Organocatalyst treatment improves variant calling and mutant detection in archival clinical samples
Abstract 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 incub...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-04-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-10301-0 |
| _version_ | 1818147512751489024 |
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| author | Leah C. Wehmas Charles E. Wood Ping Guan Mark Gosink Susan D. Hester |
| author_facet | Leah C. Wehmas Charles E. Wood Ping Guan Mark Gosink Susan D. Hester |
| author_sort | Leah C. Wehmas |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-12-11T12:36:26Z |
| format | Article |
| id | doaj.art-c82f8591c92b4bb0a366519a2cd4a31f |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-11T12:36:26Z |
| publishDate | 2022-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-c82f8591c92b4bb0a366519a2cd4a31f2022-12-22T01:07:08ZengNature PortfolioScientific Reports2045-23222022-04-0112111010.1038/s41598-022-10301-0Organocatalyst treatment improves variant calling and mutant detection in archival clinical samplesLeah C. Wehmas0Charles E. Wood1Ping Guan2Mark Gosink3Susan D. Hester4Office of Research and Development, U.S. Environmental Protection AgencyOffice of Research and Development, U.S. Environmental Protection AgencyNational Cancer InstitutePfizerOffice of Research and Development, U.S. Environmental Protection AgencyAbstract 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.https://doi.org/10.1038/s41598-022-10301-0 |
| spellingShingle | Leah C. Wehmas Charles E. Wood Ping Guan Mark Gosink Susan D. Hester Organocatalyst treatment improves variant calling and mutant detection in archival clinical samples Scientific Reports |
| title | Organocatalyst treatment improves variant calling and mutant detection in archival clinical samples |
| title_full | Organocatalyst treatment improves variant calling and mutant detection in archival clinical samples |
| title_fullStr | Organocatalyst treatment improves variant calling and mutant detection in archival clinical samples |
| title_full_unstemmed | Organocatalyst treatment improves variant calling and mutant detection in archival clinical samples |
| title_short | Organocatalyst treatment improves variant calling and mutant detection in archival clinical samples |
| title_sort | organocatalyst treatment improves variant calling and mutant detection in archival clinical samples |
| url | https://doi.org/10.1038/s41598-022-10301-0 |
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