Oatk is designed for de novo assembly of complex plant organelle genomes using PacBio HiFi data. The toolkit consists of three major tools. syncasm is a de novo HiFi read assembler using a sparse de Bruijn graph constructed from closed syncmers (Edgar, R. 2021). hmm_annotation is a HMMER wrapper for convenient annotation of organelle sequences using a pre-built HMM profile database which is available at OatkDB. pathfinder is a tool used for parsing and circularising organelle genomes from the assembled sequences combining the HMM annotations and assembly graph structure. oatk is a wrapper for running syncasm, hmm_annotation and path_finder collectively.
You need to have a C compiler, GNU make and zlib development files installed. Download the source code from this repo or with git clone https://github.com/c-zhou/oatk.git. Then type make in the source code directory to compile.
A typical run consists of three steps: HiFi read assembly with syncasm, HMM annotation with hmm_annotation and organelle genome extraction with pathfinder. You can run three components separately as detailed below. Alternatively, oatk provides a wrapper to run them in a more compact way.
Here is an example to run syncasm,
syncasm -k 1001 -c 150 -t 8 -o oatk $input_hifi
Four optional and one positional parameters are set in this example:
-k specifies the syncmer size.
-c specifies the synmcer coverage threshold. Syncmers with a coverage below the threshold will be excluded from the assembly graph. This is a crucial parameter for organelle genome assembly. A proper choice of this threshold will help filter out the nuclear genome and numts. As an empirical instruction, this parameter can be set as 5-10 times the value of nuclear sequence coverage.
-t specifies the thread number.
-o specifies the prefix of the output files.
The positional parameter specifies the input PacBio HiFi data file. The program recognises both FASTA and FASTQ format, plain and gzipped.
The command will generate several files. The oatk_utg_final.gfa is the file for the final genome assembly in GFA format.
Here is an example to run hmm_annotation,
hmm_annotation -t 4 --nhmmscan /usr/bin/nhmmscan -o oatk_utg_final.mito.txt $hmm_db_dir/angiosperm_mito.fam oatk_utg_final.gfa
hmm_annotation -t 4 --nhmmscan /usr/bin/nhmmscan -o oatk_utg_final.pltd.txt $hmm_db_dir/angiosperm_pltd.fam oatk_utg_final.gfa
Three optional and two positional parameters are set in this example:
-t specifies the thread number.
-o specifies the output file.
--nhmmscan specifies the path to the nhmmscan executable. If not specified, the program will assume it is in the environmental path.
The first positional parameter specifies the HMM profile database. Proper database file for the target species should be selected. In this example, the database for angiosperms was used. Here, 'mito' for mitochondrial and 'pltd' for chloroplast.
The second positional parameter specifies the file of sequences for annotation. The program recognises FASTA, FASTQ, and GFA format, both plain and gzipped.
Here is an example to run pathfinder,
pathfinder -m oatk_utg_final.mito.txt -p oatk_utg_final.pltd.txt -o oatk oatk_utg_final.gfa
Three optional and one positional parameters are set in this example:
-m specifies the mitochondrial annotation file. With this parameter, the program will attempt to parse the mitochondrial genome.
-p specifies the chloroplast annotation file. With this parameter, the program will attempt to parse the chloroplast genome.
-o specifies the prefix of the output files.
The positional parameter specifies the assembly graph file.
The major output files in this example include: oatk.mito.fasta and oatk.mito.gfa for mitochondrial genome assembly; and oatk.pltd.fasta and oatk.pltd.gfa for chloroplast genome assembly. If the assembly is too complicated to solve, there will also be oatk.mito.unassembled.fasta and oatk.pltd.unassembled.fasta for the unitigs.
The above three steps can be alternatively run with the oatk wrapper as,
oatk -k 1001 -c 150 -t 8 --nhmmscan /usr/bin/nhmmscan -m $hmm_db_dir/angiosperm_mito.fam -p $hmm_db_dir/angiosperm_pltd.fam -o oatk $input_hifi
The major output files are similar.
path_to_fasta is a tool used to extract FASTA sequences from GFA file with a path. For example, path_to_fasta oatk_utg_final.gfa u1+,u2-,u3-,u2+.