This workflow describes a series of steps executed to get from raw fastq files, resulting from the amplicon sequencing of sample(s), to OTU table, describing the taxonomic determination summary for the analysed sample(s). It executes on Linux command line, using a Snakemake workflow management system.

1. QC of input files
2. Trim input files
3. QC of trimmed files
4. Join forward and referse sequences
5. QC of joined sequences
6. Cluster sequences
7. Pick representative sequences
8. Detect and remove chimeric representative sequences/clusters
9. Taxonomic classification
10. Create OTU table

1. Create a directory with input data. This should be paired illumina sequences.
   • This can be a directory of symbolic links to data elsewhere on a file system.
2. In config.yaml file:
   • Verify the working directory, this is the location of the pipeline output
   • Verify input directory, this can be an absolute path or relative to the working directory
   • Verify reference fasta and taxonomy, this can be absolute paths or relative to the working directory
   • Verify that input sequences file extension
   • Verify the input_file_forward_postfix parameter corresponds to the naming of your raw files. Change it, if necessary.
3. Optional: change tools parameters/paths in config.yaml file.

For details on the workflow tools, their version, arguments used, and order of execution see Snakefile.

To check if the workflow will run correctly without executing the steps:

$ snakemake -np --configfile config.yaml

To execute the workflow:

$ snakemake --configfile config.yaml

Note: If you are not in the same directory as the Snakefile you will need the extra parameter --snakefile with the path to the Snakefile

This worflow runs on Linux. To install this workflow, either locally or on a cluster, you will need to have the following requirements installed.

• Python 3.5+
• PyYAML 5.4+
• Snakemake 3.7.1
• FastQC 0.11.5
• Trimomatic 0.36
• Qiime 1.9

Download the latest release of this project:

/~https://github.com/AAFC-MBB/snakemake-amplicon-metagenomics/releases

OR

Check out this project (requires git):

$ git clone /~https://github.com/AAFC-MBB/snakemake-workflows.git

Automated test is located in snakemake-workflows/amplicon_workflow/test/. To run the test, first download the test data to snakemake-workflows/amplicon_workflow/test/data/ directory (see README in that directory for the instructions). Before you execute the test please note, that the test runs Snakefile with the test data and therefore uses the same output directory as a regular snakemake command (default is snakemake-workflows/amplicon_workflow/data). Therefore if you already have some input or intermetiate workflow execution data in your data directory and you would like to keep it - back it up.

Execute the tests:

$ ./test.sh -clean -run

For more information about Snakemake, visit their website: https://bitbucket.org/snakemake/snakemake/wiki/Home

Oksana Korol

Christine Lowe

See License file.