A comprehensive evaluation of normalization methods for Illumina high-throughput RNA sequencing data analysis

Dillies, Marie‐Agnès; Rau, Andrea; Aubert, Julie; Hennequet-Antier, Christelle; Jeanmougin, M.; Servant, Nicolas; Keime, Céline; Marot, Guillemette; Castel, David; Estellé, Jordi; Guernec, Grégory; Jagla, Bernd; Jouneau, Luc; Laloë, Dénis; Gall, Caroline Le; Schaeffer, Brigitte; Crom, Stéphane Le; Guedj, Mickaël; Jaffrézic, Florence

doi:10.1093/bib/bbs046

Cited by 1,070 publications

(1,030 citation statements)

References 25 publications

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“…Statistical cutoffs were established at Po0.05 and P-values were corrected for false discovery rate. Because of current controversy surrounding use of reads per kilobase per million (Dillies et al, 2013), normalization methods were validated using a series of established housekeeping genes (Supplementary Figure 1). The genome plot was generated using BRIG (Alikhan et al, 2011).…”

Section: Bioinformatic Analysismentioning

confidence: 99%

Nutrients drive transcriptional changes that maintain metabolic homeostasis but alter genome architecture in Microcystis

Steffen¹,

Dearth²,

Dill³

et al. 2014

The ISME Journal

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The cyanobacterium Microcystis aeruginosa is a globally distributed bloom-forming organism that degrades freshwater systems around the world. Factors that drive its dispersion, diversification and success remain, however, poorly understood. To develop insight into cellular-level responses to nutrient drivers of eutrophication, RNA sequencing was coupled to a comprehensive metabolomics survey of M. aeruginosa sp. NIES 843 grown in various nutrient-reduced conditions. Transcriptomes were generated for cultures grown in nutrient-replete (with nitrate as the nitrogen (N) source), nitrogen-reduced (with nitrate, urea or ammonium acting as the N sources) and phosphate-reduced conditions. Extensive expression differences (up to 696 genes for urea-grown cells) relative to the control treatment were observed, demonstrating that the chemical variant of nitrogen available to cells affected transcriptional activity. Of particular note, a high number of transposase genes (up to 81) were significantly and reproducibly up-regulated relative to the control when grown on urea. Conversely, phosphorus (P) reduction resulted in a significant cessation in transcription of transposase genes, indicating that variation in nutrient chemistry may influence transcription of transposases and may impact the highly mosaic genomic architecture of M. aeruginosa. Corresponding metabolomes showed comparably few differences between treatments, suggesting broad changes to gene transcription are required to maintain metabolic homeostasis under nutrient reduction. The combined observations provide novel and extensive insight into the complex cellular interactions that take place in this important bloom-forming organism during variable nutrient conditions and highlight a potential unknown molecular mechanism that may drive Microcystis blooms and evolution.

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Section: Bioinformatic Analysismentioning

confidence: 99%

Nutrients drive transcriptional changes that maintain metabolic homeostasis but alter genome architecture in Microcystis

Steffen¹,

Dearth²,

Dill³

et al. 2014

The ISME Journal

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show abstract

“…Apart from the main sRNAbench programme, a differential expression module based on edgeR (Robinson et al, 2010) was used to generate an expression matrix of all miRNAs detected. Note that by using edgeR, sRNAbench applies implicitly TMM normalisation in the detection of differentially expressed small RNAs, which was reported to be among the most stable methods [42,43]. …”

Section: Methodsmentioning

confidence: 99%

Glycosylated extracellular vesicles released by glioblastoma cells are decorated by CCL18 allowing for cellular uptake via chemokine receptor CCR8

Berenguer

Lagerweij

Zhao

et al. 2018

J of Extracellular Vesicle

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Cancer cells release extracellular vesicles (EVs) that contain functional biomolecules such as RNA and proteins. EVs are transferred to recipient cancer cells and can promote tumour progression and therapy resistance. Through RNAi screening, we identified a novel EV uptake mechanism involving a triple interaction between the chemokine receptor CCR8 on the cells, glycans exposed on EVs and the soluble ligand CCL18. This ligand acts as bridging molecule, connecting EVs to cancer cells. We show that glioblastoma EVs promote cell proliferation and resistance to the alkylating agent temozolomide (TMZ). Using in vitro and in vivo stem-like glioblastoma models, we demonstrate that EV-induced phenotypes are neutralised by a small molecule CCR8 inhibitor, R243. Interference with chemokine receptors may offer therapeutic opportunities against EV-mediated cross-talk in glioblastoma.

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“…Overexpression of SPINK1 in PCa exhibits outlier-expression in ~10-15% of the total PCa cases (Tomlins et al, 2008). Thus, to stratify patients with increased expression of SPINK1, we sorted TCGA patients' samples on the basis of increasing SPINK1 expression (descending order), and divided the dataset into four equal parts by employing Quartile-based normalization method (73), the top 25% of the patients (N=119) corresponding to the upper quartile (QU, log2 (RPM+1)>5.468 or log2 (normalized count+1)>1.892), were assigned as SPINK1 high or SPINK1-positive patient samples and the lower quartile (QL, log2 (RPM+1)<1.124 or log2 (normalized count+1)<-2.611), were considered as SPINK1 low or SPINK1-negative samples. Also, we found about 18 patients with outlier expression of SPINK1 with log2 (RPM+1) of greater than 11.984, which were included in the heat map representation of SPINK1 positive TCGA patients in Figure 1B.…”

Section: Integrative Analyses For Tcga-prad Datamentioning

confidence: 99%

Epigenetic silencing of miRNA-338-5p and miRNA-421 drives SPINK1-positive prostate cancer

Bhatia

Yadav

Tiwari

et al. 2018

Preprint

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PurposeSerine Peptidase Inhibitor, Kazal type-1 (SPINK1) overexpression defines the second most recurrent and aggressive prostate cancer (PCa) subtype. However, the underlying molecular mechanism and pathobiology of SPINK1 in PCa remains largely unknown.Experimental DesignMicroRNA-prediction tools were employed to examine the SPINK1-3’UTR for miRNAs binding. Luciferase reporter assays were performed to confirm the SPINK1-3’UTR binding of shortlisted miR-338-5p/miR-421. Further, miR-338-5p/-421 overexpressing cancer cells (SPINK1-positive) were evaluated for oncogenic properties using cell-based functional assays and mice xenograft model. Global gene expression profiling was performed to unravel the biological pathways altered by miR-338-5p/-421. Immunohistochemistry and RNA in-situ hybridization was carried-out on PCa patients’ tissue microarray for SPINK1 and EZH2 expression respectively. Chromatin immunoprecipitation assay was performed to examine EZH2 occupancy on the miR-338-5p/-421 regulatory regions. Bisulfite sequencing and methylated DNA-immunoprecipitation was performed on PCa cell lines and patients’ specimens.ResultsWe established a critical role of miRNA-338-5p/-421 in post-transcriptional regulation of SPINK1. Ectopic expression of miRNA-338-5p/-421 in SPINK1-positive PCa cells abrogate oncogenic properties including cell-cycle progression, stemness and drug resistance, and show reduced tumor burden and distant metastases in mice model. Importantly, we show SPINK1-positive PCa patients exhibit increased EZH2 expression, suggesting its role in miRNA-338-5p/-421 epigenetic silencing. Furthermore, presence of CpG dinucleotide DNA methylation marks on the regulatory regions of miR-338-5p/-421 in SPINK1-positive PCa cells and patients’ specimens confirms epigenetic silencing.ConclusionOur findings revealed that miRNA-338-5p/-421 are epigenetically silenced in SPINK1-positive PCa, while restoring the expression of these miRNAs using epigenetic drugs or synthetic mimics could abrogate SPINK1-mediated oncogenesis.TRANSLATIONAL IMPACTWe establish a regulatory model involving the functional interplay between SPINK1, miRNA-338-5p/miRNA-421 and EZH2, thereby, revealing hitherto unknown mechanism of SPINK1 up-regulation in SPINK1-positive subtype. Our findings provide a strong rationale for the development of potential therapeutic strategies for SPINK1-positive malignancies. We demonstrate that restoring miRNA-338-5p/miRNA-421 expression using epigenetic drugs including DNMTs inhibitors in combination with HDACs or HKMTs inhibitors or miRNA synthetic mimics in SPINK1-positive prostate cancer abrogate SPINK1-mediated oncogenicity. The major findings of this study will not only advance the prostate cancer field, but will also be valuable for treatment and disease management of other SPINK1-positive malignancies.

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A comprehensive evaluation of normalization methods for Illumina high-throughput RNA sequencing data analysis

Cited by 1,070 publications

References 25 publications

Nutrients drive transcriptional changes that maintain metabolic homeostasis but alter genome architecture in Microcystis

Nutrients drive transcriptional changes that maintain metabolic homeostasis but alter genome architecture in Microcystis

Glycosylated extracellular vesicles released by glioblastoma cells are decorated by CCL18 allowing for cellular uptake via chemokine receptor CCR8

Epigenetic silencing of miRNA-338-5p and miRNA-421 drives SPINK1-positive prostate cancer

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