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<title>Re: [maker-devel] Question on ESTs and functional annotations</title>
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<p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";
color:#1F497D'>Thanks very much Carson, that is quite helpful. I will try
using the est2genome results instead of the blastn results.<o:p></o:p></span></p>
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<p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";
color:#1F497D'>I am indeed using Chado and Gbrowse, so that is what I would
like to do. I will try using InterproScan.<o:p></o:p></span></p>
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<p class=MsoNormal><b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'>From:</span></b><span
style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'> Carson Holt
[mailto:carson.holt@genetics.utah.edu] <br>
<b>Sent:</b> Monday, November 16, 2009 3:42 PM<br>
<b>To:</b> Shane Brubaker; maker-devel@yandell-lab.org<br>
<b>Subject:</b> Re: [maker-devel] Question on ESTs and functional annotations<o:p></o:p></span></p>
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<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:11.0pt;
font-family:"Calibri","sans-serif"'>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.<br>
<br>
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).<br>
<br>
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.<br>
<br>
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.<br>
<br>
Hope that helps,<br>
Carson<br>
<br>
<br>
<br>
On 11/16/09 1:45 PM, "Shane Brubaker" <<a
href="SBrubaker@Aurorabiofuels.com">SBrubaker@Aurorabiofuels.com</a>> wrote:</span><o:p></o:p></p>
<p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-size:11.0pt;
font-family:"Calibri","sans-serif"'>Hi, I had a couple of general questions.<br>
<br>
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?<br>
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?<br>
<br>
<br>
Thanks,<br>
Shane<br>
<br>
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