[Embunnik]

I had this question too and I just checked their adapters and primers to figure out the answer. My conclusion is that their design is correct:

For ChIP-exo, you typically perform single-end sequencing, not paired-end sequencing. The Illumina R1 sequencing primer anneals to the P5 site, which does contain the extra T that is normally used for A-overhang ligation. The P7 site indeed misses a T compared to regular libraries, but this is not an issue as long as you don't intend to perform PE sequencing.

I just wonder why they decided to perform blunt-end ligations... I would expect ligations with an A-overhang to be more efficient. Does anyone have any experience with this?

[TonyBrooks]

I had this question too and I just checked their adapters and primers to figure out the answer. My conclusion is that their design is correct:

For ChIP-exo, you typically perform single-end sequencing, not paired-end sequencing. The Illumina R1 sequencing primer anneals to the P5 site, which does contain the extra T that is normally used for A-overhang ligation. The P7 site indeed misses a T compared to regular libraries, but this is not an issue as long as you don't intend to perform PE sequencing.

I just wonder why they decided to perform blunt-end ligations... I would expect ligations with an A-overhang to be more efficient. Does anyone have any experience with this?

Yeah, I should have said that as it's P7, it's only an issue for PE. However, more and more of our ChIP-Seq is PE now, simply because they go on runs shared with exome samples. Seems a bit weird to remove the option for PE for the sake of one base.

The protocol I mapped out used a dA-tail, but I suppose because you exonuclease treat, this would chew up any dimer. The main reason you dA-tail and ligate is to reduce possibility of dimer, so blunt end ligation possibly isn't so much of an issue.

[pmad]

HI albireo,

The package CexoR is also available from Bioconductor for ChIP-exo peak calling (http://www.bioconductor.org/packages...tml/CexoR.html)

Pedro M

Hi, yes that is what I meant. I have performed a number of FRiP analyses (similarly to what done in the 2012 ENCODE Genome Research ChIP-seq methods paper) with very disappointing results in one case (~4-5% FRiP). This data is decent in other respects (high correlation of replicates, consensus motif present in 70% of the peaks).



I have not tested this specific peak caller, but have some experience with other methods, including Genetrack (the original peak caller used in the Pugh publication), Macs 2.0, Apex, Peakzilla and GEM, with wildly varying results. I should also add that my data is Illumina-based and was processed directly by Peconic.

[cptzantar]

FYI: Active Motif has recently released a ChIP-exo kit which contains all reagents necessary to go from DNA fragmentation, through the ChIP-exo reaction, and finishes with the generation of a library.

[jwfoley]

Indeed, they did a very nice webinar about this new product: http://epigenie.com/webinar-precisio...sing-chip-exo/

I'm surprised they're using blunt-end ligations.

[Manna]

FYI: Active Motif has recently released a ChIP-exo kit which contains all reagents necessary to go from DNA fragmentation, through the ChIP-exo reaction, and finishes with the generation of a library.

I'm using it, though for a DNA-modification, not a regular ChIP against some DNA-bound protein. Does anyone know the sequences of Active Motif's ridiculously priced indexing primers? Their tech support said they were "slightly modified from Illumina's" and proprietary. In other words, "We will now charge you $45 for $0.45 worth of stuff."