Recommended Standards for ChIP-Seq Experiments
Version 1.0
November 5, 2008
1. Matched sequenced reference sample
Deeply sequenced reference samples such as Input DNA exhibit “peaks” in thepromoter regions of many known genes. In order to identify regions of transcription
factor binding in a ChIP-Seq experiment it is necessary to have sequenced a relevant
reference sample in the same cell-line (and same cellular condition) in order to showthat the region is enriched in the ChIP sample compared to the control. For the case ofChIP-Seq experiments for different transcription factors, only one control sample needbe produced for each cell-line. The control (i.e. Input DNA) should be sequenced to atleast the same depth as the greatest depth of any matching individual transcription
factor ChIP-Seq experiment.
2. Depth of Sequencing
The required depth of sequencing needed will vary depending on the nature of thebinding of the transcription factor (number of binding sites, domain vs. point source
binding, efficiency of the antibody). Saturation for some transcription factors/chromatin
modifications might require exceptionally deep sequencing to achieve probablesaturation of all biologically relevant sites. We recognize that for many factors, we will
not have a large sample of previously known biologically relevant sites at the time of
the ChIP-Seq measurement. One of three criteria should therefore be used for depth ofsequencing:a) When the number of targets begins to saturate (i.e. approach the asymptotic
amount of identifiable targets). The criteria should be ≥ 95% of the extrapolated
total number of targets (for HeLa-S3 Pol II 12 million mapped reads yields greaterthan 95% of the approximately 30,000 extrapolated total targets).
b) If the total number of targets does not approach saturation, one should detect
99% of targets that show at least 2-fold enrichment over control with 90% of thedata.
c) If either a) or b) are not satisfied, then at least 12 million mapped reads should besequenced for human or 3-4 million mapped reads for worm or fly. Since
individual lanes of data for Solexa/Illumina ChIP-Seq samples typically generate
3-4 million mapped reads, human would correspond to 3-4 lanes and worm or fly
would correspond to one lane.
3. Number and Reproducibility of Biological Replicas
At least two biological replicates are necessary in order ensure that the experiment isreproducible. It does not seem that there will be a significant gain in information beyond
two biological replicates, when they are in reasonable agreement. The data fromreplicates can then analyzed and reported as the required independent target site lists.
We propose to require the following agreement between biological replicas (target lists
identified using common number of mapped reads):a) The number of mapped reads from different replicas should be within a factor oftwo of each other.
b) Length of target lists should be within a factor of two of each other.
c) Either of the following options
I. Intersect top fraction (40%) of target list from one replica against the entire
other target list and require a threshold amount of overlap (90%) and repeat
for the reciprocal.

ChIP-­‐seq	  Standards	  v1.0	  	  2008	  November	  II. Target lists scored using all available reads share more than 75% of targets incommon.
These standards have been recommended following an analysis by the Snyder lab of deeply
sequenced ChIP-Seq datasets for HeLa-S3 Pol II. These recommendations may be revisited
as more datasets are available.

ChIP-­‐seq	  Standards	  v1.0	  	  2008	  November