GIL .. BIOSCI/Bionet News .. Biosequences .. Software .. FTP

<!doctype html public "-//W3C//DTD W3 HTML//EN"> <html><head><style type="text/css"></style><title>RE: [Zbrafish] Mapcrosses and Positional Cloning</title></head><body> <div>Hi Tom,</div> <div><br></div> <div>I'd only add a couple of things to Becky's response:</div> <div><br></div> <div>-- It is worth contacting people at Sanger/Ensembl (there should be a feedback link on the webpage) to let them know the problems you see. Hopefully it will lead to a resolution in Zv8.</div> <div><br></div> <div>-- For propagating the next generation of mapcross in cases where you have already rough-mapped, I suggest raising an incross from an existing map pair. This ensures that your allele system does not become any more complex, and with a high likelihood your current informative markers will also be informative in the next generation. (Since WIKs are not isogenic, outcrossing to WIK could introduce new marker alleles.)</div> <div><br></div> <div>Chi-Bin Chien</div> <div><br></div> <div>At 10:33 AM -0500 11/26/07, Burdine, Rebecca D wrote:</div> <blockquote type="cite" cite>Content-class: urn:content-classes:message<br> Content-Type: multipart/alternative;<br> <x-tab>  </x-tab>boundary="----_=_NextPart_001_01C83041.B5CF2406"<br> </blockquote> <blockquote type="cite" cite><font face="Arial" size="-1" color="#0000FF">Hi Tom,</font></blockquote> <blockquote type="cite" cite> </blockquote> <blockquote type="cite" cite><font face="Arial" size="-1" color="#0000FF">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></blockquote> <blockquote type="cite" cite> </blockquote> <blockquote type="cite" cite><font face="Arial" size="-1" color="#0000FF">We also check synteny with other organisms which gives us more confidence in the Ensembl assembly or in our own.</font></blockquote> <blockquote type="cite" cite> </blockquote> <blockquote type="cite" cite><font face="Arial" size="-1" color="#0000FF">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></blockquote> <blockquote type="cite" cite> </blockquote> <blockquote type="cite" cite><font face="Arial" size="-1" color="#0000FF">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></blockquote> <blockquote type="cite" cite> </blockquote> <blockquote type="cite" cite><font face="Arial" size="-1" color="#0000FF">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></blockquote> <blockquote type="cite" cite> </blockquote> <blockquote type="cite" cite><font face="Arial" size="-1" color="#0000FF">Good luck,</font></blockquote> <blockquote type="cite" cite><font face="Arial" size="-1" color="#0000FF">Becky</font></blockquote> <blockquote type="cite" cite> </blockquote> <blockquote type="cite" cite><font face="Arial" size="-1">---------------------------------------------------</font ></blockquote> <blockquote type="cite" cite><font face="Arial" size="-1">Rebecca D. Burdine, Ph.D.</font></blockquote> <blockquote type="cite" cite><font face="Arial" size="-1">Assistant Professor</font></blockquote> <blockquote type="cite" cite><font face="Arial" size="-1">Dept. of Molecular Biology</font></blockquote> <blockquote type="cite" cite><font face="Arial" size="-1">Princeton University</font></blockquote> <blockquote type="cite" cite><font face="Arial" size="-1">Washington Road Mof 433</font></blockquote> <blockquote type="cite" cite><font face="Arial" size="-1">Princeton, NJ 08544 </font></blockquote> <blockquote type="cite" cite> </blockquote> <blockquote type="cite" cite><font face="Arial" size="-1">Phone: (609) 258-7515</font></blockquote> <blockquote type="cite" cite><font face="Arial" size="-1">Fax: (609) 258-1343</font></blockquote> <blockquote type="cite" cite><font face="Arial" size="-1">Email:</font> <a href="mailto:rburdine@princeton.edu"><font face="Arial" size="-1">rburdine@princeton.edu</font></a></blockquote> <blockquote type="cite" cite><font face="Arial" size="-1">Admin Assistant: Cathy Falk (609) 258-1604</font></blockquote> <blockquote type="cite" cite> </blockquote> <blockquote type="cite" cite><br> <blockquote> <hr></blockquote> <blockquote><font face="Tahoma" size="-1"><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</font><br> <font face="Tahoma" size="-1"></font></blockquote> <blockquote>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,</blockquote> <blockquote><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> </blockquote> <blockquote>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<br> </blockquote> </blockquote> <blockquote type="cite" cite><br> _______________________________________________<br> Zbrafish mailing list<br> Zbrafish@net.bio.net<br> http://www.bio.net/biomail/listinfo/zbrafish</blockquote> <div><br></div> </body> </html>

Send comments to us at archive@iubio.bio.indiana.edu