04 - custom curation ruleset

Automated curation and validation of mitogenome annotations is a central goal of MitoPilot. When samples are processed through the Annotate module, annotations are first generated using a combination of MITOS2 and tRNAscan-SE.

Following the initial annotation, samples are processed through an automated curation process where modifications are made in attempt to bring the annotations closer to a state that will be considered acceptable for publication by NCBI GenBank. This automated curation process is based on two important inputs to the pipeline, a high-quality reference database of protein coding genes and a set of user configurable parameters.

MitoPilot currently has curation/validation rulesets for the following groups of organisms:

• fishes
• starfish
• octocorals
• hexacorals
• dipterans
• turtles
• copepods (testing in progress)
• mammals (untested)
• lepidosaurs (untested)

We are always looking to add new taxa! If your taxonomic group is not listed here, please post an issue or contact Dan MacGuigan directly.

Curation Parameters

Curation parameters are set in the Curation Opts section of the Annotate module.

Fig. 5

Below is a full list of curation parameters.

• hit_threshold [PCG only]: Minimum percent sequencing similarity for a protein-coding gene. Any hits below this threshold will not be used for curation.
• max_overlap: Maximum proportion of gene length that can overlap with another gene.
• count: Maximum expected number of gene copies.
• max_len: Maximum expected length (bp) of the gene.
• min_len: Minimum expected length (bp) of the gene.
• overlap: Start (5’) and stop (3’) rules for overlap between genes. Start is the maximum number of base pairs that can overlap at the 5’ end of a gene. Stop is whether the 3’ end of a gene can overlap AT ALL with another gene (True/False).
• start_codons [PCG only]: Acceptable start codons.
• stop_codons [PCG only]: Acceptable stop codons, including incomplete stop codons.
• intron [PCG only]: Can this gene have introns? (True/False)

If a gene fails to meet the specified parameters, it will be flagged with a warning message.

Customization of the parameters can be achieved by providing a complete named list or by passing individual modifications to the params_TAXON_mito() function. For example, the default expected count of trnW genes could be increased to 2 and the default percent similarity of an “acceptable” protein-coding gene match could be reduced to 85 by initializing a new project with:

new_project(
    ...,
    curate_params = params_fish_mito(
        list(
            hit_threshold = 85,
            rules = list(
                trnW = list(
                    count = 2
                )
            )
        )
    )
)

The full set of default curation parameters can be viewed in the MitoPilot GUI for each sample under Curation Opts. Alternatively, one could examine the source code for a specific curation parameters function. For example, here is the source code for params_fish_mito.

At the moment, these values can not be edited for individual samples within the GUI, but this feature will eventually be added.