Overview page#

The overview page contains a summary of all regions found in all records/contigs within the input given to antiSMASH.

Region overview

At the top left of the page is the antiSMASH version information (1). Direct comparisons between antiSMASH results should use the same version for consistency, as results can change between versions.

At the top right of the page are ancillary links that may be useful. Download allows you to download various parts of the results. About links to information about antiSMASH (including publications). Help links to these documentation pages. Finally, Contact links to a page with a form to send feedback or questions to the antiSMASH developers.

Under the top bar are region buttons (2). These buttons are visible on all antiSMASH HTML pagesand can be used to quickly jump between regions. If a region is being viewed, the matching button for that region will be highlighted. Region buttons are in the form X.Y. For example, 5.7 would be the seventh region within the fifth record. Region buttons are color coded by predicted secondary metabolite type. Clicking the Overview button will bring you back to this page.

Under these buttons are a summary of each record, each starting the name of the record (3) as given in the input file. At the top of the summary is an image (4) showing where the regions are located in the record. Following that is a table with a summary of each region found in the record, with each row (5) having the following fields:

  • Region: the region number
  • Type: the product types as detected by antiSMASH (clicking the types will take you to their description in these help pages)
  • From, To: the location of the region (in nucleotides)
  • Most similar known cluster: the closest compound from the MiBIG database (clicking this will take you to the MiBIG entry), along with its type (e.g. t1pks+t3pks)
  • Similarity: a percentage of genes within the closest known compound that have a significant BLAST hit to genes within the current region

The last two columns containing comparisons to the MiBIG database will only be shown if antiSMASH was run with the KnownClusterBlast option.

Clicking anywhere in the row that is not already an external link will take you to the detailed results for that region.

The antiSMASH 5 region concept#

Currently, there is no good method available to accurately predict gene cluster borders based purely on the submitted sequence data (with the exception of the CASSIS algorithm, which is able to detect co-regulated genes in fungal genomes). In antiSMASH5, the display of gene clusters change to reflect the fact that the BGC borders are just offsets defined in the cluster detection rules, we renamed the highest level that is displayed to “Region”.

A region in antiSMASH 5 corresponds to the gene cluster annotation in antiSMASH 4 and earlier.

How are antiSMASH 5 regions defined?#

In the first step, all gene products of the analyzed sequence are searched against a database of highly conserved enzyme HMM profiles (core-enzymes), which are indicative of a specific BGC type. In a second step, pre-defined cluster rules are employed to define individual protoclusters encoded in the region. These make up a core, which is extended by its neighbourhood, which constiutes of genes encoded up- and downstream of the core.

Here is an example of a rule for detecting a protocluster for a type I PKS:

Example cluster rule

Whenever antiSMASH finds a gene coding for a protein that has as PKS_AT domain and a PKS_KS domain of various sub-types, a new “Type I PKS protocluster” feature is generated within the region; this feature comprises the core gene-product(s) that trigger the rule (i.e. the PKS encoding genes and extends the neighborhood to the left and right of the core genes by 20 kb to the left and right (as defined by the EXTENT parameter in the rule definition). The values for the different cluster types are empirically determined and generally tend to overpredict, i.e. included also adjacent genes. After the protocluster features are assigned (note: there can be multiple protocluster features within a single region), they are checked for overlaps (as defined by the CUTOFF parameter) and are grouped into several types of candidate clusters to reflect the observation that many BGCs actually comprise several classes of biosynthetic machinery. For example glycopeptides like vancomycin or balhimycin (shown) are synthesized via NRPS, but also contain a type III PKS as a precursor pathway.

Balhimycin biosynthetic gene cluster

Thus, the displayed region contains the NRPS protocluster (green bar) and a T3PKS protocluster yellow bar. As their neighbourhoods overlap, they are assigned to a candidate cluster.

Candidate cluster types#

Figure candidate clusters

Chemical hybrid#

These candidate clusters contains protoclusters which share cluster-defining CDS/genes/gene products. They will also include protoclusters within that shared range that do not share a CDS, provided that they are completely contained within the supercluster border. For example, case A in the image above.

In the example, CDS 1 and 2 define the protocluster of type A, and CDS 2 and 4 define the protocluster of type B. Since both protoclusters share a CDS that defines those protoclusters, a chemical hybrid candidate cluster is created. If CDS 4 and some later CDS were also contributing to a third protocluster type, it would be merged into this same candidate cluster. If CDS 3 defined another protocluster which did not extend past CDS 1 or 4, then that new protocluster would also be included in the chemical hybrid, under the assumption that it is relevant to the other protoclusters.

Interleaved#

These candidate clusters contain protoclusters which do not share cluster-defining CDS features, but their core locations overlap, e.g. case B in the image above.  

Neighbouring#

These candidate clusters are the weakest form of hybrid candidates. They contain protoclusters which do not match either chemical or interleaved variants, but transitively overlap in their neighbourhoods. Every protocluster in a "neighbouring candidate cluster" will also belong to one of the other kinds of candidate clusters, including the single kind, described below.

Single#

This kind of candidate clusters exists where only one protocluster is contained, e.g. case D. It exists only for internal consistency. A single candidate cluster will not exist for a protocluster which is contained in either a chemical hybrid or an interleaved candidate cluster, but will exist for each inside a neighbouring candidate. For example, both protoclusters in case C will have an independent single candidate, along with the neighbouring.

Region Results page#

Region display

In the upper panel, "Gene cluster description", information is given about each gene cluster that was detected. In the upper line, the biosynthetic type and location of the gene cluster are displayed. Underneath this title line, all genes present in a detected gene cluster are outlined. The borders of the gene clusters have been estimated using different greedily chosen cut-offs specified per gene cluster type. Genes are color coded by predicted function. Putative biosynthetic genes are colored red, transport-related genes are colored blue, and regulation-related genes are colored green. These predictions depend on the smCOG functionality an will be missing if you chose to not run smCOG predictions. Hovering over a gene with the mouse will prompt the gene name to be displayed above the gene.

Gene details#

Gene details display

Clicking the gene will provide more information on the gene: its annotation, its smCOG (secondary metabolism gene family), its location, and cross-links specific to that gene.

Detailed annotation for PKS/NRPS domains#

Region display

In the middle panel, "Detailed annotation", you can find more in-depth information on the selected gene cluster. For predicted modular polyketide synthase (PKS) and/or nonribosomal peptide synthetase (NRPS) proteins, you will find the domain annotations.

Clicking on a domain image will prompt more information to be displayed, such as the name of the detected domain, its precise location, any substrate specificites predicted, and a link to run Blast on the domain. For predicted Lantipeptide clusters, the predicted core peptide sequences of all identified prepeptides is displayed. A list of detected domains can be found here.

Identifying similar BGCs#

The tab "Homologous gene clusters", displays the top ten gene clusters from the internal antiSMASH database that are most similar to a detected gene cluster, visually aligned to it. The drop-down selection menu can be used to browse through the gene clusters. Genes with the same colour are putative homologs based on significant Blast hits between them. Similar information is provided in the "KnownCluserBlast folder", which detects BGCs included in the curated MIBiG dataset. "SubClusterBlast" provides information about conserved operons, which, for example are involved in precursor biosynthesis of non-proteinogenic amino acids.

Prediction of putative core sturctures#

Structure Prediction

In the upper side panel on the right. "Predicted core structure", a rough prediction of the overall chemical structure of a the product of a detected nonribosomal peptide or polyketide biosynthesis gene cluster is given. The second folder "PKS/NRPS monomers" displays detailed predictions for all monomers. Prediction details are available for multiple methods.

PKS type I AT domain specificities are predicted using a twenty-four amino acid signature sequence of the active site (Yadav et al., 2003), as well as with pHMMs based on the method of Minowa et al. (Minowa et al., 2007), which is also used to predict co-enzyme A ligase domain specificities. NRPS A domain specificities are predicted using both the signature sequence method and the support-vector machines-based method of NRPSPredictor2 (Rausch et al., 2005 & Röttig et al., 2011), and the method of Minowa et al. (Minowa et al., 2007). Ketoreductase domain-based stereochemistry predictions for PKSs (Starcevic et al., 2008) are also performed.

For PKS type II clusters, the side panel shows a list of the predicted starter units along with bitscores, a list of the predicted number of malonyl elongations along with bitscores, the predicted product class, a list of product molecular weights predictions for each combination of starter unit and number of malonyl elongations. Furthermore, it displays each gene/protein that is involved in the polyketide biosynthesis of the cluster. For each gene/protein the type is indicated and for some a prediction of function is displayed. More details on the predictions can be found here.

A chemical structure is displayed for NRPS and Type I PKS metabolites, which is derived by combining the results of the domain analyses.

IMPORTANT NOTE: This is a structure prediction of the core structure of the molecule; it is very likely the final molecule will be different!

Downloading results#

Main menu

In the upper right, a small list of buttons offers further functionality. The question-mark button will get you to this help page. The exclamation-mark button leads to a page explaining about antiSMASH. The downward-pointing arrow will open a menu offering to download the complete set of results from the antiSMASH run, a summary GenBank output file, or a log file for debug purposes. The GenBank file can be viewed in a genome browser such as Artemis.

Results on the public webserver are only kept for ONE month and will be deleted afterwards. It is highly recommended that you download the full result set before they expire.