Whole Transcriptome Profiling: An RNA-Seq Primer and Implications for Pharmacogenomics Research.
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ABSTRACT:
SUBMITTER: Sa ACC
PROVIDER: S-EPMC5866981 | biostudies-literature | 2018 Mar
REPOSITORIES: biostudies-literature
Publications
Whole Transcriptome Profiling: An RNA-Seq Primer and Implications for Pharmacogenomics Research.
Sá Ana Caroline C ACC Sadee Wolfgang W Johnson Julie A JA
Clinical and translational science 20171023 2
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Project description:BackgroundRNA-Seq has enabled high-throughput gene expression profiling to provide insight into the functional link between genotype and phenotype. Low quantities of starting RNA can be a severe hindrance for studies that aim to utilize RNA-Seq. To mitigate this bottleneck, whole transcriptome amplification (WTA) technologies have been developed to generate sufficient sequencing targets from minute amounts of RNA. Successful WTA requires accurate replication of transcript abundance without the loss or distortion of specific mRNAs. Here, we test the efficacy of NuGEN's Ovation RNA-Seq V2 system, which uses linear isothermal amplification with a unique chimeric primer for amplification, using white adipose tissue from standard laboratory rats (Rattus norvegicus). Our goal was to investigate potential biological artifacts introduced through WTA approaches by establishing comparisons between matched raw and amplified RNA libraries derived from biological replicates.ResultsWe found that 93% of expressed genes were identical between all unamplified versus matched amplified comparisons, also finding that gene density is similar across all comparisons. Our sequencing experiment and downstream bioinformatic analyses using the Tuxedo analysis pipeline resulted in the assembly of 25,543 high-quality transcripts. Libraries constructed from raw RNA and WTA samples averaged 15,298 and 15,253 expressed genes, respectively. Although significant differentially expressed genes (P < 0.05) were identified in all matched samples, each of these represents less than 0.15% of all shared genes for each comparison.ConclusionsTranscriptome amplification is efficient at maintaining relative transcript frequencies with no significant bias when using this NuGEN linear isothermal amplification kit under ideal laboratory conditions as presented in this study. This methodology has broad applications, from clinical and diagnostic, to field-based studies when sample acquisition, or sample preservation, methods prove challenging.
| S-EPMC4520150 | biostudies-literature
Project description:Tendons are fibrous connective tissues that transmit force between muscle and bone. Whereas the molecular and cellular mechanisms of bone and muscle development have been well studied, that of tendon development is poorly understood. Using the Scx-GFP transgenic mice, we isolated GFP(+) cells from the developing mouse limbs at E11.5, E13.5, and E15.5, respectively, and carried out whole transcriptome RNA-seq analysis. Comparing the gene expression profiles of GFP(+) and GFP(-) cells in the E13.5 limb isolated over 1,500 genes that exhibited enrichment of mRNA expression by at least 1.5-fold in the GFP(+) cells. Of these, 778 genes showed expression up-regulated by more than 1.5-fold from E11.5 to E13.5 and 516 genes showed expression up-regulated by more than 1.5-fold from E13.5 to E15.5 in the GFP(+) cell population. Interestingly, over 30 genes encoding transcription factors are among the early-activated genes in the GFP(+) cells. Whole mount and section in situ hybridization analyses showed that many of these transcription factor genes have distinct patterns of expression during limb development and identified Foxf2 expression as a specific marker for differentiated dorsal limb tendon cells. Together, these data provide a valuable resource for further investigation of the molecular mechanisms regulating tendon development.
| S-EPMC4616261 | biostudies-literature
Project description:Paired DNA and RNA profiling is increasingly employed in genomics research to uncover molecular mechanisms of disease and to explore personal genotype and phenotype correlations. Here, we introduce Simul-seq, a technique for the production of high-quality whole-genome and transcriptome sequencing libraries from small quantities of cells or tissues. We apply the method to laser-capture-microdissected esophageal adenocarcinoma tissue, revealing a highly aneuploid tumor genome with extensive blocks of increased homozygosity and corresponding increases in allele-specific expression. Among this widespread allele-specific expression, we identify germline polymorphisms that are associated with response to cancer therapies. We further leverage this integrative data to uncover expressed mutations in several known cancer genes as well as a recurrent mutation in the motor domain of KIF3B that significantly affects kinesin-microtubule interactions. Simul-seq provides a new streamlined approach for generating comprehensive genome and transcriptome profiles from limited quantities of clinically relevant samples.
| S-EPMC5734913 | biostudies-literature
Project description:BackgroundFormalin-fixed paraffin-embedded (FFPE) tissue samples are routinely archived in the course of patient care and can be linked to clinical outcomes with long-term follow-up. However, FFPE tissues have degraded RNA which poses challenges for analyzing gene expression. Next-generation sequencing (NGS) is rapidly becoming accepted as an effective tool for measuring gene expressions for research and clinical use. However, the feasibility of NGS has not been firmly established when using FFPE tissue.ResultsWe optimized strategies for whole transcriptome sequencing (RNA-seq) using FFPE tissue. Ribosomal RNA (rRNA) was successfully depleted by competitive hybridization using the Ribo-zero™ Kit (Epicentre Biotechnologies), and rRNA sequence content was less than one percent for each library. Gene expression measured by FFPE RNA-seq was compared to two different standards: RNA-seq from fresh frozen (FF) tissue and quantitative PCR (qPCR). Both FF and FFPE tumors were sequenced on an Illumina Genome Analyzer IIX with an average of 10 million reads. The distribution of FPKMs (fragments per kilobase of exon per million fragments mapped) and number of detected genes were similar between FFPE and FF. RNA-seq expressions from FF and FFPE samples from the same renal cell carcinoma (RCC) correlated highly (r = 0.919 for tumor 1 and r = 0.954 for tumor 2). On hierarchical cluster analysis, samples clustered by patient identity rather than method of preservation. TaqMan qPCR of 424 RCC-related genes correlated highly with FFPE RNA-seq expressions (r = 0.775 for FFPE tumor 1, r = 0.803 for FFPE tumor 2). Expression fold changes were considered, to assess biologic relevance of gene expressions. Expression fold changes between FFPE tumors (tumor 1/tumor 2) correlated well when comparing qPCR and RNA-seq (r = 0.890). Expression fold changes between tumors from different risk groups (our high risk RCC/The Cancer Genome Atlas, TCGA, low risk RCC) also correlated well when comparing RNA-seq from FF and FFPE tumors (r = 0.887).ConclusionsFFPE RNA-seq provides reliable genes expression data, comparable to that obtained from fresh frozen tissue. It represents a useful tool for discovery and validation of biomarkers.
| S-EPMC4298956 | biostudies-literature
Project description:To better understand the tumorigenesis and metastasis of human metastatic melanoma(MM) , we analyzed the transcriptomes of three cell lines that represent the three distinct stages of MM pathogenesis: the normal stage (HEMn), the onset of MM (A375), and the metastasis stage (A2085). Using the next generation sequencing (NGS) technology, we detected unsymmetrical expression of genes particularly in Chr9, 12, and 14 among three cell lines, an indication of the involvement of these genes in the tumorigenesis and metastasis of MM. These genes were assigned into 41 categories by their expression patterns and their biological functions were subsequently analyzed by Ingenuity Pathway Analysis software. In the top category associated with cancer, HIF1A, IL8, TERT, ONECUT1 and FOXA1 had direct interaction with the transcription factors or cytokines known to be involved in the tumorigenesis or metastasis in other malignant tumors. Our findings suggest that pathway about cytokines regulation in macrophages may play a more prominent role in the pathogenesis of MM. This study provides not only new targets for downstream mechanistic studies in the tumorigenesis and metastasis of MM, but also a new strategy to study the progression of other malignant cancers. Compare the transcriptome of 3 cell lines of human melanocyte/melanoma
| S-ECPF-GEOD-35704 | biostudies-other
Project description:Mendelian disorders with cutaneous manifestations comprise a genotypically heterogeneous group of over 1,000 diseases, and in most of them mutant genes have been identified. Mutation detection approaches in these diseases have largely focused on DNA analysis by next-generation sequencing techniques, including gene-targeted sequencing panels as well as whole-exome and whole-genome sequencing. Genome-wide homozygosity mapping (HM), based on DNA polymorphism, has also assisted in the identification of candidate genes in families with consanguinity. However, specific pathogenic variants have not been disclosed in many individual patients when analyzed by next-generation sequencing, and in particular, DNA-based analysis failed to identify many of the mutations impacting on splicing or gene expression. Whole-transcriptome sequencing by RNA sequencing (RNA-Seq), with appropriate bioinformatics, provides a robust tool to identify additional mutations to facilitate genetic diagnosis in genodermatoses. RNA-Seq can be used for variant calling and HM similar to DNA-based approaches, but it also allows for the identification of mutations that result in aberrant transcriptome expression, as displayed by heatmap analysis, and altered splicing patterns of RNA, as visualized by Sashimi plots. Thus, clinical RNA-Seq extends molecular diagnostics of rare genodermatoses, and it could provide a reliable first-tier diagnostic approach to extend mutation databases in patients with heritable skin diseases.
| S-EPMC8722382 | biostudies-literature
Project description:This report describes an improved protocol to generate stranded, barcoded RNA-seq libraries to capture the whole transcriptome. By optimizing the use of duplex specific nuclease (DSN) to remove ribosomal RNA reads from stranded barcoded libraries, we demonstrate improved efficiency of multiplexed next generation sequencing (NGS). This approach detects expression profiles of all RNA types, including miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (long non-coding RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA) without the use of gel electrophoresis. The improved protocol generates high quality data that can be used to identify differential expression in known and novel coding and non-coding transcripts, splice variants, mitochondrial genes and SNPs (single nucleotide polymorphisms).
| S-EPMC3739992 | biostudies-literature
Project description:Measuring allelic RNA expression ratios is a powerful approach for detecting cis-acting regulatory variants, RNA editing, loss of heterozygosity in cancer, copy number variation, and allele-specific epigenetic gene silencing. Whole transcriptome RNA sequencing (RNA-Seq) has emerged as a genome-wide tool for identifying allelic expression imbalance (AEI), but numerous factors bias allelic RNA ratio measurements. Here, we compare RNA-Seq allelic ratios measured in nine different human brain regions with a highly sensitive and accurate SNaPshot measure of allelic RNA ratios, identifying factors affecting reliable allelic ratio measurement. Accounting for these factors, we subsequently surveyed the variability of RNA editing across brain regions and across individuals.We find that RNA-Seq allelic ratios from standard alignment methods correlate poorly with SNaPshot, but applying alternative alignment strategies and correcting for observed biases significantly improves correlations. Deploying these methods on a transcriptome-wide basis in nine brain regions from a single individual, we identified genes with AEI across all regions (SLC1A3, NHP2L1) and many others with region-specific AEI. In dorsolateral prefrontal cortex (DLPFC) tissues from 14 individuals, we found evidence for frequent regulatory variants affecting RNA expression in tens to hundreds of genes, depending on stringency for assigning AEI. Further, we find that the extent and variability of RNA editing is similar across brain regions and across individuals.These results identify critical factors affecting allelic ratios measured by RNA-Seq and provide a foundation for using this technology to screen allelic RNA expression on a transcriptome-wide basis. Using this technology as a screening tool reveals tens to hundreds of genes harboring frequent functional variants affecting RNA expression in the human brain. With respect to RNA editing, the similarities within and between individuals leads us to conclude that this post-transcriptional process is under heavy regulatory influence to maintain an optimal degree of editing for normal biological function.
| S-EPMC3765493 | biostudies-literature
Project description:BackgroundRNA-seq and microarray are the two popular methods employed for genome-wide transcriptome profiling. Current comparison studies have shown that transcriptome quantified by these two methods correlated well. However, none of them have addressed if they complement each other, considering the strengths and the limitations inherent with them. The pivotal requirement to address this question is the knowledge of a well known data set. In this regard, HrpX regulome from pathogenic bacteria serves as an ideal choice as the target genes of HrpX transcription factor are well studied due to their central role in pathogenicity.ResultsWe compared the performance of RNA-seq and microarray in their ability to detect known HrpX target genes by profiling the transcriptome from the wild-type and the hrpX mutant strains of γ-Proteobacterium Xanthomonas citri subsp. citri. Our comparative analysis indicated that gene expression levels quantified by RNA-seq and microarray well-correlated both at absolute as well as relative levels (Spearman correlation-coefficient, rs > 0.76). Further, the expression levels quantified by RNA-seq and microarray for the significantly differentially expressed genes (DEGs) also well-correlated with qRT-PCR based quantification (rs = 0.58 to 0.94). Finally, in addition to the 55 newly identified DEGs, 72% of the already known HrpX target genes were detected by both RNA-seq and microarray, while, the remaining 28% could only be detected by either one of the methods.ConclusionsThis study has significantly advanced our understanding of the regulome of the critical transcriptional factor HrpX. RNA-seq and microarray together provide a more comprehensive picture of HrpX regulome by uniquely identifying new DEGs. Our study demonstrated that RNA-seq and microarray complement each other in transcriptome profiling.
| S-EPMC3534599 | biostudies-literature
Project description:Memory programming of cytotoxic T cells (CTLs) by inflammatory cytokines can be regulated by mammalian target of rapamycin (mTOR). We have shown that inhibition of mTOR during CTL activation leads to the enhancement of memory, but the molecular mechanisms remain largely unknown. Using high-throughput RNA-Seq, we identified genes and functions in mouse CTLs affected by mTOR inhibition through rapamycin. Of the 43,221 identified transcripts, 184 transcripts were differentially expressed after rapamycin treatment, corresponding to 128 annotated genes. Of these genes, 114 were downregulated and only 14 were upregulated. Most importantly, 50 of them are directly related to cell death and survival. In addition, several genes such as CD62L are related to migration. Furthermore, we predicted downregulation of transcriptional regulators based on the total differentially expressed genes, as well as the subset of apoptosis-related genes. Quantitative PCR confirmed the differential expressions detected in RNA-Seq. We conclude that the regulatory function of rapamycin may work through inhibition of multiple genes related to apoptosis and migration, which enhance CTL survival into memory.
| S-EPMC4198470 | biostudies-literature