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<DIV dir=ltr align=left><SPAN class=314492415-26112007><FONT face=Arial
color=#0000ff size=2>Hi Tom,</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=314492415-26112007><FONT face=Arial
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=314492415-26112007><FONT face=Arial
color=#0000ff size=2>1) Yes yes and yes. Our regions get flipped over,
flipped around, squeezed together, then spread onto different chromosomes.
What we do is rely on the MGH, HS and GAT maps to set up our region,
then turn to the Radiation hybrid maps to help flesh these out. Finally we
see how well ensemble has assembled this region. If ensembl is off, we
just blast and assemble our own contig that mimics what the genetic maps tell us
is true.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=314492415-26112007><FONT face=Arial
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=314492415-26112007><FONT face=Arial
color=#0000ff size=2>We also check synteny with other organisms which gives us
more confidence in the Ensembl assembly or in our own.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=314492415-26112007><FONT face=Arial
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=314492415-26112007><FONT face=Arial
color=#0000ff size=2>In the end we often get a region that seems reasonable and
then hunt for candidates. At times, our mutant ended up being in the
region assembled on Ensembl, but not where you would predict based on
mapping.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=314492415-26112007><FONT face=Arial
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=314492415-26112007><FONT face=Arial
color=#0000ff size=2>aside - at the last Zfish meeting, the people from the
genome project said that if they get two identical stretches that assemble to
different places, they leave both in until they can determine which is
real.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=314492415-26112007><FONT face=Arial
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=314492415-26112007><FONT face=Arial
color=#0000ff size=2>2) I think your strategy is ok. We map Wik/AB and it
has worked fine for us. The only things to keep an eye out for is that in
some of our WIK mapping we see WIK contributing two different sizes for the Z
markers. So we sometimes have three or four band patterns to score.
This can get really complicated.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=314492415-26112007><FONT face=Arial
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=314492415-26112007><FONT face=Arial
color=#0000ff size=2>Good luck,</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=314492415-26112007><FONT face=Arial
color=#0000ff size=2>Becky</FONT></SPAN></DIV>
<DIV><FONT face=Arial color=#0000ff size=2></FONT> </DIV>
<DIV align=left><FONT face=Arial
size=2>---------------------------------------------------</FONT></DIV>
<DIV align=left><FONT face=Arial size=2>Rebecca D. Burdine, Ph.D.</FONT></DIV>
<DIV align=left><FONT face=Arial size=2>Assistant Professor</FONT></DIV>
<DIV align=left><FONT face=Arial size=2>Dept. of Molecular Biology</FONT></DIV>
<DIV align=left><FONT face=Arial size=2>Princeton University</FONT></DIV>
<DIV align=left><FONT face=Arial size=2>Washington Road Mof 433</FONT></DIV>
<DIV align=left><FONT face=Arial size=2>Princeton, NJ 08544 </FONT></DIV>
<DIV align=left><FONT face=Arial size=2></FONT> </DIV>
<DIV align=left><FONT face=Arial size=2>Phone: (609) 258-7515</FONT></DIV>
<DIV align=left><FONT face=Arial size=2>Fax: (609) 258-1343</FONT></DIV>
<DIV align=left><FONT face=Arial size=2>Email: <A
href="mailto:rburdine@princeton.edu">rburdine@princeton.edu</A></FONT></DIV>
<DIV align=left><FONT face=Arial size=2>Admin Assistant: Cathy Falk (609)
258-1604</FONT></DIV>
<DIV> </DIV><BR>
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style="PADDING-LEFT: 5px; MARGIN-LEFT: 5px; BORDER-LEFT: #0000ff 2px solid; MARGIN-RIGHT: 0px">
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<HR tabIndex=-1>
<FONT face=Tahoma size=2><B>From:</B> zbrafish-bounces@oat.bio.indiana.edu
[mailto:zbrafish-bounces@oat.bio.indiana.edu] <B>On Behalf Of </B>Thomas
Bartman<BR><B>Sent:</B> Tuesday, November 20, 2007 9:40 AM<BR><B>To:</B>
Zbrafish@magpie.bio.indiana.edu<BR><B>Subject:</B> [Zbrafish] Mapcrosses and
Positional Cloning<BR></FONT><BR></DIV>
<DIV></DIV>Hi all.<BR><BR>We're having trouble with our positional cloning
project, and we hoping for some input on the following two
questions:<BR><BR>1) Are others noticing a great deal of misassembly to the
genome still? It seems that every new release of the assembly, our
markers jump all over. For example, on Zv5 we had two markers 500 kb
apart, which went to 12 megabases apart on Zv6. We kept walking, and got
down to 4 MB, but then Zv7 pushed those 7 MB apart. Furthermore, Zv7 has
three of our favorite markers in order A-B-C on the genome, but our data has
them A-C-B. <BR><BR>(as an aside, we are looking at another gene, and
have found it (all 800 bp) with the <U>identical</U> DNA sequence on two
different chromosomes on Zv7. Even if these are redundant copies, how
likely is it that they will match at 800 of 800 bp, or is this a sign of
further misassembly?)<BR><BR>2) This is the crossing strategy we used.
Are there any glaring errors in this that could be causing us problems or are
we
o.k.?<BR><BR><X-TAB> </X-TAB>a)
A female AB carrying the mutation was crossed to a male WIK. Embryos
were collected for positional cloning from this mapcross. However, we
ran out of embryo DNA and the mapcross line got old and stopped laying
well.
So,<BR><BR><X-TAB> </X-TAB>b) A
male from the above mapcross was backcrossed to a female WIK. We
continue to collect embryos from this mapcross backcross.<BR><BR>I wasn't sure
if I read somewhere if it makes a great difference if the crosses to the
mapping line are against females or males.<BR><BR>Thanks for any advice,
comments.<BR><BR>Tom<BR><BR><BR><X-SIGSEP>
<P></X-SIGSEP>Thomas Bartman, M.D., Ph.D.<BR>Divisions of Neonatology,
Pulmonary Biology, and Developmental Biology<BR>Cincinnati Children's Hospital
Medical Center<BR>3333 Burnet Ave, MLC 7009<BR>Cincinnati, OH
45229-3039<BR><BR>Office: 513-636-9902 </P></BLOCKQUOTE></BODY></HTML>
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