454 nebulization problem

[LewisStewart]

Hi all,

I am currently following the 3kb paired end library prep protocol for the 454 GS Junior. All seemed to be going well until we reached the nebulization step. The bioanalyzer trace showed us that our fragmented DNA had a peak of around 700bp (see image attached). The peak also seemed to be very low, indicating that very little DNA was present.

I have a few questions regarding this:
1. Has anyone tried increasing the quantity of DNA prior to DNA circularization at step 3.6?
2. Has anyone had similar problems with nebulization as i have experienced?
3. Has anyone tried using a Diagenode Bioruptor NGS as an alternative to nebulization and, if so, do you have a protocol for doing this?

I'd be grateful if anyone could answer these or give us any further suggestions

Cheers
Lewis

[MissDNA]

I am not sure if Jr and FLX protocols for 3 kb PE are the same. I have prepared several 3kb PE libraries for FLX, and your peak actually looks much better than several of mine. I have never seen a peak that looks like the manual! With a peak like that I would continue the protocol. Usually I proceed with 2 library amplifications, since there is enough beads for two. Then sometimes I combine the preps in the same MinElute column after melt, sometimes I carry them as 2 separate libraries till the end. They tend to sequence very well eventhough in the end there is not much library.

Increasing the amount of DNA pre circularization does not help. I have tried to increase circularization time but the improvement was minimal.

I have never tried any other nebulization method.

[Coyk]

Hi Lewis,
I've been wrestling with the 3kb paired end library protocol myself. I just did a 454jr sequencing run after having restarted from various steps in the protocol. The first time I made it to the step you mentioned, I got similar results (maybe worse) and asked the same question of the forum. A fellow member assured me that she has made many successful PE libraries having seen this very broad peak at the nebulization step. I also asked someone who works at a sequencing facility at the Institute for Genome Sciences in Baltimore and she said they use a Covaris at this step and our results didn't surprise her because nebulization tends to give much broader distribution of fragment length. I, too, would be interested to know if anyone has seen the sharp peak after nebulization we are supposed to see. Since receiving this feedback, I have not bothered with running the DNA chip at this point. I am really interested to hear about your resultant library. If you would send me a message when you've quantified it at the end of the protocol, I would greatly appreciate it. Thanks!

[MissDNA]

Hi Coyk,

You disapeared. Hasn´t stuff worked for you yet?
I answered your last PM. I will try to attach some of my traces from my nebulization peaks and final product.

[LewisStewart]

Hi guys, thanks for the replies. The protocols for the FLX and Jnr are the same. After the nebulization, we did continue, with even less success. We completed the protocol up to "Library Characterization" and i've attached our Bioanalyzer RNA trace. You'll notice that there is no peak at the desired location of around 500nt. Fluorometric measurement did not detect any cDNA in the sample, it was deemed too low to measure. Obviously, based on your replies, a problem has arisen downstream of nebulization rather than at the nebulization itself.

Again, thanks for taking the time to reply.
Lewis

[MissDNA]

Attached are the files. They correspond to 3 libraries I prepared together. The final product is the result of only one library amplification. B. emersonii did not work and I contact Roche's tech support about it. Since the 2 other preps seemed fine, the suggestion was that there was something wrong with the sample. They recommended a DNA clean up kit, the clean up was made and I was able to produce a library.
Nothe that BR3459 is not a great library but we sequenced it anyway, half plate PE and 1/2 plate SG and we got the genome almost closed.

[LewisStewart]

We think that it may have went wrong at the "Library Amplification" step (step 3.10). How many PCR cycles did you perform? We think that only 5 cycles, as indicated by the protocol, is far too few. Which DNA clean-up kit did you use and at which point did you perform this?

Thanks
Lewis

[MissDNA]

The protocol says 15 cycles, and that is what I do. The only modification I do in the whole protocol is right at the beginning, where I don´t do that Ampure after shrearing, instead I concentrade my sample using a QIAQuick column. As I mentioned before sometimes I combine the melt product for 2 libraries in one MinElute column. Other than that I follow the protocol religiously.

The clean up was performed by the person who sent us the sample. She bought the recommended kit (High Pure PCR Template Preparation Kit Cat. No. 11 796 828 001 -Roche), did the purification and sent me the sample. Then I started the protocol all over again with the purified sample.

[LewisStewart]

We have tried 15 cycles and it worked! The previous attempt (using 5 cycles) was under the supervision of the trainer from roche who insisted that the protocol was correct in telling us to perform 5 cycles. Thanks to you we now know that she was talking rubbish. Thank you.

I've attached my final library Bioanalyzer RNA trace. What do you think?

Thanks again, you've been a massive help

[MissDNA]

In which protocol says 5 cycles? Unfortunartely some Roche people have no clue about what they are saying.

Your trace is in seconds not nt but it looks the right size. I don´t think I´ve ever seen a peak that high, also there is a little shoulder but I don´t think that would be a problem. Is that the product of only one amplification?

I am glad I could help. One day I will have to start preparing 8kb and 20kb libraries and those are way more challenger. I completely forgot how to set up the elutrap.

[Coyk]

Yes, sorry MissDNA, I did disappear for a little while. I went to a Cold Spring Harbor Laboratory class (excellent) and was juggling other projects. Still am. Like I said, we got one run out of one try of the library and are still waiting to hear if it was successful.
My 3kb paired end library protocol says 5 cycles too! That's what I used! This is very illuminating. If we go through the protocol again, I will use 15 cycles. I am so relieved to hear this from you and so incredibly irritated at all the money and time that's been wasted.

[MissDNA]

Wow it is good that Lewis mentioned the number of cycles then!
The protocol I use is: Paired End Library Preparation Method Manual – 3 kb Span
GS FLX Titanium Series October 2009. It is the one up on 454 page. I looked up now there and it says 15 cycles.
I would ask for kits replacement, especially Lewis, who was under Roche´s training and heard from one of their people that 5 cycles was correct.
With so little DNA after nebulization it would be really hard get any library with only 5 cycles.

[MissDNA]

Just checked and the Jr. manual (revised June 2010) it does say 5 cycles! Over 1 yr and nobody has ever noticed the typo! Amazing.

[RCJK]

Actually it does have a 1 before the 5, there is just a big gap between them, so it may have been some formatting issue. Regardless, it would be really easy to take it as meaning only 5 cycles are needed.

[MissDNA]

I did see the the 1 now. Still Roche's trainer should have told them the correct was 15 and not insist on 5.