Overview - What is RNA-Seq?
The transcriptome is a collection of all RNA present in a cell or a population of cells at any given moment. The transcriptome is dynamic, as the levels of RNA transcripts vary during different developmental stages or in response to certain conditions.
Transcriptome sequencing or RNA-Seq is a next-generation sequencing (NGS)-based approach to profiling and analysing RNA. This technique delivers unbiased information without the need for prior knowledge of the genome or transcriptome. Transcriptome sequencing is often the method of choice for analysis of differentially expressed genes, as well as for RNA editing and profiling of allele-specific gene expression. RNA-seq can also be used to investigate splicing patterns, splicing variants, gene isoforms, single nucleotide polymorphisms and post transcriptional modifications.
Applications - What are the advantages of RNA-Seq?
Sequencing of RNA transcripts is a beneficial method for RNA profiling because it allows for:
- Digital gene expression (DGE) analysis – sensitive quantification of gene expression levels and transcriptional activity
- Identification of both known and novel transcripts
- Analysis across a wide dynamic range – detection of more differentially expressed genes with higher fold change
- Profile the RNA transcripts of nearly any organism without the need for a reference sequence
- Complete view of the entire transcriptome
Workflow - Methods & technologies for whole transcriptome sequencing
Transcriptome sequencing begins with isolating RNA and converting this RNA into so-called complementary DNA (cDNA). Total RNA or RNA types like mRNA or small RNA (link) can be interrogated. Strand-specific or random-primed libraries can be created based on how the cDNA is synthesised. Once adapters are ligated to the cDNA fragments, the cDNA undergoes single-read or paired-end sequencing. The sequencing reads are either assembled de novo or they are aligned to a reference genome or transcriptome. The reads can be quantified to evaluate differential gene expression levels or analysed further to provide data on transcript variants and posttranscriptional modifications.
Microarray vs. RNA-Seq
RNA-seq is an evolving technology, whereas microarrays are the traditional method for differential gene expression analysis. RNA-Seq offers distinct advantages over hybridisation-based methods. Next-generation sequencing of transcriptomes does not rely on pre-designed primers and probes, which eliminates content bias and provides a truly genome-wide view of the transcriptome. RNA-Seq provides the possibility of exploring coding and non-coding RNA with more sensitivity, higher dynamic range and with single-base resolution. NGS-based sequencing of cDNA can reveal complex regions like exon-exon junctions or sequence variations that cannot be profiled by hybridisation methods. Multiplexing capabilities and the ability to store and re-analyse data without repeated experiments render NGS of transcriptomes more cost-effective than microarrays.
Scientific expertise: RNA-Seq
GATC Biotech is an experienced provider of RNA-seq services. With several specialised sequencing products to serve the transcriptomics field, GATC Biotech helps answer questions regarding gene function on a daily basis. GATC Biotech uses Illumina’s or PacBio’s sequencing technologies, for flexible sequencing coverage. The RNA-seq services at GATC Biotech can help further various research objectives, from screening to discovery projects, as well as de novo transcriptome analysis and isoform sequencing (Iso-seq)-driven research.
Transcriptome analysis at GATC Biotech has helped elucidate cardiac remodelling mechanisms, post-transcriptional gene silencing, features of CD4+ T cells in coeliac disease, characteristics of photosynthetic genes in maize, the molecular diversity of snake venom, strategies for obtaining human-induced pluripotent stem-cell-derived neurons and many others.
Find here, a list of selected research articles supported by GATC Biotech’s sequencing products, including articles on transcriptomics.
>Health and the Understanding of Metabolism, Aging and Nutrition
The aim of HUMAN, a collaborative project targeting small and medium enterprises (SMEs), is to generate unique humanised mouse models. Potential targets of metabolic disease and aging are validated with innovative technology platforms, such as large-scale omics phenotyping. Of particular interest is the investigation into how disease conditions alter the post-translational modification status of transcription factors, thereby affecting physiological pathways. A combination of targeted and screening approaches is proposed to identify changes in modification states of major liver transcription factors in mouse models.
GATC Biotech's task:
GATC Biotech will describe the phenotype of humanised mouse models and induced pluripotent stem cells (iPSCs) by transcriptomic and epigenomic analysis. Deep sequencing of the transcriptome will be accomplished using Illumina’s sequencing technology, which enables the detection of sequence variants (single nucleotide polymorphisms (SNPs) and deletion-insertion polymorphisms (DIPs)) with high accuracy. Furthermore, GATC Biotech will investigate cytosine methylation as well as histone modifications that are important transcriptional regulators of gene expression.
The HUMAN project is funded by the European Union and is led by Sweden’s Karolinska Institutet.
Products related to cDNA sequencing
Are you planning a project in need of transcript sequencing? GATC Biotech offers whole-transcriptome sequencing, mRNA sequencing and small RNA sequencing services to provide in-depth analysis of differential gene activity, alternative splicing and expression of gene isoforms. You can count on our one-stop service, which combines RNA isolation, rRNA depletion, library preparation, sequencing on the state-of-the-art Illumina platform, and expert transcriptomic data analysis all in one package.
See how our INVIEW TRANSCRIPTOME product can help advance your transcriptome project or contact us (link) for more information.
If you are analysing novel isoforms, genes and gene fusion events, then see how our INVIEW TRANSCRIPTOME ISOFORM DISCOVER can enhance your research by providing full-length cDNA sequences that require no assembly.
Further reading on RNA-Seq
Han, Y., Gao, S., Muegge, K., Zhang, W., Zhou, B. Advanced Applications of RNA Sequencing and Challenges. Bioinform. Biol. Insights. 9(Suppl 1):29-46 (2015).
McGettigan, P.A. Transcriptomics in the RNA-seq era. Curr. Opin. Chem. Biol. 17 (1), 4 – 11 (2013).