TOTAL RNA SEQ
Overview - What is total RNA-Seq?
To obtain a truly complete picture of transcriptomes, all RNA transcripts of an organism need to be taken into account regardless of whether the RNA is coding or non-coding. To achieve this, the entire RNA of an organism must be isolated rather than fractions like messenger RNA (mRNA). Since ribosomal RNA (rRNA) accounts for as much as 98% of the transcriptome, the rRNA is often depleted in order to optimise the number of reads covering the actual RNA of interest. Whole-transcriptome sequencing has numerous research applications, including the discovery of novel RNA species and investigations of untranslated regions and exon boundaries.
Applications - What are the advantages of whole transcriptome sequencing?
Total RNA-Seq is an advantageous technique for researchers looking to:
- Obtain the most comprehensive genome-wide view of the transcriptome
- Map genes by UTR and by characterising exon boundaries
- Analyse splicing and alternative splicing mechanisms
- Investigate transcriptomes of human and non-human origin
Workflow - Methods & technologies for whole transcriptome sequencing
All RNA-Seq approaches, including total RNA-Seq, have similar workflows. RNA is isolated, cDNA is reverse transcribed, the cDNA is fragmented, and adapters are ligated to the cDNA fragments that finally undergo single-end or paired-end sequencing. Subsequently, the reads are aligned to a reference transcriptome or assembled de novo to reveal a detailed profile at single base resolution.
Total RNA-Seq differs from other transcriptome sequencing strategies as the method allows for sequencing of both coding and non-coding RNA. Rather than undergoing polyA+ selection that targets the mRNA species, the whole RNA is often subjected to rRNA depletion instead. As rRNA makes up a large percentage of the entire RNA, reduction of rRNA helps allocate more sequencing reads to transcripts of interest. cDNA is synthesised from the rest of the rRNA depleted RNA. Optionally, strand-specific information during downstream processing can be provided. The cDNA is then treated as in all other RNA-Seq workflows.
In isoform sequencing (Iso-Seq), full-length cDNA sequences can be generated from the 5’ end of the transcript to the polyA tail without reconstruction or a need for complicated algorithms. This approach is based on the Single-Molecule Real-Time (SMRT) technology for generation of ultra-long reads.
Total RNA-Seq vs. mRNA sequencing
Long RNA sequencing techniques, whole-transcriptome sequencing and mRNA sequencing are all powerful methods for transcriptome profiling. The objectives dictate the transcriptome sequencing approach to be used in a particular case.
Total RNA-Seq is used when all RNA transcripts of an organism must be analysed. This approach can be used for exploring all RNA species. mRNA sequencing can only be used for analysing the coding part of the genome. Since this RNA makes up only 1-4% of the transcriptome, enrichment of mRNA can greatly increase the amount of reads dedicated to exon analysis. Importantly, the sensitivity towards mRNA transcripts expressed at lower levels is also increased.
Small non-coding RNAs are analysed through a third transcriptome sequencing approach for microRNA (15-30 nucleotides) and small RNA (30 to 200 nucleotides). For information regarding small RNA sequencing, please click here.
Full-length transcripts of human, plant, animal transcriptomes can also be obtained with our Iso-Seq service.
Scientific expertise: Total RNA-Seq
GATC Biotech is a trusted provider of transcriptome sequencing services, including total RNA-Seq. With extended sequencing coverage and our excellent BioIT analysis, we can help you discover new variants and alternative splicing mechanisms in nearly any genome of interest. With GATC Biotech’s service, you can receive strand-specific data from transcriptomes without the need for well-defined reference sequences. Our expertise with the ultra-long read-length SMRT technology has recently enabled de novo transcriptome profiling and isoform characterisation by full cDNA sequence analysis. In recent years, the transcriptome sequencing services provided by GATC Biotech have helped elucidate mechanisms of enamel formation, the minimum survival requirements for Staphylococcus aureus and insecticide defence mechanisms, among many others.
Find here, a list of selected research articles supported by GATC Biotech’s sequencing products, including articles on transcriptomics.
Products related to transcriptome sequencing
Would you like to explore the effects of all RNA transcripts on gene expression in your model organism? Then enjoy the most simple and accurate method of whole-transcriptome profiling using our sample-to-data package. We provide a full range of services starting with RNA isolation from various starting materials, expert library preparation, sequencing on the Illumina or PacBio platforms, professional BioIT analysis and a final, comprehensive GATC Data Analysis report.
For better isoform profiling with full-length transcripts that require no assembly, try our INVIEW TRANSCRIPTOME ISOFORM DISCOVER.
For de novo transcriptome analysis, our NGSELECT product might be just what you need to take your research to the next level.
Further reading on whole transcriptome sequencing
Jiang, Z. et al. Whole transcriptome analysis with sequencing: methods, challenges and potential solutions. Cell Mol Life Sci. 72(18):3425-39 (2015).
Morozova, O., Hirst, M., Marra, M.A. Applications of New Sequencing Technologies for Transcriptome. Annu Rev Genomics Hum Genet. 10: 135-151 (2009).