jxshi/priset

Tool for identifying primer sequences for metabarcoding experiments given a taxonomy and a reference database.

PriSeT

Tool for assisting the search of primer sequences for metabarcoding experiments. Given a taxonomic identifier, a reference database, and a genetic target region, PriSeT identifies conserved sections suitable for PCR primers such that taxonomic coverage and separation are optimal.

Requirements

Platform	Details	Tested
	Linux 64 bit	-
	Mac OS 64 bit	High Sierra 10.12.6

R

If not installed on your system yet, install R via your standard package manager. However, for MacOS I recommend not to install via port, but download the binaries from CRAN, because I ran into installation errors when when trying to install igraph and others in an interactive R session. If you install the package from cran.r-project.org, open the R.app, go to the package installer (under Packages & Data), search for the below listed packages and click the install button. If you use R in terminal, start an inter session, install the required R packages shiny and DT for table output, and treemap and d3treeR for an interactive tree map plot.

$ R
> install.packages("shiny")
> install.packages("DT")
> install.packages("igraph")
> install.packages("treemap")

To make the treemap interactive you need d3treeR hosted on github. To download it in an interactive session, follow these steps:

> install.packages("devtools")
> library(devtools)
> install_github("d3treeR/d3treeR")

wget https://ftp.gnu.org/pub/gnu/libiconv/libiconv-1.15.tar.gz
tar -zxvf libiconv-1.15.tar.gz
cd libiconv-1.15
./configure --prefix=/usr/local
make
make install

Taxonomic Tree and Library

In order to explore the potential primer sequences hierarchically w.r.t. an existing taxonomy, the taxonomic node identifier (taxid) needs to be related to reference sequences. You can use the tool tactac to create a subset of your reference library. Calling python tactac.py --subtree <taxid> (plus password) will create three files:

• Taxonomic subtree as tuples in csv format: /subset/<taxid>/root_<taxid>.tax
• Taxonomic map of taxids assigned directly to accessions: /subset/<taxid>/root_<taxid>.acc
• Library of all sequences under <taxid>: /subset/<taxid>/root_<taxid>.fasta

Setup

Clone PriSeT and its submodules

git clone --recurse-submodules https://github.com/mariehoffmann/PriSeT.git

Compilation

1. Create a build directory and compile with cmake

cmake -DCMAKE_BUILD_TYPE=Debug ../PriSeT

2. Build

make -j

3. Call binary with the library directory containing: * Taxonomy root_<taxid>.tax * Taxid to accessions map root_<taxid>.acc * Library with reference sequences root_<taxid>.fasta

Assume lib_dir and work_dir as your library and working directories. The latter one will store the FM index, mappings, the shiny app as R script and the input table for the shiny app.

 ./priset <lib_dir> <work_dir> [--skip-idx]

Unit Tests

cd ../build
cmake -DCMAKE_C_COMPILER=/usr/local/bin/gcc -DCMAKE_CXX_COMPILER=/usr/local/bin/g++ ../PriSeT/tests/
make

References

[1] Pockrandt, C., Alzamel, M., Iliopoulos, C. S., Reinert, K.. GenMap: Fast and Exact Computation of Genome Mappability. bioRxiv, presented on RECOMB-Seq, 2019.

[2] Federhen, S. (2012). The NCBI Taxonomy database. Nucleic Acids Research, 40(D1), D136–D143. http://doi.org/10.1093/nar/gkr1178

[3] PCR primer design constraints: www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html