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<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>Ahmed,<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span lang=EN-GB
style='font-size:10.0pt;font-family:Arial;color:navy'>No I did not use the
PHRED/PHRAP/CONSED package for this (I did try it but found the TGICL suite
more appropriate for ESTs).<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span lang=EN-GB
style='font-size:10.0pt;font-family:Arial;color:navy'><a
href="http://www.tigr.org/tdb/tgi/software/">http://www.tigr.org/tdb/tgi/software/</a><o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span lang=EN-GB
style='font-size:10.0pt;font-family:Arial;color:navy'>For basecalling I used a
windows platform based caller since PHRED at that time did not support our used
dyes for sequencing.. :(<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span lang=EN-GB
style='font-size:10.0pt;font-family:Arial;color:navy'>Thereafter the TGICL basically
uses megablast to cluster all sequences into groups and than assembles it using
the cap3 assembler.<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span lang=EN-GB
style='font-size:10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span lang=EN-GB
style='font-size:10.0pt;font-family:Arial;color:navy'>Clustering and assembling
is always difficult to explain. As far as I understand the clustering of large
groups of sequences speeds up the second step; assembling. Basically you end up
with contigs (having more than one sequence) or singletons.<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span lang=EN-GB
style='font-size:10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span lang=EN-GB
style='font-size:10.0pt;font-family:Arial;color:navy'>With unigene list I mean
a list of all different genes present in my sample of 13k. Like the Gene
indices lists of species present at TIGR. <a
href="http://www.tigr.org/tdb/tgi/index.shtml">http://www.tigr.org/tdb/tgi/index.shtml</a><o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span lang=EN-GB
style='font-size:10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span lang=EN-GB
style='font-size:10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span lang=EN-GB
style='font-size:10.0pt;font-family:Arial;color:navy'>Now the next steps.<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span lang=EN-GB
style='font-size:10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span lang=EN-GB
style='font-size:10.0pt;font-family:Arial;color:navy'>Alex<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span lang=EN-GB
style='font-size:10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span lang=EN-GB
style='font-size:10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span lang=EN-GB
style='font-size:10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
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<p class=MsoNormal><b><font size=2 face=Tahoma><span style='font-size:10.0pt;
font-family:Tahoma;font-weight:bold'>Van:</span></font></b><font size=2
face=Tahoma><span style='font-size:10.0pt;font-family:Tahoma'>
ahmed.moustafa@gmail.com [mailto:ahmed.moustafa@gmail.com] <b><span
style='font-weight:bold'>Namens </span></b>Ahmed Moustafa<br>
<b><span style='font-weight:bold'>Verzonden:</span></b> donderdag 10 augustus
2006 23:53<br>
<b><span style='font-weight:bold'>Aan:</span></b> Bossers, Alex<br>
<b><span style='font-weight:bold'>Onderwerp:</span></b> Re: [BiO BB] Starting
point EST annotation/grouping</span></font><o:p></o:p></p>
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<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'><o:p> </o:p></span></font></p>
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<p class=MsoNormal><font size=3 color=red face=Arial><span style='font-size:
12.0pt;font-family:Arial;color:red'>Hi Alex,</span></font><o:p></o:p></p>
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<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'> <o:p></o:p></span></font></p>
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<p class=MsoNormal><font size=3 color=red face=Arial><span style='font-size:
12.0pt;font-family:Arial;color:red'>Regarding the steps that you mentioned to
process ESTs, I would like to ask you:</span></font><o:p></o:p></p>
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<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'> <o:p></o:p></span></font></p>
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<p class=MsoNormal><font size=3 color=red face=Arial><span style='font-size:
12.0pt;font-family:Arial;color:red'>(1) What is the difference between
clustering and assembling the ESTs?</span></font><o:p></o:p></p>
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<p class=MsoNormal><font size=3 color=red face=Arial><span style='font-size:
12.0pt;font-family:Arial;color:red'>(2) What is a unigene?</span></font><o:p></o:p></p>
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<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'> <o:p></o:p></span></font></p>
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<p class=MsoNormal><font size=3 color=red face=Arial><span style='font-size:
12.0pt;font-family:Arial;color:red'>BTW, are you using the Phred/Phrap/Consed
suite?</span></font><o:p></o:p></p>
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<p><font size=3 color=red face=Arial><span style='font-size:12.0pt;font-family:
Arial;color:red'>Thanks in advance!</span></font><o:p></o:p></p>
<p><font size=3 color=red face=Arial><span style='font-size:12.0pt;font-family:
Arial;color:red'>Ahmed</span></font><o:p></o:p></p>
<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'><o:p> </o:p></span></font></p>
<div>
<p class=MsoNormal><span class=gmailquote><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'>On 8/10/06, <b><span style='font-weight:bold'>Bossers,
Alex</span></b> <<a href="mailto:Alex.Bossers@wur.nl">Alex.Bossers@wur.nl</a>>
wrote:</span></font></span> <o:p></o:p></p>
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<p><font size=2 face=Arial><span style='font-size:10.0pt;font-family:Arial'>Dear
all,</span></font><o:p></o:p></p>
<p><font size=2 face=Arial><span style='font-size:10.0pt;font-family:Arial'> </span></font><o:p></o:p></p>
<p><font size=2 face=Arial><span lang=EN-GB style='font-size:10.0pt;font-family:
Arial'>I am looking for a point where to start in "annotating" our
groups of ESTs/genes….</span></font><o:p></o:p></p>
<p><font size=2 face=Arial><span lang=EN-GB style='font-size:10.0pt;font-family:
Arial'> </span></font><o:p></o:p></p>
<p><font size=2 face=Arial><span lang=EN-GB style='font-size:10.0pt;font-family:
Arial'>Briefly;</span></font><o:p></o:p></p>
<p><font size=2 face=Arial><span lang=EN-GB style='font-size:10.0pt;font-family:
Arial'> I
have a DB containing my 13k ESTs. </span></font><o:p></o:p></p>
<p><font size=2 face=Arial><span lang=EN-GB style='font-size:10.0pt;font-family:
Arial'> I
clustered/assembled them after cleaning using the Tiger TGICL tools.</span></font><o:p></o:p></p>
<p><font size=2 face=Arial><span lang=EN-GB style='font-size:10.0pt;font-family:
Arial'> Now I
have a "unigene list" of contigs and singletons (about 7k features).</span></font><o:p></o:p></p>
<p><font size=2 face=Arial><span lang=EN-GB style='font-size:10.0pt;font-family:
Arial'> BLAST
made it possible to "annotate" about half of the sequences further
with known (useful) information.</span></font><span lang=EN-GB> </span><o:p></o:p></p>
<p><font size=2 face=Arial><span lang=EN-GB style='font-size:10.0pt;font-family:
Arial'> </span></font><o:p></o:p></p>
<p><font size=2 face=Arial><span lang=EN-GB style='font-size:10.0pt;font-family:
Arial'>Now I want to do the following (at best using (semi) automated
conditions (web based or linux based tools));</span></font><o:p></o:p></p>
<p style='margin-left:53.25pt;text-indent:-18.0pt'><font size=2 face=Arial><span
lang=EN-GB style='font-size:10.0pt;font-family:Arial'>1.</span></font><font
size=1><span lang=EN-GB style='font-size:7.5pt'>
</span></font><font face=Arial><span lang=EN-GB style='font-family:Arial'>I
would like to know how many genes of which groups/classes are available and
which genes of my set belong to it (i.e. Apoptosis, development,
immunology,…….. This is to get an overview of the genes present and how
complete the repertoire is we finally put onto our microarray. </span></font><o:p></o:p></p>
<p style='margin-left:53.25pt;text-indent:-18.0pt'><font size=2 face=Arial><span
lang=EN-GB style='font-size:10.0pt;font-family:Arial'>2.</span></font><font
size=1><span lang=EN-GB style='font-size:7.5pt'>
</span></font><font face=Arial><span lang=EN-GB style='font-family:Arial'>Are
some of these genes normally tissue specific?</span></font><o:p></o:p></p>
<p><font size=2 face=Arial><span lang=EN-GB style='font-size:10.0pt;font-family:
Arial'> </span></font><o:p></o:p></p>
<p><font size=2 face=Arial><span lang=EN-GB style='font-size:10.0pt;font-family:
Arial'> </span></font><o:p></o:p></p>
<p><font size=2 face=Arial><span lang=EN-GB style='font-size:10.0pt;font-family:
Arial'>Any help to get me started into the right direction is appreciated,</span></font><o:p></o:p></p>
<p><font size=2 face=Arial><span lang=EN-GB style='font-size:10.0pt;font-family:
Arial'> </span></font><o:p></o:p></p>
<p><font size=2 face=Arial><span lang=EN-GB style='font-size:10.0pt;font-family:
Arial'>Alex</span></font><o:p></o:p></p>
<p><font size=2 face=Arial><span lang=EN-GB style='font-size:10.0pt;font-family:
Arial'> </span></font><o:p></o:p></p>
<p><font size=2 face="News Gothic"><span lang=EN-GB style='font-size:10.0pt;
font-family:"News Gothic"'> </span></font><o:p></o:p></p>
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