mapro: Markers for Prokaryotes

Authors

Beatriz Vieira Mourato and Bernhard Haubold

Description

This package contains shell scripts to discover and evaluate candidate marker regions in reference prokaryotes.

Set Up Analysis

Run

$ make

This constructs four scripts: genomes.sh for downloading target and neighbor genomes, markers.sh for extracting markers from the genomes, and primers.sh for designing primers from the markers and testing them. The auxiliary script driver.sh drives the analysis of one or more prokaryotes. These four scripts are located in the directory scripts and are explained in the documentation.

Run Toy Analysis

To run the toy analysis we need a small database of Enterobacter cloacae sequences, which we download from the net.

$ make ecl.db

We also need a Neighbors database, which we also get ready-made from the net.

$ make neidb

This database allows us to reproduce the analysis of bacterial taxa described in our forthcoming publication. Alternatively, you can construct the Neighbors database from scratch using the current database dump and genome files.

$ make newNeidb

We change into the directory analysis constructed with the initial make, and run the example script for generating primers.

$ cd analysis
$ bash examplePrimers.sh

Two analyses are attempted, the first for Aquifex aeolicus fails due to a lack of genomes. However, the second for Enterobacter cloacae succeeds. The markers for E. cloacae are now contained in ecl/markers.fasta, the primers in ecl/primers.fasta. To calculate the sensitivity and specificity of these markers, we run the other example script.

$ bash exampleChecking.sh

The sensitivity and specificity measures based on the taxonomy are now in ecl/scop.out, and the corrected sensitivity and specificity measures are in ecl/cops.out.

Run Full Analysis

We make markers and primers for all prokaryotes in list.txt.

$ bash ../scripts/driver.sh -n ../data/neidb < ../data/list.txt

We can use hashes, #, to comment out lines in list.txt. To check the primers, we run

$ bash ../scripts/driver.sh -n ../data/neidb -c -b <blastDb> < ../data/list.txt

We use nt as our database for checking primers. This makes checking slow, and it might be a good idea to check only primers for organisms of particular interest.

Get Results of Full Analysis

Rather than running the full analysis, the markers can be downloaded from inside the mapro directory by running

$ make results

The maker sequences for the 68 successful analyses are now contained in data/results/tag.fasta, where tag refers to strain names that can be resolved using the entries in list.txt.

Annotate Amplicons

The primers generated by the full analysis imply amplicons and it might be of interest to annotate them. This is done by changing into the analysis directory

$ cd analysis

and running the script anam.sh on the same list of taxa as the original analysis.

$ bash ../scripts/anam.sh < ../data/list.txt

Every directory that contains a pair of primers now also contains the file anam.txt with the annotations of the corresponding amplicon.

Make Documentation

To generate the documentation, run

$ make doc

The documentation is now in doc/mapro.pdf.

Run Docker container

Alternatively, there is also a docker container available. To get the docker run:

$ docker pull beatrizvm/mapro

It can then be run iteratively with:

$ docker run -h docker -it beatrizvm/mapro

Where

-h changes container's host name for easier readability (in this case to docker),
-it runs the container in a CLI interactive mode

If you want to create a link between the docker container and your own terminal, you can add a mount flag (-v). This will create and link a newly made directory ("Shared_mapro") in your home directory and the directory "Shared" within the container. Files placed within this shared directory can be accessed within/outside the docker.

For example:

$ docker run -v ~/Shared_mapro:/home/mapro/Shared -h docker -it beatrizvm/mapro

The shared directory makes it possible to access files from outside the docker or results from the analysis saved to this directory.

For practical purposes, only a small subset of nt is actually contained within the docker. It would then be possible, for example, to share the directory with a local nt database, and access it from within the docker. This would remove the need to download the nt database directly within the docker.

To exit the docker terminal, use either ctr-D or the command exit.

The directory "Extras" contains the documentation for the installed packages (neighbors, fur, prim, biobox and phylonium).

Dependencies

Apt Packages

Running mapro requires installation of a number of apt packages:

$ sudo apt install bc phylonium ncbi-blast+ primer3

It also requires the command line tool datasets

For typesetting the documentation, these extra packages are required:

$ sudo apt install noweb texlive-science texlive-pstricks texlive-latex-extra texlive-fonts-extra

Other packages

We also need the packages Neighbors, Fur, Prim, and Biobox.

License

GNU General Public License