{ "metadata": { "name": "", "signature": "sha256:6571dff426ba0700768e9fbb8d9db8013accb37383a2c71cd6eb6ef9d937a89d" }, "nbformat": 3, "nbformat_minor": 0, "worksheets": [ { "cells": [ { "cell_type": "heading", "level": 1, "metadata": {}, "source": [ "Supplemental Data and Code: Lineage and developmental patterns of DNA methylation in oysters" ] }, { "cell_type": "markdown", "metadata": {}, "source": [ "This notebook includes procedures for analyzing data desribed in \n", "the Master's thesis \"DNA methylation variation in gametes and larvae of the Pacific oyster, _Crassostrea gigas_\" by Claire Olson. \n", "\n", "---\n", " \n", "\n", "\n", "In theory, anyone should be able to reproduce our work in IPython, if they have the following software installed.\n", "* BSMAP\n", "* bedtools\n", "* R\n", "* rpy2 (interface to R from Python) \n", "\n", "The analysis was originally done on the Mac OSX operating system. \n", "\n", "The general premise of how this framework is set-up is that you will provide minimal information including a working directory and location of software. The following image denotes need for user input. \n", "\n", "The intent is to clone the github repository locally and run so accompaning scripts, etc. are available. Please note Data files that you will download and will produce are large (>20Gb).\n", "\n", "\n", "The notebook is divided into 5 sections \n", "\n", "* [Downloading data](#Downloading-Files) \n", "\n", "* [Read aligning: BSMAP](#Running-BSMAP) \n", "\n", "* [Global differences: methylkit](#Global-Differences:-methylkit) \n", "\n", "* [Differentially methylated loci: methylkit](#Differentially-methylated-loci) \n", "\n", "* [Genomic location of DMLs: bedtools](#Genomic-location-of-DMLs) \n", "\n", "\n", "Please post any comments and questions in issues." ] }, { "cell_type": "markdown", "metadata": {}, "source": [ "Before getting started, set the location o BSMAP on your computer and hit shift-enter" ] }, { "cell_type": "code", "collapsed": false, "input": [ "bsmaploc=\"/Users/Shared/Apps/bsmap-2.74/\"" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 1 }, { "cell_type": "heading", "level": 1, "metadata": {}, "source": [ "Downloading Data" ] }, { "cell_type": "code", "collapsed": false, "input": [ "#to confirm you current directory run the command and you should see a wd directory\n", "!ls\n" ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ "\u001b[31mBiGo_dev.ipynb\u001b[m\u001b[m \u001b[31mREADME.md\u001b[m\u001b[m \u001b[30m\u001b[43mscripts\u001b[m\u001b[m \u001b[30m\u001b[43mwd\u001b[m\u001b[m\r\n" ] } ], "prompt_number": 2 }, { "cell_type": "code", "collapsed": false, "input": [ "cd wd" ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ "/Volumes/web/cnidarian/olson-thesis-nb-master-12111102/wd\n" ] } ], "prompt_number": 3 }, { "cell_type": "code", "collapsed": false, "input": [ "#This command downloads a archived file including six BS-seq libraries (4.3 Gb)\n", "\n", "#!wget http://eagle.fish.washington.edu/trilobite/Crassostrea_gigas_HTSdata/BiGo_lar_fastq_mcf.tgz\n", "!curl -O http://eagle.fish.washington.edu/trilobite/Crassostrea_gigas_HTSdata/BiGo_lar_fastq_mcf.tgz \n" ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ " % Total % Received % Xferd Average Speed Time Time Time Current\r\n", " Dload Upload Total Spent Left Speed\r\n", "\r", " 0 0 0 0 0 0 0 0 --:--:-- --:--:-- --:--:-- 0" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 0 4221M 0 12.0M 0 0 45.7M 0 0:01:32 --:--:-- 0:01:32 45.8M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 1 4221M 1 51.2M 0 0 41.0M 0 0:01:42 0:00:01 0:01:41 41.1M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 2 4221M 2 85.0M 0 0 37.6M 0 0:01:52 0:00:02 0:01:50 37.6M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 2 4221M 2 105M 0 0 32.1M 0 0:02:11 0:00:03 0:02:08 32.2M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 3 4221M 3 131M 0 0 30.7M 0 0:02:17 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"output_type": "stream", "stream": "stdout", "text": [ "\r", " 99 4221M 99 4195M 0 0 25.8M 0 0:02:43 0:02:42 0:00:01 26.4M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 99 4221M 99 4221M 0 0 25.8M 0 0:02:43 0:02:43 --:--:-- 25.1M\r", "100 4221M 100 4221M 0 0 25.8M 0 0:02:43 0:02:43 --:--:-- 24.9M\r\n" ] } ], "prompt_number": 4 }, { "cell_type": "code", "collapsed": false, "input": [ "#uncompress files\n", "!tar -zxvf BiGo_lar_fastq_mcf.tgz" ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ "x mcf_M1_R1.fastq" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r\n", "x mcf_M1_R2.fastq" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r\n", "x mcf_M3_R1.fastq" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r\n", "x mcf_M3_R2.fastq" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r\n", "x mcf_T1D3_R1.fastq" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r\n", "x mcf_T1D3_R2.fastq" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r\n", "x mcf_T1D5_R1.fastq" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r\n", "x mcf_T1D5_R2.fastq" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r\n", "x mcf_T3D3_R1.fastq" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r\n", "x mcf_T3D3_R2.fastq" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r\n", "x mcf_T3D5_R1.fastq" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r\n", "x mcf_T3D5_R2.fastq" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r\n" ] } ], "prompt_number": 5 }, { "cell_type": "code", "collapsed": false, "input": [ "#Downloading the oyster genome \n", "#!wget http://eagle.fish.washington.edu/trilobite/Crassostrea_gigas_ensembl_tracks/Crassostrea_gigas.GCA_000297895.1.22.dna_sm.genome.fa\n", "!curl -O http://eagle.fish.washington.edu/trilobite/Crassostrea_gigas_ensembl_tracks/Crassostrea_gigas.GCA_000297895.1.22.dna_sm.genome.fa \n", " " ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ " % Total % Received % Xferd Average Speed Time Time Time Current\r\n", " Dload Upload Total Spent Left Speed\r\n", "\r", " 0 0 0 0 0 0 0 0 --:--:-- --:--:-- --:--:-- 0" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 2 541M 2 13.9M 0 0 16.0M 0 0:00:33 --:--:-- 0:00:33 26.2M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 9 541M 9 53.0M 0 0 30.2M 0 0:00:17 0:00:01 0:00:16 37.4M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 14 541M 14 79.9M 0 0 29.0M 0 0:00:18 0:00:02 0:00:16 33.0M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 18 541M 18 99.9M 0 0 26.6M 0 0:00:20 0:00:03 0:00:17 29.2M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 22 541M 22 123M 0 0 26.0M 0 0:00:20 0:00:04 0:00:16 28.0M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 28 541M 28 154M 0 0 26.9M 0 0:00:20 0:00:05 0:00:15 28.8M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 32 541M 32 178M 0 0 26.3M 0 0:00:20 0:00:06 0:00:14 24.9M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 36 541M 36 199M 0 0 25.8M 0 0:00:20 0:00:07 0:00:13 24.0M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 41 541M 41 225M 0 0 25.8M 0 0:00:20 0:00:08 0:00:12 25.2M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 46 541M 46 251M 0 0 24.3M 0 0:00:22 0:00:10 0:00:12 22.9M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 49 541M 49 267M 0 0 24.9M 0 0:00:21 0:00:10 0:00:11 22.6M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 56 541M 56 304M 0 0 25.9M 0 0:00:20 0:00:11 0:00:09 25.3M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 61 541M 61 330M 0 0 25.9M 0 0:00:20 0:00:12 0:00:08 26.1M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 66 541M 66 360M 0 0 26.2M 0 0:00:20 0:00:13 0:00:07 27.0M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 69 541M 69 378M 0 0 25.7M 0 0:00:21 0:00:14 0:00:07 28.8M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 74 541M 74 404M 0 0 25.7M 0 0:00:21 0:00:15 0:00:06 27.4M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 80 541M 80 437M 0 0 26.1M 0 0:00:20 0:00:16 0:00:04 26.5M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 85 541M 85 463M 0 0 26.1M 0 0:00:20 0:00:17 0:00:03 26.5M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 90 541M 90 487M 0 0 26.0M 0 0:00:20 0:00:18 0:00:02 25.4M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 96 541M 96 522M 0 0 26.4M 0 0:00:20 0:00:19 0:00:01 28.7M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", "100 541M 100 541M 0 0 26.4M 0 0:00:20 0:00:20 --:--:-- 28.8M\r\n" ] } ], "prompt_number": 6 }, { "cell_type": "heading", "level": 1, "metadata": {}, "source": [ "Running BSMAP" ] }, { "cell_type": "code", "collapsed": false, "input": [ "#bsmap will need to be downloaded if not already installed from https://code.google.com/p/bsmap/\n", "#another option in running bsmap on iPlant (iplantcollaborative.org)" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 7 }, { "cell_type": "code", "collapsed": false, "input": [ "for i in (\"M1\",\"T1D3\",\"T1D5\", \"M3\", \"T3D3\", \"T3D5\"):\n", " !{bsmaploc}bsmap \\\n", "-a mcf_{i}_R1.fastq \\\n", "-b mcf_{i}_R2.fastq \\\n", "-d Crassostrea_gigas.GCA_000297895.1.22.dna_sm.genome.fa \\\n", "-o bsmap_out_{i}.sam \\\n", "-p 4" ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ "\r\n", "BSMAP v2.74\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Start at: Thu Dec 11 11:15:36 2014\r\n", "\r\n", "Input reference file: Crassostrea_gigas.GCA_000297895.1.22.dna_sm.genome.fa" ] }, { "output_type": "stream", "stream": "stdout", "text": [ " \t(format: FASTA)\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Load in 7658 db seqs, total size 557717710 bp. 9 secs passed\r\n", "total_kmers: 43046721\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Create seed table. 25 secs passed\r\n", "max number of mismatches: read_length * 8% \tmax gap size: 0\r\n", "kmer cut-off ratio: 5e-07\r\n", "max multi-hits: 100\tmax Ns: 5\tseed size: 16\tindex interval: 4\r\n", "quality cutoff: 0\tbase quality char: '!'\r\n", "min fragment size:28\tmax fragemt size:500\r\n", "start from read #1\tend at read #4294967295\r\n", "additional alignment: T in reads => C in reference\r\n", "mapping strand (read_1): ++,-+\r\n", "mapping strand (read_2): +-,--\r\n", "Pair-end alignment(4 threads)\r\n", "Input read file #1: mcf_M1_R1.fastq" ] }, { "output_type": "stream", "stream": "stdout", "text": [ " \t(format: FASTQ)\r\n", "Input read file #2: mcf_M1_R2.fastq" ] }, { "output_type": "stream", "stream": "stdout", "text": [ " \t(format: FASTQ)\r\n", "Output file: bsmap_out_M1.sam\t (format: SAM)\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #1: \t50000 read pairs finished. 41 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #3: \t100000 read pairs finished. 42 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #2: \t150000 read pairs finished. 42 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #0: \t200000 read pairs finished. 42 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #1: \t250000 read pairs finished. 57 secs passed\r\n" ] }, { "output_type": 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finished. 706 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #3: \t7900000 read pairs finished. 708 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #1: \t7850000 read pairs finished. 708 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #0: \t7950000 read pairs finished. 712 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #3: \t8023643 read pairs finished. 714 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #2: \t8000000 read pairs finished. 719 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Total number of aligned reads: \r\n", "pairs: 3982551 (50%)\r\n", "single a: 1489442 (19%)\r\n", "single b: 1430646 (18%)\r\n", "Done.\r\n", "Finished at Thu Dec 11 11:27:35 2014\r\n", "Total time consumed: 719 secs\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r\n", "BSMAP v2.74\r\n", "Start at: Thu Dec 11 11:27:36 2014\r\n", "\r\n", "Input reference file: Crassostrea_gigas.GCA_000297895.1.22.dna_sm.genome.fa" ] }, { "output_type": "stream", "stream": "stdout", "text": [ " \t(format: FASTA)\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Load in 7658 db seqs, total size 557717710 bp. 9 secs passed\r\n", "total_kmers: 43046721\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Create seed table. 26 secs passed\r\n", "max number of mismatches: read_length * 8% \tmax gap size: 0\r\n", "kmer cut-off ratio: 5e-07\r\n", "max multi-hits: 100\tmax Ns: 5\tseed size: 16\tindex interval: 4\r\n", "quality cutoff: 0\tbase quality char: '!'\r\n", "min fragment size:28\tmax fragemt size:500\r\n", "start from read #1\tend at read #4294967295\r\n", "additional alignment: T in reads => C in reference\r\n", "mapping strand (read_1): ++,-+\r\n", "mapping strand (read_2): +-,--\r\n", "Pair-end alignment(4 threads)\r\n", "Input read file #1: mcf_T1D3_R1.fastq" ] }, { "output_type": "stream", "stream": "stdout", "text": [ " \t(format: FASTQ)\r\n", "Input read file #2: mcf_T1D3_R2.fastq \t(format: FASTQ)\r\n", "Output file: bsmap_out_T1D3.sam\t (format: SAM)\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #3: \t50000 read pairs finished. 40 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #1: \t100000 read pairs finished. 42 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #2: \t150000 read pairs finished. 42 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #0: \t200000 read pairs finished. 43 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #3: \t250000 read pairs finished. 56 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #1: \t300000 read pairs finished. 57 secs 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"output_type": "stream", "stream": "stdout", "text": [ "Total number of aligned reads: \r\n", "pairs: 2717832 (54%)\r\n", "single a: 1117376 (22%)\r\n", "single b: 1178713 (23%)\r\n", "Done.\r\n", "Finished at Thu Dec 11 11:34:34 2014\r\n", "Total time consumed: 418 secs\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r\n", "BSMAP v2.74\r\n", "Start at: Thu Dec 11 11:34:34 2014\r\n", "\r\n", "Input reference file: Crassostrea_gigas.GCA_000297895.1.22.dna_sm.genome.fa \t(format: FASTA)\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Load in 7658 db seqs, total size 557717710 bp. 10 secs passed\r\n", "total_kmers: 43046721\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Create seed table. 27 secs passed\r\n", "max number of mismatches: read_length * 8% \tmax gap size: 0\r\n", "kmer cut-off ratio: 5e-07\r\n", "max multi-hits: 100\tmax Ns: 5\tseed size: 16\tindex interval: 4\r\n", "quality cutoff: 0\tbase quality char: '!'\r\n", 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"text": [ "Thread #2: \t6150000 read pairs finished. 591 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Total number of aligned reads: \r\n", "pairs: 2725017 (44%)\r\n", "single a: 1704213 (28%)\r\n", "single b: 1415455 (23%)\r\n", "Done.\r\n", "Finished at Thu Dec 11 11:44:25 2014\r\n", "Total time consumed: 591 secs\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r\n", "BSMAP v2.74\r\n", "Start at: Thu Dec 11 11:44:26 2014\r\n", "\r\n", "Input reference file: Crassostrea_gigas.GCA_000297895.1.22.dna_sm.genome.fa \t(format: FASTA)\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Load in 7658 db seqs, total size 557717710 bp. 9 secs passed\r\n", "total_kmers: 43046721\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Create seed table. 26 secs passed\r\n", "max number of mismatches: read_length * 8% \tmax gap size: 0\r\n", "kmer cut-off ratio: 5e-07\r\n", "max multi-hits: 100\tmax Ns: 5\tseed size: 16\tindex interval: 4\r\n", "quality cutoff: 0\tbase quality char: '!'\r\n", "min fragment size:28\tmax fragemt size:500\r\n", "start from read #1\tend at read #4294967295\r\n", "additional alignment: T in reads => C in reference\r\n", "mapping strand (read_1): ++,-+\r\n", "mapping strand (read_2): +-,--\r\n", "Pair-end alignment(4 threads)\r\n", "Input read file #1: mcf_M3_R1.fastq" ] }, { "output_type": "stream", "stream": "stdout", "text": [ " \t(format: FASTQ)\r\n", "Input read file #2: mcf_M3_R2.fastq \t(format: FASTQ)\r\n", "Output file: bsmap_out_M3.sam\t (format: SAM)\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #1: \t50000 read pairs finished. 43 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #3: \t100000 read pairs finished. 43 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #0: \t150000 read pairs finished. 44 secs passed\r\n" ] }, { "output_type": "stream", 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passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #0: \t7350000 read pairs finished. 658 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #2: \t7400000 read pairs finished. 659 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #1: \t7450000 read pairs finished. 668 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #3: \t7500000 read pairs finished. 670 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #0: \t7550000 read pairs finished. 675 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #2: \t7600000 read pairs finished. 677 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #1: \t7650000 read pairs finished. 684 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #3: \t7700000 read pairs finished. 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"\r\n", "Input reference file: Crassostrea_gigas.GCA_000297895.1.22.dna_sm.genome.fa \t(format: FASTA)\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Load in 7658 db seqs, total size 557717710 bp. 9 secs passed\r\n", "total_kmers: 43046721\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Create seed table. 26 secs passed\r\n", "max number of mismatches: read_length * 8% \tmax gap size: 0\r\n", "kmer cut-off ratio: 5e-07\r\n", "max multi-hits: 100\tmax Ns: 5\tseed size: 16\tindex interval: 4\r\n", "quality cutoff: 0\tbase quality char: '!'\r\n", "min fragment size:28\tmax fragemt size:500\r\n", "start from read #1\tend at read #4294967295\r\n", "additional alignment: T in reads => C in reference\r\n", "mapping strand (read_1): ++,-+\r\n", "mapping strand (read_2): +-,--\r\n", "Pair-end alignment(4 threads)\r\n", "Input read file #1: mcf_T3D3_R1.fastq \t(format: FASTQ)\r\n", "Input read file #2: mcf_T3D3_R2.fastq \t(format: FASTQ)\r\n", "Output 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"text": [ "Thread #3: \t7100000 read pairs finished. 646 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Total number of aligned reads: \r\n", "pairs: 4047152 (57%)\r\n", "single a: 1332491 (19%)\r\n", "single b: 1205102 (17%)\r\n", "Done.\r\n", "Finished at " ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thu Dec 11 12:06:51 2014\r\n", "Total time consumed: 646 secs\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r\n", "BSMAP v2.74\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Start at: Thu Dec 11 12:06:52 2014\r\n", "\r\n", "Input reference file: Crassostrea_gigas.GCA_000297895.1.22.dna_sm.genome.fa \t(format: FASTA)\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Load in 7658 db seqs, total size 557717710 bp. 9 secs passed\r\n", "total_kmers: 43046721\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Create seed table. 26 secs passed\r\n", "max number of mismatches: read_length * 8% \tmax gap size: 0\r\n", "kmer cut-off ratio: 5e-07\r\n", "max multi-hits: 100\tmax Ns: 5\tseed size: 16\tindex interval: 4\r\n", "quality cutoff: 0\tbase quality char: '!'\r\n", "min fragment size:28\tmax fragemt size:500\r\n", "start from read #1\tend at read #4294967295\r\n", "additional alignment: T in reads => C in reference\r\n", "mapping strand (read_1): ++,-+\r\n", "mapping strand (read_2): +-,--\r\n", "Pair-end alignment(4 threads)\r\n", "Input read file #1: mcf_T3D5_R1.fastq \t(format: FASTQ)\r\n", "Input read file #2: mcf_T3D5_R2.fastq" ] }, { "output_type": "stream", "stream": "stdout", "text": [ " \t(format: FASTQ)\r\n", "Output file: bsmap_out_T3D5.sam\t (format: SAM)\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #0: \t100000 read pairs finished. 42 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #1: \t50000 read pairs finished. 42 secs passed\r\n" ] }, { "output_type": 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] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #1: \t6500000 read pairs finished. 573 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #2: \t6550000 read pairs finished. 576 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #3: \t6600000 read pairs finished. 577 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #0: \t6650000 read pairs finished. 579 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #1: \t6700000 read pairs finished. 589 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #2: \t6750000 read pairs finished. 592 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #3: \t6800000 read pairs finished. 593 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #0: \t6850000 read pairs finished. 595 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #1: \t6900000 read pairs finished. 605 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #2: \t6950000 read pairs finished. 608 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #3: \t7000000 read pairs finished. 609 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #0: \t7050000 read pairs finished. 611 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #2: \t7125800 read pairs finished. 614 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Thread #1: \t7100000 read pairs finished. 617 secs passed\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "Total number of aligned reads: \r\n", "pairs: 4092568 (57%)\r\n", "single a: 1250715 (18%)\r\n", "single b: 1133306 (16%)\r\n", "Done.\r\n", "Finished at Thu Dec 11 12:17:10 2014\r\n", "Total time consumed: 618 secs\r\n" ] } ], "prompt_number": 8 }, { "cell_type": "heading", "level": 1, "metadata": {}, "source": [ "_methratio_" ] }, { "cell_type": "markdown", "metadata": {}, "source": [ "methratio is a python script that accompanies BSMAP that determines methylation level on CpG loci" ] }, { "cell_type": "code", "collapsed": false, "input": [ "for i in (\"M1\",\"T1D3\",\"T1D5\", \"M3\", \"T3D3\", \"T3D5\"):\n", " !python {bsmaploc}methratio.py \\\n", "-d Crassostrea_gigas.GCA_000297895.1.22.dna_sm.genome.fa \\\n", "-u -z -g \\\n", "-o methratio_out_{i}.txt \\\n", "-s {bsmaploc}samtools \\\n", "bsmap_out_{i}.sam \\\n" ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 12:17:12 2014: reading reference Crassostrea_gigas.GCA_000297895.1.22.dna_sm.genome.fa ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 12:17:47 2014: reading bsmap_out_M1.sam ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "[samopen] SAM header is present: 7658 sequences.\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\t@ Thu Dec 11 12:22:52 2014: read 10000000 lines\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 12:23:18 2014: combining CpG methylation from both strands ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 12:23:42 2014: writing methratio_out_M1.txt ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 12:33:36 2014: done.\r\n", "total 8716467 valid mappings, 48671764 covered cytosines, average coverage: 1.78 fold.\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 12:33:38 2014: reading reference Crassostrea_gigas.GCA_000297895.1.22.dna_sm.genome.fa ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 12:34:19 2014: reading bsmap_out_T1D3.sam ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "[samopen] SAM header is present: 7658 sequences.\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 12:37:05 2014: combining CpG methylation from both strands ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 12:37:29 2014: writing methratio_out_T1D3.txt ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 12:43:21 2014: done.\r\n", "total 5759215 valid mappings, 26507310 covered cytosines, average coverage: 1.32 fold.\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 12:43:23 2014: reading reference Crassostrea_gigas.GCA_000297895.1.22.dna_sm.genome.fa ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 12:43:57 2014: reading bsmap_out_T1D5.sam ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "[samopen] SAM header is present: 7658 sequences.\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 12:48:21 2014: combining CpG methylation from both strands ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 12:48:45 2014: writing methratio_out_T1D5.txt ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 12:57:56 2014: done.\r\n", "total 6974208 valid mappings, 45446465 covered cytosines, average coverage: 1.54 fold.\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 12:57:58 2014: reading reference Crassostrea_gigas.GCA_000297895.1.22.dna_sm.genome.fa ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 12:58:32 2014: reading bsmap_out_M3.sam ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "[samopen] SAM header is present: 7658 sequences.\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\t@ Thu Dec 11 13:03:48 2014: read 10000000 lines\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 13:04:41 2014: combining CpG methylation from both strands ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 13:05:05 2014: writing methratio_out_M3.txt ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 13:16:00 2014: done.\r\n", "total 9773215 valid mappings, 53389861 covered cytosines, average coverage: 1.78 fold.\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 13:16:02 2014: reading reference Crassostrea_gigas.GCA_000297895.1.22.dna_sm.genome.fa ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 13:16:36 2014: reading bsmap_out_T3D3.sam ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "[samopen] SAM header is present: 7658 sequences.\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\t@ Thu Dec 11 13:21:53 2014: read 10000000 lines\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 13:22:12 2014: combining CpG methylation from both strands ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 13:22:37 2014: writing methratio_out_T3D3.txt ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 13:33:24 2014: done.\r\n", "total 8847902 valid mappings, 52255860 covered cytosines, average coverage: 1.65 fold.\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 13:33:26 2014: reading reference Crassostrea_gigas.GCA_000297895.1.22.dna_sm.genome.fa ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 13:34:00 2014: reading bsmap_out_T3D5.sam ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "[samopen] SAM header is present: 7658 sequences.\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\t@ Thu Dec 11 13:39:15 2014: read 10000000 lines\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 13:39:32 2014: combining CpG methylation from both strands ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 13:39:57 2014: writing methratio_out_T3D5.txt ...\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "@ Thu Dec 11 13:50:16 2014: done.\r\n", "total 8808414 valid mappings, 51732152 covered cytosines, average coverage: 1.69 fold.\r\n" ] } ], "prompt_number": 9 }, { "cell_type": "heading", "level": 1, "metadata": {}, "source": [ "Global Differences: methylkit" ] }, { "cell_type": "markdown", "metadata": {}, "source": [ "**Converting methratio files for methylkit**" ] }, { "cell_type": "code", "collapsed": false, "input": [ "#first methratio files are converted to filter for CG context, 3x coverage (mr3x.awk), and reformatting (mr_gg.awk.sh).\n", "#due to issue passing variable to awk, simple scripts were used (included in repository)\n", "for i in (\"M1\",\"T1D3\",\"T1D5\", \"M3\", \"T3D3\", \"T3D5\"):\n", " !echo {i}\n", " !grep \"[A-Z][A-Z]CG[A-Z]\" methratio_out_{i}CG.txt\n", " !awk -f ../scripts/mr3x.awk methratio_out_{i}CG.txt > mr3x.{i}.txt\n", " !awk -f ../scripts/mr_gg.awk.sh mr3x.{i}.txt > mkfmt_{i}.txt\n" ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ "M1\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "T1D3\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "T1D5\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "M3\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "T3D3\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "T3D5\r\n" ] } ], "prompt_number": 10 }, { "cell_type": "markdown", "metadata": {}, "source": [ "**Running R > methylkit**" ] }, { "cell_type": "code", "collapsed": false, "input": [ "%pylab inline" ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ "Populating the interactive namespace from numpy and matplotlib\n" ] } ], "prompt_number": 11 }, { "cell_type": "code", "collapsed": false, "input": [ "%load_ext rpy2.ipython\n" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 12 }, { "cell_type": "code", "collapsed": false, "input": [ "%%R\n", "#UNCOMMENT IF YOU NEED TO INSTALL PACKAGES\n", "# dependencies\n", "#install.packages( c(\"data.table\",\"devtools\"))\n", "#source(\"http://bioconductor.org/biocLite.R\")\n", "#biocLite(c(\"GenomicRanges\",\"IRanges\"))\n", "\n", "# install the development version from github\n", "#library(devtools)\n", "#install_github(\"al2na/methylKit\",build_vignettes=FALSE)\n" ], "language": "python", "metadata": {}, "outputs": [ { "metadata": {}, "output_type": "display_data", "text": [ "NULL\n" ] } ], "prompt_number": 13 }, { "cell_type": "code", "collapsed": false, "input": [ "%R library(methylKit)\n" ], "language": "python", "metadata": {}, "outputs": [ { "metadata": {}, "output_type": "pyout", "prompt_number": 14, "text": [ "array(['methylKit', 'tools', 'stats', 'graphics', 'grDevices', 'utils',\n", " 'datasets', 'methods', 'base'], \n", " dtype='|S9')" ] } ], "prompt_number": 14 }, { "cell_type": "code", "collapsed": false, "input": [ "%%R file.list <- list \n", "('mkfmt_M1.txt',\n", " 'mkfmt_T1D3.txt',\n", " 'mkfmt_T1D5.txt',\n", " 'mkfmt_M3.txt',\n", " 'mkfmt_T3D3.txt',\n", " 'mkfmt_T3D5.txt'\n", ")\n" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 15 }, { "cell_type": "code", "collapsed": false, "input": [ "%%R\n", "myobj=read(file.list,sample.id=list(\"1_sperm\",\"1_72hpf\",\"1_120hpf\",\"3_sperm\",\"3_72hpf\",\"3_120hpf\"),assembly=\"v9\",treatment=c(0,0,0,1,1,1))" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 16 }, { "cell_type": "code", "collapsed": false, "input": [ "%%R\n", "meth<-unite(myobj)\n", "#getCorrelation(meth,plot=T)\n", "hc<- clusterSamples(meth, dist=\"correlation\", method=\"ward\", plot=T)\n", "#PCA<-PCASamples(meth)" ], "language": "python", "metadata": {}, "outputs": [ { "metadata": {}, "output_type": "display_data", "text": [ "The \"ward\" method has been renamed to \"ward.D\"; note new \"ward.D2\"\n" ] }, { "metadata": {}, "output_type": "display_data", "png": 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HH/SXw8APHjxYcA+5sQKh+eC3W0+4v1BH+OCFAfWkrdTg4cgPfb75STT9UU7oCh3dfLDt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\nkcD/Ay67A1+IV3H0AAAAAElFTkSuQmCC\n" } ], "prompt_number": 17 }, { "cell_type": "heading", "level": 1, "metadata": {}, "source": [ "Differentially methylated loci" ] }, { "cell_type": "markdown", "metadata": {}, "source": [ "Determining differentially methylated loci using methylkit\n" ] }, { "cell_type": "code", "collapsed": false, "input": [ "%%R\n", "#lineage-specific DMLs\n", "#note that file.list was defined in prior section\n", "DMLobj=read(file.list,sample.id=list(\"M1\",\"T1D3\",\"T1D5\",\"M3\",\"T3D3\",\"T3D5\"),assembly=\"v9\",treatment=c(1,1,1,0,0,0), context=\"CpG\")\n", "lin<-unite(DMLobj)\n", "lin.pooled <- pool(lin, sample.ids <- c(\"lin_1\", \"lin_3\"))\n", "lin_DML.fisher <- calculateDiffMeth(lin.pooled)\n", "select(lin_DML.fisher, 1)\n", "lin_DML_p <- getData(lin_DML.fisher)\n", "lin_DML_filt <- lin_DML_p[lin_DML_p$pvalue < 0.01 & lin_DML_p$meth.diff > 25,]\n", "write.csv(lin_DML_filt,file=\"lin_DML_filt\")" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 18 }, { "cell_type": "code", "collapsed": false, "input": [ "!wc -l lin_DML_filt" ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ " 190 lin_DML_filt\r\n" ] } ], "prompt_number": 19 }, { "cell_type": "heading", "level": 3, "metadata": {}, "source": [ "Next will determine differentially methylated loci during development. This is done by doing three pairwise comparisons" ] }, { "cell_type": "code", "collapsed": false, "input": [ "%%R file.list <- list \n", "('mkfmt_M1.txt',\n", " 'mkfmt_T1D3.txt',\n", " 'mkfmt_M3.txt',\n", " 'mkfmt_T3D3.txt'\n", ")" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 20 }, { "cell_type": "code", "collapsed": false, "input": [ "%%R \n", "#Developmentally different DMLs (Males v Day3)\n", "DMLobj=read(file.list,sample.id=list(\"M1\",\"T1D3\",\"M3\",\"T3D3\"), assembly=\"v9\",treatment=c(1,0,1,0), context=\"CpG\")\n", "DevelMvD3<-unite(DMLobj)\n", "DevelMvD3.pooled <- pool(DevelMvD3, sample.ids <- c(\"Males\", \"Day3\"))\n", "DevelMvD3_DML.fisher <- calculateDiffMeth(DevelMvD3.pooled)\n", "select(DevelMvD3_DML.fisher, 1)\n", "DevelMvD3_DML_p <- getData(DevelMvD3_DML.fisher)\n", "DevelMvD3_DML_filt <- DevelMvD3_DML_p[DevelMvD3_DML_p$pvalue < 0.01 & DevelMvD3_DML_p$meth.diff > 25,]\n", "write.csv(DevelMvD3_DML_filt,file=\"DevelMvD3_DML_filt\")" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 21 }, { "cell_type": "code", "collapsed": false, "input": [ "!wc -l DevelMvD3_DML_filt" ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ " 30 DevelMvD3_DML_filt\r\n" ] } ], "prompt_number": 22 }, { "cell_type": "code", "collapsed": false, "input": [ "%%R file.list <- list \n", "('mkfmt_M1.txt',\n", " 'mkfmt_T1D5.txt',\n", " 'mkfmt_M3.txt',\n", " 'mkfmt_T3D5.txt'\n", ")" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 23 }, { "cell_type": "code", "collapsed": false, "input": [ "%%R \n", "#Developmentally different DMLs (Males v Day5)\n", "DMLobj=read(file.list,sample.id=list(\"M1\",\"T1D5\",\"M3\",\"T3D5\"), assembly=\"v9\",treatment=c(1,0,1,0), context=\"CpG\")\n", "DevelMvD5<-unite(DMLobj)\n", "DevelMvD5.pooled <- pool(DevelMvD5, sample.ids <- c(\"Males\", \"Day5\"))\n", "DevelMvD5_DML.fisher <- calculateDiffMeth(DevelMvD5.pooled)\n", "select(DevelMvD5_DML.fisher, 1)\n", "DevelMvD5_DML_p <- getData(DevelMvD5_DML.fisher)\n", "DevelMvD5_DML_filt <- DevelMvD5_DML_p[DevelMvD5_DML_p$pvalue < 0.01 & DevelMvD5_DML_p$meth.diff > 25,]\n", "write.csv(DevelMvD5_DML_filt,file=\"DevelMvD5_DML_filt\")" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 24 }, { "cell_type": "code", "collapsed": false, "input": [ "!wc -l DevelMvD5_DML_filt" ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ " 86 DevelMvD5_DML_filt\r\n" ] } ], "prompt_number": 25 }, { "cell_type": "code", "collapsed": false, "input": [ "%%R file.list <- list \n", "('mkfmt_T1D3.txt',\n", " 'mkfmt_T1D5.txt',\n", " 'mkfmt_T3D3.txt',\n", " 'mkfmt_T3D5.txt'\n", ")" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 26 }, { "cell_type": "code", "collapsed": false, "input": [ "%%R \n", "#Developmentally different DMLs (Day3 v Day5)\n", "DMLobj=read(file.list,sample.id=list(\"T1D3\",\"T1D5\",\"T3D3\",\"T3D5\"), assembly=\"v9\",treatment=c(1,0,1,0), context=\"CpG\")\n", "DevelD3vD5<-unite(DMLobj)\n", "DevelD3vD5.pooled <- pool(DevelD3vD5, sample.ids <- c(\"Day3\", \"Day5\"))\n", "DevelD3vD5_DML.fisher <- calculateDiffMeth(DevelD3vD5.pooled)\n", "select(DevelD3vD5_DML.fisher, 1)\n", "DevelD3vD5_DML_p <- getData(DevelD3vD5_DML.fisher)\n", "DevelD3vD5_DML_filt <- DevelD3vD5_DML_p[DevelD3vD5_DML_p$pvalue < 0.01 & DevelD3vD5_DML_p$meth.diff > 25,]\n", "write.csv(DevelD3vD5_DML_filt,file=\"DevelD3vD5_DML_filt\")" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 27 }, { "cell_type": "code", "collapsed": false, "input": [ "!wc -l DevelD3vD5_DML_filt" ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ " 47 DevelD3vD5_DML_filt\r\n" ] } ], "prompt_number": 28 }, { "cell_type": "code", "collapsed": false, "input": [ "#removing column titles\n", "!tail -n +2 DevelMvD5_DML_filt > DevelMvD5_DML\n", "!tail -n +2 DevelD3vD5_DML_filt > DevelD3vD5_DML " ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 29 }, { "cell_type": "code", "collapsed": false, "input": [ "#Concatenate all developmetnally different DMLs to one file\n", "!cat DevelMvD3_DML_filt DevelMvD5_DML DevelD3vD5_DML > Devel_DML_filt" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 30 }, { "cell_type": "code", "collapsed": false, "input": [ "!wc -l Devel_DML_filt" ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ " 161 Devel_DML_filt\r\n" ] } ], "prompt_number": 31 }, { "cell_type": "markdown", "metadata": {}, "source": [ "###Creating DML BED files" ] }, { "cell_type": "code", "collapsed": false, "input": [ "!tail -n +2 lin_DML_filt | awk -F, '{print $2, $3, $4, \"DML_lin\" }' | tr -d '\"' | tr ' ' \"\\t\" > lineage_dml.bed" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 32 }, { "cell_type": "code", "collapsed": false, "input": [ "!wc -l lineage_dml.bed" ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ " 189 lineage_dml.bed\r\n" ] } ], "prompt_number": 33 }, { "cell_type": "code", "collapsed": false, "input": [ "!tail -n +2 Devel_DML_filt | awk -F, '{print $2, $3, $4, \"DML_dev\" }' | tr -d '\"' | tr ' ' \"\\t\" > dev_dml.bed" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 34 }, { "cell_type": "code", "collapsed": false, "input": [ "!wc -l dev_dml.bed" ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ " 160 dev_dml.bed\r\n" ] } ], "prompt_number": 35 }, { "cell_type": "heading", "level": 1, "metadata": {}, "source": [ "Genomic location of DMLs" ] }, { "cell_type": "code", "collapsed": false, "input": [ "#In order to find location of DMLs oyster genome tracks will be downloaded\n", "#and intersectbed (bedtools suite) run\n", "#Note track with all CG's is large (~977mb)" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 36 }, { "cell_type": "code", "collapsed": false, "input": [ "cd genome_tracks\n" ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ "/Volumes/web/cnidarian/olson-thesis-nb-master-12111102/wd/genome_tracks\n" ] } ], "prompt_number": 37 }, { "cell_type": "code", "collapsed": false, "input": [ "for i in (\"exon\",\"intron\",\"TE\",\"gene\",\"1k5p_gene_promoter\",\"CG\"):\n", " !curl -O http://eagle.fish.washington.edu/trilobite/Crassostrea_gigas_v9_tracks/Cgigas_v9_{i}.gff \n", "\n" ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ " % Total % Received % Xferd Average Speed Time Time Time Current\r\n", " Dload Upload Total Spent Left Speed\r\n", "\r", " 0 0 0 0 0 0 0 0 --:--:-- --:--:-- --:--:-- 0" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", "100 11.7M 100 11.7M 0 0 21.2M 0 --:--:-- --:--:-- --:--:-- 54.3M\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ " % Total % Received % Xferd Average Speed Time Time Time Current\r\n", " Dload Upload Total Spent Left Speed\r\n", "\r", " 0 0 0 0 0 0 0 0 --:--:-- --:--:-- --:--:-- 0" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", "100 12.0M 100 12.0M 0 0 37.6M 0 --:--:-- --:--:-- --:--:-- 37.8M\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ " % Total % Received % Xferd Average Speed Time Time Time Current\r\n", " Dload Upload Total Spent Left Speed\r\n", "\r", " 0 0 0 0 0 0 0 0 --:--:-- --:--:-- --:--:-- 0" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", "100 6325k 100 6325k 0 0 51.9M 0 --:--:-- --:--:-- --:--:-- 52.7M\r\n" ] }, { "output_type": "stream", "stream": "stdout", "text": [ " % Total % Received % Xferd Average Speed 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"\r", " 80 932M 80 750M 0 0 23.9M 0 0:00:38 0:00:31 0:00:07 24.5M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 84 932M 84 783M 0 0 24.2M 0 0:00:38 0:00:32 0:00:06 25.4M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 84 932M 84 789M 0 0 23.6M 0 0:00:39 0:00:33 0:00:06 23.5M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 86 932M 86 807M 0 0 23.5M 0 0:00:39 0:00:34 0:00:05 22.3M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 90 932M 90 847M 0 0 24.0M 0 0:00:38 0:00:35 0:00:03 24.2M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 93 932M 93 876M 0 0 24.1M 0 0:00:38 0:00:36 0:00:02 25.0M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 96 932M 96 897M 0 0 24.0M 0 0:00:38 0:00:37 0:00:01 22.9M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", " 98 932M 98 914M 0 0 23.8M 0 0:00:39 0:00:38 0:00:01 25.3M" ] }, { "output_type": "stream", "stream": "stdout", "text": [ "\r", "100 932M 100 932M 0 0 23.9M 0 0:00:38 0:00:38 --:--:-- 26.8M\r\n" ] } ], "prompt_number": 38 }, { "cell_type": "code", "collapsed": false, "input": [ "cd .." ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ "/Volumes/web/cnidarian/olson-thesis-nb-master-12111102/wd\n" ] } ], "prompt_number": 39 }, { "cell_type": "code", "collapsed": false, "input": [ "for i in (\"exon\",\"intron\",\"TE\",\"1k5p_gene_promoter\"):\n", " !intersectbed \\\n", " -u \\\n", " -a lineage_dml.bed \\\n", " -b ./genome_tracks/Cgigas_v9_{i}.gff \\\n", " > {i}_intersect_DML_lin_u.txt\n", " !wc -l {i}_intersect_DML_lin_u.txt > lin{i}" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 40 }, { "cell_type": "code", "collapsed": false, "input": [ "#Concatenate counts of genomic regions into one table for lineage-specific DMLs\n", "!cat linintron linexon lin1k5p_gene_promoter linTE > lintable" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 41 }, { "cell_type": "code", "collapsed": false, "input": [ "!awk 'FNR==NR{sum+=$1;next}; {print $0,sum}' lintable{,} > lin_total" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 42 }, { "cell_type": "code", "collapsed": false, "input": [ "!awk '{print $2, $1, $3, (($1/$3)*100)}' lin_total > lineage_DMLs" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 43 }, { "cell_type": "code", "collapsed": false, "input": [ "for i in (\"exon\",\"intron\",\"TE\",\"1k5p_gene_promoter\"):\n", " !intersectbed \\\n", " -u \\\n", " -a dev_dml.bed \\\n", " -b ./genome_tracks/Cgigas_v9_{i}.gff \\\n", " > {i}_intersect_DML_dev_u.txt\n", " !wc -l {i}_intersect_DML_dev_u.txt > dev{i}" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 44 }, { "cell_type": "code", "collapsed": false, "input": [ "#Concatenate counts of genomic regions into one table for developmentally different DMLs\n", "!cat devintron devexon dev1k5p_gene_promoter devTE > devtable" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 45 }, { "cell_type": "code", "collapsed": false, "input": [ "!awk 'FNR==NR{sum+=$1;next}; {print $0,sum}' devtable{,} > dev_total" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 46 }, { "cell_type": "code", "collapsed": false, "input": [ "!awk '{print $2, $1, $3, (($1/$3)*100)}' dev_total > developmental_DMLs" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 47 }, { "cell_type": "code", "collapsed": false, "input": [ "for i in (\"exon\",\"intron\",\"TE\",\"gene\",\"1k5p_gene_promoter\"):\n", " !intersectbed \\\n", " -u \\\n", " -a ./genome_tracks/Cgigas_v9_CG.gff \\\n", " -b ./genome_tracks/Cgigas_v9_{i}.gff \\\n", " > {i}_intersect_CG_u.txt\n", " !wc -l {i}_intersect_CG_u.txt > CG{i}" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 48 }, { "cell_type": "code", "collapsed": false, "input": [ "#Concatenate counts of genomic regions into one table for all CGs in oyster genome\n", "!cat CGintron CGexon CG1k5p_gene_promoter CGTE > CGtable" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 49 }, { "cell_type": "code", "collapsed": false, "input": [ "!awk 'FNR==NR{sum+=$1;next}; {print $0,sum}' CGtable{,} > CG_total" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 50 }, { "cell_type": "code", "collapsed": false, "input": [ "!awk '{print $2, $1, $3, (($1/$3)*100)}' CG_total > all_CGs" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 51 }, { "cell_type": "code", "collapsed": false, "input": [ "!head all_CGs" ], "language": "python", "metadata": {}, "outputs": [ { "output_type": "stream", "stream": "stdout", "text": [ "intron_intersect_CG_u.txt 2815997 5128245 54.9115\r\n", "exon_intersect_CG_u.txt 1129658 5128245 22.0282\r\n", "1k5p_gene_promoter_intersect_CG_u.txt 593081 5128245 11.565\r\n", "TE_intersect_CG_u.txt 589509 5128245 11.4953\r\n" ] } ], "prompt_number": 136 }, { "cell_type": "code", "collapsed": false, "input": [ "!paste -d\" \" lineage_DMLs developmental_DMLs all_CGs > StackedBars" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 52 }, { "cell_type": "code", "collapsed": false, "input": [ "!awk '{print $4, $8, $12}' StackedBars | tr ' ' \"\\t\" > StackedBars_DMLs" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 53 }, { "cell_type": "heading", "level": 1, "metadata": {}, "source": [ "Stacked Bar Plot of DMLs versus all CGs" ] }, { "cell_type": "code", "collapsed": false, "input": [ "%%R \n", "DMLs<-as.matrix(read.table('StackedBars_DMLs', header=F))\n", "colnames(DMLs)<-c(\"Lin DMLs\",\"Devel DMLs\", \"All CpGs\")\n", "par(mar=c(5.1, 4.1, 4.1, 8.1), xpd=T)\n", "par(xpd=T, mar=par()$mar+c(0,0,0,5))\n", "barplot(as.matrix(DMLs), col=c(\"#BCCCC1\", \"#8DAB96\", \"#4A7958\", \"#2F583B\"), ylab=\"Proportion of CpG within a genomic region (%)\")\n", "legend(\"topright\",inset=c(-0.63,-0), legend=c(\"Transposable Element\", \"Promoter Region\", \"Exon\", \"Intron\"), pch=c(19,19,19), col=c(\"#2F583B\",\"#4A7958\",\"#8DAB96\",\"#BCCCC1\"))" ], "language": "python", "metadata": {}, "outputs": [ { "metadata": {}, "output_type": "display_data", "png": 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S0aJFbcGCBa6tVcH3yy+/dMv1j6bF6969uzVq1MiV/N566y0XgFTS08xsKjlqEorjjz/e\n3n77bStWrJgLsCoxNmjQwOWtdSqh67j6kaC27BNOOMF+/vnncD6aZlU/AlSaVnBX8ItMyrd58+Zu\nmzJlythdd91lU6dOjdwkxWsFTgVz76GZ5lInBXn94ND4/U2aNHEldwVjL916662m0vGf/vQnN9GL\nfsyoxC8THU8/bHQOf/nLX6x06dKm9nqt92oWYnl77fhyyg/9hdJuQPEkeUYAAQQCFGjWsKl9PHem\nHQwFXS+VKF7CGtdr5L3N9LPahr2kkqKmmFSJTNXMmvHKm8VJ1adeUhBTdbHSww8/bDfddJOdcsop\nrsr1nnvusSuvvNKtU0nZS6oifu2112zdunVu2kUFXy+pClulbo1/r2Dev39/S0pKsquuuspVZat6\n+9VXX3UP3S2ioOydl44ROQuc8lHAjUwqdapaV4EyMqkqPPI8vHUvvPBCzDZgb72eta+q2R955JHw\nYlWNe0nBVEk/YvRa16BUvHhx27dvn/shog5fqedo148PXYOftztIPvmHEnA++SC5DATys8BVF/ex\nCmXLm4KuUtnSZaxB7frWq1vPLF92kSK/T6epEplKYWpjVDBU6U9tml67poJFrKQSsKqON2/e7AKx\ngqaql5VUfewlVV2rVK12Wh1/586d3ipXra320oMHD4bzV3WzSpTPP/+8KSCqVKt8VPJVadI7Lx1E\nwdBLasdWVW1k0o+D0047ze2r/fVQoPeCZOS2GX3drl07V43tHU/V5DpnL8klraS2YVU/64eBdwyV\nflUtreTnndYx89o6AnBe+8Q4XwQKoEDJEiVt1uSP7G833GHXX9bP7h5wp705fkpC/0h7YxCovVKT\nw6hNU22RaQ2Tq4/immuusWeffdbUPqySr6qvveD48ccfu2C7a9cuVyrt2rWrq7JVsNU+2k7togqG\nKsW+8sordvnll7vr6tGjR7jEqh8HqsbW3OnaR0E58rzUpqrzV1BXkFY7dmTSNakKW7POKaktWlXj\nkaXoyO137NjhqoiVr/dQiTwyqd164sSJpm11Tmo/9zqtRW7n91olYXXW0uBLOg/9gFHVumof0kqq\nfZBnfkhp/0TJD1fINSCAQL4QUEn1ygv+lG3XUqdOHdfTWb1x1dNZwUBBUb2cVXL1S//85z9dlfH4\n8eNdRyz1fK5cubLbXNXbXrvtueeeawMHDnTBVSVatdVqHwV5BSEFV5UK1aFJ7aqq/m7ZsqXbTlW2\nCpo6NwVeBTsFXS1X0n6qMlcAVlC78cYb3XLvH1UD/+tf/3LV66py1nt1YvJK/9523nPqqmotV/6R\nDgryKvFqmUr16qH917/+1TtEhp7vvPNO10b83HPPuXNRz3C1Y0f28E59IK1XKV/Xqx8veTkVCv1y\n+b3feF6+igyc++uvv+46NOh/AL/UuWtnW3tosxUrWcxvk3y7/OCWfTb+3jGuN2ZGL7JN2xPtt2OS\nrHDR36vwMrpfftgueVuSPTPmKTvrrLMyfDmNTwi1v9X5vQo1wzvlkw0LbztiLz/7ogtGsS5Jt/go\nkCkIBp327t3rgqQ6BsWTVBJUO6dX9arORSoN6/YjVS17baCRx1TPaQWv1NWtCsoqcaojUmRS1XbF\nihVdR6zI5XqtwKxz1y09fklBXUFaPzASlZSnkkqmmU1qe9ePltQOfsdTiVmm+iGROqkjnB76AZXb\nEyXg3P4JcX4IIJCjApkNJAqMsZKqWvWIlSI7GkWuVxW4HqlTWoFTPYPTCr46lkq8aR0jdX4ZeZ9Z\nr8hj60dIPEm9oWMF33iOkRu2JQDnhk+Bc0AAgXwncNlll/lW8ea7i+WCMiVAAM4UGzshgAACaQuo\nlzAJgbQE6AWdlg7rEEAAAQQQyCYBAnA2wXJYBBBAAAEE0hIgAKelwzoEEMhVAivXr7b53y+0nzav\nz9B56R5W9az1ptRTJyXdg6sxmoNI06ZNC0+8kNn8NW61OmjpFic9NJiG7hHW7U9+9/VmJC/dQhQ5\nvnRG9mGbrAnQBpw1P/ZGAIEcEnjizWftk/mf2sHDSaEhKZPsz90vt6t69MlQ7prAQKMuKXmzF+ke\nW92bm5Pp6aeftksuuSTLWWqIy5kzZ4aPo8ErdM9y+/bt3QAb4RVxvNA9uRm9DSiOw7JpGgKUgNPA\nYRUCCOQOgU/mz7JXPn7TtuzYZpoXeP/B/fbSR2/Y18u/ifsEGzdu7EqMGr7x22+/dSNZaVAJDWah\ne1o1bZ+myNPIVj179nQjNCkTjXusxxlnnOHGKdZsQhoqUpMmKPDNnTvXnYtKoRpYQwNwaNALr2Sq\nwSY0eIZKmhrUQkMwaEKGWNP5pZ4mMb2L1LlrfGhvhCjdVxtrmj8dR+NEaxAL5Tt69Gg3U5GWq2St\nka2UZBNrukS/aQLdTvwTtwAl4LjJ2AEBBHJa4LNFX9jR5KMpst27f6/NXfqVtWv2xwQAKTaI8Wb/\n/v324YcfuiEZNT2fps1TMFTVqwbp0cAYmlVo3LhxbpQqjbmsQUJUdaygpuEjtb8GydAgLBp1SpPD\na5IF7aOS6RNPPOFGmVLA1YQN14SGqlQg/vOf/+xmPNL0fLpFKXI6P22nmYU0ZKSCucaQ1n293jSJ\nqS9Fg35omEslDUGp17oWzeqkpPGUNbCFSsY6Xx1b6/XQKFma9UlBW+NWe/MC69qVp5JG6dIPC41I\n9cEHH7gBelasWBGeJlATTmhsa00NqOkYNR0iKX4BSsDxm7EHAgjksECZUtGjLGl+4DIlo5fHOjWN\nsKX2X1VDaypBBRBv8Ae1p957770ueGkYSA0JqaCpEqJG51KAVXBSUnBWgNSMSVqvAKaSp4KqNw2h\nhme87rrr3OxImgBBx1Kw1ahYCnAauEKv05vOL3KaxNTXpAB9++232/XXXx+u0lZw1bHV7u03zZ+C\nsUrmqgbX0JGafSl10sxJftMlattY0wSmPgbvMyZACThjTmyFAAIBCvzfWRfbnMXz7Jddv4bPIjRP\nkV3S6fcSX3ihzwsFJA0FqdGT6tatm2JkKgVSL2mqwMip/RRcNXKUZi9Sipw9SMfyxkxWQPWmJ9Qx\nIqch1GuvatfLR8/pTecXOU1i5H56rXuMvTZgVYuryls/IpQUQNWWG2uavzVr1ljklIGx7lVOa7pE\nHT9y9C4FfO+6tY4UnwAl4Pi82BoBBAIQaFCzno0a+A+rXrma1ahc3U5s3Nom3zPBKpWPPfxj6lNU\nqVaD+Ku3cOphISMnJFC1rQb695KmyVOJUj2nlSK39bZJ/axjfPfdd+HFajdVdW7qlN50fhnJS8dU\nNbkmb9AcwkqqWvab5k/TEkZOYKA28NRJNQN+0yVqWzpqpRbL/HsCcObt2BMBBHJQoGndxvb6yEn2\n2sjn7dEho61+jboJz129omfPnu0CqDpTPfPMM9a8efNwD+qMZKhjaCpDdYhS8NZEMKeffrrbVSVG\nbx7grE7nF3kuandW9bLaYtOa5q9bt26us9iMGTNcSVltzKmTgrnfdImpt+V91gQIwFnzY28EEMhH\nAiqVqn1YJcV6oWn2FDzffvvtuK5QnZJUJa0gptKoqry1TEltx0OHDnVty+p5Xa1aNZePSuaaqSje\n6fy8E1NV+G233ebahTVLkG4peumll9w5qB3am+ZPPbsfeeQR10lLVeN+NQKaLlHV2jp3BW1vukQv\nP54TI8B0hBGOTEfIdIQRX4c0XzIdYZo8USvz0nSEOnm1a6oEm5WZg3bv3u06XaWetUdTDaozllfF\nnIjp/KLA//+C1NP8/fjjj6Z2YPXeVlLvbZWeP/30U/c+9T9+0yWm3i43vWc6wtz0aXAuCCCAQJwC\nmtM3K8FX2aWey9c7hdTTDKpaOruS19PbO37VqlWtY8eOrpe2fhg89dRT7nYrb33q58gOV6nX8T7r\nAlRBZ92QIyCAAAJ5QqB06dLu3l5Vj+u1BhLx7h3OExeQz06S25Dy2QfK5SCAAAJpCajdWfcvk4IX\noAQc/GfAGSCAAAIIFEABSsAF8EPnkoMR2PzzZrNthYLJPOBcD+49YGpXzemkYRo1AEfZsmWj7l+d\nP3++aVxoEgJBCeT8/xFBXSn5IhCwQPVa1a1i2xoBn0Uw2e9buTPLIyZpIoap86bbrr27Q0NQlrYr\nulxi5UqXy9AFqedv6g5JGdqRjRDIRgGqoLMRl0MjEClQqFBhK1qiWIF8FCqctZL/nn2/2aNvTbDV\nm34MDUe53dZtWW8T3n/BtDwrSZMSaEAMzUykpHGiNfiGUlqzIo0ZM8Y6derkxpfu3bu3aZIHEgLx\nChCA4xVjewQQyHGBmQtnR+W587dd9vX3GZuOUEH10UcfDT80SIWSOiMtX77cTZbw/PPPuxl+NPuR\n7pfVxAt6LF682I0hreVKurd21KhRbrALDeW4YMECdz+tW8k/CMQhQBV0HFhsigACwQjsC83/mzqp\n1Bpreert9F7DS2p0Ki9pooMrr7zSdE/u008/bX379nVV5N5QjpGzImkfzYqkUaO8WZE0m1D37t3d\n4bp27Wpr1651r/kHgXgECMDxaLEtAggEItCoVkP7Yf0qSzp8KJx/4VCV/vE1G4Tfp/VCQyv6tQF3\n7tzZqlev7oKxNztQerMiRQ5QwYxAacmzLi0BqqDT0mEdAgjkCoGTGrVywbZk8dCcukWLWflQ56tT\nT2hrTes0yvL5aTAKTZCgGYD0Wim9WZGYESjL7BwgJEAJmK8BAgjkegEFvD7n/J/9+PM623tgnx1T\ntrzVqlIzw+e9Y8eO8NjL3k66NUnjMg8cONB1vNL4z5qgXp2rNKOR2nk1raAmrs/MrEhePjwj4CdA\nAPaTYTkCCOQ6gfrVMzcFoWYLSp1efPFF+/zzz+3UU08Nt+c+++yzbkaiJ598MjwrksaE1vy68c6K\nlDo/3iOQWoAAnFqE9wggkG8EVMr1bjGKdVHqiBWZ1PnKS3/5y19syJAhUbMijR492tvEPad+n2Il\nbxBIQ4A24DRwWIUAAgVbIBGzIhVsQa4+LQHfEnBSUpItXLjQtYFoFJnjjz/eTj75ZNceEsSQcmld\nBOsQQAABBBDIawJRJWBV17z88svWvHlzu/32223GjBlulJepU6e6++Zq1aplGgVGAZqEAAIIIIAA\nApkTSFECVo/AXr16mW4snzt3ruuKn/qw6k34xBNPWJcuXdzoLzVqFMyxbVO78B4BBBBAAIF4BFKU\ngFW1/Morr9jgwYNjBl8duGLFinbXXXfZ9OnTXc/AeDJjWwQQQCCnBerWrWszZ85MN9sRI0ZQs5eu\nEhskUiAqAGvqroykUqVKmUaAISGAAAI5IaDmsQ1bN9mqjT/az9u3JDTLI0eO2H333WdHjx5N6HE5\nGAJpCaQIwLE2PHjwoA0dOtROOOEEu/TSS+3LL7+MtRnLEEAAgWwTUPD99JvP7duVS2zp6mU2f/lC\nm7PkyzRvMYp1MmeffbZp0gV1KtXwk2pOU9IsSEqtW7e2X375xTQ8pQbiqFq1qv3vf/+zjRs32h13\n3GFqcmvVqpXrG6PtlyxZYpqk4bbbbjPdL9ymTRs3eYPWkRBITyBFAI51v9xrr73mvoQKvCNHjnSD\nkqd3UNYjgAACiRRYs3Gtm3rw0JHD7rBHjh6xX3fvdCXiePJZvXq1TZkyxTTpgka3UkfTXbt2pZiC\nUIF01apVLsj+5z//sRNPPNH69Olj27dvt6+//tpuueUWN4Whhq5UvxkN6FGpUiU3k1KHDh1s+PDh\n8ZwS2xZggRQB+LfffnPDsE2bNi1Moi/jDz/8YIsWLXK/7IoVKxZexwsEEEAgJwS27/7VjianrB4+\nHArGO/bsjDv7YcOGWdOmTe2CCy5w/VgUSL2mt2OOOca8cZ7VF0bbHDp0yD777DNXAKlZs6bdcMMN\nrgTtjYxVvnx5U/uxStQqSTMzUtwfSYHdIUUA1pdQnbA0IPk555zjJqQ+77zz7JRTTnHzaK5YscLG\njh1bYLG4cAQQCEagVIlSURkXDo0PXbJ4yajl6S1QtbKX1I9FATZW0pSFSpoZSa8VfL2k4SsVuJWY\nGclT4TlegRQBWDvrV9xjjz1mqnrRFF4KwGrXUFW0fuXVq1cv3jzYHgEEEMiSQKPaDax0RBAuZIXc\n8RrWqhf3cb0Sbno7FilSxG2iaQwVbDVjkpeWLl1q9evXd28zejxvX54R8ASiArBWqMpZbSBPPfWU\nPfLII67Uqwmov/32W28/nhFAAIEcE1BJt/PJZ1qt42pa5QrHWv0ada37qedYkcK/B8msnoiCbYkS\nJVx7cOpjNWzY0AVbTdSgfjJqjluwYIGbxCH1trxHIB6BFANxaEdNx6Uef+pUoIHIdf+cRsZavHix\nKwHr9iN1YiAhgAACOSmgeYDbNm2TbVmq57NG+lOAjUwKzqoNVEes8ePHu45Xjz/+uLVo0cJ1yorc\nltcIxCOQIgDrHjiVfDXIhtJzzz1nGoKyb9++ruu9Oh3Mnz8/nuPHva3m5NyzZ48b8CPundkBAQQQ\nSCWgNlwvpe4g9eOPP3qr7MMPP7S9e/e68Q3Wr18fXq4X6t2sbbdu3Wqqkvaqndu1a2fff/99eNu2\nbdumeB9ewQsEYgikqIIuXLiwuw/u9NNPt549e7pfe+oFGJn0BUt00rjS6rqvjg7Fixd3pW91jtAv\nzIkTJyY6O46HAAIIxBRIb3Ahdbjygm/MA7AQgTgEUpSAtd9DDz1k+vWne950U3pOfNkGDRpkmzdv\ntg8++MAaNGjgfoHu3r3bli1b5obF1L12qhonIYAAAgggkF8EUpSA9+/f79p8VRJVz+e0gu/s2bPt\n559/TojDRx99ZE8//bSr5tYE2sq3QoUKrtpn3LhxFjlJdkIy5CAIIIAAAggELJAiAKv699NPPzX1\neFbHK40QE5k2bNhgb775pnXs2NGt925ej9wmM69V1ew3WLpGrFGbCwkBBBBAAIH8JJCiClq9/f7x\nj3+4e950z6+qhtUxSzeuK/hqlBh1RlAPwJYtWybMQYOgq4ehBvlQl3+NLKPgv3z5clOnLHUEIyGA\nAAIIIJCfBFIEYO/CNOC4xknVQ22za9ascUOvRY744m2biGeNtbpw4UI3B7F6KSpPlXrV7qvSdlpV\n4ZH5qypbQ8bFSgrmCu4kBBBAAAEEcoNAzAAceWLVqlUzPbI7lSwZutE+dB+el1Ty3rdvX4aDr/Zr\n0qSJ7xSJusle8x2TEieg0YH2rt4b+oxStGQkLoNcfKSD+w7wfcrFnw+nhkBeEMgVEUk9nidMmODG\nntbgH7oPuH///qapEDW4+cMPP2zqnJVe0sTbesRKChbbtm2LtYplmRSoVSd029gJFaxQ4d+HBczk\nYfLkbr+t+tU1j+TJk+ekEUAgVwjkigCseTc1AIjGnda8mmr3/e9//+tKtArIr7/+uvXr1y9XgHES\nfwiEOquHSr9mRYomZjjAP46c+19ltFkk919J7jhD3Wo4Y8YMU00YCYGsCMyaNctuvvnmrBwix/bN\nFQH43Xffta+++spVH2/ZssVNiK3OXkp33nmnGxKTAJxj3wkyQiDHBdT5U384VetFQiArAvfff781\na9YsK4fIsX19A7Bm/hg4cKAtWbLENFKVl3r06OEmaPDeJ+JZWBr+Um3A6kSl+5G9pDGoTzrpJO8t\nzwggkA8FND+vHiQECpKAbwAePXq0uxVIg49Htr9qkoZEp6FDh9q1117relvfeuutrg1YQVkjcX3+\n+efu3uRE58nxEEAAAQQQCFLANwBrRiSVgCN7JmfXiaq6WcNO/vrrr3bssce6aqhp06a5+Tc1FrRm\nYCIhgAACCCCQnwR8A/Cll15qkydPNs32kV33/0ZCqlOLgq+Sbhm66KKLIlfzGgEEEEAAgXwl4HsD\np27b0QhU1atXt0aNGrn2GbXRDB48OF8BcDEIIIAAAggEIeBbAtY0hLGmHsyONuAgLpw8EchpgeTQ\n4DJJ+wtmL9+Cet05/R0jv7wl4BuANSOSHpqWUONAqxRcunTpvHV1nC0CuUhg29Zttv3jX3PRGeXc\nqRzYu9801jwJAQT+EPANwEeOHLHrrrvOnn/+eVOpV7cl9ezZ06ZMmeLaaP84BK8QQCAjAhql7fyb\nemVk03y3zZfvzTb9TSEhgMAfAr5twJqfV6NTqXeySsErV6605ORke/DBB//Ym1cIIIAAAgggkCkB\n3wA8b948GzZsWHhEkQYNGtg999zjRqvJVE7shAACCCCAAAJhAd8AfNppp9ns2bPDG+qF3muaQBIC\nCCCAAAIIZE3Atw34sssus5YtW7oS7xlnnGELFiywRYsWUQLOmjd7I4AAAggg4AR8S8AaFEPjQPft\n29c0N+/5559vS5cutTZt2kCHAAIIIIAAAlkUiCoBt2/f3jQO9Jw5c0zDQEamZ5991jQZw7hx4yIX\n8xoBBBBAAAEE4hSICsATJkywevXqWcOGDa1bt25Rh6tYsWLUMhYggAACCCCAQHwCUQFYMxApVahQ\nwWrVqhXf0dgaAQQQQAABBDIkEBWAvb00qbGqnCOTJkzQaFgaE1rV0DVr1oxczesCJpB8NNmOHj5q\nR48cKmBXbnbowB9zZBe4i+eCEUAgIQK+Afiss86yF1980W6//XZTu/CXX35pY8aMsSFDhtjPP/9s\nGit64cKFCTmJ3HKQffv228F9+yxpj2/ftNxyqgk/j72bd8V9zC2bt9i+tWutcJFCce+b13fYt2ef\nFS3q+79PXr88zh8BBHJAwPcvyKRJk9zAG71793anoarpkiVL2vz58238+PH2zjvv2JYtW6xq1ao5\ncJo5k8Wunbvs4O79Vqy4L0vOnEgAuRzanxT3WL3qK3D65V2seMkSAZxxsFku+GieHT58ONiTIHcE\nEMjTAr6RRrchaSjKyPTDDz9Y8eLF3aJDhw5ZqVKlIlfn+dc169SycsceZyXKlszz1xLvBez4YQtj\n9caLxvYIIIBAFgR8A/A111xj3bt3t5kzZ7ppCTU0pcaD1mhYqoZW6ad8+fJZyJpdEUAAAQQQKLgC\nvo2dTZo0MQVdjYilgTg0LvSPP/5oxx9/vPXv399VQRdcNq4cAQQQQACBrAn4loB1WLXvDhgwwDZv\n3myVK1cOdzpp3rx51nJlbwQQQAABBAq4gG8JWKXekSNHWqtWraxr1672ySefuPmAt23bVsDJuHwE\nEEAAAQSyLuAbgDUi1owZM+ytt95yuXTp0sXd96vlJAQQQAABBBDImoBvAFZnqzvuuMNq1KjhcihW\nrJgNHjzYBeWsZcneCCCAAAIIIOAbgGvXrh01H/C7775r1atXRw0BBBBAAAEEsijg2wlLI2C1a9fO\npk+f7ka+6tChg60NjXr08ccfZzFLdkcAAQQQQAAB3wCse3yXLVtmr776qv3000/WqVMn9yhSpAhq\nCCCAAAIIIJBFAd8APHz4cHcb0t/+9rcsZsHuCCCAAAIIIJBawLcNuG7durZkyRKGJ0wtxnsEEEAA\nAQQSIOBbAtY4z++//74bblIdsryq53PPPdcefvjhBGTNIRBAAAEEECi4Ar4BWONAawak1EmTNJAQ\nQAABBBBAIGsCvgFYVdB6kBBAAAEEEEAg8QK+bcCJz4ojIoAAAggggIAnQAD2JHhGAAEEEEAgBwV8\nA3BycrK9/PLL9tlnn7nTeeKJJ2zixIn0is7BD4esEEAAAQTyr4BvANYkDGPHjrVq1aq5q+/YsaML\nyJMmTcq/GlwZAggggAACOSTgG4A//PBDu//++61x48buVFq0aOEC8htvvJFDp0Y2CCCAAAII5F8B\n3wCsHtDTpk1LceWzZs1y9wWnWMgbBBBAAAEEEIhbwPc2pGuvvdbOOecc++CDD+zUU0+1xYsX25Yt\nW0wlYxICCCCAAAIIZE3ANwDXrFnT5s2b52Y/WrlypV133XWmGZEKF/YtNGftTNgbAQQQQACBAiTg\nG4BlUKFCBevVq1cB4uBSEUAAAQQQyBmBqADcvn17Gz16tM2ZM8fddpT6NHr06GHjxo1LvZj3CCCA\nAAIIIBCHQFQAnjBhgtWrV88aNmxo3bp1izpUxYoVo5bllwX79u61347ss73b9+SXS8rwdWz7cXOG\nt2VDBBBAAIGsC0QFYG8CBlU/V69e3b799ls7ePBgOKfDhw+HX+e3F7/t/s0O/LLXSpQsmd8uLd3r\nObw3KTzjVbobswECCCCAQJYFogKwd8QvvvjCtf+WK1fOSkYEJJWKx4wZ422Wr57r1qtrDU5vZuUr\nVchX15WRi1k082tGOcsIFNsggAACCRLwDcCTJ0+2UaNG2TXXXJOgrDgMAggggAACCHgCvvcUaSCO\nHTt2eNvxjAACCCCAAAIJFPAtAQ8dOtROPPFEmz59ujVv3jycpdqI+/btG37PCwQQQAABBBCIX8A3\nAD/++ON24MABa9KkSYo24GLFisWfC3sggAACCCCAQAoB3wC8aNEie+ihh+ySSy5JsQNvEEAAAQQQ\nQCDrAr5twBdddJEbB/ro0aNZz4UjIIAAAggggEAKAd8AvG3bNnvttdesbNmy1qhRI2vatKl7DB48\nOMUBeIMAAggggAAC8Qv4VkGff/75dvLJJ9vOnTtt69atVqdOHdcWXKlSpfhzYQ8EEEAAAQQQSCHg\nG4Br1Khh99xzjz3//POmoKtA3LNnT5syZUqKA/AGAQQQQAABBOIX8K2Cfvrpp23VqlW2bNky2759\nu2lKwuTkZHvwwQfjz4U9EEAAAQQQQCCFgG8A1lzAw4YNs2bNmrkdGjRo4ErEs2bNSnEA3iCAAAII\nIIBA/AK+Afi0006z2bNnpzii3lepUiXFskS/0WQPjMCVaFWOhwACCCCQ2wR824Avu+wya9mypanE\ne8YZZ9iCBQtM9wZnRwk4KSnJ7r33XtP40xs3bnRV3aVLl7b69eubRuTq169fbnPjfBBAAAEEEMiS\ngG8J+Nhjj7UlS5a4YSd1L7B6RS9dutTatGmTpQxj7Txo0CD77rvv3H3Hu3fvNuW3adMme+aZZ+yp\np56yJ598MtZuLEMAAQQQQCDPCviWgHVFCsK33nprtl/cRx99ZHPnzrVq1aqF89J8xB06dLBx48bZ\niBEjbMCAAeF1vEAAAQQQQCCvC/gGYN12NHDgQFcKVhWxl3r06GGPPPKI9zYhzy1atLCZM2da7969\no473/vvvZ3u7c1SmLMiQwIb1G+zlhydZkSJFMrR9ftpo+5Ztdtegv+anS+JaEEAghwV8A/Do0aNt\n165dNn78eDcalnde2TEQx3333Wd9+vSxsWPHWsOGDa18+fIu7+XLl5s6ZU2dOtXLnudcJKA2+msH\n32BFCvu2ZOSis03sqfzv7anuu5nYo3I0BBAoSAK+AVidoVQC7ty5c7Z7aNrDhQsXumrotWvX2ubN\nm12pV9XOHTt2tEKFCmXoHDRoyBtvvBFz259//tnat28fcx0LMydQuFBh0+xYpUqXytwB8vBehQvg\nj448/HFx6gjkSgHfAHzppZe6Xsnt2rWz4447LttPvmTJkimCvaZCVNVmRoOvTvDKK6809d6Old56\n6y03mlesdSxDAAEEEEAgpwV86w7VC1lVv9WrV8/2yRh++uknu+qqq2z+/PmmSSD69+/vOmQdc8wx\ndu2111pkG3RaQCqNlSlTJuZDAb4gtlWm5cU6BBBAAIHgBHxLwBdccIG1bds26syyow1YY05rsofm\nzZvbAw884NrWdMvTwYMH7W9/+5v985//dI+ok2EBAggggAACeVTANwDXrl3b9FBSm2zlypWtaFHf\nzbN0+Z999pl9//33Vrx4cXv77bftnXfesVq1arljKvjedNNNWTo+OyOAAAIIIJDbBHyroDUYxsiR\nI61Vq1bWtWtX++STT9xsSKoiTnRq3LixvfDCC+6wZ511Vopez7oNSfMRkxBAAAEEEMhPAr4BeMKE\nCTZjxgxT5yWlLl26WM2aNU3LE50ef/xxGzNmjBt4Q/cf33HHHaae0ZqPWLMy6TYlEgIIIIAAAvlJ\nwLdOWRMvKBBqXmAldXAaPHiwqw6+6667Emqge3817eH06dNtxYoVrj24YsWKruSrITCzq+o7oRfB\nwRBAAAEEEIhDwDcAq/1XQVhVwl569913Xa9o730in3W7Ubdu3dwjkcflWAgggAACCORGAd8AfPvt\nt5vuAVapVINYaFxmDZLx8ccf58br4JwQQAABBBDIUwK+Abhq1aquWvjVV1813afbqVMn9+Be2jz1\n+XKyCCCAAAK5VMA3AN9///327LPPhk9bvZRVTax5eps2bepmKVKnLBICCCCAAAIIxC/g2wtabb8a\nPerOO+909+XqWR2xhgwZ4nooa6AOEgIIIIAAAghkTsC3BDxp0iTTCFXeFIGtW7d2AVnDRWqGJA2W\nsWXLFlNVNQkBBBBAAAEE4hPwLQEfe+yxtmrVqhRH++GHH9xoVVp46NAhK1Wq4M2CkwKENwgggAAC\nCGRSwLcEfM0111j37t1t5syZbkzoefPm2cqVK92tSaqGrlevnpu3N5P5shsCCCCAAAIFWsC3BNyk\nSRNT0NX0fhqWctiwYfbjjz/a8ccf72YrUhU0CQEEEEAAAQQyJ+BbAtbh1L47YMCAqCNr1iISAgjE\nJ7B/33776Ye18e2UT7b+ac26fHIlXAYCiRNIMwAnLhuOhAACO3fssMUzFxRIiJ2/7GA+7gL5yXPR\naQkQgNPSYR0CCRRQv4m/jbo7gUfMO4ea8sxkO3LkSN45Yc4UgRwQ8G0D9st7//79fqtYjgACCCCA\nAAIZFPAtAf/yyy9u5iP1fNYvV3XEOnDggJ166qk2ZcqUDB6ezRBAAAEEEEAgloBvCXjs2LG2b98+\nu/76661WrVpuTt7y5cvb8OHDYx2HZQgggAACCCAQh4BvAF69erUNHTrUdD/wxo0b7f/+7/9s4sSJ\nNmbMmDgOz6YIIIAAAgggEEvANwBrogXNglS2bFlLSkqy7du3W6VKldyyWAdiGQIIIIAAAghkXMC3\nDbh///5uDmANvHHRRReZJl9QINbAHCQEEEAAAQQQyJqAbwA+4YQTbMWKFe7evQ4dOthTTz1lxxxz\njF1++eVZy5G9EUAAAQQQQMB8A7BsqlWrFia65ZZbwq95gQACCCCAAAJZE/BtA87aYdkbAQQQQAAB\nBNISIACnpcM6BBBAAAEEskmAAJxNsBwWAQQQQACBtAR824B37txpAwcOtCVLlrjez95BevToYY88\n8oj3lmcEEEAAAQQQyISAbwAePXq07dq1y8aPH+/uBfaOrXuBSQgggAACCCCQNQHfAKzRr1QC7ty5\nc9ZyYG8EEEAAAQQQiBLwbQO+9NJLbfLkybZ169aonViAAAIIIIAAAlkT8A3AmzZtsqlTp1r16tWt\nUaNG1rRpU/cYPHhw1nJkbwQQQAABBBDwH4hDQ0+2bds2iog24CgSFiCAAAIIIBC3gG8bcO3atU0P\nEgIIIIAAAggkXiAqALdv397UA3rOnDlu+sHUWeo2pHHjxqVezHsEEEAAAQQQiEMgKgBPmDDB6tWr\nZw0bNrRu3bpFHapixYpRy1iAAAIIIIAAAvEJRAXg1q1buyNUqFDBatWqFd/R2BoBBBBAAAEEMiQQ\nFYC9vfbs2WO33nqrffPNN7Z3715vsZ133nlucI7wAl4ggAACCCCAQNwCvgFY7cDr16+3f//731a5\ncuXwgamCDlPwAgEEEEAAgUwL+AbgdevW2e233x6zHTjTubEjAggggAACCDgB34E4NBLWq6++akeP\nHoUKAQQQQAABBBIsEFUC7tChg+3YscNls2rVKnvzzTddZ6xChQq5Zd27d2c2pAR/CBwOAQQQQKDg\nCUQF4CeeeMIOHz7sOl4VL17cihUrFlY5ePCgWxdewAsEEEAAAQQQyJRAVBV08+bNrWXLlvb222+7\n6Qj12nts2LDBdcrKVE7shAACCCCAAAJhgagS8HPPPWcDBgxwG2gu4MhUrlw5GzVqVOQiXiOAAAII\nIIBAJgSiSsA33XSTHTp0yMaMGWNz5851r/Ve1dK7d+92cwRnIh92QQABBBBAAIEIgagSsNYVLVrU\nhgwZErEZLxFAAAEEEEAgkQJRAZjJGBLJy7EQQAABBBCILRAVgL3JGDZu3GjDhg0zjQ1dpEiR8N6M\nhBWm4AUCCCCAAAKZFogKwN5kDOXLlzfdkrR69Wo3/nPPnj3tnHPOsVKlSmU6M3ZEAAEEEEAAgd8F\nojpheTAXXHCBff7557Z06VLr0qWLvfjii1a9enUbOXKktwnPCCCAAAIIIJBJgagScORxVqxYYTNm\nzHCP2bNnW7Nmzax27dqRm/AaAQQQQAABBDIh4FsCvvPOO61p06b24YcfWt++fU3BWLclXX311ZnI\nhl0QQAABBBBAIFLAtwR8zTXXWOnSpW369OmuM1bHjh3t7LPPdo/jjjsu8hi8RgABBBBAAIE4BXxL\nwE2aNLG7777bPvvsM5s3b55VqlTJ+vfvb7fcckucWbA5AggggAACCKQW8C0Ba9zn//73vzZt2jT7\n4osv7OSTT7aHHnrILr744tTH4D0CCCCAAAIIxCngG4AnTpzo2n3V/qse0BoHmoQAAggggAACiRHw\nDcCqfiYhgAACCCCAQPYI+LYBZ092HBUBBBBAAAEEJJDrArBmXdqxYwefDgIIIIAAAvlawLcKOiev\nOikpye69916bPHmyaQzq5ORkdwtU/fr1bejQodavX78cOR3lm5x81JIOHMyR/HJTJklJh3LT6XAu\nCCCAQL4XiBmAd+3aZWvWrLETTzzRjQf93XffOYhTTjklWwbiGDRokG3evNk++OADa9CggZUpU8bN\nPbxs2TIbPHiwHThwwAYMGJDtH8aG9Rvsq4e/smIlimd7Xrktg907dtrt/bjFLLd9LpwPAgjkX4Go\nAKyxn88880wX8BSA33//fRcQFRhvvfVW04AcKpkmMn300UdulK1q1aqFD1uhQgXr0KGDjRs3zkaM\nGJEjAbhhw4Y2aPjtVvm4yuHzKCgvpr71vqkmgoQAAgggkDMCUW3AGmjjgQcesH/961/hM9Dwkw8+\n+KD9+c9/tqeeeiq8PFEvWrRoYTNnzox5OP0AqFKlSsx1LEQAAQQQQCCvCqQoAR89etQWLVrkBt+I\ndUEaijI7AvB9991nffr0sbFjx5pKoZoKUdXgy5cvN3XKmjp1aqzTYRkCCCCAAAJ5ViBFAFYnpGLF\nilnRon8sfvPNN90yXaHWZ8d8wKrqXrhwoauGXrt2rWsPVqlX7b6q8i5UqFCGgCdMmGBTpkyJue22\nbdvsjDPOiLmOhQgggAACCOS0wB+RNpRzkSJFrHXr1m4e4E6dOrlziQy4//vf/7ItiJUsWdI6d+4c\nvn51vFKbZEaDr3a84YYb3CN8kIgXr7/+uikIkxBAAAEEEMgNAlFtwCp1XnfddW4SBlVJK+3evdtG\njRrlqoIvv/zyHDlvlbyHDBmSI3mRCQIIIIAAAjktkKIErMx79eplhQsXtksuucTd/qOeyT/++KM1\nbtzYdZSqU6dOws+xUaNG9ssvv6Q4rkq/av9VIO7Zs6dpbGoSAggggAAC+UUgKgDrwhR8FfRWrFhh\nK1eudPcD16pVK9uuWcH12muvNU38oB7XSu+8845rE1bva90XTEIAAQQQQCA/CURVQXsXp7bXpk2b\n2rnnnmv79++3Q4eyb6QkdY6aP3++rVq1ylU7K+BWrlzZypYta3Xr1nWvvfPiGQEEEEAAgfwg4BuA\nFXBvvvlmFwRPOukk96xOTtkViHXr0QsvvGB/+tOfXM/njz/+OD/4cg0IIIAAAgjEFPANwLqlR8NR\nLl682Pbs2eOet2/f7gbpiHmkBC1UJy+NjKU24ciRsRJ0eA6DAAIIIIBArhCI2QasM/vqq69s2LBh\nrhpa75s0aWL33HNPjvRMVnvze++9p2xJCCCAAAII5EsB3xJw165d3UQM3tSAui930qRJ5t0fnC81\nuCgEEEAAAQRySMC3BKx7fzU7kUakat68uesNrc5Yxx9/fHi0qQULFtBDOYc+KLJBAAEEEMhfAr4B\n+MILL7R27dq5qz1y5IgbhjJyiEqtiBwlK3+xcDUIIIAAAghkr0BUANZ4z4899pht2bLFRo4c6XKf\nM2eOXX/99W555HCR2XtqHB0BBBBAAIH8KxDVBqyg++yzz7qBOLzLPuWUU2zQoEF22WWXuft1veU8\nI4AAAggggEDmBKJKwBr68ZVXXrFmzZqFj6iq55tuusndGvTGG29Y27Ztw+t4gQACCCCAAALxC6Qo\nAWvsZd37q3GfY6WzzjrLvv7661irWIYAAggggAACcQikCMAq6Z5wwgn25ZdfxjyElqs6moQAAggg\ngAACWRNIEYB1qH79+ln//v3t22+/DR9Z0xJqPl21D6t3NAkBBBBAAAEEsiYQ1QZ84403uskXVNI9\n9thj3X3AmhGpXLlyboai0047LWs5sjcCCCCAAAIIWFQAlsngwYNtwIAB9s0339i6deusVatWbijK\nIkWKQIYAAggggAACCRCIGYB13BIlSliHDh3cIwH5cAgEEEAAAQQQiBCIagOOWMdLBBBAAAEEEMgm\nAQJwNsFyWAQQQAABBNISSBGA1dtZvaCVvvvuOzfwRlo7sw4BBBBAAAEEMieQIgBr0gWNdDV//nx7\n9NFHbebMmW5MaI0L7T127dqVuZzYCwEEEEAAAQTCAik6YRUrVsxuvfVW69Gjh+3cudPN/1u4cIoY\nbZdeeqlNnjw5fABeIIAAAggggED8Aimja2j/+++/37Zt22Z33323TZ8+3fbu3ZviQfCNH5k9EEAA\nAQQQSC2QogQcufKee+5xb7dv324bNmywRo0aWenSpSM34TUCCCCAAAIIZFIgqgTsHUftweqQVbly\nZevSpYsbCatXr1528OBBbxOeEUAAAQQQQCCTAr4B+Omnn7ZVq1bZsmXLTKVgDUeZnJxsDz74YCaz\nYjcEEEAAAQQQ8AR8A/C8efNs2LBh4XmBGzRoYKqWnjVrlrcvzwgggAACCCCQSQHfAKxJF2bPnp3i\nsHpfpUqVFMt4gwACCCCAAALxC/h2wrrsssusZcuWrsR7xhln2IIFC2zRokWUgOM3Zg8EEEAAAQSi\nBHxLwJqKcMmSJda3b1/TCFnnn3++LV261Nq0aRN1EBYggAACCCCAQHwCviVgHUZBWANzkBBAAAEE\nEEAgsQK+JeDEZsPREEAAAQQQQCBSgAAcqcFrBBBAAAEEckiAAJxD0GSDAAIIIIBApIBvG7AmYxg4\ncKDriJWUlBTeRxM1PPLII+H3vEAAAQQQQACB+AV8A/Do0aNNUw+OHz/eypYtGz5ypUqVwq95gQAC\nCCCAAAKZE/ANwBs3bnQl4M6dO2fuyOyFAAIIIIAAAr4Cvm3A3ry/W7du9d2ZFQgggAACCCCQOQHf\nALxp0yabOnWqVa9e3U1F2LRpU9Nj8ODBmcuJvRBAAAEEEEAgLOBbBX3BBRdY27ZtTZ2xVAquU6eO\nlSxZ0mgDDtvxAgEEEEAAgUwL+AbgGjVquNmPnn/+eRd0FYh79uxpU6ZMyXRm7IgAAggggAACvwv4\nVkEzHzBfEQQQQAABBLJPwDcAMx9w9qFzZAQQQAABBHwDMPMB8+VAAAEEEEAg+wR824CZDzj70Dky\nAggggAACviVg5gPmy4EAAggggED2CfiWgJUl8wFnHzxHRgABBBAo2AJRAbh9+/amcaDnzJljEydO\njNLRZAzjxo2LWs4CBBBAAAEEEMi4QFQAnjBhgtWrV8+qVq1qGge6WLFi4aMdPHjQDh06FH7PCwQQ\nQAABBBDInEBUG3CzZs2sRIkS9uSTT9qOHTusefPm4cdPP/3kSseZy4q9EEAAAQQQQMATiCoBP/fc\nczZgwAC3XlMRRqZy5crZqFGjIhfxGgEEEEAAAQQyIRBVAr7ppptcNbMC7dy5c91rVTsfPnzYdu/e\n7aYozEQ+7IIAAggggAACEQJRAVjrihYtaps3b7ZPP/3Uvdb7IkWKROzGSwQQQAABBBDIikDMAKwD\n1q1b15YsWWJHjhzJyvHZFwEEEEAAAQRiCES1AXvblCpVyt5//30rX7681a5dO1wCPvfcc+3hhx/2\nNuMZAQQQQAABBDIh4BuAu3fvbq1bt446pAbnICGAAAIIIIBA1gR8A7CqoPXYvn27bdiwwRo1amSl\nS5fOWm7sjQACCCCAAAJOwLcNWG2//fr1s8qVK1uXLl1MtyD16tXLNBgHCQEEEEAAAQSyJuAbgJ9+\n+mlbtWqVLVu2zJWCV65cacnJyfbggw9mLUf2RgABBBBAAAHzDcDz5s2zYcOGmUbGUmrQoIHdc889\nNmvWrGxl0/3GGoGLhAACCCCAQH4W8A3Ap512ms2ePTvFtet9lSpVUixLxJukpCQbPny4621dvHhx\nq1SpkpUpU8ZatGgRc0KIROTJMRBAAAEEEAhSwLcT1mWXXWYtW7Z0Jd4zzjjDFixYYIsWLcqWEl3m\nV6AAABXhSURBVPCgQYPcwB8ffPCBK2kr+GrULVV/Dx482A4cOBAeHjNILPJGAAEEEEAgUQK+JWDd\nbqSBOPr27WtHjx61888/35YuXWpt2rRJVN7h43z00UemNudWrVpZ2bJlrVChQlahQgXr0KGDm/rw\nnXfeCW/LCwQQQAABBPKDgG8JWBenIKyxodetW+emKIycmjCRF6+q5pkzZ1rv3r2jDqvBQLKj2jsq\nIxYggAACCCCQgwK+AVgTMKj695lnnnHTE6qd9uqrr7bHH388xRzBiTjX++67z/r06WNjx461hg0b\nutG3du3aZcuXL3eTQEydOjVD2ezfv9/0iJV+++035jKOBcMyBBBAAIFABHwD8IQJE2zNmjW2ePFi\na9q0qa1YscJ1lHrggQdcb+hEnu2JJ55oCxcudLMvrV271rUHq9SraRFPP/30DI9H/eabb9rbb78d\n89Q0mEi7du1irmMhAggggAACOS3gG4C/+uordxuSgq9SkyZNXOAdMmRIws9x/fr1dtddd9lbb73l\n2n2ffPJJO/74410+r7zyilv+2muvpZuv2qv1iJVef/1127ZtW6xVLEMAAQQQQCDHBXw7YXXt2tWe\neOKJ8D256ok8adIk69SpU8JPUlXP1atXt/nz57sA3LFjR/vhhx8Sng8HRAABBBBAILcI+JaAdRuQ\nbgtSVXDz5s1NI2GpfVUl0ylTprjz161JumUoq0ltvKqC1gxMag8+4YQTTLMuff7551k9NPsjgAAC\nCCCQKwV8A/CFF17o2kx37txpW7dutTp16ljJkiVTXIQCZiKSAq5Kv2eeeaY73BVXXGGbNm2yHj16\n2I033piILDgGAggggAACuUrANwDXqFHDtfk+//zzbmQqBeKePXu60m+JEiUSehG61UkDf9x+++32\n17/+1R1bbc179uxxy5QvCQEEEEAAgfwk4NsGnJOTMXTr1s1Wr15t5513XgrbESNGmAbpUHU0CQEE\nEEAAgfwk4BuAc3oyBrUla+jL1Omss86y/v37p17MewQQQAABBPK0gG8AzsnJGPK0ICePAAIIIIBA\nJgR824BzcjKGTJw3uyCAAAIIIJCnBXxLwKVLl86xyRjytCAnjwACCCCAQCYEfEvAmp+3atWq9re/\n/S0Th2UXBBBAAAEEEEhLwLcEXLduXVcCPnLkSFr7sw4BBBBAAAEEMiHgWwLWIBuaCrB8+fJWu3Zt\nK1KkiDu8bgl6+OGHM5EVuyCAAAIIIICAJ+AbgLt3726tW7f2tgs/a45gEgIIIIAAAghkTSBmANaw\nkF9//bUbAKNBgwZZy4G9EUAAAQQQQCBKIKoN+IUXXnBjMk+ePNlNwuANDRm1JwsQQAABBBBAINMC\nUQH40UcftRdffNHmzJljX375pY0ZM8YOHjyY6QzYEQEEEEAAAQSiBaIC8MaNG619+/Zuy1atWrnp\nCNevXx+9J0sQQAABBBBAINMCUQE4KSnJihUrFj5gxYoV7cCBA+H3vEAAAQQQQACBrAvE7IS1e/du\n86Yc1H3AmhZQ0xEqFS9e3DRKFgkBBBBAAAEEMi8QMwA3btw4xRE1MYOXNEb0a6+95r3lGQEEEEAA\nAQQyIRAVgFeuXGnJycm+h1IJmIQAAggggAACWROICsBq8yUhgAACCCCAQPYKRHXCyt7sODoCCCCA\nAAIISIAAzPcAAQQQQACBAAQIwAGgkyUCCCCAAAIEYL4DCCCAAAIIBCBAAA4AnSwRQAABBBAgAPMd\nQAABBBBAIAABAnAA6GSJAAIIIIAAAZjvAAIIIIAAAgEIEIADQCdLBBBAAAEECMB8BxBAAAEEEAhA\ngAAcADpZIoAAAgggQADmO4AAAggggEAAAgTgANDJEgEEEEAAAQIw3wEEEEAAAQQCECAAB4BOlggg\ngAACCBCA+Q4ggAACCCAQgAABOAB0skQAAQQQQIAAzHcAAQQQQACBAAQIwAGgkyUCCCCAAAIEYL4D\nCCCAAAIIBCBAAA4AnSwRQAABBBAgAPMdQAABBBBAIAABAnAA6GSJAAIIIIAAAZjvAAIIIIAAAgEI\nEIADQCdLBBBAAAEECMB8BxBAAAEEEAhAgAAcADpZIoAAAgggQADmO4AAAggggEAAAgTgANDJEgEE\nEEAAAQIw3wEEEEAAAQQCECAAB4BOlggggAACCBCA+Q4ggAACCCAQgAABOAB0skQAAQQQQIAAzHcA\nAQQQQACBAAQIwAGgkyUCCCCAAAIEYL4DCCCAAAIIBCBAAA4AnSwRQAABBBAgAPMdQAABBBBAIAAB\nAnAA6GSJAAIIIIAAAZjvAAIIIIAAAgEIEIADQCdLBBBAAAEECMB8BxBAAAEEEAhAINcF4MOHD9uO\nHTsCoCBLBBBAAAEEck4gVwTgpKQkGz58uNWuXduKFy9ulSpVsjJlyliLFi1s4sSJOadBTggggAAC\nCOSQQNEcyifNbAYNGmSbN2+2Dz74wBo0aOCC7+7du23ZsmU2ePBgO3DggA0YMCDNY2jlmjVrbN26\ndTG3W7p0qZUoUSLmOm/hodAPgdUrVtq6NWu9RQXm+dv5i+z8zt3jut6DBw/a0kVLrHDhXPE7Lq5z\nz+rGS79dYtYnvqMcOHDQFsybH99O+WTr5UuX5ZMr4TIQSJxAoeRQStzhMnek+vXr29y5c61atWpR\nB5g3b56NGDHCpk2bFrUu9YJPPvnEvvjii9SL3futW7faKaecYldddVXM9Vr46axZNiv0KFKkiO82\n+XdFsg0dMtRKlSqV4Ut8P/SDadGiRRnePj9tWKJEcbt10K3p/qiLvObX33jdVqz4IXJRgXldvnw5\nG3DTACtWrFiBuWYuFIH0BHJFAL7wwgutT58+1rt376jz/fvf/25r1661F198MWodCxBAAAEEEMir\nArkiAC9cuNAF4HLlylnDhg2tfPnytmvXLlu+fLmpU9bUqVOtbt26edWY80YAAQQQQCBKIFcEYJ2V\n2nlVDa3SrtqDq1SpYo0aNbKOHTtaoUKFok6cBQgggAACCORlgVwTgPMyIueOAAIIIIBAvAIFr/tq\nvEJsjwACCCCAQDYIEICzAZVDIoAAAgggkJ4AATg9IdYjgAACCCCQDQIE4GxA5ZAIIIAAAgikJ0AA\nTk+I9QgggAACCGSDAAE4G1A5JAIIIIAAAukJEIDTE2I9AggggAAC2SBAAM4GVA6JAAIIIIBAegIE\n4PSEWI8AAggggEA2CBCAswGVQyKAAAIIIJCeAAE4PSHWI4AAAgggkA0CBOBsQOWQCCCAAAIIpCdA\nAE5PiPUIIIAAAghkgwABOBtQOSQCCCCAAALpCRCA0xNiPQIIIIAAAtkgQADOBlQOiUBuEdi1a1em\nT+Xo0aO2ffv2TO/PjgggkLYAAThtn/Dahx56yC699NLw+8gXDRo0sG+//TZyUZqvf/31VytUqJCV\nL1/ePSpUqGAnn3yyvfbaa+H9xo4d67YZP358eJleHD582CpXrmydOnVyy9M6rxQ7ZsObs88+20qV\nKuWuoVy5clanTh278847bf/+/dmQm1mNGjVsxYoVUcdO7zw2btzoLM8888yofa+//nq3buvWrfbT\nTz+510eOHInaLqcX3HbbbVamTBnbsWNHiqw9gy+++MJatWqVYp33Jjk52e69915r2rSpnXLKKdak\nSRPT8ZKSkrxN0nxesGCBdevWzU444QT3PatVq5b985//dN+9NHdkJQIIxCVAAI6LK/bGn3/+uftj\nFXut/9L169fb7t27XSlj9OjR7o/ku+++G96hZs2a9uabb4bf68WMGTOscOHc87HpB4KuQQ85fP31\n13bDDTekOOeceJPeecjsxx9/tM2bN4dPRz9mdM65Lem89D3o27evvfTSS3GfXr9+/Wzu3Lk2Z84c\n94Nl0aJFtmrVKvvLX/6S7rG0X69eveyuu+6y77//3pYuXWra/8MPP7Qnnngi3f3ZAAEEMi6Qe/6S\nZ/ycc92WV111la1Zs8aWLFliV199tQukxx57rLVp08YWL16c7vkWLVrUVIobNGiQ/fvf/w5vf9pp\np9kPP/xgKp15SaXknj17em/TfJ48ebIrlepcLrvssqjSVJo7x7lSJXqVgJ988kl78cUX7eeff3ZH\nmDVrlrVu3dqOOeYYV4Pwyy+/2G+//eaW7dy5M5zLwIED7a233jKV3kaOHGkqdekHyP333++WhTdM\n54XfeWj5hRdeaG+//Xb4CPoxE6tUHN4g4oU+h1NPPdVU0j/ppJNcgItYndCXCnYnnnii3Xjjjfbs\ns8/GdWz9EJK/AnelSpXcvqql+M9//mPdu3d37/Ud+8c//uFqXfSZ3X333eE8Hn30UZevV8OiFapx\nmTBhgjsnvc9JC+VHQiC/ChCAE/DJKvgeOHDAPfTHT3/4VHLo0KGDDR8+PMM56A+7/rh5qUiRInbB\nBReEg4ZKRrNnzw5XP3vbxXrW+Sio/fe//7XVq1fb3r177amnnoq1aUKXHX/88S5I6Tq2bdvmgt6w\nYcNs+fLlpqr2Bx54wMqWLeuqk9977z2X9759+1zA6Nixo+lHgwy17p133rGXX37Zvvrqq7jPMfI8\nvJ0vueSSFDUK+jHj16zg7eM963O86KKL3I8hlTBvvvlmb1XCn1944QW7/PLLXaCXzfz58zOch6qP\na9eu7YJm5E7VqlULB2B9Lmq60I+bTz75xDnLXEl5qTkkdWrRokX4x0pOWqQ+D94jkJ8ECMAJ/jTV\nrjtixAirXr26XXHFFbZ27doM56B91OanUqCXFDRUMlTSH8vTTz/dVGJOL6nEp040KuUdPHjQ/ZFV\n+2x2J+WrP/bqvKPzbt68uQtcas9UtebUqVPdKfTu3TscDLVMP1ZU0po0aZIpwDVs2NC1XV577bUu\nGMd73pHn4e3buXNn11avc9OPGZXOtSwjSeYKbmqDVvD98ssvM7Jb3NuoVkCfs0rrSn/605/iKgWr\ntkTfwfSSSrgqETdq1Mj0WXjfMTWLqMbES6qVadu2rQvK55xzjlucUxbeOfCMQH4VIAAn+JM97rjj\nwkdU0NEf+owmdQJStauCh5dUNa1SiQLz66+/7trnvHVpPZcoUcJ16lJA0zHPP//8mB2Y0jpGZtfp\nj7hKYRs2bHDV8uoEpIeqexVg1ClK1egKgKqOfuONN9yPFeWndaoi9fbR64ULF2bqVLzz8HYuVqyY\nnXvuua591fsxU7x4cW91ms8PP/ywHTp0yHVqatasWYoOc2nuGOdKlcpVW6EqaAVHVR2rFkDLMpL0\ng0ftvZE/4rSf3qtN10v6weMllXhVi6Okjl2qvfGSgrN+OPXp0yfcnJJTFt458IxAfhUgACf4k40M\nnvEeWtWukX8Ytb8CadeuXV3QmDlzpnmlkPSOrdKvqrTVO1sPlYqys9rUO5/p06ebgpp60KoHrtqx\n1R7sPVSKVE9enU+XLl3CwVAlfaV27dq5ampv+5UrV9qUKVO8w2f4OfI8IndSlbOqtlXiy2j1s/ZX\nqU8d4tSJ66abbjK1+2fHLTqqfh41apQ7R52nrkM/ZvTjKyNJP1wUbFN3LtOPHP2Y83qoK0h7SQG3\nXr167m379u1dk4cXwPX56bOJrCnIKQvv/HhGIL8KEIDj+GR1G4duIfIekZ2I4jhMeFOVanUstZeq\nF6/a4YYOHRpe771QoFDHJHUCUkBOnWKdlzo7qd1OpVCVinr06JF6t4S8V8lM16CSqzoPDRgwwHUm\nU+lfPxZUVeuVYHV9qvbUjwMlVdHfc889dsYZZ7j2YS27+OKLbeLEieGqePUE1i1Z6aW0ziNyX+Wv\n3sH6MaNbbWIl7/P1nlWLcc0117iqYLXvX3nlle5z8IJUrGNkZpna6vUD5brrrnOfmT43PWSQ0c5Y\nKuWrfffPf/5z+NY49WyWszpeqUOW0scff2ybNm0y3SesHyP6kaekDlnLli2zW265xdaGmk90S9Y3\n33zj+jKoDV8pJyxcRvyDQH4XCP0RIWVAIFQVqobZFI9QdbPbs379+smhWzWSQ52FkkMlkPDRQrfk\npHjvrQiVnFIcJ9QpKTlUWkx+//33vU2SQ9V8yaEA5d6HerYmhwJvcqgU496/8soryaEOS+51Wuel\nY9StWzc5VBpNDvV2decXziABL0Il2PB1hP7wJ8sh9Ic+OfRHO3z0xx57LDkUjJMbN26cHOoNnRy6\nfzW8LtTBKFnXruvxUiiQJodKXMmhEnJyqB04OdQJLVnbKYXayJND1ajepuHn9M4j9CMkOdShLbx9\nKMgnh9pW3fvQjwF3DVu2bElet25d+HoiP+tQVXlyqBnAnX/o3trkUBBODt02Fj5eol6E+g4k69xS\np1AgTA7VrCSHAmPYIFTCTW7ZsmXqTcPv//rXvzo/zzHU/p8c+iHh1oc6xSWHalqS9f0Ntdcnh3ru\nJ8vBS6Hq6ORQk0VyqOd6cqg2wz2HfhQk63urlBMW3rnwjEB+Fiiki8vvPzIK+vWp12uVKlUCY1Ap\nSrUFkZ170jsZr81TJenclFRroVuRVA2bF5JK8d7tSN756n5g1aSotKsOerqeWEk1FfruVK1aNdZq\nV0uRlyxiXgQLEQhQIG/8FQkQKD9kHWTwlZ9up4on+Gqf3BZ4dU5KFStW/P1FHvk3dfCNPG211afV\nCU2Dl/gFXx0nr1lEXjuvEcgNAgTg3PApcA4I5KCABmXRjyISAggEK0AVdLD+5I4AAgggUEAF6AVd\nQD94LhsBBBBAIFgBAnCw/uSOAAIIIFBABQjABfSD57IRQAABBIIVIAAH60/uCCCAAAIFVIAAXEA/\neC4bAQQQQCBYAQJwsP7kjgACCCBQQAUIwAX0g+eyEUAAAQSCFSAAB+tP7ggggAACBVSAAFxAP3gu\nGwEEEEAgWAECcLD+5I4AAgggUEAFCMAF9IPnshFAAAEEghUgAAfrT+4IIIAAAgVUgABcQD94LhsB\nBBBAIFgBAnCw/uSOAAIIIFBABQjABfSD57IRQAABBIIVIAAH60/uCCCAAAIFVIAAXEA/eC4bAQQQ\nQCBYAQJwsP7kjgACCCBQQAUIwAX0g+eyEUAAAQSCFSAAB+tP7ggggAACBVSAAFxAP3guGwEEEEAg\nWAECcLD+5I4AAgggUEAFCMAF9IPnshFAAAEEghUgAAfrT+4IIIAAAgVUgABcQD94LhsBBBBAIFgB\nAnCw/uSOAAIIIFBABQjABfSD57IRQAABBIIVIAAH60/uCCCAAAIFVIAAXEA/eC4bAQQQQCBYAQJw\nsP7kjgACCCBQQAUIwAX0g+eyEUAAAQSCFSAAB+tP7ggggAACBVSAAFxAP3guGwEEEEAgWAECcLD+\n5I4AAgggUEAFCMAF9IPnshFAAAEEghUgAAfrT+4IIIAAAgVUgABcQD94LhsBBBBAIFgBAnCw/uSO\nAAIIIFBABQjABfSD57IRQAABBIIVIAAH60/uCCCAAAIFVIAAXEA/eC4bAQQQQCBYAQJwsP7kjgAC\nCCBQQAUIwAX0g+eyEUAAAQSCFfh/aUhX7hRU+bcAAAAASUVORK5CYII=\n" } ], "prompt_number": 54 }, { "cell_type": "code", "collapsed": false, "input": [ "#Formatting family-specific DML files for stats\n", "!wc -l lineage_dml.bed > lineage_countstotal" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 209 }, { "cell_type": "code", "collapsed": false, "input": [ "!wc -l ./genome_tracks/Cgigas_v9_CG.gff > CG_countstotal" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 210 }, { "cell_type": "code", "collapsed": false, "input": [ "!cat linTE lineage_countstotal > Lineage_TEs" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 211 }, { "cell_type": "code", "collapsed": false, "input": [ "!awk '{print $1}' Lineage_TEs > Lineage_TEs_counts" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 212 }, { "cell_type": "code", "collapsed": false, "input": [ "!cat CGTE CG_countstotal > CG_TEs" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 213 }, { "cell_type": "code", "collapsed": false, "input": [ "!awk '{print $1}' CG_TEs > CG_TEs_counts" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 214 }, { "cell_type": "code", "collapsed": false, "input": [ "!paste Lineage_TEs_counts CG_TEs_counts > LinTEs_combined" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 215 }, { "cell_type": "code", "collapsed": false, "input": [ "!awk '{print $1, $2}' LinTEs_combined > Lineage_TEs_stats" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 216 }, { "cell_type": "code", "collapsed": false, "input": [ "%%R \n", "#Stats for TEs: family-specific\n", "LinStats<-read.table('Lineage_TEs_stats')\n", "chisq.test(LinStats) " ], "language": "python", "metadata": {}, "outputs": [ { "metadata": {}, "output_type": "display_data", "text": [ "\n", "\tPearson's Chi-squared test with Yates' continuity correction\n", "\n", "data: Stats\n", "X-squared = 18.6144, df = 1, p-value = 1.6e-05\n", "\n" ] } ], "prompt_number": 217 }, { "cell_type": "code", "collapsed": false, "input": [ "#formatting developmental DML files for stats\n", "!wc -l dev_dml.bed > dev_countstotal" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 218 }, { "cell_type": "code", "collapsed": false, "input": [ "!cat devTE dev_countstotal > Dev_TEs" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 219 }, { "cell_type": "code", "collapsed": false, "input": [ "!awk '{print $1}' Dev_TEs > Dev_TEs_counts" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 220 }, { "cell_type": "code", "collapsed": false, "input": [ "!paste Dev_TEs_counts CG_TEs_counts > DevTEs_combined" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 221 }, { "cell_type": "code", "collapsed": false, "input": [ "!awk '{print $1, $2}' DevTEs_combined > Dev_TEs_stats" ], "language": "python", "metadata": {}, "outputs": [], "prompt_number": 222 }, { "cell_type": "code", "collapsed": false, "input": [ "%%R \n", "#Stats for TEs: developmentally different\n", "DevStats<-read.table('Dev_TEs_stats')\n", "chisq.test(DevStats) " ], "language": "python", "metadata": {}, "outputs": [ { "metadata": {}, "output_type": "display_data", "text": [ "\n", "\tPearson's Chi-squared test with Yates' continuity correction\n", "\n", "data: DevStats\n", "X-squared = 9.5941, df = 1, p-value = 0.001952\n", "\n" ] } ], "prompt_number": 223 }, { "cell_type": "code", "collapsed": false, "input": [], "language": "python", "metadata": {}, "outputs": [] } ], "metadata": {} } ] }