Python Meetup2014 (Ying Liu)
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The New York Python Meetup: Biology & Python (05-29-2014)

The New York Python Meetup: Biology & Python (05-29-2014)

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Python Meetup2014 (Ying Liu) Presentation Transcript

  • 1. Unveiling Epigenetic Regulation with Next Generation Sequencing (NGS) and Python Ying Liu Weill Cornell Medical College The New York Python Meetup, 05-29-2014
  • 2. About Me • PhD candidate, Weill Cornell Medical College • Major Area: Stem cell epigenomics, Computational Biology • Graduation: Fall 2014 • Dream job: data scientist in biomedical informatics • Email: yingliuyl@gmail.com • LinkedIn: https://www.linkedin.com/pub/ying-liu/b/669/605
  • 3. Reprogram > 20 days (Thousands of genes change expression) Induced pluripotent stem (iPS) cells Adult cells Express pluripotent stem cell specific genes (4 genes) 2012 Nobel Prize in Physiology or Medicine Generate Pluripotent Stem Cells from Mature Adult Cells Limitation • Reprogram efficiency: 0.01 - 0.1% • Molecular mechanism is not fully understood ?
  • 4. Human Genome Project • Human genome: ~ 3 billion DNA base pairs • Complete sequence: 2003 First sequence draft: 2001
  • 5. Nature 454, 711-715 Gene Expression My project: Histone X Enriches at expressing genes Epigenetic Regulation • Epigenetics: study of heritable changes in gene activity that are NOT caused by changes in the DNA sequence • One of the major epigenetic regulators: Histone protein Histone proteins DNA
  • 6. Induced pluripotent stem (iPS) cells Adult cells 2012 Nobel Prize in Physiology or Medicine Project Detect histone X function in initiating adult cells reprogramming to iPS cells. Experiment • Collect cells at the beginning (Day 0, 3, 6, 10) and after reprogramming (iPS); • Map genome-wide histone X localization with Next Generation Sequencing (NGS); • Analyze the dynamic change of genome- wide histone X localization with Python program and framework. Reprogram > 20 days (Thousands of genes change expression) Express pluripotent stem cell specific genes (4 genes) Generate Pluripotent Stem Cells from Mature Adult Cells
  • 7. Next Generation DNA Sequencing (Illumina, Inc) Genome-wide Analysis of Epigenetic Regulation Computation analysis (by genome) Tools: Python, R, etc. Align DNA sequence to chromosome Display in genome browser (by gene) chromosome
  • 8. 10 kb Day 0 Day 3 Day 6 Day 10 Day 0 Day 3 Day 6 Day 10 Histone X K27me3 Pou5f1 Nanog Histone X Enriches Near Stem Cell Specific Genes At the Beginning of Cell Reprogramming Genome browser (IGV) Alignment output (BED format) chr1 3000062 3000113 HWI-1KL117_0134:6:2101:14893:19331#ACAGTG/A..GTG. 37 + chr1 3000113 3000164 HWI-1KL117_0134:6:2302:6790:10626#ACAGTG/A..GT.. 37 + chr1 3000146 3000197 HWI-1KL117_0134:6:2303:8145:108924#ACAGTG/A..GT.. 37 - chr1 3000154 3000205 HWI-1KL117_0134:6:2202:14995:109690#ACAGTG/A..GT.. 37 - chr1 3000241 3000292 HWI-1KL117_0134:6:1304:12589:77263#ACAGTG/A..GT.. 25 - chr1 3000311 3000362 HWI-1KL117_0134:6:1101:17212:111473#ACAGTG/A..GT.. 37 - chr1 3000334 3000385 HWI-1KL117_0134:6:2308:10385:78074#ACAGTG/A..GT.. 25 - chr1 3000385 3000436 HWI-1KL117_0134:6:2102:20734:102615#ACAGTG/A..GG.. 37 + chr1 3000498 3000549 HWI-1KL117_0134:6:1203:3146:72739#ACAGTG/A..GTG. 37 - chr1 3000538 3000589 HWI-1KL117_0134:6:1101:1921:57017#ACAGTG/A..GT.. 37 + Chrom Start End Strand
  • 9. Computational Pipeline for Genome-wide DNA Sequence Analysis Bardet AF, Stark A, Nature Protocols, 2012 Alignment Analysis (Python, Perl) • BWA • Picard • Samtools • MACS, Cistrome (X. Shirley Liu Lab) • ChIPseeqer (Olivier Elemento Lab)
  • 10. Peak Identification with Python Program: Model-based Analysis of ChIP-Seq (MACS) Zhang Y, Liu XS, et al. Genome Biology 2008 Feng J, Liu XS, et al. Nature Protocol 2012 (1) (2) Requirement: ~3 GB of RAM, 1.5 h per data set with 30 million DNA sequence reads. d: estimated DNA fragment size 5’ 3’ 3’ 5’ d • Read distribution: Poisson distribution • Use dynamic λlocal to capture local biases in the genome λlocal = max (λBG, [λregion, λ1k], λ5k, λ10k) λBG: constant estimated from the genome background λregion: estimated from the candidate region λ1k, λ5k, λ10k: estimated from 1kb, 5kb, 10kb local window in the control • p-value: default threshold is 10-5 (3) (4)
  • 11. Galaxy / Cistrome MACS integrated web-based application: http://cistrome.org/ap/
  • 12. ChIPseeqer • Graphical User Interface • Command-line http://physiology.med.cornell.edu/faculty/elemento/lab/chipseq.shtml
  • 13. 10 kb Day 0 Day 3 Day 6 Day 10 Day 0 Day 3 Day 6 Day 10 Histone X K27me3 Pou5f1 Nanog Histone X Enriches Near Stem Cell Specific Genes At the Beginning of Cell Reprogramming
  • 14. Day: 0 3 6 10 iPS E 0 3 6 10 iPS E L H L H Expression Histone X ExpressionChangeExpressionStable Pou5f1 Sox2 Cdh1 Cldn3 Jag2 Zbtb32 Elf3 Msh6 Lefty1 Piwil2 Notch4 Tjp3 Fbxo15 Cldn6 Foxh1 Zp3 Fgf15 Nodal Tdgf1 Gdf3 Nanog Fgf4 Dppa3 Histone X Enriches At Stem Cell Specific Gene Promoters Prior to Gene Expression Activation Embryonic placenta development Stem cell maintenance Response to nutrient Cell-cell signaling DNA metabolic process DNA recombination Formation of primary germ layer Chromosome organization Mesoderm development Cell fate commitment Stem cell differentiation Blastocyst formation Meiosis Sexual reproduction Thyroid hormone metabolic process Cellular response to abiotic stimulus X0 X1 X2 X3 X4 X5 X6 Embryonic placenta development, GO:0001892 Stem cell maintenance, GO:0019827 Response to nutrient, GO:0007584 Cell-cell signaling, GO:0007267 DNA metabolic process, GO:0006259 DNA recombination, GO:0006310 Formation of primary germ layer, GO:0001704 Chromosome organization, GO:0051276 Mesoderm development, GO:0007498 Cell fate commitment, GO:0045165 Stem cell differentiation, GO:0048863 Blastocyst formation, GO:0001825 Meiosis, GO:0007126 Sexual reproduction, GO:0019953 Thyroid hormone metabolic process, GO:0042403 Cellular response to abiotic stimulus, GO:0071214 5 Enrichment -5 Depletion X0 X1 X2 X3 X4 X5 X6 Embryonic p Stem cell m Response to Cell-cell s DNA metabol DNA recombi Formation o Chromosome Mesoderm de Cell fate c Stem cell d Blastocyst Meiosis, GO Sexual repr Thyroid hor Cellular re 5 Enrichment -5 Depletion Expression Active Stable Group - a b c a b c Gene Ontology Analysis a. Histone X enrich during Day 0 – 10 b. Histone X enrich in iPS (after Day 10) c. Histone X not Enrich
  • 15. Induced pluripotent stem (iPS) cells Adult cells Limitation • Reprogram efficiency: 0.01 - 0.1% • Molecular mechanism is not fully understood Our Genome-wide analysis suggests: Histone X participates in stem cell gene activation at the early stage of adult cell reprogram. Express pluripotent stem cell specific genes (4 genes) Reprogram > 20 days (Thousands of genes change expression) Generate Pluripotent Stem Cells from Mature Adult Cells