This manuscript has been accepted for publication in Genome Biology and Evolution
An up-to-date preprint can be found on BioRxiv

This repository contains the code used for the assembly and annotation of a chromosome-level, haplotype-resolved assembly of white clover (Trifolium repens). The pipeline begins with input of the Dovetail haplotype assemblies, associated AGP (i.e., “A Golden Path”) files (Download here), and linkage map data, and ends with the generation of the phased diploid assembly in FASTA format (NCBI BioProjects PRJNA957817 and PRJNA957816), the annotated haploid mapping assembly in FASTA, NCBI Sequin, and GFF3 formats (BioProject PRJNA951196), and manuscript figures.

Graphical depiction of pipeline can be found here. Docstrings for each Snakemake rule can be found here

In addition to the raw dovetail haplotype FASTAs, the following dependencies/resources need to be obtained prior to running the pipeline.

1. GeneMark-ES/ET/EP+ ver 4.71_lic needs to be obtained from here. GeneMark is licensed software and could not be included as part of the pipeline. Once downloaded, install GeneMark as recommended in their README file. Once installed, the following needs to be done:
   1. Include the .gm_key in the workflow/ directory containing the main Snakefile used for running the pipeline
   2. Point to the GeneMark and ProtHint (included with GeneMark) programs in the config file
2. A copy of the RepBase repeat library (I used RepBaseRepeatMaskerEdition-20181026.tar.gz) from here. A license is required to use this database so it could not be included. Once obtained, it needs to be included in resources/ directory
3. A copy of the InterProScan data needs to be obtained and included in the resources/ directory. The following commands can be run to download and setup the database:

wget ftp://ftp.ebi.ac.uk/pub/software/unix/iprscan/5/5.61-93.0/alt/interproscan-data-5.61-93.0.tar.gz
tar -xvzfp interproscan-data-5.61-93.0.tar.gz
chmod -R 777 interproscan-data-5.61-93.0

This pipeline requires Conda and Singularity:

A minimal installation of Conda (i.e., Miniconda) can be installed by following the instructions for your platform here

Installation of Singularity requires Admin privileges, but using Singularity to run pre-created containers does not. Installation instructions can be found here. All Singularity containers used in this pipeline are avalaible in this public repository, though they will be automatically pulled and executed by the pipeline. Assuming Conda is installed, the this repository's Conda environment can be replicated by running the following command:

conda env create -f environment.yaml -n dcg 

This will create a Conda environment named dcgcontaining a minimal set of dependencies required to run the pipeline.

After activating the environment (conda activate dcg), the pipeline can be executed from the workflow directory by running a command that looks something like:

snakemake --use-conda --use-singularity --singularity-args "--bind <path> --bind <path/to/interproscan/data>:/opt/interproscan-5.61-93.0/data" --configfile ../config/<configfile> --notemp -j <cores>

for local execution. Here, <path> is the path on the cluster from which files will be read/written (e.g., /scratch), <path/to/interproscan/data> is the full path to the interproscan data in resources/, <configfile> is one of the configfiles in the config directory that needs to be modified to match the paths on your system, and <cores> is the number of cores available for executing parallel processes.