User: Rory Stark

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Rory Stark100
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Posts by Rory Stark

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Answer: A: edgeR/DESeq for ChIP-seq analysis
... As Mark said, DiffBind provides a straightforward workflow for this type of ChIP-seq analysis, with all the statistical heavy lifting done by edgeR and/or DESeq. Regarding histone marks, we have had success using DiffBind to analyze wider regions of enrichment. For example, in Chandra et al (Mol C ...
written 6.9 years ago by Rory Stark100
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Answer: A: DiffBind - overlap between different peak callers
... Hello Paolo- There are a couple of ways of getting the consensus peaksets. If you already have added them to your DBA object (e.g. by using dba.peakset), you can retrieve the overlapping set (and/or write it to a file) using dba.pekaset with the bRetrieve and writefile options: peaks = dba.peakset ...
written 7.1 years ago by Rory Stark100
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Comment: C: DiffBind - sample sheet for multiple replicates and peak
... Hello António- Looking closer, I now see the problem with your original sample sheet: you have a column named Peak.caller that should be named PeakCaller. Changing the column name should allow DiffBind to differentiate the peaksets based on the peak caller name. Cheers- Rory From: Rory Stark > ...
written 7.1 years ago by Rory Stark100
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Answer: A: DiffBind - sample sheet for multiple replicates and peak
... Hello Ant?nio- Regarding your sample sheet, you should be able to use the same sample ID and the same read files for each sample, and specify the peak caller as MACS and QuEST as in your initial sample sheet. This should load two sets of peak for each sample, differing only peak caller name (as Dif ...
written 7.1 years ago by Rory Stark100
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Comment: C: edgeR package : question about 'exactTest' results
... Hi helen- Addressing first the use of pre-defined regions instead of peak callers, this is possible with DiffBind. The easiest way to to write out the regions in a single file (chr, start, end, 1) and specify the same file for all the samples. Alternatively, if you are creating the region file dyna ...
written 7.7 years ago by Rory Stark100
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Answer: A: edgeR package : question about 'exactTest' results
... Hi helen- After calling exactTest, you can call topTags with the result. topTags takes a parameter, n, which is the number of results to return -- if you set it to the total number of sites you are comparing, you will get them all back with their associated stats. I end up doing something like: &g ...
written 7.7 years ago by Rory Stark100
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Comment: C: Fwd: differential binding question
... I agree with Mark that fitting a GLM using a design matrix with both factors (the condition and whether an IP was done) is the most straighforward way of analyzing this dataset. Reegarding peak calling, you could use the RNA-Seq data to determine the relevant regions as in a standard RNA-Seq experi ...
written 7.8 years ago by Rory Stark100
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Comment: C: Fwd: differential binding question
... That's the big question Mali! The more I think about it, the less confident I am that it will work. As I understand it you want to control for transcripts whose expression may change but whose affinity (rate at which the protein binds) stays the same. Without the control, higher expression = more t ...
written 7.8 years ago by Rory Stark100
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Answer: A: Fwd: differential binding question
... Hi Mali- You can try this pretty easily using DiffBind. I suggest calling peaks on each IP separately (each IP and its matching RNA-Seq control) and read these four peaksets into DiffBind (you could also use two peak callers and read in all eight peaksets to identify more potential sites). DiffBind ...
written 7.8 years ago by Rory Stark100
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Comment: C: Using DESeq with ChIP-seq data - all or non-redundant reads?
... Ian- Given that you are using DESeq to look at differential binding, the relative magnitudes of the peaks is crucial to the calculations. Removing duplicates will clip the dynamic range such that no peak can have a greater magnitude than the read length. In our case, where we use 36-base single-end ...
written 8.0 years ago by Rory Stark100

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