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Orientation

The training environment contains all the software, code and data necessary to work through this training course, so you don’t need to install anything yourself. However, you do need a (free) account to log in, and you should take a few minutes to familiarize yourself with the interface.

If you have not yet done so, please follow the Environment Setup directions before going any further.

Materials provided

For the purpose of the course, we’ll be working in the TaxoFlow directory, where you will find all the code files, test data and accessory files you will need. To move into it, run the following command:

cd TaxoFlow

Focus only on the TaxoFlow folder

The repository many other files and directories that may distract you, so just run this command on the terminal to open only the folder where the files are located: 

code .

Before we go any further, we are going to download some files that are too large to be permanently stored within the GitHub repository. Specifically, this is a set of files that constitute the database required by Kraken2 and Bracken.

Run the following commands in that exact order and wait until all of them are finished:

mkdir -p data/krakendb && cd "$_"
wget --no-check-certificate --no-proxy 'https://zenodo.org/api/records/17708950/files/krakendb.tar.gz/content'
tar -xvzf content
rm -r content
cd -

Briefly, this creates a directory called krakendb under data/ and moves into it. Then, it downloads an archive file with wget, unpacks its contents with tar, and deletes the original archive file. Finally, it moves you back up to the original TaxoFlow/ directory.

Now, we need to retrieve the indexed genome to remove host reads. We are going to use the Arabidopsis thaliana genome assembly TAIR10 (only for an educational perspective) that has been already indexed. There more available indexed genomes here or you can build your own index. Run the following commands then:

mkdir -p data/genome && cd "$_"
wget --no-check-certificate --no-proxy 'https://genome-idx.s3.amazonaws.com/bt/TAIR10.zip'
unzip TAIR10.zip
rm -r TAIR10.zip
cd -

This code block follows the same procedure as for downloading the Kraken2 database.

Now, let’s take a look of the files contained in this directory with the tree command:

tree . -L 3

Here you should see the following directory structure:

Materials provided within Taxoflow’s directory
Directory contents
.
├── assets
│   ├── taxoflow_logo.png
│   └── workflow_taxoflow.png
├── data
│   ├── genome
│   │   └── TAIR10
│   ├── krakendb
│   │   ├── database250mers.kmer_distrib
│   │   ├── database250mers.kraken
│   │   ├── database.kraken
│   │   ├── hash.k2d
│   │   ├── names.dmp
│   │   ├── nodes.dmp
│   │   ├── opts.k2d
│   │   ├── seqid2taxid.map
│   │   └── taxo.k2d
│   ├── samples
│   │   ├── ERR2143768
│   │   ├── ERR2143770
│   │   └── ERR2143774
│   └── samplesheet.csv
├── LICENSE
├── multi
│   ├── bin
│   │   └── report.Rmd
│   ├── main.nf
│   ├── modules
│   │   ├── bowtie2.nf
│   │   ├── bracken.nf
│   │   ├── fastqc.nf
│   │   ├── knit_phyloseq.nf
│   │   ├── kraken2.nf
│   │   ├── kraken_biom.nf
│   │   ├── kReport2Krona.nf
│   │   ├── ktImportText.nf
│   │   ├── multiqc.nf
│   │   └── trimgalore.nf
│   ├── nextflow.config
│   └── taxoflow.nf
├── README.md
└── single
    ├── bin
    │   └── report.Rmd
    ├── main.nf
    ├── modules
    │   ├── bowtie2.nf
    │   ├── bracken.nf
    │   ├── fastqc.nf
    │   ├── kraken2.nf
    │   ├── kReport2Krona.nf
    │   ├── ktImportText.nf
    │   └── trimgalore.nf
    ├── nextflow.config
    └── taxoflow.nf

This a summarized description of the files and directories found:

  • main.nf is the file we are going to invoke with the world-famous nextflow run command.
  • taxoflow.nf is where all the magic happens, it stores the order of execution of tasks and how data should be handled.
  • nextflow.config: it manages different directives for workflow execution.
  • modules is a really important folder since here we find dedicated files per each process of the pipeline.
  • bin is the directory where we store customized scripts that can be run within a given process.
  • data contains input data and related resources:
  • An indexed genome within the genome folder representing the host genome to which we want to map the reads for contamination removal.
  • krakendb is a directory that contains the Kraken2 database necessary for both taxonomic annotation and species abundance re-estimation.
  • samplesheet.csv lists the IDs and paths of the example data files, for processing in batches.
  • samples directory is where the raw sequences are stored. The names correspond to accession numbers that you can search on the Sequence Read Archive

Note

Don’t panic if this feels like a lot. This is just a glimpse of the material, and we are going to dig into each necessary file for the analysis in due time.

Now, to begin the course, click on the arrow in the bottom right corner of this page.