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BMC Genomics

, 14:638

Prokaryote microbial genomics

Abstract

BackgroundStudies of protein association with DNA on a genome wide scale are possible through methods like ChIP-Chip or ChIP-Seq. Massive problems with false positive signals in our own experiments motivated us to revise the standard ChIP-Chip protocol. Analysis of chromosome wide binding of the alternative sigma factor σ in Escherichia coli with this new protocol resulted in detection of only a subset of binding sites found in a previous study by Wade and colleagues. We suggested that the remainder of binding sites detected in the previous study are likely to be false positives. In a recent article the Wade group claimed that our conclusion is wrong and that the disputed sites are genuine σ binding sites. They further claimed that the non-detection of these sites in our study was due to low data quality.

Results-discussionWe respond to the criticism of Wade and colleagues and discuss some general questions of ChIP-based studies. We outline why the quality of our data is sufficient to derive meaningful results. Specific points are: i the modifications we introduced into the standard ChIP-Chip protocol do not necessarily result in a low dynamic range, ii correlation between ChIP-Chip replicates should not be calculated based on the whole data set as done in transcript analysis, iii control experiments are essential for identifying false positives. Suggestions are made how ChIP-based methods could be further optimized and which alternative approaches can be used to strengthen conclusions.

ConclusionWe appreciate the ongoing discussion about the ChIP-Chip method and hope that it helps other scientist to analyze and interpret their results. The modifications we introduced into the ChIP-Chip protocol are a first step towards reducing false positive signals but there is certainly potential for further optimization. The discussion about the σ binding sites in question highlights the need for alternative approaches and further investigation of appropriate methods for verification.

KeywordsChIP-Chip ChIP-Seq False-positive σ seqA AbbreviationsDSTDisputed σ targets

ChIPChromatin immunoprecipitation

ChIP-ChipChromatin immunoprecipitation with microarray technology

ChIP-SeqChromatin immunoprecipitation with next generation sequencing technology

RNAPRNA polymerase.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2164-14-638 contains supplementary material, which is available to authorized users.

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Author: Daniel Schindler - Torsten Waldminghaus

Source: https://link.springer.com/







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