{
"cells": [
{
"cell_type": "markdown",
"metadata": {},
"source": [
"# ARTIC pipeline example\n",
"\n",
"This is a brief run through of the ARTIC pipeline. It covers:\n",
"\n",
"* installing the pipeline\n",
"* what files you need\n",
"* what commands to use\n",
"* what output is produced\n",
"\n",
"***\n",
"\n",
"## Using this notebook\n",
"\n",
"We are using a [jupyter notebook](https://jupyter.org/) for this example so that we can host it on [Binder](mybinder.org). If you want to run the commands for yourself on the command line, you will need to remove the leading `!` which is before all the code in this notebook (which is telling jupyter to execute a system command).\n",
"\n",
"To run this notebook, you can click on each cell and press Run. Be sure to wait for the cell to complete before moving on to the next one. It might take a minute or so for each cell to complete.\n",
"\n",
"## Installing the pipeline\n",
"\n",
"If you are using this notebook via Binder, or the GitHub repository, the pipeline is already installed. For reference though, you can install the pipeline from conda:\n",
"\n",
"```\n",
"conda create -n artic-env\n",
"conda activate artic-env\n",
"conda install -c bioconda -c conda-forge artic\n",
"```\n",
"\n",
"We are using version 1.2.1, check you are on the same:"
]
},
{
"cell_type": "code",
"execution_count": 2,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"artic 1.2.1\r\n"
]
}
],
"source": [
"!artic -v"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"## Data\n",
"\n",
"To begin, we need some data. This repository already has some data for you to use, which was generated from a SARS-COV-2 positive control sample at the University of Birmingham. If you want to obtain the data for yourself, you can run the following:\n",
"\n",
"```\n",
"wget http://artic.s3.climb.ac.uk/BHAM-Run88-PTC.fastq\n",
"```\n",
"\n",
"This test data is only the FASTQ reads from the positive control sample. We have already basecalled, demuxed and filtered them from the original FAST5 data for this sample.\n",
"\n",
"> Because we only have FASTQ data, this example will use the **medaka** workflow of the ARTIC pipeline. This is because the **nanopolish** version requires FAST5 data as well as FASTQ.\n",
"\n",
"\n",
"To run the **medaka** workflow ARTIC pipeline on this data, we need to know:\n",
"\n",
"* what version of the ARTIC primer scheme was used\n",
" * version 3\n",
"* what [medaka model](https://github.com/nanoporetech/medaka#models) to use\n",
" * use `{pore}_{device}_{caller variant}_{caller version}`\n",
" * r941_min_high_g351\n",
"\n",
"As well as the FASTQ reads, we will also need:\n",
"\n",
"* primer scheme (BED format)\n",
"* reference sequence (FASTA format)\n",
"\n",
"### Primer scheme and reference sequence\n",
"\n",
"Although the ARTIC pipeline will download these for us, we can also get them for ourselves in order to familiarise ourselves with them:"
]
},
{
"cell_type": "code",
"execution_count": 3,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"[21:41:48] [artic-tools::get_scheme] starting primer scheme downloader\n",
"[21:41:48] [artic-tools::get_scheme] \trequested scheme:\tscov2\n",
"[21:41:48] [artic-tools::get_scheme] \trequested version:\t3\n",
"[21:41:48] [artic-tools::get_scheme] fetching manifest file\n",
"[21:41:48] [artic-tools::get_scheme] \tARTIC manifest URL:\thttps://raw.githubusercontent.com/artic-network/primer-schemes/master/schemes_manifest.json\n",
"[21:41:50] [artic-tools::get_scheme] \tARTIC repository DOI:\t10.5281/zenodo.4004423\n",
"[21:41:50] [artic-tools::get_scheme] finding primer scheme\n",
"[21:41:50] [artic-tools::get_scheme] \tfound requested scheme:\tsars-cov-2 (using alias scov2)\n",
"[21:41:50] [artic-tools::get_scheme] downloading primer scheme\n",
"[21:41:51] [artic-tools::get_scheme] \tsaving primers to:\tscov2.v3.primer.bed\n",
"[21:41:51] [artic-tools::get_scheme] \tsaving reference to:\tscov2.v3.reference.fasta\n",
"[21:41:51] [artic-tools::get_scheme] comparing checksums\n",
"[21:41:51] [artic-tools::get_scheme] \tsha256 for primers:\t6e98d7d5d1c6edac8ef0bac70d698e0828ae42bafe8f3bda0a6257d00ce414b5\n",
"[21:41:51] [artic-tools::get_scheme] \tsha256 for reference:\tb09a4a3d6824dc4a9f3a17d480f3335f73cb1507897f6dad0de871e8f00d8637\n"
]
}
],
"source": [
"!artic-tools get_scheme --schemeVersion 3 scov2"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"This will have downloaded the primer scheme (`scov2.v3.primer.bed`) and the reference sequence (`scov2.v3.reference.fasta`). You can get some information on the primer scheme using artic-tools:"
]
},
{
"cell_type": "code",
"execution_count": 4,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"[21:41:56] [artic-tools::validate_scheme] starting primer scheme validator\r\n",
"[21:41:56] [artic-tools::validate_scheme] reading scheme\r\n",
"[21:41:56] [artic-tools::validate_scheme] collecting scheme stats\r\n",
"[21:41:56] [artic-tools::validate_scheme] \tprimer scheme file:\tscov2.v3.primer.bed\r\n",
"[21:41:56] [artic-tools::validate_scheme] \treference sequence:\tMN908947.3\r\n",
"[21:41:56] [artic-tools::validate_scheme] \tnumber of pools:\t2\r\n",
"[21:41:56] [artic-tools::validate_scheme] \tnumber of primers:\t218 (includes 22 alts)\r\n",
"[21:41:56] [artic-tools::validate_scheme] \tminimum primer size:\t22\r\n",
"[21:41:56] [artic-tools::validate_scheme] \tmaximum primer size:\t57\r\n",
"[21:41:56] [artic-tools::validate_scheme] \tnumber of amplicons:\t98\r\n",
"[21:41:56] [artic-tools::validate_scheme] \tmean amplicon size:\t343\r\n",
"[21:41:56] [artic-tools::validate_scheme] \tmaximum amplicon size:\t375\r\n",
"[21:41:56] [artic-tools::validate_scheme] \tscheme ref. span:\t30-29866\r\n",
"[21:41:56] [artic-tools::validate_scheme] \tscheme overlaps:\t12.850247%\r\n"
]
}
],
"source": [
"!artic-tools validate_scheme scov2.v3.primer.bed"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"The primer scheme file is in a BED format, where the columns equate to the following:\n",
"\n",
"\n",
"| column | name | type | description |\n",
"| :----- | :--------- | :----------- | :-------------------------------------------------------- |\n",
"| 1 | chrom | string | primer reference sequence |\n",
"| 2 | chromStart | int | starting position of the primer in the reference sequence |\n",
"| 3 | chomEnd | int | ending position of the primer in the reference sequence |\n",
"| 4 | name | string | primer name |\n",
"| 5 | primerPool | int | primer pool* |\n",
"| 6 | strand | string (+/-) | primer direction |\n",
"\n",
"* column 5 in the BED spec is an int for score, whereas here we are using it to denote primerPool.\n",
"\n",
"If you want to look at the primer scheme file, we can do that here with some Python:"
]
},
{
"cell_type": "code",
"execution_count": 5,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
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"MN908947.3\t320\t342\tnCoV-2019_2_LEFT\t2\t+\n",
"MN908947.3\t704\t726\tnCoV-2019_2_RIGHT\t2\t-\n",
"MN908947.3\t642\t664\tnCoV-2019_3_LEFT\t1\t+\n",
"MN908947.3\t1004\t1028\tnCoV-2019_3_RIGHT\t1\t-\n",
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"MN908947.3\t3771\t3795\tnCoV-2019_13_LEFT\t1\t+\n",
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"MN908947.3\t4054\t4077\tnCoV-2019_14_LEFT\t2\t+\n",
"MN908947.3\t4044\t4068\tnCoV-2019_14_LEFT_alt4\t2\t+\n",
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"MN908947.3\t4402\t4424\tnCoV-2019_14_RIGHT_alt2\t2\t-\n",
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"MN908947.3\t5230\t5259\tnCoV-2019_18_LEFT\t2\t+\n",
"MN908947.3\t5257\t5287\tnCoV-2019_18_LEFT_alt2\t2\t+\n",
"MN908947.3\t5620\t5644\tnCoV-2019_18_RIGHT\t2\t-\n",
"MN908947.3\t5620\t5643\tnCoV-2019_18_RIGHT_alt1\t2\t-\n",
"MN908947.3\t5563\t5586\tnCoV-2019_19_LEFT\t1\t+\n",
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"MN908947.3\t6526\t6548\tnCoV-2019_21_RIGHT_alt0\t1\t-\n",
"MN908947.3\t6466\t6495\tnCoV-2019_22_LEFT\t2\t+\n",
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"MN908947.3\t8249\t8275\tnCoV-2019_28_LEFT\t2\t+\n",
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"MN908947.3\t11927\t11949\tnCoV-2019_39_RIGHT\t1\t-\n",
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"MN908947.3\t13007\t13029\tnCoV-2019_44_LEFT_alt3\t2\t+\n",
"MN908947.3\t13378\t13400\tnCoV-2019_44_RIGHT\t2\t-\n",
"MN908947.3\t13363\t13385\tnCoV-2019_44_RIGHT_alt0\t2\t-\n",
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"MN908947.3\t13307\t13336\tnCoV-2019_45_LEFT_alt2\t1\t+\n",
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"MN908947.3\t13961\t13984\tnCoV-2019_46_RIGHT_alt2\t2\t-\n",
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"MN908947.3\t16485\t16510\tnCoV-2019_54_RIGHT\t2\t-\n",
"MN908947.3\t16416\t16444\tnCoV-2019_55_LEFT\t1\t+\n",
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"MN908947.3\t16748\t16770\tnCoV-2019_56_LEFT\t2\t+\n",
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"MN908947.3\t17381\t17406\tnCoV-2019_58_LEFT\t2\t+\n",
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"MN908947.3\t17674\t17697\tnCoV-2019_59_LEFT\t1\t+\n",
"MN908947.3\t18036\t18062\tnCoV-2019_59_RIGHT\t1\t-\n",
"MN908947.3\t17966\t17993\tnCoV-2019_60_LEFT\t2\t+\n",
"MN908947.3\t18324\t18348\tnCoV-2019_60_RIGHT\t2\t-\n",
"MN908947.3\t18253\t18275\tnCoV-2019_61_LEFT\t1\t+\n",
"MN908947.3\t18650\t18672\tnCoV-2019_61_RIGHT\t1\t-\n",
"MN908947.3\t18596\t18618\tnCoV-2019_62_LEFT\t2\t+\n",
"MN908947.3\t18957\t18979\tnCoV-2019_62_RIGHT\t2\t-\n",
"MN908947.3\t18896\t18918\tnCoV-2019_63_LEFT\t1\t+\n",
"MN908947.3\t19275\t19297\tnCoV-2019_63_RIGHT\t1\t-\n",
"MN908947.3\t19204\t19232\tnCoV-2019_64_LEFT\t2\t+\n",
"MN908947.3\t19591\t19616\tnCoV-2019_64_RIGHT\t2\t-\n",
"MN908947.3\t19548\t19570\tnCoV-2019_65_LEFT\t1\t+\n",
"MN908947.3\t19911\t19939\tnCoV-2019_65_RIGHT\t1\t-\n",
"MN908947.3\t19844\t19866\tnCoV-2019_66_LEFT\t2\t+\n",
"MN908947.3\t20231\t20255\tnCoV-2019_66_RIGHT\t2\t-\n",
"MN908947.3\t20172\t20200\tnCoV-2019_67_LEFT\t1\t+\n",
"MN908947.3\t20542\t20572\tnCoV-2019_67_RIGHT\t1\t-\n",
"MN908947.3\t20472\t20496\tnCoV-2019_68_LEFT\t2\t+\n",
"MN908947.3\t20867\t20890\tnCoV-2019_68_RIGHT\t2\t-\n",
"MN908947.3\t20786\t20813\tnCoV-2019_69_LEFT\t1\t+\n",
"MN908947.3\t21146\t21169\tnCoV-2019_69_RIGHT\t1\t-\n",
"MN908947.3\t21075\t21104\tnCoV-2019_70_LEFT\t2\t+\n",
"MN908947.3\t21427\t21455\tnCoV-2019_70_RIGHT\t2\t-\n",
"MN908947.3\t21357\t21386\tnCoV-2019_71_LEFT\t1\t+\n",
"MN908947.3\t21716\t21743\tnCoV-2019_71_RIGHT\t1\t-\n",
"MN908947.3\t21658\t21682\tnCoV-2019_72_LEFT\t2\t+\n",
"MN908947.3\t22013\t22038\tnCoV-2019_72_RIGHT\t2\t-\n",
"MN908947.3\t21961\t21990\tnCoV-2019_73_LEFT\t1\t+\n",
"MN908947.3\t22324\t22346\tnCoV-2019_73_RIGHT\t1\t-\n",
"MN908947.3\t22262\t22290\tnCoV-2019_74_LEFT\t2\t+\n",
"MN908947.3\t22626\t22650\tnCoV-2019_74_RIGHT\t2\t-\n",
"MN908947.3\t22516\t22542\tnCoV-2019_75_LEFT\t1\t+\n",
"MN908947.3\t22877\t22903\tnCoV-2019_75_RIGHT\t1\t-\n",
"MN908947.3\t22797\t22819\tnCoV-2019_76_LEFT\t2\t+\n",
"MN908947.3\t22798\t22821\tnCoV-2019_76_LEFT_alt3\t2\t+\n",
"MN908947.3\t23192\t23214\tnCoV-2019_76_RIGHT\t2\t-\n",
"MN908947.3\t23189\t23212\tnCoV-2019_76_RIGHT_alt0\t2\t-\n",
"MN908947.3\t23122\t23144\tnCoV-2019_77_LEFT\t1\t+\n",
"MN908947.3\t23500\t23522\tnCoV-2019_77_RIGHT\t1\t-\n",
"MN908947.3\t23443\t23466\tnCoV-2019_78_LEFT\t2\t+\n",
"MN908947.3\t23822\t23847\tnCoV-2019_78_RIGHT\t2\t-\n",
"MN908947.3\t23789\t23812\tnCoV-2019_79_LEFT\t1\t+\n",
"MN908947.3\t24145\t24169\tnCoV-2019_79_RIGHT\t1\t-\n",
"MN908947.3\t24078\t24100\tnCoV-2019_80_LEFT\t2\t+\n",
"MN908947.3\t24443\t24467\tnCoV-2019_80_RIGHT\t2\t-\n",
"MN908947.3\t24391\t24416\tnCoV-2019_81_LEFT\t1\t+\n",
"MN908947.3\t24765\t24789\tnCoV-2019_81_RIGHT\t1\t-\n",
"MN908947.3\t24696\t24721\tnCoV-2019_82_LEFT\t2\t+\n",
"MN908947.3\t25052\t25076\tnCoV-2019_82_RIGHT\t2\t-\n",
"MN908947.3\t24978\t25003\tnCoV-2019_83_LEFT\t1\t+\n",
"MN908947.3\t25347\t25369\tnCoV-2019_83_RIGHT\t1\t-\n",
"MN908947.3\t25279\t25301\tnCoV-2019_84_LEFT\t2\t+\n",
"MN908947.3\t25646\t25673\tnCoV-2019_84_RIGHT\t2\t-\n",
"MN908947.3\t25601\t25623\tnCoV-2019_85_LEFT\t1\t+\n",
"MN908947.3\t25969\t25994\tnCoV-2019_85_RIGHT\t1\t-\n",
"MN908947.3\t25902\t25924\tnCoV-2019_86_LEFT\t2\t+\n",
"MN908947.3\t26290\t26315\tnCoV-2019_86_RIGHT\t2\t-\n",
"MN908947.3\t26197\t26219\tnCoV-2019_87_LEFT\t1\t+\n",
"MN908947.3\t26566\t26590\tnCoV-2019_87_RIGHT\t1\t-\n",
"MN908947.3\t26520\t26542\tnCoV-2019_88_LEFT\t2\t+\n",
"MN908947.3\t26890\t26913\tnCoV-2019_88_RIGHT\t2\t-\n",
"MN908947.3\t26835\t26857\tnCoV-2019_89_LEFT\t1\t+\n",
"MN908947.3\t26838\t26860\tnCoV-2019_89_LEFT_alt2\t1\t+\n",
"MN908947.3\t27202\t27227\tnCoV-2019_89_RIGHT\t1\t-\n",
"MN908947.3\t27190\t27215\tnCoV-2019_89_RIGHT_alt4\t1\t-\n",
"MN908947.3\t27141\t27164\tnCoV-2019_90_LEFT\t2\t+\n",
"MN908947.3\t27511\t27533\tnCoV-2019_90_RIGHT\t2\t-\n",
"MN908947.3\t27446\t27471\tnCoV-2019_91_LEFT\t1\t+\n",
"MN908947.3\t27825\t27854\tnCoV-2019_91_RIGHT\t1\t-\n",
"MN908947.3\t27784\t27808\tnCoV-2019_92_LEFT\t2\t+\n",
"MN908947.3\t28145\t28172\tnCoV-2019_92_RIGHT\t2\t-\n",
"MN908947.3\t28081\t28104\tnCoV-2019_93_LEFT\t1\t+\n",
"MN908947.3\t28442\t28464\tnCoV-2019_93_RIGHT\t1\t-\n",
"MN908947.3\t28394\t28416\tnCoV-2019_94_LEFT\t2\t+\n",
"MN908947.3\t28756\t28779\tnCoV-2019_94_RIGHT\t2\t-\n",
"MN908947.3\t28677\t28699\tnCoV-2019_95_LEFT\t1\t+\n",
"MN908947.3\t29041\t29063\tnCoV-2019_95_RIGHT\t1\t-\n",
"MN908947.3\t28985\t29007\tnCoV-2019_96_LEFT\t2\t+\n",
"MN908947.3\t29356\t29378\tnCoV-2019_96_RIGHT\t2\t-\n",
"MN908947.3\t29288\t29316\tnCoV-2019_97_LEFT\t1\t+\n",
"MN908947.3\t29665\t29693\tnCoV-2019_97_RIGHT\t1\t-\n",
"MN908947.3\t29486\t29510\tnCoV-2019_98_LEFT\t2\t+\n",
"MN908947.3\t29836\t29866\tnCoV-2019_98_RIGHT\t2\t-\n",
"\n"
]
}
],
"source": [
"with open(\"scov2.v3.primer.bed\", 'r') as f:\n",
" print(f.read())"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"## Running the pipeline\n",
"\n",
"Now we have the primer scheme, reference sequence and our FASTQ data. We can run the pipeline!"
]
},
{
"cell_type": "code",
"execution_count": 6,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"\u001b[33m\u001b[22mcould not find primer scheme and reference sequence, attempting to download\u001b[39m\u001b[22m\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22m artic-tools get_scheme scov2 --schemeVersion 3\n",
"[21:42:18] [artic-tools::get_scheme] starting primer scheme downloader\n",
"[21:42:18] [artic-tools::get_scheme] \trequested scheme:\tscov2\n",
"[21:42:18] [artic-tools::get_scheme] \trequested version:\t3\n",
"[21:42:18] [artic-tools::get_scheme] fetching manifest file\n",
"[21:42:18] [artic-tools::get_scheme] \tARTIC manifest URL:\thttps://raw.githubusercontent.com/artic-network/primer-schemes/master/schemes_manifest.json\n",
"[21:42:19] [artic-tools::get_scheme] \tARTIC repository DOI:\t10.5281/zenodo.4004423\n",
"[21:42:19] [artic-tools::get_scheme] finding primer scheme\n",
"[21:42:19] [artic-tools::get_scheme] \tfound requested scheme:\tsars-cov-2 (using alias scov2)\n",
"[21:42:19] [artic-tools::get_scheme] downloading primer scheme\n",
"[21:42:19] [artic-tools::get_scheme] \tsaving primers to:\tscov2.v3.primer.bed\n",
"[21:42:19] [artic-tools::get_scheme] \tsaving reference to:\tscov2.v3.reference.fasta\n",
"[21:42:19] [artic-tools::get_scheme] comparing checksums\n",
"[21:42:19] [artic-tools::get_scheme] \tsha256 for primers:\t6e98d7d5d1c6edac8ef0bac70d698e0828ae42bafe8f3bda0a6257d00ce414b5\n",
"[21:42:19] [artic-tools::get_scheme] \tsha256 for reference:\tb09a4a3d6824dc4a9f3a17d480f3335f73cb1507897f6dad0de871e8f00d8637\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22m artic-tools validate_scheme ./primer-schemes/scov2/V3/scov2.scheme.bed\n",
"[21:42:19] [artic-tools::validate_scheme] starting primer scheme validator\n",
"[21:42:19] [artic-tools::validate_scheme] reading scheme\n",
"[21:42:19] [artic-tools::validate_scheme] collecting scheme stats\n",
"[21:42:19] [artic-tools::validate_scheme] \tprimer scheme file:\t./primer-schemes/scov2/V3/scov2.scheme.bed\n",
"[21:42:19] [artic-tools::validate_scheme] \treference sequence:\tMN908947.3\n",
"[21:42:19] [artic-tools::validate_scheme] \tnumber of pools:\t2\n",
"[21:42:19] [artic-tools::validate_scheme] \tnumber of primers:\t218 (includes 22 alts)\n",
"[21:42:19] [artic-tools::validate_scheme] \tminimum primer size:\t22\n",
"[21:42:19] [artic-tools::validate_scheme] \tmaximum primer size:\t57\n",
"[21:42:19] [artic-tools::validate_scheme] \tnumber of amplicons:\t98\n",
"[21:42:19] [artic-tools::validate_scheme] \tmean amplicon size:\t343\n",
"[21:42:19] [artic-tools::validate_scheme] \tmaximum amplicon size:\t375\n",
"[21:42:19] [artic-tools::validate_scheme] \tscheme ref. span:\t30-29866\n",
"[21:42:19] [artic-tools::validate_scheme] \tscheme overlaps:\t12.850247%\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22mminimap2 -a -x map-ont -t 2 ./primer-schemes/scov2/V3/scov2.reference.fasta ../data/BHAM-Run88-PTC.fastq.gz | samtools view -bS -F 4 - | samtools sort -o my_example.sorted.bam -\n",
"[M::mm_idx_gen::0.002*2.10] collected minimizers\n",
"[M::mm_idx_gen::0.003*2.02] sorted minimizers\n",
"[M::main::0.004*2.01] loaded/built the index for 1 target sequence(s)\n",
"[M::mm_mapopt_update::0.004*1.94] mid_occ = 3\n",
"[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 1\n",
"[M::mm_idx_stat::0.004*1.88] distinct minimizers: 5587 (99.93% are singletons); average occurrences: 1.004; average spacing: 5.332\n",
"[M::worker_pipeline::11.622*1.58] mapped 124656 sequences\n",
"[M::main] Version: 2.17-r941\n",
"[M::main] CMD: minimap2 -a -x map-ont -t 2 ./primer-schemes/scov2/V3/scov2.reference.fasta ../data/BHAM-Run88-PTC.fastq.gz\n",
"[M::main] Real time: 11.624 sec; CPU: 18.317 sec; Peak RSS: 0.165 GB\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22msamtools index my_example.sorted.bam\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22malign_trim --normalise 100 ./primer-schemes/scov2/V3/scov2.scheme.bed --start --remove-incorrect-pairs --report my_example.alignreport.txt < my_example.sorted.bam 2> my_example.alignreport.er | samtools sort -T my_example - -o my_example.trimmed.rg.sorted.bam\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22malign_trim --normalise 100 ./primer-schemes/scov2/V3/scov2.scheme.bed --remove-incorrect-pairs --report my_example.alignreport.txt < my_example.sorted.bam 2> my_example.alignreport.er | samtools sort -T my_example - -o my_example.primertrimmed.rg.sorted.bam\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22msamtools index my_example.trimmed.rg.sorted.bam\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22msamtools index my_example.primertrimmed.rg.sorted.bam\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22mmedaka consensus --model r941_min_high_g351 --threads 2 --chunk_len 800 --chunk_ovlp 400 --RG 1 my_example.trimmed.rg.sorted.bam my_example.1.hdf\n",
"[21:43:15 - medaka] Reads will be filtered to only those with RG tag: 1\n",
"[21:43:15 - Predict] Processing region(s): MN908947.3:0-29903\n",
"[21:43:15 - Predict] Setting tensorflow threads to 2.\n",
"[21:43:15 - Predict] Processing 1 long region(s) with batching.\n",
"[21:43:15 - Predict] Using model: /Users/willrowe/opt/miniconda3/envs/artic-pipeline-example/lib/python3.6/site-packages/medaka/data/r941_min_high_g351_model.hdf5.\n",
"[21:43:15 - ModelLoad] Building model with cudnn optimization: False\n",
"[21:43:16 - DLoader] Initializing data loader\n",
"[21:43:16 - Sampler] Initializing sampler for consensus of region MN908947.3:0-29903.\n",
"[21:43:16 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 30-409.\n",
"[21:43:16 - Feature] Processed MN908947.3:30.0-409.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 642-1027.\n",
"[21:43:16 - Feature] Processed MN908947.3:642.0-1027.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 1242-1650.\n",
"[21:43:16 - Feature] Processed MN908947.3:1242.0-1650.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 1868-2268.\n",
"[21:43:16 - Feature] Processed MN908947.3:1868.0-2268.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 2504-2903.\n",
"[21:43:16 - Feature] Processed MN908947.3:2504.0-2903.0 (median depth 177.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 3144-3530.\n",
"[21:43:16 - Feature] Processed MN908947.3:3144.0-3530.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 3771-4163.\n",
"[21:43:16 - Feature] Processed MN908947.3:3771.0-4163.0 (median depth 192.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 4294-4689.\n",
"[21:43:16 - Feature] Processed MN908947.3:4294.0-4689.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 4939-5320.\n",
"[21:43:16 - Feature] Processed MN908947.3:4939.0-5320.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 5563-5956.\n",
"[21:43:16 - Feature] Processed MN908947.3:5563.0-5956.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 6167-6549.\n",
"[21:43:16 - Feature] Processed MN908947.3:6167.0-6549.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 6718-7116.\n",
"[21:43:16 - Feature] Processed MN908947.3:6718.0-7116.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 7305-7693.\n",
"[21:43:16 - Feature] Processed MN908947.3:7305.0-7693.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 7943-8340.\n",
"[21:43:16 - Feature] Processed MN908947.3:7943.0-8340.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 8595-8982.\n",
"[21:43:16 - Feature] Processed MN908947.3:8595.0-8982.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 9204-9584.\n",
"[21:43:16 - Feature] Processed MN908947.3:9204.0-9584.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 9784-10170.\n",
"[21:43:16 - Feature] Processed MN908947.3:9784.0-10170.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 10362-10762.\n",
"[21:43:16 - Feature] Processed MN908947.3:10362.0-10762.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 10999-11393.\n",
"[21:43:16 - Feature] Processed MN908947.3:10999.0-11393.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 11555-11948.\n",
"[21:43:16 - Feature] Processed MN908947.3:11555.0-11948.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 12110-12489.\n",
"[21:43:16 - Feature] Processed MN908947.3:12110.0-12489.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 12710-13095.\n",
"[21:43:16 - Feature] Processed MN908947.3:12710.0-13095.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 13307-13698.\n",
"[21:43:16 - Feature] Processed MN908947.3:13307.0-13698.0 (median depth 197.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 13918-14298.\n",
"[21:43:16 - Feature] Processed MN908947.3:13918.0-14298.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 14545-14925.\n",
"[21:43:16 - Feature] Processed MN908947.3:14545.0-14925.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 15171-15559.\n",
"[21:43:16 - Feature] Processed MN908947.3:15171.0-15559.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 15827-16208.\n",
"[21:43:16 - Feature] Processed MN908947.3:15827.0-16208.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 16416-16832.\n",
"[21:43:16 - Feature] Processed MN908947.3:16416.0-16832.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 17065-17451.\n",
"[21:43:16 - Feature] Processed MN908947.3:17065.0-17451.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 17674-18061.\n",
"[21:43:16 - Feature] Processed MN908947.3:17674.0-18061.0 (median depth 162.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 18253-18671.\n",
"[21:43:16 - Feature] Processed MN908947.3:18253.0-18671.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 18896-19296.\n",
"[21:43:16 - Feature] Processed MN908947.3:18896.0-19296.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 19548-19938.\n",
"[21:43:16 - Feature] Processed MN908947.3:19548.0-19938.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 20172-20571.\n",
"[21:43:16 - Feature] Processed MN908947.3:20172.0-20571.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 20786-21168.\n",
"[21:43:16 - Feature] Processed MN908947.3:20786.0-21168.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 21357-21742.\n",
"[21:43:16 - Feature] Processed MN908947.3:21357.0-21742.0 (median depth 119.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 21961-22345.\n",
"[21:43:16 - Feature] Processed MN908947.3:21961.0-22345.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 22516-22902.\n",
"[21:43:16 - Feature] Processed MN908947.3:22516.0-22902.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 23122-23521.\n",
"[21:43:16 - Feature] Processed MN908947.3:23122.0-23521.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 23789-24168.\n",
"[21:43:16 - Feature] Processed MN908947.3:23789.0-24168.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 24391-24788.\n",
"[21:43:16 - Feature] Processed MN908947.3:24391.0-24788.2 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 24978-25368.\n",
"[21:43:16 - Feature] Processed MN908947.3:24978.0-25368.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 25601-25993.\n",
"[21:43:16 - Feature] Processed MN908947.3:25601.0-25993.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 26197-26589.\n",
"[21:43:16 - Feature] Processed MN908947.3:26197.0-26589.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 26835-27226.\n",
"[21:43:16 - Feature] Processed MN908947.3:26835.0-27226.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 27446-27853.\n",
"[21:43:16 - Feature] Processed MN908947.3:27446.0-27853.0 (median depth 91.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 28081-28463.\n",
"[21:43:16 - Feature] Processed MN908947.3:28081.0-28463.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 28677-29062.\n",
"[21:43:16 - Feature] Processed MN908947.3:28677.0-29062.0 (median depth 200.0)\n",
"[21:43:16 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 29288-29692.\n",
"[21:43:16 - Feature] Processed MN908947.3:29288.0-29692.0 (median depth 200.0)\n",
"[21:43:16 - Sampler] Took 0.15s to make features.\n",
"[21:43:16 - Sampler] Region MN908947.3:2504.0-2903.0 (794 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:16 - Sampler] Region MN908947.3:3771.0-4163.0 (771 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:16 - Sampler] Region MN908947.3:4939.0-5320.0 (770 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:16 - Sampler] Region MN908947.3:6167.0-6549.0 (771 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:16 - Sampler] Region MN908947.3:8595.0-8982.0 (778 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:16 - Sampler] Region MN908947.3:10999.0-11393.0 (757 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:16 - Sampler] Region MN908947.3:12110.0-12489.0 (748 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:16 - Sampler] Region MN908947.3:12710.0-13095.0 (793 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:16 - Sampler] Region MN908947.3:13918.0-14298.0 (777 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:16 - Sampler] Region MN908947.3:17674.0-18061.0 (750 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:16 - Sampler] Region MN908947.3:20786.0-21168.0 (797 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:16 - Sampler] Region MN908947.3:21357.0-21742.0 (714 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:16 - Sampler] Region MN908947.3:22516.0-22902.0 (787 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:16 - Sampler] Region MN908947.3:23789.0-24168.0 (773 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:16 - Sampler] Region MN908947.3:27446.0-27853.0 (656 positions) is smaller than inference chunk length 800, quarantining.\n"
]
},
{
"name": "stdout",
"output_type": "stream",
"text": [
"[21:43:17 - PWorker] All done, 15 remainder regions.\n",
"[21:43:17 - Predict] Processing 15 short region(s).\n",
"[21:43:17 - ModelLoad] Building model with cudnn optimization: False\n",
"[21:43:18 - DLoader] Initializing data loader\n",
"[21:43:18 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:18 - Sampler] Initializing sampler for consensus of region MN908947.3:2504-2904.\n",
"[21:43:18 - Feature] Processed MN908947.3:2504.0-2903.0 (median depth 177.0)\n",
"[21:43:18 - Sampler] Took 0.01s to make features.\n",
"[21:43:19 - PWorker] All done, 0 remainder regions.\n",
"[21:43:19 - DLoader] Initializing data loader\n",
"[21:43:19 - Sampler] Initializing sampler for consensus of region MN908947.3:3771-4164.\n",
"[21:43:19 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:19 - Feature] Processed MN908947.3:3771.0-4163.0 (median depth 192.0)\n",
"[21:43:19 - Sampler] Took 0.02s to make features.\n",
"[21:43:20 - PWorker] All done, 0 remainder regions.\n",
"[21:43:20 - DLoader] Initializing data loader\n",
"[21:43:20 - Sampler] Initializing sampler for consensus of region MN908947.3:4939-5321.\n",
"[21:43:20 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:20 - Feature] Processed MN908947.3:4939.0-5320.0 (median depth 200.0)\n",
"[21:43:20 - Sampler] Took 0.02s to make features.\n",
"[21:43:20 - PWorker] All done, 0 remainder regions.\n",
"[21:43:20 - DLoader] Initializing data loader\n",
"[21:43:20 - Sampler] Initializing sampler for consensus of region MN908947.3:6167-6550.\n",
"[21:43:20 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:21 - Feature] Processed MN908947.3:6167.0-6549.0 (median depth 200.0)\n",
"[21:43:21 - Sampler] Took 0.01s to make features.\n",
"[21:43:21 - PWorker] All done, 0 remainder regions.\n",
"[21:43:21 - DLoader] Initializing data loader\n",
"[21:43:21 - Sampler] Initializing sampler for consensus of region MN908947.3:8595-8983.\n",
"[21:43:21 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:21 - Feature] Processed MN908947.3:8595.0-8982.0 (median depth 200.0)\n",
"[21:43:21 - Sampler] Took 0.02s to make features.\n",
"[21:43:21 - PWorker] All done, 0 remainder regions.\n",
"[21:43:21 - DLoader] Initializing data loader\n",
"[21:43:21 - Sampler] Initializing sampler for consensus of region MN908947.3:10999-11394.\n",
"[21:43:21 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:21 - Feature] Processed MN908947.3:10999.0-11393.0 (median depth 200.0)\n",
"[21:43:21 - Sampler] Took 0.02s to make features.\n",
"[21:43:21 - PWorker] All done, 0 remainder regions.\n",
"[21:43:21 - DLoader] Initializing data loader\n",
"[21:43:21 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:21 - Sampler] Initializing sampler for consensus of region MN908947.3:12110-12490.\n",
"[21:43:21 - Feature] Processed MN908947.3:12110.0-12489.0 (median depth 200.0)\n",
"[21:43:21 - Sampler] Took 0.02s to make features.\n",
"[21:43:22 - PWorker] All done, 0 remainder regions.\n",
"[21:43:22 - DLoader] Initializing data loader\n",
"[21:43:22 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:22 - Sampler] Initializing sampler for consensus of region MN908947.3:12710-13096.\n",
"[21:43:22 - Feature] Processed MN908947.3:12710.0-13095.0 (median depth 200.0)\n",
"[21:43:22 - Sampler] Took 0.02s to make features.\n",
"[21:43:22 - PWorker] All done, 0 remainder regions.\n",
"[21:43:22 - DLoader] Initializing data loader\n",
"[21:43:22 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:22 - Sampler] Initializing sampler for consensus of region MN908947.3:13918-14299.\n",
"[21:43:22 - Feature] Processed MN908947.3:13918.0-14298.0 (median depth 200.0)\n",
"[21:43:22 - Sampler] Took 0.03s to make features.\n",
"[21:43:23 - PWorker] All done, 0 remainder regions.\n",
"[21:43:23 - DLoader] Initializing data loader\n",
"[21:43:23 - Sampler] Initializing sampler for consensus of region MN908947.3:17674-18062.\n",
"[21:43:23 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:23 - Feature] Processed MN908947.3:17674.0-18061.0 (median depth 162.0)\n",
"[21:43:23 - Sampler] Took 0.01s to make features.\n",
"[21:43:23 - PWorker] All done, 0 remainder regions.\n",
"[21:43:23 - DLoader] Initializing data loader\n",
"[21:43:23 - Sampler] Initializing sampler for consensus of region MN908947.3:20786-21169.\n",
"[21:43:23 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:23 - Feature] Processed MN908947.3:20786.0-21168.0 (median depth 200.0)\n",
"[21:43:23 - Sampler] Took 0.03s to make features.\n",
"[21:43:24 - PWorker] All done, 0 remainder regions.\n",
"[21:43:24 - DLoader] Initializing data loader\n",
"[21:43:24 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:24 - Sampler] Initializing sampler for consensus of region MN908947.3:21357-21743.\n",
"[21:43:24 - Feature] Processed MN908947.3:21357.0-21742.0 (median depth 119.0)\n",
"[21:43:24 - Sampler] Took 0.03s to make features.\n",
"[21:43:25 - PWorker] All done, 0 remainder regions.\n",
"[21:43:25 - DLoader] Initializing data loader\n",
"[21:43:25 - Sampler] Initializing sampler for consensus of region MN908947.3:22516-22903.\n",
"[21:43:25 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:25 - Feature] Processed MN908947.3:22516.0-22902.0 (median depth 200.0)\n",
"[21:43:25 - Sampler] Took 0.03s to make features.\n",
"[21:43:25 - PWorker] All done, 0 remainder regions.\n",
"[21:43:25 - DLoader] Initializing data loader\n",
"[21:43:25 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:25 - Sampler] Initializing sampler for consensus of region MN908947.3:23789-24169.\n",
"[21:43:26 - Feature] Processed MN908947.3:23789.0-24168.0 (median depth 200.0)\n",
"[21:43:26 - Sampler] Took 0.03s to make features.\n",
"[21:43:26 - PWorker] All done, 0 remainder regions.\n",
"[21:43:26 - DLoader] Initializing data loader\n",
"[21:43:26 - Sampler] Initializing sampler for consensus of region MN908947.3:27446-27854.\n",
"[21:43:26 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:26 - Feature] Processed MN908947.3:27446.0-27853.0 (median depth 91.0)\n",
"[21:43:26 - Sampler] Took 0.04s to make features.\n",
"[21:43:27 - PWorker] All done, 0 remainder regions.\n",
"[21:43:27 - Predict] Finished processing all regions.\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22mmedaka variant ./primer-schemes/scov2/V3/scov2.reference.fasta my_example.1.hdf my_example.1.vcf\n",
"[21:43:32 - DataIndex] Loaded 1/1 (100.00%) sample files.\n",
"[21:43:32 - Variants] Processing MN908947.3:0-.\n",
"[21:43:32 - TrimOverlap] MN908947.3:56.1-409.0 and MN908947.3:642.0-984.3 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:32 - TrimOverlap] MN908947.3:683.2-1027.0 and MN908947.3:1242.0-1613.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:32 - TrimOverlap] MN908947.3:1274.0-1650.0 and MN908947.3:1868.0-2236.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:32 - TrimOverlap] MN908947.3:1894.0-2268.0 and MN908947.3:2504.0-2903.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:32 - TrimOverlap] MN908947.3:2504.0-2903.0 and MN908947.3:3144.0-3514.3 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:32 - TrimOverlap] MN908947.3:3155.0-3530.0 and MN908947.3:3771.0-4163.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:32 - TrimOverlap] MN908947.3:3771.0-4163.0 and MN908947.3:4294.0-4678.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:32 - TrimOverlap] MN908947.3:4303.5-4689.0 and MN908947.3:4939.0-5320.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:32 - TrimOverlap] MN908947.3:4939.0-5320.0 and MN908947.3:5563.0-5920.2 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:32 - TrimOverlap] MN908947.3:5599.1-5956.0 and MN908947.3:6167.0-6549.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:32 - TrimOverlap] MN908947.3:6167.0-6549.0 and MN908947.3:6718.0-7076.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:32 - TrimOverlap] MN908947.3:6757.1-7116.0 and MN908947.3:7305.0-7669.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:32 - TrimOverlap] MN908947.3:7328.0-7693.0 and MN908947.3:7943.0-8311.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:7965.2-8340.0 and MN908947.3:8595.0-8982.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:8595.0-8982.0 and MN908947.3:9204.0-9558.0 cannot be concatenated as there is no overlap and they do not abut.\n"
]
},
{
"name": "stdout",
"output_type": "stream",
"text": [
"[21:43:33 - TrimOverlap] MN908947.3:9229.1-9584.0 and MN908947.3:9784.0-10152.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:9801.0-10170.0 and MN908947.3:10362.0-10750.3 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:10374.0-10762.0 and MN908947.3:10999.0-11393.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:10999.0-11393.0 and MN908947.3:11555.0-11925.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:11576.0-11948.0 and MN908947.3:12110.0-12489.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:12110.0-12489.0 and MN908947.3:12710.0-13095.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:12710.0-13095.0 and MN908947.3:13307.0-13670.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:13336.2-13698.0 and MN908947.3:13918.0-14298.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:13918.0-14298.0 and MN908947.3:14545.0-14918.3 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:14551.1-14925.0 and MN908947.3:15171.0-15553.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:15175.1-15559.0 and MN908947.3:15827.0-16202.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:15833.3-16208.0 and MN908947.3:16416.0-16802.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:16452.0-16832.0 and MN908947.3:17065.0-17401.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:17115.1-17451.0 and MN908947.3:17674.0-18061.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:17674.0-18061.0 and MN908947.3:18253.0-18604.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:18316.1-18671.0 and MN908947.3:18896.0-19255.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:18934.1-19296.0 and MN908947.3:19548.0-19920.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:19559.0-19938.0 and MN908947.3:20172.0-20554.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:20189.0-20571.0 and MN908947.3:20786.0-21168.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:20786.0-21168.0 and MN908947.3:21357.0-21742.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:21357.0-21742.0 and MN908947.3:21961.0-22340.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:21968.0-22345.0 and MN908947.3:22516.0-22902.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:22516.0-22902.0 and MN908947.3:23122.0-23497.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:23154.1-23521.0 and MN908947.3:23789.0-24168.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:23789.0-24168.0 and MN908947.3:24391.0-24755.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:24419.1-24788.2 and MN908947.3:24978.0-25356.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:24986.3-25368.0 and MN908947.3:25601.0-25948.9 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:25632.1-25993.0 and MN908947.3:26197.0-26516.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:26274.3-26589.0 and MN908947.3:26835.0-27188.2 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:26863.1-27226.0 and MN908947.3:27446.0-27853.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:27446.0-27853.0 and MN908947.3:28081.0-28445.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:28102.0-28463.0 and MN908947.3:28677.0-29035.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:33 - TrimOverlap] MN908947.3:28705.2-29062.0 and MN908947.3:29288.0-29642.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22mmedaka consensus --model r941_min_high_g351 --threads 2 --chunk_len 800 --chunk_ovlp 400 --RG 2 my_example.trimmed.rg.sorted.bam my_example.2.hdf\n",
"[21:43:37 - medaka] Reads will be filtered to only those with RG tag: 2\n",
"[21:43:37 - Predict] Processing region(s): MN908947.3:0-29903\n",
"[21:43:37 - Predict] Setting tensorflow threads to 2.\n",
"[21:43:37 - Predict] Processing 1 long region(s) with batching.\n",
"[21:43:37 - Predict] Using model: /Users/willrowe/opt/miniconda3/envs/artic-pipeline-example/lib/python3.6/site-packages/medaka/data/r941_min_high_g351_model.hdf5.\n",
"[21:43:37 - ModelLoad] Building model with cudnn optimization: False\n",
"[21:43:39 - DLoader] Initializing data loader\n",
"[21:43:39 - Sampler] Initializing sampler for consensus of region MN908947.3:0-29903.\n",
"[21:43:39 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 320-725.\n",
"[21:43:39 - Feature] Processed MN908947.3:320.0-725.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 943-1336.\n",
"[21:43:39 - Feature] Processed MN908947.3:943.0-1336.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 1573-1963.\n",
"[21:43:39 - Feature] Processed MN908947.3:1573.0-1963.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 2181-2591.\n",
"[21:43:39 - Feature] Processed MN908947.3:2181.0-2591.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 2826-3209.\n",
"[21:43:39 - Feature] Processed MN908947.3:2826.0-3209.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 3460-3852.\n",
"[21:43:39 - Feature] Processed MN908947.3:3460.0-3852.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 4044-4449.\n",
"[21:43:39 - Feature] Processed MN908947.3:4044.0-4449.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 4636-5016.\n",
"[21:43:39 - Feature] Processed MN908947.3:4636.0-5016.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 5230-5643.\n",
"[21:43:39 - Feature] Processed MN908947.3:5230.0-5643.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 5867-6271.\n",
"[21:43:39 - Feature] Processed MN908947.3:5867.0-6271.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 6466-6872.\n",
"[21:43:39 - Feature] Processed MN908947.3:6466.0-6872.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 7035-7414.\n",
"[21:43:39 - Feature] Processed MN908947.3:7035.0-7414.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 7626-8018.\n",
"[21:43:39 - Feature] Processed MN908947.3:7626.0-8018.0 (median depth 117.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 8249-8660.\n",
"[21:43:39 - Feature] Processed MN908947.3:8249.0-8660.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 8888-9270.\n",
"[21:43:39 - Feature] Processed MN908947.3:8888.0-9270.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 9477-9857.\n",
"[21:43:39 - Feature] Processed MN908947.3:9477.0-9857.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 10076-10458.\n",
"[21:43:39 - Feature] Processed MN908947.3:10076.0-10458.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 10666-11073.\n",
"[21:43:39 - Feature] Processed MN908947.3:10666.0-11073.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 11306-11692.\n",
"[21:43:39 - Feature] Processed MN908947.3:11306.0-11692.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 11863-12255.\n",
"[21:43:39 - Feature] Processed MN908947.3:11863.0-12255.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 12417-12801.\n",
"[21:43:39 - Feature] Processed MN908947.3:12417.0-12801.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 13005-13399.\n",
"[21:43:39 - Feature] Processed MN908947.3:13005.0-13399.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 13599-13983.\n",
"[21:43:39 - Feature] Processed MN908947.3:13599.0-13983.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 14207-14600.\n",
"[21:43:39 - Feature] Processed MN908947.3:14207.0-14600.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 14865-15245.\n",
"[21:43:39 - Feature] Processed MN908947.3:14865.0-15245.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 15481-15885.\n",
"[21:43:39 - Feature] Processed MN908947.3:15481.0-15885.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 16118-16509.\n",
"[21:43:39 - Feature] Processed MN908947.3:16118.0-16509.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 16748-17151.\n",
"[21:43:39 - Feature] Processed MN908947.3:16748.0-17151.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 17381-17760.\n",
"[21:43:39 - Feature] Processed MN908947.3:17381.0-17760.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 17966-18347.\n",
"[21:43:39 - Feature] Processed MN908947.3:17966.0-18347.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 18596-18978.\n",
"[21:43:39 - Feature] Processed MN908947.3:18596.0-18978.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 19204-19615.\n",
"[21:43:39 - Feature] Processed MN908947.3:19204.0-19615.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 19844-20254.\n",
"[21:43:39 - Feature] Processed MN908947.3:19844.0-20254.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 20472-20889.\n",
"[21:43:39 - Feature] Processed MN908947.3:20472.0-20889.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 21075-21454.\n",
"[21:43:39 - Feature] Processed MN908947.3:21075.0-21454.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 21658-22037.\n",
"[21:43:39 - Feature] Processed MN908947.3:21658.0-22037.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 22262-22649.\n",
"[21:43:39 - Feature] Processed MN908947.3:22262.0-22649.0 (median depth 34.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 22797-23213.\n",
"[21:43:39 - Feature] Processed MN908947.3:22797.0-23213.0 (median depth 121.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 23443-23846.\n",
"[21:43:39 - Feature] Processed MN908947.3:23443.0-23846.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 24078-24466.\n",
"[21:43:39 - Feature] Processed MN908947.3:24078.0-24466.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 24696-25075.\n",
"[21:43:39 - Feature] Processed MN908947.3:24696.0-25075.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 25279-25672.\n",
"[21:43:39 - Feature] Processed MN908947.3:25279.0-25672.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 25902-26314.\n",
"[21:43:39 - Feature] Processed MN908947.3:25902.0-26314.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 26520-26912.\n",
"[21:43:39 - Feature] Processed MN908947.3:26520.0-26912.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 27141-27532.\n",
"[21:43:39 - Feature] Processed MN908947.3:27141.0-27532.0 (median depth 200.0)\n"
]
},
{
"name": "stdout",
"output_type": "stream",
"text": [
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 27784-28171.\n",
"[21:43:39 - Feature] Processed MN908947.3:27784.0-28171.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 28394-28778.\n",
"[21:43:39 - Feature] Processed MN908947.3:28394.0-28778.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 28985-29377.\n",
"[21:43:39 - Feature] Processed MN908947.3:28985.0-29377.0 (median depth 200.0)\n",
"[21:43:39 - Feature] Pileup counts do not span requested region, requested MN908947.3:0-29903, received 29486-29865.\n",
"[21:43:39 - Feature] Processed MN908947.3:29486.0-29865.0 (median depth 200.0)\n",
"[21:43:39 - Sampler] Took 0.14s to make features.\n",
"[21:43:39 - Sampler] Region MN908947.3:4044.0-4449.0 (794 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:39 - Sampler] Region MN908947.3:4636.0-5016.0 (784 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:39 - Sampler] Region MN908947.3:7035.0-7414.0 (759 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:39 - Sampler] Region MN908947.3:7626.0-8018.0 (687 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:39 - Sampler] Region MN908947.3:12417.0-12801.0 (778 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:39 - Sampler] Region MN908947.3:14865.0-15245.0 (779 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:39 - Sampler] Region MN908947.3:17381.0-17760.0 (793 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:39 - Sampler] Region MN908947.3:21075.0-21454.0 (799 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:39 - Sampler] Region MN908947.3:22262.0-22649.0 (488 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:39 - Sampler] Region MN908947.3:22797.0-23213.0 (687 positions) is smaller than inference chunk length 800, quarantining.\n",
"[21:43:42 - PWorker] All done, 10 remainder regions.\n",
"[21:43:42 - Predict] Processing 10 short region(s).\n",
"[21:43:42 - ModelLoad] Building model with cudnn optimization: False\n",
"[21:43:44 - DLoader] Initializing data loader\n",
"[21:43:44 - Sampler] Initializing sampler for consensus of region MN908947.3:4044-4450.\n",
"[21:43:44 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:44 - Feature] Processed MN908947.3:4044.0-4449.0 (median depth 200.0)\n",
"[21:43:44 - Sampler] Took 0.01s to make features.\n",
"[21:43:44 - PWorker] All done, 0 remainder regions.\n",
"[21:43:44 - DLoader] Initializing data loader\n",
"[21:43:44 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:44 - Sampler] Initializing sampler for consensus of region MN908947.3:4636-5017.\n",
"[21:43:44 - Feature] Processed MN908947.3:4636.0-5016.0 (median depth 200.0)\n",
"[21:43:44 - Sampler] Took 0.02s to make features.\n",
"[21:43:45 - PWorker] All done, 0 remainder regions.\n",
"[21:43:45 - DLoader] Initializing data loader\n",
"[21:43:45 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:45 - Sampler] Initializing sampler for consensus of region MN908947.3:7035-7415.\n",
"[21:43:45 - Feature] Processed MN908947.3:7035.0-7414.0 (median depth 200.0)\n",
"[21:43:45 - Sampler] Took 0.02s to make features.\n",
"[21:43:45 - PWorker] All done, 0 remainder regions.\n",
"[21:43:45 - DLoader] Initializing data loader\n",
"[21:43:45 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:45 - Sampler] Initializing sampler for consensus of region MN908947.3:7626-8019.\n",
"[21:43:45 - Feature] Processed MN908947.3:7626.0-8018.0 (median depth 117.0)\n",
"[21:43:45 - Sampler] Took 0.02s to make features.\n",
"[21:43:45 - PWorker] All done, 0 remainder regions.\n",
"[21:43:45 - DLoader] Initializing data loader\n",
"[21:43:45 - Sampler] Initializing sampler for consensus of region MN908947.3:12417-12802.\n",
"[21:43:45 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:45 - Feature] Processed MN908947.3:12417.0-12801.0 (median depth 200.0)\n",
"[21:43:45 - Sampler] Took 0.02s to make features.\n",
"[21:43:46 - PWorker] All done, 0 remainder regions.\n",
"[21:43:46 - DLoader] Initializing data loader\n",
"[21:43:46 - Sampler] Initializing sampler for consensus of region MN908947.3:14865-15246.\n",
"[21:43:46 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:46 - Feature] Processed MN908947.3:14865.0-15245.0 (median depth 200.0)\n",
"[21:43:46 - Sampler] Took 0.03s to make features.\n",
"[21:43:46 - PWorker] All done, 0 remainder regions.\n",
"[21:43:46 - DLoader] Initializing data loader\n",
"[21:43:46 - Sampler] Initializing sampler for consensus of region MN908947.3:17381-17761.\n",
"[21:43:46 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:46 - Feature] Processed MN908947.3:17381.0-17760.0 (median depth 200.0)\n",
"[21:43:46 - Sampler] Took 0.01s to make features.\n",
"[21:43:46 - PWorker] All done, 0 remainder regions.\n",
"[21:43:46 - DLoader] Initializing data loader\n",
"[21:43:46 - Sampler] Initializing sampler for consensus of region MN908947.3:21075-21455.\n",
"[21:43:46 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:46 - Feature] Processed MN908947.3:21075.0-21454.0 (median depth 200.0)\n",
"[21:43:46 - Sampler] Took 0.01s to make features.\n",
"[21:43:46 - PWorker] All done, 0 remainder regions.\n",
"[21:43:46 - DLoader] Initializing data loader\n",
"[21:43:46 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:46 - Sampler] Initializing sampler for consensus of region MN908947.3:22262-22650.\n",
"[21:43:47 - Feature] Processed MN908947.3:22262.0-22649.0 (median depth 34.0)\n",
"[21:43:47 - Sampler] Took 0.01s to make features.\n",
"[21:43:47 - PWorker] All done, 0 remainder regions.\n",
"[21:43:47 - DLoader] Initializing data loader\n",
"[21:43:47 - PWorker] Running inference for 0.0M draft bases.\n",
"[21:43:47 - Sampler] Initializing sampler for consensus of region MN908947.3:22797-23214.\n",
"[21:43:47 - Feature] Processed MN908947.3:22797.0-23213.0 (median depth 121.0)\n",
"[21:43:47 - Sampler] Took 0.02s to make features.\n",
"[21:43:48 - PWorker] All done, 0 remainder regions.\n",
"[21:43:48 - Predict] Finished processing all regions.\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22mmedaka variant ./primer-schemes/scov2/V3/scov2.reference.fasta my_example.2.hdf my_example.2.vcf\n",
"[21:43:53 - DataIndex] Loaded 1/1 (100.00%) sample files.\n",
"[21:43:53 - Variants] Processing MN908947.3:0-.\n",
"[21:43:53 - TrimOverlap] MN908947.3:366.2-725.0 and MN908947.3:943.0-1291.2 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:989.1-1336.0 and MN908947.3:1573.0-1958.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:1578.0-1963.0 and MN908947.3:2181.0-2546.2 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:2219.1-2591.0 and MN908947.3:2826.0-3193.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:2838.0-3209.0 and MN908947.3:3460.0-3810.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:3498.2-3852.0 and MN908947.3:4044.0-4449.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:4044.0-4449.0 and MN908947.3:4636.0-5016.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:4636.0-5016.0 and MN908947.3:5230.0-5630.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:5250.0-5643.0 and MN908947.3:5867.0-6256.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:5877.4-6271.0 and MN908947.3:6466.0-6858.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:6479.1-6872.0 and MN908947.3:7035.0-7414.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:7035.0-7414.0 and MN908947.3:7626.0-8018.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:7626.0-8018.0 and MN908947.3:8249.0-8622.0 cannot be concatenated as there is no overlap and they do not abut.\n"
]
},
{
"name": "stdout",
"output_type": "stream",
"text": [
"[21:43:53 - TrimOverlap] MN908947.3:8291.0-8660.0 and MN908947.3:8888.0-9263.2 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:8893.3-9270.0 and MN908947.3:9477.0-9794.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:9540.1-9857.0 and MN908947.3:10076.0-10449.2 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:10083.0-10458.0 and MN908947.3:10666.0-11056.2 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:10679.1-11073.0 and MN908947.3:11306.0-11672.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:11325.2-11692.0 and MN908947.3:11863.0-12246.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:11872.3-12255.0 and MN908947.3:12417.0-12801.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:12417.0-12801.0 and MN908947.3:13005.0-13368.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:13027.1-13399.0 and MN908947.3:13599.0-13975.3 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:13604.2-13983.0 and MN908947.3:14207.0-14580.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:14230.0-14600.0 and MN908947.3:14865.0-15245.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:14865.0-15245.0 and MN908947.3:15481.0-15862.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:15501.1-15885.0 and MN908947.3:16118.0-16483.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:16145.3-16509.0 and MN908947.3:16748.0-17112.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:16790.0-17151.0 and MN908947.3:17381.0-17760.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:17381.0-17760.0 and MN908947.3:17966.0-18321.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:17985.0-18347.0 and MN908947.3:18596.0-18972.2 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:18599.2-18978.0 and MN908947.3:19204.0-19540.3 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:19272.1-19615.0 and MN908947.3:19844.0-20219.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:19868.5-20254.0 and MN908947.3:20472.0-20865.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:20497.1-20889.0 and MN908947.3:21075.0-21454.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:21075.0-21454.0 and MN908947.3:21658.0-22024.2 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:21669.1-22037.0 and MN908947.3:22262.0-22649.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:22262.0-22649.0 and MN908947.3:22797.0-23213.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:22797.0-23213.0 and MN908947.3:23443.0-23765.2 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:23510.2-23846.0 and MN908947.3:24078.0-24445.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:53 - TrimOverlap] MN908947.3:24092.1-24466.0 and MN908947.3:24696.0-25044.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:54 - TrimOverlap] MN908947.3:24720.1-25075.0 and MN908947.3:25279.0-25642.0 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:54 - TrimOverlap] MN908947.3:25302.0-25672.0 and MN908947.3:25902.0-26253.2 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:54 - TrimOverlap] MN908947.3:25954.2-26314.0 and MN908947.3:26520.0-26895.3 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:54 - TrimOverlap] MN908947.3:26539.2-26912.0 and MN908947.3:27141.0-27522.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:54 - TrimOverlap] MN908947.3:27152.1-27532.0 and MN908947.3:27784.0-28160.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:54 - TrimOverlap] MN908947.3:27795.1-28171.0 and MN908947.3:28394.0-28756.9 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:54 - TrimOverlap] MN908947.3:28418.2-28778.0 and MN908947.3:28985.0-29352.2 cannot be concatenated as there is no overlap and they do not abut.\n",
"[21:43:54 - TrimOverlap] MN908947.3:29015.1-29377.0 and MN908947.3:29486.0-29859.1 cannot be concatenated as there is no overlap and they do not abut.\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22martic_vcf_merge my_example ./primer-schemes/scov2/V3/scov2.scheme.bed 2> my_example.primersitereport.txt 1:my_example.1.vcf 2:my_example.2.vcf\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22mbgzip -f my_example.merged.vcf\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22mtabix -p vcf my_example.merged.vcf.gz\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22martic-tools check_vcf --dropPrimerVars --dropOverlapFails --vcfOut my_example.merged.filtered.vcf my_example.merged.vcf.gz ./primer-schemes/scov2/V3/scov2.scheme.bed 2> my_example.vcfreport.txt\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22mmv my_example.merged.filtered.vcf my_example.merged.vcf\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22mbgzip -f my_example.merged.vcf\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22mtabix -f -p vcf my_example.merged.vcf.gz\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22mlongshot -P 0 -F -A --no_haps --bam my_example.primertrimmed.rg.sorted.bam --ref ./primer-schemes/scov2/V3/scov2.reference.fasta --out my_example.merged.vcf --potential_variants my_example.merged.vcf.gz\n",
"\n",
"2021-01-14 21:43:56 Automatically determining max read coverage.\n",
"2021-01-14 21:43:56 Estimating mean read coverage...\n",
"2021-01-14 21:43:56 Total reference positions: 29903\n",
"2021-01-14 21:43:56 Total bases in bam: 6503829\n",
"2021-01-14 21:43:56 Mean read coverage: 217.50\n",
"2021-01-14 21:43:56 Min read coverage set to 6.\n",
"2021-01-14 21:43:56 Max read coverage set to 291.\n",
"2021-01-14 21:43:56 Estimating alignment parameters...\n",
"2021-01-14 21:43:56 Done estimating alignment parameters.\n",
"\n",
" Transition Probabilities:\n",
" match -> match: 0.983\n",
" match -> insertion: 0.007\n",
" match -> deletion: 0.010\n",
" deletion -> match: 0.647\n",
" deletion -> deletion: 0.353\n",
" insertion -> match: 0.673\n",
" insertion -> insertion: 0.327\n",
"\n",
" Emission Probabilities:\n",
" match (equal): 0.987\n",
" match (not equal): 0.004\n",
" insertion: 1.000\n",
" deletion: 1.000\n",
"\n",
" GENOTYPE PRIORS:\n",
" REF G1/G2 PROB\n",
" C D/I 0.0000000000015624993116907664\n",
" G A/A 0.00016666692910805806\n",
" G D/I 0.0000000000015624993116907664\n",
" T T/T 0.9984401606149776\n",
" A C/D 0.0000000004166671407128834\n",
" A A/T 0.0003333338549006453\n",
" C A/A 0.00016666692910805806\n",
" G T/T 0.00016666692910805806\n",
" C C/T 0.0003333338549006453\n",
" T A/T 0.0003333338549006453\n",
" G G/I 0.000001250002950856874\n",
" T D/I 0.0000000000015624993116907664\n",
" C D/D 0.000006249988919003825\n",
" T G/D 0.0000000004166671407128834\n",
" C A/T 0.00000011111137065235802\n",
" C G/D 0.0000000004166671407128834\n",
" G T/I 0.0000000004166671407128834\n",
" C C/C 0.9984401606149776\n",
" A G/G 0.00016666692910805806\n",
" A A/I 0.000001250002950856874\n",
" A A/C 0.0003333338549006453\n",
" T T/I 0.000001250002950856874\n",
" G G/D 0.000001250002950856874\n",
" T C/G 0.00000011111137065235802\n",
" G C/G 0.0003333338549006453\n",
" G A/I 0.0000000004166671407128834\n",
" G C/I 0.0000000004166671407128834\n",
" C T/D 0.0000000004166671407128834\n",
" G A/C 0.00000011111137065235802\n",
" A I/I 0.000006249988919003825\n",
" A G/T 0.00000011111137065235802\n",
" C G/I 0.0000000004166671407128834\n",
" T A/C 0.00000011111137065235802\n",
" T A/I 0.0000000004166671407128834\n",
" C A/G 0.00000011111137065235802\n",
" T T/D 0.000001250002950856874\n",
" A C/T 0.00000011111137065235802\n",
" A A/D 0.000001250002950856874\n",
" A T/T 0.00016666692910805806\n",
" A D/I 0.0000000000015624993116907664\n",
" G A/T 0.00000011111137065235802\n",
" G G/G 0.9984401606149776\n",
" G C/T 0.00000011111137065235802\n",
" G I/I 0.000006249988919003825\n",
" T A/D 0.0000000004166671407128834\n",
" T C/D 0.0000000004166671407128834\n",
" T I/I 0.000006249988919003825\n",
" A C/G 0.00000011111137065235802\n",
" C C/G 0.0003333338549006453\n",
" C A/D 0.0000000004166671407128834\n",
" C G/T 0.00000011111137065235802\n",
" T D/D 0.000006249988919003825\n",
" A A/G 0.0003333338549006453\n",
" T C/I 0.0000000004166671407128834\n",
" A D/D 0.000006249988919003825\n",
" T A/A 0.00016666692910805806\n",
" T G/I 0.0000000004166671407128834\n",
" G G/T 0.0003333338549006453\n",
" C G/G 0.00016666692910805806\n",
" G T/D 0.0000000004166671407128834\n",
" C C/D 0.000001250002950856874\n",
" A G/D 0.0000000004166671407128834\n",
" C A/C 0.0003333338549006453\n",
" C A/I 0.0000000004166671407128834\n",
" T G/T 0.0003333338549006453\n",
" A T/D 0.0000000004166671407128834\n",
" G C/D 0.0000000004166671407128834\n",
" G A/D 0.0000000004166671407128834\n",
" C T/I 0.0000000004166671407128834\n",
" C C/I 0.000001250002950856874\n",
" T C/T 0.0003333338549006453\n",
" A C/I 0.0000000004166671407128834\n",
" A C/C 0.00016666692910805806\n",
" C I/I 0.000006249988919003825\n",
" G D/D 0.000006249988919003825\n",
" A A/A 0.9984401606149776\n",
" A G/I 0.0000000004166671407128834\n",
" C T/T 0.00016666692910805806\n",
" T C/C 0.00016666692910805806\n",
" G C/C 0.00016666692910805806\n",
" T A/G 0.00000011111137065235802\n",
" T G/G 0.00016666692910805806\n",
" A T/I 0.0000000004166671407128834\n",
" G A/G 0.0003333338549006453\n",
"2021-01-14 21:43:56 Reading potential variants from input VCF...\n",
"2021-01-14 21:43:56 13 potential variants identified.\n",
"2021-01-14 21:43:56 Generating haplotype fragments from reads...\n"
]
},
{
"name": "stdout",
"output_type": "stream",
"text": [
"2021-01-14 21:43:56 10% of variants processed...\n",
"2021-01-14 21:43:56 20% of variants processed...\n",
"2021-01-14 21:43:56 30% of variants processed...\n",
"2021-01-14 21:43:56 40% of variants processed...\n",
"2021-01-14 21:43:56 50% of variants processed...\n",
"2021-01-14 21:43:57 60% of variants processed...\n",
"2021-01-14 21:43:57 70% of variants processed...\n",
"2021-01-14 21:43:57 80% of variants processed...\n",
"2021-01-14 21:43:57 100% of variants processed.\n",
"2021-01-14 21:43:57 Calling initial genotypes using pair-HMM realignment...\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22martic_vcf_filter --medaka my_example.merged.vcf my_example.pass.vcf my_example.fail.vcf\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22mbgzip -f my_example.pass.vcf\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22mtabix -p vcf my_example.pass.vcf.gz\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22martic_make_depth_mask --store-rg-depths ./primer-schemes/scov2/V3/scov2.reference.fasta my_example.primertrimmed.rg.sorted.bam my_example.coverage_mask.txt\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22martic_mask ./primer-schemes/scov2/V3/scov2.reference.fasta my_example.coverage_mask.txt my_example.fail.vcf my_example.preconsensus.fasta\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22mbcftools consensus -f my_example.preconsensus.fasta my_example.pass.vcf.gz -m my_example.coverage_mask.txt -o my_example.consensus.fasta\n",
"Note: the --sample option not given, applying all records regardless of the genotype\n",
"Applied 9 variants\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22martic_fasta_header my_example.consensus.fasta \"my_example/ARTIC/medaka\"\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22mcat my_example.consensus.fasta ./primer-schemes/scov2/V3/scov2.reference.fasta > my_example.muscle.in.fasta\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22mmuscle -in my_example.muscle.in.fasta -out my_example.muscle.out.fasta\n",
"\n",
"MUSCLE v3.8.1551 by Robert C. Edgar\n",
"\n",
"http://www.drive5.com/muscle\n",
"This software is donated to the public domain.\n",
"Please cite: Edgar, R.C. Nucleic Acids Res 32(5), 1792-97.\n",
"\n",
"my_example.muscle.in 2 seqs, lengths min 29903, max 29903, avg 29903\n",
"00:00:00 2 MB(0%) Iter 1 100.00% K-mer dist pass 1\n",
"00:00:00 2 MB(0%) Iter 1 100.00% K-mer dist pass 2\n",
"00:00:15 970 MB(6%) Iter 1 100.00% Align node \n",
"00:00:15 970 MB(6%) Iter 1 100.00% Root alignment\n",
"\u001b[32m\u001b[22mRunning: \u001b[39m\u001b[22martic_get_stats --scheme ./primer-schemes/scov2/V3/scov2.scheme.bed --align-report my_example.alignreport.txt --vcf-report my_example.vcfreport.txt my_example\n"
]
}
],
"source": [
"!artic minion \\\n",
" --normalise 100 \\\n",
" --threads 2 \\\n",
" --medaka \\\n",
" --medaka-model r941_min_high_g351 \\\n",
" --strict \\\n",
" --read-file ../data/BHAM-Run88-PTC.fastq.gz \\\n",
" scov2/V3 \\\n",
" my_example"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"That's it! Let's have a quick run through of the parameters we used so that we can understand what was happening.\n",
"\n",
"|parameter|explanation|\n",
"|:--------|:----------|\n",
"|`normalise`| This caps amplicon coverage to 200 reads, used mainly to speed up the pipeline run. |\n",
"|`threads`| This sets the number of CPU threads to use during the pipeline. We set this to 2 here as that is the limit on Binder, but if you are playing along at home you can increase this to make things run a bit more quickly. |\n",
"|`medaka`| This tells the ARTIC pipeline to use the **medaka** workflow|\n",
"|`medaka-model`| This specifies which model to use for the **medaka** program calls.|\n",
"|`strict`| This runs an additional filtering of reported variants, checking them in overlap regions of the primer scheme to see if they are artifacts reported in only one primer pool.|\n",
"|`read-file`| This tells the pipeline where to find the reads.|\n",
"|`scov/V3`| This specifies the name of the primer scheme and the version to use. If it isn't found locally, the pipeline will try finding it in the ARTIC primer scheme repository.|\n",
"|`my_exmple`| The name to give this pipeline run, all output will have this prepended to the filenames.|\n",
"\n",
"\n",
"## Pipeline output\n",
"\n",
"### Files\n",
"\n",
"Now it is time to check what the pipeline has produced for us. The files we are most interested in are:\n",
"\n",
"|filename|description|\n",
"|:-------|:----------|\n",
"|`my_example.trimmed.rg.sorted.bam`| the post-processed alignment of reads to the reference genome. |\n",
"|`my_example.primertrimmed.rg.sorted.bam` | the post-processed alignment with additional softmasking to exclude primer sequences. |\n",
"|`my_example.vcfreport.txt` | a report evaluating reported variants against the primer scheme. |\n",
"|`my_example.pass.vcf.gz` | detected variants that passed filters. |\n",
"|`my_example.consensus.fasta` | the consensus sequence for the input sample. |\n",
"|`my_example..muscle.out.fasta` | an alignment of the consensus sequence against the reference sequence. |\n",
"\n",
"### QC report\n",
"\n",
"Before we look at these files, we can use MultiQC to check our amplicon coverage (pre-normalisation to 100) and if we have any variants reported that may be a result of contamination. Use the ARTIC MultiQC plugin:"
]
},
{
"cell_type": "code",
"execution_count": 7,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"\u001b[1;30m[INFO ]\u001b[0m multiqc : This is MultiQC v1.9\n",
"\u001b[1;30m[INFO ]\u001b[0m multiqc : Template : default\n",
"\u001b[1;30m[INFO ]\u001b[0m multiqc : Searching : /Users/willrowe/Desktop/artic-pipeline-example/notebooks\n",
"\u001b[?25lSearching 37 files.. [####################################] 100% \u001b[?25h\n",
"\u001b[1;30m[INFO ]\u001b[0m custom_content : custom_data_lineplot: Found 1 samples (linegraph)\n",
"\u001b[1;30m[INFO ]\u001b[0m custom_content : custom_data_json_table: Found 1 samples (table)\n",
"\u001b[1;30m[INFO ]\u001b[0m multiqc : Compressing plot data\n",
"\u001b[1;30m[INFO ]\u001b[0m multiqc : Report : multiqc_report.html\n",
"\u001b[1;30m[INFO ]\u001b[0m multiqc : Data : multiqc_data\n",
"\u001b[1;30m[INFO ]\u001b[0m multiqc : MultiQC complete\n"
]
}
],
"source": [
"!multiqc ."
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"This will have produced a report HTML file called [multiqc_report.html](multiqc_report.html). You can click on that link or use the following code to view it in this notebook:"
]
},
{
"cell_type": "code",
"execution_count": 8,
"metadata": {},
"outputs": [
{
"data": {
"text/html": [
"\n",
" \n",
" "
],
"text/plain": [
""
]
},
"execution_count": 8,
"metadata": {},
"output_type": "execute_result"
}
],
"source": [
"from IPython.display import IFrame\n",
"IFrame(src='./multiqc_report.html', width=600, height=400)"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"Use the report to see if any amplicons are marked as low coverage due to insufficient reads being assigned. Also use the report to see if there are any overlap variant fails. This is when a variant is idenified within an amplicon overlap region of the scheme but is found in only one amplicon.\n",
"\n",
"### VCF files\n",
"\n",
"As mentioned above, the file containing the filtered variants is `my_example.pass.vcf.gz`. Let's take a look:"
]
},
{
"cell_type": "code",
"execution_count": 9,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"chromosome\tpos\tref\talt\n",
"MN908947.3\t2618\tA\t[G]\n",
"MN908947.3\t8782\tC\t[T]\n",
"MN908947.3\t18488\tT\t[C]\n",
"MN908947.3\t21846\tC\t[T]\n",
"MN908947.3\t23605\tT\t[G]\n",
"MN908947.3\t26354\tT\t[A]\n",
"MN908947.3\t28144\tT\t[C]\n",
"MN908947.3\t29366\tC\t[T]\n",
"MN908947.3\t29596\tA\t[G]\n"
]
}
],
"source": [
"# import pyVCF and open the variant file\n",
"import vcf\n",
"vcf_reader = vcf.Reader(filename=\"my_example.pass.vcf.gz\")\n",
"\n",
"# print the variants\n",
"print(\"chromosome\\tpos\\tref\\talt\")\n",
"for record in vcf_reader:\n",
" print(\"{}\\t{}\\t{}\\t{}\" .format(record.CHROM, record.POS, record.REF, record.ALT))"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"### FASTA files\n",
"\n",
"We also have the consensus sequence and the alignment files to look out. Here is an example of how we can look at them with Python:"
]
},
{
"cell_type": "code",
"execution_count": 10,
"metadata": {},
"outputs": [
{
"name": "stdout",
"output_type": "stream",
"text": [
"alignment length= 29903\n",
"\n",
"my_example/ARTIC/medaka\n",
"NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGATCTGTTCTCTAAACGAACTTTAAAATCTGTGTGGCTGTCACTCGGCTGCATGCTTAGTGCACTCACGCAGTATAATTAATAACTAATTACTGTCGTTGACAGGACACGAGTAACTCGTCTATCTTCTGCAGGCTGCTTACGGTTTCGTCCGTGTTGCAGCCGATCATCAGCACATCTAGGTTTCGTCCGGGTGTGACCGAAAGGTAAGATGGAGAGCCTTGTCCCTGGTTTCAACGAGAAAACACACGTCCAACTCAGTTTGCCTGTTTTACAGGTTCGCGACGTGCTCGTACGTGGCTTTGGAGACTCCGTGGAGGAGGTCTTATCAGAGGCACGTCAACATCTTAAAGATGGCACTTGTGGCTTAGTAGAAGTTGAAAAAGGCGTTTTGCCTCAACTTGAACAGCCCTATGTGTTCATCAAACGTTCGGATGCTCGAACTGCACCTCATGGTCATGTTATGGTTGAGCTGGTAGCAGAACTCGAAGGCATTCAGTACGGTCGTAGTGGTGAGACACTTGGTGTCCTTGTCCCTCATGTGGGCGAAATACCAGTGGCTTACCGCAAGGTTCTTCTTCGTAAGAACGGTAATAAAGGAGCTGGTGGCCATAGTTACGGCGCCGATCTAAAGTCATTTGACTTAGGCGACGAGCTTGGCACTGATCCTTATGAAGATTTTCAAGAAAACTGGAACACTAAACATAGCAGTGGTGTTACCCGTGAACTCATGCGTGAGCTTAACGGAGGGGCATACACTCGCTATGTCGATAACAACTTCTGTGGCCCTGATGGCTACCCTCTTGAGTGCATTAAAGACCTTCTAGCACGTGCTGGTAAAGCTTCATGCACTTTGTCCGAACAACTGGACTTTATTGACACTAAGAGGGGTGTATACTGCTGCCGTGAACATGAGCATGAAATTGCTTGGTACACGGAACGTTCTGAAAAGAGCTATGAATTGCAGACACCTTTTGAAATTAAATTGGCAAAGAAATTTGACACCTTCAATGGGGAATGTCCAAATTTTGTATTTCCCTTAAATTCCATAATCAAGACTATTCAACCAAGGGTTGAAAAGAAAAAGCTTGATGGCTTTATGGGTAGAATTCGATCTGTCTATCCAGTTGCGTCACCAAATGAATGCAACCAAATGTGCCTTTCAACTCTCATGAAGTGTGATCATTGTGGTGAAACTTCATGGCAGACGGGCGATTTTGTTAAAGCCACTTGCGAATTTTGTGGCACTGAGAATTTGACTAAAGAAGGTGCCACTACTTGTGGTTACTTACCCCAAAATGCTGTTGTTAAAATTTATTGTCCAGCATGTCACAATTCAGAAGTAGGACCTGAGCATAGTCTTGCCGAATACCATAATGAATCTGGCTTGAAAACCATTCTTCGTAAGGGTGGTCGCACTATTGCCTTTGGAGGCTGTGTGTTCTCTTATGTTGGTTGCCATAACAAGTGTGCCTATTGGGTTCCACGTGCTAGCGCTAACATAGGTTGTAACCATACAGGTGTTGTTGGAGAAGGTTCCGAAGGTCTTAATGACAACCTTCTTGAAATACTCCAAAAAGAGAAAGTCAACATCAATATTGTTGGTGACTTTAAACTTAATGAAGAGATCGCCATTATTTTGGCATCTTTTTCTGCTTCCACAAGTGCTTTTGTGGAAACTGTGAAAGGTTTGGATTATAAAGCATTCAAACAAATTGTTGAATCCTGTGGTAATTTTAAAGTTACAAAAGGAAAAGCTAAAAAAGGTGCCTGGAATATTGGTGAACAGAAATCAATACTGAGTCCTCTTTATGCATTTGCATCAGAGGCTGCTCGTGTTGTACGATCAATTTTCTCCCGCACTCTTGAAACTGCTCAAAATTCTGTGCGTGTTTTACAGAAGGCCGCTATAACAATACTAGATGGAATTTCACAGTATTCACTGAGACTCATTGATGCTATGATGTTCACATCTGATTTGGCTACTAACAATCTAGTTGTAATGGCCTACATTACAGGTGGTGTTGTTCAGTTGACTTCGCAGTGGCTAACTAACATCTTTGGCACTGTTTATGAAAAACTCAAACCCGTCCTTGATTGGCTTGAAGAGAAGTTTAAGGAAGGTGTAGAGTTTCTTAGAGACGGTTGGGAAATTGTTAAATTTATCTCAACCTGTGCTTGTGAAATTGTCGGTGGACAAATTGTCACCTGTGCAAAGGAAATTAAGGAGAGTGTTCAGACATTCTTTAAGCTTGTAAATAAATTTTTGGCTTTGTGTGCTGACTCTATCATTATTGGTGGAGCTAAACTTAAAGCCTTGAATTTAGGTGAAACATTTGTCACGCACTCAAAGGGATTGTACAGAAAGTGTGTTAAATCCAGAGAAGAAACTGGCCTACTCATGCCTCTAAAAGCCCCAAAAGAAATTATCTTCTTAGAGGGAGAAACACTTCCCACAGAAGTGTTAACAGAGGAAGTTGTCTTGAAAACTGGTGATTTACAACCATTAGAACAACCTACTAGTGAAGCTGTTGAAGCTCCATTGGTTGGTACACCAGTTTGTGTTAACGGGCTTATGTTGCTCGAAATCAAAGACACAGAAAAGTACTGTGCCCTTGCACCTAATATGATGGTAACAAACAATACCTTCACACTCAAAGGCGGTGCACCAACAAAGGTTACTTTTGGTGATGACACTGTGATAGAAGTGCAAGGTTACAAGAGTGTGAATATCACTTTTGAACTTGATGAAAGGATTGATAAAGTACTTAATGAGAAGTGCTCTGCCTATACAGTTGAACTCGGTACAGAAGTAAATGAGTTCGCCTGTGTTGTGGCAGATGCTGTCATAAAAACTTTGCAACCAGTATCTGAATTACTTACACCACTGGGCATTGATTTAGATGAGTGGAGTATGGCTACATACTACTTATTTGATGAGTCTGGTGAGTTTAAATTGGCTTCACATATGTATTGTTCTTTCTACCCTCCAGATGAGGATGAAGAAGAAGGTGATTGTGAAGAAGAAGAGTTTGAGCCATCAACTCAATATGAGTATGGTACTGAAGATGATTACCAAGGTAAACCTTTGGAATTTGGTGCCACTTCTGCTGCTCTTCAACCTGAAGAAGAGCAAGAAGAAGATTGGTTAGATGATGATAGTCAACAAACTGTTGGTCAACAAGACGGCAGTGAGGACAATCAGACAACTACTATTCAAACAATTGTTGAGGTTCAACCTCAATTAGAGATGGAACTTACACCAGTTGTTCAGACTATTGAAGTGAATAGTTTTAGTGGTTATTTAAAACTTACTGACAATGTATACATTAAAAATGCAGACATTGTGGAAGAAGCTAAAAAGGTAAAACCAACAGTGGTTGTTAATGCAGCCAATGTTTACCTTAAACATGGAGGAGGTGTTGCAGGAGCCTTAAATAAGGCTACTAACAATGCCATGCAAGTTGAATCTGATGATTACATAGCTACTAATGGACCACTTAAAGTGGGTGGTAGTTGTGTTTTAAGCGGACACAATCTTGCTAAACACTGTCTTCATGTTGTCGGCCCAAATGTTAACAAAGGTGAAGACATTCAACTTCTTAAGAGTGCTTATGAAAATTTTAATCAGCACGAAGTTCTACTTGCACCATTATTATCAGCTGGTATTTTTGGTGCTGACCCTATACATTCTTTAAGAGTTTGTGTAGATACTGTTCGCACAAATGTCTACTTAGCTGTCTTTGATAAAAATCTCTATGACAAACTTGTTTCAAGCTTTTTGGAAATGAAGAGTGAAAAGCAAGTTGAACAAAAGATCGCTGAGATTCCTAAAGAGGAAGTTAAGCCATTTATAACTGAAAGTAAACCTTCAGTTGAACAGAGAAAACAAGATGATAAGAAAATCAAAGCTTGTGTTGAAGAAGTTACAACAACTCTGGAAGAAACTAAGTTCCTCACAGAAAACTTGTTACTTTATATTGACATTAATGGCAATCTTCATCCAGATTCTGCCACTCTTGTTAGTGACATTGACATCACTTTCTTAAAGAAAGATGCTCCATATATAGTGGGTGATGTTGTTCAAGAGGGTGTTTTAACTGCTGTGGTTATACCTACTAAAAAGGCTGGTGGCACTACTGAAATGCTAGCGAAAGCTTTGAGAAAAGTGCCAACAGACAATTATATAACCACTTACCCGGGTCAGGGTTTAAATGGTTACACTGTAGAGGAGGCAAAGACAGTGCTTAAAAAGTGTAAAAGTGCCTTTTACATTCTACCATCTATTATCTCTAATGAGAAGCAAGAAATTCTTGGAACTGTTTCTTGGAATTTGCGAGAAATGCTTGCACATGCAGAAGAAACACGCAAATTAATGCCTGTCTGTGTGGAAACTAAAGCCATAGTTTCAACTATACAGCGTAAATATAAGGGTATTAAAATACAAGAGGGTGTGGTTGATTATGGTGCTAGATTTTACTTTTACACCAGTAAAACAACTGTAGCGTCACTTATCAACACACTTAACGATCTAAATGAAACTCTTGTTACAATGCCACTTGGCTATGTAACACATGGCTTAAATTTGGAAGAAGCTGCTCGGTATATGAGATCTCTCAAAGTGCCAGCTACAGTTTCTGTTTCTTCACCTGATGCTGTTACAGCGTATAATGGTTATCTTACTTCTTCTTCTAAAACACCTGAAGAACATTTTATTGAAACCATCTCACTTGCTGGTTCCTATAAAGATTGGTCCTATTCTGGACAATCTACACAACTAGGTATAGAATTTCTTAAGAGAGGTGATAAAAGTGTATATTACACTAGTAATCCTACCACATTCCACCTAGATGGTGAAGTTATCACCTTTGACAATCTTAAGACACTTCTTTCTTTGAGAGAAGTGAGGACTATTAAGGTGTTTACAACAGTAGACAACATTAACCTCCACACGCAAGTTGTGGACATGTCAATGACATATGGACAACAGTTTGGTCCAACTTATTTGGATGGAGCTGATGTTACTAAAATAAAACCTCATAATTCACATGAAGGTAAAACATTTTATGTTTTACCTAATGATGACACTCTACGTGTTGAGGCTTTTGAGTACTACCACACAACTGATCCTAGTTTTCTGGGTAGGTACATGTCAGCATTAAATCACACTAAAAAGTGGAAATACCCACAAGTTAATGGTTTAACTTCTATTAAATGGGCAGATAACAACTGTTATCTTGCCACTGCATTGTTAACACTCCAACAAATAGAGTTGAAGTTTAATCCACCTGCTCTACAAGATGCTTATTACAGAGCAAGGGCTGGTGAAGCTGCTAACTTTTGTGCACTTATCTTAGCCTACTGTAATAAGACAGTAGGTGAGTTAGGTGATGTTAGAGAAACAATGAGTTACTTGTTTCAACATGCCAATTTAGATTCTTGCAAAAGAGTCTTGAACGTGGTGTGTAAAACTTGTGGACAACAGCAGACAACCCTTAAGGGTGTAGAAGCTGTTATGTACATGGGCACACTTTCTTATGAACAATTTAAGAAAGGTGTTCAGATACCTTGTACGTGTGGTAAACAAGCTACAAAATATCTAGTACAACAGGAGTCACCTTTTGTTATGATGTCAGCACCACCTGCTCAGTATGAACTTAAGCATGGTACATTTACTTGTGCTAGTGAGTACACTGGTAATTACCAGTGTGGTCACTATAAACATATAACTTCTAAAGAAACTTTGTATTGCATAGACGGTGCTTTACTTACAAAGTCCTCAGAATACAAAGGTCCTATTACGGATGTTTTCTACAAAGAAAACAGTTACACAACAACCATAAAACCAGTTACTTATAAATTGGATGGTGTTGTTTGTACAGAAATTGACCCTAAGTTGGACAATTATTATAAGAAAGACAATTCTTATTTCACAGAGCAACCAATTGATCTTGTACCAAACCAACCATATCCAAACGCAAGCTTCGATAATTTTAAGTTTGTATGTGATAATATCAAATTTGCTGATGATTTAAACCAGTTAACTGGTTATAAGAAACCTGCTTCAAGAGAGCTTAAAGTTACATTTTTCCCTGACTTAAATGGTGATGTGGTGGCTATTGATTATAAACACTACACACCCTCTTTTAAGAAAGGAGCTAAATTGTTACATAAACCTATTGTTTGGCATGTTAACAATGCAACTAATAAAGCCACGTATAAACCAAATACCTGGTGTATACGTTGTCTTTGGAGCACAAAACCAGTTGAAACATCAAATTCGTTTGATGTACTGAAGTCAGAGGACGCGCAGGGAATGGATAATCTTGCCTGCGAAGATCTAAAACCAGTCTCTGAAGAAGTAGTGGAAAATCCTACCATACAGAAAGACGTTCTTGAGTGTAATGTGAAAACTACCGAAGTTGTAGGAGACATTATACTTAAACCAGCAAATAATAGTTTAAAAATTACAGAAGAGGTTGGCCACACAGATCTAATGGCTGCTTATGTAGACAATTCTAGTCTTACTATTAAGAAACCTAATGAATTATCTAGAGTATTAGGTTTGAAAACCCTTGCTACTCATGGTTTAGCTGCTGTTAATAGTGTCCCTTGGGATACTATAGCTAATTATGCTAAGCCTTTTCTTAACAAAGTTGTTAGTACAACTACTAACATAGTTACACGGTGTTTAAACCGTGTTTGTACTAATTATATGCCTTATTTCTTTACTTTATTGCTACAATTGTGTACTTTTACTAGAAGTACAAATTCTAGAATTAAAGCATCTATGCCGACTACTATAGCAAAGAATACTGTTAAGAGTGTCGGTAAATTTTGTCTAGAGGCTTCATTTAATTATTTGAAGTCACCTAATTTTTCTAAACTGATAAATATTATAATTTGGTTTTTACTATTAAGTGTTTGCCTAGGTTCTTTAATCTACTCAACCGCTGCTTTAGGTGTTTTAATGTCTAATTTAGGCATGCCTTCTTACTGTACTGGTTACAGAGAAGGCTATTTGAACTCTACTAATGTCACTATTGCAACCTACTGTACTGGTTCTATACCTTGTAGTGTTTGTCTTAGTGGTTTAGATTCTTTAGACACCTATCCTTCTTTAGAAACTATACAAATTACCATTTCATCTTTTAAATGGGATTTAACTGCTTTTGGCTTAGTTGCAGAGTGGTTTTTGGCATATATTCTTTTCACTAGGTTTTTCTATGTACTTGGATTGGCTGCAATCATGCAATTGTTTTTCAGCTATTTTGCAGTACATTTTATTAGTAATTCTTGGCTTATGTGGTTAATAATTAATCTTGTACAAATGGCCCCGATTTCAGCTATGGTTAGAATGTACATCTTCTTTGCATCATTTTATTATGTATGGAAAAGTTATGTGCATGTTGTAGACGGTTGTAATTCATCAACTTGTATGATGTGTTACAAACGTAATAGAGCAACAAGAGTCGAATGTACAACTATTGTTAATGGTGTTAGAAGGTCCTTTTATGTCTATGCTAATGGAGGTAAAGGCTTTTGCAAACTACACAATTGGAATTGTGTTAATTGTGATACATTCTGTGCTGGTAGTACATTTATTAGTGATGAAGTTGCGAGAGACTTGTCACTACAGTTTAAAAGACCAATAAATCCTACTGACCAGTCTTCTTACATCGTTGATAGTGTTACAGTGAAGAATGGTTCCATCCATCTTTACTTTGATAAAGCTGGTCAAAAGACTTATGAAAGACATTCTCTCTCTCATTTTGTTAACTTAGACAACCTGAGAGCTAATAACACTAAAGGTTCATTGCCTATTAATGTTATAGTTTTTGATGGTAAATCAAAATGTGAAGAATCATCTGCAAAATCAGCGTCTGTTTACTACAGTCAGCTTATGTGTCAACCTATACTGTTACTAGATCAGGCATTAGTGTCTGATGTTGGTGATAGTGCGGAAGTTGCAGTTAAAATGTTTGATGCTTACGTTAATACGTTTTCATCAACTTTTAACGTACCAATGGAAAAACTCAAAACACTAGTTGCAACTGCAGAAGCTGAACTTGCAAAGAATGTGTCCTTAGACAATGTCTTATCTACTTTTATTTCAGCAGCTCGGCAAGGGTTTGTTGATTCAGATGTAGAAACTAAAGATGTTGTTGAATGTCTTAAATTGTCACATCAATCTGACATAGAAGTTACTGGCGATAGTTGTAATAACTATATGCTCACCTATAACAAAGTTGAAAACATGACACCCCGTGACCTTGGTGCTTGTATTGACTGTAGTGCGCGTCATATTAATGCGCAGGTAGCAAAAAGTCACAACATTGCTTTGATATGGAACGTTAAAGATTTCATGTCATTGTCTGAACAACTACGAAAACAAATACGTAGTGCTGCTAAAAAGAATAACTTACCTTTTAAGTTGACATGTGCAACTACTAGACAAGTTGTTAATGTTGTAACAACAAAGATAGCACTTAAGGGTGGTAAAATTGTTAATAATTGGTTGAAGCAGTTAATTAAAGTTACACTTGTGTTCCTTTTTGTTGCTGCTATTTTCTATTTAATAACACCTGTTCATGTCATGTCTAAACATACTGACTTTTCAAGTGAAATCATAGGATACAAGGCTATTGATGGTGGTGTCACTCGTGACATAGCATCTACAGATACTTGTTTTGCTAACAAACATGCTGATTTTGACACATGGTTTAGTCAGCGTGGTGGTAGTTATACTAATGACAAAGCTTGCCCATTGATTGCTGCAGTCATAACAAGAGAAGTGGGTTTTGTCGTGCCTGGTTTGCCTGGCACGATATTACGCACAACTAATGGTGACTTTTTGCATTTCTTACCTAGAGTTTTTAGTGCAGTTGGTAACATCTGTTACACACCATCAAAACTTATAGAGTACACTGACTTTGCAACATCAGCTTGTGTTTTGGCTGCTGAATGTACAATTTTTAAAGATGCTTCTGGTAAGCCAGTACCATATTGTTATGATACCAATGTACTAGAAGGTTCTGTTGCTTATGAAAGTTTACGCCCTGACACACGTTATGTGCTCATGGATGGCTCTATTATTCAATTTCCTAACACCTACCTTGAAGGTTCTGTTAGAGTGGTAACAACTTTTGATTCTGAGTACTGTAGGCACGGCACTTGTGAAAGATCAGAAGCTGGTGTTTGTGTATCTACTAGTGGTAGATGGGTACTTAACAATGATTATTACAGATCTTTACCAGGAGTTTTCTGTGGTGTAGATGCTGTAAATTTACTTACTAATATGTTTACACCACTAATTCAACCTATTGGTGCTTTGGACATATCAGCATCTATAGTAGCTGGTGGTATTGTAGCTATCGTAGTAACATGCCTTGCCTACTATTTTATGAGGTTTAGAAGAGCTTTTGGTGAATACAGTCATGTAGTTGCCTTTAATACTTTACTATTCCTTATGTCATTCACTGTACTCTGTTTAACACCAGTTTACTCATTCTTACCTGGTGTTTATTCTGTTATTTACTTGTACTTGACATTTTATCTTACTAATGATGTTTCTTTTTTAGCACATATTCAGTGGATGGTTATGTTCACACCTTTAGTACCTTTCTGGATAACAATTGCTTATATCATTTGTATTTCCACAAAGCATTTCTATTGGTTCTTTAGTAATTACCTAAAGAGACGTGTAGTCTTTAATGGTGTTTCCTTTAGTACTTTTGAAGAAGCTGCGCTGTGCACCTTTTTGTTAAATAAAGAAATGTATCTAAAGTTGCGTAGTGATGTGCTATTACCTCTTACGCAATATAATAGATACTTAGCTCTTTATAATAAGTACAAGTATTTTAGTGGAGCAATGGATACAACTAGCTACAGAGAAGCTGCTTGTTGTCATCTCGCAAAGGCTCTCAATGACTTCAGTAACTCAGGTTCTGATGTTCTTTACCAACCACCACAAACCTCTATCACCTCAGCTGTTTTGCAGAGTGGTTTTAGAAAAATGGCATTCCCATCTGGTAAAGTTGAGGGTTGTATGGTACAAGTAACTTGTGGTACAACTACACTTAACGGTCTTTGGCTTGATGACGTAGTTTACTGTCCAAGACATGTGATCTGCACCTCTGAAGACATGCTTAACCCTAATTATGAAGATTTACTCATTCGTAAGTCTAATCATAATTTCTTGGTACAGGCTGGTAATGTTCAACTCAGGGTTATTGGACATTCTATGCAAAATTGTGTACTTAAGCTTAAGGTTGATACAGCCAATCCTAAGACACCTAAGTATAAGTTTGTTCGCATTCAACCAGGACAGACTTTTTCAGTGTTAGCTTGTTACAATGGTTCACCATCTGGTGTTTACCAATGTGCTATGAGGCCCAATTTCACTATTAAGGGTTCATTCCTTAATGGTTCATGTGGTAGTGTTGGTTTTAACATAGATTATGACTGTGTCTCTTTTTGTTACATGCACCATATGGAATTACCAACTGGAGTTCATGCTGGCACAGACTTAGAAGGTAACTTTTATGGACCTTTTGTTGACAGGCAAACAGCACAAGCAGCTGGTACGGACACAACTATTACAGTTAATGTTTTAGCTTGGTTGTACGCTGCTGTTATAAATGGAGACAGGTGGTTTCTCAATCGATTTACCACAACTCTTAATGACTTTAACCTTGTGGCTATGAAGTACAATTATGAACCTCTAACACAAGACCATGTTGACATACTAGGACCTCTTTCTGCTCAAACTGGAATTGCCGTTTTAGATATGTGTGCTTCATTAAAAGAATTACTGCAAAATGGTATGAATGGACGTACCATATTGGGTAGTGCTTTATTAGAAGATGAATTTACACCTTTTGATGTTGTTAGACAATGCTCAGGTGTTACTTTCCAAAGTGCAGTGAAAAGAACAATCAAGGGTACACACCACTGGTTGTTACTCACAATTTTGACTTCACTTTTAGTTTTAGTCCAGAGTACTCAATGGTCTTTGTTCTTTTTTTTGTATGAAAATGCCTTTTTACCTTTTGCTATGGGTATTATTGCTATGTCTGCTTTTGCAATGATGTTTGTCAAACATAAGCATGCATTTCTCTGTTTGTTTTTGTTACCTTCTCTTGCCACTGTAGCTTATTTTAATATGGTCTATATGCCTGCTAGTTGGGTGATGCGTATTATGACATGGTTGGATATGGTTGATACTAGTTTGTCTGGTTTTAAGCTAAAAGACTGTGTTATGTATGCATCAGCTGTAGTGTTACTAATCCTTATGACAGCAAGAACTGTGTATGATGATGGTGCTAGGAGAGTGTGGACACTTATGAATGTCTTGACACTCGTTTATAAAGTTTATTATGGTAATGCTTTAGATCAAGCCATTTCCATGTGGGCTCTTATAATCTCTGTTACTTCTAACTACTCAGGTGTAGTTACAACTGTCATGTTTTTGGCCAGAGGTATTGTTTTTATGTGTGTTGAGTATTGCCCTATTTTCTTCATAACTGGTAATACACTTCAGTGTATAATGCTAGTTTATTGTTTCTTAGGCTATTTTTGTACTTGTTACTTTGGCCTCTTTTGTTTACTCAACCGCTACTTTAGACTGACTCTTGGTGTTTATGATTACTTAGTTTCTACACAGGAGTTTAGATATATGAATTCACAGGGACTACTCCCACCCAAGAATAGCATAGATGCCTTCAAACTCAACATTAAATTGTTGGGTGTTGGTGGCAAACCTTGTATCAAAGTAGCCACTGTACAGTCTAAAATGTCAGATGTAAAGTGCACATCAGTAGTCTTACTCTCAGTTTTGCAACAACTCAGAGTAGAATCATCATCTAAATTGTGGGCTCAATGTGTCCAGTTACACAATGACATTCTCTTAGCTAAAGATACTACTGAAGCCTTTGAAAAAATGGTTTCACTACTTTCTGTTTTGCTTTCCATGCAGGGTGCTGTAGACATAAACAAGCTTTGTGAAGAAATGCTGGACAACAGGGCAACCTTACAAGCTATAGCCTCAGAGTTTAGTTCCCTTCCATCATATGCAGCTTTTGCTACTGCTCAAGAAGCTTATGAGCAGGCTGTTGCTAATGGTGATTCTGAAGTTGTTCTTAAAAAGTTGAAGAAGTCTTTGAATGTGGCTAAATCTGAATTTGACCGTGATGCAGCCATGCAACGTAAGTTGGAAAAGATGGCTGATCAAGCTATGACCCAAATGTATAAACAGGCTAGATCTGAGGACAAGAGGGCAAAAGTTACTAGTGCTATGCAGACAATGCTTTTCACTATGCTTAGAAAGTTGGATAATGATGCACTCAACAACATTATCAACAATGCAAGAGATGGTTGTGTTCCCTTGAACATAATACCTCTTACAACAGCAGCCAAACTAATGGTTGTCATACCAGACTATAACACATATAAAAATACGTGTGATGGTACAACATTTACTTATGCATCAGCATTGTGGGAAATCCAACAGGTTGTAGATGCAGATAGTAAAATTGTTCAACTTAGTGAAATTAGTATGGACAATTCACCTAATTTAGCATGGCCTCTTATTGTAACAGCTTTAAGGGCCAATTCTGCTGTCAAATTACAGAATAATGAGCTTAGTCCTGTTGCACTACGACAGATGTCTTGTGCTGCCGGTACTACACAAACTGCTTGCACTGATGACAATGCGTTAGCTTACTACAACACAACAAAGGGAGGTAGGTTTGTACTTGCACTGTTATCCGATTTACAGGATTTGAAATGGGCTAGATTCCCTAAGAGTGATGGAACTGGTACTATCTATACAGAACTGGAACCACCTTGTAGGTTTGTTACAGACACACCTAAAGGTCCTAAAGTGAAGTATTTATACTTTATTAAAGGATTAAACAACCTAAATAGAGGTATGGTACTTGGTAGTTTAGCTGCCACAGTACGTCTACAAGCTGGTAATGCAACAGAAGTGCCTGCCAATTCAACTGTATTATCTTTCTGTGCTTTTGCTGTAGATGCTGCTAAAGCTTACAAAGATTATCTAGCTAGTGGGGGACAACCAATCACTAATTGTGTTAAGATGTTGTGTACACACACTGGTACTGGTCAGGCAATAACAGTTACACCGGAAGCCAATATGGATCAAGAATCCTTTGGTGGTGCATCGTGTTGTCTGTACTGCCGTTGCCACATAGATCATCCAAATCCTAAAGGATTTTGTGACTTAAAAGGTAAGTATGTACAAATACCTACAACTTGTGCTAATGACCCTGTGGGTTTTACACTTAAAAACACAGTCTGTACCGTCTGCGGTATGTGGAAAGGTTATGGCTGTAGTTGTGATCAACTCCGCGAACCCATGCTTCAGTCAGCTGATGCACAATCGTTTTTAAACGGGTTTGCGGTGTAAGTGCAGCCCGTCTTACACCGTGCGGCACAGGCACTAGTACTGATGTCGTATACAGGGCTTTTGACATCTACAATGATAAAGTAGCTGGTTTTGCTAAATTCCTAAAAACTAATTGTTGTCGCTTCCAAGAAAAGGACGAAGATGACAATTTAATTGATTCTTACTTTGTAGTTAAGAGACACACTTTCTCTAACTACCAACATGAAGAAACAATTTATAATTTACTTAAGGATTGTCCAGCTGTTGCTAAACATGACTTCTTTAAGTTTAGAATAGACGGTGACATGGTACCACATATATCACGTCAACGTCTTACTAAATACACAATGGCAGACCTCGTCTATGCTTTAAGGCATTTTGATGAAGGTAATTGTGACACATTAAAAGAAATACTTGTCACATACAATTGTTGTGATGATGATTATTTCAATAAAAAGGACTGGTATGATTTTGTAGAAAACCCAGATATATTACGCGTATACGCCAACTTAGGTGAACGTGTACGCCAAGCTTTGTTAAAAACAGTACAATTCTGTGATGCCATGCGAAATGCTGGTATTGTTGGTGTACTGACATTAGATAATCAAGATCTCAATGGTAACTGGTATGATTTCGGTGATTTCATACAAACCACGCCAGGTAGTGGAGTTCCTGTTGTAGATTCTTATTATTCATTGTTAATGCCTATATTAACCTTGACCAGGGCTTTAACTGCAGAGTCACATGTTGACACTGACTTAACAAAGCCTTACATTAAGTGGGATTTGTTAAAATATGACTTCACGGAAGAGAGGTTAAAACTCTTTGACCGTTATTTTAAATATTGGGATCAGACATACCACCCAAATTGTGTTAACTGTTTGGATGACAGATGCATTCTGCATTGTGCAAACTTTAATGTTTTATTCTCTACAGTGTTCCCACCTACAAGTTTTGGACCACTAGTGAGAAAAATATTTGTTGATGGTGTTCCATTTGTAGTTTCAACTGGATACCACTTCAGAGAGCTAGGTGTTGTACATAATCAGGATGTAAACTTACATAGCTCTAGACTTAGTTTTAAGGAATTACTTGTGTATGCTGCTGACCCTGCTATGCACGCTGCTTCTGGTAATCTATTACTAGATAAACGCACTACGTGCTTTTCAGTAGCTGCACTTACTAACAATGTTGCTTTTCAAACTGTCAAACCCGGTAATTTTAACAAAGACTTCTATGACTTTGCTGTGTCTAAGGGTTTCTTTAAGGAAGGAAGTTCTGTTGAATTAAAACACTTCTTCTTTGCTCAGGATGGTAATGCTGCTATCAGCGATTATGACTACTATCGTTATAATCTACCAACAATGTGTGATATCAGACAACTACTATTTGTAGTTGAAGTTGTTGATAAGTACTTTGATTGTTACGATGGTGGCTGTATTAATGCTAACCAAGTCATCGTCAACAACCTAGACAAATCAGCTGGTTTTCCATTTAATAAATGGGGTAAGGCTAGACTTTATTATGATTCAATGAGTTATGAGGATCAAGATGCACTTTTCGCATATACAAAACGTAATGTCATCCCTACTATAACTCAAATGAATCTTAAGTATGCCATTAGTGCAAAGAATAGAGCTCGCACCGTAGCTGGTGTCTCTATCTGTAGTACTATGACCAATAGACAGTTTCATCAAAAATTATTGAAATCAATAGCCGCCACTAGAGGAGCTACTGTAGTAATTGGAACAAGCAAATTCTATGGTGGTTGGCACAACATGTTAAAAACTGTTTATAGTGATGTAGAAAACCCTCACCTTATGGGTTGGGATTATCCTAAATGTGATAGAGCCATGCCTAACATGCTTAGAATTATGGCCTCACTTGTTCTTGCTCGCAAACATACAACGTGTTGTAGCTTGTCACACCGTTTCTATAGATTAGCTAATGAGTGTGCTCAAGTATTGAGTGAAATGGTCATGTGTGGCGGTTCACTATATGTTAAACCAGGTGGAACCTCATCAGGAGATGCCACAACTGCTTATGCTAATAGTGTTTTTAACATTTGTCAAGCTGTCACGGCCAATGTTAATGCACTTTTATCTACTGATGGTAACAAAATTGCCGATAAGTATGTCCGCAATTTACAACACAGACTTTATGAGTGTCTCTATAGAAATAGAGATGTTGACACAGACTTTGTGAATGAGTTTTACGCATATTTGCGTAAACATTTCTCAATGATGATACTCTCTGACGATGCTGTTGTGTGTTTCAATAGCACTTATGCATCTCAAGGTCTAGTGGCTAGCATAAAGAACTTTAAGTCAGTTCTTTATTATCAAAACAATGTTTTTATGTCTGAAGCAAAATGTTGGACTGAGACTGACCTTACTAAAGGACCTCATGAATTTTGCTCTCAACATACAATGCTAGTTAAACAGGGTGATGATTATGTGTACCTTCCTTACCCAGATCCATCAAGAATCCTAGGGGCCGGCTGTTTTGTAGATGATATCGTAAAAACAGATGGTACACTTATGATTGAACGGTTCGTGTCTTTAGCTATAGATGCTTACCCACTTACTAAACATCCTAATCAGGAGTATGCTGATGTCTTTCATTTGTACTTACAATACATAAGAAAGCTACATGATGAGTTAACAGGACACATGTTAGACATGTATTCTGTTATGCTTACTAATGATAACACTTCAAGGTATTGGGAACCTGAGTTTTATGAGGCTATGTACACACCGCATACAGTCTTACAGGCTGTTGGGGCTTGTGTTCTTTGCAATTCACAGACTTCATTAAGATGTGGTGCTTGCATACGTAGACCATTCTTATGTTGTAAATGCTGTTACGACCATGTCATATCAACATCACATAAATTAGTCTTGTCTGTTAATCCGTATGTTTGCAATGCTCCAGGTTGTGATGTCACAGATGTGACTCAACTTTACTTAGGAGGTATGAGCTATTATTGTAAATCACATAAACCACCCATTAGTTTTCCATTGTGTGCTAATGGACAAGTTTTTGGTTTATATAAAAATACATGTGTTGGTAGCGATAATGTTACTGACTTTAATGCAATTGCAACATGTGACTGGACAAATGCTGGTGATTACATTTTAGCTAACACCTGTACTGAAAGACTCAAGCTTTTTGCAGCAGAAACGCTCAAAGCTACTGAGGAGACATTTAAACTGTCTTATGGTATTGCTACTGTACGTGAAGTGCTGTCTGACAGAGAATTACATCTTTCATGGGAAGTTGGTAAACCTAGACCACCACTTAACCGAAATTATGTCTTTACTGGTTATCGTGTAACTAAAAACAGTAAAGTACAAATAGGAGAGTACACCTTTGAAAAAGGTGACTATGGTGATGCTGTTGTTTACCGAGGTACAACAACTTACAAATTAAATGTTGGTGATTATTTTGTGCTGACATCACATACAGTAATGCCATTAAGTGCACCTACACTAGTGCCACAAGAGCACTATGTTAGAATTACTGGCTTATACCCAACACTCAATATCTCAGATGAGTTTTCTAGCAATGTTGCAAATTATCAAAAGGTTGGTATGCAAAAGTATTCTACACTCCAGGGACCACCTGGTACTGGTAAGAGTCATTTTGCTATTGGCCTAGCTCTCTACTACCCTTCTGCTCGCATAGTGTATACAGCTTGCTCTCATGCCGCTGTTGATGCACTATGTGAGAAGGCATTAAAATATTTGCCTATAGATAAATGTAGTAGAATTATACCTGCACGTGCTCGTGTAGAGTGTTTTGATAAATTCAAAGTGAATTCAACATTAGAACAGTATGTCTTTTGTACTGTAAATGCATTGCCTGAGACGACAGCAGATATAGTTGTCTTTGATGAAATTTCAATGGCCACAAATTATGATTTGAGTGTTGTCAATGCCAGATTACGTGCTAAGCACTATGTGTACATTGGCGACCCTGCTCAATTACCTGCACCACGCACATTGCTAACTAAGGGCACACTAGAACCAGAATATTTCAATTCAGTGTGTAGACTTATGAAAACTATAGGTCCAGACATGTTCCTCGGAACTTGTCGGCGTTGTCCTGCTGAAATTGTTGACACTGTGAGTGCTTTGGTTTATGATAATAAGCTTAAAGCACATAAAGACAAATCAGCTCAATGCTTTAAAATGTTTTATAAGGGTGTTATCACGCATGATGTTTCATCTGCAATTAACAGGCCACAAATAGGCGTGGTAAGAGAATTCCTTACACGTAACCCTGCTTGGAGAAAAGCTGTCTTTATTTCACCTTATAATTCACAGAATGCTGTAGCCTCAAAGATTTTGGGACTACCAACTCAAACTGTTGATTCATCACAGGGCTCAGAATATGACTATGTCATATTCACTCAAACCACTGAAACAGCTCACTCTTGTAATGTAAACAGATTTAATGTTGCTATTACCAGAGCAAAAGTAGGCATACTTTGCATAATGTCTGATAGAGACCTTTATGACAAGTTGCAATTTACAAGTCTTGAAATTCCACGTAGGAATGTGGCAACTTTACAAGCTGAAAATGTAACAGGACTCTTTAAAGATTGTAGTAAGGTAATCACTGGGTTACATCCTACACAGGCACCTACACACCTCAGTGTTGACACTAAATTCAAAACTGAAGGTTTATGTGTTGACATACCTGGCATACCTAAGGACATGACCTATAGAAGACTCATCTCTATGATGGGTTTTAAAATGAATTATCAAGTTAATGGTTACCCTAACATGTTTATCACCCGCGAAGAAGCTATAAGACATGTACGTGCATGGATTGGCTTCGATGTCGAGGGGTGTCATGCTACTAGAGAAGCTGTTGGTACCAATTTACCTTTACAGCTAGGTTTTTCTACAGGTGTTAACCTAGTTGCTGTACCTACAGGTTATGTTGATACACCTAATAATACAGATTTTTCCAGAGTTAGTGCTAAACCACCGCCTGGAGATCAATTTAAACACCTCACACCACTTATGTACAAAGGACTTCCTTGGAATGTAGTGCGTATAAAGATTGTACAAATGTTAAGTGACACACTTAAAAATCTCTCTGACAGAGTCGTATTTGTCTTATGGGCACATGGCTTTGAGTTGACATCTATGAAGTATTTTGTGAAAATAGGACCTGAGCGCACCTGTTGTCTATGTGATAGACGTGCCACATGCTTTTCCACTGCTTCAGACACTTATGCCTGTTGGCATCATTCTATTGGATTTGATTACGTCTATAATCCGTTTATGATTGATGTTCAACAATGGGGTTTTACAGGTAACCTACAAAGCAACCATGATCTGTATTGTCAAGTCCATGGTAATGCACATGTAGCTAGTTGTGATGCAATCATGACTAGGTGTCTAGCTGTCCACGAGTGCTTTGTTAAGCGTGTTGACTGGACTATTGAATATCCTATAATTGGTGATGAACTGAAGATTAATGCGGCTTGTAGAAAGGTTCAACACATGGTTGTTAAAGCTGCATTATTAGCAGACAAATTCCCAGTTCTTCACGACATTGGTAACCCTAAAGCTATTAAGTGTGTACCTCAAGCTGATGTAGAATGGAAGTTCTATGATGCACAGCCTTGTAGTGACAAAGCTTATAAAATAGAAGAATTATTCTATTCTTATGCCACACATTCTGACAAATTCACAGATGGTGTATGCCTATTTTGGAATTGCAATGTCGATAGATATCCTGCTAATTCCATTGTTTGTAGATTTGACACTAGAGTGCTATCTAACCTTAACTTGCCTGGTTGTGATGGTGGCAGTTTGTATGTAAATAAACATGCATTCCACACACCAGCTTTTGATAAAAGTGCTTTTGTTAATTTAAAACAATTACCATTTTTCTATTACTCTGACAGTCCATGTGAGTCTCATGGAAAACAAGTAGTGTCAGATATAGATTATGTACCACTAAAGTCTGCTACGTGTATAACACGTTGCAATTTAGGTGGTGCTGTCTGTAGACATCATGCTAATGAGTACAGATTGTATCTCGATGCTTATAACATGATGATCTCAGCTGGCTTTAGCTTGTGGGTTTACAAACAATTTGATACTTATAACCTCTGGAACACTTTTACAAGACTTCAGAGTTTAGAAAATGTGGCTTTTAATGTTGTAAATAAGGGACACTTTGATGGACAACAGGGTGAAGTACCAGTTTCTATCATTAATAACACTGTTTACACAAAAGTTGATGGTGTTGATGTAGAATTGTTTGAAAATAAAACAACATTACCTGTTAATGTAGCATTTGAGCTTTGGGCTAAGCGCAACATTAAACCAGTACCAGAGGTGAAAATACTCAATAATTTGGGTGTGGACATTGCTGCTAATACTGTGATCTGGGACTACAAAAGAGATGCTCCAGCACATATATCTACTATTGGTGTTTGTTCTATGACTGACATAGCCAAGAAACCAACTGAAACGATTTGTGCACCACTCACTGTCTTTTTTGATGGTAGAGTTGATGGTCAAGTAGACTTATTTAGAAATGCCCGTAATGGTGTTCTTATTACAGAAGGTAGTGTTAAAGGTTTACAACCATCTGTAGGTCCCAAACAAGCTAGTCTTAATGGAGTCACATTAATTGGAGAAGCCGTAAAAACACAGTTCAATTATTATAAGAAAGTTGATGGTGTTGTCCAACAATTACCTGAAACTTACTTTACTCAGAGTAGAAATTTACAAGAATTTAAACCCAGGAGTCAAATGGAAATTGATTTCTTAGAATTAGCTATGGATGAATTCATTGAACGGTATAAATTAGAAGGCTATGCCTTCGAACATATCGTTTATGGAGATTTTAGTCATAGTCAGTTAGGTGGTTTACATCTACTGATTGGACTAGCTAAACGTTTTAAGGAATCACCTTTTGAATTAGAAGATTTTATTCCTATGGACAGTACAGTTAAAAACTATTTCATAACAGATGCGCAAACAGGTTCATCTAAGTGTGTGTGTTCTGTTATTGATTTATTACTTGATGATTTTGTTGAAATAATAAAATCCCAAGATTTATCTGTAGTTTCTAAGGTTGTCAAAGTGACTATTGACTATACAGAAATTTCATTTATGCTTTGGTGTAAAGATGGCCATGTAGAAACATTTTACCCAAAATTACAATCTAGTCAAGCGTGGCAACCGGGTGTTGCTATGCCTAATCTTTACAAAATGCAAAGAATGCTATTAGAAAAGTGTGACCTTCAAAATTATGGTGATAGTGCAACATTACCTAAAGGCATAATGATGAATGTCGCAAAATATACTCAACTGTGTCAATATTTAAACACATTAACATTAGCTGTACCCTATAATATGAGAGTTATACATTTTGGTGCTGGTTCTGATAAAGGAGTTGCACCAGGTACAGCTGTTTTAAGACAGTGGTTGCCTACGGGTACGCTGCTTGTCGATTCAGATCTTAATGACTTTGTCTCTGATGCAGATTCAACTTTGATTGGTGATTGTGCAACTGTACATACAGCTAATAAATGGGATCTCATTATTAGTGATATGTACGACCCTAAGACTAAAAATGTTACAAAAGAAAATGACTCTAAAGAGGGTTTTTTCACTTACATTTGTGGGTTTATACAACAAAAGCTAGCTCTTGGAGGTTCCGTGGCTATAAAGATAACAGAACATTCTTGGAATGCTGATCTTTATAAGCTCATGGGACACTTCGCATGGTGGACAGCCTTTGTTACTAATGTGAATGCGTCATCATCTGAAGCATTTTTAATTGGATGTAATTATCTTGGCAAACCACGCGAACAAATAGATGGTTATGTCATGCATGCAAATTACATATTTTGGAGGAATACAAATCCAATTCAGTTGTCTTCCTATTCTTTATTTGACATGAGTAAATTTCCCCTTAAATTAAGGGGTACTGCTGTTATGTCTTTAAAAGAAGGTCAAATCAATGATATGATTTTATCTCTTCTTAGTAAAGGTAGACTTATAATTAGAGAAAACAACAGAGTTGTTATTTCTAGTGATGTTCTTGTTAACAACTAAACGAACAATGTTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGTTAATCTTACAACCAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCATTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCAGGATGTTAACTGCACAGAAGTCCCTGTTGCTATTCATGCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGTGCAGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCCGCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTCTATTGCCATACCCACAAATTTTACTATTAGTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAGACATCAGTAGATTGTACAATGTACATTTGTGGTGATTCAACTGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTTTGTACACAATTAAACCGTGCTTTAACTGGAATAGCTGTTGAACAAGACAAAAACACCCAAGAAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCACCAATTAAAGATTTTGGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAAGCAAGAGGTCATTTATTGAAGATCTACTTTTCAACAAAGTGACACTTGCAGATGCTGGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGCTAGAGACCTCATTTGTGCACAAAAGTTTAACGGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAATGATTGCTCAATACACTTCTGCACTGTTAGCGGGTACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATTTAATAGTGCTATTGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGAAAACTTCAAGATGTGGTCAACCAAAATGCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAATTAGAGCTGCAGAAATCAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGTCAGAGTGTGTACTTGGACAATCAAAAAGAGTTGATTTTTGTGGAAAGGGCTATCATCTTATGTCCTTCCCTCAGTCAGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAAAAGAACTTCACAACTGCTCCTGCCATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAGGTGTCTTTGTTTCAAATGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAACCACAAATCATTACTACAGACAACACATTTGTGTCTGGTAACTGTGATGTTGTAATAGGAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGACATCTCTGGCATTAATGCTTCAGTTGTAAACATTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAATTATGCTTTGCTGTATGACCAGTTGCTGTAGTTGTCTCAAGGGCTGTTGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGACTCTGAGCCAGTGCTCAAAGGAGTCAAATTACATTACACATAAACGAACTTATGGATTTGTTTATGAGAATCTTCACAATTGGAACTGTAACTTTGAAGCAAGGTGAAATCAAGGATGCTACTCCTTCAGATTTTGTTCGCGCTACTGCAACGATACCGATACAAGCCTCACTCCCTTTCGGATGGCTTATTGTTGGCGTTGCACTTCTTGCTGTTTTTCAGAGCGCTTCCAAAATCATAACCCTCAAAAAGAGATGGCAACTAGCACTCTCCAAGGGTGTTCACTTTGTTTGCAACTTGCTGTTGTTGTTTGTAACAGTTTACTCACACCTTTTGCTCGTTGCTGCTGGCCTTGAAGCCCCTTTTCTCTATCTTTATGCTTTAGTCTACTTCTTGCAGAGTATAAACTTTGTAAGAATAATAATGAGGCTTTGGCTTTGCTGGAAATGCCGTTCCAAAAACCCATTACTTTATGATGCCAACTATTTTCTTTGCTGGCATACTAATTGTTACGACTATTGTATACCTTACAATAGTGTAACTTCTTCAATTGTCATTACTTCAGGTGATGGCACAACAAGTCCTATTTCTGAACATGACTACCAGATTGGTGGTTATACTGAAAAATGGGAATCTGGAGTAAAAGACTGTGTTGTATTACACAGTTACTTCACTTCAGACTATTACCAGCTGTACTCAACTCAATTGAGTACAGACACTGGTGTTGAACATGTTACCTTCTTCATCTACAATAAAATTGTTGATGAGCCTGAAGAACATGTCCAAATTCACACAATCGACGGTTCATCCGGAGTTGTTAATCCAGTAATGGAACCAATTTATGATGAACCGACGACGACTACTAGCGTGCCTTTGTAAGCACAAGCTGATGAGTACGAACTTATGTACTCATTCGTTTCGGAAGAGACAGGTACGTTAATAGTTAATAGCGTACTTCTTTTTCTTGCTTTCGTGGTATTCTTGCTAGTTACACTAGCCATCCTTACTGCGCATCGATTGTGTGCGTACTGCTGCAATATTGTTAACGTGAGTCTTGTAAAACCTTCTTTTTACGTTTACTCTCGTGTTAAAAATCTGAATTCTTCTAGAGTTCCTGATCTTCTGGTCTAAACGAACTAAATATTATATTAGTTTTTCTGTTTGGAACTTTAATTTTAGCCATGGCAGATTCCAACGGTACTATTACCGTTGAAGAGCTTAAAAAGCTCCTTGAACAATGGAACCTAGTAATAGGTTTCCTATTCCTTACATGGATTTGTCTTCTACAATTTGCCTATGCCAACAGGAATAGGTTTTTGTATATAATTAAGTTAATTTTCCTCTGGCTGTTATGGCCAGTAACTTTAGCTTGTTTTGTGCTTGCTGCTGTTTACAGAATAAATTGGATCACCGGTGGAATTGCTATCGCAATGGCTTGTCTTGTAGGCTTGATGTGGCTCAGCTACTTCATTGCTTCTTTCAGACTGTTTGCGCGTACGCGTTCCATGTGGTCATTCAATCCAGAAACTAACATTCTTCTCAACGTGCCACTCCATGGCACTATTCTGACCAGACCGCTTCTAGAAAGTGAACTCGTAATCGGAGCTGTGATCCTTCGTGGACATCTTCGTATTGCTGGACACCATCTAGGACGCTGTGACATCAAGGACCTGCCTAAAGAAATCACTGTTGCTACATCACGAACGCTTTCTTATTACAAATTGGGAGCTTCGCAGCGTGTAGCAGGTGACTCAGGTTTTGCTGCATACAGTCGCTACAGGATTGGCAACTATAAATTAAACACAGACCATTCCAGTAGCAGTGACAATATTGCTTTGCTTGTACAGTAAGTGACAACAGATGTTTCATCTCGTTGACTTTCAGGTTACTATAGCAGAGATATTACTAATTATTATGAGGACTTTTAAAGTTTCCATTTGGAATCTTGATTACATCATAAACCTCATAATTAAAAATTTATCTAAGTCACTAACTGAGAATAAATATTCTCAATTAGATGAAGAGCAACCAATGGAGATTGATTAAACGAACATGAAAATTATTCTTTTCTTGGCACTGATAACACTCGCTACTTGTGAGCTTTATCACTACCAAGAGTGTGTTAGAGGTACAACAGTACTTTTAAAAGAACCTTGCTCTTCTGGAACATACGAGGGCAATTCACCATTTCATCCTCTAGCTGATAACAAATTTGCACTGACTTGCTTTAGCACTCAATTTGCTTTTGCTTGTCCTGACGGCGTAAAACACGTCTATCAGTTACGTGCCAGATCAGTTTCACCTAAACTGTTCATCAGACAAGAGGAAGTTCAAGAACTTTACTCTCCAATTTTTCTTATTGTTGCGGCAATAGTGTTTATAACACTTTGCTTCACACTCAAAAGAAAGACAGAATGATTGAACTTTCATTAATTGACTTCTATTTGTGCTTTTTAGCCTTTCTGCTATTCCTTGTTTTAATTATGCTTATTATCTTTTGGTTCTCACTTGAACTGCAAGATCATAATGAAACTTGTCACGCCTAAACGAACATGAAATTTCTTGTTTTCTTAGGAATCATCACAACTGTAGCTGCATTTCACCAAGAATGTAGTTTACAGTCATGTACTCAACATCAACCATATGTAGTTGATGACCCGTGTCCTATTCACTTCTATTCTAAATGGTATATTAGAGTAGGAGCTAGAAAATCAGCACCTTTAATTGAATTGTGCGTGGATGAGGCTGGTTCTAAATCACCCATTCAGTACATCGATATCGGTAATTATACAGTTTCCTGTTCACCTTTTACAATTAATTGCCAGGAACCTAAATTGGGTAGTCTTGTAGTGCGTTGTTCGTTCTATGAAGACTTTTTAGAGTATCATGACGTTCGTGTTGTTTTAGATTTCATCTAAACGAACAAACTAAAATGTCTGATAATGGACCCCAAAATCAGCGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGATTCAACTGGCAGTAACCAGAATGGAGAACGCAGTGGGGCGCGATCAAAACAACGTCGGCCCCAAGGTTTACCCAATAATACTGCGTCTTGGTTCACCGCTCTCACTCAACATGGCAAGGAAGACCTTAAATTCCCTCGAGGACAAGGCGTTCCAATTAACACCAATAGCAGTCCAGATGACCAAATTGGCTACTACCGAAGAGCTACCAGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCCCTATGGTGCTAACAAAGACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAGCAGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTCATCACGTAGTCGCAACAGTTCAAGAAATTCAACTCCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGTACTGCCACTAAAGCATACAATGTAACACAAGCTTTCGGCAGACGTGGTCCAGAACAAACCCAAGGAAATTTTGGGGACCAGGAACTAATCAGACAAGGAACTGATTACAAACATTGGCCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAATGTCGCGCATTGGCATGGAAGTCACACCTTCGGGAACGTGGTTGACCTACACAGGTGCCATCAAATTGGATGACAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATAAGCATATTGACGCATACAAAACATTCCCATCAACAGAGCCTAAAAAGGACAAAAAGAAGAAGGCTGATGAAACTCAAGCCTTACCGCAGAGACAGAAGAAACAGCAAACTGTGACTCTTCTTCCTGCTGCAGATTTGGATGATTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGACTCAACTCAGGCCTAAACTCATGCAGACCACACAAGGCAGATGGGCTATATAAACGTTTTCGCTTTTCCGTTTACGATGTATAGTCTACTCTTGTGCAGAATGAATTCTCGTAACTACATAGCACAAGTAGATGTAGTTAACTTTAATCTCACATAGCAATCTTTAATCAGTGTGTAACATTAGGGAGGACTTGAAAGAGCCACCACATTTTCACCGAGGCCACGCGGAGTACGATCGAGTGTACAGTGAACAATGCTAGGGAGAGCTGCCTATATGGAAGAGCCCTAATGTGTAAAATTAATTTTAGTAGTGCTATCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN\n",
"MN908947.3\n",
"ATTAAAGGTTTATACCTTCCCAGGTAACAAACCAACCAACTTTCGATCTCTTGTAGATCTGTTCTCTAAACGAACTTTAAAATCTGTGTGGCTGTCACTCGGCTGCATGCTTAGTGCACTCACGCAGTATAATTAATAACTAATTACTGTCGTTGACAGGACACGAGTAACTCGTCTATCTTCTGCAGGCTGCTTACGGTTTCGTCCGTGTTGCAGCCGATCATCAGCACATCTAGGTTTCGTCCGGGTGTGACCGAAAGGTAAGATGGAGAGCCTTGTCCCTGGTTTCAACGAGAAAACACACGTCCAACTCAGTTTGCCTGTTTTACAGGTTCGCGACGTGCTCGTACGTGGCTTTGGAGACTCCGTGGAGGAGGTCTTATCAGAGGCACGTCAACATCTTAAAGATGGCACTTGTGGCTTAGTAGAAGTTGAAAAAGGCGTTTTGCCTCAACTTGAACAGCCCTATGTGTTCATCAAACGTTCGGATGCTCGAACTGCACCTCATGGTCATGTTATGGTTGAGCTGGTAGCAGAACTCGAAGGCATTCAGTACGGTCGTAGTGGTGAGACACTTGGTGTCCTTGTCCCTCATGTGGGCGAAATACCAGTGGCTTACCGCAAGGTTCTTCTTCGTAAGAACGGTAATAAAGGAGCTGGTGGCCATAGTTACGGCGCCGATCTAAAGTCATTTGACTTAGGCGACGAGCTTGGCACTGATCCTTATGAAGATTTTCAAGAAAACTGGAACACTAAACATAGCAGTGGTGTTACCCGTGAACTCATGCGTGAGCTTAACGGAGGGGCATACACTCGCTATGTCGATAACAACTTCTGTGGCCCTGATGGCTACCCTCTTGAGTGCATTAAAGACCTTCTAGCACGTGCTGGTAAAGCTTCATGCACTTTGTCCGAACAACTGGACTTTATTGACACTAAGAGGGGTGTATACTGCTGCCGTGAACATGAGCATGAAATTGCTTGGTACACGGAACGTTCTGAAAAGAGCTATGAATTGCAGACACCTTTTGAAATTAAATTGGCAAAGAAATTTGACACCTTCAATGGGGAATGTCCAAATTTTGTATTTCCCTTAAATTCCATAATCAAGACTATTCAACCAAGGGTTGAAAAGAAAAAGCTTGATGGCTTTATGGGTAGAATTCGATCTGTCTATCCAGTTGCGTCACCAAATGAATGCAACCAAATGTGCCTTTCAACTCTCATGAAGTGTGATCATTGTGGTGAAACTTCATGGCAGACGGGCGATTTTGTTAAAGCCACTTGCGAATTTTGTGGCACTGAGAATTTGACTAAAGAAGGTGCCACTACTTGTGGTTACTTACCCCAAAATGCTGTTGTTAAAATTTATTGTCCAGCATGTCACAATTCAGAAGTAGGACCTGAGCATAGTCTTGCCGAATACCATAATGAATCTGGCTTGAAAACCATTCTTCGTAAGGGTGGTCGCACTATTGCCTTTGGAGGCTGTGTGTTCTCTTATGTTGGTTGCCATAACAAGTGTGCCTATTGGGTTCCACGTGCTAGCGCTAACATAGGTTGTAACCATACAGGTGTTGTTGGAGAAGGTTCCGAAGGTCTTAATGACAACCTTCTTGAAATACTCCAAAAAGAGAAAGTCAACATCAATATTGTTGGTGACTTTAAACTTAATGAAGAGATCGCCATTATTTTGGCATCTTTTTCTGCTTCCACAAGTGCTTTTGTGGAAACTGTGAAAGGTTTGGATTATAAAGCATTCAAACAAATTGTTGAATCCTGTGGTAATTTTAAAGTTACAAAAGGAAAAGCTAAAAAAGGTGCCTGGAATATTGGTGAACAGAAATCAATACTGAGTCCTCTTTATGCATTTGCATCAGAGGCTGCTCGTGTTGTACGATCAATTTTCTCCCGCACTCTTGAAACTGCTCAAAATTCTGTGCGTGTTTTACAGAAGGCCGCTATAACAATACTAGATGGAATTTCACAGTATTCACTGAGACTCATTGATGCTATGATGTTCACATCTGATTTGGCTACTAACAATCTAGTTGTAATGGCCTACATTACAGGTGGTGTTGTTCAGTTGACTTCGCAGTGGCTAACTAACATCTTTGGCACTGTTTATGAAAAACTCAAACCCGTCCTTGATTGGCTTGAAGAGAAGTTTAAGGAAGGTGTAGAGTTTCTTAGAGACGGTTGGGAAATTGTTAAATTTATCTCAACCTGTGCTTGTGAAATTGTCGGTGGACAAATTGTCACCTGTGCAAAGGAAATTAAGGAGAGTGTTCAGACATTCTTTAAGCTTGTAAATAAATTTTTGGCTTTGTGTGCTGACTCTATCATTATTGGTGGAGCTAAACTTAAAGCCTTGAATTTAGGTGAAACATTTGTCACGCACTCAAAGGGATTGTACAGAAAGTGTGTTAAATCCAGAGAAGAAACTGGCCTACTCATGCCTCTAAAAGCCCCAAAAGAAATTATCTTCTTAGAGGGAGAAACACTTCCCACAGAAGTGTTAACAGAGGAAGTTGTCTTGAAAACTGGTGATTTACAACCATTAGAACAACCTACTAGTGAAGCTGTTGAAGCTCCATTGGTTGGTACACCAGTTTGTATTAACGGGCTTATGTTGCTCGAAATCAAAGACACAGAAAAGTACTGTGCCCTTGCACCTAATATGATGGTAACAAACAATACCTTCACACTCAAAGGCGGTGCACCAACAAAGGTTACTTTTGGTGATGACACTGTGATAGAAGTGCAAGGTTACAAGAGTGTGAATATCACTTTTGAACTTGATGAAAGGATTGATAAAGTACTTAATGAGAAGTGCTCTGCCTATACAGTTGAACTCGGTACAGAAGTAAATGAGTTCGCCTGTGTTGTGGCAGATGCTGTCATAAAAACTTTGCAACCAGTATCTGAATTACTTACACCACTGGGCATTGATTTAGATGAGTGGAGTATGGCTACATACTACTTATTTGATGAGTCTGGTGAGTTTAAATTGGCTTCACATATGTATTGTTCTTTCTACCCTCCAGATGAGGATGAAGAAGAAGGTGATTGTGAAGAAGAAGAGTTTGAGCCATCAACTCAATATGAGTATGGTACTGAAGATGATTACCAAGGTAAACCTTTGGAATTTGGTGCCACTTCTGCTGCTCTTCAACCTGAAGAAGAGCAAGAAGAAGATTGGTTAGATGATGATAGTCAACAAACTGTTGGTCAACAAGACGGCAGTGAGGACAATCAGACAACTACTATTCAAACAATTGTTGAGGTTCAACCTCAATTAGAGATGGAACTTACACCAGTTGTTCAGACTATTGAAGTGAATAGTTTTAGTGGTTATTTAAAACTTACTGACAATGTATACATTAAAAATGCAGACATTGTGGAAGAAGCTAAAAAGGTAAAACCAACAGTGGTTGTTAATGCAGCCAATGTTTACCTTAAACATGGAGGAGGTGTTGCAGGAGCCTTAAATAAGGCTACTAACAATGCCATGCAAGTTGAATCTGATGATTACATAGCTACTAATGGACCACTTAAAGTGGGTGGTAGTTGTGTTTTAAGCGGACACAATCTTGCTAAACACTGTCTTCATGTTGTCGGCCCAAATGTTAACAAAGGTGAAGACATTCAACTTCTTAAGAGTGCTTATGAAAATTTTAATCAGCACGAAGTTCTACTTGCACCATTATTATCAGCTGGTATTTTTGGTGCTGACCCTATACATTCTTTAAGAGTTTGTGTAGATACTGTTCGCACAAATGTCTACTTAGCTGTCTTTGATAAAAATCTCTATGACAAACTTGTTTCAAGCTTTTTGGAAATGAAGAGTGAAAAGCAAGTTGAACAAAAGATCGCTGAGATTCCTAAAGAGGAAGTTAAGCCATTTATAACTGAAAGTAAACCTTCAGTTGAACAGAGAAAACAAGATGATAAGAAAATCAAAGCTTGTGTTGAAGAAGTTACAACAACTCTGGAAGAAACTAAGTTCCTCACAGAAAACTTGTTACTTTATATTGACATTAATGGCAATCTTCATCCAGATTCTGCCACTCTTGTTAGTGACATTGACATCACTTTCTTAAAGAAAGATGCTCCATATATAGTGGGTGATGTTGTTCAAGAGGGTGTTTTAACTGCTGTGGTTATACCTACTAAAAAGGCTGGTGGCACTACTGAAATGCTAGCGAAAGCTTTGAGAAAAGTGCCAACAGACAATTATATAACCACTTACCCGGGTCAGGGTTTAAATGGTTACACTGTAGAGGAGGCAAAGACAGTGCTTAAAAAGTGTAAAAGTGCCTTTTACATTCTACCATCTATTATCTCTAATGAGAAGCAAGAAATTCTTGGAACTGTTTCTTGGAATTTGCGAGAAATGCTTGCACATGCAGAAGAAACACGCAAATTAATGCCTGTCTGTGTGGAAACTAAAGCCATAGTTTCAACTATACAGCGTAAATATAAGGGTATTAAAATACAAGAGGGTGTGGTTGATTATGGTGCTAGATTTTACTTTTACACCAGTAAAACAACTGTAGCGTCACTTATCAACACACTTAACGATCTAAATGAAACTCTTGTTACAATGCCACTTGGCTATGTAACACATGGCTTAAATTTGGAAGAAGCTGCTCGGTATATGAGATCTCTCAAAGTGCCAGCTACAGTTTCTGTTTCTTCACCTGATGCTGTTACAGCGTATAATGGTTATCTTACTTCTTCTTCTAAAACACCTGAAGAACATTTTATTGAAACCATCTCACTTGCTGGTTCCTATAAAGATTGGTCCTATTCTGGACAATCTACACAACTAGGTATAGAATTTCTTAAGAGAGGTGATAAAAGTGTATATTACACTAGTAATCCTACCACATTCCACCTAGATGGTGAAGTTATCACCTTTGACAATCTTAAGACACTTCTTTCTTTGAGAGAAGTGAGGACTATTAAGGTGTTTACAACAGTAGACAACATTAACCTCCACACGCAAGTTGTGGACATGTCAATGACATATGGACAACAGTTTGGTCCAACTTATTTGGATGGAGCTGATGTTACTAAAATAAAACCTCATAATTCACATGAAGGTAAAACATTTTATGTTTTACCTAATGATGACACTCTACGTGTTGAGGCTTTTGAGTACTACCACACAACTGATCCTAGTTTTCTGGGTAGGTACATGTCAGCATTAAATCACACTAAAAAGTGGAAATACCCACAAGTTAATGGTTTAACTTCTATTAAATGGGCAGATAACAACTGTTATCTTGCCACTGCATTGTTAACACTCCAACAAATAGAGTTGAAGTTTAATCCACCTGCTCTACAAGATGCTTATTACAGAGCAAGGGCTGGTGAAGCTGCTAACTTTTGTGCACTTATCTTAGCCTACTGTAATAAGACAGTAGGTGAGTTAGGTGATGTTAGAGAAACAATGAGTTACTTGTTTCAACATGCCAATTTAGATTCTTGCAAAAGAGTCTTGAACGTGGTGTGTAAAACTTGTGGACAACAGCAGACAACCCTTAAGGGTGTAGAAGCTGTTATGTACATGGGCACACTTTCTTATGAACAATTTAAGAAAGGTGTTCAGATACCTTGTACGTGTGGTAAACAAGCTACAAAATATCTAGTACAACAGGAGTCACCTTTTGTTATGATGTCAGCACCACCTGCTCAGTATGAACTTAAGCATGGTACATTTACTTGTGCTAGTGAGTACACTGGTAATTACCAGTGTGGTCACTATAAACATATAACTTCTAAAGAAACTTTGTATTGCATAGACGGTGCTTTACTTACAAAGTCCTCAGAATACAAAGGTCCTATTACGGATGTTTTCTACAAAGAAAACAGTTACACAACAACCATAAAACCAGTTACTTATAAATTGGATGGTGTTGTTTGTACAGAAATTGACCCTAAGTTGGACAATTATTATAAGAAAGACAATTCTTATTTCACAGAGCAACCAATTGATCTTGTACCAAACCAACCATATCCAAACGCAAGCTTCGATAATTTTAAGTTTGTATGTGATAATATCAAATTTGCTGATGATTTAAACCAGTTAACTGGTTATAAGAAACCTGCTTCAAGAGAGCTTAAAGTTACATTTTTCCCTGACTTAAATGGTGATGTGGTGGCTATTGATTATAAACACTACACACCCTCTTTTAAGAAAGGAGCTAAATTGTTACATAAACCTATTGTTTGGCATGTTAACAATGCAACTAATAAAGCCACGTATAAACCAAATACCTGGTGTATACGTTGTCTTTGGAGCACAAAACCAGTTGAAACATCAAATTCGTTTGATGTACTGAAGTCAGAGGACGCGCAGGGAATGGATAATCTTGCCTGCGAAGATCTAAAACCAGTCTCTGAAGAAGTAGTGGAAAATCCTACCATACAGAAAGACGTTCTTGAGTGTAATGTGAAAACTACCGAAGTTGTAGGAGACATTATACTTAAACCAGCAAATAATAGTTTAAAAATTACAGAAGAGGTTGGCCACACAGATCTAATGGCTGCTTATGTAGACAATTCTAGTCTTACTATTAAGAAACCTAATGAATTATCTAGAGTATTAGGTTTGAAAACCCTTGCTACTCATGGTTTAGCTGCTGTTAATAGTGTCCCTTGGGATACTATAGCTAATTATGCTAAGCCTTTTCTTAACAAAGTTGTTAGTACAACTACTAACATAGTTACACGGTGTTTAAACCGTGTTTGTACTAATTATATGCCTTATTTCTTTACTTTATTGCTACAATTGTGTACTTTTACTAGAAGTACAAATTCTAGAATTAAAGCATCTATGCCGACTACTATAGCAAAGAATACTGTTAAGAGTGTCGGTAAATTTTGTCTAGAGGCTTCATTTAATTATTTGAAGTCACCTAATTTTTCTAAACTGATAAATATTATAATTTGGTTTTTACTATTAAGTGTTTGCCTAGGTTCTTTAATCTACTCAACCGCTGCTTTAGGTGTTTTAATGTCTAATTTAGGCATGCCTTCTTACTGTACTGGTTACAGAGAAGGCTATTTGAACTCTACTAATGTCACTATTGCAACCTACTGTACTGGTTCTATACCTTGTAGTGTTTGTCTTAGTGGTTTAGATTCTTTAGACACCTATCCTTCTTTAGAAACTATACAAATTACCATTTCATCTTTTAAATGGGATTTAACTGCTTTTGGCTTAGTTGCAGAGTGGTTTTTGGCATATATTCTTTTCACTAGGTTTTTCTATGTACTTGGATTGGCTGCAATCATGCAATTGTTTTTCAGCTATTTTGCAGTACATTTTATTAGTAATTCTTGGCTTATGTGGTTAATAATTAATCTTGTACAAATGGCCCCGATTTCAGCTATGGTTAGAATGTACATCTTCTTTGCATCATTTTATTATGTATGGAAAAGTTATGTGCATGTTGTAGACGGTTGTAATTCATCAACTTGTATGATGTGTTACAAACGTAATAGAGCAACAAGAGTCGAATGTACAACTATTGTTAATGGTGTTAGAAGGTCCTTTTATGTCTATGCTAATGGAGGTAAAGGCTTTTGCAAACTACACAATTGGAATTGTGTTAATTGTGATACATTCTGTGCTGGTAGTACATTTATTAGTGATGAAGTTGCGAGAGACTTGTCACTACAGTTTAAAAGACCAATAAATCCTACTGACCAGTCTTCTTACATCGTTGATAGTGTTACAGTGAAGAATGGTTCCATCCATCTTTACTTTGATAAAGCTGGTCAAAAGACTTATGAAAGACATTCTCTCTCTCATTTTGTTAACTTAGACAACCTGAGAGCTAATAACACTAAAGGTTCATTGCCTATTAATGTTATAGTTTTTGATGGTAAATCAAAATGTGAAGAATCATCTGCAAAATCAGCGTCTGTTTACTACAGTCAGCTTATGTGTCAACCTATACTGTTACTAGATCAGGCATTAGTGTCTGATGTTGGTGATAGTGCGGAAGTTGCAGTTAAAATGTTTGATGCTTACGTTAATACGTTTTCATCAACTTTTAACGTACCAATGGAAAAACTCAAAACACTAGTTGCAACTGCAGAAGCTGAACTTGCAAAGAATGTGTCCTTAGACAATGTCTTATCTACTTTTATTTCAGCAGCTCGGCAAGGGTTTGTTGATTCAGATGTAGAAACTAAAGATGTTGTTGAATGTCTTAAATTGTCACATCAATCTGACATAGAAGTTACTGGCGATAGTTGTAATAACTATATGCTCACCTATAACAAAGTTGAAAACATGACACCCCGTGACCTTGGTGCTTGTATTGACTGTAGTGCGCGTCATATTAATGCGCAGGTAGCAAAAAGTCACAACATTGCTTTGATATGGAACGTTAAAGATTTCATGTCATTGTCTGAACAACTACGAAAACAAATACGTAGTGCTGCTAAAAAGAATAACTTACCTTTTAAGTTGACATGTGCAACTACTAGACAAGTTGTTAATGTTGTAACAACAAAGATAGCACTTAAGGGTGGTAAAATTGTTAATAATTGGTTGAAGCAGTTAATTAAAGTTACACTTGTGTTCCTTTTTGTTGCTGCTATTTTCTATTTAATAACACCTGTTCATGTCATGTCTAAACATACTGACTTTTCAAGTGAAATCATAGGATACAAGGCTATTGATGGTGGTGTCACTCGTGACATAGCATCTACAGATACTTGTTTTGCTAACAAACATGCTGATTTTGACACATGGTTTAGCCAGCGTGGTGGTAGTTATACTAATGACAAAGCTTGCCCATTGATTGCTGCAGTCATAACAAGAGAAGTGGGTTTTGTCGTGCCTGGTTTGCCTGGCACGATATTACGCACAACTAATGGTGACTTTTTGCATTTCTTACCTAGAGTTTTTAGTGCAGTTGGTAACATCTGTTACACACCATCAAAACTTATAGAGTACACTGACTTTGCAACATCAGCTTGTGTTTTGGCTGCTGAATGTACAATTTTTAAAGATGCTTCTGGTAAGCCAGTACCATATTGTTATGATACCAATGTACTAGAAGGTTCTGTTGCTTATGAAAGTTTACGCCCTGACACACGTTATGTGCTCATGGATGGCTCTATTATTCAATTTCCTAACACCTACCTTGAAGGTTCTGTTAGAGTGGTAACAACTTTTGATTCTGAGTACTGTAGGCACGGCACTTGTGAAAGATCAGAAGCTGGTGTTTGTGTATCTACTAGTGGTAGATGGGTACTTAACAATGATTATTACAGATCTTTACCAGGAGTTTTCTGTGGTGTAGATGCTGTAAATTTACTTACTAATATGTTTACACCACTAATTCAACCTATTGGTGCTTTGGACATATCAGCATCTATAGTAGCTGGTGGTATTGTAGCTATCGTAGTAACATGCCTTGCCTACTATTTTATGAGGTTTAGAAGAGCTTTTGGTGAATACAGTCATGTAGTTGCCTTTAATACTTTACTATTCCTTATGTCATTCACTGTACTCTGTTTAACACCAGTTTACTCATTCTTACCTGGTGTTTATTCTGTTATTTACTTGTACTTGACATTTTATCTTACTAATGATGTTTCTTTTTTAGCACATATTCAGTGGATGGTTATGTTCACACCTTTAGTACCTTTCTGGATAACAATTGCTTATATCATTTGTATTTCCACAAAGCATTTCTATTGGTTCTTTAGTAATTACCTAAAGAGACGTGTAGTCTTTAATGGTGTTTCCTTTAGTACTTTTGAAGAAGCTGCGCTGTGCACCTTTTTGTTAAATAAAGAAATGTATCTAAAGTTGCGTAGTGATGTGCTATTACCTCTTACGCAATATAATAGATACTTAGCTCTTTATAATAAGTACAAGTATTTTAGTGGAGCAATGGATACAACTAGCTACAGAGAAGCTGCTTGTTGTCATCTCGCAAAGGCTCTCAATGACTTCAGTAACTCAGGTTCTGATGTTCTTTACCAACCACCACAAACCTCTATCACCTCAGCTGTTTTGCAGAGTGGTTTTAGAAAAATGGCATTCCCATCTGGTAAAGTTGAGGGTTGTATGGTACAAGTAACTTGTGGTACAACTACACTTAACGGTCTTTGGCTTGATGACGTAGTTTACTGTCCAAGACATGTGATCTGCACCTCTGAAGACATGCTTAACCCTAATTATGAAGATTTACTCATTCGTAAGTCTAATCATAATTTCTTGGTACAGGCTGGTAATGTTCAACTCAGGGTTATTGGACATTCTATGCAAAATTGTGTACTTAAGCTTAAGGTTGATACAGCCAATCCTAAGACACCTAAGTATAAGTTTGTTCGCATTCAACCAGGACAGACTTTTTCAGTGTTAGCTTGTTACAATGGTTCACCATCTGGTGTTTACCAATGTGCTATGAGGCCCAATTTCACTATTAAGGGTTCATTCCTTAATGGTTCATGTGGTAGTGTTGGTTTTAACATAGATTATGACTGTGTCTCTTTTTGTTACATGCACCATATGGAATTACCAACTGGAGTTCATGCTGGCACAGACTTAGAAGGTAACTTTTATGGACCTTTTGTTGACAGGCAAACAGCACAAGCAGCTGGTACGGACACAACTATTACAGTTAATGTTTTAGCTTGGTTGTACGCTGCTGTTATAAATGGAGACAGGTGGTTTCTCAATCGATTTACCACAACTCTTAATGACTTTAACCTTGTGGCTATGAAGTACAATTATGAACCTCTAACACAAGACCATGTTGACATACTAGGACCTCTTTCTGCTCAAACTGGAATTGCCGTTTTAGATATGTGTGCTTCATTAAAAGAATTACTGCAAAATGGTATGAATGGACGTACCATATTGGGTAGTGCTTTATTAGAAGATGAATTTACACCTTTTGATGTTGTTAGACAATGCTCAGGTGTTACTTTCCAAAGTGCAGTGAAAAGAACAATCAAGGGTACACACCACTGGTTGTTACTCACAATTTTGACTTCACTTTTAGTTTTAGTCCAGAGTACTCAATGGTCTTTGTTCTTTTTTTTGTATGAAAATGCCTTTTTACCTTTTGCTATGGGTATTATTGCTATGTCTGCTTTTGCAATGATGTTTGTCAAACATAAGCATGCATTTCTCTGTTTGTTTTTGTTACCTTCTCTTGCCACTGTAGCTTATTTTAATATGGTCTATATGCCTGCTAGTTGGGTGATGCGTATTATGACATGGTTGGATATGGTTGATACTAGTTTGTCTGGTTTTAAGCTAAAAGACTGTGTTATGTATGCATCAGCTGTAGTGTTACTAATCCTTATGACAGCAAGAACTGTGTATGATGATGGTGCTAGGAGAGTGTGGACACTTATGAATGTCTTGACACTCGTTTATAAAGTTTATTATGGTAATGCTTTAGATCAAGCCATTTCCATGTGGGCTCTTATAATCTCTGTTACTTCTAACTACTCAGGTGTAGTTACAACTGTCATGTTTTTGGCCAGAGGTATTGTTTTTATGTGTGTTGAGTATTGCCCTATTTTCTTCATAACTGGTAATACACTTCAGTGTATAATGCTAGTTTATTGTTTCTTAGGCTATTTTTGTACTTGTTACTTTGGCCTCTTTTGTTTACTCAACCGCTACTTTAGACTGACTCTTGGTGTTTATGATTACTTAGTTTCTACACAGGAGTTTAGATATATGAATTCACAGGGACTACTCCCACCCAAGAATAGCATAGATGCCTTCAAACTCAACATTAAATTGTTGGGTGTTGGTGGCAAACCTTGTATCAAAGTAGCCACTGTACAGTCTAAAATGTCAGATGTAAAGTGCACATCAGTAGTCTTACTCTCAGTTTTGCAACAACTCAGAGTAGAATCATCATCTAAATTGTGGGCTCAATGTGTCCAGTTACACAATGACATTCTCTTAGCTAAAGATACTACTGAAGCCTTTGAAAAAATGGTTTCACTACTTTCTGTTTTGCTTTCCATGCAGGGTGCTGTAGACATAAACAAGCTTTGTGAAGAAATGCTGGACAACAGGGCAACCTTACAAGCTATAGCCTCAGAGTTTAGTTCCCTTCCATCATATGCAGCTTTTGCTACTGCTCAAGAAGCTTATGAGCAGGCTGTTGCTAATGGTGATTCTGAAGTTGTTCTTAAAAAGTTGAAGAAGTCTTTGAATGTGGCTAAATCTGAATTTGACCGTGATGCAGCCATGCAACGTAAGTTGGAAAAGATGGCTGATCAAGCTATGACCCAAATGTATAAACAGGCTAGATCTGAGGACAAGAGGGCAAAAGTTACTAGTGCTATGCAGACAATGCTTTTCACTATGCTTAGAAAGTTGGATAATGATGCACTCAACAACATTATCAACAATGCAAGAGATGGTTGTGTTCCCTTGAACATAATACCTCTTACAACAGCAGCCAAACTAATGGTTGTCATACCAGACTATAACACATATAAAAATACGTGTGATGGTACAACATTTACTTATGCATCAGCATTGTGGGAAATCCAACAGGTTGTAGATGCAGATAGTAAAATTGTTCAACTTAGTGAAATTAGTATGGACAATTCACCTAATTTAGCATGGCCTCTTATTGTAACAGCTTTAAGGGCCAATTCTGCTGTCAAATTACAGAATAATGAGCTTAGTCCTGTTGCACTACGACAGATGTCTTGTGCTGCCGGTACTACACAAACTGCTTGCACTGATGACAATGCGTTAGCTTACTACAACACAACAAAGGGAGGTAGGTTTGTACTTGCACTGTTATCCGATTTACAGGATTTGAAATGGGCTAGATTCCCTAAGAGTGATGGAACTGGTACTATCTATACAGAACTGGAACCACCTTGTAGGTTTGTTACAGACACACCTAAAGGTCCTAAAGTGAAGTATTTATACTTTATTAAAGGATTAAACAACCTAAATAGAGGTATGGTACTTGGTAGTTTAGCTGCCACAGTACGTCTACAAGCTGGTAATGCAACAGAAGTGCCTGCCAATTCAACTGTATTATCTTTCTGTGCTTTTGCTGTAGATGCTGCTAAAGCTTACAAAGATTATCTAGCTAGTGGGGGACAACCAATCACTAATTGTGTTAAGATGTTGTGTACACACACTGGTACTGGTCAGGCAATAACAGTTACACCGGAAGCCAATATGGATCAAGAATCCTTTGGTGGTGCATCGTGTTGTCTGTACTGCCGTTGCCACATAGATCATCCAAATCCTAAAGGATTTTGTGACTTAAAAGGTAAGTATGTACAAATACCTACAACTTGTGCTAATGACCCTGTGGGTTTTACACTTAAAAACACAGTCTGTACCGTCTGCGGTATGTGGAAAGGTTATGGCTGTAGTTGTGATCAACTCCGCGAACCCATGCTTCAGTCAGCTGATGCACAATCGTTTTTAAACGGGTTTGCGGTGTAAGTGCAGCCCGTCTTACACCGTGCGGCACAGGCACTAGTACTGATGTCGTATACAGGGCTTTTGACATCTACAATGATAAAGTAGCTGGTTTTGCTAAATTCCTAAAAACTAATTGTTGTCGCTTCCAAGAAAAGGACGAAGATGACAATTTAATTGATTCTTACTTTGTAGTTAAGAGACACACTTTCTCTAACTACCAACATGAAGAAACAATTTATAATTTACTTAAGGATTGTCCAGCTGTTGCTAAACATGACTTCTTTAAGTTTAGAATAGACGGTGACATGGTACCACATATATCACGTCAACGTCTTACTAAATACACAATGGCAGACCTCGTCTATGCTTTAAGGCATTTTGATGAAGGTAATTGTGACACATTAAAAGAAATACTTGTCACATACAATTGTTGTGATGATGATTATTTCAATAAAAAGGACTGGTATGATTTTGTAGAAAACCCAGATATATTACGCGTATACGCCAACTTAGGTGAACGTGTACGCCAAGCTTTGTTAAAAACAGTACAATTCTGTGATGCCATGCGAAATGCTGGTATTGTTGGTGTACTGACATTAGATAATCAAGATCTCAATGGTAACTGGTATGATTTCGGTGATTTCATACAAACCACGCCAGGTAGTGGAGTTCCTGTTGTAGATTCTTATTATTCATTGTTAATGCCTATATTAACCTTGACCAGGGCTTTAACTGCAGAGTCACATGTTGACACTGACTTAACAAAGCCTTACATTAAGTGGGATTTGTTAAAATATGACTTCACGGAAGAGAGGTTAAAACTCTTTGACCGTTATTTTAAATATTGGGATCAGACATACCACCCAAATTGTGTTAACTGTTTGGATGACAGATGCATTCTGCATTGTGCAAACTTTAATGTTTTATTCTCTACAGTGTTCCCACCTACAAGTTTTGGACCACTAGTGAGAAAAATATTTGTTGATGGTGTTCCATTTGTAGTTTCAACTGGATACCACTTCAGAGAGCTAGGTGTTGTACATAATCAGGATGTAAACTTACATAGCTCTAGACTTAGTTTTAAGGAATTACTTGTGTATGCTGCTGACCCTGCTATGCACGCTGCTTCTGGTAATCTATTACTAGATAAACGCACTACGTGCTTTTCAGTAGCTGCACTTACTAACAATGTTGCTTTTCAAACTGTCAAACCCGGTAATTTTAACAAAGACTTCTATGACTTTGCTGTGTCTAAGGGTTTCTTTAAGGAAGGAAGTTCTGTTGAATTAAAACACTTCTTCTTTGCTCAGGATGGTAATGCTGCTATCAGCGATTATGACTACTATCGTTATAATCTACCAACAATGTGTGATATCAGACAACTACTATTTGTAGTTGAAGTTGTTGATAAGTACTTTGATTGTTACGATGGTGGCTGTATTAATGCTAACCAAGTCATCGTCAACAACCTAGACAAATCAGCTGGTTTTCCATTTAATAAATGGGGTAAGGCTAGACTTTATTATGATTCAATGAGTTATGAGGATCAAGATGCACTTTTCGCATATACAAAACGTAATGTCATCCCTACTATAACTCAAATGAATCTTAAGTATGCCATTAGTGCAAAGAATAGAGCTCGCACCGTAGCTGGTGTCTCTATCTGTAGTACTATGACCAATAGACAGTTTCATCAAAAATTATTGAAATCAATAGCCGCCACTAGAGGAGCTACTGTAGTAATTGGAACAAGCAAATTCTATGGTGGTTGGCACAACATGTTAAAAACTGTTTATAGTGATGTAGAAAACCCTCACCTTATGGGTTGGGATTATCCTAAATGTGATAGAGCCATGCCTAACATGCTTAGAATTATGGCCTCACTTGTTCTTGCTCGCAAACATACAACGTGTTGTAGCTTGTCACACCGTTTCTATAGATTAGCTAATGAGTGTGCTCAAGTATTGAGTGAAATGGTCATGTGTGGCGGTTCACTATATGTTAAACCAGGTGGAACCTCATCAGGAGATGCCACAACTGCTTATGCTAATAGTGTTTTTAACATTTGTCAAGCTGTCACGGCCAATGTTAATGCACTTTTATCTACTGATGGTAACAAAATTGCCGATAAGTATGTCCGCAATTTACAACACAGACTTTATGAGTGTCTCTATAGAAATAGAGATGTTGACACAGACTTTGTGAATGAGTTTTACGCATATTTGCGTAAACATTTCTCAATGATGATACTCTCTGACGATGCTGTTGTGTGTTTCAATAGCACTTATGCATCTCAAGGTCTAGTGGCTAGCATAAAGAACTTTAAGTCAGTTCTTTATTATCAAAACAATGTTTTTATGTCTGAAGCAAAATGTTGGACTGAGACTGACCTTACTAAAGGACCTCATGAATTTTGCTCTCAACATACAATGCTAGTTAAACAGGGTGATGATTATGTGTACCTTCCTTACCCAGATCCATCAAGAATCCTAGGGGCCGGCTGTTTTGTAGATGATATCGTAAAAACAGATGGTACACTTATGATTGAACGGTTCGTGTCTTTAGCTATAGATGCTTACCCACTTACTAAACATCCTAATCAGGAGTATGCTGATGTCTTTCATTTGTACTTACAATACATAAGAAAGCTACATGATGAGTTAACAGGACACATGTTAGACATGTATTCTGTTATGCTTACTAATGATAACACTTCAAGGTATTGGGAACCTGAGTTTTATGAGGCTATGTACACACCGCATACAGTCTTACAGGCTGTTGGGGCTTGTGTTCTTTGCAATTCACAGACTTCATTAAGATGTGGTGCTTGCATACGTAGACCATTCTTATGTTGTAAATGCTGTTACGACCATGTCATATCAACATCACATAAATTAGTCTTGTCTGTTAATCCGTATGTTTGCAATGCTCCAGGTTGTGATGTCACAGATGTGACTCAACTTTACTTAGGAGGTATGAGCTATTATTGTAAATCACATAAACCACCCATTAGTTTTCCATTGTGTGCTAATGGACAAGTTTTTGGTTTATATAAAAATACATGTGTTGGTAGCGATAATGTTACTGACTTTAATGCAATTGCAACATGTGACTGGACAAATGCTGGTGATTACATTTTAGCTAACACCTGTACTGAAAGACTCAAGCTTTTTGCAGCAGAAACGCTCAAAGCTACTGAGGAGACATTTAAACTGTCTTATGGTATTGCTACTGTACGTGAAGTGCTGTCTGACAGAGAATTACATCTTTCATGGGAAGTTGGTAAACCTAGACCACCACTTAACCGAAATTATGTCTTTACTGGTTATCGTGTAACTAAAAACAGTAAAGTACAAATAGGAGAGTACACCTTTGAAAAAGGTGACTATGGTGATGCTGTTGTTTACCGAGGTACAACAACTTACAAATTAAATGTTGGTGATTATTTTGTGCTGACATCACATACAGTAATGCCATTAAGTGCACCTACACTAGTGCCACAAGAGCACTATGTTAGAATTACTGGCTTATACCCAACACTCAATATCTCAGATGAGTTTTCTAGCAATGTTGCAAATTATCAAAAGGTTGGTATGCAAAAGTATTCTACACTCCAGGGACCACCTGGTACTGGTAAGAGTCATTTTGCTATTGGCCTAGCTCTCTACTACCCTTCTGCTCGCATAGTGTATACAGCTTGCTCTCATGCCGCTGTTGATGCACTATGTGAGAAGGCATTAAAATATTTGCCTATAGATAAATGTAGTAGAATTATACCTGCACGTGCTCGTGTAGAGTGTTTTGATAAATTCAAAGTGAATTCAACATTAGAACAGTATGTCTTTTGTACTGTAAATGCATTGCCTGAGACGACAGCAGATATAGTTGTCTTTGATGAAATTTCAATGGCCACAAATTATGATTTGAGTGTTGTCAATGCCAGATTACGTGCTAAGCACTATGTGTACATTGGCGACCCTGCTCAATTACCTGCACCACGCACATTGCTAACTAAGGGCACACTAGAACCAGAATATTTCAATTCAGTGTGTAGACTTATGAAAACTATAGGTCCAGACATGTTCCTCGGAACTTGTCGGCGTTGTCCTGCTGAAATTGTTGACACTGTGAGTGCTTTGGTTTATGATAATAAGCTTAAAGCACATAAAGACAAATCAGCTCAATGCTTTAAAATGTTTTATAAGGGTGTTATCACGCATGATGTTTCATCTGCAATTAACAGGCCACAAATAGGCGTGGTAAGAGAATTCCTTACACGTAACCCTGCTTGGAGAAAAGCTGTCTTTATTTCACCTTATAATTCACAGAATGCTGTAGCCTCAAAGATTTTGGGACTACCAACTCAAACTGTTGATTCATCACAGGGCTCAGAATATGACTATGTCATATTCACTCAAACCACTGAAACAGCTCACTCTTGTAATGTAAACAGATTTAATGTTGCTATTACCAGAGCAAAAGTAGGCATACTTTGCATAATGTCTGATAGAGACCTTTATGACAAGTTGCAATTTACAAGTCTTGAAATTCCACGTAGGAATGTGGCAACTTTACAAGCTGAAAATGTAACAGGACTCTTTAAAGATTGTAGTAAGGTAATCACTGGGTTACATCCTACACAGGCACCTACACACCTCAGTGTTGACACTAAATTCAAAACTGAAGGTTTATGTGTTGACATACCTGGCATACCTAAGGACATGACCTATAGAAGACTCATCTCTATGATGGGTTTTAAAATGAATTATCAAGTTAATGGTTACCCTAACATGTTTATCACCCGCGAAGAAGCTATAAGACATGTACGTGCATGGATTGGCTTCGATGTCGAGGGGTGTCATGCTACTAGAGAAGCTGTTGGTACCAATTTACCTTTACAGCTAGGTTTTTCTACAGGTGTTAACCTAGTTGCTGTACCTACAGGTTATGTTGATACACCTAATAATACAGATTTTTCCAGAGTTAGTGCTAAACCACCGCCTGGAGATCAATTTAAACACCTCATACCACTTATGTACAAAGGACTTCCTTGGAATGTAGTGCGTATAAAGATTGTACAAATGTTAAGTGACACACTTAAAAATCTCTCTGACAGAGTCGTATTTGTCTTATGGGCACATGGCTTTGAGTTGACATCTATGAAGTATTTTGTGAAAATAGGACCTGAGCGCACCTGTTGTCTATGTGATAGACGTGCCACATGCTTTTCCACTGCTTCAGACACTTATGCCTGTTGGCATCATTCTATTGGATTTGATTACGTCTATAATCCGTTTATGATTGATGTTCAACAATGGGGTTTTACAGGTAACCTACAAAGCAACCATGATCTGTATTGTCAAGTCCATGGTAATGCACATGTAGCTAGTTGTGATGCAATCATGACTAGGTGTCTAGCTGTCCACGAGTGCTTTGTTAAGCGTGTTGACTGGACTATTGAATATCCTATAATTGGTGATGAACTGAAGATTAATGCGGCTTGTAGAAAGGTTCAACACATGGTTGTTAAAGCTGCATTATTAGCAGACAAATTCCCAGTTCTTCACGACATTGGTAACCCTAAAGCTATTAAGTGTGTACCTCAAGCTGATGTAGAATGGAAGTTCTATGATGCACAGCCTTGTAGTGACAAAGCTTATAAAATAGAAGAATTATTCTATTCTTATGCCACACATTCTGACAAATTCACAGATGGTGTATGCCTATTTTGGAATTGCAATGTCGATAGATATCCTGCTAATTCCATTGTTTGTAGATTTGACACTAGAGTGCTATCTAACCTTAACTTGCCTGGTTGTGATGGTGGCAGTTTGTATGTAAATAAACATGCATTCCACACACCAGCTTTTGATAAAAGTGCTTTTGTTAATTTAAAACAATTACCATTTTTCTATTACTCTGACAGTCCATGTGAGTCTCATGGAAAACAAGTAGTGTCAGATATAGATTATGTACCACTAAAGTCTGCTACGTGTATAACACGTTGCAATTTAGGTGGTGCTGTCTGTAGACATCATGCTAATGAGTACAGATTGTATCTCGATGCTTATAACATGATGATCTCAGCTGGCTTTAGCTTGTGGGTTTACAAACAATTTGATACTTATAACCTCTGGAACACTTTTACAAGACTTCAGAGTTTAGAAAATGTGGCTTTTAATGTTGTAAATAAGGGACACTTTGATGGACAACAGGGTGAAGTACCAGTTTCTATCATTAATAACACTGTTTACACAAAAGTTGATGGTGTTGATGTAGAATTGTTTGAAAATAAAACAACATTACCTGTTAATGTAGCATTTGAGCTTTGGGCTAAGCGCAACATTAAACCAGTACCAGAGGTGAAAATACTCAATAATTTGGGTGTGGACATTGCTGCTAATACTGTGATCTGGGACTACAAAAGAGATGCTCCAGCACATATATCTACTATTGGTGTTTGTTCTATGACTGACATAGCCAAGAAACCAACTGAAACGATTTGTGCACCACTCACTGTCTTTTTTGATGGTAGAGTTGATGGTCAAGTAGACTTATTTAGAAATGCCCGTAATGGTGTTCTTATTACAGAAGGTAGTGTTAAAGGTTTACAACCATCTGTAGGTCCCAAACAAGCTAGTCTTAATGGAGTCACATTAATTGGAGAAGCCGTAAAAACACAGTTCAATTATTATAAGAAAGTTGATGGTGTTGTCCAACAATTACCTGAAACTTACTTTACTCAGAGTAGAAATTTACAAGAATTTAAACCCAGGAGTCAAATGGAAATTGATTTCTTAGAATTAGCTATGGATGAATTCATTGAACGGTATAAATTAGAAGGCTATGCCTTCGAACATATCGTTTATGGAGATTTTAGTCATAGTCAGTTAGGTGGTTTACATCTACTGATTGGACTAGCTAAACGTTTTAAGGAATCACCTTTTGAATTAGAAGATTTTATTCCTATGGACAGTACAGTTAAAAACTATTTCATAACAGATGCGCAAACAGGTTCATCTAAGTGTGTGTGTTCTGTTATTGATTTATTACTTGATGATTTTGTTGAAATAATAAAATCCCAAGATTTATCTGTAGTTTCTAAGGTTGTCAAAGTGACTATTGACTATACAGAAATTTCATTTATGCTTTGGTGTAAAGATGGCCATGTAGAAACATTTTACCCAAAATTACAATCTAGTCAAGCGTGGCAACCGGGTGTTGCTATGCCTAATCTTTACAAAATGCAAAGAATGCTATTAGAAAAGTGTGACCTTCAAAATTATGGTGATAGTGCAACATTACCTAAAGGCATAATGATGAATGTCGCAAAATATACTCAACTGTGTCAATATTTAAACACATTAACATTAGCTGTACCCTATAATATGAGAGTTATACATTTTGGTGCTGGTTCTGATAAAGGAGTTGCACCAGGTACAGCTGTTTTAAGACAGTGGTTGCCTACGGGTACGCTGCTTGTCGATTCAGATCTTAATGACTTTGTCTCTGATGCAGATTCAACTTTGATTGGTGATTGTGCAACTGTACATACAGCTAATAAATGGGATCTCATTATTAGTGATATGTACGACCCTAAGACTAAAAATGTTACAAAAGAAAATGACTCTAAAGAGGGTTTTTTCACTTACATTTGTGGGTTTATACAACAAAAGCTAGCTCTTGGAGGTTCCGTGGCTATAAAGATAACAGAACATTCTTGGAATGCTGATCTTTATAAGCTCATGGGACACTTCGCATGGTGGACAGCCTTTGTTACTAATGTGAATGCGTCATCATCTGAAGCATTTTTAATTGGATGTAATTATCTTGGCAAACCACGCGAACAAATAGATGGTTATGTCATGCATGCAAATTACATATTTTGGAGGAATACAAATCCAATTCAGTTGTCTTCCTATTCTTTATTTGACATGAGTAAATTTCCCCTTAAATTAAGGGGTACTGCTGTTATGTCTTTAAAAGAAGGTCAAATCAATGATATGATTTTATCTCTTCTTAGTAAAGGTAGACTTATAATTAGAGAAAACAACAGAGTTGTTATTTCTAGTGATGTTCTTGTTAACAACTAAACGAACAATGTTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGTTAATCTTACAACCAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATAATGAAAATGGAACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATCTATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCATGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCAGGATGTTAACTGCACAGAAGTCCCTGTTGCTATTCATGCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGTGCAGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCCTCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTCTATTGCCATACCCACAAATTTTACTATTAGTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAGACATCAGTAGATTGTACAATGTACATTTGTGGTGATTCAACTGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTTTGTACACAATTAAACCGTGCTTTAACTGGAATAGCTGTTGAACAAGACAAAAACACCCAAGAAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCACCAATTAAAGATTTTGGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAAGCAAGAGGTCATTTATTGAAGATCTACTTTTCAACAAAGTGACACTTGCAGATGCTGGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGCTAGAGACCTCATTTGTGCACAAAAGTTTAACGGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAATGATTGCTCAATACACTTCTGCACTGTTAGCGGGTACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATTTAATAGTGCTATTGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGAAAACTTCAAGATGTGGTCAACCAAAATGCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCAAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAATTAGAGCTGCAGAAATCAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGTCAGAGTGTGTACTTGGACAATCAAAAAGAGTTGATTTTTGTGGAAAGGGCTATCATCTTATGTCCTTCCCTCAGTCAGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAGAAAAGAACTTCACAACTGCTCCTGCCATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAGGTGTCTTTGTTTCAAATGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAACCACAAATCATTACTACAGACAACACATTTGTGTCTGGTAACTGTGATGTTGTAATAGGAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGACATCTCTGGCATTAATGCTTCAGTTGTAAACATTCAAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAATTATGCTTTGCTGTATGACCAGTTGCTGTAGTTGTCTCAAGGGCTGTTGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGACTCTGAGCCAGTGCTCAAAGGAGTCAAATTACATTACACATAAACGAACTTATGGATTTGTTTATGAGAATCTTCACAATTGGAACTGTAACTTTGAAGCAAGGTGAAATCAAGGATGCTACTCCTTCAGATTTTGTTCGCGCTACTGCAACGATACCGATACAAGCCTCACTCCCTTTCGGATGGCTTATTGTTGGCGTTGCACTTCTTGCTGTTTTTCAGAGCGCTTCCAAAATCATAACCCTCAAAAAGAGATGGCAACTAGCACTCTCCAAGGGTGTTCACTTTGTTTGCAACTTGCTGTTGTTGTTTGTAACAGTTTACTCACACCTTTTGCTCGTTGCTGCTGGCCTTGAAGCCCCTTTTCTCTATCTTTATGCTTTAGTCTACTTCTTGCAGAGTATAAACTTTGTAAGAATAATAATGAGGCTTTGGCTTTGCTGGAAATGCCGTTCCAAAAACCCATTACTTTATGATGCCAACTATTTTCTTTGCTGGCATACTAATTGTTACGACTATTGTATACCTTACAATAGTGTAACTTCTTCAATTGTCATTACTTCAGGTGATGGCACAACAAGTCCTATTTCTGAACATGACTACCAGATTGGTGGTTATACTGAAAAATGGGAATCTGGAGTAAAAGACTGTGTTGTATTACACAGTTACTTCACTTCAGACTATTACCAGCTGTACTCAACTCAATTGAGTACAGACACTGGTGTTGAACATGTTACCTTCTTCATCTACAATAAAATTGTTGATGAGCCTGAAGAACATGTCCAAATTCACACAATCGACGGTTCATCCGGAGTTGTTAATCCAGTAATGGAACCAATTTATGATGAACCGACGACGACTACTAGCGTGCCTTTGTAAGCACAAGCTGATGAGTACGAACTTATGTACTCATTCGTTTCGGAAGAGACAGGTACGTTAATAGTTAATAGCGTACTTCTTTTTCTTGCTTTCGTGGTATTCTTGCTAGTTACACTAGCCATCCTTACTGCGCTTCGATTGTGTGCGTACTGCTGCAATATTGTTAACGTGAGTCTTGTAAAACCTTCTTTTTACGTTTACTCTCGTGTTAAAAATCTGAATTCTTCTAGAGTTCCTGATCTTCTGGTCTAAACGAACTAAATATTATATTAGTTTTTCTGTTTGGAACTTTAATTTTAGCCATGGCAGATTCCAACGGTACTATTACCGTTGAAGAGCTTAAAAAGCTCCTTGAACAATGGAACCTAGTAATAGGTTTCCTATTCCTTACATGGATTTGTCTTCTACAATTTGCCTATGCCAACAGGAATAGGTTTTTGTATATAATTAAGTTAATTTTCCTCTGGCTGTTATGGCCAGTAACTTTAGCTTGTTTTGTGCTTGCTGCTGTTTACAGAATAAATTGGATCACCGGTGGAATTGCTATCGCAATGGCTTGTCTTGTAGGCTTGATGTGGCTCAGCTACTTCATTGCTTCTTTCAGACTGTTTGCGCGTACGCGTTCCATGTGGTCATTCAATCCAGAAACTAACATTCTTCTCAACGTGCCACTCCATGGCACTATTCTGACCAGACCGCTTCTAGAAAGTGAACTCGTAATCGGAGCTGTGATCCTTCGTGGACATCTTCGTATTGCTGGACACCATCTAGGACGCTGTGACATCAAGGACCTGCCTAAAGAAATCACTGTTGCTACATCACGAACGCTTTCTTATTACAAATTGGGAGCTTCGCAGCGTGTAGCAGGTGACTCAGGTTTTGCTGCATACAGTCGCTACAGGATTGGCAACTATAAATTAAACACAGACCATTCCAGTAGCAGTGACAATATTGCTTTGCTTGTACAGTAAGTGACAACAGATGTTTCATCTCGTTGACTTTCAGGTTACTATAGCAGAGATATTACTAATTATTATGAGGACTTTTAAAGTTTCCATTTGGAATCTTGATTACATCATAAACCTCATAATTAAAAATTTATCTAAGTCACTAACTGAGAATAAATATTCTCAATTAGATGAAGAGCAACCAATGGAGATTGATTAAACGAACATGAAAATTATTCTTTTCTTGGCACTGATAACACTCGCTACTTGTGAGCTTTATCACTACCAAGAGTGTGTTAGAGGTACAACAGTACTTTTAAAAGAACCTTGCTCTTCTGGAACATACGAGGGCAATTCACCATTTCATCCTCTAGCTGATAACAAATTTGCACTGACTTGCTTTAGCACTCAATTTGCTTTTGCTTGTCCTGACGGCGTAAAACACGTCTATCAGTTACGTGCCAGATCAGTTTCACCTAAACTGTTCATCAGACAAGAGGAAGTTCAAGAACTTTACTCTCCAATTTTTCTTATTGTTGCGGCAATAGTGTTTATAACACTTTGCTTCACACTCAAAAGAAAGACAGAATGATTGAACTTTCATTAATTGACTTCTATTTGTGCTTTTTAGCCTTTCTGCTATTCCTTGTTTTAATTATGCTTATTATCTTTTGGTTCTCACTTGAACTGCAAGATCATAATGAAACTTGTCACGCCTAAACGAACATGAAATTTCTTGTTTTCTTAGGAATCATCACAACTGTAGCTGCATTTCACCAAGAATGTAGTTTACAGTCATGTACTCAACATCAACCATATGTAGTTGATGACCCGTGTCCTATTCACTTCTATTCTAAATGGTATATTAGAGTAGGAGCTAGAAAATCAGCACCTTTAATTGAATTGTGCGTGGATGAGGCTGGTTCTAAATCACCCATTCAGTACATCGATATCGGTAATTATACAGTTTCCTGTTTACCTTTTACAATTAATTGCCAGGAACCTAAATTGGGTAGTCTTGTAGTGCGTTGTTCGTTCTATGAAGACTTTTTAGAGTATCATGACGTTCGTGTTGTTTTAGATTTCATCTAAACGAACAAACTAAAATGTCTGATAATGGACCCCAAAATCAGCGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGATTCAACTGGCAGTAACCAGAATGGAGAACGCAGTGGGGCGCGATCAAAACAACGTCGGCCCCAAGGTTTACCCAATAATACTGCGTCTTGGTTCACCGCTCTCACTCAACATGGCAAGGAAGACCTTAAATTCCCTCGAGGACAAGGCGTTCCAATTAACACCAATAGCAGTCCAGATGACCAAATTGGCTACTACCGAAGAGCTACCAGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCCCTATGGTGCTAACAAAGACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAGCAGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTCATCACGTAGTCGCAACAGTTCAAGAAATTCAACTCCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGTACTGCCACTAAAGCATACAATGTAACACAAGCTTTCGGCAGACGTGGTCCAGAACAAACCCAAGGAAATTTTGGGGACCAGGAACTAATCAGACAAGGAACTGATTACAAACATTGGCCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAATGTCGCGCATTGGCATGGAAGTCACACCTTCGGGAACGTGGTTGACCTACACAGGTGCCATCAAATTGGATGACAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATAAGCATATTGACGCATACAAAACATTCCCACCAACAGAGCCTAAAAAGGACAAAAAGAAGAAGGCTGATGAAACTCAAGCCTTACCGCAGAGACAGAAGAAACAGCAAACTGTGACTCTTCTTCCTGCTGCAGATTTGGATGATTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGACTCAACTCAGGCCTAAACTCATGCAGACCACACAAGGCAGATGGGCTATATAAACGTTTTCGCTTTTCCGTTTACGATATATAGTCTACTCTTGTGCAGAATGAATTCTCGTAACTACATAGCACAAGTAGATGTAGTTAACTTTAATCTCACATAGCAATCTTTAATCAGTGTGTAACATTAGGGAGGACTTGAAAGAGCCACCACATTTTCACCGAGGCCACGCGGAGTACGATCGAGTGTACAGTGAACAATGCTAGGGAGAGCTGCCTATATGGAAGAGCCCTAATGTGTAAAATTAATTTTAGTAGTGCTATCCCCATGTGATTTTAATAGCTTCTTAGGAGAATGACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA\n"
]
}
],
"source": [
"from Bio import AlignIO\n",
"alignment = AlignIO.read(\"my_example.muscle.out.fasta\", \"fasta\")\n",
"print(\"alignment length= {}\\n\" .format(alignment.get_alignment_length()))\n",
"for record in alignment:\n",
" print(\"{}\\n{}\".format(record.id, record.seq))"
]
},
{
"cell_type": "markdown",
"metadata": {},
"source": [
"## Questions\n",
"\n",
"Now we have reached the end of the example, here are some questions for you to try. You might need to add a few more cells below so that you can use the files and Python that we used earlier to help answer them:\n",
"\n",
"* how many amplicons were classed as low coverage and what was the reported coverage for these?\n",
"* how many variants were identified that were subsequently marked as FAIL?\n",
"* how many variants were incorporated into the consensus genome?\n",
"* how many Ns are in the consensus genome and why might they be there?"
]
},
{
"cell_type": "code",
"execution_count": null,
"metadata": {},
"outputs": [],
"source": []
}
],
"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": "3.7.6"
}
},
"nbformat": 4,
"nbformat_minor": 4
}