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Lesson 3
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lesson_3.ipynb
{
"cells": [
{
"cell_type": "markdown",
"metadata": {},
"source": [
"# Γενετική ανάλυση μέσω του προγράμματος plink\n",
"Σε αυτή τη διάλεξη θα μάθουμε να κάνουμε τυπικές επεξεργασίες σε πραγματικά δεδομένα γενετικής. Ως κύριο πρόγραμμα θα χρησημοποιήσουμε το [plink](http://zzz.bwh.harvard.edu/plink/) το οποίο είναι ο \"ελβετικός σουγιάς\" για τέτοιου είδους αναλύσεις. Για δεδομένα θα χρησημοποιήσουμε το χρωμόσωμα 22 από το 1000 Genomes Project.\n",
"\n",
"Το [1000 Genomes Project](https://en.wikipedia.org/wiki/1000_Genomes_Project) (1KGP για συντομία) είναι ίσως το δεύτερο πιο σημαντικό project στη γενετική μετά το [Human Genome Project](https://en.wikipedia.org/wiki/Human_Genome_Project) (HGP). Αν το HGP δημιούργησε το πρώτο πλήρες γονιδίωμα του ανθρώπου, το 1KGP ήταν το πρώτο που μελέτησε τη γενετική ετερογένεια ενός μεγάλου μέρους του ανθρώπινου πληθυσμού. Στη τελική του φάση, το 1KGP είχε αλληλουχίσει το γονιδίωμα από 2504 ανθρώπους οι οποίοι ανήκουν σε 26 διαφορετικούς πληθυσμούς.\n",
"\n",
"Ας παίξουμε λίγο με τα δεδομένα από το 1KGP!\n",
"\n",
"Τα δεδομένα από τη τελευταία (τρίτη) φάση του 1KGP βρίσκονται σε αυτό το link: ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/\n",
"\n",
"Αυτό το site (στη πραγματικότητα ftp site αλλά αυτό δεν έχει σημασία) περιέχει όλες τις μεταλλάξεις (variants) που εντοπίστηκαν. Όλα τα variants περιγράφονται σε [VCF](https://en.wikipedia.org/wiki/Variant_Call_Format) φορμάτ και είναι συμπιεσμένα με το πρόγραμμα gzip. Κάθε αρχείο έχει τη κατάλληξη .vcf.gz και υπάρχει ένα αρχείο για κάθε χρωμόσωμα. Ας \"παίξουμε\" με το μικρότερο χρωμόσωμα το οποίο είναι το 22.\n",
"\n",
"Αρχικά κατεβάζουμε το αρχείο. **ΠΡΟΣΟΧΗ** Το αρχείο αυτό, αν και συμπιεσμένο, είναι 205 ΜΒ:"
]
},
{
"cell_type": "code",
"execution_count": 1,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"--2018-05-01 18:18:32-- ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/ALL.chr22.phase3_shapeit2_mvncall_integrated_v5a.20130502.genotypes.vcf.gz\n",
" => ‘1kgp_chr22.vcf.gz’\n",
"Resolving ftp.1000genomes.ebi.ac.uk (ftp.1000genomes.ebi.ac.uk)... 193.62.192.8\n",
"Connecting to ftp.1000genomes.ebi.ac.uk (ftp.1000genomes.ebi.ac.uk)|193.62.192.8|:21... connected.\n",
"Logging in as anonymous ... Logged in!\n",
"==> SYST ... done. ==> PWD ... done.\n",
"==> TYPE I ... done. ==> CWD (1) /vol1/ftp/release/20130502 ... done.\n",
"==> SIZE ALL.chr22.phase3_shapeit2_mvncall_integrated_v5a.20130502.genotypes.vcf.gz ... 214453750\n",
"==> PASV ... done. ==> RETR ALL.chr22.phase3_shapeit2_mvncall_integrated_v5a.20130502.genotypes.vcf.gz ... done.\n",
"Length: 214453750 (205M) (unauthoritative)\n",
"\n",
"ALL.chr22.phase3_sh 100%[===================>] 204.52M 7.92MB/s in 24s \n",
"\n",
"2018-05-01 18:18:56 (8.47 MB/s) - ‘1kgp_chr22.vcf.gz’ saved [214453750]\n",
"\n"
]
}
],
"source": [
"!wget -O 1kgp_chr22.vcf.gz ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/ALL.chr22.phase3_shapeit2_mvncall_integrated_v5a.20130502.genotypes.vcf.gz"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"Η πληροφορία για τις περιοχές και τους πληθυσμούς που ανήκει κάθε sample υπάρχει στο αρχείο που βρίσκεται σε αυτό το link: ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/integrated_call_samples_v3.20130502.ALL.panel . Ας το κατεβάσουμε:"
]
},
{
"cell_type": "code",
"execution_count": 2,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"--2018-05-01 18:19:02-- ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/integrated_call_samples_v3.20130502.ALL.panel\n",
" => ‘sample_information.csv’\n",
"Resolving ftp.1000genomes.ebi.ac.uk (ftp.1000genomes.ebi.ac.uk)... 193.62.192.8\n",
"Connecting to ftp.1000genomes.ebi.ac.uk (ftp.1000genomes.ebi.ac.uk)|193.62.192.8|:21... connected.\n",
"Logging in as anonymous ... Logged in!\n",
"==> SYST ... done. ==> PWD ... done.\n",
"==> TYPE I ... done. ==> CWD (1) /vol1/ftp/release/20130502 ... done.\n",
"==> SIZE integrated_call_samples_v3.20130502.ALL.panel ... 55156\n",
"==> PASV ... done. ==> RETR integrated_call_samples_v3.20130502.ALL.panel ... done.\n",
"Length: 55156 (54K) (unauthoritative)\n",
"\n",
"integrated_call_sam 100%[===================>] 53.86K --.-KB/s in 0.03s \n",
"\n",
"2018-05-01 18:19:02 (1.51 MB/s) - ‘sample_information.csv’ saved [55156]\n",
"\n"
]
}
],
"source": [
"!wget -O sample_information.csv ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/integrated_call_samples_v3.20130502.ALL.panel"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"Ας \"φορτώσουμε\" αυτό το αρχείο μέσω pandas:"
]
},
{
"cell_type": "code",
"execution_count": 14,
"metadata": {
"collapsed": true
},
"outputs": [],
"source": [
"import pandas as pd\n",
"samples = pd.read_csv('sample_information.csv', sep=r'\\s+')"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"Ας τυπώσουμε τις πρώτες 10 γραμμές:"
]
},
{
"cell_type": "code",
"execution_count": 15,
"metadata": {},
"outputs": [
{
"data": {
"text/html": [
"<div>\n",
"<style>\n",
" .dataframe thead tr:only-child th {\n",
" text-align: right;\n",
" }\n",
"\n",
" .dataframe thead th {\n",
" text-align: left;\n",
" }\n",
"\n",
" .dataframe tbody tr th {\n",
" vertical-align: top;\n",
" }\n",
"</style>\n",
"<table border=\"1\" class=\"dataframe\">\n",
" <thead>\n",
" <tr style=\"text-align: right;\">\n",
" <th></th>\n",
" <th>sample</th>\n",
" <th>pop</th>\n",
" <th>super_pop</th>\n",
" <th>gender</th>\n",
" </tr>\n",
" </thead>\n",
" <tbody>\n",
" <tr>\n",
" <th>0</th>\n",
" <td>HG00096</td>\n",
" <td>GBR</td>\n",
" <td>EUR</td>\n",
" <td>male</td>\n",
" </tr>\n",
" <tr>\n",
" <th>1</th>\n",
" <td>HG00097</td>\n",
" <td>GBR</td>\n",
" <td>EUR</td>\n",
" <td>female</td>\n",
" </tr>\n",
" <tr>\n",
" <th>2</th>\n",
" <td>HG00099</td>\n",
" <td>GBR</td>\n",
" <td>EUR</td>\n",
" <td>female</td>\n",
" </tr>\n",
" <tr>\n",
" <th>3</th>\n",
" <td>HG00100</td>\n",
" <td>GBR</td>\n",
" <td>EUR</td>\n",
" <td>female</td>\n",
" </tr>\n",
" <tr>\n",
" <th>4</th>\n",
" <td>HG00101</td>\n",
" <td>GBR</td>\n",
" <td>EUR</td>\n",
" <td>male</td>\n",
" </tr>\n",
" <tr>\n",
" <th>5</th>\n",
" <td>HG00102</td>\n",
" <td>GBR</td>\n",
" <td>EUR</td>\n",
" <td>female</td>\n",
" </tr>\n",
" <tr>\n",
" <th>6</th>\n",
" <td>HG00103</td>\n",
" <td>GBR</td>\n",
" <td>EUR</td>\n",
" <td>male</td>\n",
" </tr>\n",
" <tr>\n",
" <th>7</th>\n",
" <td>HG00105</td>\n",
" <td>GBR</td>\n",
" <td>EUR</td>\n",
" <td>male</td>\n",
" </tr>\n",
" <tr>\n",
" <th>8</th>\n",
" <td>HG00106</td>\n",
" <td>GBR</td>\n",
" <td>EUR</td>\n",
" <td>female</td>\n",
" </tr>\n",
" <tr>\n",
" <th>9</th>\n",
" <td>HG00107</td>\n",
" <td>GBR</td>\n",
" <td>EUR</td>\n",
" <td>male</td>\n",
" </tr>\n",
" </tbody>\n",
"</table>\n",
"</div>"
],
"text/plain": [
" sample pop super_pop gender\n",
"0 HG00096 GBR EUR male\n",
"1 HG00097 GBR EUR female\n",
"2 HG00099 GBR EUR female\n",
"3 HG00100 GBR EUR female\n",
"4 HG00101 GBR EUR male\n",
"5 HG00102 GBR EUR female\n",
"6 HG00103 GBR EUR male\n",
"7 HG00105 GBR EUR male\n",
"8 HG00106 GBR EUR female\n",
"9 HG00107 GBR EUR male"
]
},
"execution_count": 15,
"metadata": {},
"output_type": "execute_result"
}
],
"source": [
"samples[:10]"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"Τώρα ας εγκαταστήσουμε το plink:"
]
},
{
"cell_type": "code",
"execution_count": 3,
"metadata": {
"collapsed": true
},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"Reading package lists... Done\n",
"Building dependency tree \n",
"Reading state information... Done\n",
"Suggested packages:\n",
" zip\n",
"The following NEW packages will be installed:\n",
" unzip\n",
"0 upgraded, 1 newly installed, 0 to remove and 113 not upgraded.\n",
"Need to get 158 kB of archives.\n",
"After this operation, 530 kB of additional disk space will be used.\n",
"Get:1 http://eu-central-1.ec2.archive.ubuntu.com/ubuntu xenial/main amd64 unzip amd64 6.0-20ubuntu1 [158 kB]\n",
"Fetched 158 kB in 0s (12.5 MB/s)\n",
"Selecting previously unselected package unzip.\n",
"(Reading database ... 51121 files and directories currently installed.)\n",
"Preparing to unpack .../unzip_6.0-20ubuntu1_amd64.deb ...\n",
"Unpacking unzip (6.0-20ubuntu1) ...\n",
"Processing triggers for mime-support (3.59ubuntu1) ...\n",
"Processing triggers for man-db (2.7.5-1) ...\n",
"Setting up unzip (6.0-20ubuntu1) ...\n"
]
}
],
"source": [
"!sudo apt-get install -y unzip"
]
},
{
"cell_type": "code",
"execution_count": 4,
"metadata": {
"collapsed": true
},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"--2018-05-01 18:19:20-- http://www.cog-genomics.org/static/bin/plink180410/plink_linux_x86_64.zip\n",
"Resolving www.cog-genomics.org (www.cog-genomics.org)... 54.243.100.91, 23.21.104.182, 50.16.250.62\n",
"Connecting to www.cog-genomics.org (www.cog-genomics.org)|54.243.100.91|:80... connected.\n",
"HTTP request sent, awaiting response... 200 OK\n",
"Length: 2285389 (2.2M) [application/zip]\n",
"Saving to: ‘plink_linux_x86_64.zip’\n",
"\n",
"plink_linux_x86_64. 100%[===================>] 2.18M 3.48MB/s in 0.6s \n",
"\n",
"2018-05-01 18:19:21 (3.48 MB/s) - ‘plink_linux_x86_64.zip’ saved [2285389/2285389]\n",
"\n"
]
}
],
"source": [
"!wget http://www.cog-genomics.org/static/bin/plink180410/plink_linux_x86_64.zip"
]
},
{
"cell_type": "code",
"execution_count": 5,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"Archive: plink_linux_x86_64.zip\n",
" inflating: plink \n",
" inflating: LICENSE \n",
" inflating: toy.ped \n",
" inflating: toy.map \n",
" inflating: prettify \n"
]
}
],
"source": [
"!unzip plink_linux_x86_64.zip"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"Για αρχή θα αποσυμπιέσουμε το VCF αρχείο:"
]
},
{
"cell_type": "code",
"execution_count": 6,
"metadata": {},
"outputs": [],
"source": [
"!gunzip 1kgp_chr22.vcf.gz "
]
},
{
"cell_type": "markdown",
"metadata": {
"collapsed": true
},
"source": [
"Τώρα θα το μετατρέψουμε από VCF στο φορμάτ του plink (το οποίο ονομάζεται BED/BIM/FAM)"
]
},
{
"cell_type": "code",
"execution_count": 9,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"PLINK v1.90b5.4 64-bit (10 Apr 2018) www.cog-genomics.org/plink/1.9/\n",
"(C) 2005-2018 Shaun Purcell, Christopher Chang GNU General Public License v3\n",
"Logging to 1kgp_chr22.vcf.log.\n",
"Options in effect:\n",
" --make-bed\n",
" --out 1kgp_chr22.vcf\n",
" --recode\n",
" --vcf 1kgp_chr22.vcf\n",
"\n",
"1998 MB RAM detected; reserving 999 MB for main workspace.\n",
"\r",
"--vcf: 1k variants complete.\r",
"--vcf: 2k variants complete.\r",
"--vcf: 3k variants complete.\r",
"--vcf: 4k variants complete.\r",
"--vcf: 5k variants complete.\r",
"--vcf: 6k variants complete.\r",
"--vcf: 7k variants complete.\r",
"--vcf: 8k variants complete.\r",
"--vcf: 9k variants complete.\r",
"--vcf: 10k variants complete.\r",
"--vcf: 11k variants complete.\r",
"--vcf: 12k variants complete.\r",
"--vcf: 13k variants complete.\r",
"--vcf: 14k variants complete.\r",
"--vcf: 15k variants complete.\r",
"--vcf: 16k variants complete.\r",
"--vcf: 17k variants complete.\r",
"--vcf: 18k variants complete.\r",
"--vcf: 19k variants complete.\r",
"--vcf: 20k variants complete.\r",
"--vcf: 21k variants complete.\r",
"--vcf: 22k variants complete.\r",
"--vcf: 23k variants complete.\r",
"--vcf: 24k variants complete.\r",
"--vcf: 25k variants complete.\r",
"--vcf: 26k variants complete.\r",
"--vcf: 27k variants complete.\r",
"--vcf: 28k variants complete.\r",
"--vcf: 29k variants complete.\r",
"--vcf: 30k variants complete.\r",
"--vcf: 31k variants complete.\r",
"--vcf: 32k variants complete.\r",
"--vcf: 33k variants complete.\r",
"--vcf: 34k variants complete.\r",
"--vcf: 35k variants complete.\r",
"--vcf: 36k variants complete.\r",
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"--vcf: 38k variants complete.\r",
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"--vcf: 202k variants complete.\r",
"--vcf: 203k variants complete.\r",
"--vcf: 204k variants complete.\r",
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"--vcf: 1100k variants complete.\r",
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"--vcf: 1102k variants complete.\r",
"--vcf: 1103k variants complete.\r",
"--vcf: 1kgp_chr22.vcf-temporary.bed + 1kgp_chr22.vcf-temporary.bim +\n",
"1kgp_chr22.vcf-temporary.fam written.\n",
"1103547 variants loaded from .bim file.\n",
"2504 people (0 males, 0 females, 2504 ambiguous) loaded from .fam.\n",
"Ambiguous sex IDs written to 1kgp_chr22.vcf.nosex .\n",
"Using 1 thread.\n",
"Before main variant filters, 2504 founders and 0 nonfounders present.\n",
"Calculating allele frequencies... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\b\b done.\n",
"Total genotyping rate is 0.99984.\n",
"1103547 variants and 2504 people pass filters and QC.\n",
"Note: No phenotypes present.\n",
"--make-bed to 1kgp_chr22.vcf.bed + 1kgp_chr22.vcf.bim + 1kgp_chr22.vcf.fam ...\n",
"0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\bdone.\n",
"--recode ped to 1kgp_chr22.vcf.ped + 1kgp_chr22.vcf.map ... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\n"
]
},
{
"name": "stderr",
"output_type": "stream",
"text": [
"Error: File write failure.\n"
]
}
],
"source": [
"%%bash\n",
"./plink --vcf 1kgp_chr22.vcf \\\n",
" --make-bed --recode \\\n",
" --out 1kgp_chr22"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"Παρατηρούμε ότι το αρχείο μας έχει 1103547 γονότυπους από 2504 ανθρώπους. (Αγνοήστε το λάθος)\n",
"\n",
"Ας αφαιρέσουμε τους γονότυπους που έχουν Allele Frequency μικρότερο από 5%:"
]
},
{
"cell_type": "code",
"execution_count": 11,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"PLINK v1.90b5.4 64-bit (10 Apr 2018) www.cog-genomics.org/plink/1.9/\n",
"(C) 2005-2018 Shaun Purcell, Christopher Chang GNU General Public License v3\n",
"Logging to 1kgp_chr22_maf.log.\n",
"Options in effect:\n",
" --bfile 1kgp_chr22\n",
" --maf 0.05\n",
" --make-bed\n",
" --out 1kgp_chr22_maf\n",
" --recode\n",
"\n",
"1998 MB RAM detected; reserving 999 MB for main workspace.\n",
"1103547 variants loaded from .bim file.\n",
"2504 people (0 males, 0 females, 2504 ambiguous) loaded from .fam.\n",
"Ambiguous sex IDs written to 1kgp_chr22_maf.nosex .\n",
"Using 1 thread.\n",
"Before main variant filters, 2504 founders and 0 nonfounders present.\n",
"Calculating allele frequencies... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\b\b done.\n",
"Total genotyping rate is 0.99984.\n",
"991032 variants removed due to minor allele threshold(s)\n",
"(--maf/--max-maf/--mac/--max-mac).\n",
"112515 variants and 2504 people pass filters and QC.\n",
"Note: No phenotypes present.\n",
"--make-bed to 1kgp_chr22_maf.bed + 1kgp_chr22_maf.bim + 1kgp_chr22_maf.fam ...\n",
"0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\bdone.\n",
"--recode ped to 1kgp_chr22_maf.ped + 1kgp_chr22_maf.map ... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\bdone.\n"
]
}
],
"source": [
"%%bash\n",
"\n",
"./plink --bfile 1kgp_chr22 \\\n",
" --maf 0.05 \\\n",
" --make-bed --recode \\\n",
" --out 1kgp_chr22_maf"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"Παρατηρούμε ότι από τους 1103547 γονότυπους φιλτράραμε τα 991032 και κρατήσαμε τα 112515. Ας φιτράρουμε και τους γονότυπους που έχουν Hardy Weinberg μικρότερο από 0.0001:"
]
},
{
"cell_type": "code",
"execution_count": 13,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"PLINK v1.90b5.4 64-bit (10 Apr 2018) www.cog-genomics.org/plink/1.9/\n",
"(C) 2005-2018 Shaun Purcell, Christopher Chang GNU General Public License v3\n",
"Logging to 1kgp_chr22_maf_hwe.log.\n",
"Options in effect:\n",
" --bfile 1kgp_chr22_maf\n",
" --hwe 0.0001\n",
" --make-bed\n",
" --out 1kgp_chr22_maf_hwe\n",
" --recode\n",
"\n",
"1998 MB RAM detected; reserving 999 MB for main workspace.\n",
"112515 variants loaded from .bim file.\n",
"2504 people (0 males, 0 females, 2504 ambiguous) loaded from .fam.\n",
"Ambiguous sex IDs written to 1kgp_chr22_maf_hwe.nosex .\n",
"Using 1 thread.\n",
"Before main variant filters, 2504 founders and 0 nonfounders present.\n",
"Calculating allele frequencies... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\b\b done.\n",
"Total genotyping rate is 0.99861.\n",
"--hwe: 54981 variants removed due to Hardy-Weinberg exact test.\n",
"57534 variants and 2504 people pass filters and QC.\n",
"Note: No phenotypes present.\n",
"--make-bed to 1kgp_chr22_maf_hwe.bed + 1kgp_chr22_maf_hwe.bim +\n",
"1kgp_chr22_maf_hwe.fam ... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\bdone.\n",
"--recode ped to 1kgp_chr22_maf_hwe.ped + 1kgp_chr22_maf_hwe.map ... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\bdone.\n"
]
},
{
"name": "stderr",
"output_type": "stream",
"text": [
"Warning: --hwe observation counts vary by more than 10%. Consider using\n",
"--geno, and/or applying different p-value thresholds to distinct subsets of\n",
"your data.\n"
]
}
],
"source": [
"%%bash\n",
"./plink --bfile 1kgp_chr22_maf \\\n",
" --hwe 0.0001 \\\n",
" --make-bed --recode \\\n",
" --out 1kgp_chr22_maf_hwe"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"Παρατηρούμε οτι από τους 112515 γονότυπους έμειναν 57534. Αφαίρεσε πολλά επειδή τα δεδομένα μας αποτελούντε από πολλούς πληθυσμούς. \n",
"\n",
"Ας δημιουργήσουμε ένα dataset που θα έχει μόνο τους Άγγλους:"
]
},
{
"cell_type": "code",
"execution_count": 20,
"metadata": {},
"outputs": [],
"source": [
"# Για αρχή φτιάχνουμε ένα αρχείο το οποίο θα έχει τα sample IDs των Άγγλων:\n",
"GBR = samples[samples['pop'] == 'GBR']['sample'].tolist()\n",
"with open('GBR.txt', 'w') as f:\n",
" f.write('\\n'.join(['{} {}'.format(x,x) for x in GBR]) + '\\n')"
]
},
{
"cell_type": "code",
"execution_count": 22,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"PLINK v1.90b5.4 64-bit (10 Apr 2018) www.cog-genomics.org/plink/1.9/\n",
"(C) 2005-2018 Shaun Purcell, Christopher Chang GNU General Public License v3\n",
"Logging to 1kgp_chr22_maf_GBR.log.\n",
"Options in effect:\n",
" --bfile 1kgp_chr22_maf\n",
" --keep GBR.txt\n",
" --make-bed\n",
" --out 1kgp_chr22_maf_GBR\n",
" --recode\n",
"\n",
"1998 MB RAM detected; reserving 999 MB for main workspace.\n",
"112515 variants loaded from .bim file.\n",
"2504 people (0 males, 0 females, 2504 ambiguous) loaded from .fam.\n",
"Ambiguous sex IDs written to 1kgp_chr22_maf_GBR.nosex .\n",
"--keep: 91 people remaining.\n",
"Using 1 thread.\n",
"Before main variant filters, 91 founders and 0 nonfounders present.\n",
"Calculating allele frequencies... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\b\b done.\n",
"Total genotyping rate in remaining samples is 0.99895.\n",
"112515 variants and 91 people pass filters and QC.\n",
"Note: No phenotypes present.\n",
"--make-bed to 1kgp_chr22_maf_GBR.bed + 1kgp_chr22_maf_GBR.bim +\n",
"1kgp_chr22_maf_GBR.fam ... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\bdone.\n",
"--recode ped to 1kgp_chr22_maf_GBR.ped + 1kgp_chr22_maf_GBR.map ... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\bdone.\n"
]
}
],
"source": [
"%%bash\n",
"# Κρατάμε μόνο τους Αγγλους\n",
"./plink --bfile 1kgp_chr22_maf \\\n",
" --keep GBR.txt \\\n",
" --make-bed --recode \\\n",
" --out 1kgp_chr22_maf_GBR"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"Ας αφαιρέσουμε τους γονότυπους οι οποίοι συνδέονται με άλλους γονότυπους μέσω Linkage Disequilibrium. Για να το κάνει αυτο παίρνει ένα \"παράθυρο\" από 50 γονότυπους. Στη συνέχεια αφαιρεί όλους τους γονότυπους μέσα σε αυτό το παράθυρο των οποίων το $r^2$ του Linkage Disequilibrium (όπως το υπολογίσαμε στη προηγούμενη διάλεξη) μεταξύ τους (pairwise) είναι μεγαλύτερο από 0.2. Όταν τελειώσει μετακινεί το \"παράθυρο\" 5 θέσεις πιο κάτω:"
]
},
{
"cell_type": "code",
"execution_count": 23,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"PLINK v1.90b5.4 64-bit (10 Apr 2018) www.cog-genomics.org/plink/1.9/\n",
"(C) 2005-2018 Shaun Purcell, Christopher Chang GNU General Public License v3\n",
"Logging to 1kgp_chr22_maf_GBR_LD.log.\n",
"Options in effect:\n",
" --bfile 1kgp_chr22_maf_GBR\n",
" --indep-pairwise 50 5 0.5\n",
" --out 1kgp_chr22_maf_GBR_LD\n",
"\n",
"1998 MB RAM detected; reserving 999 MB for main workspace.\n",
"112515 variants loaded from .bim file.\n",
"91 people (0 males, 0 females, 91 ambiguous) loaded from .fam.\n",
"Ambiguous sex IDs written to 1kgp_chr22_maf_GBR_LD.nosex .\n",
"Using 1 thread.\n",
"Before main variant filters, 91 founders and 0 nonfounders present.\n",
"Calculating allele frequencies... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\b\b done.\n",
"Total genotyping rate is 0.99895.\n",
"112515 variants and 91 people pass filters and QC.\n",
"Note: No phenotypes present.\n",
"\r",
"0%\r",
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"Pruned 94335 variants from chromosome 22, leaving 18180.\n",
"Pruning complete. 94335 of 112515 variants removed.\n",
"Writing...\r",
"Marker lists written to 1kgp_chr22_maf_GBR_LD.prune.in and\n",
"1kgp_chr22_maf_GBR_LD.prune.out .\n"
]
}
],
"source": [
"%%bash\n",
"./plink --bfile 1kgp_chr22_maf_GBR \\\n",
" --indep-pairwise 50 5 0.5 \\\n",
" --out 1kgp_chr22_maf_GBR_LD"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"Παρατηρούμε ότι από τους 112515 γονότυπους αφαίρεσε ως εξαρτημένους μέσω LD τους 94335. Μπορούμε να τους αφαιρέσουμε ως εξής:"
]
},
{
"cell_type": "code",
"execution_count": 24,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"PLINK v1.90b5.4 64-bit (10 Apr 2018) www.cog-genomics.org/plink/1.9/\n",
"(C) 2005-2018 Shaun Purcell, Christopher Chang GNU General Public License v3\n",
"Logging to 1kgp_chr22_maf_GBR_LD.log.\n",
"Options in effect:\n",
" --bfile 1kgp_chr22_maf_GBR\n",
" --extract 1kgp_chr22_maf_GBR_LD.prune.in\n",
" --make-bed\n",
" --out 1kgp_chr22_maf_GBR_LD\n",
" --recode\n",
"\n",
"1998 MB RAM detected; reserving 999 MB for main workspace.\n",
"112515 variants loaded from .bim file.\n",
"91 people (0 males, 0 females, 91 ambiguous) loaded from .fam.\n",
"Ambiguous sex IDs written to 1kgp_chr22_maf_GBR_LD.nosex .\n",
"--extract: 18187 variants remaining.\n",
"Using 1 thread.\n",
"Before main variant filters, 91 founders and 0 nonfounders present.\n",
"Calculating allele frequencies... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\b\b done.\n",
"Total genotyping rate is 0.997813.\n",
"18187 variants and 91 people pass filters and QC.\n",
"Note: No phenotypes present.\n",
"--make-bed to 1kgp_chr22_maf_GBR_LD.bed + 1kgp_chr22_maf_GBR_LD.bim +\n",
"1kgp_chr22_maf_GBR_LD.fam ... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\bdone.\n",
"--recode ped to 1kgp_chr22_maf_GBR_LD.ped + 1kgp_chr22_maf_GBR_LD.map ... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\bdone.\n"
]
},
{
"name": "stderr",
"output_type": "stream",
"text": [
"Warning: At least 5 duplicate IDs in --extract file.\n"
]
}
],
"source": [
"%%bash\n",
"./plink --bfile 1kgp_chr22_maf_GBR \\\n",
" --extract 1kgp_chr22_maf_GBR_LD.prune.in \\\n",
" --recode --make-bed \\\n",
" --out 1kgp_chr22_maf_GBR_LD"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"Άραγε υπάρχουν συγγενείς ανάμεσα στους Άγγλους; Με τη παράμετρο --genome μπορεί να ελέγξει όλα τα δυνατά ζευγάρια για τυχών γενετικές συγγένειες:"
]
},
{
"cell_type": "code",
"execution_count": 26,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"PLINK v1.90b5.4 64-bit (10 Apr 2018) www.cog-genomics.org/plink/1.9/\n",
"(C) 2005-2018 Shaun Purcell, Christopher Chang GNU General Public License v3\n",
"Logging to 1kgp_chr22_maf_GBR_LD_RELATED.log.\n",
"Options in effect:\n",
" --bfile 1kgp_chr22_maf_GBR_LD\n",
" --genome full\n",
" --out 1kgp_chr22_maf_GBR_LD_RELATED\n",
"\n",
"1998 MB RAM detected; reserving 999 MB for main workspace.\n",
"18187 variants loaded from .bim file.\n",
"91 people (0 males, 0 females, 91 ambiguous) loaded from .fam.\n",
"Ambiguous sex IDs written to 1kgp_chr22_maf_GBR_LD_RELATED.nosex .\n",
"Using 1 thread.\n",
"Before main variant filters, 91 founders and 0 nonfounders present.\n",
"Calculating allele frequencies... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\b\b done.\n",
"Total genotyping rate is 0.997813.\n",
"18187 variants and 91 people pass filters and QC.\n",
"Note: No phenotypes present.\n",
"\r",
"1152 markers complete.\r",
"2304 markers complete.\r",
"3456 markers complete.\r",
"4608 markers complete.\r",
"5760 markers complete.\r",
"6912 markers complete.\r",
"8064 markers complete.\r",
"9216 markers complete.\r",
"10368 markers complete.\r",
"11520 markers complete.\r",
"12672 markers complete.\r",
"13824 markers complete.\r",
"14976 markers complete.\r",
"16128 markers complete.\r",
"17280 markers complete.\r",
"18187 markers complete.\r",
"IBD calculations complete. \n",
"\r",
"Writing... 20%\r",
"Writing... 42%\r",
"Writing... 63%\r",
"Writing... 86%\r",
"Writing... 99%\r",
"Finished writing 1kgp_chr22_maf_GBR_LD_RELATED.genome .\n"
]
}
],
"source": [
"%%bash\n",
"./plink --bfile 1kgp_chr22_maf_GBR_LD \\\n",
" --genome full \\\n",
" --out 1kgp_chr22_maf_GBR_LD_RELATED"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"Το αποτέλεσμα είναι ένα αρχείο το οποίο στις γραμμές περιέχει όλα τα δυνατα ζευγάρια. Η πιο σημαντική στήλη είναι η PI_HAT η οποία περιέχει το [coefficient of relationship](https://en.wikipedia.org/wiki/Coefficient_of_relationship). Οι τιμές αυτής της στήλης παίρνουν τιμές από το 1 (μονοζυγωτικοί δίδυμοι) μέχρι το 0 (καμία συγγένεια). Μπορούμε να βγάλουμε συμπέρασμα για τη συγγένεια σύμφωνα με [τον πίνακα της σελίδας](https://en.wikipedia.org/wiki/Coefficient_of_relationship#Human_relationships)\n",
"\n",
"Για παράδειγμα ας δείξουμε όλα τα ζευγάρια με PI_HAT > 0.2:"
]
},
{
"cell_type": "code",
"execution_count": 33,
"metadata": {},
"outputs": [
{
"data": {
"text/html": [
"<div>\n",
"<style>\n",
" .dataframe thead tr:only-child th {\n",
" text-align: right;\n",
" }\n",
"\n",
" .dataframe thead th {\n",
" text-align: left;\n",
" }\n",
"\n",
" .dataframe tbody tr th {\n",
" vertical-align: top;\n",
" }\n",
"</style>\n",
"<table border=\"1\" class=\"dataframe\">\n",
" <thead>\n",
" <tr style=\"text-align: right;\">\n",
" <th></th>\n",
" <th>IID1</th>\n",
" <th>IID2</th>\n",
" <th>PI_HAT</th>\n",
" </tr>\n",
" </thead>\n",
" <tbody>\n",
" <tr>\n",
" <th>1179</th>\n",
" <td>HG00112</td>\n",
" <td>HG00123</td>\n",
" <td>0.3561</td>\n",
" </tr>\n",
" <tr>\n",
" <th>1470</th>\n",
" <td>HG00116</td>\n",
" <td>HG00120</td>\n",
" <td>0.3977</td>\n",
" </tr>\n",
" <tr>\n",
" <th>2158</th>\n",
" <td>HG00127</td>\n",
" <td>HG00148</td>\n",
" <td>0.2381</td>\n",
" </tr>\n",
" <tr>\n",
" <th>3690</th>\n",
" <td>HG00238</td>\n",
" <td>HG00240</td>\n",
" <td>0.3133</td>\n",
" </tr>\n",
" </tbody>\n",
"</table>\n",
"</div>"
],
"text/plain": [
" IID1 IID2 PI_HAT\n",
"1179 HG00112 HG00123 0.3561\n",
"1470 HG00116 HG00120 0.3977\n",
"2158 HG00127 HG00148 0.2381\n",
"3690 HG00238 HG00240 0.3133"
]
},
"execution_count": 33,
"metadata": {},
"output_type": "execute_result"
}
],
"source": [
"GBR_Related = pd.read_csv('1kgp_chr22_maf_GBR_LD_RELATED.genome', sep=r'\\s+')\n",
"GBR_Related[GBR_Related['PI_HAT'] > 0.2][['IID1','IID2', 'PI_HAT']]"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"Εδώ φίανεται για παρλαδειγμα ότι το δείγμα HG00127 και το δείγμα HG00148 φαίνεται να είναι ξαδέλφια! **ΠΡΟΣΟΧΗ** Αυτή η ανάλυση δεν ευσταθεί για νομικούς λόγους (μεγάλη πιθανότητα λάθους). Μία πολύ καλή παρουσίαση για την ανάλυση που γίνεται για την επαλήθευση συγγένειας η οποία χρησιμοποιείται ως νομικό τεκμήριο βρίσκεται εδώ: https://www.promega.com/-/media/files/resources/conference-proceedings/ishi-15/parentage-and-mixture-statistics-workshop/parentagestatisticsinnon-typicalcases.pdf?la=en"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"Τέλος, μπορούμε να κάνουμε ένα PCA plot το οποία δείχνει τις γενετικές αποστάσεις μεταξύ πληθυσμών. Για αρχή ας πάρουμε τα LD ανεξάρτητα που βρίκαμε για τους Άγγλους και να τα εφαρμόσουμε για όλους τους πληθυσμούς:"
]
},
{
"cell_type": "code",
"execution_count": 35,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"PLINK v1.90b5.4 64-bit (10 Apr 2018) www.cog-genomics.org/plink/1.9/\n",
"(C) 2005-2018 Shaun Purcell, Christopher Chang GNU General Public License v3\n",
"Logging to 1kgp_chr22_maf_LD.log.\n",
"Options in effect:\n",
" --bfile 1kgp_chr22_maf\n",
" --extract 1kgp_chr22_maf_GBR_LD.prune.in\n",
" --make-bed\n",
" --out 1kgp_chr22_maf_LD\n",
" --recode\n",
"\n",
"1998 MB RAM detected; reserving 999 MB for main workspace.\n",
"112515 variants loaded from .bim file.\n",
"2504 people (0 males, 0 females, 2504 ambiguous) loaded from .fam.\n",
"Ambiguous sex IDs written to 1kgp_chr22_maf_LD.nosex .\n",
"--extract: 18187 variants remaining.\n",
"Using 1 thread.\n",
"Before main variant filters, 2504 founders and 0 nonfounders present.\n",
"Calculating allele frequencies... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\b\b done.\n",
"Total genotyping rate is 0.997566.\n",
"18187 variants and 2504 people pass filters and QC.\n",
"Note: No phenotypes present.\n",
"--make-bed to 1kgp_chr22_maf_LD.bed + 1kgp_chr22_maf_LD.bim +\n",
"1kgp_chr22_maf_LD.fam ... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\bdone.\n",
"--recode ped to 1kgp_chr22_maf_LD.ped + 1kgp_chr22_maf_LD.map ... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\bdone.\n"
]
},
{
"name": "stderr",
"output_type": "stream",
"text": [
"Warning: At least 5 duplicate IDs in --extract file.\n"
]
}
],
"source": [
"%%bash\n",
"./plink --bfile 1kgp_chr22_maf \\\n",
" --extract 1kgp_chr22_maf_GBR_LD.prune.in \\\n",
" --recode --make-bed \\\n",
" --out 1kgp_chr22_maf_LD"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"Τώρα μπορούμε να τρέξουμε το PCA ως εξής (κάνουμε reduce σε 2 διαστάσεις):"
]
},
{
"cell_type": "code",
"execution_count": 36,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"PLINK v1.90b5.4 64-bit (10 Apr 2018) www.cog-genomics.org/plink/1.9/\n",
"(C) 2005-2018 Shaun Purcell, Christopher Chang GNU General Public License v3\n",
"Logging to 1kgp_chr22_maf_LD_PCA.log.\n",
"Options in effect:\n",
" --bfile 1kgp_chr22_maf_LD\n",
" --out 1kgp_chr22_maf_LD_PCA\n",
" --pca 2\n",
"\n",
"1998 MB RAM detected; reserving 999 MB for main workspace.\n",
"18187 variants loaded from .bim file.\n",
"2504 people (0 males, 0 females, 2504 ambiguous) loaded from .fam.\n",
"Ambiguous sex IDs written to 1kgp_chr22_maf_LD_PCA.nosex .\n",
"Using 1 thread.\n",
"Before main variant filters, 2504 founders and 0 nonfounders present.\n",
"Calculating allele frequencies... 0%\b\b1%\b\b2%\b\b3%\b\b4%\b\b5%\b\b6%\b\b7%\b\b8%\b\b9%\b\b10%\b\b\b11%\b\b\b12%\b\b\b13%\b\b\b14%\b\b\b15%\b\b\b16%\b\b\b17%\b\b\b18%\b\b\b19%\b\b\b20%\b\b\b21%\b\b\b22%\b\b\b23%\b\b\b24%\b\b\b25%\b\b\b26%\b\b\b27%\b\b\b28%\b\b\b29%\b\b\b30%\b\b\b31%\b\b\b32%\b\b\b33%\b\b\b34%\b\b\b35%\b\b\b36%\b\b\b37%\b\b\b38%\b\b\b39%\b\b\b40%\b\b\b41%\b\b\b42%\b\b\b43%\b\b\b44%\b\b\b45%\b\b\b46%\b\b\b47%\b\b\b48%\b\b\b49%\b\b\b50%\b\b\b51%\b\b\b52%\b\b\b53%\b\b\b54%\b\b\b55%\b\b\b56%\b\b\b57%\b\b\b58%\b\b\b59%\b\b\b60%\b\b\b61%\b\b\b62%\b\b\b63%\b\b\b64%\b\b\b65%\b\b\b66%\b\b\b67%\b\b\b68%\b\b\b69%\b\b\b70%\b\b\b71%\b\b\b72%\b\b\b73%\b\b\b74%\b\b\b75%\b\b\b76%\b\b\b77%\b\b\b78%\b\b\b79%\b\b\b80%\b\b\b81%\b\b\b82%\b\b\b83%\b\b\b84%\b\b\b85%\b\b\b86%\b\b\b87%\b\b\b88%\b\b\b89%\b\b\b90%\b\b\b91%\b\b\b92%\b\b\b93%\b\b\b94%\b\b\b95%\b\b\b96%\b\b\b97%\b\b\b98%\b\b\b99%\b\b\b\b done.\n",
"Total genotyping rate is 0.997566.\n",
"18187 variants and 2504 people pass filters and QC.\n",
"Note: No phenotypes present.\n",
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"[extracting eigenvalues and eigenvectors]\r",
"--pca: Results saved to 1kgp_chr22_maf_LD_PCA.eigenval and\n",
"1kgp_chr22_maf_LD_PCA.eigenvec .\n"
]
}
],
"source": [
"%%bash\n",
"./plink --bfile 1kgp_chr22_maf_LD \\\n",
" --pca 2 \\\n",
" --out 1kgp_chr22_maf_LD_PCA"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"Μπορούμε να κάνουμε plot τα αποτελέσματα του PCA με τον παρακάτω κώδικα:"
]
},
{
"cell_type": "code",
"execution_count": 64,
"metadata": {
"scrolled": false
},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"Plotting: EUR\n",
"Plotting: EAS\n",
"Plotting: AMR\n",
"Plotting: SAS\n",
"Plotting: AFR\n"
]
},
{
"data": {
"image/png": 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/aXgzov2FiIiIDA2Z2H05ETgY9rgRuCLRNT1VsU+BcUCrMeYK4ClgCvCVRFUy\nY8wqYBVARUVFBoadGd7h33fNX+VPJz6z65/ZdWxXv9977ri5bGjaQHfjf2KMwbVuRPsLVctERESG\njkxUykyc56IrXgmvsda+Z62dC1wG3G+MKY73Idba1dbaamtt9fjx4/s14Ex5s+HX3P/2d/iX3T/n\nyR2r/fVdt8+9o9/vXegUMnlUhd+537UujnEwGIwxjC4alYHvQERERHJFJkJZIzA57PEkoCnRNcaY\nAmAM0BZ+gbW2FjgJXJyBMWVdXVstj2//KUEbxGLpcrsimrc6pu+/tQbDtRV/zLKKayOmRb8444s4\nxsG1Lk/uWK0pTBERkSEkE9OXm4CZxpipwCFgJXBr1DWvArcDG4GbgfXWWttzz8GeKc0pwCygIQNj\nyqq6tlqeq3suYpelYxxGF43ihfpfcPTUEXq7q9XbHOC1ufAW84efY7mzdSfWWk1hioiIDEH9DmU9\ngepe4E1CLTGestbuMsb8DbDZWvsq8DPgWWPMXkIVspU9ty8F7jPGdAEucLe1trW/Y8qmurZavvvO\nA3S5Xf5zARPgizO+yJM7VtPtdhNwAjjGidsaI5EFFyxi+pip7Di6k5LiEv/56BMFCpwCut1uCpwC\nPwSqLYaIiEj+y8gxS9bafwf+Peq5vw77dQfw5Tj3PQs8m4kxDJSdrTvpdrv9qtaCCxZx6+xb/edd\nXKxrGT9iPEdOHUn7fY21/PKDX/qtM7a0bObhqx6NCVvLKq4FYPrY6X4ILHAK1NFfREQkz+nsy17y\n2l94YejK8iXsbN3Jqa52/xqL5eipo71635qjNRGPgzYYMT3pVei8zwX8EKipTBERkfynUNZL4eu8\nRheN4skdq+lyu+I2i3VwcHH79DkGE3HAeHglrtsNdQ0JD4c6jFxERCS/KZT1gbfO64X6X/hTmdG8\ndWbvHf4vDrU3Ju3oH+8EgPKREyMqX9EVumUV17Ks4lp/E4CqZCIiIvlNoayXvGax80rn+UEpulJm\nMMwvnc/Le1/GtakrZfEC28SREyMeR+/E9EKYwpiIiMjQkJEDyQfaYB1IHr2u6/tLHwbwpzI3N29m\nU/N/9XnK0uPg8Oh/+9+9DlzhgVFhTUREJDcM2IHk55LodV07W3fy5apb/NeAfgcyiGw8m27QihcY\nFcxERETyh0JZL0Sv65pXOi8iDMU9TCqFqaOnUhAo5IOP6/3num036w+sA0g7aMULjAplIiIi+UOh\nrBfirevyFvu7uBhrIhbtO8ah/LyJNLYfTPieHx7/EAcnZrH/7z76LZB+24t4gVFERETyh0JZL0V3\n2I8OQ3fNX8W+T/YBoUavHx1BXLyrAAAgAElEQVRv4Cfbfpz0PeNNeXbbbnYdez90ADkGxzhJg1ai\njQAiIiKSHxTK+imdMDR33Fx2H9vtV8ICBLDYlOvPDp44W2EzJvXcaHRgFBERkfyhUNYH0YvvE4Wh\n8PVmAROgekI1Y4edz/Sx09nSspk/HP5D2p8ZdINaJyYiIjKEKZT1Um92OYYvvsfCzPNnMa90XsyB\n5vGax4YzGK0TExERGeIUynqpN7sc4y2+X39gHZ1uZ8y10UcyXVQyl4rRFUwfO53jnSe0TkxERGSI\nUyjrpXR2OYZPb4avNwP4bcNvYq4vdApZdMElvNd8djqzqGAYyyquVRATERE5RyiU9VKqhf3xpje9\nBrMv1P8iZnH/heeV86WZXwKICGXbjmxld+v7agIrIiJyjnBSXyLRZpfM4ctVt8QNS/GmNz3zSucR\nMIGI65tPHubJHav9NhrhutyuiPtFRERk6FIoyzBvetPBiZnenF0yh4evepTrKq9n5tgqf4F/t9vN\nwRMHYt4rVW8yERERGTp0IHkWpHNeZUS7DCdA0A1GTG0aDHcvvIfPV14/UMMWERGRLEj3QHKFskHk\nhbejp47wZsMbflsMg+FLM7/EiMKR2nUpIiKS59INZVroP4i8prN1bbWsP7COLrcLxzh8ccYXeW3f\na2n1QhMREZGhQaEsB0Tv6OxNLzQREREZGhTKckR41ezoqSM4xgFLxGaBdNaqiYiISH5SKMsh0Yv/\nPzfl834D2d4c7yQiIiL5Ry0xckj4tGXQDTJ+xAV+8ErW/0xERETynyplgyx8SnJe6TwCTgDrWgJO\nIKJHWTrHO4mIiEj+UigbBF4QG100iid3rPaD1l3zV+G1KLHW8tHxhog1ZMmOdxIREZH8plA2wMLX\nhhljcK3rd/V/t2mD/zhogzy+/adYayPWkCmMiYiIDE1aUzbAwteGudbFMY5/JNOV5Uv8I5oc4+Ba\nV2vIREREzhGqlA2w6LVhd81fxfHOE/6U5JTRlXGnNtNdQ6a2GSIiIvlJxywNgnSDU28DltpmiIiI\n5B4ds5TD0lkb1peKl04CEBERyV8KZTmorxUvtc0QERHJXwplOaivFS+1zRAREclfCmWDKNEUZX8q\nXmqbISIikp8UygZIdABLNkWpipeIiMi5R6FsAMQLYKmmKFXxEhERObeoeewAiBfAvClKr3GsFuWL\niIic21QpGwDx1ohpilJERETCqXnsAFGnfRERkXOTmsfmGK0RExERkWS0pkxEREQkByiUiYiIiOQA\nhTIRERGRHKBQJiIiIpIDFMpEREREcoBCmYiIiEgOUCgTERERyQEKZSIiIiI5QKFMREREJAcolImI\niIjkAIUyERERkRygUCYiIiKSAxTKRERERHKAQpmIiIhIDlAoExEREckBCmUiIiIiOUChTERERCQH\nKJTlsoMb4e1HQl9FRERkSMtIKDPGXGeM2WOM2WuMuS/O68OMMc/3vP6eMaay5/nPGmO2GGN29nxd\nlonxDAkHN8Iz18L6B0NfFcxERESGtH6HMmNMAPgJcD1wEfDfjTEXRV12J/CxtXYG8Bjwdz3PtwLL\nrbXzgNuBZ/s7niGj4S0IdoINhr42vDXYIxIREZEsykSl7HJgr7V2v7W2E1gL3Bh1zY3AMz2/fhG4\n1hhjrLU11tqmnud3AcXGmGEZGFP+q7wGAkVgAqGvldcM9ohEREQkiwoy8B4TgYNhjxuBKxJdY63t\nNsZ8CowjVCnz3ATUWGvPZGBM+W/yYrh9XahCVnlN6LGIiIgMWZkIZSbOc7Y31xhj5hKa0vxcwg8x\nZhWwCqCioqL3o8xHkxcrjImIiJwjMjF92QhMDns8CWhKdI0xpgAYA7T1PJ4EvAzcZq3dl+hDrLWr\nrbXV1trq8ePHZ2DYIiIiIrkjE6FsEzDTGDPVGFMErARejbrmVUIL+QFuBtZba60xZizwb8D91tp3\nMzAWERERkbzU71Bmre0G7gXeBGqBX1hrdxlj/sYY84Wey34GjDPG7AX+AvDaZtwLzAAeNMZs6/nn\ngv6OSURERCTfGGujl3/lvurqart58+bBHoaIiIhISsaYLdba6lTXqaO/iIiISA5QKBMRERHJAQpl\nIiIiIjlAoUxEREQkByiUiYiIiOQAhTIRERGRHKBQJiIiIpIDFMpEREREcoBCmYiIiEgOUCgTERER\nyQEKZSIiIiI5QKFMREREJAcolImIiIjkAIUyERERkRygUCYiIiKSAxTKRERERHKAQpmIiIhIDlAo\nExEREckBCmUiIiIiOUChTERERCQHKJTli4Mb4e1HQl9FRERkyCkY7AFIGg5uhGeuhWAnBIrg9nUw\nefFgj0pEREQySJWyfNDwViiQ2WDoa8Nbgz0iERERyTCFsnxQeU2oQmYCoa+V1wz2iERERCTDNH2Z\nDyYvDk1ZNrwVCmSauhQRERlyFMryxeTFCmMiIiJDmKYvRURERHKAQpmIiIhIDlAoExEREckBCmXZ\noEavIiIi0kta6J9pvW30enCjdlWKiIiIQlnGxWv0mihsqVO/iIiI9ND0Zab1ptGrOvWLiIhID1XK\nMq03jV69AOdVytSpX0RE5JylUJYN6TZ6Vad+ERER6aFQNtjUqV9ERETQmrLsUmsMERERSZMqZdmS\nzs5KtcMQERGRHgpl2ZKqNYbaYYiIiEgYTV9mS6rWGGqHISIiImFUKcuWVDsr1Q5DREREwiiUZVOy\nnZVqhyEiIiJhFMoGk9phiIiISA+tKRMRERHJAQplIiIiIjlAoUxEREQkByiUiYiIiOQAhbJs01FL\nIiIikgbtvswmde0XERGRNKlSlk3q2i8iIiJpUijLplRHLYmIiIj00PRlJhzcGL8zv7r2i4iISJoU\nyvor1boxde0XEclLLS0tNDU1UV5eTllZ2WAPR84BCmX9FW/dmEKYiEhea2lp4fXXX8d1XRzH4YYb\nblAwk6xTKOut6KlKb92YVykLXzcWfi1oGlNEJE80NTXhui7WWlzXpampSaFMsk6hrDcSTVXGWzcW\nfq1TAFhwg+AEYNHXYMFtCmciIjmqvLwcx3H8Sll5eflgD0nOAQplvZFoqjLeurGIa4Nnnw8GYfMT\nsO2ZUJjzrlUFTUQkZ5SVlXHDDTdoTZkMKIWy3kg2VenxpiyHjwtVyMIDmc+G3mP7z0PhTM1lRURy\nTllZmcKYDCiFst5I1eIienpz5vVQ96vIa0wg9DVQFPqqTQIiIiJChkKZMeY64EdAAPgna+2jUa8P\nA34OXAocA1ZYaxuMMeOAF4HLgKettfdmYjxZlazFRfT05sgJZytrAIFhcP3/hdPHzlbZwitlai4r\nIiJyzup3KDPGBICfAJ8FGoFNxphXrbW7wy67E/jYWjvDGLMS+DtgBdABPAhc3PNPfoue3lxwW+if\n7T8PvR5vcX945Q1Ch5drfZmIiMg5JxOVssuBvdba/QDGmLXAjUB4KLsReKjn1y8CPzbGGGvtSeAd\nY8yMDIxj8CWa3kwWsLzKmw4vFxEROadl4uzLicDBsMeNPc/FvcZa2w18CozLwGfnnsmL4ar7Q79+\n+5FQ2Ap3cGPk897j7T/X4eUiIiLnsExUykyc52wfrkn+IcasAlYBVFRU9ObWgZeo6hX9/HV/D298\n82wvMycALlpfJiIi56Rz/WirTISyRmBy2ONJQFOCaxqNMQXAGKCtNx9irV0NrAaorq7uVaAbcIn6\nmUU/X/sSBM+AdcG1cOkqGFOhNWUiInLO8Y62CgaDGGNYunQpc+bMGexhDahMTF9uAmYaY6YaY4qA\nlcCrUde8Ctze8+ubgfXW2twOVv3hLfg3gciqV/TzExaGAhmEvk5YFJr6VCATEZFzTFNTE8Ge3p7W\nWt555x1aWloGeVQDq9+VMmtttzHmXuBNQi0xnrLW7jLG/A2w2Vr7KvAz4FljzF5CFbKV3v3GmAZg\nNFBkjPki8LmonZv5J9mC//DnG94ilIvd0NfTxwZnvCIiIlmS7pRkeXk5xhi8mo21lvr6+j5NY+br\nNKjJx4JVdXW13bx582APo/+041JERIYwb0rSO0P0hhtuSBqSamtrefvtt/3H0dOYXtgqLi6mo6Mj\nInSFv7Zhw4a0P3MgGGO2WGurU12njv6DKdUJASIiInmsqakJ13Wx1uK6Lk1NTUkD0vHjxyMee9OY\nJSUltLW18c477xBeTAoEAixZsoTW1lb27NkTUWUD0vrMXKJQlk3eOZjJAleyEwJERERyRF+mBMvL\ny3Ecx69alZeXJ33/7du3xzxvreX3v/89n3zyScxrwWAworLmMSbU9CHVZ+YahbJsyeOpydb6Glp2\nbaJs7mWUVi0a7OGIiMgg6+00pKesrIwlS5awf/9+pk2bFjPVGB7w6uvrE75PvECWiFc9i57ezAcK\nZdmSqC1Gjmutr2H9336NYHcngYIilj34lIKZiMg5Ltk0ZLIKWktLC++++y6u63L48GFKSkoAeO21\n1/yAt3z5csrKyjh9+nTEvaNHj2bUqFEcOnQoZjwTJkygubk54jnHcZg1axZVVVV5FcTCKZT1Vaqp\nyeHjwBiwTl41g23ZtYlgdye4Lm53Fy27NimUiYic4xJNQ8brLVZSUuKHtPr6elw31PrJdV02bNjA\niBEjIp7zKmQNDQ0Rn3n8+HHOP/98/3M9V111FSUlJX6w81x55ZV539dMoawvUk1NHtwY6tRv3VCX\n/uv+Pi+qZABlcy8jUFCE292FU1BI2dzLBntIIiIyyMrKyrjhhhtiKmLxeot5vzbGcN5550W8z9Gj\nR/31XuESTV1+9NFHEY8dx/GvHzVqFJ9++qn/Wmtrax+/u9yhUJaO6KpYqqlJ/3UXMKH+Y+ks+s8B\npcM7WLb8j2g5NYKyJTepSiYiIkAomEVPC8brLeax1tLe3h7zPl5gs9biOA5VVVVJ15OFc103Zgem\nZ8+ePXk9dQkKZanFq4p5nfm956KnJqNfHz4uPxb993yvpcFOSgNFMPxPBntEIiJ5I18blvZHWVkZ\nS5cu9YNSeEBLxHEcJk+ezIgRIyJC1J49eyKmI6N5FbZE72+tzav2F/EolKUSryp21f3J+4vF69yf\nD4v+82WcIiI5JtnuxHwIa/0Z45w5cygpKWHbtm0cO3YsbnUsnOu6/rTkxx9/zMyZM2ltbWXs2LGc\nPHmSzs7OuMFrzJgxOI5DW1vk0dnx2l/U1tb6Oz7zaZ2ZQlkqiapiqfqLRb+erLKWyEBPeaaqAIqI\nSFyJdif2tZXEQOrNGBOFt127dsWs/0pHc3NzzC7KRBK1xZgyZQrDhw+nqqoKgN/85jf+pgFv52a8\nYJaLYVmhLJVMdN2Pfg+Atx9J/n6D0edMJwyIiKQl+gd6ot2Jve1on42xpZLuGBOFt9raWvbu3Rtz\nfTpTmZnw0Ucf4TgOpaWlbNiwwd944Nm/f39MKMvVsKxQlo5MdN333iPdsDVYU4k6YUBEJKlEP9Dj\n7U7sTUf7bI4tmVRj9EJee3u7H3iCwaAf3jZt2hT3fadPnx43rGWatZZgMMj27dtjAhlAaWlpzHOD\nEZbToVA20NINW5pKFBHJSYl+oMfbnZgorA302JJJNsbwkBfdyqK4uJh/+7d/o6OjI+Y9jTExzWCz\nLfrcTE9RUVHMcwMdltOlUJYp6a7/Sjds9XUqMU9ab4iI5Kve/kCPF9YS6e86p76GjURjjO5DFq62\ntjZhbzDHcZg2bRqHDx9OuqNyIHR2dsY8N9BhOV0KZZnQ2/VfC28PfV1wW+82C2R6HCIi0mvZ+oGe\niXVO6Y4t3fBXXFwc8Th8nViiQFZYWMioUaM4ePDgoAcygB07dlBZWRm3ipkrYcyjUJYJ6U5JRoem\nBbcNzjhERKRfsvEDPVPrnMK77Yc/9vQm/EVPTY4ZM4aCgoKk3fO7urpoa2uLaV0xUKZMmcKBAwci\nGtrmypqxVBTKMiGdKcmDG+Gth6D7DOBmJzRVXhM61inYc7yT1qGJZFUubqmX/JWpdU6pQldvwl/4\nmCBxW4pcsWDBAoqKihg7diw7duzAWksgEIj4vczlP7cKZZmQav2XXyHrCWRk85ByE/VVRLIhV7fU\nS/7K1LRoqtDVm/BXVlbGrFmzqK2t7dNYBtKUKVN4//33/e9r6dKldHR0UFxcTFNTE21tbbS2trJn\nzx7/iKdc+3OrUJYpidZ/RVfIjAPT/hiueSjzi/cb3gK3G7Chr1mavmytr6Fl1ybK5l6mszHlnJWr\nW+olv2ViWjRV6Opt+Kuqqkp5BNJgM8YwYsSIiD+THR0dlJeX8/rrr8dtlZGLf24VyrIpXoXMKYDz\npyW+PrzBbMNboXMz3/hmeov3B6CNRmt9Dev/9msEuzsJFBSx7MGnFMzknJSrW+pF0gld8cJfomm9\nXK6WjRw5knHjxlFRUUFra2vMkUveX57iycU/twpl2eQvvO+pkJVXQ/N22PIkbHvmbMA6uBG2/xxq\n/jlU4XIKCFW7gmBM6H6bxjq0AejI37JrE8HuTnBdgl2d7HzhJ8z78j3+a6qeybkiV7fUy7krOlQl\n6sqfTj+yWbNm+ccWNTU1UVpaOmAd+ntjzJgxVFRURHTynzBhAldccQUA7e3tMeN2HMf//nLtz61C\nWTZFV64uvASatkTujoRQNa27A+j5jybopXoL1gkt2sekV/3Kckf+srmXESgoItgVCpvNOzdyZHeo\nm7Prdqt6JueUXNxSL0NTqsXp6axxTHZNfX19RD+y2tpa6urq/DDjOM6AdejvjUOHDtHU1BQRupqb\nm2loaGDnzp1+yJwzZw6lpaX+lGau/rlVKMumeGdebnsmcnrRq6Z5gcwLX16lLFAE1/09nD6WEw1h\nS6sWsezBp9j5wk9o3rkRrIsb7Aq9aC1udxctuzYplImIZEiiMBUe1NJZ45js0PQ9e/bEfG540HFd\nlw8//DDr32tfxKveffDBB/60pfd6vEPJc41CWTbFW6Afb3rRq6Y5AVj0tbP9y7b/PPS1bN6gh7Fw\npVWLmPflezhatwW3uwvjBACwbhCnoJCyuZcN8ghFRIaOeGEKiAhqS5YsSbnGMdmh6elMS8ZbLD9Y\npkyZQmNjI8FgMO606ujRozl16tQgja7vFMqyJVF3/fCDyd9+JBTO4gW1gxvPVtXC159Ff8YgHank\nVcy8dWQQu6ZMuzQlJ+joMclz8cJUdFDr6OhIa42jt04sfD2V9/65FLpSWbhwIRUVFbzzzjsxgWzK\nlCksXLiQ1157zf89877vXKdQli3JuuvHC2xX3Z/+/YneYxCCWXjYCv+1dmlKTsiBPyci/ZVoU0l0\nUEvWyT96CjQ8pHjv/95779Hc3DyA31nfLFiwgLKyMrZt2xYTyIwxVFRUUFZWxvLly/NuI45CWbZE\nL/IfPu5sZSyd45D8+8+Edm4OHxf5eo4fqRS+S1PrzGTQ5PifE5F0RW8qiRfUki3kT2fNWUtLS7/G\neN5553Hq1Kms7tCcOHEiRUVF1NbWcuDAgZjXrbVs2LCBkpKSvNyIo1CWLeGL/KN7jV3396n7iU1e\nHLru3+4Otcn49Z9Hri3LcE+yTE41ttbXcLK1CccpwKJ1ZjKIBqB3n0i2pNpxGR06ki3k91pDQPz+\nXPX19SnDlFeF+uijj+K+PmrUKBzH4cSJExHPV1ZW0tDQkM63nJK32xLiL/CH3GwKmy6Fsmzy1o+9\n/Ujk39ZPH0uvn1hzTegeCN23/ednr81gT7JMTjWGv5fjFDB92c1MvfpGVcm0rmlwDEDvPpFsSGfH\nZXToiF4bVlxcHNN/bPbs2Wn15xo7dixTpkyhqanJP3zccZyk9ySa+kx2eHlfeGEsUd+0XGwKmy6F\nsoEQ76Dw/vQTC/8BH70WrQ8yOdUY/l6WICNKyxXItK5pcGW5d59INqSz4zK6F1lZWRlLlizxF79v\n2LCBqqoq/30g1AE/XiALP0rJcRzmzZvHu+++GxPmtm/f3uvvpb29vY+/C8lZaxk7dmzEIekTJ06k\nuro6L6tkoFA2gPpwUPiERbGP0/0B34vKjNcQ1u3u6vdUY6r3Oid3ZGpdkwwhqabUJDPS2XHpBbXw\nfx8dHR3+e3h9usIrSl71LPrfYfTC+Pr6+pg+X0DEOi5vOnPEiBE0NTXx6aefZvc3JY6CggICgYDf\nGmPatGl5/d+lQtlA6OtB4aePhRb5e8c0nT6W3g/4XlZmSqsWccnt93Hwvd8y+YrP9josRQet8FYZ\n2pGJ1jXJkJFO13jJjHR2XBYXF8f8+4gOc6WlpdTV1QGhcPX222/7IS0QCHDDDTfQ1tbG/v37mTZt\nGosWhf4/Oboidvr06Zh+ZtZaDhw4gOM4XHzxxX2qoqXiOI7/mfGmKr2GsOHVQW+Rfz5SKBsIff2h\nXHkNBIbF3pfqvXpZmWmtr2HrM48S7DrD0d1/YOywTii7OK2KVqKg5d8TVrFr2bXz3NyRqXVNMkSk\ns4NPMiNRNSs8qIUfjRQMBtm8eTPV1dX+NcXFxezfvz8mzHiPg8Eg27Zt8xfuHzp0CAgFneHDh0fc\nM3z4cMrLy/1A53211hIMBuns7GTBggV88MEHGWnaGn5+Zfj3G34oemVlJXPmzKGmpsZ/Lt//u1Qo\nGwh9/aGc6L5U79XLENiyaxPBrjM9xyR1s/9f76OhfRzBYDBlRSvperSoil3Z1U9kbJo072hdkwwB\niTrCS2Ylq0h6Oy7jHY106NAhmpub/YrZ66+/nrQhrDEmJkDV1dUxZ86cmDVmqZqv1tbWZuzAcmMM\n559/vh/GvOodnD2j0xjD5MmTgaH136VC2UDp6w/lePeleq9ehsCyuZcRCDi43d04xmKsS7C7q2e2\nNXlFK+kasqiKXSmNCac2RST3JZpSk8xK9xzLeAEofK2ZtyYMYPz48QQCgYgdkl7AO3r0qP9ca2sr\nLS0tNDQ0MHz4cEaNGsUVV1xBWVkZNTU1SacSMxXIjDHs2bMHa21EKI23kcGbqhwq/10qlA1VvQiB\npVWLWPZn36Hlhb+ibHg7OAV8eHI8btBNWdFKtoYsXsWudPKi7IQxtZwQGRD52JBzIGRyA0Q6lZ/w\na7z+Y16I8a73Klfe2Zj19fURoay5uZmjR48yYcKEiOfDO/ufPHmSXbt2+dOh2TyOacGCBRQVFdHe\n3k5dXV3cUBq9kcF7baj8d6lQNlhyLESUXvUVSitn+GNadro4aUUrenF/3KA1UGupkm1syLHfZxEZ\nejK9ASKdyk/0NRC5CzO6O39bW1vMdCeE1pWdf/75HD161B//8ePHI67Zu3cvxhgcx2Hq1Kns3bs3\n5ffgVbzCq3UAM2bMoLCwkNbW1ogKHcDOnTtZvnw5gL/7MzqUDqWpyngUygZDJvtWZTJ0hFXXSiFh\nRSvpLsro8QzEWqpEGxvUH0wka9Qa46xsbIBIp/IT7+il8DGFTzXW1dXFBCRPaWkppaWl/g7M48eP\nx+yk9Bb079u3L63xjxkzJm6LjH379vGFL3yBqqoqXnnllYjXvN+7RYsWJQylQ2mqMh6FssGQqb5V\ngxQ6Ei7uTzKerPYnS7SxQf3BRLJCrTEi5Ur1JjwoR09vJuuq39ra6i/qb2pqYvbs2ZSUlNDW1hZx\nXbKF/NHXjxkzJqKpq8da6wev6OOXjDH+71104Iz+S8BQ/e9NoWwwZKpvVRZDR7IQlXBxf8NbtJ5w\naDk5hrLzzlDaM56s9ydLNE2q/mAiWaHWGJFyoXoTLyh7Y/LWaMUTCAQ4depURKPY2tpaHMfxe4QZ\nY5g1axalpaVs2LAhZk2Z4zhcddVVEf3OSkpKOHDgQEyICw+tkydPjghl8+fPj/t7dy79JUChbDBk\naq1VlkJHshDlhbVLbr+PMyc+jQhtrUxi/UcTCVpDoNWyjEmU0v9jnNKqsiXapar+YCIZlyuVoVwy\n2NWbpqamiJ5lXjUqvH1GdECaOHEi06ZNixvYrLXMnj2bkSNHRqxT81pjeF+jq1deM1eA2bNnR/QV\nGz9+PEuWLIm7aB+gqKgo4fd2rvwlQKFssGRirVWWQkeiEJWq4tXSepogAcDiEqCl9TSl9O8Yp35X\n2dQfTCTjcqEydK5KtJavuLg44rrox9ECgQDTpk2LW/kC/NMAvOAUXa3yDjVP9u++qqoqYsF+eCCD\nULgPBAIpw/259JcAhbJ8l6HQEV6NShSiUlW8yuZeRqBwWMx9SdtmpJDJw9JFJHMGuzJ0LqqtrfV7\ndHlHJEGoknTkyJGIa8OrUNE9zUaPHs2CBQvo6OiICGTGGObPn09RURHFxcVs2LDBD0KTJ0/2r023\nWpUqvKcb7s+lvwQolOWTvu60THFfvGpUvBCVquKVLHwlbJuRQiYPSxcRyVctLS1+IIPQFGV9fX3E\nUUse71zMmpqaiEX/3nXHjx9nw4YNXHzxxRH3WWspKipi0aJF1NTUREyHJlqQn0qq8J5uuD9X/hKg\nUJYv+rrTMo374lWj5v7pqpgQlSh0pdWzrI/6U2U756gnm8igymabkOhql9cwNl6bi8mTJ0dUubxF\n/5s3b/bPtwwGg+zYsSPivkAg4IetZNOfs2bN8teY1dfXA/jTmdHUOqV3FMryRcNbEDwD1g19TXen\nZRo7NHtTjYoOXelsCuhvmMp00BuS1JNNZFBleodgdJjx1l955z4uXbqUkpKSmEpZIBBgxIgRMQvj\nFy1aRHV1Nc3NzRE7LT0TJ06kurraH/OBAwcixuOFQG89WUtLC6+99pr/Xnv27GH58uX+/V5gi3dc\nkiSmUJYvho8LBTIIfR0+Lr370tihGV6NGjZqDC27NvnPJ+IFrpOtTX3aFBDvvVQJ6wf1ZBMZVJnc\nIZgo4MVbV+U9V1xcTEdHB+Xl5RH9wqIXxnu7Jr32Ft5nhAeylpaWmFBWUVHBBRdc4H92TU1NRJUu\n/Hv2xh8eFof6rslMUSjLVdFTUaePAQ7ghr6ePpb6Hkh7h6YXhqKDFBB3utK7znEKcJwCLMFebQrw\nZL2H2blCPdlEYgzk1AaPjt8AACAASURBVFkmdwiGB7xgMMjmzZv90BRvsXyi6pQxxt/xGB6UvEpb\neKDz1qdVVVXFnSpduHBhzM5J7/uFyPDnjT/cUN81mSkKZbko3lRU5TVQMCzxD91k01dp7tCMDlK7\nX/kZTTW/x7pBAoXD/MAUfp0lSPml1xA808HkKz6b9qaARJ+p3ZV9pJ5sIhEGuuFoJncIRi/MP3To\nEM3NzUm/h+jpRE9rays1NTW0t7f772et5e2332bBggVUVlbGTENeeeWVMVOl8cLg8uXLI9aUAdTU\n1PgHl3unCcyaNSvhmjOJpFA2GFItyI43FXXV/cl/6GZg+io8SBknQNPWt7Buz+6brk4/MMVe93ts\nMMiR3ZsYW1HlrwFb9uBTtGx4ibIRpygd3pHyM7W7sp/Uk03ENxgNRzO1Q9ALeOEL81N9D14/sHDG\nmIiqWfQxSdu3b+eTTz6JmYZsbW1Nu1VFeJUuPAQvWbLEn05VGEufQtlAS2dBdqKpqGQ/dMPvcQrg\n0wOhz+rFD+nwtWWnWpvYu+4F/zXjOHH7jh3bu4NDm9cB4HZ38uHvX/ErXaXDOyht/D+hMdU/Hvd7\n1e5KEcmGfG84WlZWFrEw3/8e0txlPXbsWC688ELq6ur8IFZRUcFHH30Ucd2pU6cipiEhVC2rqqpi\n0aL0//84OgR3dHT06n4JUSgbaOlUtPoyFeXds/3nUPMUbF4NW/8J/p+fQPWqtIfnVbla62v48D9f\nIdjViXEcqr/23bh9x/7ryYci7o84xCPO99p6ujgmgGl3pYhk2lBoOBrzPXTuT/iX+qqqqogAdvz4\ncebNmxcRTBcuXMjYsWPZvn27/xne4ePvvvuuf2i5d2h4b37P8j0E5wqFsoGW7oLsvkxFTV7cE4S6\nARdcF/79Xiib1+v3SreCNe3qG/nwrZdxg104gUKmXX3j2RejvtdWJmlRv4gMmHxtOBq9QcH/Ht5+\nKuFf6svKyiLOmrTW0tHRERNMy8rKGD16tH9wuHdW5ZVXXhkx/djbUDUUQnAuUCgbaNlekF15DThO\nKJBB6A9vH9sjpFPBKq1axLXfezp+eIv6Xls279SifhGRJOIdpeQHnBR/qY8+azIm1PWYM2cOc+bM\noaWlxe/6n4lQla8hOJcolA2GZFWw3nZlj75+8uLQlOW/3xsKZIFhmW+PEPWZXrAK728W0XvsqvsB\nKDtd3O9F/eppJiJDVbyjlCKmEVP8pb6srIwlS5b4VbBkASnR7lSFqsGlUJZL0u3K7oWi4ePgjW+e\nvf66vw/1L6u8Bu74fXaqcWFjbO0YScusexk2aR5b/vlhP2xdescDbH3m0ZhpynSmRJOFLvU0E5F8\nEj0NmapvWrz+YDHTiAn+Uh/do6y5uZmSkpKEIWswdqdKagpluSSdTQDhwc044AYBF7rP9FTH3LOB\nrqdCla60qlA9Y2w9Wcj6A2UEP3wJY37lt85wuzvZt/6lhNOUyaZEU4Uu9TQTkXwRr0VE9HmU0SEo\n3lFK6QSldDroxzu2SQvzc49CWS5JZxPA9p9DdwdgwVowgHVC68hssOdszJ5AB2lXy/xA1HUG4wSo\n/tp3mfHHK/zX/LDWM8aW0+cRtKGz0KwNRrzX8PMvIHDwg15PU6YKXeppJiL5IroStX///pSVqb6u\n60rVQb83xzbJ4FIoyyWpNgEc3Bhqd+E3nnDBmlBfssXfgvf+4WygGz4u7QOqW+tr2PnCTwh2ngEs\nNtjN5qe+z9iKUIfmmOrV7eso2/gC5le/wQYjA5lTUMRFN97JRTfemfIszb2/e56D7/2WyVd8lhl/\nvCJl6FJPMxFJx0Aer5ToM6MrUdOmTYvtORZHX9Z1hX9WvA76iaYqtYYs92QklBljrgN+BASAf7LW\nPhr1+jDg58ClwDFghbW2oee1+4E7gSDwDWvtm5kYU95Ktgmg4a2e6cpwNlQdKx4bGejS7PAfXiEL\n7zJmg0H2//4Vzistj61e/ekqSicv5vxtt9C2b6d/z8gJU1h8zyMRYSnRdOTe3z3Ppp4eZ8073gVg\nxh+v4JLb7/ODWrzQpZ5mIpLMQB+vlOwzoytRJSUlWQmLqapemqrMH/0OZcaYAPAT4LNAI7DJGPOq\ntXZ32GV3Ah9ba2cYY1YCfwesMMZcBKwE5gLlwO+MMVU2ej5MQiK69gcAA2732anO6ECXRj80f8rQ\n2tD7AaFwZvnwrZe59I4HElavyuZeHhHK5iy/I+01YPvWvxQxjn3rX2JsRZW/QeBo3Rb/yCYZYL3d\nASySQ+JVhbzns1U5i/7M+vp6//PCu9pnszKV7L01VZk/MlEpuxzYa63dD2CMWQvcCISHshuBh3p+\n/SLwY2OM6Xl+rbX2DPChMWZvz/ttzMC4hp7o6U1I/MMzwVRo9GL+6CnDCQuu5NCm9YDFukHOnPg0\n7pRha30N9W/8K5hQkCuddak/3emJNx3pfb5TOCzi2uHnX5A0xKkVxgBJdwewSI6KrgoVFxdnvXIW\nPX3o7YAcqEpdOjRVmR8yEcomAgfDHjcCVyS6xlrbbYz5FBjX8/wfou6dGO9DjDGrgFUQOr/rnBVd\nDUv2A9N7rWfRf+vp4sjpxFV/QSmNLFv1F7S0nvarYM3b340IUuFHL+16eTVlcy+LqrBB654trP/b\nr0VMUUavAQNY97++2nOQuRMx1PJFVzG2oipuVU6tMAZQBg62FxlM0VWhdFo/9HcNWvhntre3+8cd\nqdWE9FYmQpmJ85xN85p07g09ae1qYDVAdXV13GskSlTVo2XSX5ytRHV10vLCX1E6rpXSQBGlt6+D\nyaHgVfnfbsQAU6++MaJSFR6MLrn9PgIFRaG1aDa0EzTejsnwNWCbnnwIt7sTwG+hAYBxOHPi04QL\n+dUKYwClewyYyCBLFqSiq0LJ1lNlag2a95levzCvatbe3k5LS8ugbTiQ/JKJUNYITA57PAloSnBN\nozGmABgDtKV5r/RVVNWjbMSps5UoB8qGn+h57Yx/WHh48Joado5ldDDypjU//P0r7H/rZawbTNmm\nIiZJOwEM4BQUMmzUGL8KN/dPIw9QVyuMAZTtY8BEMqA3QSrVeqpMN1H1Ps9r5FpXV0d9ff2gbjiQ\n/JGJULYJmGmMmQocIrRw/9aoa14Fbie0VuxmYL211hpjXgWeM8b8H0IL/WcC/5WBMQnEVD1KF3+Z\nZQu/EqpEOU2U1vyv0HXWheHjklakEgWjEaXlXHrHA5w58WnK9V7Rh5dfescDnGncybDTh9j69A8I\nBoNxpyfVCmOAJdsBLJIDmpqa/EapMUcRxZFsPVU2diaWlZX53fkHchpTXfrzX79DWc8asXuBNwm1\nxHjKWrvLGPM3wGZr7av/P3tvHh5HfaZr37+qXiR5txu3d2wDwokhICySETkGx0BCWIbhsGX5gkOS\nQ3KG5LsyOSczkAkfZLIxk1wzmQmZJEwCMVkBE8YJYQnY2BgQxLJlAsS2sC2MbdltS7Zl2ZJ6qfp9\nf1R39aJqqVtSS93Se1+Xj9zd1dXV8uTUw/s+7/MCPwV+njTyH8URbiSPewRnKCAB3C6Tl8OIR9Uj\nRDIvbNO3AQOwnZ89HYSXXpG3IpUpjIKTpvCXtT+lbesGZ2muvzCfV6i2jmW3fiWdTXb2AnjlVt6M\nTMCKzwBU3vakRGEIgpCiqqqq38fFUKrJxNGIoejvM6WtWRkMS06Z1vpJ4Mmc5/6/jL/3Ajfmee83\ngW8Ox3UIHuSreixcAb5glncoNL//ilTqsWPWj7nPW/FYQT6v9pbm7MiLaz5AyIoRrtaYajo2hrQn\nBUEYkN7e3n4fF0spJhNHI4Yi32dKW7NykET/8Uoe71C+ilQqkuJUexu2Fc96TRlG1qRkPlHXpz3a\nXUPIDIAyWDT1JPrsv2bxVbcVVhGTLK1s5PchVAiFVmz6Oy61I7IcwlALGTiIRCI0NzePiDjzEpjS\n1qwcRJRVCqW46RboHcqcvDQMH8ow0VYCwN2TmYrM6C+6Irz0QpQy0NigFMF557L52Cr2/OUFbFtj\nbn2FxVfdlu8y0kiWVjby+xAqhEIrNgMdVy5hqPmuM1OoAaNepZJE/8pBRFklMJI3XQ/xl1nh0lic\nsfIGN89k2qJ3Ee3qdCtkqeOseJQ9G9dmrVXa8eRDrphL7dd0dmc6c5kFx12Ua5bWaFWryvX3IQg5\nFJoZ1tTU5Br5+1vePdrVnnzbAzJFWG1t7ahXqcpFxAoDI6KsEvC66aae708AFCsS8oi/3MnLVH6Z\nV3aZYfiwbSdUtnXD4yy+5FqOv9Pi7rnMJCXQAFCqcD9ZOWZpjWa1qhx/H4LgwUAVm1TlKSXIgLKu\n7Hh9n1yhBv3npI0U5SBihYERUVYJ5N50q2cMLAAGIxLyVFwKDXWNdnWyeMV17HruEUBjWwlef/QH\nxE6d8Pw4ZfrAtlEGLLrw4n79ZNletTLM0hrNapVkiwkVQqGZYSnmzp1LfX192YqJfN8nU4TV1tZS\nW1srVSqhIESUVQK5N91CBEDmMYleeO2hgW/WA1RcTrW30bpxLUCfvZnKMOlub2PaondhBoJY8Rho\nm0Ovv4wyzD4fdfr/uJra899D5NEvE64+SejUaqi+xfOyvL1qZZalNdrVKskWEyqEYjLDChVkoxn3\nkPt98gk1EWNCIYgoK0e82o65N92BBMDCFWD4IOXZan4Azrtl4F2ZeZaYZ8Zg7NnwOJfe/TMAFl18\nLcf376Zj51Z2PfcwyvQxp+4SThxspevAHncF0+S5Z3DiwG73o95pfJrasMXSGe1JcWlmicvMylhF\nrFmSapUgDJnBeJ/KMe5BWoXCYBFRVm4M1HZMCbYrvgc9HfkFwPwGqLsVmn4MaLCtwlpqHhWXyJub\ns2IwbCtO68a1tL6w1q2IpdBWggNN67LerwyTuctWZIkybVm0Huhhz8EwStssmt5DKCku8+3ZTFXk\nTrW30d7SXJ7CTMSYIAyJYgWNxD0IYwkRZeVGf63J/gSbV3XtvFtg2+rBtdQyzhdeeiGG6XcrZYbp\nR4NTvcoQZJ4oxey65Rx7e0fO87DrT5vAngzAnpMhLu2pIkRfr9rR1u0svPhaejvbOdi8id3rH+Xt\nF9YWtEVAEISxzXDEPUjavVAuiCgrN/rzJuUTbPnE2mBbajnnC61ax6V3/4w9G9eiwF1U3rrhcWw7\njseq8SwObM6snCnneK2d75HEtiy3LZnrVXM+J+FknNkWaD3ybUwJZxWEsmSocQ/l2P4Uxi8iysqN\n/oRUPsHWX3VtMC01j/OFlt+ZJYDaW5qdvyhQho8ZZ51P+86t6cqZMnDFVwYTZy3gZOSdPs8rZWS1\nJVPTnt3tbexa/6iTkaacVihaj+w6JglnFcqQsV7dKeb7DSU5v9Ttz7H+7yQMLyLKypF8QiqfYBvu\nyb8Czhd5czO2nQCt0bbF1HlnUPfxL9G6cS09x9vpOXaYo7tfz36TUrzrmlvZuvpe7EQclELbNmgb\nbVvsfu5h9jz/GPWf+ipnXnazm4XW+sJaNyPtglV3EO3q9FzjVDIknFUoM8Z6dWcw32+wv5NSpt2P\n9X8nYfgRUVZpeAm24Z78K+B84aUXZgXF7n7+MRZdci2LLrm2z8LyFMEpMzjaut0VVqfa29i97pGs\nY1JJ/1MX1Lp7OL0y0tpbmnnz8ftHRpyNdtyFIOQw1s3tg/l+g/2dlDLtfqz/OwnDj4iysUJKrO1r\nhE3fHro4y1etS3qrQgtXMPv85e6kpbYSboaZlyADiB5vZ/dzD9PqCziRGpE3aH3OwkYlj3B+atvO\n8ovlLkkfaMfmsCNxF0KZMdZ3GQ7m+w3ld1KqCIux/u8kDD8iysYSpfY+Jc/f3mUQ6fl3CK3Mejmy\nfTOTZy8a8DS25Zj0w/Edru8/jcIwfdl+sRyT/ajklg1X3IUMDAjDwFjfZZjv+/Xnzyr172Qw3rCx\n/u8kDD8iyiqJ/m7o+xphwz2QiAJ2abxPb2+gvctg/d65WFphtL8GhulkoAFdB/bQdXCvY/J3ozKc\nnZa2bbnHGaZj0o+8/A62VmQrs5xJTg+hmbuLc8QM/0NFBgaEYWQ0A0pHwrye+/0K8WeV6ncyFG+Y\nBMkKxSCirFIYKKNs9aVgJQUZRnHep0KrNwtXEOn5d6ykkNI2TF/87mxDv23lvEkz/68+yL5X/oht\nWyjDZNmtX0lXtp7+bXISU2WcwmmF7tm4FnWwmUWnDELV2ZOgXj6zskcGBoQxwGiZ10fTn1XKz5bp\nTCETEWWVQn83dPc126lSLb4MVtxT2A2/mOrN/AbCN34H80f/jG1pDH+AM1Zez/G9O/P6yAAO/2Wz\nM22ZJNrV6a5RmnXuRRz680vZb9A2u9avcQXeHjWXSxfsJzQx5ixjp6/PrCKQgQFhDJBPoAyHuHDP\nYXYQ7tyc9R+Ko+nPKtVny3SmkIuIskqhvxt67muFCjLoK/Zee8itmrX3VPWpRoWWf4KV4XP6PL97\n/WMY/iBHd/05KcDSbcieo5GMD9TseuYh/vybf8u5kBxzWUbFzdYGkZ4JhCb0wtNfhPC53qunyt2n\nJQMDwhjAS6AMh7hwz2FZGDrG1R0PEN74dfc/FEfTn1Wqz5bpTCEXEWWVQn839KHc7DMFnWFC84Ng\nJ2jvncj6fQuwLKvPhGNmlaq9pZmtq+91JyHPvvITHHt7B8HJ0zjasoWuw23JD0pPVnYfa896zvmr\nchL7te0ExOJMdAIYpkG4ptupBBazeqockf2YQoXjJVCam5uzxEVLS8uAAia3suYKFMDGpC1wJuFT\nrVn/e8/0Z410268U3jCZzhRyEVFWSfR3Qx/szT5T0HW+A1v+C7RF5KQfKxEHDVY8yp6Naz3bhZmT\nkFY8xo4/rHaElTJcUZWugmVHX2RimD60tkE76f7Lbv0KR1u3O2udlpxBaONnwTL7VglfewgSvc75\nxaflUCmVQ6FiyRUomeJCKcXOnTvRWuetmnlV1txzWBYGFnNiu/KHVw9TZW60vVwynSnkIqJMyM44\nSy4wD0+MY3SY2Jaza3LP848BsPiSa7OqZN3tbY4AUzqZ0O8cr0lPX2YLshTp56afcS7TF72bXese\nTe61jNHWvImLv3xf+vCFZ/YVGvsancpequ1pmOLTqrTKoTAmyBQXJ0+eZMeOHf225LzadnV1dWmB\nYnYQ7vxU3v+wGGrbr5y8XDKdKWQiokzI5vxVAITOu4XFTz/DruceATTaSrB73SO8/cJaVt71AIAT\n4BqLkhJFylAow+9h+u9bGXOfUwZnrLze+bsbowEHmtax67mHOfOym50nvCqBb28AO6Mad9aVznOp\n48cjMuEpjBKZ+ydbWlr6bcnla9tlC5TL8n5W7vurqqoK3nkZiURoamrCshzfqni5hHJCRNl4I19r\nK7fCct4tLLrkWlpfWIsVjzqxFVq7Ya3gtDUzzfnatphTfwltWzY4FbM+KKafcQ5Hd7+Rfp/WbF19\nLwsvvrbP0bvXP5YWZUlSU5vhpRcSyvLD+eCtJ2Hn78d1hSgy5ULaJl3JnN7thO0DUjksJdIm9qSQ\nltxQ23aZ76+qquLll18uqOqVqpClBBkgXi6hrBBRNhYo9ObQX2vLo8KSygNr3biWPRseR9tWTlhr\nnzh+eo4eRuv0c2YgmKymAWjCS99L57630kIPR+gpyAmdBcMfzNpv6bleycMPV1SFaAzdWCORCE9s\n3os98WqMSVdx9fvOIFzh36lskTZxvxTSkhtq2y71/twhg/6qXqm2Z4q5c+dSX18vVTKhbBBRVukU\nc3Por7WVJ3IjNWm56JJr+8RgzDq3ITtjTOvsIFnIEGQO/prJrLzrAbb96l85sr0JAGWYTFv0Lk5b\ncoH7HIbJ0V1/pr1lqyvAPNcrXXdbHz9cwRlgY+zGmj29ZtBmzUBuNSVC2sRlQzETjLnHiiATyg0R\nZZVOMTeH/rLO5jfAFd+D7Y/Bu67vc46UOGtvaebNx+8nOGkKkTdfLfpy27Zt4uBrm9LiC9B2gqYH\nvpExrQlVk6fT29kBOi3A+l2vNJhYkDF2Y5Xx+hFEgoCHTLHTj7nHZz4utBUq045CuaMyW02VQn19\nvW5qahr4wPFAsdWeQj1lHufJbB9mR16UEGVgmD4WrbiOxZc4vrNi1itledByjx9jlTIojzH/ccMY\nan2PNMVOP+Yef9FFFxXsIxOEckAptUVrXT/QcVIpq3SKrRDlyzMroGqU2T50IjCyPWClYPKcRXQd\nbGX3cw+zZ/2jnPHei1k0t5pQ9bkDvtfTg5YpzMZgwr6M148gEgQ8aIqNtMg9fs+ePZKEL4xJRJSN\nBYbj5jBAOyaVSWYYPmydcMWYMkzH2F8icXbiwG5SmWbattj1yvO0Ks3K5gcI/e1TnqugUnh60HKr\nZXJjFYQRp9hWe+7xixcv5tChQ9KqF8YcIsoEh36qRpkVJ8PwMX3x0qShX6O1Zu6yD9Bz7DBGIEjH\nzuY8cRjDhcLS8HpkMvOfuJ+tL2/LWwnr14MmCMKoUai3qz/f2PTp06VVL4w5RJSNJQbjccl9j8f7\nstqWWFRPnwm7ky9qm7bmjenVSsMlyPq0RnXWz0Onaoi88KdkqoaNFY/SmrMKKlRb505tFupBEwRh\nZBio1e7lO6urqyv4/YJQiYgoK2eKEVmDMa0X+J7cilPVlFCGaMpcreQ9NGJW1WD1dhf0lR0UNaHZ\ndB854D7OzkNLLTfXKMNICjPNng2PsyhjDRRkL08XBKFyGOoqJUGoRIzRvgAhDynBtP4u5+e+xv6P\n9zLqD0SB70lVnM696f9l5V0PsPiSazH9AVAGyjBQhun8MU3P92cJMsOk5rS5A1yYzhBkmWSva1Km\nyZwLVoBKiTTL3TYgDBP7GmHTtwf+vz+hYolEIjQ3NxOJREb7UrJI+ciUUuIbE8YNUikrV4rN0BpM\nblIR78mtOF2w6o5ktpiFMgzmXLCCOXXL2fzTr0M/LUwF3oJLJf/7oN+BgXS1TJk+6j/1VaYuqOXQ\nn18S31gpGIORIUI25bSYOxcv35lEvghjHRFl5UqxImuw8Q7JBeScd0ve92RmfYHjMetub0v6xzTa\ntmhr3sjkOQvzCLLs/ZieeIkxZbirmFJVsrlL65hx7oosj5j4xkrEGAvXFfpS7i3CTN9YOQtIQRgu\nRJSVK7kiC5w2Un+Cq5h4B48F5F7kTl4C2HYCw/A5xn7tiCxtW+zZ8HjOu1NesL47MgdEGYTOvoD2\nHemQYKUM3v2RL/cRXmXtG6vkgFFJrc+mkv8t81BJWyDKXUAKwnAgoqycSYmsUrSRCqyCZGV96bjz\npNbYOs6UBWfTuXeH+1z0xNGs9856z0XJ3ZgpQZbtCesPZRgEJ07Jeq7idk/09+9WCTf4MRiuO2jG\naCu3ktYOVZKAFITBIqKsEihFG6nAKkjm5KUTFGs765W05sS+t8isglmTZ5OYNg/fsf1MCihmvvu9\nSVHWjxjLsxVAWwlQyXBat+WpvQNgy5V8/26VdIMv13DdlKitngE9HaUXjWO4lVsp0RKVJCAFYbCI\nKKsEStFGKrAKkpv11bpxLbuee4RUcKwyTdCaxJTZdJ/3N6BMYtqC5t/Suf+tgi7FEV42ubWwtq0b\nWXDRh9n74hPOE1oT7z6RdYyX361svGX5/t3G8A1+RHBFbTQp6A3wBUsrbqWVWxZUioAUhMEioqwS\nKFUbqcAqSK5nq/WFtVjxGMowWHLVKvw1kzmYCLKnowcMA2xITJ3L3peezDiLYm79Sg5u24SdiCWf\nUpj+ABesuoOjrdvZ8/xjWUvOtZWg6+DerGvZ8YfVzLvwUkK1dXn9bp57LkeDfP9ucoMfGq6oTVVY\n7dKLW2nlCoIwAkhOWaUwvwGW31lYIGwJc6VCtXVcsOoOJ7TVtmh5+peEl17IBB11Kj+2DdrCd2y/\nMzmpDFAGZiDInLrlOdOXilnveT9TF9QyITQH7dHGTHS1o4x0/pm2LFo3rgVy/G5WHDsRz9pzWRZ4\n/bulbvArv17erctyJSVqUzEqGCMjbgv936AgCMIgkUrZWGKEvErRrk5HQGmNnYjTunEtJ/fvpqbt\ngOspM08cdA7WGmX6uGDVHRxr3Z4tyrTNgaZ1HNy2iWXXfgRDQVqWOa3ME4fbUGZq0tNpcaaS+3P9\nbs4pLe+8snIz1perV6sSyKxajZSnTBgUkismCMUhoqzSyRQbI+RVSokhKxZFa5td69eAbWFCWoxl\noG2btm2bOLKj2fN8diJG2x9/CHY1oFDKoMqI02P5AAW2zcQ5i+g6sCd5Pie5f+l1t2X53SCPp6yS\njPVCYYioLXskV0wQikdEWSWTKzau+N6IeJVCtXXUXvFxtv/up05BS/e/hFwBBzav6/eYnriBrRWg\n0FonBRmARhkmJw+9kz6fYboiLNfv5ukjqzRjfblV9QRhEEiumCAUj4iySiZXbPR0FG5GHuKN/9jb\nOzye9QiJVYrA5OlEO9vznksZBmdMP0XnwSCWTp3HidGYPn8R089+L7vWP+qeb3bdctczVpCZv5KM\n9VLVGz5E3I4qkismCMUjoqyS8RIbhbR1huHGP/99lyczyJL0yRtLB8ZGT3TkPY8yTOo/fRdTgzEW\nPr+GI0e6OHE43QKtnrWYRZdcS+sLa13v2MHmTRzY8nzhU5aVNDlXaVW9cqXMxO1gvVWV7MmSXDFB\nKB4RZZVMf2KjvypBSW78OuvvCo1GJXdX5qDSz89ZtgKA5374L+5y80yBd7B5E+/+60+73rFT7W3s\nXv9o1pRlKh6j34yySvEgVVJVr5wpobgtVigN1luV+76LLrqI3t7eAT+3nISc5IoJQnGIKKt0vMTG\nQFWCYbjx73v12b5PKoUCpgW7meSPsbdrSp9DJsw6ne7D+92dmQc2r8vym2nbckRbxuOU2DrV3kZv\nZ7sziam06y3LzCsrm4yywVJJVb1ypkTidjACa7Deqsz3WZbFiy++CNDv54q5XhAqGxFlY5GBqgTD\ncOP3bF/aFho4LxoHQwAAIABJREFU2lvN0d5qz/edOrTX8/lclOlzUv6VIt59gnVf+2Q6dDaHrLyy\njOrZsDAavqRKqeqVMyUSt4MRWIP1VmW+D0Anq8v9fW4lmOvLqZInCOWGiLKxSCFVgiHe+KcuqGVu\n/aX0HDtM9fSZHGhan/Gqh+G/CEx/kHnvvYy9L/0BbSXY/vsHvfdjZlTRUnllnhllg6XMfElCkZRA\n3A5GYA3WW5X5vqqqKl5++eUBPzd1fZblVKKrqqoK/3IjgFTyBKF/RJSNRUrcAnPbhfEopmlwxvl1\nHGjKPUrl/NV78bgX8957Ge+8/FTaj+b5PuUKsNz9nMNWJRPTvZDDUATWYMRH5vumT58+4OeGw2Eu\nuugiXnzxRbTWvPzyy0yfPr1shE8lVPIEYTQRUTZWKWELLPLmZqx4FLTGSiQ4uv4+QnOW0H6gzfsN\nWnNazSl6ak7nZPvhAc9/cNuLOeuYclHMes9FnHvj7a4Ay80rGxbEdC94MFrm9UI/t7e31/17uQkf\nickQhP4RUSYUTXDSFJKpsQC0Hp/IsqptdBByJi6V0Wfq8kh3FXQPLMgAYieP539RGZj+QJYg6xcv\nT1ihPjEx3QtFUC5eqVILn6F8T4nJEIT+UdorsqDMqa+v101Nffplwgjgti5jvTiiTKHQnDuznXD1\nKSI9Ewkv/wTHp7+PzT/5p2Tav3NcVkuzSJTpY8lVq/DXTC68RenlCQPxiQnDzrB4pYZxqKRUAlE8\nYYIwOJRSW7TW9QMdJ5Wyccxg/j9ud9IRSIksw+cnPDFBqCpOaPIpaLiR0PwGpi6oJfLyYwR7DrD1\npS1YiYR3btmAKOo/9VXOvOzm4t7m5QmD8vOJSfJ8xVOoVyrv/+aGeaikVC1W8YQJQmkRUTZOyfov\nXgVXzz+OWTOLSHtPv5Wo8NILMQwftu0IM2WYXHDrVyEY482mpwjXf5hQ8maS6fOaemkzrRvXusvL\ni0NztHV78V8ynyesnHxiMuE5JiikZdhvlalMh0pyRaR4wgShtIgoG6e0bX8F20qgUdi2xa6tL3Lg\ntR1YmJj+ICtv+xKhzpedg8+7xb1BhGrrWLziOnY99wgpT9nR1u1sfWG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ok+pM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\nIiKSAErKRERERBLg/wERP54+ceoBzAAAAABJRU5ErkJggg==\n",
"text/plain": [
"<matplotlib.figure.Figure at 0x7fe4c8bda5c0>"
]
},
"metadata": {},
"output_type": "display_data"
}
],
"source": [
"import matplotlib.pyplot as plt\n",
"import numpy as np\n",
"\n",
"super_populations = samples['super_pop'].unique().tolist()\n",
"\n",
"color_list = plt.cm.Set1(np.linspace(0, 1, len(super_populations)))\n",
"\n",
"pca_samples = []\n",
"dim_1 = []\n",
"dim_2 = []\n",
"\n",
"with open('1kgp_chr22_maf_LD_PCA.eigenvec') as f:\n",
" for l in f:\n",
" ls = l.replace('\\n', '').split()\n",
" pca_samples.append(ls[0])\n",
" dim_1.append(float(ls[2]))\n",
" dim_2.append(float(ls[3]))\n",
"\n",
"legends = []\n",
"legends_names = []\n",
"fig, ax = plt.subplots(figsize=(10,10))\n",
"for sp_index, sp in enumerate(super_populations):\n",
" print ('Plotting:', sp)\n",
" sp_samples = samples[samples['super_pop'] == sp]['sample'].tolist()\n",
" \n",
" these_data = [(x,y) for x,y,z in zip(dim_1, dim_2, pca_samples) if z in sp_samples]\n",
" \n",
" leg, = ax.plot([x[0] for x in these_data], [x[1] for x in these_data], '.', c = color_list[sp_index])\n",
" legends.append(leg)\n",
" legends_names.append(sp)\n",
" \n",
"plt.legend(legends, legends_names)\n",
"plt.show()"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"Το αποτέλεσμα είναι πολύ κοντά στο αποτέλεσμα που βγάζει το google για \"[1000 Genomes PCA](https://www.google.com/search?q=1000+genomes+PCA&client=firefox-b-ab&source=lnms&tbm=isch&sa=X&biw=1280&bih=640#imgrc=rNEE-Q8SgYYnvM)\""
]
}
],
"metadata": {
"kernelspec": {
"display_name": "Python 3",
"language": "python",
"name": "python3"
},
"language_info": {
"codemirror_mode": {
"name": "ipython",
"version": 3
},
"file_extension": ".py",
"mimetype": "text/x-python",
"name": "python",
"nbconvert_exporter": "python",
"pygments_lexer": "ipython3",
"version": "3.6.3"
}
},
"nbformat": 4,
"nbformat_minor": 2
}
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