[Zbrafish] Mapcrosses and Positional Cloning

[Chi-Bin Chien]

Hi Tom,

I'd only add a couple of things to Becky's response:

-- 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.

-- 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.)

Chi-Bin Chien

At 10:33 AM -0500 11/26/07, Burdine, Rebecca D wrote:
>Content-class: urn:content-classes:message
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>Hi Tom,
>
>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.
>
>We also check synteny with other organisms which gives us more 
>confidence in the Ensembl assembly or in our own.
>
>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.
>
>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.
>
>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.
>
>Good luck,
>Becky
>
>---------------------------------------------------
>Rebecca D. Burdine, Ph.D.
>Assistant Professor
>Dept. of Molecular Biology
>Princeton University
>Washington Road Mof 433
>Princeton, NJ 08544 
>
>Phone: (609) 258-7515
>Fax: (609) 258-1343
>Email: <mailto:rburdine from princeton.edu>rburdine from princeton.edu
>Admin Assistant: Cathy Falk (609) 258-1604
>
>
>
>From: zbrafish-bounces from oat.bio.indiana.edu 
>[mailto:zbrafish-bounces from oat.bio.indiana.edu] On Behalf Of Thomas 
>Bartman
>Sent: Tuesday, November 20, 2007 9:40 AM
>To: Zbrafish from magpie.bio.indiana.edu
>Subject: [Zbrafish] Mapcrosses and Positional Cloning
>
>Hi all.
>
>We're having trouble with our positional cloning project, and we 
>hoping for some input on the following two questions:
>
>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. 
>
>(as an aside, we are looking at another gene, and have found it (all 
>800 bp) with the identical 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?)
>
>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.?
>
>	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,
>
>	b) A male from the above mapcross was backcrossed to a female 
>WIK.  We continue to collect embryos from this mapcross backcross.
>
>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.
>
>Thanks for any advice, comments.
>
>Tom
>
>Thomas Bartman, M.D., Ph.D.
>Divisions of Neonatology, Pulmonary Biology, and Developmental Biology
>Cincinnati Children's Hospital Medical Center
>3333 Burnet Ave, MLC 7009
>Cincinnati, OH  45229-3039
>
>Office: 513-636-9902
>
>
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