Minimum Coverage for ChIP-Seq & RNA-Seq

canis · 07-08-2009, 05:56 AM

Hi all,

Can anyone please suggest what should be the minimum Coverage and Read Depth required to get meaningful results for ChIP-Seq and RNA-Seq analyses.

Thanks,
Veer

SGPSling · 07-14-2009, 01:56 AM

I don't think this question has been examined carefully to date. You may want to read the recent paper of Rozowsky et al. Nat. Biotech 27 (1) 66-75. The bottom line is different transcription factor has different saturation depth. We simply need ChIP seq data of more transcription factor to see if there is a generalized pattern for different class of TFs.

Anyone can add more reference discussed the same matter will be really appreciated.

canis · 07-16-2009, 03:52 AM

Thanks SGPSling I will go through the paper Rozowsky et al. Nat. Biotech 27 (1) 66-75. My understanding of what you have posted is that the saturation depth can not be predicted in advance , it will depend on the sample used. Am I correct in my understanding?

james hadfield · 07-16-2009, 04:25 AM

SGPSling is correct but I would say 1 lane for ChIP-Seq and 2-3 for mRNA-Seq are good ballparks.

SGPSling · 07-16-2009, 07:22 PM

Hi, Canis: I would say the saturation depth depends on the TF you are studying. In the paper I quoted, the identified peaks saturated arount 3-4 M reads for PolII so that one lane would be enough. But for STAT1, it didn't reach plateau even when they sequenced 24 M reads.

For most of the ChIP seq papers published so far, they usually got 1-2 M reads. So James is right that people usually do one lane for each sample in a ChIP seq experiment at this moment.

As for now, we cannot predict the saturation depth for ChIP. But I believe with more results published in the future, one migth be able to do so. At this stage, we simply need more data to improve our knowledge.

SGPSling · 07-16-2009, 07:25 PM

Sorry, don't know where the thumbs down icon come from in my last post. It is not what I want.

ECO · 07-16-2009, 08:19 PM

Quote:

Originally Posted by SGPSling

Sorry, don't know where the thumbs down icon come from in my last post. It is not what I want.

Fixed!

james hadfield · 07-17-2009, 02:34 AM

I said one lanes should do for ChIP-Seq and that comment is based on us geting up to 20M reads from a single lane. We routinely get over 10M reads and (I am a little gobsmacked by this) saw a sinlge control lane generate 0.97Gbp from a 42 cycle run!
If you only get 1-2M reads then you need more lanes!
James.

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07-08-2009, 05:56 AM	#1
canis Junior Member Location: bangalore Join Date: Sep 2008 Posts: 3	Minimum Coverage for ChIP-Seq & RNA-Seq Hi all, Can anyone please suggest what should be the minimum Coverage and Read Depth required to get meaningful results for ChIP-Seq and RNA-Seq analyses. Thanks, Veer

07-14-2009, 01:56 AM	#2
SGPSling Junior Member Location: Singapore Join Date: Jun 2009 Posts: 8	I don't think this question has been examined carefully to date. You may want to read the recent paper of Rozowsky et al. Nat. Biotech 27 (1) 66-75. The bottom line is different transcription factor has different saturation depth. We simply need ChIP seq data of more transcription factor to see if there is a generalized pattern for different class of TFs. Anyone can add more reference discussed the same matter will be really appreciated.

07-16-2009, 03:52 AM	#3
canis Junior Member Location: bangalore Join Date: Sep 2008 Posts: 3	Thanks SGPSling I will go through the paper Rozowsky et al. Nat. Biotech 27 (1) 66-75. My understanding of what you have posted is that the saturation depth can not be predicted in advance , it will depend on the sample used. Am I correct in my understanding?

07-16-2009, 04:25 AM	#4
james hadfield Moderator Cambridge, UK Community Forum Location: Cambridge, UK Join Date: Feb 2008 Posts: 221	SGPSling is correct but I would say 1 lane for ChIP-Seq and 2-3 for mRNA-Seq are good ballparks.

07-16-2009, 07:22 PM	#5
SGPSling Junior Member Location: Singapore Join Date: Jun 2009 Posts: 8	Hi, Canis: I would say the saturation depth depends on the TF you are studying. In the paper I quoted, the identified peaks saturated arount 3-4 M reads for PolII so that one lane would be enough. But for STAT1, it didn't reach plateau even when they sequenced 24 M reads. For most of the ChIP seq papers published so far, they usually got 1-2 M reads. So James is right that people usually do one lane for each sample in a ChIP seq experiment at this moment. As for now, we cannot predict the saturation depth for ChIP. But I believe with more results published in the future, one migth be able to do so. At this stage, we simply need more data to improve our knowledge.

07-16-2009, 07:25 PM	#6
SGPSling Junior Member Location: Singapore Join Date: Jun 2009 Posts: 8	Sorry, don't know where the thumbs down icon come from in my last post. It is not what I want.

07-17-2009, 02:34 AM	#8
james hadfield Moderator Cambridge, UK Community Forum Location: Cambridge, UK Join Date: Feb 2008 Posts: 221	I said one lanes should do for ChIP-Seq and that comment is based on us geting up to 20M reads from a single lane. We routinely get over 10M reads and (I am a little gobsmacked by this) saw a sinlge control lane generate 0.97Gbp from a 42 cycle run! If you only get 1-2M reads then you need more lanes! James.