ChIP-Seq

Table Of Contents

1. Measuring Enrichment Prior to Library Prep
2. Control
3. Read Redundancy

1.  Measuring Enrichment Prior to Library Prep

Test your antibody and sample prep before making a library. We recommend that the enrichment ratio \( {{C^+/I^+}\over{C^-/I^-}} \ge 10 \). Where \( C^+ \) is ChIP at positive control, \( I^+ \) is input at positive control, \( C^- \) is ChIP at negative control, and \( I^- \) is input at negative control.

To do this, you need at least two primer pairs, a positive control (+) and a negative control (-) which you measure on the ChIP and input samples using Q RT-PCR. See the figure to the right.

2.  Control

We strongly recommend that you sequence an \textcolor{red}{input} library for each condition. Note that in the figure, the input track has a strong peak at the same place as the ChIP peak. The strength of inputs peaks or bias is dependent on the state of the cells as well as chromatin preparation conditions.

3.  Read Redundancy

Like most Illumina library preps, ChIP-Seq libraries usually require a number of rounds of PCR. PCR creates copies of DNA fragments which are statistically dependant on each other. During data analysis it is important to reduce dependant fragments to a single fragment when possible. This can reduce the read count dramatically for some libraries. However duplicate fragments can also be produced in ways that are statistically independant and so can be left in during the analysis, if the analysis is able to identify which is which.

Usually DNA is fragmented by sonication for ChIP-Seq libraries which produces largely random breakage. So in regions with low coverage, duplicated reads are more likely to come from PCR copies. As the coverage gets deeper, the chance that a duplicated read is from an independent fragment increases, though only if the library has sufficient complexity. In enriched areas, i.e., peaks, it is quite possible to see duplicate reads that are independant.

Since removing duplicated reads can dramatically reduce the effective depth of sequencing it is important to assess the level of duplication and take steps to reduce it if necessary and if possible. However, the amount of material available, the size of the target's footprint in the genome, and the effectiveness of the anitbody may limit what is possible. The table below covers most of the common scenarios.