Kraken_pathogen.ipynb

{
  "nbformat": 4,
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  "metadata": {
    "colab": {
      "name": "Kraken-microbiom.ipynb",
      "provenance": [],
      "toc_visible": true,
      "include_colab_link": true
    },
    "kernelspec": {
      "name": "python3",
      "display_name": "Python 3"
    }
  },
  "cells": [
    {
      "cell_type": "markdown",
      "metadata": {
        "id": "view-in-github",
        "colab_type": "text"
      },
      "source": [
        "<a href=\"https://colab.research.google.com/github/martin-steinegger/kraken-protocol/blob/main/Kraken_pathogen.ipynb\" target=\"_parent\"><img src=\"https://colab.research.google.com/assets/colab-badge.svg\" alt=\"Open In Colab\"/></a>"
      ]
    },
    {
      "cell_type": "markdown",
      "metadata": {
        "id": "APwVpqYJBx3p"
      },
      "source": [
        "#Metagenome analysis using the Kraken software suite - Pathogen Identification\n",
        "\n",
        "This notebook executes the \"Pathogen Identification\" workflow described in the manuscript \"Metagenome analysis using the Kraken software suite\". To execute this notebook click \"Runtime\" -> \"Run all\"'. Running this whole notebook takes approx. **30-40 minutes**! To see the code click \"Show code\" under the section of interest."
      ]
    },
    {
      "cell_type": "code",
      "metadata": {
        "id": "zCcLpdTan0lx",
        "cellView": "form"
      },
      "source": [
        "#@title Install Software using Conda\n",
        "!wget https://repo.continuum.io/miniconda/Miniconda3-4.5.4-Linux-x86_64.sh\n",
        "!chmod +x Miniconda3-4.5.4-Linux-x86_64.sh\n",
        "!./Miniconda3-4.5.4-Linux-x86_64.sh -b -f -p /usr/local\n",
        "\n",
        "!conda create -yqn tutorial -c conda-forge -c bioconda kraken2 krakentools bracken\n",
        "# in a shell you would run the following, however we want the binaries to be available in this notebook\n",
        "# source activate tutorial\n",
        "conda_path = !source activate tutorial && echo $CONDA_PREFIX:$PATH\n",
        "%set_env PATH=$conda_path"
      ],
      "execution_count": null,
      "outputs": []
    },
    {
      "cell_type": "code",
      "metadata": {
        "id": "oVQBxDeHoDI6",
        "cellView": "form"
      },
      "source": [
        "#@title Download Samples\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/071/SRR12486971/SRR12486971_1.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/071/SRR12486971/SRR12486971_2.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/072/SRR12486972/SRR12486972_1.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/072/SRR12486972/SRR12486972_2.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/074/SRR12486974/SRR12486974_1.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/074/SRR12486974/SRR12486974_2.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/078/SRR12486978/SRR12486978_1.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/078/SRR12486978/SRR12486978_2.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/079/SRR12486979/SRR12486979_1.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/079/SRR12486979/SRR12486979_2.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/081/SRR12486981/SRR12486981_1.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/081/SRR12486981/SRR12486981_2.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/083/SRR12486983/SRR12486983_1.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/083/SRR12486983/SRR12486983_2.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/088/SRR12486988/SRR12486988_1.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/088/SRR12486988/SRR12486988_2.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/089/SRR12486989/SRR12486989_1.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/089/SRR12486989/SRR12486989_2.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/090/SRR12486990/SRR12486990_1.fastq.gz\n",
        "!wget -qnc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/090/SRR12486990/SRR12486990_2.fastq.gz\n",
        "\n",
        "\n",
        "for sample in [\"SRR12486971\", \"SRR12486972\", \"SRR12486974\", \"SRR12486978\", \"SRR12486983\", \"SRR12486988\", \"SRR12486989\", \"SRR12486990\"]: \n",
        "  !gunzip {sample}_1.fastq.gz \n",
        "  !gunzip {sample}_2.fastq.gz"
      ],
      "execution_count": null,
      "outputs": []
    },
    {
      "cell_type": "code",
      "metadata": {
        "id": "a2R65tFYohTY",
        "cellView": "form"
      },
      "source": [
        "#@title Download Reference Database\n",
        "!mkdir protocol_db\n",
        "!wget -qnc https://genome-idx.s3.amazonaws.com/kraken/k2_standard_eupath_20201202.tar.gz\n",
        "!tar xvfz k2_standard_eupath_20201202.tar.gz -C protocol_db"
      ],
      "execution_count": null,
      "outputs": []
    },
    {
      "cell_type": "code",
      "metadata": {
        "id": "X7w0PTT2qUIm",
        "cellView": "form"
      },
      "source": [
        "#@title Classify Reads using Kraken2\n",
        "for sample in [\"SRR12486971\", \"SRR12486972\", \"SRR12486974\", \"SRR12486978\", \"SRR12486979\", \"SRR12486981\", \"SRR12486983\", \"SRR12486988\", \"SRR12486989\", \"SRR12486990\"]: \n",
        "  !kraken2 --db protocol_db --report {sample}.k2report --report-minimizer-data --minimum-hit-groups 3 {sample}_1.fastq {sample}_2.fastq > {sample}.kraken2"
      ],
      "execution_count": null,
      "outputs": []
    },
    {
      "cell_type": "code",
      "metadata": {
        "id": "s7dUekmH8-B-",
        "cellView": "form"
      },
      "source": [
        "#@title Download k2reports\n",
        "!zip report.zip *.k2report\n",
        "from google.colab import files\n",
        "files.download('report.zip') \n",
        "  "
      ],
      "execution_count": null,
      "outputs": []
    },
    {
      "cell_type": "markdown",
      "metadata": {
        "id": "cUcrzBfsJGa3"
      },
      "source": [
        "##Visualizing Sample using Pavian"
      ]
    },
    {
      "cell_type": "code",
      "metadata": {
        "id": "VUK3Dm15rXCf"
      },
      "source": [
        "#Visualizing Sample using Pavian\n",
        "from IPython.display import IFrame\n",
        "IFrame('https://fbreitwieser.shinyapps.io/pavian/', width='100%', height=600)"
      ],
      "execution_count": null,
      "outputs": []
    },
    {
      "cell_type": "code",
      "metadata": {
        "id": "-Afa4tMi-PEU",
        "cellView": "form"
      },
      "source": [
        "#@title Extract Reads from Pathogens (extract_kraken_reads.py)\n",
        "for (sample,taxid) in [(\"SRR12486971\",\"723287\"), (\"SRR12486972\",\"5059\"), (\"SRR12486974\",\"5476\"), (\"SRR12486978\", \"1774\"), (\"SRR12486983\", \"10298\"), (\"SRR12486988\", \"61605\"), (\"SRR12486989\", \"1311\"), (\"SRR12486990\", \"1280\")]: \n",
        "  !extract_kraken_reads.py -k {sample}.kraken2 --include-children -s {sample}_1.fastq -s2 {sample}_2.fastq -t {taxid} -r {sample}.k2report -o {sample}_A.algerae.tid{taxid}.1.fa -o2 {sample}_A.algerae.tid{taxid}.2.fa "
      ],
      "execution_count": null,
      "outputs": []
    }
  ]
}