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=August= ==August 4th - August 10th== * Received CpG island probe code from Dr. Zhang. * Need to make modifications discussed in meeting: * Necessary modifications: ** '''Remove bisulfite conversion function - DONE''' *** Create dummy function that simply does not do a replacement, but creates fwd and rev strands *** Commented code to find/replace *** Question: parts we are looking at; do they have N's in them? Probably shouldn't. ** '''Remove CG score penalty - DONE''' *** in function getProbeList *** remove section starting with "my $CG_penalty" *** set CG_penalty as always zero for quick fix, remove CG finding code later ** '''Add function to check sequence uniqueness in human genome (build hash table ahead of time, check Dr. Zhang's functions)''' *** Looks like function oligoFreq does this; currently just uses log of each side *** Modify probe score calculation to favor segment B being more unique *** See function getProbeList ** '''Modify score calculation so that Tm of segment A should be high and Tm of segment B should be low''' *** At the moment, Tm score is used in p_score calculation *** Simply modify to adjust so that A has high Tm, B has low Tm *** See function getProbeList *** Specific thresholds? ** '''Exon database''' *** Ask about our current UCSC data; it comes in format on PGP server, or is this modified? *** Found Exon-Intron Database; uses GenBank *** Show Dr. Zhang the readme ==August 11th - August 17th== * Will copy from notebook ==August 18th - August 24th== * Will copy from Notebook ==August 27th== *Have code to read and analyze Cosmic set data; file is [[Media:ReadCosmic_20080827.txtβ|"readCosmic.pl"]] *'''Comparison of Cosmic set data to Dr. Li's data (Harvard Collaborator)''' **Dr. Li's data showed that the last base pair of the ligation arm was important. ***Dr. Li checked average expression for each base. ***G was preferred for ligation, followed by A, C, and T. ***[[Image:08_22_2008_JinLi_Ligation_Arm_Base_Dist.png]] **Average expression was calculated using the [[Media:MakeHist_20080827.txt|"makeHist.pl"]] file. ***This file averages the abundance of the probes that used each base. **The results were slightly different. ***For the COSMIC set, it was found that for the proximal ligation arm, G was preferred, followed by C, A, and T. ***This makes intuitive sense, as the bond between G and C is stronger than the bond between A and T. ***The results were also much less pronounced than Dr. Li's results; the difference in base performance is much smaller. {|border="1" cellpadding="5" cellspacing="0" | align="center" colspan="21" | '''Cosmic Set Results''' |- |'''Base:'''||A||T||G||C |- |'''Proximal Extension Arm:'''||98.2276826484018||105.4814504701790||97.8751164168995||105.1120477176380 |- |'''Distal Extension Arm:'''||104.9745183977170||107.4126226040130||106.5526921141540||88.8525735059634 |- |'''Proximal Ligation Arm:'''||95.1374247734938||83.7531848897751||135.360909000299||107.876484056988 |- |'''Distal Ligation Arm:'''||103.1547820103830||113.0664396095970||96.5690024678530||96.7110820814283 |} *[[Image:ProxDist_Base_Cosmic.png]] **Analyze these results later; perhaps add in a preference on the Proximal Ligation Arm to favor G or C over A or T in the score. *'''Implementation of new probe set algorithm.''' **Algorithm is summarized above. **Implement in Perl using hash table in getCpGIslands.pl file. *'''Use R to analyze Cosmic Data Set''' ** Downloaded and installed GNU R and R-commander (GUI) ***Can load CSV and tab-separated files. **''lm'' command can do a linear model fit to the data. ***May need to take the log of several pieces of data however in order to improve fit (if data is exponentially correlated). ***Initial ''lm'' results for Cosmic set show a very poor correlation...probably need to take log of several pieces of data set. **Earlier, Dr. Zhang's analysis on CES22K set used the following factors: ***ProbeDist: distribution of oligo abundance determined by Solexa sequencing. - '''Don't have this for the Cosmic set''' ***H1_Tm, H2_Tm: H1 (ligation arm), H2 (extension arm), 12bp sub-sequences of H1 and H2. - '''Normalized for the Cosmic set; very close to 62 C in all cases''' ***H1R_dist, H2L_dist: K-mer distribution of H1&H2 12bp sub-sequences in the human genome. - '''Have this''' ***Core_25bp_genomic_homology: weighted score of the 25bp homologous sequences in the human genome, mapped by SOAP with <=3bp mismatches. - '''Not sure what this is...ask Dr. Zhang''' ==August 28th== *TO DO TODAY: **Take log of various factors in R, see if this affects ''lm'' results. **See if any other combinations of factors produce better fits in R. **Implement algorithm to select probes in Perl. *'''R Analysis of Cosmic Data Set''' **Tried taking the log of all factors aside from Tm, checked fit. **Fit is still not very good ***Very low R^2 value ***Checked by plotting in Octave; fit does not match up to data well at all. **Perhaps a different, non-linear, non-logarithmic model is needed? **One issue with the Cosmic Data Set is that there is no data for oligonucleotide bias on the chip. ***Agilent's chips have some inherent bias in what molecules are present and not present. ***This bias was measured for the CES22k set that Dr. Zhang worked on but ''not'' for the Cosmic set. ***This may be causing issues with fitting a curve to the Cosmic set, as this bias cannot be used in the fit. **Came up with some ideas while working from home. ***Perhaps instead of log or linear fit, use a normal distribution type fit? ***Could fit a normal distribution to each factor and try and standardize it; this might result in a better overall fit. ***I am currently looking into how to easily do this in R or Octave; shouldn't be too hard to do a fit this way. **Received data for CES22K set ***A preliminary look at the data in Octave makes it seem that R^2 values will remain very low. ***Confirmed that fit is not that great in Octave. ***However, data for bias is present; this means that this can be factored into the analysis. ***Work on this tomorrow. ==August 29th== *TO DO TODAY: **Make some slides for Cosmic and CES22k sets to illustrate findings for linear and log fits **Hopefully find a good way to fit normal distributions to the data; create some plots for this **Test CES22k set for proximal and distal bases as well (just as with Dr. Li's data and Cosmic set above) *'''Analysis of Cosmic and CES22k Data Set''' **R^2 value of fit is very low due to large number of data points. ***Idea from Dr. Zhang; check high and low end points for input factors (maybe outside 1 std. deviation?) ***This might increase Signal-to-Noise ratio for the data. **Was doing some reading on R linear model modules; stumbled across ''effects'' module. ***''effects'' uses results from an ''lm'' command and generates "effect plots" ***It generates the effects of a set of variables on an output variable in individual graph form ***Red lines in graphs represent a 95% confidence interval ***A paper on the ''effects'' module can be found here: http://www.jstatsoft.org/v08/i15/paper ***'''Need to investigate this module and make sure that any assumptions it makes are valid for the data we are using.''' ****It seemed that it was being used in a variety of applications online that used linear models. **Used the ''effects'' module on Dr. Zhang's earlier CES22k fit to determine effects of various factors on expression output. ***The results for this are very interesting, and provide a much nicer picture than just the fit data. ---- *'''CES22K SET:'''[[Image:CES22K Effect Plots.png]] ---- *'''COSMIC SET:'''[[Image:Cosmic Effect Plots.png]] ---- *'''Proximal and Distal Arm Base Comparison''' **The above analysis (using [[Media:MakeHist_CES.txt|makeHist_CES.pl]]) was repeated for the CES22k set. **The results are below. {|border="1" cellpadding="5" cellspacing="0" | align="center" colspan="21" | '''CES22k Set Results''' |- |'''Base:'''||A||T||G||C |- |'''Proximal Extension Arm:'''||305.94||326.29||236.49||247.1 |- |'''Distal Extension Arm:'''||283.08||290.35||263.52||257.27 |- |'''Proximal Ligation Arm:'''||244.76||163.81||402.33||253.13 |- |'''Distal Ligation Arm:'''||280.15||303.46||245.07||263.64 |} *[[Image:ProxDist_Base_CES22k.png]] **Similar to Cosmic set results; G and C are preferred at the Proximal Ligation site over A and T. **Differences are more pronounced, like Dr. Li's data. **Some differences are present in the other bases as well...possibly analyze for significance (in Dr. Li's data, Cosmic data, and this data)? *'''Slides to illustrate findings so far''' **Will make tonight (8-29) and email out.
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