[Zbrafish] Mapcrosses and Positional Cloning

[Burdine, Rebecca D]

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: 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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