SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
Illumina Chip-seq libraray prep temreis Introductions 0 06-29-2011 06:52 PM
Problems with Invitrogen Size Select E-gels for Illumina RNA-Seq Library Sample Prep? Jerry Glenn Sample Prep / Library Generation 0 04-18-2011 07:14 AM
Chip-seq sample prep for bacteria icanper Sample Prep / Library Generation 2 07-30-2010 02:34 AM
ChIP seq sample prep for Illumina athos Epigenetics 2 12-01-2008 10:01 PM
Chip-seq sample prep protocols for 454? What_Da_Seq Epigenetics 5 08-02-2008 12:25 AM

Reply
 
Thread Tools
Old 09-19-2008, 01:02 AM   #1
athos
Junior Member
 
Location: uk

Join Date: Sep 2008
Posts: 9
Default ChIP seq sample prep for Illumina

Hi Everyone, I'm hoping someone can give some advice on ChIP enriched DNA. Does anyone try and quantify this by nanodrop/picogreen before starting the Illumina sample prep protocol (the protocol recommends starting with approximately 10ng DNA)? We have had difficulty in quantifying samples reliably. Is anyone doing ChIP seq regularly that can help?
Thanks.
athos is offline   Reply With Quote
Old 09-19-2008, 04:03 AM   #2
Chipper
Senior Member
 
Location: Sweden

Join Date: Mar 2008
Posts: 324
Default

Yesy, we do but it does not tell you much since you do not know what is contaminaition and or background DNA. Usually PCR or qPCR will be more informative, if you have a good positive control.
Chipper is offline   Reply With Quote
Old 09-21-2008, 11:58 PM   #3
athos
Junior Member
 
Location: uk

Join Date: Sep 2008
Posts: 9
Default

thanks Chipper. Can I just ask - do you normally follow the Illumina sample prep protocol as is, or do you ever do the size selection after the enrichment PCR?
Cheers.

Last edited by athos; 09-22-2008 at 12:00 AM.
athos is offline   Reply With Quote
Old 09-22-2008, 03:39 AM   #4
Chipper
Senior Member
 
Location: Sweden

Join Date: Mar 2008
Posts: 324
Default

Hi, I have not done the sample prep myself and not seen the protocol but I thought size selection was supposed to be after enrichment as it makes more sense to me. Otherwise the risk of contamination by ladders or other saples increases and you will probably lose a lot of fragments during size selection. This protocol uses amplification first: http://www.genomecenter.ucdavis.edu/...brary_prep.pdf
Chipper is offline   Reply With Quote
Old 09-24-2008, 10:43 AM   #5
cjohns
Junior Member
 
Location: Seattle

Join Date: Sep 2008
Posts: 7
Default

Here is what I found on our Chip-Seq:

Neither Nano-Drop nor bioanalyzer nor Pico Green quantitation of our IP products agree so we stopped trying to quantitate them. We use 1/3 of the IP product for the sample prep protocol (the same amount we used for Chip-chip and it gives us good results)

The gel purification step is much trickier than their protocol states. If you run the gel for 60 min as they state and cut out 200+/- 25 bp you will get a huge amount of adapter complexes in your final sample that will show up as a big peak at about 120bp on bioanalyzer results after amplification. They will also make up about 50% of your sequences on the machine. What we do is run a much longer gel for at slow speeds for 5+hours and cut out 180-300 bp and then continue with protocol as is.

Amplifying after size selection has not led to any perceivable contamination by ladders (of course, leave empty lanes between samples and ladders).
cjohns is offline   Reply With Quote
Old 09-24-2008, 11:15 AM   #6
Chipper
Senior Member
 
Location: Sweden

Join Date: Mar 2008
Posts: 324
Default

Hi,
thanks for the heads up on the gel purification. How many cells do you generally use? I ment to say that contamination can be an issue if you dont have the adapters on when running the gel, but it only a few thousand such reads in our runs so not a real problem.
Chipper is offline   Reply With Quote
Old 12-01-2008, 03:08 PM   #7
jawdekar
Junior Member
 
Location: LA

Join Date: Oct 2008
Posts: 1
Default ChIP-Seq help

hi,

can anyone tell me if I can enrich the adapter-modified DNA fragments by PCR first followed by the size selection of the library. This is because I am starting will approximately 10ng of ChIP'ed DNA and it seems unlikely that I can visualize 10ng on a gel for me to excise DNA in the ~200bp range.

thanks
jawdekar is offline   Reply With Quote
Old 12-03-2008, 08:40 AM   #8
tomv
Junior Member
 
Location: Cambridge MA

Join Date: Aug 2008
Posts: 8
Default

You can definitely gel purify after amplification. This is what we have done for a long time. We are just now testing whether its better to do it before, after or before AND after but the results are not in yet. I'd be interested to know if anyone else has tested this.
tomv is offline   Reply With Quote
Old 12-09-2008, 07:30 AM   #9
alere
Junior Member
 
Location: U.K.

Join Date: Jul 2008
Posts: 3
Default ChIP seq enrichment

Hi everyone,
I'm preparing the ChIP enriched DNA for the ChIP seq.
I'm wondering if is possible (and better) do the size selection directly on the ChIP sample before the amplification step as the ILLUMINA protocol recommend. Is possible to see any band on a 2% gel loading the ChIP from 20x 10 6 cells?? In this case the adaptor ligation and the library amplification step will more specific leading to an effective 200bp long fragment enrichment.
Thanks.
alere is offline   Reply With Quote
Old 06-18-2009, 05:30 PM   #10
captainentropy
Member
 
Location: San Francisco Bay Area

Join Date: Mar 2009
Posts: 89
Default

athos, I agree with cjohns comment. I use Qubit for what I suspect will be really low concentration samples and Nanodrop for post-PCR quantitation. I've compared Qubit and Nanodrop on low [DNA] samples and they're usually close in numbers but percentage-wise they aren't even close (eg. Qubit says 6 ng/ul, Nanodrop says 4 ng/ul). Also, the tech in the Bioanalyzer facility says not to trust the Bioanalyzer for quantitation purposes as it has ~20% CV!

jawdekar, yes, you can definitely amplify after the ligation step. However, be warned that you may get massive amplification of concatemerized adapters that weren't removed in the clean-up step (Qiagen minelute). Thus if you want to amplify at this point it's important to use the right amount of adapters in the ligation step. As the Illumina protocol is written, using a 1:10 dilution is typically waaaay too many adapters. I use 1:30 as does another person I know that gets beautiful libraries (and that's ChIPed DNA from flies so there is tons of DNA per ChIP to start with). Basically you don't want your sample DNA to be the limiting factor but too many unligated adapters are problematic as they often become the preferred template for amplification and ultimately sequencing.
captainentropy is offline   Reply With Quote
Old 01-21-2010, 06:40 PM   #11
xenia.zhang
Member
 
Location: ma

Join Date: Jan 2010
Posts: 10
Default

Hi all,

I have a couple chipseq libraries that show really good qPCR enrichment both before and after library construction, but sequencing results show very low signal, which resembles some other libraries where antibody is not optimal, though I know the Ab I use is a very good one. Then I start to suspect I have low ChIP enriched DNA for library construction, and tried to quantify concentration on high sensitivity bioanalyzer DNA kit. Unfortunately, for most of them, I can't detect anything, though the kit claims to be able to detect as low as 5pg/ul.

I wonder whether anyone else has this problem of quantifying chip enriched DNA with bioanalyzer high sensitivity dna kit, even with good Chiped dna samples? Then I wouldn't worry too much. Or it's really problem of my sample?

Or does any of you have any experience in this similar good qPCR, bad sequencing situation?
Million thanks to all!



-Sherry
xenia.zhang is offline   Reply With Quote
Old 01-21-2010, 06:41 PM   #12
xenia.zhang
Member
 
Location: ma

Join Date: Jan 2010
Posts: 10
Default

BTW, immunoprecipitation itself does work. About half of the peaks callled are real. The others are basically either background or 'peaks' around 30bp, which are false positives.
xenia.zhang is offline   Reply With Quote
Old 01-25-2010, 11:19 AM   #13
captainentropy
Member
 
Location: San Francisco Bay Area

Join Date: Mar 2009
Posts: 89
Default

from what I've been told about the Bioanalyzer is that it's not very good for quantitation purposes. I use it just to see what my library looks like (is there a peak around 80 or 125 bp indicating adapter carryover). According to Illumina, even if you see nothing on the bioanalyzer you may be able to generate clusters for sequencing. Even when I've had a tiny peak on the bioanalyzer chromatogram the sequencing was successful. For quantitation I use either a Nanodrop or Qubit. Qubit is more sensitive at low DNA concentrations. I have not done qPCR on my samples but the guy who does my sequencing does qPCR (on my samples) and I've never been told there was a problem.

What exactly do you mean when you say "About half of the peaks callled are real." Are you saying that the half that are "real" passed QC and uniquely mapped to the genome? I assume you have a bioanalyzer chromatogram? Was there a big peak around 70 or 124 bp? If so that is due to adapter carryover in the size selection step. I had that happen a couple of times and one time much less than half the reads mapped.
captainentropy is offline   Reply With Quote
Old 01-25-2010, 11:47 AM   #14
zhaoj
Junior Member
 
Location: philadelphia

Join Date: Jan 2010
Posts: 5
Default

Hi Sherry
I had that problem before, I suspect the quantity of DNA was low in my CHIP sample (hard to quantitate), so I combined several ChIP and worked fine in sequencing.
zhaoj is offline   Reply With Quote
Old 01-25-2010, 03:32 PM   #15
xenia.zhang
Member
 
Location: ma

Join Date: Jan 2010
Posts: 10
Default

Hi zhaoj,

Thanks for your answer! That's also what i suspect. By combining several ChIP, do you combine at the final elution step, or you actually use more chromatin prep?

THanks.



-Sherry
xenia.zhang is offline   Reply With Quote
Old 01-25-2010, 03:36 PM   #16
xenia.zhang
Member
 
Location: ma

Join Date: Jan 2010
Posts: 10
Default

captainentropy,

Thanks for your reply!

Actually I'm at the step before library construction. I figure I have to have decent amount of ChIP enriched DNA before I start doing library construction.

'half of the peaks are real' means, for example, I got 300 peaks from peak calling software (MACS/Cisgenome, etc). I manually checked these peaks and compared with other lab members' similar result and only about half of these peaks seem real. The other peaks are just background peaks that also exist in a control sample, or are too short to be real.

Hope this is clear?

thanks.


-Sherry
xenia.zhang is offline   Reply With Quote
Old 03-19-2010, 03:10 PM   #17
hon
Junior Member
 
Location: san fran

Join Date: Sep 2009
Posts: 9
Default

Hi Sherry,

Have you figured your problem yet on low peak signal? Recently I have been in similar situations that couple of my histone ChIP and Anti-HA Tag ChIP are not working out quite well, and like you said resembling to those ChIP of inefficient antibody. I kind of in line with zhaoj who suggest that the ChIP DNA used for making library is not enough. Previously I chip-seq two samples, K4me3 and K27me3, both of them working out really fine, although I only use 1 millions cells for chip assay. I thought I could get similar success by using same number of cells for one transcriptional factor ChIP (HA tagged), and one histone ChIP (K79me2), however the sequencing results turned out are not good, e.g. not many peaks, and high FDR (30%). Similar to you, I am quite confident that the antibody should be ok, as I checked it by QPCR to show good enrichments on my known targets. While I am repeating the ChIP seq with more cells and waiting for the results, I wonder there could be other problems that cause the failure of ChIP seq?

For your bioanalyzer questions, I have tried the high sensitivity kit that can not detect the ChIP DNA, I guess because the quantity of ChIP DNA is really low, plus that they are in smear (not a single band), so even let's say if you have 5ng total but the smear may have 1000 bands, each band end up has 5pg, is already reach the limit of detection. In other aspect I guess all people using bioanalyser should pay attention is the bioanalyser is very sensitive to salt that means if there's minimal salt in your ChIP DNA that will ruin the measurement, I have seen this many times already, especially for phenol purified ChIP DNA which is not very clean and always contains a lot of salt. Hope this helps.

Quote:
Originally Posted by xenia.zhang View Post
Hi all,

I have a couple chipseq libraries that show really good qPCR enrichment both before and after library construction, but sequencing results show very low signal, which resembles some other libraries where antibody is not optimal, though I know the Ab I use is a very good one. Then I start to suspect I have low ChIP enriched DNA for library construction, and tried to quantify concentration on high sensitivity bioanalyzer DNA kit. Unfortunately, for most of them, I can't detect anything, though the kit claims to be able to detect as low as 5pg/ul.

I wonder whether anyone else has this problem of quantifying chip enriched DNA with bioanalyzer high sensitivity dna kit, even with good Chiped dna samples? Then I wouldn't worry too much. Or it's really problem of my sample?

Or does any of you have any experience in this similar good qPCR, bad sequencing situation?
Million thanks to all!



-Sherry
hon is offline   Reply With Quote
Old 03-19-2010, 05:29 PM   #18
xenia.zhang
Member
 
Location: ma

Join Date: Jan 2010
Posts: 10
Default

Hi Hon,

Thanks for your suggestion. Current my explanation regarding your question is still not enough ChIP DNA. For histone modification, you don't need lots of starting cell since its so abundant and widespread. But for transcription factor, it's much weaker and rare. I know people in my lab who use half a million cell to do transcription factor chipseq. Although he did get result, total peak number compared with those using at least 20million cells is very small, and signal is not strong. Current my way is to use every chromatin prep for 2 ChIP, instead of 5 as before, and pool 2 chiped DNA together for library construction. I consulted experienced lab and they said that they've been successfully constructed library with 1-3ng of ChIPed DNA, but it's not always successful.

I suggest you can use picogreen (from invitrogen) to quantify your ChIPed DNA. I tried once, and although I haven't been able to get linear standard curve, my DNA is in the range, and after calculation, is around 3ng per ChIP (that's why I choose to combine two chips). The sequencing result seems to be fine now.

yah. That's my answer. Hope it will help!

good luck!
Sherry




Quote:
Originally Posted by hon View Post
Hi Sherry,

Have you figured your problem yet on low peak signal? Recently I have been in similar situations that couple of my histone ChIP and Anti-HA Tag ChIP are not working out quite well, and like you said resembling to those ChIP of inefficient antibody. I kind of in line with zhaoj who suggest that the ChIP DNA used for making library is not enough. Previously I chip-seq two samples, K4me3 and K27me3, both of them working out really fine, although I only use 1 millions cells for chip assay. I thought I could get similar success by using same number of cells for one transcriptional factor ChIP (HA tagged), and one histone ChIP (K79me2), however the sequencing results turned out are not good, e.g. not many peaks, and high FDR (30%). Similar to you, I am quite confident that the antibody should be ok, as I checked it by QPCR to show good enrichments on my known targets. While I am repeating the ChIP seq with more cells and waiting for the results, I wonder there could be other problems that cause the failure of ChIP seq?

For your bioanalyzer questions, I have tried the high sensitivity kit that can not detect the ChIP DNA, I guess because the quantity of ChIP DNA is really low, plus that they are in smear (not a single band), so even let's say if you have 5ng total but the smear may have 1000 bands, each band end up has 5pg, is already reach the limit of detection. In other aspect I guess all people using bioanalyser should pay attention is the bioanalyser is very sensitive to salt that means if there's minimal salt in your ChIP DNA that will ruin the measurement, I have seen this many times already, especially for phenol purified ChIP DNA which is not very clean and always contains a lot of salt. Hope this helps.
xenia.zhang is offline   Reply With Quote
Old 03-19-2010, 11:14 PM   #19
hon
Junior Member
 
Location: san fran

Join Date: Sep 2009
Posts: 9
Default

So you said you combined 2 ChIP assay DNA to make library which works, do you know that equal to how many cells? Dose your transcriptional factor bind to a lot of genomic targets? I found if a TF doesn't have many targets may require even more cells. Right now I don't even bother to measure the ChIP DNA as I think that can not guarantee the ChIP seq will work. Even you ChIP with IgG which can give several ng of ChIP DNA that will make a good library but not give you any significant peaks. Seems people more incline to use more than 10 millions cells for TF ChIP, while histone ChIP can be in million range.
hon is offline   Reply With Quote
Old 10-12-2010, 08:11 AM   #20
saikumarkv
Member
 
Location: Cincinnati

Join Date: Dec 2009
Posts: 11
Default

Hi everyone,
In a previous experiment I used some MIRA enriched DNA (methylated) for a single read library prep (using Illumina Chip-seq kit) and sequencing, which worked very well. Right now I have lots of ChIP samples to sequence of various amounts (2-20ng). We decided we will use PE adapters for all sample preps from here on. I'm testing the NEBNext DNA sample prep kit and Illumina PE sample prep kits with different amounts of DNA (1-10ng). The question I have is whether anyone adopted the PE sample prep kit (where they use lot more reagents and enzymes) to the ChiIP-sample prep kit. Of course I will change the adapter and primer concentrations. I'm also testing TUFTS custom adapters and primers they they were gracious to share. Thanks in advance.
saikumarkv is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 02:06 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO