Transcriptional Profiling of Hypoxia-Regulated Non-coding RNAs in Human Primary Endothelial Cells

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