Verdant FAQ

1. Do I need to log in to use Verdant?

You do not need a personal User ID. We have provided a general user ID, but uploaded data and analyses under this ID will be deleted after 72 hours.

User: verdant_public_user
Password: Verdant1

2. My upload doesn't seem to be working. What's wrong?

Currently, plastomes must be in a zipped directory, and each plastome must be in its own file. If they are not, then Verdant will not proceed with the upload. You should be seeing an error message if this is the case. If you do not see an error message, either check your e-mail or contact us.
Alternatively, if you have uploaded a sample belonging to an order that is not present in the database, then Verdant will not have references to choose for annotation. We are putting together a set of genes to use in these cases, but the annotations will most likely be questionable.

3. My upload seemed to work, but I am not able to see my data.

There are few possibilities for why you might not see your data.

  • Plastome sequences should be in FASTA format.
  • FASTA ID should be in the specific format explained on either the Upload Plastomes page or in the Tutorial. Verdant uses a taxonomic system to organize data and will ignore entries that are not properly formatted.
  • Plastome IDs should not be exact copies of what is already in Verdant. If species name, accession, and source are already found in Verdant, the upload will be ignored. This is to prevent redundancy in the database.

4. Why is my annotation missing a number of genes?

There are a few possible explanations for this.

  • Assembly issues may be causing the annotation of a gene to not be accepted. If a stop codon is introduced into a protein-coding gene sequence, and the result are ORFs less than 70% the original length of the gene, then annotation will likely fail. In these cases, even if annotation of the gene is completed, it may still have a premature stop codon.
  • The naming scheme for the FASTA file is very important. Verdant uses these values to identify references from the existing database. If incorrect values are used, then Verdant will choose inappropriate references for annotation. If you are testing the accuracy of Verdant by uploading a sequence that is already in the database, alter the "Accession" value to represent your test. Keep all other taxonomic information consistent with the true taxonomy of the organism
  • Verdant will sometimes make mistakes. If a gene is completely missing, the references for that gene may have not met the threshold of similarity to be used to complete annotation. The most efficient way to fix this is to download the annotation of your plastome, alter the annotation by adding the missing gene, and reupload the annotation directly. Sequencher or Geneious are useful tools for annotating missing genes. Once the updated annotation is added, make sure to delete the old annotation from the server. You can then upload other plastomes with a more accurate and similar plastome that will be used for reference.

5. Why didn’t my search turn up any results?

If you click on “Click Here to Access Your Files” and see only the Parent Directory, there are several possible explanations.

  • First, you may not have given Verdant a valid gene or gene region to search. For example, if you tried trnL-F, nothing will happen because it isn’t on the list of available genome features. To find the list, go back to the project page and look under the heading Search the Project. There is a line “For a full list of possible regions to search, click here.” and “here” is a hyperlink. The list can also be found here.
  • Second, you may not have specified whether you want simply all the FASTA files for the features or whether you wanted an alignment. Go back to the project page, look under the search window and click on FASTA or ALIGNMENT.
  • Third, you may not have waited long enough. Because of server limitations at CyVerse, alignments of many taxa may take a while, as will tree constructions. Wait until you get an email from Verdant telling you that your search is ready.

6. Why does it take so long to produce a tree?

Verdant is designed for automated chloroplast annotations and alignment and is not meant to replace a tree-searching program on a cluster. It produces only the RAxML tree for convenience. Because the time required for the analysis scales with the number of plastomes, attempts to produce a tree will take a while. We recommend in these cases that you download the alignment and do a rigorous phylogenetic analysis on another resource such as CIPRES or your institutional server.

7. My search returned sequences files, but some of the taxa are blank. What's going on?

Not all annotated features or regions between features are present in all taxa. Lineages and species with gene loss, such as Cuscuta and other parasitic plants, and analyses including taxa with inversions will create instances where not all taxa share an orthologous segment of the chloroplast genome. In these cases, we have included the missing taxa in the output as blank. This is to inform the user that this region is missing in this taxon. In alignments, these are treated as gaps.

8. My sequence and alignment files have numbers at the beginning of them. What do they mean?

Verdant uses identifying numbers at the beginning of file names to track information about the sequences in the file. Different numbers have different meanings dependent on the type of search implemented.

  • Numbers 0-XXX: When given a range, Verdant breaks it into annotated features and identifies the inter-feature regions based on the surrounding features. The alignments are conducted on these pieces to increase accuracy. Alignments are concatenated back together in the order they were identified from the original chloroplast genome sequences. This is where the numbers come into play. They represent the order in which the features were identified so they can be concatenated together properly.
  • Number 8888: When given a range, alignments are created by concatenated individually aligned features. However, an unaligned sequence of the range pulled directly from the genomes is given. This file begins with 8888.
  • Number 9999: Single genes are returned with 9999 at the beginning of the file name.

8. I have found full plastomes in GenBank that are not in the publicly available database in Verdant. Why are they not included?

We use stringent filtering criteria (see McKain et al., supplementary information) to decide which publicly available plastomes to include. These criteria include:

  • Quadripartite structure in the format Large single copy--Inverted repeat B--Small single copy--Inverted repeat A. If sequences were not present in this format, they were ignored. Though this selects out some chloroplast genomes that are naturally lacking inverted repeats, it was easy way to remove many questionable chloroplast genomes from GenBank sequences.
  • Removal of chloroplast genomes with annotations of non-canonical chloroplast genes. Using a verified list of known angiosperm chloroplast genes, the remaining GenBank sequences were filtered to remove sequences that had species-specific, annotated ORFs. This was for consistency across all angiosperms.
  • The filtering processes identified 164 chloroplast genomes that fit these criteria, which have been added to Verdant. Users are welcome to add more plastomes from GenBank.

9. Can I use Verdant to make annotations for GenBank submission?

You can download the tbl format annotations and make GenBank submissions using existing for programs for submission (e.g. tbl2asn).

10. Can I delete my plastomes from Verdant?

Yes, you can, but only if you have not made them public yet. To delete an entry, go to the ADD/REMOVE page for a project and proceed to the Select Individual Taxa page. Identify the entry you would like to delete and scroll to the right. You will see the option "DELETE FROM SERVER".

11. My annotations are not correct. Is there something I can do to fix this?

We recommend uploading a trusted annotation for your taxa in this case. If your family is not represented in the VVerdant database or your lineage is highly diverged from its closest relatives in Verdant, the annotation process may not be able to identify identities to gene features. Users can upload annotations directly to Verdant under the Upload Annotations tab.

12. Is it possible that genes exist in my chloroplast genomes that were not annotated?

It is very possible, espeically if the reference chloroplast genomes do not possess genes that are present in your chloroplast genomes. Users can download intergenic regions from their chloroplast genomes using the "Full" keyword in the Search tool. Users can then BLAST these seqeunces to identify possible gene space. Users may also write to Verdant administrators and we can provide unannotated ORFs from chloroplast genomes.

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