QuestionsCategory: ChIP-Seq Data AnalysisControl Types: Under what conditions is it better to use the Input versus IgG control for ChIP-Seq?
stonelakeak@nih.gov Staff asked 5 months ago

Under what conditions is it better to use the Input versus IgG control for ChIP-Seq?

1 Answers
stonelakeak@nih.gov Staff answered 5 months ago

Input and IgG account for different types of biases inherent in the data. IgG is important for identifying non-specific binding of the beads used for pull-down. IgG can also help identify non-specific binding of an IgG antibody although each IgG antibody will have different non-specific binding patterns. Input samples tell more about the three-dimensional structure of the chromosomes. They are especially important for dealing with variations in the fragmentation step of the ChIP protocol as certain regions of the genome are more likely to shear than others based upon their structure and GC content. As a result, there will be certain regions of the genome that will often only have long fragments that will never end up in the sequencing reaction, and others that will tend to be too short for sequencing. During the pull-down step, fragments bound by the protein of interest will be enriched for, but the underlying biases of the fragmentation step will remain. Also, the enrichment step isn’t perfect and there is often a large number of reads sequenced that may not actually have bene bound by the protein of interest. Therefore, in my experience, the input sample tends to be the better control in most cases, even though it has its own limitations. – answered by Tovah Markowitz, Paul Schaughency, Vishal Koparde.