Date: Fri, 30 Dec 2005 05:50:32 -0500
Organization: Yale Bioinformatics (http://bioinfo.mbb.yale.edu)
Subject: Summary of pseudogene call on Wed. 21-Dec at 11 AM EDT  [PGENE]

Hi,

I very much enjoyed the call before the holidays.  I hope everyone is
enjoying their holiday time.  Below I have attached a summary of the
last call .

cheers, marK

##
## Summary of 21-Dec-05 Call
##

* Main topics of discussion

1. Understanding Alex's RACE results

2. Deyou's MSA analysis and how it can be integrated into either G&T
or MSA paper.

3. What is the list of pgenes that will be presented to the world as a
freeze on a certain date and a time framework to keep to a scheduled
paper publication date in March 06.

** RACE

* Results

Alex completed RACE experiments of the four pools A,B,C and D. (See
attached email below.)  Pool A covers pseudogenes for which specific
primers could be designed. So analysis of Pool A gives the cleanest
interpretable results with no gene overlap and cross hyb issues.  Pool
C is done with primers that could potentially prime with parental gene
if ENCODE array has the parent gene.

 From the initial analysis, it looks like a very small number of pgenes
are transcribed.  For starters, the analysis of pool A will provide
the minimal set of transcribed pgenes which will then have to be
studied carefully.

So in a nutshell we have to do the following:

a. Generate a list of pgene RACE frags; are there probes in region
that are highly likely to be transcribed?
b. In addition to pool A, pools B and D have to be
analyzed. Processing steps will include looking for extensions say 5kB
away from the primer and see if there are probes in the region unique
to a pseudogene.
c. Clone the RACE products and sequence to unambiguously verify the
transcribed pgene.

* What do we need from Alex/Phil?

1. The exact list of pgenes that were picked for the RACE experiments.

2. File containing the pseudomedians needed from Phil

* ToDos

France will do an analysis of the race frags similar to her analysis
on genes.

Deyou will look in some detail at race frag results after we can
correctly interpret them.

** snoRNAs

Tom introduced the topic of snoRNAs and pgenes. He suggested that we
look to see if there were snoRNA in non-processed pgenes. Potentially
the role of pgenes could be to replicate elements such as snoRNA ( in
introns) rather than protein coding diversity. See
http://lowelab.ucsc.edu/snoRNAdb/ .

** Deyou's MSA analysis

Indicates that most pgenes have been created after mouse/human
split. (See pdf in ppt directory.) Not clear if this would go in G&T
or MSA paper or in a future paper...

** Future Paper

It was agreed that while it may be included in one of the ENCODE
papers, an expanded detailed paper would be written later.

** List of pgenes to be presented to the outside world

It was agreed that we stick with the list of pgenes that have been
looked over by Adam (wherever there was no consensus). The only
remaining bit is for Adam to look at the pgenes that are specific to
Yontao's analysis and include them after manual inspection. This will
be the list that will be put out for the general public.

* So Adam has the following chores:

1. Include Yontao's pgenes in current set after careful inspection.

2.  Associate each pgene with its parent gene and location of parent
gene on the genome.  

3.  Tag each pgene as either processed or non-processed.

** Timeline

1. A final set of pgenes that does not differentiate between GENCODE
and ENCODE pgenes should be ready by Jan 7.
2. France will redo analysis of transfrags and tars shortly thereafter.
3. A rough draft of paper should be in works by Jan.7
4. This gives one month, the month of Feb to polish up the paper.
5. Paper ready to go out by March.


 -------- Original Message --------
Subject: FW: pooling pseudogenes for Affy
Subject: pooling pseudogenes for Affy


Dear Philipp, Tom,

We are at the finishing stage with the 5'pseudoRACE in 12 tissues. I
will bring the samples with me next week and drop them on Wednesday at
your lab before moving to USCS, thus it is time to think about the best
way to pool the pseudoRACE fragments. This is what I propose:

We have tested 49 non-processed pseudogenes, 26 with specific primers (5
to 14 mismatches/25mer, between pseudogene and parental gene) and 23
with "common primers (0 to 3 mismatches/25mer, between pseudogene and
parental gene).

Similarly, we have tested 109 processed pseudogenes (in fact we test 125
but sometimes we are dealing with primers recognizing multiple
pseudogenes), 25 with specific primers (7 to 14 mismatches/25mer,
between pseudogene and parental gene) and 84 with "common primers (0 to
3 mismatches/25mer, between pseudogene and parental gene).  

I suggest:

1. to pool all the specific reactions (26 non-processed and 25
processed) 2. to pool the "common" non-processed (23 reactions; 8 with
no mismatch, 7 with 1 mismatch, 7 with 2 mismatches and 1 with 3
mismatches) 3. to separate in two pools the "common" processed (total of
84 reactions, in the first pool of 48, the reactions done with primers
with no mismatches, in the second pool the 36 reactions with mismatches
(1 to 3 mismatches)

What do you think of this conservative stance? We are looking at a total
of 48 ENCODE affymetrix chips.

Alexandre