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<P><IMG SRC="GeneSeqerTutorial_files/pgdb.gif" ALT="NSF Plant Genome Database" WIDTH=150 HEIGHT=100 ALIGN=bottom>
<DIV id=left><B><U>What Is This?</U></B><BR>
</DIV></P>
</TD>
<TD VALIGN=top>
<P><BR>
</P>
<H2><CENTER>GeneSeqer@PlantGDB - guided
tutorial</CENTER></H2>
<P>
<HR SIZE=1>
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<td align="center"><a href="/cgi-bin/PlantGDBblast">BLAST@PlantGDB</a></td>
<td align="center"><a href="http://www.plantgdb.org/cgi-bin/GeneSeqer.cgi">GeneSeqer@PlantGDB</a></td>
<td align="center"><a href="http://www.zmdb.iastate.edu/cgi-bin/download?head=header&foot=footer&dir=/ZmDB">Download</a></td>
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<P>This step-by-step tutorial covers the basic concepts
behind setting up a GeneSeqer@PlantGDB gene structure
analysis, understanding the results of an analysis, and
applying these results to further investigations. This
tutorial illustrates the individual steps necessary to
use the <A HREF="http://www.plantgdb.org/cgi-bin/GeneSeqer.cgi">http://www.plantgdb.org/cgi-bin/GeneSeqer.cgi</A>
web service and tips to make its use more efficient and
rewarding.</P>
<P><BR>
</P>
<H3><A HREF="http://www.plantgdb.org/tutorial/#Setup"><FONT COLOR="#000000">Setting
up GeneSeqer@PlantGDB</FONT></A></H3>
<UL>
<LI><A HREF="http://www.plantgdb.org/tutorial/#Plan">Step
1. Getting started</A></LI>
<LI><A HREF="http://www.plantgdb.org/tutorial/#EnterG">Step
2. Entering genomic (target) sequence</A></LI>
<LI><A HREF="http://www.plantgdb.org/tutorial/#EnterT">Step
3. Entering transcribed (query) sequence(s)</A></LI>
<LI><A HREF="http://www.plantgdb.org/tutorial/#Choose">Step
4. Choosing parameters and options</A></LI>
<LI><A HREF="http://www.plantgdb.org/tutorial/#Submit">Step
5. Submission</A></LI>
</UL>
<P> </P>
<H3><A HREF="http://www.plantgdb.org/tutorial/#Results"><FONT COLOR="#000000">Interpreting
the results</FONT></A></H3>
<UL>
<LI><A HREF="http://www.plantgdb.org/tutorial/#Picture">The
summary graphic</A></LI>
<LI><A HREF="http://www.plantgdb.org/tutorial/#Alignment">The
alignment file</A></LI>
<LI><A HREF="http://www.plantgdb.org/tutorial/#Prediction">The
predicted gene structures and ORFs</A></LI>
</UL>
<P> </P>
<H3><A HREF="http://www.plantgdb.org/tutorial/#Refinement"><FONT COLOR="#000000">Further
investigation & refined analysis</FONT></A></H3>
<UL>
<LI><A HREF="http://www.plantgdb.org/tutorial/#Detailed">http://www.plantgdb.org/tutorial/#Detailed</A></LI>
<LI><A HREF="http://www.plantgdb.org/tutorial/#HomologPa">http://www.plantgdb.org/tutorial/#HomologPa</A></LI>
</UL>
<P><BR>
<BR>
<HR SIZE=1>
</P>
</TD>
<TD>
<P></P>
</TD>
</TR></TBODY>
</TABLE>
<A NAME=Setup></A></P>
<H3><FONT COLOR="#660033">Setting up
GeneSeqer@PlantGDB</FONT></H3>
<P><A NAME=Plan></A></P>
<H4><FONT COLOR="#007766">Step 1. Getting started</FONT></H4>
<P>The <A HREF="http://www.plantgdb.org/cgi-bin/GeneSeqer.cgi" TARGET="_blank">http://www.plantgdb.org/cgi-bin/GeneSeqer.cgi</A>
web service is intended primarily for the purpose of performing
spliced alignment of query sequences (sequences representing
transcribed genes, i.e. ESTs, cDNAs, and proteins) with a target
sequence (genomic DNA). The results of such an alignment are
useful in many studies. Because there are a number of questions
which may be addressed by spliced alignment, the process by which
you use GeneSeqer@PlantGDB may vary. For instance, your choice of
input sequences can be determined based on similarity to other
sequences (both genomic and transcribed) or you may already
possess an uncharacterized sequence you wish to know more about.
In this example, the later case applies. The form elements found
in this tutorial are identical to those of the standard web
service. You may wish to <A HREF="http://www.plantgdb.org/cgi-bin/GeneSeqer.cgi" TARGET=tutorial>http://www.plantgdb.org/cgi-bin/GeneSeqer.cgi</A>
and follow along in parallel with this tutorial however this is
not required.</P>
<P><BR>
<BR>
<A NAME=EnterG></A></P>
<H4><FONT COLOR="#007766">Step 2. Entering genomic (target)
sequence</FONT></H4>
<P>To demonstrate a typical use of this service, we have choosen
to characterize a segment of genomic sequence from <I>Sorghum
bicolor</I> (<A HREF="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=nucleotide&cmd=search&term=AF503433" TARGET="_blank">GenBank
accession AF503433</A>). This bacterial artificial chromosome
(BAC) represents approximately 142,000 bases of genomic sequence.
While this sequence could be pasted into the large text area below
or even saved to a local file and uploaded, we have choosen to
simply enter the accession number in the appropriate field as
shown. To finish this step, we select the <U>GenBank</U> format. A
detailed description of optional formats can be found at the
<A HREF="http://bioinformatics.iastate.edu/bioinformatics2go/gs/help.html#FORMAT" TARGET="_blank">Select
format</A> link.<BR>
<BR>
<TABLE WIDTH="50%" align=center><TBODY>
<TR>
<TD>
<P><TABLE WIDTH="50%" align=center><TBODY>
<TR>
<TD>
<UL>
<LI> </LI>
<LI>
<H3><FONT COLOR="#FF0000">Enter sequence
accesion number</FONT></H3></LI>
<LI>
<H3><FONT COLOR="#FF0000">Select appropriate
format</FONT></H3></LI>
</UL>
</TD>
</TR></TBODY>
</TABLE>
</P>
</TD>
</TR>
<TR>
<TD>
<P>
<HR>
</P>
<P> </P>
<H2><CENTER>STEP 2: Input genomic
DNA sequence</CENTER></H2>
<P>Sequences should be in the one-letter-code
({a,b,c,g,h,k,m,n,r,s,t,u,w,y}), upper or lower case; all
other characters are ignored during input. Multiple
sequence input is accepted in <A HREF="http://bioinformatics.iastate.edu/bioinformatics2go/gs/help.html#FASTA">FASTA</A>
format (sequences separated by identifier lines of the
form “>SQ;name_of_sequence comments”) or in
<A HREF="http://bioinformatics.iastate.edu/bioinformatics2go/gs/help.html#GENBANK">GenBank</A>
format.</P>
<P><I>Paste your genomic DNA sequence here:</I><BR>
<TEXTAREA NAME="_gdnap" ROWS=5 COLS=82 WRAP=virtual></TEXTAREA><BR>
<I>... or upload your sequence file (specify file
name):</I><BR>
<BR>
<FONT COLOR="#FF0000"><I>... or type in the GenBank
accession number of your sequence:</I><BR>
<INPUT TYPE=text NAME=acc VALUE="AF503433" SIZE=50></FONT><BR>
<A HREF="http://bioinformatics.iastate.edu/bioinformatics2go/gs/help.html#FORMAT">Select
format</A>:
<INPUT TYPE=radio NAME="_l" VALUE="plain ">plain
<INPUT TYPE=radio NAME="_l" VALUE="FASTA ">FASTA
<FONT COLOR="#FF0000"><INPUT TYPE=radio NAME="_l" VALUE=GenBank CHECKED>GenBank</FONT><BR>
<A HREF="http://bioinformatics.iastate.edu/bioinformatics2go/gs/help.html#SNAME">Sequence
name</A>: <INPUT TYPE=text NAME="_f" VALUE="" SIZE=30>
(optional, used in <A HREF="http://bioinformatics.iastate.edu/bioinformatics2go/gs/help.html#PLAIN">plain
sequence format</A> only)<BR>
<A HREF="http://bioinformatics.iastate.edu/bioinformatics2go/gs/help.html#FROMTO">From
position</A>: <INPUT TYPE=text NAME="_a" VALUE="" SIZE=10>
<A HREF="http://bioinformatics.iastate.edu/bioinformatics2go/gs/help.html#FROMTO">to
position</A>: <INPUT TYPE=text NAME="_b" VALUE="" SIZE=10>
<A HREF="http://bioinformatics.iastate.edu/bioinformatics2go/gs/help.html#STRAND">Strand</A>:
<INPUT TYPE=radio NAME="_r" VALUE="original ">original
<INPUT TYPE=radio NAME="_r" VALUE="reverse ">reverse
<INPUT TYPE=radio NAME="_r" VALUE=both CHECKED>both
<HR>
</P>
</TD>
</TR></TBODY>
</TABLE>
</P>
<P><BR>
<BR>
<A NAME=EnterT></A></P>
<H4><FONT COLOR="#007766">Step 3. Entering transcribed (query)
sequence(s)</FONT></H4>
<P>While an exhaustive alignment of "All Plant" ESTs and cDNAs is
possible, the size of our genomic sequence in this example calls
for a more efficient approach. Generally, when characterizing a
large genomic sequence (as is being demonstrated), the detection
of genic regions is the primary goal. Only after their detection,
are these regions looked at in greater detail. Note the options
for alignment of specific data types as well as individual or
logical species groups. These functions as well as alignment of
sequences of your own choosing are discussed in the <A HREF="http://www.plantgdb.org/tutorial/#Refinement">refined
analysis</A> section later in this tutorial.<BR>
For now, we choose to align only the representative TUG collection
of all plants. This sequence collection represents the Tentative
Unique Gene clusters assembled using the <A HREF="http://www.plantgdb.org/ESTCluster/progress.html" TARGET="_blank">PlantGDB
contiging method</A>.<BR>
<BR>
<TABLE WIDTH="50%" align=center><TBODY>
<TR>
<TD>
<P><TABLE WIDTH="50%" align=center><TBODY>
<TR>
<TD>
<UL>
<LI> </LI>
<LI>
<H3><FONT COLOR="#FF0000">Select "All Plants"
TUG database</FONT></H3></LI>
</UL>
</TD>
</TR></TBODY>
</TABLE>
</P>
</TD>
</TR>
<TR>
<TD>
<P>
<HR>
</P>
<H2><CENTER>STEP 3: Select or
input cDNA/EST sequences</CENTER></H2>
<P>Spliced Alignment: The output will show an optimal
threading of a significantly matching cDNA/EST sequence
into the genomic DNA by aligning putative exons only and
displaying putative introns as (long) gaps in the
cDNA/EST. You can supply your own EST/cDNA sequence as
well as your own protein sequence to do the spliced
alignment. However, your own EST/cDNA sequence can NOT be
combined with our pre-processed databases (EST, TUG, or
cDNA).</P>
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<TABLE CELLPADDING=4><TBODY>
<TR>
<TH>
<P>Database</P>
</TH>
<TH>
<P>EST</P>
</TH>
<TH>
<P>TUG</P>
</TH>
<TH>
<P>cDNA</P>
</TH>
<!-- <th align="LEFT">Protein</th> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><I>Shortcut</I></P>
</TD>
<TD>
<P></P>
</TD>
<TD>
<P></P>
</TD>
<TD>
<P></P>
</TD>
</TR>
<TR>
<TD>
<P><A HREF="http://www.zmdb.iastate.edu/PlantGDB/">All
Plants</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=AllPLANTest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=AllPLANTtug CHECKED></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=AllPLANTcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='AllPLANTprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.zmdb.iastate.edu/PlantGDB/">All
Monocots</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=AllMONOCOTest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=AllMONOCOTtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=AllMONOCOTcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='AllMONOCOTprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.zmdb.iastate.edu/PlantGDB/">All
Dicots</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=AllDICOTest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=AllDICOTtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=AllDICOTcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='AllDICOTprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.zmdb.iastate.edu/PlantGDB/">All
Grasses</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=AllGRASSest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=AllGRASStug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=AllGRASScdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='AllGRASSprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD COLSPAN=4 HEIGHT=22>
<P></P>
</TD>
</TR>
<TR>
<TD>
<P></P>
</TD>
<TD>
<P></P>
</TD>
<TD>
<P></P>
</TD>
<TD>
<P></P>
</TD>
</TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?id=3702" TARGET="_blank">Arabidopsis
thaliana</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=ATest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=ATtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=ATcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='ATprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?id=4498" TARGET="_blank">Avena
sativa</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=ASest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=AStug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=AScdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='ASprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?id=3555" TARGET="_blank">Beta
vulgaris subsp. vulgaris</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=BVSVest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=BVSVtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=BVSVcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='BVSVprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THISOPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?id=3847" TARGET="_blank">Glycine
max</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=GMest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=GMtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=GMcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='GMprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?id=29729" TARGET="_blank">Gossypium
arboreum</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=GAest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=GAtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=GAcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='GAprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?id=3635" TARGET="_blank">Gossypium
hirsutum</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=GHest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=GHtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=GHcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='GHprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?mode=Info&id=4513&lvl=3&nucl=1&keep=1&srchmode=1&unlock" TARGET="_blank">Hordeum
vulgare</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=HVest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=HVtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=HVcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='HVprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?mode=Info&id=34305&lvl=3&nucl=1&keep=1&srchmode=1&unlock" TARGET="_blank">Lotus
japonicus</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=LJest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=LJtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=LJcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='LJprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?id=4081" TARGET="_blank">Lycopersicon
esculentum</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=LEest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=LEtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=LEcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='LEprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?id=62890" TARGET="_blank">Lycopersicon
hirsutum</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=LHest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=LHtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=LHcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='LHprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?id=28526" TARGET="_blank">Lycopersicon
pennellii</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=LPest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=LPtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=LPcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='LPprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?id=3197" TARGET="_blank">Marchantia
polymorpha</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=MPest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=MPtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=MPcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='MPprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?mode=Info&id=3879&lvl=3&nucl=1&keep=1&srchmode=1&unlock" TARGET="_blank">Medicago
sativa</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=MSest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=MStug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=MScdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='MSprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?id=3880" TARGET="_blank">Medicago
truncatula</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=MTest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=MTtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=MTcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='MTprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?id=3554" TARGET="_blank">Mesembryanthemum
crystallinum</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=MCest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=MCtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=MCcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='MCprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?id=4530" TARGET="_blank">Oryza
sativa</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=OSest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=OStug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=OScdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='OSprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?id=3352" TARGET="_blank">Pinus
taeda</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=PTest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=PTtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=PTcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='PTprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?id=3352" TARGET="_blank">Populus
tremula X Populus tremuloides</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=PTPTest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=PTPTtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=PTPTcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='PTPTprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THISOPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?mode=Info&id=4550&lvl=3&nucl=1&keep=1&srchmode=1&unlock" TARGET="_blank">Secale
cereale</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=SCest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=SCtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=SCcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='SCprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?mode=Info&id=4113&lvl=3&keep=1&srchmode=1&unlock" TARGET="_blank">Solanum
tuberosum</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=STest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=STtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=STcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='STprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?id=4558" TARGET="_blank">Sorghum
bicolor</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=SBest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=SBtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=SBcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='SBprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?id=132711" TARGET="_blank">Sorghum
propinquum</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=SPest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=SPtug></P>
</TD>
<TD>
<P></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='SPprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?mode=Info&id=4565&lvl=3" TARGET="_blank">Triticum
aestivum</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=TAest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=TAtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=TAcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='TAprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR>
<TR>
<TD>
<P><A HREF="http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?id=4577" TARGET="_blank">Zea
mays</A></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=ZMest></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=ZMtug></P>
</TD>
<TD>
<P><INPUT TYPE=checkbox NAME="_d" VALUE=ZMcdna></P>
</TD>
<!-- <td><input type='checkbox' name='_d' value='ZMprotein'></td> WAIT UNTIL GENESEQER2 TO ACTIVEATE THIS OPTION --></TR></TBODY>
</TABLE>
<SCRIPT LANGUAGE=javascript><!--
if(document.getElementById || document.all)
toggleElement(hider_list);
//--></SCRIPT>
<TABLE CELLPADDING=4><TBODY>
<TR>
<TD>
<P><I>OR paste your own
</I><B><I>EST/cDNA</I></B><I> sequence(s) here
in </I><A HREF="http://bioinformatics.iastate.edu/bioinformatics2go/gs/help.html#FORMAT"><I>FASTA</I></A><I>
format</I><BR>
<TEXTAREA NAME="_estp" ROWS=5 COLS=60 WRAP=virtual></TEXTAREA></P>
</TD>
<TD>
<P><I>... and/or paste your own
</I><B><I>Protein</I></B><I> sequence(s) here in
</I><A HREF="http://bioinformatics.iastate.edu/bioinformatics2go/gs/help.html#FORMAT"><I>FASTA</I></A><I>
format</I><BR>
<TEXTAREA NAME="_trgpp" ROWS=5 COLS=60 WRAP=virtual></TEXTAREA></P>
</TD>
</TR>
<TR>
<TD>
<P><I>... and/or upload your
</I><B><I>EST/cDNA</I></B><I> sequence file
(specify file name):</I><BR>
</P>
</TD>
<TD>
<P><I>... and/or upload your
</I><B><I>Protein</I></B><I> sequence file
(specify file name):</I><BR>
</P>
</TD>
</TR></TBODY>
</TABLE>
<HR>
</P>
</TD>
</TR></TBODY>
</TABLE>
</P>
<P><BR>
<BR>
<A NAME=Choose></A></P>
<H4><FONT COLOR="#007766">Step 4. Choosing parameters and
options</FONT></H4>
<P>We now choose the maize splicing model parameter, being the
most closely related model available.<BR>
<BR>
<TABLE WIDTH="50%" align=center><TBODY>
<TR>
<TD>
<P><TABLE WIDTH="50%" align=center><TBODY>
<TR>
<TD>
<UL>
<LI> </LI>
<LI>
<H3><FONT COLOR="#FF0000">Select maize splice
site model</FONT></H3></LI>
</UL>
</TD>
</TR></TBODY>
</TABLE>
</P>
</TD>
</TR>
<TR>
<TD>
<P>
<HR>
</P>
<H2><CENTER>STEP 4: Select splice
site model</CENTER></H2>
<P><A HREF="http://bioinformatics.iastate.edu/bioinformatics2go/gs/help.html#SPECIES">Species</A>
(selects species-specific splice site model)
<SELECT NAME="_s">
<OPTION VALUE=Arabidopsis>Arabidopsis
<OPTION VALUE=maize SELECTED>maize
<OPTION VALUE=generic>generic
</SELECT>
<HR>
</P>
</TD>
</TR></TBODY>
</TABLE>
</P>
<P><BR>
<BR>
<A NAME=Submit></A></P>
<H4><FONT COLOR="#007766">Step 5. Submission</FONT></H4>
<P>And finally our sequences are submitted. For a large sequence
such as this, analysis may take as long as 30 min. Thus results
are available via email as well. The results of this demonstration
have been cached and are therefore immediately available by
clicking the submit button.<BR>
<BR>
<TABLE WIDTH="50%" align=center><TBODY>
<TR>
<TD>
<P><TABLE WIDTH="50%" align=center><TBODY>
<TR>
<TD>
<UL>
<LI> </LI>
<LI>
<H3><FONT COLOR="#FF0000">Submit for
processing</FONT></H3></LI>
</UL>
</TD>
</TR></TBODY>
</TABLE>
</P>
</TD>
</TR>
<TR>
<TD>
<P>
<HR>
</P>
<H2><CENTER>STEP 4: Submit
job</CENTER></H2>
<P>Select "Submit" to send the job to the server. By
default, output will be posted to your browser. You may
select to have the ouput sent to you by email instead.
Selection of this option is advised in the rare case that
output posting is slow due to server overload.</P>
<P><A NAME=SUBMITNOW></A><INPUT TYPE=checkbox NAME=Click VALUE=on>Click
here to send the output to this email address:
<INPUT TYPE=text NAME=email VALUE="" SIZE=25><BR>
<INPUT TYPE=checkbox NAME=HTML VALUE=on>HTML formatted
output [default: simple text].This option will
not work if your mailer wraps long lines.</P>
<P><SCRIPT LANGUAGE=JavaScript><!--
function showCachedResults(){
window.location="#Results";
window.open("./SB_GSQexample/SBtugGSQ.html","SbBxGSQ");
return 0;
}
--></SCRIPT>
<INPUT TYPE=button NAME=Button VALUE="Submit" onclick="showCachedResults();">
<INPUT TYPE=reset VALUE="Reset">
<INPUT TYPE=hidden NAME=".cgifields" VALUE="_l"><INPUT TYPE=hidden NAME=".cgifields" VALUE=Click><INPUT TYPE=hidden NAME=".cgifields" VALUE=HTML><INPUT TYPE=hidden NAME=".cgifields" VALUE="_r"></FORM>
<HR>
</P>
</TD>
</TR></TBODY>
</TABLE>
</P>
<P><BR>
<BR>
<A NAME=Results></A></P>
<H3><FONT COLOR="#660033">Interpreting the results</FONT></H3>
<P>If neccessary, click <A HREF="http://www.plantgdb.org/tutorial/SB_GSQexample/SBtugGSQ.html" TARGET=SbBxGSQ>here</A>
to open the results of the GeneSeqer@PlantGDB analysis described
above.<BR>
<BR>
<B>NOTE:</B> For large files, most browsers will take a while to
correctly process all location tags. This may cause links on the
graphic to appear non-functional. After the browser has completely
loaded the page however, all links will be fully functional.</P>
<P><TABLE CELLSPACING=5><TBODY>
<TR>
<TD VALIGN=top>
<P><BR>
<IMG SRC="GeneSeqerTutorial_files/GSQdisplay.png" WIDTH=500 HEIGHT=500 ALIGN=bottom hspace=10></P>
</TD>
<TD>
<P><A NAME=Picture></A></P>
<H4><FONT COLOR="#007766">The summary graphic</FONT></H4>
<P>Large genomic sequences are broken into fragments of
60000 bases for visualization. Each of these segments can
be viewed by selecting it in the drop down menu on the
left of the results window. The corresponding graphic
summary for each segment is displayed in the upper pane
of the results window. The summary graphic is clickable;
by selecting a structure (colored arrow) within the
graphic, the alignment file in the lower pane will be
scrolled to the appropriate section dealing with the
element represented by your selection. Colored arrows
represent aligned sequences and predicted gene structures
according to their unique color. In this example, red
arrows represent predicted open reading frames; green
arrows represent possible gene structures (possibly
alternative structures); and blue arrows represent the
alignment of EST or cDNA sequences. For all arrow
drawings exons are represented as colored rectangles
connect by thin lines which depict introns. A legend as
to the color scheme is shown when you move your cursor
over the "PREDICTION SUMMARY" title above the
graphic.</P>
<P><BR>
<A NAME=Alignment></A></P>
<H4><FONT COLOR="#007766">The alignment file</FONT></H4>
<P>The alignment file found in the lower pane of the
results window is the heart of the GeneSeqer@PlantGDB
output. This text shows the base-to-base alignment of the
expressed sequence(s) with the genomic DNA. Predicted
introns are shown as strings of periods '.'. Score
statistics for the alignment quality as well as the
predicted splice site quality are shown for each aligned
sequence. In addition, links to the source of each
sequence are provided above their respective
alignments.</P>
</TD>
</TR></TBODY>
</TABLE>
<BR>
<A NAME=Prediction></A></P>
<H4><FONT COLOR="#007766">The predicted gene structures and
ORFs</FONT></H4>
<P>The culmination of the GeneSeqer@PlantGDB analysis is the
prediction of an accurate gene structure. The quality of this
prediction can be assessed by prediction of a probable open
reading frame (ORF) and comparison to know proteins. Predicted
ORFs are shown as red arrows in the summary graphic. Additionaly,
the longest ORF as well as its translation frame is displayed in
the alignment file. The NCBI blastp link following the translated
ORF sequence in the alignment file will allow you to more easily
find putative homologs for this putative gene.<BR>
<BR>
<BR>
<BR>
<A NAME=Refinement></A></P>
<H3><FONT COLOR="#660033">Further investigation & refined
analysis</FONT></H3>
<P><A NAME=Detailed></A></P>
<H4><FONT COLOR="#007766">Detailed (Refined) annotation using
GeneSeqer@PlantGDB</FONT></H4>
<P>Interesting gene regions found through the process described
above can be further refined through various methods. One such
method, demonstrated in this paragraph, involves a detailed look
at the evidence (ESTs and cDNAs) supporting a given gene
structure. Through the spliced alignment of "All Plants" ESTs and
cDNAs to the restricted region, insight into possible alternative
gene structures, polymorphisms, and differential transcription is
made possible. To demonstrate this concept, we have choosen the
15kb region extending from base 7500 to base 22500 of the
<I>Sorghum bicolor</I> BAC analyzed above. The results are
available <A HREF="http://www.plantgdb.org/tutorial/SB_GSQexample/SBdetailedGSQ.html" TARGET=GSQxSBdetail>here</A>.
This analysis was done in the same manor as above with the
exceptions that the 7500 to 22500 range was input in step 2 and
the "All Plants" EST and cDNA options were choosen in step 3.</P>
<P><BR>
<TABLE CELLSPACING=5><TBODY>
<TR>
<TD VALIGN=top>
<P><IMG SRC="GeneSeqerTutorial_files/SBdetailedGSQ.png" X-CLARIS-USEIMAGEWIDTH X-CLARIS-USEIMAGEHEIGHT ALIGN=bottom hspace=10></P>
</TD>
<TD VALIGN=top>
<P>As shown by the summary graphic, three distinct gene
regions have been characterized. These three gene regions
putatively represent a mitochondrial carrier protein,
subunit 1 of a cleavage stimulation factor, and a serine
threonine kinase based on BlastP queries with the NCBI
non-redundant database as described in the next section.
Interestingly, spliced alignment of non-native (non
<I>Sorghum</I>) transcripts alone are responsible for the
characterization of the mitochondiral carrier protein in
the 7800 to 11800 region shown to the left. Also
noteworthy is the apparent alternative gene structure
represented by an exon in the 9438 to 9477 region of this
gene. The native transcript presumably encoded by this
gene region is assumed to lack this exon or to express it
as an alternatively spliced product due to the low local
alignment similarity of the homologous sequence
alignments. Investigation as to the origin of the
transcripts corresponding to each gene structure reveal
two (2) transcripts arising from monocotyledons
(<I>Secale cereale</I> (rye) gi:10093099; <I>Oryza
sativa</I> (rice) gi:27547342) and two (2) transcripts
arising from dicotyledons (<I>Solanum tuberosum</I>
(potato) gi:17074557l <I>Lycopersicon esculentum</I>
(tomato) gi:18260535). In this example, the gene
structure lacking the exon in question is supported by
spliced alignment of the monocot homologs and thus as
assumed before most likely represents the native
<I>Sorghum</I> gene transcript.</P>
</TD>
</TR></TBODY>
</TABLE>
<BR>
<A NAME=HomologPa></A></P>
<H4><FONT COLOR="#007766">Homologous protein alignment using
GeneSeqer@PlantGDB</FONT></H4>
<P>Determining the complete gene structure, representing the
entire coding region, of a gene is in some cases not possible
using the alignment of transcribed sequences alone. As mentioned
above, inclusion of homologous transcripts can increase the
coverage of these alignments but is not always sufficient to
produce a complete gene structure. For this reason,
GeneSeqer@PlantGDB includes an interface allowing the alignment of
homologous proteins. These homologs may be determined through the
use of the NCBI blastp link provided in the ORF section of the web
service results. The results shown <A HREF="http://www.plantgdb.org/tutorial/SB_GSQexample/SBprotGSQ.html" TARGET=GSQxSBprot>here</A>
represent such alignments in the 7800 to 11800 region of the
<I>Sorghum</I> BAC used throughout this demonstration.</P>
<P><BR>
<TABLE CELLSPACING=5><TBODY>
<TR>
<TD VALIGN=top>
<P>The 10 putatively homologous proteins aligned in this
example were obtained using the external BlastP link
provided in the GeneSeqer@PlantGDB text results. Each ORF
prediction found in the text results is followed by this
link to facilitate searches against the NCBI
non-redundant database. In this example, all 10 proteins
demonstrated e-values of at most 8e-66. As was shown in
the preceding section, homologous alignments of two (2)
putative <I>Arabidopsis thaliana</I> proteins suggest an
alternative gene structure while alignment of the other
eight (8) protein sequences confers the predicted native
gene structure.</P>
</TD>
<TD VALIGN=top>
<P><IMG SRC="GeneSeqerTutorial_files/SBprotGSQ.png" X-CLARIS-USEIMAGEWIDTH X-CLARIS-USEIMAGEHEIGHT ALIGN=bottom hspace=10></P>
</TD>
</TR></TBODY>
</TABLE>
<BR>
<BR>
<BR>
<HR SIZE=1 WIDTH="90%">
<FONT FACE="times new roman, garamond, arial, tahoma" COLOR="#FFFFFF">
<TABLE BORDER=0 CELLSPACING=30 CELLPADDING=2 align=center><TBODY>
<TR>
<TD VALIGN=bottom>
<P><A HREF="http://www.plantgdb.org/AtGDB/index.html"><IMG SRC="GeneSeqerTutorial_files/AtGDB_logo.png" WIDTH=75 HEIGHT=50 BORDER=0 ALIGN=bottom><BR>
AtGDB</A></P>
</TD>
<TD VALIGN=bottom>
<P><A HREF="http://www.zmdb.iastate.edu/"><IMG SRC="GeneSeqerTutorial_files/image001.gif" WIDTH=75 HEIGHT=50 BORDER=0 ALIGN=bottom><BR>
ZmDB</A></P>
</TD>
<TD VALIGN=bottom>
<P><A HREF="http://www.plantgdb.org/"><IMG SRC="GeneSeqerTutorial_files/PlantGDB.gif" WIDTH=75 HEIGHT=50 BORDER=0 ALIGN=bottom><BR>
PlantGDB</A></P>
</TD>
<TD VALIGN=bottom>
<P><A HREF="http://gremlin1.zool.iastate.edu/~volker/"><IMG SRC="GeneSeqerTutorial_files/BGlogo.jpg" WIDTH=42 HEIGHT=50 BORDER=0 ALIGN=bottom><BR>
Brendel Group</A></P>
</TD>
<TD VALIGN=bottom>
<P><A HREF="http://www.plantsciences.iastate.edu/"><IMG SRC="GeneSeqerTutorial_files/PSI.gif" WIDTH=52 HEIGHT=50 BORDER=0 ALIGN=bottom><BR>
Plant Sciences Institute</A></P>
</TD>
<TD VALIGN=bottom>
<P><A HREF="http://www.iastate.edu/"><IMG SRC="GeneSeqerTutorial_files/isu.gif" WIDTH=75 HEIGHT=50 BORDER=0 ALIGN=bottom><BR>
Iowa State University</A></P>
</TD>
<TD VALIGN=bottom>
<P><A HREF="http://www.nsf.gov/"><IMG SRC="GeneSeqerTutorial_files/nsflogo.gif" WIDTH=50 HEIGHT=50 BORDER=0 ALIGN=bottom><BR>
NSF Plant Genome Research</A></P>
</TD>
</TR></TBODY>
</TABLE>
</TD></TR></TABLE></FONT></P>
<P>
</FORM></P>
</BODY>
</HTML>
See more files for this project here