[maker-devel] Question on ESTs and functional annotations

[Carson Holt]

1. The BLASTN ESTs are the BLASTN results of aligning the EST library you provided during a MAKER run.  These are filtered using the thresholds you set in the maker_bopts.ctl file for coverage, identity, etc.  I think the default coverage is 80% and the default identity is 85%.  So all alignments should contain at least 80% of the EST provided with 85% identity.  You can tighten and loosen these thresholds as needed since you do expect a certain amount of sequencing error in the ESTs.

Positive BLASTN results actually get realigned using Exonerate which gives a higher quality alignment that is splice site aware.  BLASTN alignments are recorded more for informative purposes.  The important alignments are actually the Exonerate est2genome alignments which are derived from the BLASTN alignments.  Note, you will sometimes see a BLASTN alignment on one strand while the Exonerate alignment will be on the other, this is because splice sites allow you to correctly determine the strand which is not actually inherent from the EST sequence (except for Sanger sequence).

2. For functional annotation, I like to use InterProScan from the EBI.  It can be used to identify protein domains and related GO functional categories for a gene.  InterProScan takes a while to run though, as it is an extremely extensive analysis.  The current version of MAKER includes a few accessory scripts for adding InterProScan results into the final annotation set.  The main script is ipr_update_gff.  It will add functional tags to the gene models in accordance to GFF3 format.  You can then dump these GFF3 files into a Chado database and run things like GBrowse off of it.

If you are using GBrowse for viewing your data, you can set it up to link out to the appropriate external sites.  It is not entirely simple to do, but they do provide documentation.  You will also need to be familiar with SQL for some of the more advanced options.

Hope that helps,
Carson



On 11/16/09 1:45 PM, "Shane Brubaker" <SBrubaker at Aurorabiofuels.com> wrote:

Hi, I had a couple of general questions.

1.  What are the EST blastn results that are found in the MAKER gff3 output?  I thought these might be a track of my ESTs mapped onto the genome, but they seem to contain pieces of the EST but not the entire thing?
2.  What is a good way to add functional annotation to the gene results? I have a track of protein matches, I used Swissprot as my set of proteins.  So I can see proteins and I can look up my Swissprot accession number and find out more about that gene.  But what I really want is to click on my actual gene models (from augustus or snap) and then have some functional annotation on that page which has been transferred onto the gene, as well as some links out to the appropriate external sites.  Is there a good general approach for doing that?


Thanks,
Shane

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