Daniel Klimes (daniel.s.klimes@gmail.com)
Dantools is a tool used for comparing genome sequences between divergent organisms, such as different species. It does this by creating a pseudogenome, a modification of a reference genome to match the sequence of another. In doing so, a list of genetic variants between given species is produced. Given a GFF file, these variants can be annotated by their positions relative tofeatures. Additionally, by keeping track of position shifts from indels, the annotations of the original reference can be extended to the new species' genome. This allows for the alignment of RNA-Seq data from divergent species against a set of genomes with unified annotations. Critically, Dantools does not require either genome to be well assembled, and can even accept an RNA-Seq .fastq input.
Dantools in its base form accepts two genomes as input: the base and the source. In its first step, Dantools breaks the source genome into sequence "fragments", 50-10,000 base pieces of the genome and its reverse complement. These fragments are then aligned against the base genome using HISAT2 with lowered alignment stringency. Freebayes is used to tabulate variants, which are then used to modify the base genome. This process of alignment, variant collection, and base modification is repeated until a satisfactory "modified" genome is created.
After the final iteration, a new VCF file is generated using optimized Needleman-Wunsch algorithms. This VCF can optionally be labeled by its relative position within features and flanking regions. In the final step, the input GFF (if provided) is then shifted to accomodate indel mutations that were created in the process of modification.
Because of the flexibility of inputs, Dantools can accept RNA-Seq reads in fastq format and minimally-assembled genomes.
In future iterations of Dantools, functionality will be added for: -Determining genome rearrangements -Translating nucleic acid to amino acid changes
One may wish to keep this in its own tree, perl provides a number of mechanisms to do so, here is an example of how one might install dantools in a self-contained tree with the github-downloaded source in src/
mkdir -p dantools/202406
cd dantools/202406
git clone https://github.com/elsayed-lab/dantools.git
mv dantools src
## tell MakeMaker and Module::Build to use this tree
## All prerequisite perl libraries will go to dantools/202406/lib/perl5
export PERL5LIB="$(pwd)/lib/perl5:${PERL5LIB}"
export PERL_MM_OPT="INSTALL_BASE=$(pwd)"
export PERL_MB_OPT="--install_base $(pwd)"
## If you have App::cpanminus installed
cpanm ExtUtils::AutoInstall inc::Module::Install File::ShareDir::Install
## If not, then a similar invocation of cpan will work,
## you might alway want to check out local::lib
cd src
perl Makefile.PL && make && make install
## Depending on your system, you may need to manually install some dependencies
cpanm Moo::Role Parallel::ForkManager
make && make install
Dantools relies on a set of software packages not listed in the makefile to perform some of its tasks:
Comparisons begin with the dantools pseudogenome command, which generates the pseudogenome and, more importantly, a VCF file that describes the variants in the source genome relative to the base. Invocations differ by source data format:
Source is a genome in fasta format:
dantools pseudogen -b base.fasta -s source.fasta
Source is an already fragmented genome in fasta format:
dantools pseudogen -b base.fasta -s source.fasta --fragment no
Source is unpaired RNA-Seq reads in fastq format:
dantools pseudogen -b base.fasta --reads-u source.fastq
Source is paired-end RNA-Seq reads in fastq format:
dantools pseudogen -b base.fasta -1 mate1.fastq -2 mate2.fastq
Because many indels were applied during the pseudogenome creation process, the base genome features need to be shifted in their relative positions:
dantools shift -v variants.vcf -f base.gff
Now, RNA-Seq data aligned against the pseudogenome can be counted with the shifted GFF file.
If one wants to label the variants according to a GFF file:
dantools label -v variants.vcf -f base.gff --features five_prime_UTR,CDS,three_prime_UTR
These variants can optionally be translated into amino acid changes and scored by a mutation scoring matrix.
Additional functionalities exist and are listed with the simple command:
dantools