Comparison of Accuracy of Whole-Exome Sequencing with Formalin-Fixed Paraffin-Embedded and Fresh Frozen Tissue Samples

Abstract

Formalin fixing with paraffin embedding (FFPE) has been a standard sample preparation method for decades, and archival FFPE samples are still very useful resources. Nonetheless, the use of FFPE samples in cancer genome analysis using next-generation sequencing, which is a powerful technique for the identification of genomic alterations at the nucleotide level, has been challenging due to poor DNA quality and artificial sequence alterations. In this study, we performed whole-exome sequencing of matched frozen samples and FFPE samples of tissues from 4 cancer patients and compared the next-generation sequencing data obtained from these samples. The major differences between data obtained from the 2 types of sample were the shorter insert size and artificial base alterations in the FFPE samples. A high proportion of short inserts in the FFPE samples resulted in overlapping paired reads, which could lead to overestimation of certain variants; >20% of the inserts in the FFPE samples were double sequenced. A large number of soft clipped reads was found in the sequencing data of the FFPE samples, and about 30% of total bases were soft clipped. The artificial base alterations, C>T and G>A, were observed in FFPE samples only, and the alteration rate ranged from 200 to 1,200 per 1M bases when sequencing errors were removed. Although high-confidence mutation calls in the FFPE samples were compatible to that in the frozen samples, caution should be exercised in terms of the artifacts, especially for low-confidence calls. Despite the clearly observed artifacts, archival FFPE samples can be a good resource for discovery or validation of biomarkers in cancer research based on whole-exome sequencing.

Superresolution Imaging of Clinical Formalin Fixed Paraffin Embedded Breast Cancer with Single Molecule Localization Microscopy

“…These preserved tissues have the potential to improve our understanding of diseases, especially in oncology where biopsies and resected specimens are routinely maintained in clinical practice. Use of fresh frozen tissue has long been considered the “gold-standard” for molecular assays as proteins and genetic material are preserved in their native state234. However, ice crystal formation is common, creating artifacts that are not seen in FFPE preserved tissues5.…”

Mutational profiles of persistent/recurrent laryngeal squamous cell carcinoma

“…Our comparison of mutational rates in primary vs persistent/recurrent LSCC needs to be interpreted in this light. However, several recent studies have demonstrated high (ie, > 90%) concordance rates in detection of SNVs and CNVs in coding genomic regions between matched fresh‐frozen and FFPE tumor samples . In addition, comparison of amplicon‐based, targeted exome sequencing to TCGA dataset is a well‐established practice in the HNSCC literature .…”

“…However, several recent studies have demonstrated high (ie, > 90%) concordance rates in detection of SNVs and CNVs in coding genomic regions between matched freshfrozen and FFPE tumor samples. [28][29][30] In addition, comparison of amplicon-based, targeted exome sequencing to TCGA dataset is a well-established practice in the HNSCC literature. 11,13 Thus, we believe that these differences in DNA sequencing methodology do not significantly diminish the validity of our comparisons and conclusions.…”

Circulating tumour DNA: a minimally invasive biomarker for tumour detection and stratification

“…Studies have shown that formalin fixation causes DNA denaturation, base alteration and production of sequence artefact (Douglas and Rogers 1998;Oh et al 2015). Gallegos Ruiz and colleagues (2007) have observed mutational artefacts in epidermal growth factor receptor (EGFR) gene resulting from formalin fixation and paraffin embedding of lung tissue sample.…”

“…Therefore, formalin-processed tissue specimens might not accurately reflect the genetic alterations present in the tumour tissue. The use of fresh frozen tissue for processing might overcome these limitations, but it is restricted by logistic issues such as availability of liquid nitrogen and requirement of specialised equipment for processing and storage of frozen tissue (Budczies et al 2011;Oh et al 2015).…”