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-   -   Anyone using ChIP-exo? (http://seqanswers.com/forums/showthread.php?t=38980)

jwfoley 12-05-2013 11:27 AM

Anyone using ChIP-exo?
 
From the data analysis side, ChIP-exo (PMID: 22153082) looks much better than standard ChIP-seq - not because of the fine resolution (who really cares?), but just because of the much improved signal-to-noise. However, the only papers I've seen with it are by the same authors, and there are very few threads here about it, and I've heard rumors that it's not really working for anyone but the people who invented it.

Any anecdotes? Has anyone gotten it to work with the published protocol (PMID: 23026909) or some variant?

FiReaNG3L 12-06-2013 01:25 AM

It's working for me - it's not an easy technique as it is labor intensive and requires a lot of care, but at least for our application the results are well worth it. I'm using a slightly modified version adapted for Illumina.

jwfoley 12-06-2013 05:09 AM

Thanks. Is the Illumina version basically the same thing but with the universal Illumina adapter? Which steps are more difficult than standard ChIP-seq?

FiReaNG3L 12-06-2013 07:55 AM

My version is a custom one I implemented before their Methods paper got published - I never tested their solution.

It's not more difficult, it's just more labor intensive. It's basically a Chip + a library prep on the same day. If you follow their protocol to the letter, its 30 washes of 5 min, plus the enzymatic steps. The proper analysis also requires more work if you want to use the extra information the technique gives you.

Depending on your final goal, it could not be worth the extra work / costs or (as in my case) it could be essential. From your first post I would say if you don't really care about the fine resolution, you're better off with Chip-seq.

cptzantar 12-16-2013 09:50 AM

I've been trying to get chip-exo to work for about 2 months with little success. Would you be willing to share your protocol for chip-exo to see how it differs from what I am using?

Thanks.

james hadfield 12-16-2013 10:00 AM

We've one group making use of ChIP-exo the peaks look beautiful, are base-pair accurate for binding analysis and probably mean we only need 25% of the read depth. However the protocol is definitley not standardised, green-fingers seem to help and until it is really robust we'll keep running normal ChIP-seq for most work.

Mammooth 01-09-2014 05:10 AM

Hi!

I'm also working with the published protocol, for about... err.. half a year or so. I can't say with much success. I already changed a lot of things to make it more sound, but it seems there is still much work ahead.
Would any of you be so kind to provide a working protocol? :)
Of course any little help are welcome.

jwfoley 01-09-2014 05:19 AM

To those who report the protocol's failing: how? At what step do you get unexpected results?

james hadfield 01-22-2014 05:53 AM

Forgot to direct you to this: Development of an Illumina-based ChIP-exonuclease method provides insight into FoxA1-DNA binding properties.

http://genomebiology.com/2013/14/12/R147/abstract

albireo 01-27-2014 03:38 AM

I have been analysing illumina-based human Chip-exo data for a while - in my experience, the results are very TF-dependant.

At best, results (in terms of peak resolution and SN ratio) are almost within quoted specs.

At worst, results are comparable to ChIP-seq, with the added complication that a proper analysis will be lengthier (most off-the-shelf ChIP-seq analysis techniques will not work properly with ChIP-exo).

jwfoley 01-27-2014 04:58 AM

Thanks, albireo. Just to be clear, are you saying that in the worse cases, you have a ChIP-seq-like SN ratio, i.e. there is high background throughout the genome?

FYI I tested UniPeak (doi:10.1186/1471-2164-14-720) on the ChIP-exo data from the original paper and it seems to perform nicely with one or two non-default settings. Let me know if you'd like more information.

albireo 01-27-2014 05:32 AM

Quote:

Originally Posted by jwfoley (Post 130790)
Thanks, albireo. Just to be clear, are you saying that in the worse cases, you have a ChIP-seq-like SN ratio, i.e. there is high background throughout the genome?

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).

Quote:

Originally Posted by jwfoley (Post 130790)
FYI I tested UniPeak (doi:10.1186/1471-2164-14-720) on the ChIP-exo data from the original paper and it seems to perform nicely with one or two non-default settings. Let me know if you'd like more information.

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.

jwfoley 01-27-2014 05:48 AM

Quote:

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).
Interesting. The way ChIP-exo digests unprotected DNA, this sounds like it would come from a nonspecific antibody. My favorite negative control for ChIP-seq is a knockdown/knockout of the antibody target, though that's often biologically infeasible. I don't suppose you have that for your experiment?

albireo 01-27-2014 07:12 AM

The antibody should be sound.

We've been wondering if it is more a case of the exonuclease failing to stop at the crosslinking site, while stopping at various points before. This could be due to misfolding and be stricly dependant on the TF being analysed.

This exonuclease-stop hypothesis seems to be supported by the high variability in peak width I'm observing (which I verified via cross-correlation approaches to look at the start-stop distance - these fail to show anything apart from the phantom peak, suggesting there is no consensus start-stop distance). The fact that our TF binds in several configurations (monomeric/dimeric) does not help.

TonyBrooks 01-28-2014 03:34 AM

Quote:

Originally Posted by james hadfield (Post 130359)
Forgot to direct you to this: Development of an Illumina-based ChIP-exonuclease method provides insight into FoxA1-DNA binding properties.

http://genomebiology.com/2013/14/12/R147/abstract

Thanks for that James. We're about to look into ChIP-Exo and designed our own primers, but we may just use these instead. Am I right in thinking that there is a T missing from their P7-exo adapters though? They are doing blunt-end ligation, but the Illumina R1 and R2 sequencing primers end in T due to the dA-tail ligation generally employed.

Embunnik 02-25-2014 01:35 PM

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 02-26-2014 12:57 AM

Quote:

Originally Posted by Embunnik (Post 133707)
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 08-02-2014 04:06 PM

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


Quote:

Originally Posted by albireo (Post 130797)
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 11-14-2014 01:24 PM

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 11-14-2014 04:52 PM

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.


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