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RNA sequencing and transcriptome arrays analyses show opposing results for alternative splicing in patient derived samples.

ABSTRACT: RNA sequencing (RNA-seq) and microarrays are two transcriptomics techniques aimed at the quantification of transcribed genes and their isoforms. Here we compare the latest Affymetrix HTA 2.0 microarray with Illumina 2000 RNA-seq for the analysis of patient samples - normal lung epithelium tissue and squamous cell carcinoma lung tumours. Protein coding mRNAs and long non-coding RNAs (lncRNAs) were included in the study.Both platforms performed equally well for protein-coding RNAs, however the stochastic variability was higher for the sequencing data than for microarrays. This reduced the number of differentially expressed genes and genes with predictive potential for RNA-seq compared to microarray data. Analysis of this variability revealed a lack of reads for short and low abundant genes; lncRNAs, being shorter and less abundant RNAs, were found especially susceptible to this issue. A major difference between the two platforms was uncovered by analysis of alternatively spliced genes. Investigation of differential exon abundance showed insufficient reads for many exons and exon junctions in RNA-seq while the detection on the array platform was more stable. Nevertheless, we identified 207 genes which undergo alternative splicing and were consistently detected by both techniques.Despite the fact that the results of gene expression analysis were highly consistent between Human Transcriptome Arrays and RNA-seq platforms, the analysis of alternative splicing produced discordant results. We concluded that modern microarrays can still outperform sequencing for standard analysis of gene expression in terms of reproducibility and cost.

SUBMITTER: Nazarov PV

PROVIDER: S-EPMC5461714 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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RNA sequencing and transcriptome arrays analyses show opposing results for alternative splicing in patient derived samples.

Nazarov Petr V PV Muller Arnaud A Kaoma Tony T Nicot Nathalie N Maximo Cristina C Birembaut Philippe P Tran Nhan L NL Dittmar Gunnar G Vallar Laurent L

BMC genomics 20170606 1

<h4>Background</h4>RNA sequencing (RNA-seq) and microarrays are two transcriptomics techniques aimed at the quantification of transcribed genes and their isoforms. Here we compare the latest Affymetrix HTA 2.0 microarray with Illumina 2000 RNA-seq for the analysis of patient samples - normal lung epithelium tissue and squamous cell carcinoma lung tumours. Protein coding mRNAs and long non-coding RNAs (lncRNAs) were included in the study.<h4>Results</h4>Both platforms performed equally well for p ...[more]

PMID: 28587590

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Project description:Heat stress (HS) severely impacts the productivity and welfare of dairy cows. Investigating the biological mechanisms underlying HS response is crucial for developing effective mitigation and breeding strategies. Therefore, we evaluated the changes in milk yield, physiological indicators, blood biochemical parameters, and alternative splicing (AS) patterns of lactating Holstein cows during thermoneutral (TN, N = 19) and heat-stress (HS, N = 17) conditions. There was a significant (p < 0.05) decline in milk yield as physiological indicators increased after exposure to natural HS conditions. The levels of eight out of 13 biochemical parameters of HS were also significantly altered in the presence of HS (p < 0.05). These results demonstrate that HS negatively influences various biological processes of Holstein cows. Furthermore, we investigated AS events based on the RNA-seq data from blood samples. With HS, five common types of AS events were generally increased by 6.7-38.9%. A total of 3470 AS events corresponding to 3143 unique genes were differentially alternatively spliced (DSGs) (p-adjusted < 0.05) between TN and HS groups. The functional annotation results show that the majority of DSGs are involved in mRNA splicing and spliceosomal complex, followed by enrichment in immune and metabolic processes. Eighty-seven out of 645 differentially expressed genes (DEGs) (fold change ≥ 1.5 and false discovery rate < 0.05) overlapped with DSGs. Further analyses showed that 20 of these genes were significantly enriched for the RNA splicing, RNA binding, and RNA transport. Among them, two genes (RBM25 and LUC7L3) had strong interrelation and co-expression pattern with other genes and were identified as candidate genes potentially associated with HS responses in dairy cows. In summary, AS plays a crucial role in changing the transcriptome diversity of heat-stress-related genes in multiple biological pathways and provides a different regulation mechanism from DEGs.

Project description:IntroductionPeanut (Arachis hypogaea L.), also called groundnut is an important oil and cash crop grown widely in the world. The annual global production of groundnuts has increased to approximately 50 million tons, which provides a rich source of vegetable oils and proteins for humans. Low temperature (non-freezing) is one of the major factors restricting peanut growth, yield, and geographic distribution. Since the complexity of cold-resistance trait, the molecular mechanism of cold tolerance and related gene networks were largely unknown in peanut.MethodsIn this study, comparative transcriptomic analysis of two peanut cultivars (SLH vs. ZH12) with differential cold tolerance under low temperature (10°C) was performed using Oxford Nanopore Technology (ONT) platform.Results and discussionAs a result, we identified 8,949 novel gene loci and 95,291 new/novel isoforms compared with the reference database. More differentially expressed genes (DEGs) were discovered in cold-sensitive cultivar (ZH12) than cold-tolerant cultivar (SLH), while more alternative splicing events were found in SLH compared to ZH12. Gene Ontology (GO) analyses of the common DEGs showed that the "response to stress", "chloroplast part", and "transcription factor activity" were the most enriched GO terms, indicating that photosynthesis process and transcription factors play crucial roles in cold stress response in peanut. We also detected a total of 708 differential alternative splicing genes (DASGs) under cold stress compared to normal condition. Intron retention (IR) and exon skipping (ES) were the most prevalent alternative splicing (AS) events. In total, 4,993 transcription factors and 292 splicing factors were detected, many of them had differential expression levels and/or underwent AS events in response to cold stress. Overexpression of two candidate genes (encoding trehalose-6-phosphatephosphatases, AhTPPs) in yeast improves cold tolerance. This study not only provides valuable resources for the study of cold resistance in peanut but also lay a foundation for genetic modification of cold regulators to enhance stress tolerance in crops.

Project description:BackgroundBRCA1 is a key protein in cell network, involved in DNA repair pathways and cell cycle. Recently, the ENIGMA consortium has reported a high number of alternative splicing (AS) events at this locus in blood-derived samples. However, BRCA1 splicing pattern in breast tissue samples is unknown. Here, we provide an accurate description of BRCA1 splicing events distribution in breast tissue samples.MethodsBRCA1 splicing events were scanned in 70 breast tumor samples, 4 breast samples from healthy individuals and in 72 blood-derived samples by capillary electrophoresis (capillary EP). Molecular subtype was identified in all tumor samples. Splicing events were considered predominant if their relative expression level was at least the 10% of the full-length reference signal.Results54 BRCA1 AS events were identified, 27 of them were annotated as predominant in at least one sample. Δ5q, Δ13, Δ9, Δ5 and ▼1aA were significantly more frequently annotated as predominant in breast tumor samples than in blood-derived samples. Predominant splicing events were, on average, more frequent in tumor samples than in normal breast tissue samples (P = 0.010). Similarly, likely inactivating splicing events (PTC-NMDs, Non-Coding, Δ5 and Δ18) were more frequently annotated as predominant in tumor than in normal breast samples (P = 0.020), whereas there were no significant differences for other splicing events (No-Fs) frequency distribution between tumor and normal breast samples (P = 0.689).ConclusionsOur results complement recent findings by the ENIGMA consortium, demonstrating that BRCA1 AS, despite its tremendous complexity, is similar in breast and blood samples, with no evidences for tissue specific AS events. Further on, we conclude that somatic inactivation of BRCA1 through spliciogenic mutations is, at best, a rare mechanism in breast carcinogenesis, albeit our data detects an excess of likely inactivating AS events in breast tumor samples.

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RNA sequencing and transcriptome arrays analyses show opposing results for alternative splicing in patient derived samples.

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RNA sequencing and transcriptome arrays analyses show opposing results for alternative splicing in patient derived samples.

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