Evaluation of DNA and RNA quality from archival formalin‐fixed paraffin‐embedded tissue for next‐generation sequencing – Retrospective study in Japanese single institution

Abstract

Genetic analysis on formalin‐fixed paraffin‐embedded (FFPE) tissue specimens has become a mainstream method, from conventional direct sequencing to comprehensive analysis using next‐generation sequencing (NGS). In this study, we evaluated the quality of DNA and RNA extracted from FFPE sections, derived from surgical specimens of different tumor types. Electrophoresis was performed using a 4200 TapeStation to evaluate DNA and RNA fragmentation. DNA Ct values were higher and significantly increased over a period of 4 years compared with those from cell lines or frozen tissues. The RNA integrity number equivalent (RIN) ranged from 1 to 4.1 and DV200 ranged from 7.3 to 81%. Twelve of the 108 cases were analyzed by NGS using the AmpliSeq Cancer HotSpot Panel v2 on a Miniseq system. A sufficient number of reads and coverage were obtained in all cases. Our results revealed that NGS analysis was sufficient for FFPE‐derived DNA within 4 years of preservation. Conversely, approximately 20% of the RNA derived from FFPE within 4 years from the collection could be inappropriate for gene analysis based on RIN and DV200. It was suggested that FFPE would be adequate for genetic analysis, although it is desirable to store frozen specimens for the tumor tissues to be subjected to genetic analysis.

Targeted Next-Generation Sequencing of Cancer-Related Genes in a Norwegian Patient Cohort With Head and Neck Squamous Cell Carcinoma Reveals Novel Actionable Mutations and Correlations With Pathological Parameters

“…They noted in particular an increase in false positive SNVs in FFPE tissue after prolonged storage for more than 3-4 years (34). A similar observation has also been made by others (35). On the other hand, Kerick and co-workers have shown that smaller targeted NGS panels using PCR amplification and an increased coverage will reduce the impact of FFPE induced false positive SNVs in the work up of NGS analysis (36).…”

A clinicopathologic and molecular analysis of five cases of bronchiolar adenoma with rare mutations

“…AmpliSeq for Illumina Custom RNA Fusion Panel (Illumina, Sandiego, CA, USA) was used to detect the following ALK fusion partners: PTPN3 , C2orf44 , CLIP4 , CLTC , EML4 , FN1 , HIP1 , KIF5B , KLC1 , MSN , MYH9 , NCOA1 , NPM1 , PPFIBP1 , PRKAR1A , RNF213 , SQSTM1 , TFG , TPM1 , TPM3 , TPM4 , TPR , and VCL . For details of the method, see the previous report with some modifications 39 . Briefly, the sample was analyzed by the BaseSpace RNA amplicon v2.0.1.…”

“…For details of the method, see the previous report with some modifications. 39 Briefly, the sample was analyzed by the BaseSpace RNA amplicon v2.0.1. on the BaseSpace Sequencing Hub platform (Illumina).…”

Pure somatic pathogenic variation profiles for patients with serrated polyposis syndrome

“…However, NGS analysis in our study was successful even with aged FFPE samples, probably because the amplicon for analysis was designed to be as small as 200 bp, even if the nucleic acid was degraded. [25][26][27] Thus, it is important to consider the best sample from which to obtain DNA to match the progress of genomic analysis techniques, such as long-read sequencing. [28] Many groups have reported the in uence of pathological genetic variations of BRAF, such as c.1799T > A and p.Val600Glu, on the progression of HPs, SSLs, TSAs, and KRAS pathogenic variants for HPs and TSAs, but these analyses were performed among patients with heterogeneous germline backgrounds.…”