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Trinity College Dublin, The University of Dublin GE3M25: Data Analysis, Class 4 Karsten Hokamp, PhD Genetics TCD, 07/12/2015 http://bioinf.gen.tcd.ie/GE3M25/project
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Trinity College Dublin, The University of Dublin GE3M25 Data Handling Module Content Python Programming Bioinformatics ChIP-Seq analysis http://bioinf.gen.tcd.ie/GE3M25/project
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Trinity College Dublin, The University of Dublin Poject report hand-in: 23/12/2015 or 10/01/2016 ?
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Trinity College Dublin, The University of Dublin Class 4: Project overview Read mapping Peak detection Visualisation http://bioinf.gen.tcd.ie/GE3M25/project
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Trinity College Dublin, The University of Dublin ChIP-Seq Different sets of genes are expressed under different conditions Regulated through transcription factors that bind to promoters Binding can be captured by ChIP Bound sequences are revealed through NGS
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Trinity College Dublin, The University of Dublin
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ChIP-Seq Analysis Goal
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Trinity College Dublin, The University of Dublin Steps in this class: 1.Download FastQ data set (ChIP-Seq of TF in yeast) 2.Read mapping (Bowtie2) 3.Generate indexed and sorted BAM file 4.Peak calling 5.Visualisation (IGV) 6.Store BAM and index files GE3M25 Project
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Trinity College Dublin, The University of Dublin Data Download: Start here: bioinf.gen.tcd.ie/GE3M25 GE3M25 Project
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Trinity College Dublin, The University of Dublin Data Download: bioinf.gen.tcd.ie/GE3M25/project GE3M25 Project Optional control file for improved results
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Trinity College Dublin, The University of Dublin Data Download: bioinf.gen.tcd.ie/GE3M25/project/data GE3M25 Project
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Trinity College Dublin, The University of Dublin Steps in this class: 1.Download FastQ data set (ChIP-Seq of TF in yeast) ✔ 2.Read mapping (Bowtie2) 3.Generate indexed and sorted BAM file 4.Peak calling 5.Visualisation (IGV) 6.Store BAM and index files GE3M25 Project
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Trinity College Dublin, The University of Dublin Installing Bowtie Start here: bioinf.gen.tcd.ie/GE3M25/project GE3M25 Project
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Trinity College Dublin, The University of Dublin Installing Bowtie Switch to Terminal, unpack GE3M25 Project
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Trinity College Dublin, The University of Dublin Generate Index Download reference sequence from bioinf.gen.tcd.ie/GE3M25/project GE3M25 Project
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Trinity College Dublin, The University of Dublin Generate Index Run bowtie2-build GE3M25 Project./bowtie2-build S288C_reference_sequence_R64-2-1_20150113.fsa yeast programreference sequencename for index
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Trinity College Dublin, The University of Dublin Generate Index Run bowtie2-build GE3M25 Project./bowtie2-build S288C_reference_sequence_R64-2-1_20150113.fsa.txt yeast programreference sequencename for index Added by browser?
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Trinity College Dublin, The University of Dublin Generate Index Check output files GE3M25 Project
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Trinity College Dublin, The University of Dublin Read mapping Run bowtie2 GE3M25 Project./bowtie2 -x yeast -U 13222222.ChIP.fastq.gz -p 4 -S 13222222.sam programFastQ fileindex name output use of four CPU cores
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Trinity College Dublin, The University of Dublin Read mapping Run bowtie2 GE3M25 Project./bowtie2 -x yeast -U 13222222.ChIP.fastq.gz -p 4 > 13222222.sam program FastQ file index name output to screen redirected to file use of four CPU cores
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Trinity College Dublin, The University of Dublin GE3M25 Project Read mapping Output summary
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Trinity College Dublin, The University of Dublin GE3M25 Project Read mapping - other options (see Bowtie2 manual)
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Trinity College Dublin, The University of Dublin Steps in this class: 1.Download FastQ data set (ChIP-Seq of TF in yeast) ✔ 2.Read mapping (Bowtie2) ✔ 3.Generate indexed and sorted BAM file 4.Peak calling 5.Visualisation (IGV) 6.Store BAM and index files GE3M25 Project
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Trinity College Dublin, The University of Dublin GE3M25 Project Installing samtools Start here: bioinf.gen.tcd.ie/GE3M25/project Download and chmod +x samtools in Terminal
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Trinity College Dublin, The University of Dublin GE3M25 Project./samtools view -b -S 13222222.sam > 13222222.bam program input file command output Paramters -b output in binary format -S input in SAM format Sorting and indexing - Change from SAM to BAM format redirection of output to file
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Trinity College Dublin, The University of Dublin GE3M25 Project./samtools view -b -S -@ 4 13222222.sam > 13222222.bam Sorting and indexing - Change from SAM to BAM format use 4 cores for compression
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Trinity College Dublin, The University of Dublin GE3M25 Project./samtools sort -@ 4 13222222.bam 13222222.sorted program input file command output prefix use all four cores Sorting and indexing - Sort BAM file.bam suffix will be added
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Trinity College Dublin, The University of Dublin GE3M25 Project./samtools index 13222222.sorted.bam program input file command Sorting and indexing - create index file with.bai index will be created
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Trinity College Dublin, The University of Dublin Sorting and indexing Check output files GE3M25 Project
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Trinity College Dublin, The University of Dublin Steps in this class: 1.Download FastQ data set (ChIP-Seq of TF in yeast) ✔ 2.Read mapping (Bowtie2) ✔ 3.Generate indexed and sorted BAM file ✔ 4.Peak calling 5.Visualisation (IGV) 6.Store BAM and index files GE3M25 Project
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Trinity College Dublin, The University of Dublin GE3M25 Project Installing macs pip install macs --user In the Terminal: find ~/ -iname '*macs*' Find the location of the tool: /Users/kahokamp//Library/Python/2.7/bin/macs same as ~/Library/Python/2.7/bin/macs
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Trinity College Dublin, The University of Dublin GE3M25 Project Peak calling
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Trinity College Dublin, The University of Dublin GE3M25 Project Peak calling path/to/macs -t 13222222.sorted.bam -n yeast_macs -g 12000000 program treatment file output prefix genome size replace with path to macs
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Trinity College Dublin, The University of Dublin GE3M25 Project Peak calling
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Trinity College Dublin, The University of Dublin GE3M25 Project Peak calling
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Trinity College Dublin, The University of Dublin GE3M25 Project Peak calling pairing model
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Trinity College Dublin, The University of Dublin GE3M25 Project Peak calling Check output
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Trinity College Dublin, The University of Dublin GE3M25 Project hide first 19 rows sort by column G or H
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Trinity College Dublin, The University of Dublin GE3M25 Project
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Trinity College Dublin, The University of Dublin Steps in this class: 1.Download FastQ data set (ChIP-Seq of TF in yeast) ✔ 2.Read mapping (Bowtie2) ✔ 3.Generate indexed and sorted BAM file ✔ 4.Peak calling 5.Visualisation (IGV) 6.Store BAM and index files GE3M25 Project
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Trinity College Dublin, The University of Dublin GE3M25 Project 1. Download IGV (local copy on bioinf) 2. Unpack (on the command line_: unzip IGV_2.3.66.app.zip 3. Start by double-click in Finder 4. Load S. cerevisiae (sacCer3) genome 5. Load BAM file Visualisation with IGV (Integrated Genome Viewer)
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Trinity College Dublin, The University of Dublin GE3M25 Project pick a region with a peak navigate there in IGV
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Trinity College Dublin, The University of Dublin GE3M25 Project
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Trinity College Dublin, The University of Dublin Steps in this class: 1.Download FastQ data set (ChIP-Seq of TF in yeast) ✔ 2.Read mapping (Bowtie2) ✔ 3.Generate indexed and sorted BAM file ✔ 4.Peak calling 5.Visualisation (IGV) ✔ 6.Store BAM and index files GE3M25 Project
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Trinity College Dublin, The University of Dublin GE3M25 Project Storage of BAM file Upload.bam, bam.bai and MACS files through bioinf.gen.tcd.ie/GE3M25/project
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Trinity College Dublin, The University of Dublin Optional steps in this class: 1. Download and map Input file 2. Run MACS with Input file as control 3. Change parameters in Bowtie2, MACS 4. Trim FastQ data 5. Compare results GE3M25 Project
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Trinity College Dublin, The University of Dublin Don't forget to log out!
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