Impact of replacing nucleic acid co-extraction reagents on performance of NGS for concurrent detection of DNA and RNA in formalin-fixed paraffin-embedded tissues
ZHAO Panpan
XU Jie
HUANG Jianjiang
Abstract：Objective: To investigate the impact of replacing nucleic acid co-extraction reagents on the results of next-generation sequencing (NGS) for simultaneous DNA and RNA detection in formalin-fixed paraffin-embedded (FFPE) tissues. Methods: FFPE samples from the Department of Pathology, Guangdong Provincial People's Hospital, were collected from August 2023 to May 2024. Nine cases with NGS results for DNA and RNA co-detection were selected. After sectioning the wax blocks, the samples were divided into two groups and extracted using either the original manual extraction kit or a newly introduced automated extraction kit for co-extraction of DNA and RNA. Subsequently, NGS library construction and sequencing were performed on the extracted nucleic acids, followed by bioinformatics analysis of the sequencing data. Cases with differing results were selected for repeated experiments to verify the findings. Results: The DNA and RNA extracted using both kits met the standards for NGS library construction. In terms of variant detection, the mutation results from the manual extraction group were consistent with previous pathology department results. The fusion mutations detected by the automated extraction group were consistent with those from the manual group. However, there were differences between the manual and automated groups in the detection of single nucleotide variants (SNVs), insertions/deletions (INDELs), and copy number variations (CNVs). The repeated experiments confirmed that changes in the extraction reagents could lead to false positive mutations and differences in CNV determination. Conclusion: Replacing the nucleic acid co-extraction reagents can affect the NGS detection results of DNA and RNA in FFPE samples, especially regarding CNVs and INDELs. When updating or replacing extraction reagents or making other process-related changes in clinical testing, it is necessary to validate the performance of the new reagents using previously tested samples.
Keywords：Nucleic acid co-extractionFormalin-fixed paraffin-embedded tissuesNext-generation sequencingDNA sequencingRNA sequencing
Publication Date：2025-09-28
Online Publishing Date：2025-11-13(First online date of this platform, not the publication date of the document)
Pages：5( 1202-1206 )