Beatriz Vieira Mourato and Bernhard Haubold
This package contains shell scripts to discover and evaluate candidate marker regions in reference prokaryotes.
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
doc/mapro.pdf.
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.
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.
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.
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.
To generate the documentation, run
$ make doc
The documentation is now in doc/mapro.pdf.
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).
Running mapro requires installation of a number of apt packages:
$ sudo apt install bc phylonium ncbi-blast+ primer3 noweb
It also requires the command line tool datasets
For the documentation these extra packages are required:
$ sudo apt install texlive-science texlive-pstricks texlive-latex-extra texlive-fonts-extra
We also need the packages Neighbors, Fur, Prim, and Biobox.
For applications see:
- Mourato, B. V., Tsers, I., Denker, S., Klötzl, F., & Haubold, B. (2024). Marker discovery in the large. Bioinformatics, 1-7.