Standard gene expression profiling provides you a fast and affordable analysis on protein-coding RNA. With Total Transcriptome, you extend your vision towards all transcripts, including long non-coding RNA (lncRNA) and circular RNA (circRNA) species.
Total Transcriptome
Identify all RNA species, including partially degraded RNA
Why analyse total transcriptome?
Discuss your project with me
Comprehensive view of transcription profiles
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.
Service workflow
1. Initial meeting
When you consider using one of our services, our scientific team will help choosing the optimal project design. Each service can be customized by adding options. You will receive an overview of project budget and deliverables, so you can make an informed choice. Once you return your Purchase Order form, your project manager will contact you to discuss the next steps.
2. Sample delivery
Sample specifications for total transcriptome are:
- Optimal total RNA amount: ≥500 ng / sample
- Minimal RNA quantity: 100 ng
- Minimal volume of 25 μl/sample
- Quality RIN ≥3 / RQN ≥3 and OD260/280 ~1.8-2.2 ; OD260/230 ~1.8-2.2
For low input samples, please refer to our ultra-low input RNAseq service.
3. Sample entry QC
After sample arrival, Entry-QC is performed to determinate the sample quality and quantity.
A report with QC results will be provided and results and progression of the project can be discussed.
4. Library prep/QC and sequencing
Sequencing NovaSeq PE150
Gene-expression analysis:
- Standard read depth 30M/ sample.
- Removal of Globin RNA possible.
- Unique Molecular Identifier tags (UMIs).
This amount is sufficient for performing a statistically sound transcriptome analysis. Contact customer support if you require another amount of reads per sample.
5. Data QC
The raw data that is generated by the sequencer must be divided to obtain one data file set per sample for each sequencing run. This is called demultiplexing.
Your project manager checks the data quality by analyzing the quality metrics of the run. Final inspection of the data takes place and the dataset is transferred onto our portal. It is also possible to receive your data on hard disk.
6. Reporting
You receive your Project Report together with your data. It contains an overview of the experimental methods used, sample specifications, and a summary of all technical information for publication. Deliverables:
- Gene-expression analysis (raw data)
- Stranded sequencing of all RNA transcripts, including rRNA depletion
- Quality score Q30 of ≥80% for PE 150 reads
- Count table (optional)
- Differential gene expression analysis (optional)
- PCA plot, heatmap, pathway analysis (optional)
Deliverables overview
- 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.
Structural variant analysis
To determine structural variation such as isoforms, splice variants and gene-fusions we advise a sequencing depth of 50 – 100 million reads per sample.
Service specifications
We have summarized key information about our total transcriptome service into a service specification sheet.
Let's get the conversation started for your next NGS project
Please either fill in this form or email us directly at info@genomescan.nl
and we will get in touch with you to discuss your requirements
