Seamless Whole Transcriptome RNA-Seq Solutions for FFPE Samples
Whole Transcriptome FFPE
Get the best sequencing results from your FFPE samples with Lexogen’s fast and flexible whole transcriptome solutions for FFPE material! CORALL FFPE Whole Transcriptome RNA-Seq provides the optimal coverage for FFPE samples of varying quantity and quality and is perfectly suited for analyzing even poor FFPE and Biobank samples with a DV200<10 %.
The CORALL FFPE Whole Transcriptome kit enables fast and cost-efficient generation of stranded, UMI-labelled, and unique dual indexed libraries with adjustable insert size. Bundles are available including rRNA depletion for human / mouse / rat ribosomal transcripts for a convenient complete workflow solution.
Performance
CORALL FFPE RNA-Seq provides a fast and fragmentation-free end-to-end whole transcriptome workflow with exceptional performance on challenging FFPE samples, including low input and low DV200 FFPE RNA and has been validated on various FFPE tissues from different organisms.
Validated Performance on various FFPE Tissues
CORALL FFPE Whole Transcriptome RNA-Seq was validated on various human FFPE tissues, including liver, lung, brain, and kidney as well as tumor and normal tissues (Fig. 1) of varying qualities (DV200 between 77 % and 45 %).
Figure 1 | Read Mapping Statistics for CORALL FFPE libraries generated from different human FFPE tissues: liver (DV200 = 66 %), lung tumor (DV200 = 64 %), brain normal tissue (DV200 = 76 % and 77 %), brain tumor (DV200= 47 % and 66 %), kidney normal tissue (DV200 = 50 %), and kidney tumor (DV200 = 45 %).
Excellent Performance Across a Wide Range of Input RNA Amounts
The majority of reads generated by CORALL FFPE library preparation from 5 ng – 200 ng mouse liver FFPE RNA present uniquely mapping reads (Fig. 2A). As a results, CORALL FFPE provides excellent gene detection across a wide range of FFPE input RNA amounts even at low sequencing depth (Fig. 2B).
Figure 2 | Basic Mapping Stats (A) and Gene Detection at a threshold of CPM>5 (B) for CORALL FFPE libraries generated from 200 ng, 50 ng, and 5 ng mouse liver FFPE RNA (DV200 = 79 %) at 8.5 M reads per sample.
Benchmarked Performance
CORALL FFPE Whole Transcriptome RNA-Seq performance and gene detection matches the performance of competitor protocols (Fig. 3) with ~95 % of genes commonly detected between both protocols (Fig. 4).
Figure 3| Read mapping statistics (A), FastQC duplicate detection (B), and gene detection at a threshold of CPM>10 (C) for CORALL FFPE and competitor libraries generated from 10 ng mouse liver FFPE RNA (DV200 = 79 %) following ribo-depletion at 15 M paired end reads per sample (2×75).
Figure 4 | Gene detection overlap. The Venn diagram illustrates the overlap of detected genes between CORALL FFPE RNA-Seq and Competitor N at normalized expression levels of >10 CPM.
Improved Transcript Body Coverage Even at Low Sequencing Depth
CORALL FFPE RNA-Seq generates transcriptome-wide smooth, uniform read coverage. Coverage was assessed at low sequencing depth of 1.2 M reads per sample and shows improved evenness compared to competitor protocols (Fig. 5).
Figure 5 | Accumulated transcript body coverage (whole transcriptome). Coverage across all transcripts was generated using the geneBody_coverage.py tool provided by RSeQC (transcripts length normalized to 100 %). CORALL FFPE coverage is compared to competitor N at 1.2 M single reads per sample.
Robust Detection of Upregulation of Cancer Marker Transcripts in Tumor FFPE Samples
CORALL FFPE detects upregulation of tumor marker transcripts as exemplified by elevated coverage of Chemokine Receptor CXCR4 in kidney tumor FFPE samples. CXCR4 is associated with an increased risk and the incidence of renal cell carcinoma and its progression (Fig. 6).
Figure 6 | Coverage of CRCX4 from human kidney tumor and matched normal FFPE tissue. Libraries were prepared from 20 ng FFPE RNA extracted with SPLIT One-Step FFPE RNA Extraction following ribo-depletion with RiboCop HMR V2. Samples were sequenced at 8.5 M paired end reads per sample and mapped against the human reference genome (hsa_GRCh38.94).
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