Quick Start Demo

This demo and tutorial will walk you through getting Prost! running on a provided zebrafish test dataset.


Prost! is designed to be easily run from the command line. A basic familiarity of the command line and optionally the ability to edit a configuration file are all that is required to Prost!.

Before continuing, make sure you’ve met the System and software dependencies.

Dependencies Not Provided in Demo

Due to size constraints, a BBMap database of the zebrafish reference genome is not included. Instructions on how to download and build a BBMap database of the zebrafish genome are included below.

Dependencies Provided in Demo

This demo provides several dependencies via the GitHub repository https://github.com/uoregon-postlethwait/prost-demo. If you wish to run Prost! against a different dataset, you’ll likely need to provide some or all of these requirements. The following dependencies are provided:

  • An appropriate Prost! configuration file (prost.config).
  • A list of high throughput sequencing fasta format files of samples (samples_filelist) and the corresponding samples files (samples/sample.brain1.fa, samples/sample.heart2.fa, and samples/sample.ovary3.fa).
  • Annotation FASTA files:
    • A FASTA file of mature microRNAs in miRBase release 21 (fa/mature_miRBase21.fa.gz).
    • A FASTA file of microRNA hairpins in miRBase release 21 (fa/hairpin_miRBase21.fa.gz).
    • A FASTA file of “other RNAs” (e.g. snoRNAs) found in zebrafish, provided by Ensembl BioMart (fa/BioMart_Dre_otherRNA.fa.gz).
  • A script (scripts/setup.sh) which uncompresses the sample and annotation FASTA files described above.

Quick Start

  1. Make sure you’ve met the following requirements:

  2. If you have a slow internet connection, you may wish to begin downloading the zebrafish reference genome (see step 5).

  3. Download the Prost! demo.

    • You can do this manually by navigating your browser to the prost-demo Releases page and clicking on the latest release link.

    • Or you do it automatically with the following command:

      curl -SLOJ $(curl -s https://api.github.com/repos/uoregon-postlethwait/prost-demo/releases/latest | grep zipball_url | head -n 1 | cut -d\" -f4)
  4. Extract the Prost! demo and cd into the demo directory:

    # Replace X.X.X with the version of the demo.
    unzip prost-demo-X.X.X.zip
    cd prost-demo-X.X.X
  5. Run the prost-demo’s setup script. This script uncompresses the samples files as well as builds the annotation BBMap databases. Run the setup script like so:

    sh scripts/setup.sh
  6. Download and extract the zebrafish reference genome. (Alternatively, if you already have a copy of the zebrafish assembly available, you may use that instead. It doesn’t need to be the exact version shown below. Just give bbmap.sh the full PATH to the ref= argument in the following step.):

    # Using wget:
    wget ftp://ftp.ensembl.org/pub/current_fasta/danio_rerio/dna/Danio_rerio.GRCz10.dna.toplevel.fa.gz
    # Or using curl:
    curl -O ftp://ftp.ensembl.org/pub/current_fasta/danio_rerio/dna/Danio_rerio.GRCz10.dna.toplevel.fa.gz
  7. Build a BBMap database of the zebrafish reference genome:

    # We've found a k-mer length of 7 or 8 works best for these data:
    bbmap.sh k=7 path=BBMap/Danio_rerio.GRCz10.dna.toplevel ref=Danio_rerio.GRCz10.dna.toplevel.fa.gz
  8. Run Prost!:

    # The demo defaults to using 4 threads.
    # Alternatively, you may run Prost! on more CPUs if they are available.
    prost --max-threads 12

Prost Logging Output in Demo

If everything went well, Prost! will output the following to both the console (i.e. STDOUT), as well as to prost.log, and it should look something like this:

∴ prost --max-threads 12
Prost! version 0.7.3.                            5:00:41 PM PDT on Apr 16, 2015.

Reading in all fasta files...
reading file1 (sample.brain1.fa)...
    83157/83157 so far... done. (elapsed time: 0.2s)
reading file2 (sample.heart2.fa)...
    24039/24039 so far... done. (elapsed time: 0.1s)
reading file3 (sample.ovary3.fa)...
    18492/18492 so far... done. (elapsed time: 0.1s)
Rejecting low reads seqs... done. (elapsed time: 0.0s)
Writing fasta search file... done. (elapsed time: 0.0s)
Alignments... done. (elapsed time: 77.9s (or 1m17s))
Designation step ONE...
        Reading BBMap hits from file...
            244/244 so far... done. (elapsed time: 0.0s)
Designation step ONE... done. (elapsed time: 0.0s)
Normalization: calculating per-sample totals.... done. (elapsed time: 0.0s)
Normalization: normalizing read counts.... done. (elapsed time: 0.0s)
Designation step TWO...
    14/14 so far... done. (elapsed time: 0.0s)
        Reading BBMap hits from file...
            1113/1113 so far... done. (elapsed time: 0.1s)
        Reading BBMap hits from file...
            2478/2478 so far... done. (elapsed time: 0.1s)
        Reading BBMap hits from file...
            238/238 so far... done. (elapsed time: 0.0s)
Annotation... done. (elapsed time: 0.2s)
Binning by Annotation... done. (elapsed time: 0.0s)
Writing output file...
    65/65 so far... done. (elapsed time: 0.1s)
Writing comressed output file...
    4/4 so far... done. (elapsed time: 0.1s)
Writing annotation-comressed output file...
    0 so far... done. (elapsed time: 0.1s)
Writing seed-comressed output file...
    0 so far... done. (elapsed time: 0.1s)
Writing mirror miRs output file... done. (elapsed time: 0.1s)
Writing arm switch output file... done. (elapsed time: 0.1s)
Writing no_hits output file...
    65/65 so far... done. (elapsed time: 0.1s)
Generating Excel Spreadsheet... done. (elapsed time: 0.2s)
Total Prost running time: 81.2s (or 1m21s).

Prost Excel Output

Once complete, Prost! produces an Excel spreadsheet with several tabs (as well as several tab separated value (TSV) files which are identical to the Excel tabs, minus formatting). The by_gen_loc bin is a good place to start. Please see the documentation for descriptions of each tab and column.

Adapting the Demo for Your Dataset

You can adapt this demo to easily run Prost! on your own dataset. To do so, you’ll need to edit the configuration file prost.config. In particular, you’ll need to edit these fields in the General section:

  • species - to specify your species
  • samples_filelist - to point to your file list of samples; alternatively, you can simply edit the file samples_filelist in the current directory.

You may also need to edit the db fields in the Alignment sections if you are using different genome or annotation databases.

Below is a snippet of the configuration file that shows you roughly what will need to be edited (some additional fields are shown below for context, but do not need to be edited). (Note that you can also download an example Prost! configuration file here: prost.config.example).

species: dre
samples_filelist: samples_filelist
mature_mir_annotation_fasta: BBMap/mature_miRBase21.fa

db: BBMap/Danio_rerio.GRCz10.dna.toplevel

type: MirbaseMirAnnotation
db: BBMap/mature_miRBase21.fa

type: MirbaseHairpinAnnotation
db: BBMap/hairpin_miRBase21.fa

type: BiomartOtherRNAAnnotation
db: BBMap/BioMart_Dre79_otherRNA.fa

After you have made those changes, simply run Prost! again: