2/ .. can be used to highlight the importance of short read alignment parameters and proper documentation of data analysis procedures. ... as well as the importance of analysing replicates if you actually have them.
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3/ Figure 2c shows impressive appearance of acetylation peaks upon ethanol exposure. Similar peaks can be found in the supplement. here a reconstruction based on deposited bigwig filespic.twitter.com/6qfKjm9Y8B
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4/ However, their profiles show the pooled signal from 3 replicates and I was wondering about the robustness of peak appearance, do all replicates have these peaks?
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5/ So I reanalyzed the data based on the M&M in the paper and to my surprise, I did not get these nice peaks. not at all. not in the Fstl1 locus (on top the reanalyzed data, on the bottom the profile deposited at GEO)pic.twitter.com/zTsCH4e27f
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7/ Needless to mention that in the individual samples these peaks were not present as well.
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8/ In the papers' M&M is stated that they used bowtie 1 with paramters -m 1 and --best, which i used as well. But in their GEO description they write that they used STAR and parameters --outFilterMultimapNmax 20 --outFilterMismatchNmax 999
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9/ so ran the pipeline again with STAR and parameters from GEO entry, and got the peaks! Great! Fstl1 locuspic.twitter.com/hVWvdHFz04
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11/ But: this STAR alignment kept multiple mapping reads (up to 20) a feature to avoid in ChIP-Seq analysis. These are most likely artifacts!
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12/ if they would have looked at/analyzed replicates it would have been obvious as e.g. can be seen in the Cep152 locus: Not only the occurence of the peak is inconsistent, also the peak summit location is slightly shifted between replicates.pic.twitter.com/JvkPrGLEme
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13/ Had a quick look and don't think this affects their peak calls in general but point is: these - most likely - artifactual peaks look impressive and therefore i guess they were chosen as 'represenative' examples. they are representative of a problem.
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