Hi all, As the subject suggests, I'm getting some weird artifacts in ChIP-chip data I'm looking at (Aiglent 244k human promoter). This is my first foray into ChIP-chip analysis, so I'm sorry if these are naive questions. Instead of painting a picture w/ a 1000 words, I figure it would be best to drop a few pictures online if someone has some time to look at them and can offer some suggestion as to what might be happening. I have 3 replicates experiments for two different Transcription Factors. In both cases, two of the three replicates look real screwy, and 1 of them (for both TFs) looks somehow "ok". The plots shown below are using the r/gProcessedSignals that from Agilent's feature extraction software, so their error-corrected/bg-subtracted is being used. I've also removed the control probes. For TF1: The "faux" microarray image looks fine. Here's pic of the Log2 ratios between the red/green channels: http://cbio.mskcc.org/~lianos/files/bioconductor/bad1.Log2Ratio.png Here's the funky MA plot for the same replicate expt: http://cbio.mskcc.org/~lianos/files/bioconductor/bad1.png Faux microarray image for "better" replicate of same TF: http://cbio.mskcc.org/~lianos/files/bioconductor/better1.Log2Ratio.png MA plot for the better replicate of TF1: http://cbio.mskcc.org/~lianos/files/bioconductor/better1.png You can see that at low the low-A values of the plot, there is a pretty significant density of probes that skew the M values quite high. They are also there in a smaller amount on the rep2 picture, (but below the M=0 line). Does anyone have an intuition as to what might be causing this? I don't have die-swap experiments, so I'm not sure if it's dye bias. Even so, is there such thing as intensity-specific dye-bias (to explain it only happening at low values of A (roughly A < 7)? The effect is even more exacerbated in TF2: Here's a "weird" replicate: http://cbio.mskcc.org/~lianos/files/bioconductor/bad2.png Here's a more reasonable replicate for TF2: http://cbio.mskcc.org/~lianos/files/bioconductor/better2.png The faux log2-ratio images for these arrays look much like the other ones, ie. there are no obvious smudges, etc. I'm going to check out what probes correspond to those dark spots in the low-A range, but in the meantime I was wondering if anybody had any suggestions. Thanks for any help, -steve -- Steve Lianoglou Graduate Student: Physiology, Biophysics and Systems Biology Weill Cornell Medical College of Cornell University http://cbio.mskcc.org/~lianos

Hi, should I say welcome to the "microarray" world, or have you this far been so fortunate not seeing these "artifacts" before? [no sarcasm]. The MA artifacts you illustrate are probably one of the most frequently observed systematic effects when comparing two channels/arrays as you do. I'm going to keep my answer short and precise: It *is* because your (non-logged) signals have *offset* that you observe these artifact. You will probably hear heaps of other reasons, but if you stick with the above you are better off. Several people spent a lot of time on this issue and there are many normalization methods addressing the offset problem in different ways (and yet more that try to treat the symptom but not the cause). I will not tell you what normalization method is the best (it probably depend on platform and quality of data etc). I also spent "some" (read tons) time trying to understand the bits and details of this, so although a bit biased, my sincer suggestion is for you to have look at: H. Bengtsson and O. H?ssjer, Methodological study of affine transformations of gene expression data with proposed robust non-parametric multi-dimensional normalization method, BMC Bioinformatics, 2006, 7:100. URL: http://www.biomedcentral.com/1471-2105/7/100/ H. Bengtsson, G. J?nsson and J. Vallon-Christersson, Calibration and assessment of channel-specific biases in microarray data with extended dynamical range, BMC Bioinformatics, 2004, 5:177. URL: http://www.biomedcentral.com/1471-2105/5/177/ It was our hope when we wrote those that if read carefully they would bring better understanding of the problem. In the end of the day (or week) you will see that what happens is very simple and intuitive. Cheers Henrik On Wed, Sep 24, 2008 at 10:43 AM, Steve Lianoglou <mailinglist.honeypot at="" gmail.com=""> wrote: > Hi all, > > As the subject suggests, I'm getting some weird artifacts in ChIP- chip data > I'm looking at (Aiglent 244k human promoter). This is my first foray into > ChIP-chip analysis, so I'm sorry if these are naive questions. > > Instead of painting a picture w/ a 1000 words, I figure it would be best to > drop a few pictures online if someone has some time to look at them and can > offer some suggestion as to what might be happening. > > I have 3 replicates experiments for two different Transcription Factors. In > both cases, two of the three replicates look real screwy, and 1 of them (for > both TFs) looks somehow "ok". The plots shown below are using the > r/gProcessedSignals that from Agilent's feature extraction software, so > their error-corrected/bg-subtracted is being used. I've also removed the > control probes. > > For TF1: > The "faux" microarray image looks fine. Here's pic of the Log2 ratios > between the red/green channels: > http://cbio.mskcc.org/~lianos/files/bioconductor/bad1.Log2Ratio.png > > Here's the funky MA plot for the same replicate expt: > http://cbio.mskcc.org/~lianos/files/bioconductor/bad1.png > > Faux microarray image for "better" replicate of same TF: > http://cbio.mskcc.org/~lianos/files/bioconductor/better1.Log2Ratio.png > > MA plot for the better replicate of TF1: > http://cbio.mskcc.org/~lianos/files/bioconductor/better1.png > > You can see that at low the low-A values of the plot, there is a pretty > significant density of probes that skew the M values quite high. They are > also there in a smaller amount on the rep2 picture, (but below the M=0 > line). > > Does anyone have an intuition as to what might be causing this? I don't have > die-swap experiments, so I'm not sure if it's dye bias. Even so, is there > such thing as intensity-specific dye-bias (to explain it only happening at > low values of A (roughly A < 7)? > > The effect is even more exacerbated in TF2: > Here's a "weird" replicate: > http://cbio.mskcc.org/~lianos/files/bioconductor/bad2.png > > Here's a more reasonable replicate for TF2: > http://cbio.mskcc.org/~lianos/files/bioconductor/better2.png > > The faux log2-ratio images for these arrays look much like the other ones, > ie. there are no obvious smudges, etc. > > I'm going to check out what probes correspond to those dark spots in the > low-A range, but in the meantime I was wondering if anybody had any > suggestions. > > Thanks for any help, > -steve > > -- > Steve Lianoglou > Graduate Student: Physiology, Biophysics and Systems Biology > Weill Cornell Medical College of Cornell University > > http://cbio.mskcc.org/~lianos > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor >