>Peter Park, Harvard Medical School – “Statistical Issues in ChIP-Seq and its Application to Dosage Compensation in Drosophila”

>(brief overview of ChIP-Seq, epigenomics again)

ChIP-Seq not always cost-competitive yet. (can’t do it at the same cost as chip-chip)

Issues in analysis:Generate tags, align, remove anomalus, assemble, subtract background, determine binding position, check sequencing depth.

Map tags in strand specific manner: (Like directional flag in Findpeaks). Scoring tags accounting for that profile. Can be incorporated into peak caller.

Do something called Cross-correlation analysis. (look at peaks in both directions.) use this to rescue more tags. Peaks get better if you add good data, and worse if you add bad data. Use it to learn something about histone modification marks. (Tolstorukov et al, Genome Research).

How deep to sequence? 10-12M reads is current. That’s one lane on illumina, but is it enough? What quality metric is important? Clearly this depends on the marks you’re seeing (narrow vs broad, noise, etc). Brings you to saturation analysis? Show no saturation for STAT1, CTCF, NRSF. [not a surprise, we knew that a year ago… We’re already using this analysis method, however, as you add new reads, you add new sites, so you have to threshold to make sure you don’t keep adding new peaks that are insignificant. Oh, he just said that. Ok, then.]

Talking about using “fold enrichment” to show saturation. This allows you to estimate how many tags you need to get a certain tag enrichment ratio.

See paper they published last year.

Next topic: Dosage compensation.

(Background on what dosage compensation is.)

In drosophila, the X chromosome is up-regulated in XY, unlike in humans, where the 2nd copy of the X is quashed in the XX genotype. Several models available. Some evidence that there’s something specific and sequence related. Can’t find anything easily in ChIP based methods – just too much information. Comparing ChIP-seq, you get sharp enrichment, whereas on ChIP-chip, you don’t see it. Seems to be saturation issue (dynamic range) on ChIP-chip, and the sharp enrichments are important.
You get specific motifs.

Deletion and mutation analysis. The motif is necessary and sufficient.

Some issues: Motif on X is enriched, but only by 2-fold. Why is X so much upregulated, then? Seems Histone H3 signals depleted over the entry sites on X chr. May also be other things going on, which aren’t known.

Refs: Alekseyenko et al., Cell, 2008 and Sural et al., Nat Str Mol Bio, 2008

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