Protocol: Optimized CRISPR-Cas9 system for genome editing in zebrafish.

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Mut-Seq pipeline installation

  1. Requirements

    • Python 3.2 (or more recent). A convenient Python installer like Anaconda can be used.
    • Download and install the GMAP read aligner.
  2. Download Mut-Seq package. Do not unzip it; this Python ZIP application contains Mut-Seq script and dependencies and can be used with Python directly (see example below). If needed, source code is available under the MPLv2 licence.

Mut-Seq example

  1. Download and unzip example source with:

    unzip -d mutseq_example example.zip
    

    This archive contains:

    1. loci.csv with the loci description
    2. seq_run1 folder with sequencing reads as FASTQ files
    3. output/report.txt is a precomputed report. A similar report should be obtained at the end of this tutorial.
  2. Start Mut-Seq analysis with:

    python3 mutseq.pyz -l mutseq_example/loci.csv -d mutseq_example -o report.txt --processor 4 --min_allele_reads 10
    

    If you didn’t download mutseq.pyz (see previous section) to the current folder, change the path to mutseq.pyz in the command above.

  3. While running, output should look like:

    14:10 - Parsing mutseq_example/loci.csv: 1 loci and 1 sample(s) found.
    14:10 - Creating index in .
    14:10 - Aligning seq_run1
    14:10 - Loading file(s): mutseq_example/seq_run1/AG00792_R1_001.fastq.gz
    14:10 - Starting GMAP (seq_run1,R1) with ['gmap', '--dir', '.', '--db', 'index', '--min-intronlength', '1600', '--allow-close-indels', '2', '--npaths', '1', '--ordered', '--input-buffer-size', '12000', '--batch', '4', '--format', 'samse', '--nthreads', '4']
    14:10 - Loading file(s): mutseq_example/seq_run1/AG00792_R2_001.fastq.gz
    14:10 - Starting GMAP (seq_run1,R2) with ['gmap', '--dir', '.', '--db', 'index', '--min-intronlength', '1600', '--allow-close-indels', '2', '--npaths', '1', '--ordered', '--input-buffer-size', '12000', '--batch', '4', '--format', 'samse', '--nthreads', '4']
    14:11 - Detecting allele in seq_run1
    14:11 - Writing report in report.txt
    
  4. Results will be in report.txt. It should be identical to the report in output/ folder from the example archive.