Abstract
The National Cancer Institute conducted the Biospecimen Pre-analytical Variables (BPV) study to determine the effects of formalin fixation and delay to fixation (DTF) on the analysis of nucleic acids. By performing whole transcriptome sequencing and small RNA profiling on matched snap-frozen and FFPE specimens exposed to different delays to fixation, this study aimed to determine acceptable delays to fixation and proper workflow for accurate and reliable Next-Generation Sequencing (NGS) analysis of FFPE specimens. In comparison to snap-freezing, formalin fixation changed the relative proportions of intronic/exonic/untranslated RNA captured by RNA-seq for most genes. The effects of DTF on NGS analysis were negligible. In 80% of specimens, a subset of RNAs was found to differ between snap-frozen and FFPE specimens in a consistent manner across tissue groups; this subset was unaffected in the remaining 20% of specimens. In contrast, miRNA expression was generally stable across various formalin fixation protocols, but displayed increased variability following a 12 h delay to fixation.
Highlights
As Next-Generation Sequencing (NGS) technologies have become more affordable, reliable and powerful, they have been increasingly used to determine clinical diagnosis, to guide treatment and predict prognostic outcome (Reviewed in[1])
We report on differences in whole transcriptome RNA sequencing (RNA-seq) and miRNA expression profiling that are attributable to formalin-fixation and delays to formalin fixation (DTF)
The vast majority of libraries were of sufficient quality to conduct 50b PE sequencing and analysis on Illumina HiSeq 2500 instrumentation with a read depth ranging from 50 M to 120 M clusters (Supplementary Table 2), and most specimens sequenced to a depth of ~70 M clusters
Summary
As Next-Generation Sequencing (NGS) technologies have become more affordable, reliable and powerful, they have been increasingly used to determine clinical diagnosis, to guide treatment and predict prognostic outcome (Reviewed in[1]). The concordance between FFPE and frozen specimens was affected by RNA quality[3,12], which is known to be influenced by a number of preanalytical factors including post-mortem interval, delay to fixation (i.e. cold ischemia time), fixation duration, temperature, and storage conditions (Reviewed in[13]). Variation in these preanalytical factors is typical in the clinical laboratories where FFPE biospecimens are generated, but how these factors affect NGS results is unclear. This study provides guidance on best-practice methodologies for FFPE tissue handling and processing as well as analytical methods to enable the accurate and reliable detection of clinically relevant expression-related endpoints using NGS- based platforms
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