Hi Anna- Before running DESeq, DiffBind sets all counts to a minimum value of 1. Not sure if that is in the documentation anywhere! Cheers- Rory On 11/09/2013 17:47, "Anna Poetsch" <anna.poetsch at="" cancer.org.uk=""> wrote: >Dear Rory, > >I attended the class about ChIP-Seq data analysis at EBI recently, where >you introduced us to the Diffbind package. There is still something that >I do not quite understand. It is about the correction for Input reads: >How is the correction for input done? Are the input counts just >subtracted after normalisation? If so, how does the package deal with >resulting negative counts, which would not be accepted by DESeq? Since >there is a normalisation step in between, the counts would not be >integers anymore, but DESeq needs integers. How is this solved? >I really tried to solve these questions looking at all the documentation >I could find and tried but failed to understand the source code. It would >be really nice, if you could help me with this. > >Kind regards, >Anna Poetsch

Actually DiffBind passes only positive integers to DESeq, and the normalization step is done by DESeq itself. If you also use edgeR, edgeR does a separate normalization, with each package using the values they normalized directly. You can see the (non-integer) normalized values after an analysis us setting bCounts=TRUE in a call to dba.report. -Rory On 11/09/2013 17:52, "Anna Poetsch" <anna.poetsch at="" cancer.org.uk=""> wrote: >Are the count values then also rounded to integers again? >________________________________________ >From: Rory Stark [Rory.Stark at cruk.cam.ac.uk] >Sent: Wednesday, September 11, 2013 5:50 PM >To: Anna Poetsch >Cc: bioconductor at r-project.org >Subject: Re: Question about Diffbind input control > >Hi Anna- > >Before running DESeq, DiffBind sets all counts to a minimum value of 1. >Not sure if that is in the documentation anywhere! > >Cheers- >Rory > >On 11/09/2013 17:47, "Anna Poetsch" <anna.poetsch at="" cancer.org.uk=""> wrote: > >>Dear Rory, >> >>I attended the class about ChIP-Seq data analysis at EBI recently, where >>you introduced us to the Diffbind package. There is still something that >>I do not quite understand. It is about the correction for Input reads: >>How is the correction for input done? Are the input counts just >>subtracted after normalisation? If so, how does the package deal with >>resulting negative counts, which would not be accepted by DESeq? Since >>there is a normalisation step in between, the counts would not be >>integers anymore, but DESeq needs integers. How is this solved? >>I really tried to solve these questions looking at all the documentation >>I could find and tried but failed to understand the source code. It would >>be really nice, if you could help me with this. >> >>Kind regards, >>Anna Poetsch > > >NOTICE AND DISCLAIMER >This e-mail (including any attachments) is intended for the above- named >person(s). If you are not the intended recipient, notify the sender >immediately, delete this email from your system and do not disclose or >use for any purpose. > >We may monitor all incoming and outgoing emails in line with current >legislation. We have taken steps to ensure that this email and >attachments are free from any virus, but it remains your responsibility >to ensure that viruses do not adversely affect you. >Cancer Research UK >Registered charity in England and Wales (1089464), Scotland (SC041666) >and the Isle of Man (1103) >A company limited by guarantee. Registered company in England and Wales >(4325234) and the Isle of Man (5713F). >Registered Office Address: Angel Building, 407 St John Street, London >EC1V 4AD.