Creating custom rule sets

synthaser uses a central rule file which controls which domains it will save in domain architecture predictions, as well as how sequences get classified based on those domain architectures.

The default rule file distributed in the package is meant for the analysis of fungal megasynthases (polyketide synthases, fatty acid synthases and nonribosomal peptide synthetases). However, this can be repurposed for the analysis of any other multidomain protein very easily using our web application.

What follows is a brief description of how synthaser uses the rule file at the various stages of its workflow.

Identifying domain ‘islands’

The problem synthaser aims to solve is best demonstrated in the following image:

This is the result of a CD-Search run using a hybrid polyketide synthase-nonribosomal peptide synthetase sequence as a query. Within this visualisation, you can see all of the domain hits found in the sequence during the search, as well as their positions within the sequence.

These domain hits very clearly fall into distinct ‘islands’ (indicated by blue boxes) with other, related domain hits. In this image, the domain islands correspond to the domain architecture of the synthase, KS-AT-DH-MT-KR-ACP-C-A-T-R.

While this is easy to figure out manually by looking at the visualisation, we often want to analyse larger numbers of these sequences (e.g. when looking at full fungal genomes), which quickly makes this manual approach prohibitive. This is where synthaser comes in.

Creating domain types

The first element of the synthaser rule file is the list of domain ‘types’ that we want synthaser to find in the CD-Search results. Each domain type is given a name as well as a list of specific domain families which correspond to the type.

Thinking back to the domain islands example above, we may wish to create a domain type called KS, which consists of the domain families PKS and PKS_KS (the top-scoring domain families in the search). This would indicate to the synthaser that whenever it finds hits for either PKS or PKS_KS domains, they should be saved and categorised as KS domains.

These can be edited in the leftmost pane in the web application:

As mentioned, we first give the domain type a name (e.g. C), then we choose a list of specific domain families (e.g. C_PKS-NRPS, Condensation). You can directly search for families in this box by accession or name.

Creating classification rules

Once synthaser has identified the domains in a query sequence and predicted its domain architecture, the next task is classification. As with the domain identification step, while we can easily determine domain architecture directly from the visual output and tell what type of synthase we have, the difficulty comes when we want to analyse more than a single sequence at a time.

These can be edited in the Classification rules pane of the web application:

This picture shows the rule for classifying a PKS-NRPS sequence. It consists of:

1. A name, given to a sequence when the rule is evaluated succesfully (Hyrid PKS-NRPS).
2. A list of domain types chosen from a multi-selection box populated by domain types created in the Domain types pane (KS, A, C, ACP, TR).
3. An evaluation expression which checks for presence of key domains for this rule (0 for KS, 1 for A and 2 for C). This rule will be satisfied if a sequence has a KS domain AND either an A OR C domain.
4. A renaming rule which specifies that a domain type should be renamed in a specific circumstance due to convention (a TR domain, if found after a C or A domain, is renamed to R).

The only thing this rule does not have is domain filters, which would control the specific domain families that should be saved for a domain type. In the fungal ruleset, this is key to being able to distinguish between KS domains from fatty acid synthases and polyketide synthases, for example.

Classification rule evaluation hierarchy

Once you have established all of your classification rules, you need to define the order in which they should be evaluated. This can be done in the Rule hierarchy pane:

This list is automatically populated by the rules that you created in the Classification rules pane; by default, they will all be on the same evaluation level.

Rules are evaluated in top-to-bottom order according to this hierarchy. They can be rearranged by simply clicking and dragging the grey handle to the left of the rule name and dropping them where you want them. This could simply mean reordering rules on the same level (e.g. moving NRPS before Hybrid PKS-NRPS), or making certain rules children of others (e.g. if the PKS rule is successfully evaluated, only then does synthaser try to evaluate the Type I rule). synthaser supports arbitrary levels of nesting.

Using the rule file

Once you’re happy with your rule set, click the Save rules button in the top-right of the web application. This wll download a JSON format file containing the domain types, classification rules and hierarchy, that you can pass directly to synthaser for it to use instead of the fungal ruleset.

For example, given a custom rule file, my_ruleset.json:

synthaser search -qf sequences.fa --rule_file my_ruleset.json

If you ever want to modify your rule set, this can also be done in the web application by loading your rule file with the Load rules button, making your tweaks, then clicking Save rules to generate a new file.