Total Transcriptome

Identify all RNA species, including partially degraded RNA

Comprehensive view of transcription profiles

Standard gene-expression analysis is performed on protein-coding RNA, since this provides you the fastest and affordable method to visualize gene-expression. However, with Total Transcriptome, you extend your vision towards all transcripts, including long non-coding RNA (lncRNA) and cicular RNA (circRNA) species.

Total RNA sequencing helps you to overcome common causes for RNA degradation, such as FFPE-fixation and laser-capture methods. Additionally, precious samples from a cohort might not be handled optimally, blocking the only source to obtain transcriptome profiles for your study. Our scientists have set up validated procedures to analyze these challenging samples. This includes clinical-grade sample isolation from FFPE material and tailored data-analysis.

A ribosomal RNA (rRNA) depletion step is usually required, since the 16S and 18S ribosomal subunits make up ~60-90% of all RNA species present in your sample. You make better use of the sequencing space by removing them at the sample preparation phase. What remains is messenger RNA, and many other regulatory types of RNA.

Working seamlessly with you on your project:

Product specifications 1 01 Product page   Total Transcriptome
  • Measurement of all RNA transcripts present
  • rRNA depletion and globin reduction are optional
  • Challenging samples: The average RNA transcript size must exceed 50 bp to allow for unique mapping and analysis. Check your (column-)isolation procedure to determine from which size (bp) the RNA fragments are retained.
Sample specifications 2 01 Product page   Total Transcriptome
  • Optimal total RNA input: 100 ng / sample
  • Ideally deliver ≥250 ng RNA to allow for QC and normalization
  • Minimally validated RNA input: 10 ng
  • For RIN / RQN of all qualities
Deliverables 3 01 Product page   Total Transcriptome
  • Stranded sequencing of all RNA transcripts, including rRNA depletion
  • Quality score Q30 of ≥80% for PE 150 reads
  • TAT: 3 weeks
Sequencing 4 01 Product page   Total Transcriptome
  • Sequencing on NovaSeq 6000 PE150
  • Gene-expression analysis: 40 million reads

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This amount is sufficient for performing a statistically sound transcriptome analysis. It is a  more accurate and complete alternative for micro-array sequencing. Contact customer support if you require another amount of reads per sample.

  • Structural variant analysis

To determine all structural variants, such as isoforms, splice variants and gene-fusions we advise a sequencing depth of 80 – 120 million reads per sample. You will receive a full transcriptome analysis, as wells as a reliable overview of SNPs and structural rearrangements.

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Data quality 5 01 Product page   Total Transcriptome

The specifications of the data-set that you will recieve are listed in your personal quotation. Generally, the sequencing quality score is Q30≥80% for PE 150 reads (Illumina’s official guarantee ≥75%). Our average score of 2018 was Q30≥90%.

Data analysis 6 01 Product page   Total Transcriptome
  • Gene-expression analysis (raw data)
  • Count table
  • PCA plot, heatmap, pathway analysis

We have summarized key information about our total transcriptome service into a product specification sheet, which you can download here.

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Discuss you project with Andre

We have over 15 years of experience in the tanscriptomics field. Talk with us about best practices and how we can help your research.

Photo André rond 01 Product page   Total Transcriptome

Andre Wijfjes

Manager Customer Support

+31 71 568 1036