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Transcription microvariability detection by NGS Institute of Immunology Centre de Recherche Public-Santé National Public Health Laboratory Luxembourg Fleur.

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Presentation on theme: "Transcription microvariability detection by NGS Institute of Immunology Centre de Recherche Public-Santé National Public Health Laboratory Luxembourg Fleur."— Presentation transcript:

1 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

2 Hypothalamic-pituitary-adrenal axis HPA-axis  reactions to stress  many physiological processes Glucocorticoids  Nervous & Immune system  Learning and memory  Stress response  … Glucocorticoid receptor (GR)  Negative feedback HPA axis  Ubiquitous presence  Expression levels vary  Complicated Structure

3  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.

4  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 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: Practical read number: Turner et al., Biochem Pharmacol 2010

6 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

7  Control Gene: ADRB2R  only 1 exon  Highly reproducible  Identical TSS selection Technical Confirmation B. A.C % 99.75% 99.83%

8 Multiple Cell Lines: Reproducibility – A549 B.A % 17.42%  0.64% Seq/TSS  0.54% Seq/TSS 78.08% 17.26% 78.59% 17.42% Proximal Promoter: 1D, 1J, 1E, 1B, 1F and 1C 78.07% 78.59%  0.82% Seq/TSS  0.81% Seq/TSS TSS’s of A549 Replicate 1 TSS’s of A549 Replicate 2

9 A549 MCF-7 A.C. B. Multiple Cell Lines: Shared, but DE TSSs Tcells Daudi MCF7 A549

10 Frequency (%) TSS Variability is Reduced on Stimulation  Daudi: High Variability TSS’s 62.88% 34.30% 71.81% 27.02%

11 11.49% 84.99% 11.05% 86.44% TSS’s of Dex Replicate 1 TSS’s of Dex Replicate % 11.49%  0.53% Seq/TSS  0.82% Seq/TSS %  1.03% Seq/TSS  1.01% Seq/TSS B.A.  Daudi + Stimuli Dexamethasone 5-aza-2’ deoxycytidine Interferon- Ɣ  Reproducible Variability  Altered TSS’s selection TSS Variability is Reduced on Stimulation - Dex

12 A.C. C.C. Daudi Dex B. Daudi AZA10 IFN Dex TSS variability is Reduced on Stimulation

13 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.

14 LNSI, CRP-Santé, Luxembourg: Claude P. Muller Jonathan D. Turner Sara Vernocchi Oliver Hunewald Steffi Schmitz Regina Brunnhöfer Anne Molitor Funding: Acknowledgments

15  Control Gene: ADRB2R  2 approaches: Manually TopHat →Similar Results Analysis Technique: Manual - TopHat B. A.


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