Etchevers:Notebook/STRA6 in eye development/2009/07/15: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(Autocreate 2009/07/15 Entry for Etchevers:Notebook/STRA6_in_eye_development)
 
No edit summary
Line 6: Line 6:
| colspan="2"|
| colspan="2"|


==Title==
==Running Findpeaks finally==
This entry will describe today's efforts.
First, we see that all five sequencing results are extremely similar when the .bed files are visualized on the UCSC Genome Browser. So much so we wondered if any of these unintended possibilities happened:


* Chromatin conformation in this particular preparation made some places more likely to non-specifically IP than others. In which case we are REALLY sorry not to have performed an IgG IP and sequenced that as well. Though could do pair-wise comparisons between banks.
* All four transcription factors recognize exactly the same thing. Highly unlikely, esp for non-specific background, or in combination with above.
* Something else was sequenced and the libraries for which we saw and approved the nature of clones were not the same as what was sequenced. Highly unlikely if not impossible.
* A single IP was separately bar-coded five times and sequenced, then separated with bioinformatics. Highly unlikely.
* All five samples were mixed before separate labeling with the bar codes. Also highly unlikely.
* Each sample had all five bar codes. Highly unlikely if not impossible.
Given that Anthony pushes for an IgG control and discourages using the simulated controls, we conclude that the first option is the most likely, but that ChIP-Seq analyses are sufficiently new that this sort of good conceptualization of the experiment from the get-go is not yet a standard.
So, will try to perform the following pair-wise combinations. The first is to compare OTX2-1 and OTX2-2 to see if these two are more similar than to any of the other TF ChIPs. I REALLY hope so.
'''OTX2-1 vs:'''
* OTX2-2
* RAX
* SOX2
* PAX6
'''RAX vs:'''
* OTX2-1
* OTX2-2
* SOX2
* PAX6
'''SOX2 vs:'''
* OTX2-1
* OTX2-2
* RAX
* PAX6
All the combinations won't be performed because we think that most will pair with PAX6 and possibly SOX2. When I say "pair" I mean use the top one [http://vancouvershortr.wiki.sourceforge.net/FP4Control as the "control"] and each subsequent bank as the sample.
Questions to ask Fasteris:
What is their empirical experience with the comparison of multiple IPs from a given chromatin preparation? Same sort of near-identical distributions of reads on the human genome. Is the range of peak sizes comparable? Is there a way to filter certain parts of the genome, not just repeated sequences, but perhaps exons even, to avoid some regions that are particularly "sticky" and precipitable from one experiment to another, across human chromatin generally?
Are all the coverage-gap-filtered peaks in all five banks actually the same or not?
*'''[[User:Etchevers|Heather]] 07:12, 15 July 2009 (EDT)''':


<!-- ## Do not edit below this line unless you know what you are doing. ## -->
<!-- ## Do not edit below this line unless you know what you are doing. ## -->
|}
|}

Revision as of 04:12, 15 July 2009

Genetics of human eye development <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Running Findpeaks finally

First, we see that all five sequencing results are extremely similar when the .bed files are visualized on the UCSC Genome Browser. So much so we wondered if any of these unintended possibilities happened:

  • Chromatin conformation in this particular preparation made some places more likely to non-specifically IP than others. In which case we are REALLY sorry not to have performed an IgG IP and sequenced that as well. Though could do pair-wise comparisons between banks.
  • All four transcription factors recognize exactly the same thing. Highly unlikely, esp for non-specific background, or in combination with above.
  • Something else was sequenced and the libraries for which we saw and approved the nature of clones were not the same as what was sequenced. Highly unlikely if not impossible.
  • A single IP was separately bar-coded five times and sequenced, then separated with bioinformatics. Highly unlikely.
  • All five samples were mixed before separate labeling with the bar codes. Also highly unlikely.
  • Each sample had all five bar codes. Highly unlikely if not impossible.

Given that Anthony pushes for an IgG control and discourages using the simulated controls, we conclude that the first option is the most likely, but that ChIP-Seq analyses are sufficiently new that this sort of good conceptualization of the experiment from the get-go is not yet a standard.

So, will try to perform the following pair-wise combinations. The first is to compare OTX2-1 and OTX2-2 to see if these two are more similar than to any of the other TF ChIPs. I REALLY hope so.

OTX2-1 vs:

  • OTX2-2
  • RAX
  • SOX2
  • PAX6

RAX vs:

  • OTX2-1
  • OTX2-2
  • SOX2
  • PAX6

SOX2 vs:

  • OTX2-1
  • OTX2-2
  • RAX
  • PAX6

All the combinations won't be performed because we think that most will pair with PAX6 and possibly SOX2. When I say "pair" I mean use the top one as the "control" and each subsequent bank as the sample.

Questions to ask Fasteris: What is their empirical experience with the comparison of multiple IPs from a given chromatin preparation? Same sort of near-identical distributions of reads on the human genome. Is the range of peak sizes comparable? Is there a way to filter certain parts of the genome, not just repeated sequences, but perhaps exons even, to avoid some regions that are particularly "sticky" and precipitable from one experiment to another, across human chromatin generally?

Are all the coverage-gap-filtered peaks in all five banks actually the same or not?

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Etchevers:Notebook/STRA6_in_eye_development/2009/07/15&oldid=324388"