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Organocatalyst treatment improves variant calling and mutant detection in archival clinical samples
Citation:
Wehmas, L., C. Wood, P. Guan, M. Gosink, AND S. Hester. Organocatalyst treatment improves variant calling and mutant detection in archival clinical samples. Scientific Data. Springer Nature, New York, NY, 12:6509, (2022). https://doi.org/10.1038/s41598-022-10301-0
Impact/Purpose:
Recently, we demonstrated that relatively simple changes to standard RNA isolation protocols using an organocatalyst with extended heated incubation (ORGΔ) significantly improved the quality of RNA-sequencing (RNA-seq) data from FFPE samples (Wehmas et al. 2018, 2019). In the present study, we hypothesized that use of this same method would also enhance FFPE DNA data quality, increasing detection and reliability of variant calls for mutation analyses of tumor samples. Furthermore, as RNA transcripts are essentially an edited, condensed version of the expressed genome, we predicted that variants in RNA-seq data that are also present in the gene coding regions of DNA-seq data could be used to filter out formalin-induced sequence artifacts in DNA. To test these ideas, we analyzed paired frozen (FR) and FFPE samples from human kidney and ovarian cancer specimens collected through the Biospecimen Pre-analytical Variables (BPV) program, a National Cancer Institute (NCI)-sponsored study that systematically assessed the effects of pre-analytical factors on the molecular profile of biospecimens (Carithers et al. 2019). We applied ORGΔ to FFPE RNA and DNA samples prior to total RNA-seq and DNA (exome) sequencing (DNA-seq), respectively, and analyzed the results relative to FR sample controls to detect changes in variant analysis. Our results show that ORGΔ improved FFPE Exome-seq data quality in several ways, including higher-quality SNP calls. These findings should have important applications in translational science and precision medicine.
Description:
Formalin fixation of biological specimens damages nucleic acids and limits their use in genomic analyses. Previously, we showed that RNA isolation with an organocatalyst (2-amino-5-methylphenyl phosphonic acid, used to speed up reversal of formalin-induced adducts) and extended heated incubation (ORGΔ) improved RNA-sequencing data from formalin-fixed paraffin-embedded (FFPE) tissue samples. The primary goal of this study was to evaluate whether ORGΔ treatment improves DNA-sequencing data from clinical FFPE samples. We isolated RNA and DNA ± ORGΔ from paired FFPE and frozen human renal and ovarian carcinoma specimens collected as part of the National Cancer Institute Biospecimen Pre-analytical Variables program. Tumor types were microscopically confirmed from adjacent tissue sections. Following extraction, DNA was fragmented and sequenced and differences were compared between frozen and FFPE sample pairs. Treatment with ORGΔ improved concurrent SNP calls in FFPE DNA compared to non-ORGΔ FFPE samples and enhanced confidence in SNP calls for all FFPE DNA samples, beyond that of matched frozen samples. In general, the concordant SNPs identified in paired frozen and FFPE DNA samples agreed for both genotype and homozygosity vs. heterozygosity of calls regardless of ORGΔ treatment. The increased confidence in ORGΔ FFPE DNA variant calls relative to the matched frozen DNA suggests a novel application of this method. With further optimization, this method may improve quality of DNA-sequencing data in FFPE as well as frozen tissue samples.
URLs/Downloads:
DOI: Organocatalyst treatment improves variant calling and mutant detection in archival clinical samples
https://pubmed.ncbi.nlm.nih.gov/35443772/