FFPE RNA-Seq OverviewThe fixation, embedding, and often long storage periods of FFPE samples can lead to high RNA degradation, making it very difficult to perform reliable, reproducible gene expression profiling with polyA RNA-Seq or ribosome-depleted RNA-Seq. Novogene offers state-of-the-art capture-based FFPE RNA-Seq for FFPE and other low-quality samples. This workflow enables researchers to apply the power of next-generation sequencing to gene expression profiling of typical clinical samples. Results from such efforts are increasingly more important for the development of powerful new treatment modalities, such as checkpoint inhibitors and personalized cancer vaccines.
The Novogene Advantage
- Comprehensive validation from FFPE RNA isolation to library construction to sequencing.
- Unsurpassed data quality with higher Q30 score, higher clean data percentage, higher mRNA mapping reads, and lower rRNA mapping reads than polyA+ RNA-seq. Typical comparison is shown in the Novogene Data tab.
- Extensive experience with hundreds of clinical FFPE samples analyzed.
- Comprehensive data analysis using widely accepted mainstream software and mature in-house pipelines. Our professional bioinformatics team offers standard, advanced, and customized analysis tailored to our clients’ needs. Raw data and analysis reports can be delivered via hard drives, FTP server, or Novogene’s cloud platform.
- HiSeq X platform, paired-end 150 bp
- NovaSeq 6000 platform, paired-end 150 bp
Data Quality Guarantee
- We guarantee that ≥ 80% of bases have a sequencing quality score ≥ Q30, which exceeds Illumina’s official guarantee of ≥ 75%.
FFPE Sample Requirements
|Quality||Agilent 2100 Bioanalyzer||Qubit|
|Too Degraded||<30% with fragments larger than 500nt||100ng|
|<30% without fragments larger than 500nt||Not recommended|
- Within 20 calendar days from verification of sample quality without data analysis
- The turnaround time for data analysis is project-dependent
Recommended Sequencing Depth
- 40 M reads, pair-end 150 bp
Analysis PipelinePipeline for RNA-Seq expression profiling
Pipeline for advanced transcriptome analysis
TruSeq Access RNA Sequencing DataBy using eight tissue FFPE samples, we compared the performance of three commercial RNA extraction kits: QIAGEN RNeasy FFPE Kit (hereafter referred to as “QIAGEN”), RecoverAll Total Nucleic Acid Isolation Kit for FFPE (hereafter referred to as “LIFE”), and MagMAX™ FFPE DNA/RNA Ultra Kit (hereafter referred to as “ABI”). Based on our results shown in Tables 1-3 and Figure 1, we selected the QIAGEN kit for our TruSeq Access RNA-Seq service due to its overall superiority in the quantity/quality of extracted RNA and the required experiment time. For the subsequent library construction, we were successful using as little as 20 ng of total RNA extracted from FFPE samples with DV200 > 30 (Table 4). For samples of lower quality (DV200 ≤ 30), however, the success rate was low, even with 100 ng of total RNA. Therefore, we do not recommend using such samples for this assay (Table 5). In the final step of RNA sequencing, Access RNA-Seq performed significantly better than polyA+ RNA-Seq with a higher Q30 score, higher clean data percentage, higher mRNA mapping reads, and lower rRNA mapping reads (Tables 6-7 and Figure 2).
Table 1. Quantity of the extracted RNA of the 8 FFPE samples from 3 commercial isolation kits, measured by Qubit Fluorometer.
Figure 1. Quantity of the extracted RNA of the 8 FFPE samples from 3 commercial isolation kits, measured by Qubit Fluorometer.
* The DV200 quality value represents the percentage of RNA fragments above 200 nucleotides.
Table 2.Quality of the extracted RNA of the 8 FFPE samples from 3 commercial isolation kits, measured by Agilent 2100 Bioanalyzer. The color legend shows different DV200 ranges.
Table 3. Considering the overall quantity, quality and experiment time of the 3 commercial isolation kits, QIAGEN RNeasy FFPE Kit (QIAGEN) was chosen for the TruSeq Access RNA-Seq service at Novogene.
Table 4. Library construction test by using 6 FFPE samples in different DV200 ranges (DV200 > 30), with lowest total RNA input of 20 ng.
Table 5. Library construction test by using 100 ng of total RNA from FFPE samples with DV200 values ≤ 30. Because of significant failure rate, such samples are not recommended for Access RNA sequencing.
Table 6. Comparison of sequencing quality using the same FFPE samples but different isolation protocols and different RNA library construction methods (polyA+ RNA-seq and Access RNA-seq).
Figure 2. Compared to polyA+ RNA-Seq, Access RNA-Seq shows higher Q30 score, higher clean data percentage, higher mRNA mapping reads, and lower rRNA mapping reads.
Table 7. Examples of data quality metrics from FFPE samples recently analyzed with Access RNA-seq.