Gene expression, single nucleotide variant and fusion transcript discovery in archival material from breast tumors

Nadine Norton, Zhifu D Sun, Yan Asmann, Daniel J. Serie, Brian M. Necela, Aditya Bhagwate, Jin Jen, Bruce W. Eckloff, Krishna R Kalari, Kevin J. Thompson, Jennifer M. Carr, Jennifer M. Kachergus, Xochiquetzal J. Geiger, Edith A. Perez, E Aubrey Thompson

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Advantages of RNA-Seq over array based platforms are quantitative gene expression and discovery of expressed single nucleotide variants (eSNVs) and fusion transcripts from a single platform, but the sensitivity for each of these characteristics is unknown. We measured gene expression in a set of manually degraded RNAs, nine pairs of matched fresh-frozen, and FFPE RNA isolated from breast tumor with the hybridization based, NanoString nCounter (226 gene panel) and with whole transcriptome RNA-Seq using RiboZeroGold ScriptSeq V2 library preparation kits. We performed correlation analyses of gene expression between samples and across platforms. We then specifically assessed whole transcriptome expression of lincRNA and discovery of eSNVs and fusion transcripts in the FFPE RNA-Seq data. For gene expression in the manually degraded samples, we observed Pearson correlations of >0.94 and >0.80 with NanoString and ScriptSeq protocols, respectively. Gene expression data for matched fresh-frozen and FFPE samples yielded mean Pearson correlations of 0.874 and 0.783 for NanoString (226 genes) and ScriptSeq whole transcriptome protocols respectively, p<2×10-16. Specifically for lincRNAs, we observed superb Pearson correlation (0.988) between matched fresh-frozen and FFPE pairs. FFPE samples across NanoString and RNA-Seq platforms gave a mean Pearson correlation of 0.838. In FFPE libraries, we detected 53.4% of high confidence SNVs and 24% of high confidence fusion transcripts. Sensitivity of fusion transcript detection was not overcome by an increase in depth of sequencing up to 3-fold (increase from ∼56 to ∼159 million reads). Both NanoString and ScriptSeq RNA-Seq technologies yield reliable gene expression data for degraded and FFPE material. The high degree of correlation between NanoString and RNA-Seq platforms suggests discovery based whole transcriptome studies from FFPE material will produce reliable expression data. The RiboZeroGold ScriptSeq protocol performed particularly well for lincRNA expression from FFPE libraries, but detection of eSNV and fusion transcripts was less sensitive.

Original languageEnglish (US)
Article numbere81925
JournalPLoS One
Volume8
Issue number11
DOIs
StatePublished - Nov 22 2013

Fingerprint

Gene expression
breast neoplasms
Tumors
Fusion reactions
Nucleotides
nucleotides
RNA
Breast Neoplasms
Gene Expression
gene expression
transcriptome
Long Noncoding RNA
Transcriptome
Libraries
Genes
sampling
messenger RNA
Genetic Association Studies
hybridization
genes

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Gene expression, single nucleotide variant and fusion transcript discovery in archival material from breast tumors. / Norton, Nadine; Sun, Zhifu D; Asmann, Yan; Serie, Daniel J.; Necela, Brian M.; Bhagwate, Aditya; Jen, Jin; Eckloff, Bruce W.; Kalari, Krishna R; Thompson, Kevin J.; Carr, Jennifer M.; Kachergus, Jennifer M.; Geiger, Xochiquetzal J.; Perez, Edith A.; Thompson, E Aubrey.

In: PLoS One, Vol. 8, No. 11, e81925, 22.11.2013.

Research output: Contribution to journalArticle

Norton, N, Sun, ZD, Asmann, Y, Serie, DJ, Necela, BM, Bhagwate, A, Jen, J, Eckloff, BW, Kalari, KR, Thompson, KJ, Carr, JM, Kachergus, JM, Geiger, XJ, Perez, EA & Thompson, EA 2013, 'Gene expression, single nucleotide variant and fusion transcript discovery in archival material from breast tumors', PLoS One, vol. 8, no. 11, e81925. https://doi.org/10.1371/journal.pone.0081925
Norton, Nadine ; Sun, Zhifu D ; Asmann, Yan ; Serie, Daniel J. ; Necela, Brian M. ; Bhagwate, Aditya ; Jen, Jin ; Eckloff, Bruce W. ; Kalari, Krishna R ; Thompson, Kevin J. ; Carr, Jennifer M. ; Kachergus, Jennifer M. ; Geiger, Xochiquetzal J. ; Perez, Edith A. ; Thompson, E Aubrey. / Gene expression, single nucleotide variant and fusion transcript discovery in archival material from breast tumors. In: PLoS One. 2013 ; Vol. 8, No. 11.
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