0_resources.ipynb

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    "tags": []
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   "source": [
    "# 0. Resources\n",
    "Running PUMATAC requires several files."
   ]
  },
  {
   "cell_type": "markdown",
   "id": "b0c8b758-a394-4430-b3c8-1d8e2b74d71f",
   "metadata": {
    "execution": {
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   "source": [
    "## 1. PUMATAC\n",
    "Clone the latest version of the pipeline from github"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 1,
   "id": "d11ba5ac-3a33-4880-9302-a5cc7d63bf86",
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     "name": "stdout",
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     "text": [
      "cloning PUMATAC\n",
      "Cloning into 'PUMATAC'...\n",
      "remote: Enumerating objects: 761, done.\u001b[K\n",
      "remote: Counting objects: 100% (761/761), done.\u001b[K\n",
      "remote: Compressing objects: 100% (589/589), done.\u001b[K\n",
      "remote: Total 761 (delta 179), reused 693 (delta 128), pack-reused 0\u001b[K\n",
      "Receiving objects: 100% (761/761), 623.70 KiB | 7.25 MiB/s, done.\n",
      "Resolving deltas: 100% (179/179), done.\n"
     ]
    }
   ],
   "source": [
    "if [ ! -d PUMATAC ]; then\n",
    "    echo \"cloning PUMATAC\"\n",
    "    git clone https://github.com/aertslab/PUMATAC\n",
    "else\n",
    "    echo \"pulling PUMATAC\"\n",
    "    cd PUMATAC\n",
    "    git pull\n",
    "    cd ..\n",
    "fi"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "47530163-91f5-40e9-a37c-6c630dce90cb",
   "metadata": {},
   "source": [
    "## 2. PUMATAC dependencies\n",
    "Necessary files such as annotated regions are downloaded from https://resources.aertslab.org/papers/PUMATAC_dependencies/, the rest of this notebook only documents how they were generated."
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
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      "\n",
      "2023-04-25 16:55:07 (1.88 MB/s) - ‘PUMATAC_dependencies/genomes/index.html?C=D;O=D’ saved [1274/1274]\n",
      "\n",
      "--2023-04-25 16:55:07--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/hg38_bwamem2/?C=N;O=D\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
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      "\n",
      "--2023-04-25 16:55:07--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/hg38_bwamem2/?C=M;O=A\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
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      "\n",
      "--2023-04-25 16:55:07--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/hg38_bwamem2/?C=S;O=A\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
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      "\n",
      "--2023-04-25 16:55:07--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/hg38_bwamem2/?C=D;O=A\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
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      "\n",
      "--2023-04-25 16:55:07--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/hg38_bwamem2/genome.fa\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
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      "\n",
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      "\n",
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      "\n",
      "--2023-04-25 16:55:35--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/hg38_bwamem2/genome.fa.0123\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
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      "\n",
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      "\n",
      "--2023-04-25 16:56:30--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/hg38_bwamem2/genome.fa.amb\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
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      "\n",
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      "\n",
      "--2023-04-25 16:56:30--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/hg38_bwamem2/genome.fa.ann\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
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      "\n",
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      "\n",
      "--2023-04-25 16:56:30--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/hg38_bwamem2/genome.fa.bwt.2bit.64\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
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      "\n",
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      "\n",
      "2023-04-25 16:57:57 (110 MB/s) - ‘PUMATAC_dependencies/genomes/hg38_bwamem2/genome.fa.bwt.2bit.64’ saved [10074748356/10074748356]\n",
      "\n",
      "--2023-04-25 16:57:58--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/hg38_bwamem2/genome.fa.pac\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 774980637 (739M) [application/x-ns-proxy-autoconfig]\n",
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      "\n",
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      "\n",
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      "\n",
      "--2023-04-25 16:58:05--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/mm10_bwamem2/?C=N;O=D\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
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      "\n",
      "--2023-04-25 16:58:05--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/mm10_bwamem2/?C=M;O=A\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
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      "\n",
      "--2023-04-25 16:58:05--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/mm10_bwamem2/?C=S;O=A\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
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      "\n",
      "--2023-04-25 16:58:05--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/mm10_bwamem2/?C=D;O=A\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 2148 (2.1K) [text/html]\n",
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      "\n",
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      "\n",
      "--2023-04-25 16:58:05--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/mm10_bwamem2/genome.fa\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 2776387546 (2.6G)\n",
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      "\n",
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      "\n",
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      "\n",
      "--2023-04-25 16:58:30--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/mm10_bwamem2/genome.fa.0123\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 5461743548 (5.1G)\n",
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      "\n",
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      "\n",
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      "\n",
      "--2023-04-25 16:59:19--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/mm10_bwamem2/genome.fa.amb\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 11144 (11K)\n",
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      "\n",
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      "\n",
      "2023-04-25 16:59:19 (32.5 MB/s) - ‘PUMATAC_dependencies/genomes/mm10_bwamem2/genome.fa.amb’ saved [11144/11144]\n",
      "\n",
      "--2023-04-25 16:59:19--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/mm10_bwamem2/genome.fa.ann\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 2645 (2.6K)\n",
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      "\n",
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      "\n",
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      "\n",
      "--2023-04-25 16:59:19--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/mm10_bwamem2/genome.fa.bwt.2bit.64\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 8875333328 (8.3G)\n",
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      "\n",
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      "\n",
      "2023-04-25 17:00:36 (110 MB/s) - ‘PUMATAC_dependencies/genomes/mm10_bwamem2/genome.fa.bwt.2bit.64’ saved [8875333328/8875333328]\n",
      "\n",
      "--2023-04-25 17:00:36--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/genomes/mm10_bwamem2/genome.fa.pac\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 682717945 (651M) [application/x-ns-proxy-autoconfig]\n",
      "Saving to: ‘PUMATAC_dependencies/genomes/mm10_bwamem2/genome.fa.pac’\n",
      "\n",
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      "\n",
      "2023-04-25 17:00:42 (113 MB/s) - ‘PUMATAC_dependencies/genomes/mm10_bwamem2/genome.fa.pac’ saved [682717945/682717945]\n",
      "\n",
      "--2023-04-25 17:00:42--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/jupyter_kernels/?C=N;O=A\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
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      "\n",
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      "\n",
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      "\n",
      "--2023-04-25 17:00:42--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/jupyter_kernels/?C=M;O=D\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 1105 (1.1K) [text/html]\n",
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      "\n",
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      "\n",
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      "\n",
      "--2023-04-25 17:00:42--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/jupyter_kernels/?C=S;O=D\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
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      "\n",
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      "\n",
      "--2023-04-25 17:00:42--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/jupyter_kernels/?C=D;O=D\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 1105 (1.1K) [text/html]\n",
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      "\n",
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      "\n",
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      "\n",
      "--2023-04-25 17:00:42--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/nextflow/?C=N;O=A\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 1527 (1.5K) [text/html]\n",
      "Saving to: ‘PUMATAC_dependencies/nextflow/index.html?C=N;O=A’\n",
      "\n",
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      "\n",
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      "\n",
      "--2023-04-25 17:00:42--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/nextflow/?C=M;O=D\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 1527 (1.5K) [text/html]\n",
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      "\n",
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      "\n",
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      "\n",
      "--2023-04-25 17:00:42--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/nextflow/?C=S;O=D\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 1527 (1.5K) [text/html]\n",
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      "\n",
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      "\n",
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      "\n",
      "--2023-04-25 17:00:42--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/nextflow/?C=D;O=D\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 1527 (1.5K) [text/html]\n",
      "Saving to: ‘PUMATAC_dependencies/nextflow/index.html?C=D;O=D’\n",
      "\n",
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      "\n",
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      "\n",
      "--2023-04-25 17:00:42--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/regions/?C=N;O=A\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 2136 (2.1K) [text/html]\n",
      "Saving to: ‘PUMATAC_dependencies/regions/index.html?C=N;O=A’\n",
      "\n",
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      "\n",
      "2023-04-25 17:00:42 (206 MB/s) - ‘PUMATAC_dependencies/regions/index.html?C=N;O=A’ saved [2136/2136]\n",
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      "\n",
      "FINISHED --2023-04-25 17:00:52--\n",
      "Total wall clock time: 7m 8s\n",
      "Downloaded: 194 files, 44G in 6m 50s (110 MB/s)\n"
     ]
    }
   ],
   "source": [
    "wget -r -np -nH --cut-dirs 2 -R index.html https://resources.aertslab.org/papers/PUMATAC/PUMATAC_dependencies/"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 2,
   "id": "fc35961b-f205-4e13-85d0-544780ff86b9",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2023-05-04T08:55:24.940554Z",
     "iopub.status.busy": "2023-05-04T08:55:24.940291Z",
     "iopub.status.idle": "2023-05-04T08:55:25.060018Z",
     "shell.execute_reply": "2023-05-04T08:55:25.059472Z",
     "shell.execute_reply.started": "2023-05-04T08:55:24.940537Z"
    },
    "tags": []
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "PUMATAC_dependencies/\n",
      "├── cache\n",
      "│   ├── broadinstitute-gatk-4.2.0.0.img\n",
      "│   ├── cache\n",
      "│   ├── cflerin-seurat-4.0.3-plus.copy.sif\n",
      "│   ├── cflerin-seurat-4.0.3-plus.sif\n",
      "│   ├── seurat.sh\n",
      "│   ├── vibsinglecellnf-bap-2021-04-27-3b48f4b.img\n",
      "│   ├── vibsinglecellnf-bwamaptools-bwa-mem2-2.2.1-zlibng-2.0.6.img\n",
      "│   ├── vibsinglecellnf-popscle-2021-05-05-da70fc7.img\n",
      "│   ├── vibsinglecellnf-singlecelltoolkit-2022-04-15-16314db.img\n",
      "│   ├── vibsinglecellnf-singlecelltoolkit-2022-07-07-0638c1d.img\n",
      "│   ├── vibsinglecellnf-sinto-0.7.3.1.img\n",
      "│   └── vibsinglecellnf-trimgalore-0.6.6.img\n",
      "├── calculate_saturation_from_fragments.py\n",
      "├── data\n",
      "│   ├── fixedcells_cto_merged.tsv\n",
      "│   └── fixedcells_general_statistics.tsv\n",
      "├── genomes\n",
      "│   ├── hg38_bwamem2\n",
      "│   └── mm10_bwamem2\n",
      "├── jupyter_kernels\n",
      "│   └── 20230419_pycistopic.sif\n",
      "├── nextflow\n",
      "│   ├── nextflow-21.04.3-all\n",
      "│   ├── nextflow-21.10.4-all\n",
      "│   └── nextflow-22.10.7-all\n",
      "├── regions\n",
      "│   ├── 1000Genomes.wgs.GRCH38.sites.minAF-0.1.freemuxlet-scATAC.vcf.gz\n",
      "│   ├── V2.hg38-rDHS-Unfiltered.blacklisted.bed\n",
      "│   ├── V2.mm10-rDHS-Unfiltered.blacklisted.bed\n",
      "│   ├── hg38-blacklist.v2.bed\n",
      "│   └── mm10-blacklist.v2.bed\n",
      "├── single_cell_toolkit\n",
      "│   ├── README.md\n",
      "│   ├── add_corrected_barcode_to_read_name.sh\n",
      "│   ├── barcard\n",
      "│   ├── barcode_10x_scatac_fastqs.sh\n",
      "│   ├── calculate_saturation_10x_samples.py\n",
      "│   ├── calculate_saturation_from_fragments.py\n",
      "│   ├── correct_barcode_from_fastq.seq\n",
      "│   ├── correct_barcode_from_fastq.sh\n",
      "│   ├── create_fragments_file\n",
      "│   ├── create_scifi_rna_barcode_fastq_for_star_solo.sh\n",
      "│   ├── extract_and_correct_biorad_barcode_in_fastq.seq\n",
      "│   ├── extract_and_correct_biorad_barcode_in_fastq.sh\n",
      "│   ├── extract_and_correct_scalebio_atac_barcode_from_fastq.seq\n",
      "│   ├── extract_and_correct_scalebio_atac_barcode_from_fastq.sh\n",
      "│   ├── extract_and_correct_scifi_rna_barcode_from_fastq.seq\n",
      "│   ├── extract_hydrop_atac_barcode_from_R2_fastq.sh\n",
      "│   ├── filter_star_solo_raw_count_matrix.py\n",
      "│   ├── fix_sra_fastq.sh\n",
      "│   ├── mark_or_remove_duplicates_per_barcode.sh\n",
      "│   ├── run_seq_program.sh\n",
      "│   ├── seq_lib\n",
      "│   └── test\n",
      "└── whitelists\n",
      "    ├── 20200227_HyDrop-ATAC_96x96x96_full_barcodes_no_underscore.txt.gz\n",
      "    ├── 20200227_HyDrop-ATAC_96x96x96_full_barcodes_no_underscore_REVCOMP.txt.gz\n",
      "    ├── 737K-arc-v1.REVCOMP.txt.gz\n",
      "    ├── 737K-arc-v1.txt.gz\n",
      "    ├── 737K-cratac-v1.REVCOMP.txt.gz\n",
      "    ├── 737K-cratac-v1.txt.gz\n",
      "    ├── hydrop_384x384.REVCOMP.txt.gz\n",
      "    ├── hydrop_384x384.txt.gz\n",
      "    └── s3atac_1.whitelist.txt.gz\n",
      "\n",
      "15 directories, 50 files\n"
     ]
    }
   ],
   "source": [
    "tree -L 2 PUMATAC_dependencies/"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "fa12007c-23e9-4675-b4c3-f1c3852f99ff",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2023-04-20T09:17:38.037034Z",
     "iopub.status.busy": "2023-04-20T09:17:38.036752Z",
     "iopub.status.idle": "2023-04-20T09:17:38.141173Z",
     "shell.execute_reply": "2023-04-20T09:17:38.140550Z",
     "shell.execute_reply.started": "2023-04-20T09:17:38.037016Z"
    }
   },
   "source": [
    "## 3. Jupyter kernels\n",
    "For the jupyter notebooks, we need specific versions for some python packages. I have included a singularity image that contains the right versions:"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 2,
   "id": "d5db5cb0-a66c-4d0f-8dea-5c4ab4cdb5ed",
   "metadata": {
    "tags": []
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "/data2/florian/x000_PUMATAC_dependencies/jupyter_kernels/20230504_pycistopic.sif\n"
     ]
    }
   ],
   "source": [
    "KERNEL_PATH=(\"/data2/florian/x000_PUMATAC_dependencies/jupyter_kernels/20230504_pycistopic.sif\")\n",
    "ls $KERNEL_PATH"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "d5aecef8-ae22-4c72-a739-e9bcecd5dac6",
   "metadata": {},
   "source": [
    "We now need to redirect Jupyter to this singularity image. Kernels are usually located in `~/.local/share/jupyter/kernels/`:"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 3,
   "id": "9df63474-c434-4c8f-8c06-64df683beea6",
   "metadata": {
    "tags": []
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "20231114_scenicplus\n",
      "bash\n",
      "cistopic_20230504\n",
      "python3\n"
     ]
    }
   ],
   "source": [
    "KERNELS_DIR=/home/florian/miniforge3/share/jupyter/kernels\n",
    "ls -1 $KERNELS_DIR"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 4,
   "id": "55741521-4029-4074-9940-43e3acef0750",
   "metadata": {},
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "/home/florian/miniforge3/share/jupyter/kernels\n"
     ]
    }
   ],
   "source": [
    "echo $KERNELS_DIR"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "f22b79f0-8fa0-4e6b-a243-c45e025d1ba8",
   "metadata": {},
   "source": [
    "Create a new directory (`cistopic_20230504`) and add the `kernel.json` file. Make sure that the path to the singularity iamge is updated.:"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 5,
   "id": "17e9121d-bf81-419b-99ea-c9b8783a26e9",
   "metadata": {
    "tags": []
   },
   "outputs": [],
   "source": [
    "if [ ! -d $KERNELS_DIR/cistopic_20230504/ ]\n",
    "then\n",
    "    echo \"making new kernels dir\"\n",
    "    mkdir $KERNELS_DIR/cistopic_20230504/\n",
    "fi"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 6,
   "id": "1362fb1b-3f6c-47d3-a7e3-cc96cef7a7b9",
   "metadata": {},
   "outputs": [],
   "source": [
    "NEWKERNELNAME=cistopic_20230504"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "8efb8488-da5e-46e2-a54e-7ea33dbe6dd8",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2023-04-20T14:10:32.187148Z",
     "iopub.status.busy": "2023-04-20T14:10:32.186952Z",
     "iopub.status.idle": "2023-04-20T14:10:32.294153Z",
     "shell.execute_reply": "2023-04-20T14:10:32.293315Z",
     "shell.execute_reply.started": "2023-04-20T14:10:32.187133Z"
    }
   },
   "source": [
    "Then, we will write a new `kernel.json` file there that redirects jupyter to our `KERNEL_PATH`. The `kernel.json` file also requires you to define which mounts it should access (since it executes a `singularity exec` command)."
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 7,
   "id": "bee69141-9624-4563-a8dd-c9fd916ca7c3",
   "metadata": {
    "tags": []
   },
   "outputs": [],
   "source": [
    "MOUNTS=/data,/data2,/data2/florian/tmp/:/tmp"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 8,
   "id": "bc953e64-e572-438d-9070-873c5728809f",
   "metadata": {
    "tags": []
   },
   "outputs": [],
   "source": [
    "cp kernel_template.json kernel.json"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 12,
   "id": "a69dfa34-ec66-4d0b-92b4-a3ed80b1fd09",
   "metadata": {
    "tags": []
   },
   "outputs": [],
   "source": [
    "sed -i \"s+/lustre1/project/stg_00090/scatac_benchmark/0_resources/cistopic_image/20230419_pycistopic.sif+$KERNEL_PATH+g\" \"kernel.json\"\n",
    "sed -i \"s+/staging,/lustre1,/data,/vsc-hard-mounts,/scratch,/scratch/leuven/330/vsc33042/tmp/:/tmp,/local_scratch+$MOUNTS+g\" \"kernel.json\"\n",
    "sed -i \"s+20230419_pycistopic.sif+$NEWKERNELNAME+g\" \"kernel.json\""
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 13,
   "id": "360152a3-9992-4d82-8c1c-68a90a2fa4f2",
   "metadata": {
    "tags": []
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "{\n",
      "    \"argv\": [\n",
      "     \"singularity\",\n",
      "     \"exec\",\n",
      "     \"-B\",\n",
      "     \"/data,/data2,/data2/florian/tmp/:/tmp\",\n",
      "     \"/data2/florian/x000_PUMATAC_dependencies/jupyter_kernels/20230504_pycistopic.sif\",\n",
      "     \"ipython\",\n",
      "     \"kernel\",\n",
      "     \"-f\",\n",
      "     \"{connection_file}\"\n",
      "    ],\n",
      "    \"display_name\": \"cistopic_20230504\",\n",
      "    \"language\": \"python\"\n",
      "}\n"
     ]
    }
   ],
   "source": [
    "cat kernel.json"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "7b347a72-32b9-4c25-939a-d3549589e04d",
   "metadata": {},
   "source": [
    "If all is well, the following should return our jupyter `kernel.json`:"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 14,
   "id": "17353bf2-de26-4469-8222-e53b41e5a2d9",
   "metadata": {
    "tags": []
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "{\n",
      "    \"argv\": [\n",
      "     \"singularity\",\n",
      "     \"exec\",\n",
      "     \"-B\",\n",
      "     \"/data,/data2,/data2/florian/tmp/:/tmp\",\n",
      "     \"/data2/florian/x000_PUMATAC_dependencies/jupyter_kernels/20230504_pycistopic.sif\",\n",
      "     \"ipython\",\n",
      "     \"kernel\",\n",
      "     \"-f\",\n",
      "     \"{connection_file}\"\n",
      "    ],\n",
      "    \"display_name\": \"cistopic_20230504\",\n",
      "    \"language\": \"python\"\n",
      "}\n"
     ]
    }
   ],
   "source": [
    "cat kernel.json > $KERNELS_DIR/cistopic_20230504/kernel.json\n",
    "cat $KERNELS_DIR/cistopic_20230504/kernel.json"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "14cb6b13-c4b2-4afc-b545-223dacd6d639",
   "metadata": {},
   "source": [
    "## 4. Example FASTQ (optional)\n",
    "Example FASTQ used to test this pipeline can be found at https://resources.aertslab.org/papers/"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 3,
   "id": "211b0a1a-0ff5-494e-8d1c-ad9640c130e3",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2023-04-25T14:46:00.616640Z",
     "iopub.status.busy": "2023-04-25T14:46:00.616391Z",
     "iopub.status.idle": "2023-04-25T14:52:09.911378Z",
     "shell.execute_reply": "2023-04-25T14:52:09.910841Z",
     "shell.execute_reply.started": "2023-04-25T14:46:00.616624Z"
    },
    "tags": []
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "--2023-04-25 16:46:00--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/\n",
      "Resolving resources.aertslab.org (resources.aertslab.org)... 134.58.50.9\n",
      "Connecting to resources.aertslab.org (resources.aertslab.org)|134.58.50.9|:443... connected.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 3523 (3.4K) [text/html]\n",
      "Saving to: ‘PUMATAC_example_fastq/index.html.tmp’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   3.44K  --.-KB/s    in 0s      \n",
      "\n",
      "2023-04-25 16:46:00 (213 MB/s) - ‘PUMATAC_example_fastq/index.html.tmp’ saved [3523/3523]\n",
      "\n",
      "Loading robots.txt; please ignore errors.\n",
      "--2023-04-25 16:46:00--  https://resources.aertslab.org/robots.txt\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 404 Not Found\n",
      "2023-04-25 16:46:00 ERROR 404: Not Found.\n",
      "\n",
      "--2023-04-25 16:46:00--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/?C=N;O=D\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 3523 (3.4K) [text/html]\n",
      "Saving to: ‘PUMATAC_example_fastq/index.html?C=N;O=D’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   3.44K  --.-KB/s    in 0s      \n",
      "\n",
      "2023-04-25 16:46:00 (280 MB/s) - ‘PUMATAC_example_fastq/index.html?C=N;O=D’ saved [3523/3523]\n",
      "\n",
      "--2023-04-25 16:46:00--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/?C=M;O=A\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 3523 (3.4K) [text/html]\n",
      "Saving to: ‘PUMATAC_example_fastq/index.html?C=M;O=A’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   3.44K  --.-KB/s    in 0s      \n",
      "\n",
      "2023-04-25 16:46:00 (293 MB/s) - ‘PUMATAC_example_fastq/index.html?C=M;O=A’ saved [3523/3523]\n",
      "\n",
      "--2023-04-25 16:46:00--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/?C=S;O=A\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 3523 (3.4K) [text/html]\n",
      "Saving to: ‘PUMATAC_example_fastq/index.html?C=S;O=A’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   3.44K  --.-KB/s    in 0s      \n",
      "\n",
      "2023-04-25 16:46:00 (307 MB/s) - ‘PUMATAC_example_fastq/index.html?C=S;O=A’ saved [3523/3523]\n",
      "\n",
      "--2023-04-25 16:46:00--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/?C=D;O=A\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 3523 (3.4K) [text/html]\n",
      "Saving to: ‘PUMATAC_example_fastq/index.html?C=D;O=A’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   3.44K  --.-KB/s    in 0s      \n",
      "\n",
      "2023-04-25 16:46:00 (307 MB/s) - ‘PUMATAC_example_fastq/index.html?C=D;O=A’ saved [3523/3523]\n",
      "\n",
      "--2023-04-25 16:46:00--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/BIO_ddseq_4__R1.LIBDS.fastq.gz\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 7563240378 (7.0G) [application/x-gzip]\n",
      "Saving to: ‘PUMATAC_example_fastq/BIO_ddseq_4__R1.LIBDS.fastq.gz’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   7.04G   112MB/s    in 65s     \n",
      "\n",
      "2023-04-25 16:47:05 (111 MB/s) - ‘PUMATAC_example_fastq/BIO_ddseq_4__R1.LIBDS.fastq.gz’ saved [7563240378/7563240378]\n",
      "\n",
      "--2023-04-25 16:47:05--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/BIO_ddseq_4__R2.LIBDS.fastq.gz\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 3267349864 (3.0G) [application/x-gzip]\n",
      "Saving to: ‘PUMATAC_example_fastq/BIO_ddseq_4__R2.LIBDS.fastq.gz’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   3.04G   112MB/s    in 28s     \n",
      "\n",
      "2023-04-25 16:47:34 (110 MB/s) - ‘PUMATAC_example_fastq/BIO_ddseq_4__R2.LIBDS.fastq.gz’ saved [3267349864/3267349864]\n",
      "\n",
      "--2023-04-25 16:47:34--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/EPF_hydrop_1__R1.LIBDS.fastq.gz\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 4023774227 (3.7G) [application/x-gzip]\n",
      "Saving to: ‘PUMATAC_example_fastq/EPF_hydrop_1__R1.LIBDS.fastq.gz’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   3.75G   111MB/s    in 34s     \n",
      "\n",
      "2023-04-25 16:48:09 (111 MB/s) - ‘PUMATAC_example_fastq/EPF_hydrop_1__R1.LIBDS.fastq.gz’ saved [4023774227/4023774227]\n",
      "\n",
      "--2023-04-25 16:48:09--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/EPF_hydrop_1__R2.LIBDS.fastq.gz\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 2515119027 (2.3G) [application/x-gzip]\n",
      "Saving to: ‘PUMATAC_example_fastq/EPF_hydrop_1__R2.LIBDS.fastq.gz’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   2.34G   111MB/s    in 22s     \n",
      "\n",
      "2023-04-25 16:48:30 (111 MB/s) - ‘PUMATAC_example_fastq/EPF_hydrop_1__R2.LIBDS.fastq.gz’ saved [2515119027/2515119027]\n",
      "\n",
      "--2023-04-25 16:48:30--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/EPF_hydrop_1__R3.LIBDS.fastq.gz\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 3986089150 (3.7G) [application/x-gzip]\n",
      "Saving to: ‘PUMATAC_example_fastq/EPF_hydrop_1__R3.LIBDS.fastq.gz’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   3.71G   112MB/s    in 35s     \n",
      "\n",
      "2023-04-25 16:49:05 (109 MB/s) - ‘PUMATAC_example_fastq/EPF_hydrop_1__R3.LIBDS.fastq.gz’ saved [3986089150/3986089150]\n",
      "\n",
      "--2023-04-25 16:49:05--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/OHS_s3atac_1__R1.LIBDS.fastq.gz\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 5281970908 (4.9G) [application/x-gzip]\n",
      "Saving to: ‘PUMATAC_example_fastq/OHS_s3atac_1__R1.LIBDS.fastq.gz’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   4.92G   112MB/s    in 45s     \n",
      "\n",
      "2023-04-25 16:49:51 (112 MB/s) - ‘PUMATAC_example_fastq/OHS_s3atac_1__R1.LIBDS.fastq.gz’ saved [5281970908/5281970908]\n",
      "\n",
      "--2023-04-25 16:49:51--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/OHS_s3atac_1__R2.LIBDS.fastq.gz\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 944812182 (901M) [application/x-gzip]\n",
      "Saving to: ‘PUMATAC_example_fastq/OHS_s3atac_1__R2.LIBDS.fastq.gz’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>] 901.04M   112MB/s    in 8.0s    \n",
      "\n",
      "2023-04-25 16:49:59 (112 MB/s) - ‘PUMATAC_example_fastq/OHS_s3atac_1__R2.LIBDS.fastq.gz’ saved [944812182/944812182]\n",
      "\n",
      "--2023-04-25 16:49:59--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/OHS_s3atac_1__R3.LIBDS.fastq.gz\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 5164240677 (4.8G) [application/x-gzip]\n",
      "Saving to: ‘PUMATAC_example_fastq/OHS_s3atac_1__R3.LIBDS.fastq.gz’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   4.81G   112MB/s    in 45s     \n",
      "\n",
      "2023-04-25 16:50:44 (110 MB/s) - ‘PUMATAC_example_fastq/OHS_s3atac_1__R3.LIBDS.fastq.gz’ saved [5164240677/5164240677]\n",
      "\n",
      "--2023-04-25 16:50:44--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/VIB_10xv2_1__R1.LIBDS.fastq.gz\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 3770256614 (3.5G) [application/x-gzip]\n",
      "Saving to: ‘PUMATAC_example_fastq/VIB_10xv2_1__R1.LIBDS.fastq.gz’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   3.51G   111MB/s    in 33s     \n",
      "\n",
      "2023-04-25 16:51:17 (109 MB/s) - ‘PUMATAC_example_fastq/VIB_10xv2_1__R1.LIBDS.fastq.gz’ saved [3770256614/3770256614]\n",
      "\n",
      "--2023-04-25 16:51:17--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/VIB_10xv2_1__R2.LIBDS.fastq.gz\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 1944035765 (1.8G) [application/x-gzip]\n",
      "Saving to: ‘PUMATAC_example_fastq/VIB_10xv2_1__R2.LIBDS.fastq.gz’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   1.81G   112MB/s    in 18s     \n",
      "\n",
      "2023-04-25 16:51:35 (102 MB/s) - ‘PUMATAC_example_fastq/VIB_10xv2_1__R2.LIBDS.fastq.gz’ saved [1944035765/1944035765]\n",
      "\n",
      "--2023-04-25 16:51:35--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/VIB_10xv2_1__R3.LIBDS.fastq.gz\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 3793794636 (3.5G) [application/x-gzip]\n",
      "Saving to: ‘PUMATAC_example_fastq/VIB_10xv2_1__R3.LIBDS.fastq.gz’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   3.53G   111MB/s    in 34s     \n",
      "\n",
      "2023-04-25 16:52:09 (108 MB/s) - ‘PUMATAC_example_fastq/VIB_10xv2_1__R3.LIBDS.fastq.gz’ saved [3793794636/3793794636]\n",
      "\n",
      "--2023-04-25 16:52:09--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/?C=N;O=A\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 3523 (3.4K) [text/html]\n",
      "Saving to: ‘PUMATAC_example_fastq/index.html?C=N;O=A’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   3.44K  --.-KB/s    in 0s      \n",
      "\n",
      "2023-04-25 16:52:09 (262 MB/s) - ‘PUMATAC_example_fastq/index.html?C=N;O=A’ saved [3523/3523]\n",
      "\n",
      "--2023-04-25 16:52:09--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/?C=M;O=D\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 3523 (3.4K) [text/html]\n",
      "Saving to: ‘PUMATAC_example_fastq/index.html?C=M;O=D’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   3.44K  --.-KB/s    in 0s      \n",
      "\n",
      "2023-04-25 16:52:09 (169 MB/s) - ‘PUMATAC_example_fastq/index.html?C=M;O=D’ saved [3523/3523]\n",
      "\n",
      "--2023-04-25 16:52:09--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/?C=S;O=D\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 3523 (3.4K) [text/html]\n",
      "Saving to: ‘PUMATAC_example_fastq/index.html?C=S;O=D’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   3.44K  --.-KB/s    in 0s      \n",
      "\n",
      "2023-04-25 16:52:09 (303 MB/s) - ‘PUMATAC_example_fastq/index.html?C=S;O=D’ saved [3523/3523]\n",
      "\n",
      "--2023-04-25 16:52:09--  https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/?C=D;O=D\n",
      "Reusing existing connection to resources.aertslab.org:443.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 3523 (3.4K) [text/html]\n",
      "Saving to: ‘PUMATAC_example_fastq/index.html?C=D;O=D’\n",
      "\n",
      "PUMATAC_example_fas 100%[===================>]   3.44K  --.-KB/s    in 0s      \n",
      "\n",
      "2023-04-25 16:52:09 (302 MB/s) - ‘PUMATAC_example_fastq/index.html?C=D;O=D’ saved [3523/3523]\n",
      "\n",
      "FINISHED --2023-04-25 16:52:09--\n",
      "Total wall clock time: 6m 9s\n",
      "Downloaded: 20 files, 39G in 6m 7s (110 MB/s)\n"
     ]
    }
   ],
   "source": [
    "wget -r -np -nH --cut-dirs 2 -R index.html https://resources.aertslab.org/papers/PUMATAC/PUMATAC_example_fastq/"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 4,
   "id": "5a77ebd9-b459-4b5b-b216-1b74bb42c712",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2023-04-25T14:52:09.912401Z",
     "iopub.status.busy": "2023-04-25T14:52:09.912206Z",
     "iopub.status.idle": "2023-04-25T14:53:44.924884Z",
     "shell.execute_reply": "2023-04-25T14:53:44.924390Z",
     "shell.execute_reply.started": "2023-04-25T14:52:09.912385Z"
    },
    "tags": []
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "0c8b78a5687d117f71d04a090eeffa80  PUMATAC_example_fastq/BIO_ddseq_4__R1.LIBDS.fastq.gz\n",
      "6a5319b566331e1c2cb92e0950863075  PUMATAC_example_fastq/BIO_ddseq_4__R2.LIBDS.fastq.gz\n",
      "0a9f8d48581694a85dc5b12a54fe7e59  PUMATAC_example_fastq/EPF_hydrop_1__R1.LIBDS.fastq.gz\n",
      "9001b4deff9eef9d6c605eadae7dff92  PUMATAC_example_fastq/EPF_hydrop_1__R2.LIBDS.fastq.gz\n",
      "b3300d28ed425417dc21d796d7a8e623  PUMATAC_example_fastq/EPF_hydrop_1__R3.LIBDS.fastq.gz\n",
      "9fa3339b8aea6b7b2f9ef4d4bf73a086  PUMATAC_example_fastq/OHS_s3atac_1__R1.LIBDS.fastq.gz\n",
      "f08b6c814abb4d3c25329dbc2f6a67ab  PUMATAC_example_fastq/OHS_s3atac_1__R2.LIBDS.fastq.gz\n",
      "448652e81c27d2000fca275bc7f94990  PUMATAC_example_fastq/OHS_s3atac_1__R3.LIBDS.fastq.gz\n",
      "dbc5f0fd828f9f81a9f2b77642395c0b  PUMATAC_example_fastq/VIB_10xv2_1__R1.LIBDS.fastq.gz\n",
      "2e00f84113992335632517f67703adf9  PUMATAC_example_fastq/VIB_10xv2_1__R2.LIBDS.fastq.gz\n",
      "9d39e83afc86000c8fda42da4e69e4db  PUMATAC_example_fastq/VIB_10xv2_1__R3.LIBDS.fastq.gz\n",
      "eb2c490a8c21c1ba9a602d0300fad076  PUMATAC_example_fastq/index.html.tmp\n",
      "ddeca777b5a429eb42308c80a3b7691f  PUMATAC_example_fastq/index.html?C=D;O=A\n",
      "87770b39885d7ccfe97e70f9886b9071  PUMATAC_example_fastq/index.html?C=D;O=D\n",
      "2c7529fee9a0a90c8bd717712c8ffe55  PUMATAC_example_fastq/index.html?C=M;O=A\n",
      "3638adc575f4e02513c6992845feee0a  PUMATAC_example_fastq/index.html?C=M;O=D\n",
      "eb2c490a8c21c1ba9a602d0300fad076  PUMATAC_example_fastq/index.html?C=N;O=A\n",
      "87770b39885d7ccfe97e70f9886b9071  PUMATAC_example_fastq/index.html?C=N;O=D\n",
      "a75127c29d2d0defc703b3da7ffe2130  PUMATAC_example_fastq/index.html?C=S;O=A\n",
      "09bb9a81e390607f4aa34640bec1277d  PUMATAC_example_fastq/index.html?C=S;O=D\n"
     ]
    }
   ],
   "source": [
    "md5sum PUMATAC_example_fastq/*"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "977bad50-f498-4fb2-b2b6-338d21bd97cf",
   "metadata": {},
   "source": [
    "# 5. Gert Hulselman's single_cell_toolkit (Optional)"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 13,
   "id": "44a87848-6929-4ba0-9a8f-5facf3ec7a17",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2023-04-25T15:00:53.798318Z",
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     "shell.execute_reply": "2023-04-25T15:00:56.622089Z",
     "shell.execute_reply.started": "2023-04-25T15:00:53.798304Z"
    },
    "tags": []
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "pulling single_cell_toolkit\n",
      "remote: Enumerating objects: 9, done.\u001b[K\n",
      "remote: Counting objects: 100% (9/9), done.\u001b[K\n",
      "remote: Compressing objects: 100% (3/3), done.\u001b[K\n",
      "remote: Total 6 (delta 4), reused 5 (delta 3), pack-reused 0\u001b[K\n",
      "Unpacking objects: 100% (6/6), 707 bytes | 21.00 KiB/s, done.\n",
      "From https://github.com/aertslab/single_cell_toolkit\n",
      "   df625f0..ef1962c  master     -> origin/master\n",
      "Updating df625f0..ef1962c\n",
      "error: Your local changes to the following files would be overwritten by merge:\n",
      "\tcalculate_saturation_from_fragments.py\n",
      "Please commit your changes or stash them before you merge.\n",
      "Aborting\n"
     ]
    }
   ],
   "source": [
    "if [ ! -d PUMATAC_dependencies/single_cell_toolkit ]; then\n",
    "    echo \"cloning single_cell_toolkit\"\n",
    "    cd PUMATAC_dependencies\n",
    "    git clone https://github.com/aertslab/single_cell_toolkit\n",
    "    cd ..\n",
    "else\n",
    "    cd PUMATAC_dependencies\n",
    "    echo \"pulling single_cell_toolkit\"\n",
    "    cd single_cell_toolkit\n",
    "    git pull\n",
    "    cd ..\n",
    "fi"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "2af0aeb8-d4f0-4b1b-89f5-b6e11850b538",
   "metadata": {},
   "source": [
    "# Documentation\n",
    "How the dependencies were generated or downloaded\n",
    "### a. NextFlow\n",
    "NextFlow 21.04.03 can be downloaded here: https://github.com/nextflow-io/nextflow/releases/tag/v21.04.3"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 33,
   "id": "d9e7ee6b-f38b-4c78-a1be-f820f705daa1",
   "metadata": {
    "execution": {
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     "shell.execute_reply": "2023-04-18T09:27:05.595105Z",
     "shell.execute_reply.started": "2023-04-18T09:27:05.389832Z"
    },
    "tags": []
   },
   "outputs": [],
   "source": [
    "wget -P resources/nextflow/ https://github.com/nextflow-io/nextflow/releases/download/v21.04.3/nextflow-21.04.3-all"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "3a6e3788-192a-497e-8fcb-7d1a5d2bd5b8",
   "metadata": {},
   "source": [
    "### b. A bwa-mem2 genome index for your species\n",
    "This genome index can be generated from a `.fasta` file using `bwa-mem2`. First, follow the github tutorial to install `bwa-mem2`: https://github.com/bwa-mem2/bwa-mem2  \n",
    "Then, generate a genome index as follows:"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "ce3b3b3c-ec30-4e5e-b8f6-dec154d64bd1",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2023-04-17T12:40:23.750627Z",
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     "shell.execute_reply.started": "2023-04-17T12:40:23.750611Z"
    }
   },
   "source": [
    "### c. Barcode whitelists for 10x Genomics and HyDrop\n",
    "For 10x Genomics, you can get the whitelist for multiome and ATAC from here:  \n",
    "https://kb.10xgenomics.com/hc/en-us/articles/115004506263-What-is-a-barcode-whitelist-  \n",
    "https://kb.10xgenomics.com/hc/en-us/articles/4412343032205-Where-can-I-find-the-barcode-whitelist-s-for-Single-Cell-Multiome-ATAC-GEX-product-  \n",
    "  \n",
    "For HyDrop, the whitelist can be sourced from https://github.com/aertslab/hydrop_data_analysis.\n",
    "For ddSEQ, a whitelist-agnostic strategy is employed.  \n",
    "For s3-ATAC, a custom whitelist should be compiled based on the indices you used.  \n",
    "If you wish to implement your own technique, a tutorial will be added."
   ]
  },
  {
   "cell_type": "markdown",
   "id": "d7cf2d65-3cf3-4ffe-8ca2-fd6a8466b93a",
   "metadata": {
    "tags": []
   },
   "source": [
    "### d. Regions for fragment counting\n",
    "If you also want to employ our QC diagnostics tool, you will require regions to count `.fragments.tsv.gz` files in. There are three possible strategies:\n",
    "1. Align your data in a bulk ATAC-seq fashion, perform de-novo peak calling on the aggregate alignment, and use these peak regions to generate your count matrix. This strategy can miss regions that are only accessible in rare cell types.\n",
    "2. Use a region set from a previous experiment on your sample.\n",
    "3. Use the ENCODE SCREEN regions: https://screen.encodeproject.org/. We subtract the ENCODE blacklist () from the ENCODE SCREEN regions."
   ]
  },
  {
   "cell_type": "markdown",
   "id": "4ffd8a59-14c5-4d35-b8ff-eb1edc72d212",
   "metadata": {},
   "source": [
    "# Compiling a hybrid mouse/human genome"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "id": "508c59db-8e6b-4dd9-a22a-c3e4d685f613",
   "metadata": {},
   "outputs": [],
   "source": [
    "#################### SETUP ####################\n",
    "\n",
    "\n",
    "human_genome=\"GRCh38\"\n",
    "mouse_genome=\"GRCm39\"\n",
    "version=\"2024-A\"\n",
    "\n",
    "\n",
    "build=\"GRCh38_and_GRCm39_GENCODEv44-GENCODEvm33\"\n",
    "mkdir -p \"$build\"\n",
    "\n",
    "\n",
    "# Download source files if they do not exist in reference_sources/ folder\n",
    "source=\"reference_sources\"\n",
    "mkdir -p \"$source\"\n",
    "\n",
    "\n",
    "# Using release 109 for GRCh38 instead of release 110 -- release 110 moved from GRCh38.p13 to GRCh38.p14,\n",
    "# which unmasked the pseudo-autosomal region. This causes ambiguous mappings to PAR locus genes.\n",
    "human_fasta_url=\"http://ftp.ensembl.org/pub/release-109/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz\"\n",
    "human_fasta_in=\"${source}/Homo_sapiens.GRCh38.dna.primary_assembly.fa\"\n",
    "human_gtf_url=\"http://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_44/gencode.v44.primary_assembly.annotation.gtf.gz\"\n",
    "human_gtf_in=\"${source}/gencode.v44.primary_assembly.annotation.gtf\"\n",
    "mouse_fasta_url=\"http://ftp.ensembl.org/pub/release-110/fasta/mus_musculus/dna/Mus_musculus.GRCm39.dna.primary_assembly.fa.gz\"\n",
    "mouse_fasta_in=\"${source}/Mus_musculus.GRCm39.dna.primary_assembly.fa\"\n",
    "mouse_gtf_url=\"http://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M33/gencode.vM33.primary_assembly.annotation.gtf.gz\"\n",
    "mouse_gtf_in=\"${source}/gencode.vM33.primary_assembly.annotation.gtf\"\n",
    "\n",
    "\n",
    "if [ ! -f \"$human_fasta_in\" ]; then\n",
    "    curl -sS \"$human_fasta_url\" | zcat > \"$human_fasta_in\"\n",
    "fi\n",
    "if [ ! -f \"$human_gtf_in\" ]; then\n",
    "    curl -sS \"$human_gtf_url\" | zcat > \"$human_gtf_in\"\n",
    "fi\n",
    "if [ ! -f \"$mouse_fasta_in\" ]; then\n",
    "    curl -sS \"$mouse_fasta_url\" | zcat > \"$mouse_fasta_in\"\n",
    "fi\n",
    "if [ ! -f \"$mouse_gtf_in\" ]; then\n",
    "    curl -sS \"$mouse_gtf_url\" | zcat > \"$mouse_gtf_in\"\n",
    "fi\n",
    "\n",
    "\n",
    "# String patterns used for both genomes\n",
    "ID=\"(ENS(MUS)?[GTE][0-9]+)\\.([0-9]+)\"\n",
    "\n",
    "\n",
    "BIOTYPE_PATTERN=\\\n",
    "\"(protein_coding|protein_coding_LoF|lncRNA|\\\n",
    "IG_C_gene|IG_D_gene|IG_J_gene|IG_LV_gene|IG_V_gene|\\\n",
    "IG_V_pseudogene|IG_J_pseudogene|IG_C_pseudogene|\\\n",
    "TR_C_gene|TR_D_gene|TR_J_gene|TR_V_gene|\\\n",
    "TR_V_pseudogene|TR_J_pseudogene)\"\n",
    "GENE_PATTERN=\"gene_type \\\"${BIOTYPE_PATTERN}\\\"\"\n",
    "TX_PATTERN=\"transcript_type \\\"${BIOTYPE_PATTERN}\\\"\"\n",
    "READTHROUGH_PATTERN=\"tag \\\"readthrough_transcript\\\"\"\n",
    "\n",
    "# Process FASTA -- translate chromosome names\n",
    "human_fasta_modified=\"$build/$(basename \"$human_fasta_in\").modified\"\n",
    "cat \"$human_fasta_in\" \\\n",
    "    | sed -E 's/^>(\\S+).*/>\\1 \\1/' \\\n",
    "    | sed -E 's/^>([0-9]+|[XY]) />chr\\1 /' \\\n",
    "    | sed -E 's/^>MT />chrM /' \\\n",
    "    > \"$human_fasta_modified\"\n",
    "\n",
    "\n",
    "# Process GTF -- split Ensembl IDs from version suffixes\n",
    "human_gtf_modified=\"$build/$(basename \"$human_gtf_in\").modified\"\n",
    "cat \"$human_gtf_in\" \\\n",
    "    | sed -E 's/gene_id \"'\"$ID\"'\";/gene_id \"\\1\"; gene_version \"\\3\";/' \\\n",
    "    | sed -E 's/transcript_id \"'\"$ID\"'\";/transcript_id \"\\1\"; transcript_version \"\\3\";/' \\\n",
    "    | sed -E 's/exon_id \"'\"$ID\"'\";/exon_id \"\\1\"; exon_version \"\\3\";/' \\\n",
    "    > \"$human_gtf_modified\"\n",
    "\n",
    "\n",
    "# Process GTF -- filter based on gene/transcript tags\n",
    "cat \"$human_gtf_modified\" \\\n",
    "    | awk '$3 == \"transcript\"' \\\n",
    "    | grep -E \"$GENE_PATTERN\" \\\n",
    "    | grep -E \"$TX_PATTERN\" \\\n",
    "    | grep -Ev \"$READTHROUGH_PATTERN\" \\\n",
    "    | sed -E 's/.*(gene_id \"[^\"]+\").*/\\1/' \\\n",
    "    | sort \\\n",
    "    | uniq \\\n",
    "    > \"${build}/gene_allowlist\"\n",
    "\n",
    "\n",
    "human_gtf_filtered=\"${build}/$(basename \"$human_gtf_in\").filtered\"\n",
    "grep -E \"^#\" \"$human_gtf_modified\" > \"$human_gtf_filtered\"\n",
    "grep -Ff \"${build}/gene_allowlist\" \"$human_gtf_modified\" \\\n",
    "    | awk -F \"\\t\" '$1 != \"chrY\" || $1 == \"chrY\" && $4 >= 2752083 && $4 < 56887903 && !/ENSG00000290840/' \\\n",
    "    >> \"$human_gtf_filtered\"\n",
    "\n",
    "\n",
    "#################### MOUSE ####################\n",
    "# Please see the GRCm39-2024-A build documentation for details on these steps.\n",
    "\n",
    "\n",
    "# Process FASTA -- translate chromosome names\n",
    "mouse_fasta_modified=\"$build/$(basename \"$mouse_fasta_in\").modified\"\n",
    "cat \"$mouse_fasta_in\" \\\n",
    "    | sed -E 's/^>(\\S+).*/>\\1 \\1/' \\\n",
    "    | sed -E 's/^>([0-9]+|[XY]) />chr\\1 /' \\\n",
    "    | sed -E 's/^>MT />chrM /' \\\n",
    "    > \"$mouse_fasta_modified\"\n",
    "\n",
    "\n",
    "# Process GTF -- split Ensembl IDs from version suffixes\n",
    "mouse_gtf_modified=\"$build/$(basename \"$mouse_gtf_in\").modified\"\n",
    "cat \"$mouse_gtf_in\" \\\n",
    "    | sed -E 's/gene_id \"'\"$ID\"'\";/gene_id \"\\1\"; gene_version \"\\3\";/' \\\n",
    "    | sed -E 's/transcript_id \"'\"$ID\"'\";/transcript_id \"\\1\"; transcript_version \"\\3\";/' \\\n",
    "    | sed -E 's/exon_id \"'\"$ID\"'\";/exon_id \"\\1\"; exon_version \"\\3\";/' \\\n",
    "    > \"$mouse_gtf_modified\"\n",
    "\n",
    "\n",
    "# Process GTF -- filter based on gene/transcript tags\n",
    "cat \"$mouse_gtf_modified\" \\\n",
    "    | awk '$3 == \"transcript\"' \\\n",
    "    | grep -E \"$GENE_PATTERN\" \\\n",
    "    | grep -E \"$TX_PATTERN\" \\\n",
    "    | grep -Ev \"$READTHROUGH_PATTERN\" \\\n",
    "    | sed -E 's/.*(gene_id \"[^\"]+\").*/\\1/' \\\n",
    "    | sort \\\n",
    "    | uniq \\\n",
    "    > \"${build}/gene_allowlist\"\n",
    "\n",
    "\n",
    "mouse_gtf_filtered=\"${build}/$(basename \"$mouse_gtf_in\").filtered\"\n",
    "grep -E \"^#\" \"$mouse_gtf_modified\" > \"$mouse_gtf_filtered\"\n",
    "grep -Ff \"${build}/gene_allowlist\" \"$mouse_gtf_modified\" \\\n",
    "    >> \"$mouse_gtf_filtered\"\n",
    "\n",
    "\n",
    "#################### MKREF ####################\n",
    "\n",
    "\n",
    "cellranger mkref --ref-version=\"$version\" \\\n",
    "    --genome=\"$human_genome\" --fasta=\"$human_fasta_modified\" --genes=\"$human_gtf_filtered\" \\\n",
    "    --genome=\"$mouse_genome\" --fasta=\"$mouse_fasta_modified\" --genes=\"$mouse_gtf_filtered\" \\\n",
    "    --nthreads=16\n"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "id": "1c21fdf4-9a79-4dcd-b933-afdc3acc84c0",
   "metadata": {},
   "outputs": [],
   "source": [
    "\n",
    "cellranger mkref --ref-version=\"$version\" \\\n",
    "    --genome=\"$human_genome\" --fasta=\"$human_fasta_modified\" --genes=\"$human_gtf_filtered\" \\\n",
    "    --genome=\"$mouse_genome\" --fasta=\"$mouse_fasta_modified\" --genes=\"$mouse_gtf_filtered\" \\\n",
    "    --nthreads=16\n"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "2e5f7d78-43db-41cf-9b48-a7ede72021bf",
   "metadata": {},
   "source": [
    "### The `PUMATAC_dependencies` directory should look like so:"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 48,
   "id": "6ee581e8-0319-441e-ab62-269f621fecde",
   "metadata": {
    "execution": {
     "iopub.execute_input": "2023-04-18T12:23:17.700963Z",
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     "shell.execute_reply": "2023-04-18T12:23:17.814288Z",
     "shell.execute_reply.started": "2023-04-18T12:23:17.700946Z"
    },
    "tags": []
   },
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "PUMATAC_dependencies\n",
      "├── cache\n",
      "│   ├── broadinstitute-gatk-4.2.0.0.img\n",
      "│   ├── cache\n",
      "│   ├── cflerin-seurat-4.0.3-plus.copy.sif\n",
      "│   ├── cflerin-seurat-4.0.3-plus.sif\n",
      "│   ├── seurat.sh\n",
      "│   ├── vibsinglecellnf-bap-2021-04-27-3b48f4b.img\n",
      "│   ├── vibsinglecellnf-bwamaptools-bwa-mem2-2.2.1-zlibng-2.0.6.img\n",
      "│   ├── vibsinglecellnf-popscle-2021-05-05-da70fc7.img\n",
      "│   ├── vibsinglecellnf-singlecelltoolkit-2022-04-15-16314db.img\n",
      "│   ├── vibsinglecellnf-singlecelltoolkit-2022-07-07-0638c1d.img\n",
      "│   ├── vibsinglecellnf-sinto-0.7.3.1.img\n",
      "│   └── vibsinglecellnf-trimgalore-0.6.6.img\n",
      "├── genomes\n",
      "│   ├── hg38_bwamem2\n",
      "│   └── mm10_bwamem2\n",
      "├── nextflow\n",
      "│   ├── nextflow-21.04.3-all\n",
      "│   ├── nextflow-21.10.4-all\n",
      "│   └── nextflow-22.10.7-all\n",
      "├── regions\n",
      "│   ├── 1000Genomes.wgs.GRCH38.sites.minAF-0.1.freemuxlet-scATAC.vcf.gz\n",
      "│   ├── V2.hg38-rDHS-Unfiltered.blacklisted.bed\n",
      "│   ├── V2.mm10-rDHS-Unfiltered.blacklisted.bed\n",
      "│   ├── hg38-blacklist.v2.bed\n",
      "│   └── mm10-blacklist.v2.bed\n",
      "└── whitelists\n",
      "    ├── 20200227_HyDrop-ATAC_96x96x96_full_barcodes_no_underscore.txt.gz\n",
      "    ├── 20200227_HyDrop-ATAC_96x96x96_full_barcodes_no_underscore_REVCOMP.txt.gz\n",
      "    ├── 737K-arc-v1.REVCOMP.txt.gz\n",
      "    ├── 737K-arc-v1.txt.gz\n",
      "    ├── 737K-cratac-v1.REVCOMP.txt.gz\n",
      "    ├── 737K-cratac-v1.txt.gz\n",
      "    ├── hydrop_384x384.REVCOMP.txt.gz\n",
      "    └── hydrop_384x384.txt.gz\n",
      "\n",
      "8 directories, 27 files\n"
     ]
    }
   ],
   "source": [
    "tree -L 2 PUMATAC_dependencies"
   ]
  }
 ],
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