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Accurate RNA Sequencing From Formalin-Fixed Cancer Tissue to Represent High-Quality Transcriptome From Frozen Tissue

PURPOSE: Accurate transcriptional sequencing (RNA-seq) from formalin-fixed paraffin-embedded (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 R...

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Autores principales: Li, Jialu, Fu, Chunxiao, Speed, Terence P., Wang, Wenyi, Symmans, W. Fraser
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society of Clinical Oncology 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5976456/
https://www.ncbi.nlm.nih.gov/pubmed/29862382
http://dx.doi.org/10.1200/PO.17.00091
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author Li, Jialu
Fu, Chunxiao
Speed, Terence P.
Wang, Wenyi
Symmans, W. Fraser
author_facet Li, Jialu
Fu, Chunxiao
Speed, Terence P.
Wang, Wenyi
Symmans, W. Fraser
author_sort Li, Jialu
collection PubMed
description PURPOSE: Accurate transcriptional sequencing (RNA-seq) from formalin-fixed paraffin-embedded (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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spelling pubmed-59764562018-05-30 Accurate RNA Sequencing From Formalin-Fixed Cancer Tissue to Represent High-Quality Transcriptome From Frozen Tissue Li, Jialu Fu, Chunxiao Speed, Terence P. Wang, Wenyi Symmans, W. Fraser JCO Precis Oncol Original Reports PURPOSE: Accurate transcriptional sequencing (RNA-seq) from formalin-fixed paraffin-embedded (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. American Society of Clinical Oncology 2018-01-26 /pmc/articles/PMC5976456/ /pubmed/29862382 http://dx.doi.org/10.1200/PO.17.00091 Text en © 2018 by American Society of Clinical Oncology https://creativecommons.org/licenses/by/4.0/ Licensed under the Creative Commons Attribution 4.0 License:https://creativecommons.org/licenses/by/4.0/
spellingShingle Original Reports
Li, Jialu
Fu, Chunxiao
Speed, Terence P.
Wang, Wenyi
Symmans, W. Fraser
Accurate RNA Sequencing From Formalin-Fixed Cancer Tissue to Represent High-Quality Transcriptome From Frozen Tissue
title Accurate RNA Sequencing From Formalin-Fixed Cancer Tissue to Represent High-Quality Transcriptome From Frozen Tissue
title_full Accurate RNA Sequencing From Formalin-Fixed Cancer Tissue to Represent High-Quality Transcriptome From Frozen Tissue
title_fullStr Accurate RNA Sequencing From Formalin-Fixed Cancer Tissue to Represent High-Quality Transcriptome From Frozen Tissue
title_full_unstemmed Accurate RNA Sequencing From Formalin-Fixed Cancer Tissue to Represent High-Quality Transcriptome From Frozen Tissue
title_short Accurate RNA Sequencing From Formalin-Fixed Cancer Tissue to Represent High-Quality Transcriptome From Frozen Tissue
title_sort accurate rna sequencing from formalin-fixed cancer tissue to represent high-quality transcriptome from frozen tissue
topic Original Reports
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5976456/
https://www.ncbi.nlm.nih.gov/pubmed/29862382
http://dx.doi.org/10.1200/PO.17.00091
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