Project description:Advantages of RNA-Seq over array based platforms are quantitative gene expression and discovery of expressed single nucleotide variants (eSNVs) and fusion transcripts from a single platform, but the sensitivity for each of these characteristics is unknown. We measured gene expression in a set of manually degraded RNAs, nine pairs of matched fresh-frozen, and FFPE RNA isolated from breast tumor with the hybridization based, NanoString nCounter, (226 gene panel) and with whole transcriptome RNA-Seq using RiboZeroGold ScriptSeq V2 library preparation kits. We performed correlation analyses of gene expression between samples and across platforms. We then specifically assessed whole transcriptome expression of lincRNA and discovery of eSNVs and fusion transcripts in the FFPE RNA-Seq data. For gene expression in the manually degraded samples, we observed Pearson correlation of >0.94 and >0.80 with NanoString and ScriptSeq protocols respectively. Gene expression data for matched fresh-frozen and FFPE samples yielded mean Pearson correlations of 0.874 and 0.783 for NanoString (226 genes) and ScriptSeq whole transcriptome protocols respectively. Specifically for lincRNAs, we observed superb Pearson correlation (0.988) between matched fresh-frozen and FFPE pairs. FFPE samples across NanoString and RNA-Seq platforms gave a mean Pearson correlation of 0.838. In FFPE libraries, we detected 53.4% of high confidence SNVs and 24% of high confidence fusion transcripts. Sensitivity of fusion transcript detection was not overcome by an increase in depth of sequencing up to 3-fold (increase from ~56 to ~159 million reads). Both NanoString and ScriptSeq RNA-Seq technologies yield reliable gene expression data for degraded and FFPE material. The high degree of correlation between NanoString and RNA-Seq platforms suggests discovery based whole transciptome studies from FFPE material will produce reliable expression data. The RiboZeroGold ScriptSeq protocol performed particularly well for lincRNA expression from FFPE libraries but detection of eSNV and fusion transcripts was less sensitive.

Project description:Advantages of RNA-Seq over array based platforms are quantitative gene expression and discovery of expressed single nucleotide variants (eSNVs) and fusion transcripts from a single platform, but the sensitivity for each of these characteristics is unknown. We measured gene expression in a set of manually degraded RNAs, nine pairs of matched fresh-frozen, and FFPE RNA isolated from breast tumor with the hybridization based, NanoString nCounter, (226 gene panel) and with whole transcriptome RNA-Seq using RiboZeroGold ScriptSeq V2 library preparation kits. We performed correlation analyses of gene expression between samples and across platforms. We then specifically assessed whole transcriptome expression of lincRNA and discovery of eSNVs and fusion transcripts in the FFPE RNA-Seq data. For gene expression in the manually degraded samples, we observed Pearson correlation of >0.94 and >0.80 with NanoString and ScriptSeq protocols respectively. Gene expression data for matched fresh-frozen and FFPE samples yielded mean Pearson correlations of 0.874 and 0.783 for NanoString (226 genes) and ScriptSeq whole transcriptome protocols respectively. Specifically for lincRNAs, we observed superb Pearson correlation (0.988) between matched fresh-frozen and FFPE pairs. FFPE samples across NanoString and RNA-Seq platforms gave a mean Pearson correlation of 0.838. In FFPE libraries, we detected 53.4% of high confidence SNVs and 24% of high confidence fusion transcripts. Sensitivity of fusion transcript detection was not overcome by an increase in depth of sequencing up to 3-fold (increase from ~56 to ~159 million reads). Both NanoString and ScriptSeq RNA-Seq technologies yield reliable gene expression data for degraded and FFPE material. The high degree of correlation between NanoString and RNA-Seq platforms suggests discovery based whole transciptome studies from FFPE material will produce reliable expression data. The RiboZeroGold ScriptSeq protocol performed particularly well for lincRNA expression from FFPE libraries but detection of eSNV and fusion transcripts was less sensitive. We performed RNASeq on RNA from nine matched pairs of fresh-frozen and FFPE tissues from breast cancer patients. The goal was to test the RiboZeroGold ScriptSeq complete low input library preparation kit for degraded RNA samples.

Project description:For the effectiveness of targeted immunotherapy, which is currently one of the most promising treatments for brain tumors, it is necessary to know specific tumor-associated antigens (TAA). This project aimed to validate the suitability of formalin-fixed and paraffin-embedded (FFPE) material instead of fresh-frozen material for RNA sequencing and downstream TAA identification. Purpose: Detect potential oligodendroglioma-associated antigens using both fresh-frozen and FFPE brain tissues; assess the suitability of the FFPE material for TAA identification. Results: A comparative analysis based on the RNA-seq of canine oligodendroglioma showed that formalin fixation of the samples had a significant effect on the RNA-seq library in terms of quality. Read distribution analysis showed that the FFPE samples contained fewer reads mapping to exonic regions and were enriched with reads mapped to introns and intergenic regions, while the fresh-frozen samples were mostly enriched in reads mapping to exons. Mismatch profile and SNVs calling analyses revealed some substitution artefacts present in the FFPE samples and absent in the corresponding fresh-frozen samples. However, according to the Principal Component Analysis, 36% of the variance between the samples could be explained by the types of conservation, while 43% of the variance could still be attributed to different expression profiles between oligodendroglioma and control conditions. A differential expression analysis of fresh-frozen oligodendroglioma versus fresh-frozen control samples identified 62 potential TAA strongly up-regulated in tumor tissue. The same analysis using the FFPE samples showed 80% of these genes (49/62) also to be differentially expressed. Comparative analysis showed good agreement between FFPE and fresh-frozen RNA-seq libraries in terms of the accuracy of gene expression measurements indicating that archived FFPE tissue can be used for identification of TAA candidates by RNA-seq providing a wealthy source of clinical samples for research. The following 10 genes encoding cell surface proteins have been identified by both approaches as potential TAA candidates based on their strong overexpression in oligodendrogliomas: PDGFRA, NOTCH1, DLL1, GPER1, TNR, IQGAP3, CD44, ERBB3, BCAS1 and ROR2. Future projects still need to investigate their suitability as TAA for targeted immunotherapy. Conclusion: Formalin fixation of the samples had a significant impact on the RNA-seq quality in terms of the accuracy of gene expression measurements, as well as the quality of the nucleotide sequences. Nevertheless, the comparative analysis showed good agreement between FFPE and fresh-frozen RNA-seq libraries.

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