TY - JOUR
T1 - Comparison of clinical targeted next-generation sequence data from formalin-fixed and fresh-frozen tissue specimens
AU - Spencer, David H.
AU - Sehn, Jennifer K.
AU - Abel, Haley J.
AU - Watson, Mark A.
AU - Pfeifer, John D.
AU - Duncavage, Eric J.
N1 - Funding Information:
Supported by the Washington University Department of Pathology and the Department of Genetics . The Genome Technology Access Center (Washington University School of Medicine), which assisted with genomic analysis, is partially supported by National Cancer Institute Cancer Center support grant P30 CA91842 to the Siteman Cancer Center and by Institute of Clinical and Translational Sciences/Clinical & Translational Science Awards grant UL1RR024992 from the National Center for Research Resources, a component of the NIH, and the NIH Roadmap for Medical Research.
PY - 2013/9
Y1 - 2013/9
N2 - Next-generation sequencing (NGS) has emerged as a powerful technique for the detection of genetic variants in the clinical laboratory. NGS can be performed using DNA from FFPE tissue, but it is unknown whether such specimens are truly equivalent to unfixed tissue for NGS applications. To address this question, we performed hybridization-capture enrichment and multiplexed Illumina NGS for 27 cancer-related genes using DNA from 16 paired fresh-frozen and routine FFPE lung adenocarcinoma specimens and conducted extensive comparisons between the sequence data from each sample type. This analysis revealed small but detectable differences between FFPE and frozen samples. Compared with frozen samples, NGS data from FFPE samples had smaller library insert sizes, greater coverage variability, and an increase in C to T transitions that was most pronounced at CpG dinucleotides, suggesting interplay between DNA methylation and formalin-induced changes; however, the error rate, library complexity, enrichment performance, and coverage statistics were not significantly different. Comparison of base calls between paired samples demonstrated concordances of >99.99%, with 96.8% agreement in the single-nucleotide variants detected and >98% accuracy of NGS data when compared with genotypes from an orthogonal single-nucleotide polymorphism array platform. This study demonstrates that routine processing of FFPE samples has a detectable but negligible effect on NGS data and that these samples can be a reliable substrate for clinical NGS testing.
AB - Next-generation sequencing (NGS) has emerged as a powerful technique for the detection of genetic variants in the clinical laboratory. NGS can be performed using DNA from FFPE tissue, but it is unknown whether such specimens are truly equivalent to unfixed tissue for NGS applications. To address this question, we performed hybridization-capture enrichment and multiplexed Illumina NGS for 27 cancer-related genes using DNA from 16 paired fresh-frozen and routine FFPE lung adenocarcinoma specimens and conducted extensive comparisons between the sequence data from each sample type. This analysis revealed small but detectable differences between FFPE and frozen samples. Compared with frozen samples, NGS data from FFPE samples had smaller library insert sizes, greater coverage variability, and an increase in C to T transitions that was most pronounced at CpG dinucleotides, suggesting interplay between DNA methylation and formalin-induced changes; however, the error rate, library complexity, enrichment performance, and coverage statistics were not significantly different. Comparison of base calls between paired samples demonstrated concordances of >99.99%, with 96.8% agreement in the single-nucleotide variants detected and >98% accuracy of NGS data when compared with genotypes from an orthogonal single-nucleotide polymorphism array platform. This study demonstrates that routine processing of FFPE samples has a detectable but negligible effect on NGS data and that these samples can be a reliable substrate for clinical NGS testing.
UR - http://www.scopus.com/inward/record.url?scp=84882261506&partnerID=8YFLogxK
U2 - 10.1016/j.jmoldx.2013.05.004
DO - 10.1016/j.jmoldx.2013.05.004
M3 - Article
C2 - 23810758
AN - SCOPUS:84882261506
SN - 1525-1578
VL - 15
SP - 623
EP - 633
JO - Journal of Molecular Diagnostics
JF - Journal of Molecular Diagnostics
IS - 5
ER -