Dataset Information

Comparison of stranded and non-stranded RNA-seq transcriptome profiling and investigation of gene overlap.

ABSTRACT: While RNA-sequencing (RNA-seq) is becoming a powerful technology in transcriptome profiling, one significant shortcoming of the first-generation RNA-seq protocol is that it does not retain the strand specificity of origin for each transcript. Without strand information it is difficult and sometimes impossible to accurately quantify gene expression levels for genes with overlapping genomic loci that are transcribed from opposite strands. It has recently become possible to retain the strand information by modifying the RNA-seq protocol, known as strand-specific or stranded RNA-seq. Here, we evaluated the advantages of stranded RNA-seq in transcriptome profiling of whole blood RNA samples compared with non-stranded RNA-seq, and investigated the influence of gene overlaps on gene expression profiling results based on practical RNA-seq datasets and also from a theoretical perspective.Our results demonstrated a substantial impact of stranded RNA-seq on transcriptome profiling and gene expression measurements. As many as 1751 genes in Gencode Release 19 were identified to be differentially expressed when comparing stranded and non-stranded RNA-seq whole blood samples. Antisense and pseudogenes were significantly enriched in differential expression analyses. Because stranded RNA-seq retains strand information of a read, we can resolve read ambiguity in overlapping genes transcribed from opposite strands, which provides a more accurate quantification of gene expression levels compared with traditional non-stranded RNA-seq. In the human genome, it is not uncommon to find genomic loci where both strands encode distinct genes. Among the over 57,800 annotated genes in Gencode release 19, there are an estimated 19 % (about 11,000) of overlapping genes transcribed from the opposite strands. Based on our whole blood mRNA-seq datasets, the fraction of overlapping nucleotide bases on the same and opposite strands were estimated at 2.94 % and 3.1 %, respectively. The corresponding theoretical estimations are 3 % and 3.6 %, well in agreement with our own findings.Stranded RNA-seq provides a more accurate estimate of transcript expression compared with non-stranded RNA-seq, and is therefore the recommended RNA-seq approach for future mRNA-seq studies.

SUBMITTER: Zhao S

PROVIDER: S-EPMC4559181 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Comparison of stranded and non-stranded RNA-seq transcriptome profiling and investigation of gene overlap.

Zhao Shanrong S Zhang Ying Y Gordon William W Quan Jie J Xi Hualin H Du Sarah S von Schack David D Zhang Baohong B

BMC genomics 20150903

<h4>Background</h4>While RNA-sequencing (RNA-seq) is becoming a powerful technology in transcriptome profiling, one significant shortcoming of the first-generation RNA-seq protocol is that it does not retain the strand specificity of origin for each transcript. Without strand information it is difficult and sometimes impossible to accurately quantify gene expression levels for genes with overlapping genomic loci that are transcribed from opposite strands. It has recently become possible to retai ...[more]

PMID: 26334759

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Project description:RNA-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.We 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.Transcriptome 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.

Dataset Information

Comparison of stranded and non-stranded RNA-seq transcriptome profiling and investigation of gene overlap.

Publications

Comparison of stranded and non-stranded RNA-seq transcriptome profiling and investigation of gene overlap.

Similar Datasets

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