Presentation on theme: "Transcription microvariability detection by NGS Institute of Immunology Centre de Recherche Public-Santé National Public Health Laboratory Luxembourg Fleur."— Presentation transcript:
Transcription microvariability detection by NGS Institute of Immunology Centre de Recherche Public-Santé National Public Health Laboratory Luxembourg Fleur A. D. Leenen, Oliver Hunewald, Sara Vernocchi, Stephanie Schmitz, Regina Brunnhöfer, Anne Molitor, Jonathan D. Turner and Claude P. Muller
human GR gene 10 exons 9 translated exons 9 alternative 1 st exons All 5’UTR Removed after splicing ATG translation start in exon 2 Translational regulation CpG island Methylation Environmental influence on expression The NR3C1 gene Turner et al., Biochem Pharmacol 2010 Cao-Lei et al., Hum Genet 2011 A. B.B.
Variable expression Hippocampus expresses all the exons and splice variants 1-B, 1-C and 1-H are the most widely expressed 1-D only in the hippocampus Complex 5’ UTR involved in the regulation of the GR transcription 1-B 1-C 3 1-C 2 1-C total 1-D 1-E 1-F 1-H GR total -actin Turner and Muller, J Mol Endo 2005 Tissue specific expression
5’RACE adapted to NGS Daudi A549 T cells MCF-7 100nM Dex 100U IFNγ 10μM AZA Cell Line + StimulusMultiple Cell Lines 1. Dephosphorylation 2. Cap removal 3. Ligation RNA oligo Exon 1xExon 2 Exon 1xExon 2 OligoExon 1xExon 2 OligoExon 1xExon 2 4. Gene specific RT 5. 1st round PCR 6. Nested PCR Shearing Library preparation Emulsion PCR NGS Sequencing (PGM Ion Torrent, life technologies) Tot. RNA extraction Region of Interest: 27kb Coverage: 20x Theoretical read number: 540000 Practical read number: 250000 Turner et al., Biochem Pharmacol 2010
Data Analysis NGS data Read Selection Remove ligated oligo sequence Mapping & TSS position retrieval (TopHat + own script) Selected TSSs Occurrence / Frequency Selected TSS’s per Cell Line and Conditions Sequence Frequencies per TSS Shared TSS’s Differentially expressed TSS’s Use of existing Software packages (NOISeq) Instead of gene, TSS as entity Between 2 Cell Lines or Conditions ± 25 most important TSS’s for differentiation between Cell Lines/Conditions Normalisation (TMM) OligoExon 1xExon 2
Control Gene: ADRB2R only 1 exon Highly reproducible Identical TSS selection Technical Confirmation B. A.C. 99.82% 99.75% 99.83%
A.C. C.C. Daudi Dex B. Daudi AZA10 IFN Dex TSS variability is Reduced on Stimulation
Conclusion Majority Reads Majority alternative 1 st exons Proximal Promoter CpG island transcription factors can bind at different positions within such a promoter site. The selection of TSS’s varies according to external influences.
LNSI, CRP-Santé, Luxembourg: Claude P. Muller Jonathan D. Turner Sara Vernocchi Oliver Hunewald Steffi Schmitz Regina Brunnhöfer Anne Molitor Funding: Acknowledgments
Control Gene: ADRB2R 2 approaches: Manually TopHat →Similar Results Analysis Technique: Manual - TopHat B. A.