TY - JOUR
T1 - Accurate RNA sequencing from formalin-fixed cancer tissue to represent high-quality transcriptome from frozen tissue
AU - Li, Jialu
AU - Fu, Chunxiao
AU - Speed, Terence P.
AU - Wang, Wenyi
AU - Symmans, W. Fraser
N1 - Publisher Copyright:
© 2019 American Society of Clinical Oncology.
PY - 2018
Y1 - 2018
N2 - Purpose Accurate transcriptional sequencing (RNA-seq) from formalin-fixed paraffinembedded (FFPE) tumor samples presents an important challenge for translational research and diagnostic development. In addition, there are now several different protocols to prepare a sequencing library from total RNA. We evaluated the accuracy of RNA-seq data generated from FFPE samples in terms of expression profiling. Methods We designed a biospecimen study to directly compare gene expression results from different protocols to prepare libraries for RNA-seq from human breast cancer tissues, with randomization to fresh frozen (FF) or FFPE conditions. The protocols were compared using multiple computational methods to assess alignment of reads to a reference genome, the uniformity and continuity of coverage, the variance and correlation of overall gene expression, patterns of measuring coding sequence, phenotypic patterns of gene expression, and measurements from representative multigene signatures. Results The principal determinant of variance in gene expression was use of exon capture probes, followed by the conditions of preservation (FF v FFPE) and phenotypic differences between breast cancers. One protocol, with RNase H-based ribosomal RNA depletion, exhibited the least variability of gene expression measurements and strongest correlation between FF and FFPE samples and was generally representative of the transcriptome from standard FF RNA-seq protocols. Conclusion Method of RNA-seq library preparation from FFPE samples had a marked effect on the accuracy of gene expression measurement compared with matched FF samples. Nevertheless, some protocols produced highly concordant expression data from FFPE RNA-seq data, compared with RNA-seq results from matched frozen samples.
AB - Purpose Accurate transcriptional sequencing (RNA-seq) from formalin-fixed paraffinembedded (FFPE) tumor samples presents an important challenge for translational research and diagnostic development. In addition, there are now several different protocols to prepare a sequencing library from total RNA. We evaluated the accuracy of RNA-seq data generated from FFPE samples in terms of expression profiling. Methods We designed a biospecimen study to directly compare gene expression results from different protocols to prepare libraries for RNA-seq from human breast cancer tissues, with randomization to fresh frozen (FF) or FFPE conditions. The protocols were compared using multiple computational methods to assess alignment of reads to a reference genome, the uniformity and continuity of coverage, the variance and correlation of overall gene expression, patterns of measuring coding sequence, phenotypic patterns of gene expression, and measurements from representative multigene signatures. Results The principal determinant of variance in gene expression was use of exon capture probes, followed by the conditions of preservation (FF v FFPE) and phenotypic differences between breast cancers. One protocol, with RNase H-based ribosomal RNA depletion, exhibited the least variability of gene expression measurements and strongest correlation between FF and FFPE samples and was generally representative of the transcriptome from standard FF RNA-seq protocols. Conclusion Method of RNA-seq library preparation from FFPE samples had a marked effect on the accuracy of gene expression measurement compared with matched FF samples. Nevertheless, some protocols produced highly concordant expression data from FFPE RNA-seq data, compared with RNA-seq results from matched frozen samples.
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U2 - 10.1200/PO.17.00091
DO - 10.1200/PO.17.00091
M3 - Article
C2 - 29862382
AN - SCOPUS:85083939051
SN - 2473-4284
SP - 1
EP - 9
JO - JCO Precision Oncology
JF - JCO Precision Oncology
IS - 2
ER -