After ChIP optimization the TruSeq LIB prep is not working anymore: peak at HMW!

[EmiVig]

Hi all,

this is my fisrt post and I would like first of all to say HELLO to everybody!!
I'm a PhD student, and I'm stuck with ChIP (on histone modifications and TF with mouse renal tissue) library prep using Illumina TruSeq ChIP sample kit.
About two months ago, the LIB prep protocol was working fine (20ng input, size selection uising AMPure XP beads instead of agarose gel, 18 cycle of PCR post-size selection, final peak at 200-400bp), indeed I sequenced 2 samples, but the sequencing data were really noisy: I lost a lot of reads in noise all around the gemone. Therefore, I re-optimzed the chromatin sonication, in order to avoid ChIPing down long fragments which I might have then introduced into my library. Indeed the ChIP bioanlyzer trace look much better now (most of the material is about 200-300bp). Unfortunately, the Library prep is not working amymore now, even if I didn't modify anything of the protocol. The Bioanalyzer showed me a small peak at 200-400bp and a broad peak at high MW (see attached LIB7-9-11).
I read several threads about that (the peak might be PCR artifacts), therefore: I reduced the input from 20 to 5 and 10ng (18 PCR cycles) and 15ng (15 PCR cycles) but nothing changed...

What else could I do?! I would really appreciate any kind of suggestions!
I'm sorry for the long post, but I tried to give you a clear overview of the problem!
THANKS!
Emi

[nucacidhunter]

I wonder if you have Bioanalyser traces of input DNA for those libraries and also what method you used to quantify them.

[EmiVig]

Hi nucacidhunter,
thanks for replying!
Yes, I have it, but I can't attached it by now, since I don't have the file with me at home...I'll upload it tomorrow morning. Anyway, I think that the ChIP inputs for those libraries are quite fine: most of the ChIPed fragments are between 150-400, and part of it stands at high MW. I use the Qubit for quantifying the ChIP samples. I also thought that I might have some problems with the quantification, but the Qubit should be fine for that purpose...one thing that came in my mind is: might the GlycoBlue affect the quantification?
Additionally: it seems like the size selections didn't work...but that sounds strange to me, because just 2 months ago, using the same protocol, the selection perfectly worked!
Do you have something in mind?
Best,
Emi

[kalyankpy]

I regularly do NGS library prep and routinely use AMPure beads. In my experience I have always noticed that library size increases by 120 bp upon PCR and smaller fragments from the sonication amplify better than the larger fragments.

No offence intended but I would recommed to check the input DNA on bioanalyzer again and compare it with the final library prep. It would be easier to troubleshoot if you could provide the bioanalyzer plot for the input and the bead ratio u are using after PCR for purification (or else where if used)

In my experience, 18 cycles of amplification with 20ng input means the PCR needs to tweaked for efficiency (in fact there are better PCR reagents than Truseq reagents).

[nucacidhunter]

Do you have something in mind?

Quantity, size range and purity (260/280 absorbance) of input DNA are important factors for successful ChIP library prep. Presence of potential inhibitors could cause failure or result in non-optimal library. Using 1.8x bead to re-purify input DNA just before end repair reaction would decrease or eliminate inhibitors. I also would like to see non-cleaned PCR product Bioanalyser trace if there is one.

[EmiVig]

Hi, I´ve attached the Bioanalyzer of ChIP samples. The first 3 samples correspond to some old ChIPs which ended up in a successful LIB-prep (see second attached Bioanalyzer, LIBK4m1, LIBK4m3, LIBK27m3). As you might see, there are a lot of long fragments, therefore I optimized the ChIP and they now look like the ChIP1-3-5-7-9-11 (there are still long fragments, but at least they look better...).
The size selection protocl is the following one:
- 100ul DNA mixed with 75ul beads
- incubate 15min at RT
- save 170ul supernatants and mix with 25ul beads
- incubate 15min at RT
- wash twice with 80% ethanol
- 18 cycles PCR
Thanks,
Emilia

[kalyankpy]

I have looked at your bioanalyzer plots. I noticed your previous working ChIP library and compared it with your present PCR amplified library. In my opinion all your reagents are working fine and your PCR reagents are also working fine!
Your plots reminded me of my previous observation with few of my libraries. Whenever libraries are overamplified (or PCR reagents become limiting) somehow it strangely influences the fragment length and they show up like bigger fragments. this could happen if the template concentration is very high or the volume of PCR reaction is low!

One thing you may consider is to lower the number of PCR cycles. Try doing some test PCRs at 10, 12 and 15 cycles. If you can not spare your library, try lowering the template volume and keep the amplification same. I am confident that you will see a different fragment lengths in your bioanalyzer.

I hope you have not used the entire library for amplification. Let us know if lowering the amplification cycles or lowering the template concentration has worked.

[nucacidhunter]

Hi Emilia
As you have noted specificity of your ChIP data is mostly depend on pull down step and library prep will have less effect. TruSeq kit has been optimised for 5-10 ng input and for a narrowly size-selected fragment after ligation. If inputs are higher that may affect results by decreasing number of fragments that have adapters in both ends. Also, by using beads for size-selection more of adapter ligated DNA is maintained for PCR step in comparison with narrow gel based size-selection recommended in the Illumina guide which will influence efficiency of PCR as kalyankpy has mentioned. To find out if PCR reagents are limiting factor and cause of observed large amplicons, you can set up a no template control (NTC) PCR reaction along with your library ones and after completion run them on TapeStation (or Bioanalyser) before any clean-up. Ideally you would want to see some of unused primers in the traces and their location can be identified based on NTC trace. It is always possible to do less cycles than intended numbers and take a sample for checking yield and size on an instrument and if yields are low, to put the same reactions immediately back into thermocycler for a few more cycles. Following formula can be used to calculate amount of amplicons: PCR volume/elution volume X ng/ul of amplicons region from instrument. Then additional required cycles can be worked out by rule of thumb expecting doubling for each additional cycle. For sequencing normally 20 ul of cleaned PCR product in a concentration above 4 ng/ul would be suffice.

[EmiVig]

Hi kalyankpy and nucacidhunter,
thanks a lot for your suggestions!Really helpful! It sounds like the PCR setting is the problem...therefore, I´ll try today using less input ChIP and also fewer PCR cycles...hopefully this time it will work. I´ll keep you updated!

[Chipper]

Hi kalyankpy and nucacidhunter,
thanks a lot for your suggestions!Really helpful! It sounds like the PCR setting is the problem...therefore, I´ll try today using less input ChIP and also fewer PCR cycles...hopefully this time it will work. I´ll keep you updated!

You can just re-amplify your old library (e.g take 10% and run 4 cycles with new primers) and check it on a standard agarose gel, if fragments are of expected size it should be fine for sequencing. But don't expect high enrichment at specific loci for k4me1 and k27me3, they are expected to be wide-spread.

[EmiVig]

Hi kalyankpy, nucacidhunter and Chipper,
sorry, I´ve been absent for a while, because I have been testing different condition for my LIBprep, but...it didn´t really worked out so well yet...I still have a second band at 500-700bp. It seems that the reason is not the high n° of cycles causing the depletion of primers... I´ll sum up briefly what I did:
- I switched to size selection with agarose gel, in order to selected a more narrow fraction of material at the right size;
- I decreased the input ChIP DNA till 2 and 5ng;
- I reduced the number of PCR cycles until 8;
Nothing really changed...then, I tried to denaturate at 95°C for 4min one of my library and the HMW band went back to the right position (see first loaded sample in the attached gel); then I tried to modify the PCR setting, thinking that it might be a problem of annealing temp. or elongation (2nd lane: 12 cycle with suggested Illumina PCR protocol; 3rd lane: 62°C of annealing temp instead of 60 with 18cycles; 4th: 20sec elongation and 3min final elongation, 18 cycles; 5th: normal PCR protocol, using half of the primer cocktail thinking that I might have too much primer that form strange structures with my fragments..). I´m sorry for the quality of the gel...anyway, I still have the HMW band, but in the 3rd-4th and 5th lane, I could at least noticed a smaller band...(the first sample has a correct size...the others are a bit too small probably...these are libraries after PCR which I haven´t cleaned up)
I was thinking...do I have too much ssDNA in my ChIP which might disturb so much my PCR? Would be helpful to increase the initial denaturation step of the PCR from 30sec to 3min at 95°C and also the denat step form 10 to 20sec during the cycles? Additionally: might be that I also have too much ligated adaptors which give me problems? I found that some people are diluting the adaptor 1:10...
I´m really getting crazy...

[nucacidhunter]

In this case I would not suggest modifying PCR. To make it simple, I do not see any problem with number two. If yield after clean-up is low (60-80 ng<), as I have posted above you can do few more cycles. If you have done size selection after adapter ligation and PCR amplicon is larger than your cut size, it could be just an issue with gel size selection and would not affect your results. If size selection was before adapter ligation, you would expect 120-130 base longer PCR product.

[EmiVig]

Hi nucacidhunter, thanks for your reply.
The problem is that the number 2 should be smaller...I did a cut at 150-200 bp after the adaptor ligation, just before the PCR enrichment step; and after the PCR , I got it double...this means that I still have the "bubble products" problem (or whatever it is) using only 12 cycles of PCR...Why is it so?!

[nucacidhunter]

By cutting 150-200 bp after adapter ligation, the insert would be 20-70 bp which is short. There are multiple consequence for such short inserts: 1) wasting sequencing data because it will sequence into adapter and you will have to trim them; 2) mapping such short reads back to genome will be difficult; 3) if 1x bead is used for PCR clean up, around 1/2 of library will be washed away along with dimers. If you still have some aliquot of size selected DNA, I would like to see Bioanalyser trace of size selected DNA and clean PCR Product.