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 "cells": [
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "## Identification and assembly practical"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "How do we know what our sample is?"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "There are a few easy ways ... \n",
    "\n",
    "   - BLASTing reads\n",
    "   - OneCodex\n",
    "   - Kraken (What's in my Pot?)\n",
    "\n",
    "\n"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "For E. coli we will use the file:\n",
    "    \n",
    "MAP006-1.pass.2D.poRe.fastq\n",
    "\n",
    "For the new genome use:\n",
    "\n",
    "pc1_shigella.tar\n",
    "\n",
    "Start with E. coli!, then move onto Shigella."
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "metadata": {
    "collapsed": true
   },
   "outputs": [],
   "source": []
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "## miniasm"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "We will start by assembling E. coli with miniasm."
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "metadata": {
    "collapsed": true
   },
   "outputs": [],
   "source": [
    "# Install minimap and miniasm (requiring gcc and zlib)\n",
    "git clone https://github.com/lh3/minimap && (cd minimap && make)\n",
    "git clone https://github.com/lh3/miniasm && (cd miniasm && make)\n",
    "# Overlap\n",
    "minimap/minimap -Sw5 -L100 -m0 -t8 MAP006-1.pass.2D.poRe.fastq MAP006-1.pass.2D.poRe.fastq | gzip -1 > reads.paf.gz\n",
    "# Layout\n",
    "miniasm/miniasm -f MAP006-1.pass.2D.poRe.fastq reads.paf.gz > contigs.gfa"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "We need to convert the unitigs file into a FASTA file:"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "metadata": {
    "collapsed": true
   },
   "outputs": [],
   "source": [
    "awk '/^S/{print \">\"$2\"\\n\"$3}' contigs.gfa | fold > contigs.fa"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "Let's use QUAST to explore the assembly quality"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "metadata": {
    "collapsed": true
   },
   "outputs": [],
   "source": [
    "wget \"http://downloads.sourceforge.net/project/quast/quast-3.2.tar.gz?r=http%3A%2F%2Fsourceforge.net%2Fprojects%2Fquast%2Ffiles%2F&ts=1450193070&use_mirror=netcologne\" -O quast.tar.gz"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "metadata": {
    "collapsed": true
   },
   "outputs": [],
   "source": [
    "tar xvfz quast.tar.gz"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "metadata": {
    "collapsed": true
   },
   "outputs": [],
   "source": [
    "sudo apt-get install python-matplotlib"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "metadata": {
    "collapsed": true
   },
   "outputs": [],
   "source": [
    "quast-3.2/quast.py -R NC_000913.3.fa contigs.fa"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "Download the PDF report from ``quast_results/latest``"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "## Hybrid assembly"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "Convert the FASTQ file to FASTA (of course, you could have output FASTA via poretools or poRe)"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "metadata": {
    "collapsed": true
   },
   "outputs": [],
   "source": [
    "seqtk seq -A MAP006-1.pass.2D.poRe.fastq > MAP006-1.pass.2D.poRe.fasta"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "Run Spades using the nanopore data and Illumina data."
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "metadata": {
    "collapsed": true
   },
   "outputs": [],
   "source": [
    "spades.py --only-assembler -k 21,51,71 -1 MiSeq/SRR2627019_1.fastq.gz -2 MiSeq/SRR2627019_2.fastq.gz --nanopore MAP006-1.pass.2D.poRe.fasta -o SPADES &"
   ]
  }
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