ChIP-seq Inputs in Differential Enrichment Christoffer Flensburg and Alicia Oshlack

1. Choosing background fraction α� 3. Use inputs at all? �

ACKNOWLEDGMENTS�

2. Choosing inputs Bi�

Summary��

Aim: Improving input usage in Differential Enrichment Analysis.

1.  Choice of background fraction α.�

2.  Choice of input Bi.�

3.  Should the input be used at all? �

Aim: Improving Differential Enrichment Analysis �

Sir = Tir !!BirS = True ChIP enrichment. T = Total number of measured reads in ChIP. B = Number of reads in background sample. α = Fraction of background reads in ChIP. i = sample r = region of genome

Differential enrichment analysis of histone modifications between groups of ChIP-seq samples is made considerably more involved due to large and varying background components in the ChIP-seq read density. Our aim is to: •  Model the total number of reads over a

region of interest r in sample i (Tir) as a superposition of a background (αBir) and an enrichment component (Sir).

•  Subtract the background using an input Bi scaled by α ≤ 1.

•  Study choices of α and Bi through FDRs and standard deviations from a Limma-Voom pipeline.

Find bestαand Bi�

In this section we examine a differential enrichment analysis on data from the NIH roadmap epigenomics project. Brain is known to have a different epigenetic profile to liver1, and the H3K36me3 mark over the gene body (r) provides large numbers of reads on many genes. Each tissue has 3 replicates Tir, with i going from 1 to 6, and each replicate has a corresponding input sample that we use as background Bir. We run the DE pipeline on Sir, and plot how the number of differentially enriched genes (FDR < 0.05) (fig a) and standard deviations vary with α (fig b).

α0.0 0.2 0.4 0.6 0.8 1.0

500

1000

1500

2000

#(FD

R  <  0.05)  

In this example, the largest statistical power is reached at a background fraction α well below 1, and a full input subtraction (α = 1) adds so much noise that only about 20% of the DE genes at α = 0.28 remain significant. This loss of power at full subtraction is a recurring behaviour, and can also be seen in section 2 and 3.

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●●std  de

v  

log(cpm)  

Best results atα<1�Don’t have to use matching inputs�

It is not always the matching input from the same sample that is the most similar to the background in the ChIP. Below are correlations of binned read counts between epigenetic marks in different tissues and their matching inputs, and two single inputs of high and low quality correlated against the same ChIP data.

Most DE genes at α = 0.28.

Few DE genes at α ~ 1.

•  α = 1 Very noisy.

•  α = 0.28 Less noise at high counts.

•  α = 0 No input

subtraction.

Inpu

t  vs  IP  

correla>

on  

0.0

0.2

0.4

0.6

0.8

●

●

●

●

●

●

● ●

●

●

●

H3K27ac H3K27me3 H3K36me3 H3K4me1 H3K4me3 H3K9ac H3K9me3

The high quality input correlates more strongly with the ChIPs than

the matching input. Even from different tissues!

§  Matching inputs §  High quality input §  Low quality input

As a high quality input correlates more strongly with the ChIP data than the matching input, we performed a differential analysis like the one in section 1, but with Bi as a single input for all i. We analysed muscle against liver, and compared matching inputs to two high quality inputs.

#(FD

R  <  0.05)  

0.0 0.2 0.4 0.6 0.8 1.0

0200

400

600

800

1000

α

– Matching input – Liver input – Brain input

Single liver input doing better than matching inputs!

Single brain input doing as well as matching inputs!

The brain input provides as much power as matching inputs, despite of coming from a very different tissue, and the liver input provides even more power than the matching inputs.

REFERENCES��1. Jiang Zhu, Mazhar Adli, et al. Genome-wide Chromatin State Transitions Associated with Developmental and Environmental Cues. Cell 152, 642–654, January 31, 2013�2. Rory Stark, Gordon Brown. DiffBind : differential binding analysis of ChIP-Seq peak data. Bioconductor http://bioconductor.org/packages/release/bioc/vignettes/DiffBind/inst/doc/DiffBind.pdf�3. Xin Zeng, et al. jMOSAiCS: joint analysis of multiple ChIP-seq datasets. Genome Biology 2013, 14:R38.�4. Zhen Shao, et al. MAnorm: a robust model for quantitave comparison of ChIP-Seq data sets. Genome Biology 2012, 13:R16.�5. Yanchun Bao, et al. Accounting for immunoprecipitation efficiencies in the statistical analysis of ChIP-seq data. BMC Bioinformatics 2013, 14:169��

FOR MORE INFORMATION ON THESE AND OTHER STUDIES PLEASE VISIT WWW.MCRI.EDU.AU�

The inputs compensate for the background, but they also introduce more noise, and often the trade-off is not beneficial. An example of this can be seen below, for a differential analysis between brain and skeletal muscle, again for the H3K36me3 mark, and with the same two high quality inputs used in section 2.

α

#(FD

R  <  0.05)  

0.0 0.2 0.4 0.6 0.8 1.0

0500

1000

1500

– Matching input – Liver input – Brain input

α = 0 gives most power.

Sometimes not worth it �

We’ve looked at many datasets from the roadmap epigenetics project and have found that often the inputs do not provide any advantage. The situation may change with a more detailed approach, such as in [2-5], but it is always important to ensure that the input is used in the best way, and that it is actually improving the analysis.

A study like this is not possible without large public datasets. On this poster, the NIH roadmap epigenomics project was used: http://www.roadmapepigenomics.org/

a)  H3K36me3  Brain  vs  Liver  

H3K36me3  Muscle  vs  Liver  

H3K36me3  Muscle  vs  Brain  

b)  H3K36me3  Brain  vs  Liver