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Evaluation of Targeted Sequencing for Transcriptional Analysis of Archival Formalin-Fixed Paraffin-Embedded (FFPE) Samples
Citation:
Hester, S., L. Wehmas, AND C. Wood. Evaluation of Targeted Sequencing for Transcriptional Analysis of Archival Formalin-Fixed Paraffin-Embedded (FFPE) Samples. Society of Toxicology Annual Meeting, San Antonio, TX, March 11 - 15, 2018.
Impact/Purpose:
Tissue archives at the EPA and elsewhere span a large array of studies covering thousands of compounds. Opening up these studies to new methods of analysis should have broad application across NHEERL and ORD programs involved in chemical prioritization and risk assessment. This access would facilitate extrapolation across models and enable other scientific organizations (including chemical/pesticide registrants) to generate mechanistic pathway-based information without the need for additional de novo mechanistic studies, reducing animal usage and streamlining the risk assessment process. However, improved analytical methods are still needed when working with archival samples, which are typically stored as formalin-fixed paraffin-embedded (FFPE) blocks. Recently, a lower-cost targeted sequencing platform called TempO-Seq was developed for gene expression profiling without the need for RNA isolation or pre-amplification. In this study, we compared genomic responses between TempO-Seq and standard whole-genome sequencing (RNA-Seq) in FFPE and paired frozen samples. Our findings show strong correlation between TempO-Seq and RNA-Seq platforms, supporting the idea that targeted sequencing can be used as a more cost-efficient and streamlined alternative method for analysis of RNA expression in FFPE samples.
Description:
Next-generation sequencing provides unprecedented access to genomic information in archival FFPE tissue samples. However, costs and technical challenges related to RNA isolation and enrichment limit use of whole-genome RNA-sequencing for large-scale studies of FFPE specimens. Recently, a targeted sequencing platform called TempO-Seq® was developed for gene expression profiling without the need for RNA isolation or pre-amplification. In this study, we compared genomic responses between the new TempO-Seq® mouse array (containing 3045 probes targeting 2756 genes) and the Illumina-based whole-genome platform (RNA-Seq) in FFPE and paired frozen (FROZ) samples. Archived liver specimens were analyzed from control mice (CON) and mice treated with 600 ppm of phenobarbital (PB) for 7 days (n=6/group). Portions of each liver were (a) snap-frozen and (b) fixed in 10% buffered formalin for 18 hours before processing to FFPE blocks. Quality sequencing metrics and expression of the pre-selected PB response genes Cyp2b10 and Cyp3a11 were evaluated. RNA-seq used a higher read depth (68.5 ± 1.4 million/sample) than TempO-Seq® (2.4 ± 0.1 million/sample) but provided similar alignment to the mouse genome (86% vs. 81%, respectively). Total mapped reads were 52% lower for FFPE vs. FROZ samples on RNA-Seq but similar for TempO-Seq® (2% higher for FFPE). On RNA-Seq, total reads mapped to Cyp2b10 were ~2,374-fold higher for PB (23,739 ± 1,647) vs. controls (≤10) in FROZ and ~829-fold higher in FFPE (≤10 in CON vs. 8,292 ± 547 in PB), indicating a signal loss of ~65%. For Cyp3a11, total reads were 78% lower in PB-treated FFPE vs. FROZ samples, but fold-change values were comparable: 58-fold higher in FROZ (1754 ± 441 in CON vs. 102,297 ± 13,747 in PB) and 68-fold higher in FFPE (327 ± 77 in CON vs. 22,294 ± 3,276 in PB). Using TempO-Seq®, Cyp2b10 reads were ~2,373-fold higher in PB mice (23,728 ± 1,067 reads) vs. controls (≤10) in FROZ and ~2,201-fold higher in FFPE (≤10 in CON vs. 22,008 ± 2,197 in PB), while Cyp3a11 reads were 49-fold higher in FROZ (29 ± 13 in CON vs. 1409 ± 86 in PB) and 50-fold higher in FFPE (67 ± 11 in CON vs. 3347 ± 503 in PB). In FFPE samples, counts for Cyp2b10 and Cyp3a11 correlated strongly between TempO-Seq® and RNA-Seq platforms (R2 > 0.96, P < 10-7 for both markers). These findings support the idea that targeted sequencing can be used as a streamlined alternative method for analysis of RNA expression in FFPE samples. This abstract does not reflect U.S. EPA policy.