Paired end library, 3kb woes

[Coyk]

Hello all,
I have made two attempts so far to make it through the paired end library protocol (Rev. June 2010 is the manual I've been referencing). I have made it through the circularization adaptor ligation, library span size selection, and fill-in reaction (up to section 3.5). After that there is a quantification step that I've done twice and both times gotten 22ng/ul which is more than enough to proceed. Apparently, though, I tend to lose everything by the time I run it on a 7500 chip after circularization and nebulization. The two most obvious possibilities to me are either the circularization adaptors aren't ligating properly or the circularization isn't happening and the subsequent DNase step chews my sample to bits. I was wondering if this has happened to anyone else and if there are any suggestions for troubleshooting.

Thanks!

[MissDNA]

I have prepared my fair share of 3kbPE libraries, and after circularization I NEVER saw a nice peak, as it shows in the manual, on the DNA 7500 chip. Most of the times I have an almost flat line. Still I continue with the protocol and most of the times I get a library because further on the protocol there is a library amplification step. Usually what I do is continue with 2 amplifications, instead of only one, and I combine the product of the 2 amplification in the final column purification step.
This week I prepared 2 libraries like that.
I really wonder if someone really sees nice peaks after circularization, and what can be done to improve circularization. Once per Roche´s tech support recommendation I increased the time of the circularization a bit. I went as far as 40 min only, but I am not sure if helped.

[Coyk]

Well this gives me much hope! Thank you so much. I will take up the charge again and hope for the best.

[MissDNA]

Cool. Let me know how it goes.
The 2 libraries I prepared this week, titrated very well. Hopefully sequencing will be good.

[newmoon]

You should check Cre recombinase activity using the control in the kit. It's not a stable enzyme.

[MissDNA]

I´ve used different lots of Cre recombinase and I always see an almost flat line after nebulization. However, as I stated before I end up with library. I never checked its stability and since it takes forever to get enzymes in Brazil is hard to keep changing lots.
I sequenced the libraries I made last week. For one of them we got almost 700k reads and median read legth of 418, which is excellent for PE. The other library was bad (barely 10e08 molec./ul) but since we were in a hurry we sequenced anyway. We got about 400k reads with median lenght of 382, most of the reads failed in the mix+dot.

[Dun]

Same problem here. During three rounds of trying, I NEVER see any peak after circularization, nor after 500bp fragmentation (I use Covaris). Absolutely no peak.
BTW: I didn't use their 'carrier DNA'

Basically, I didn't see anything after circularzation until PCR, which contains 25 cycles and yields some fragments ranging from 100~700bp. Although I've made 3 PE libraries, I am just simply not trust them, so I didn't put them into emPCR.

I desperately need some direction.
How do you guys do QC for circularzation and final PE library? THX

[MissDNA]

Why don´t you follow the protocol, using nebulization, carrier DNA and 15 cycles? Even though some libraries to not look perfect on the RNA chip, they tend to sequence well. I have prepared and sequenced over 30 3kb PE libraries, which good results.

We sequenced another 3kb PE library today. I will see the results tomorrow morning. The library was definitely not the greatest.

Could you post the agilent trace from your libraries?

[Dun]

thx MissDNA

So it's OK to have flat line after circularization?

The story after that is a little complicated. since we are trying to put RLMID adaptors and use rapid library protocol, so the fragments I was mentioned are dsDNA.
I am wondering how does the DNA trace look like right after PCR in original assay.

[MissDNA]

I see. I have never tried that and I am not really sure that would work.
I have posted in some other thread, traces after nebulization where there is an almost flat line and then the final library in the expected size range. I´ve never seen a nice peak after nebulization.
Do you have the trace of an HS chip?
It would be actually interesting to know if your libraries would sequence well.

[MissDNA]

My traces are on this thread: http://seqanswers.com/forums/showthread.php?t=16183

[Dun]

Many thx. I will check out that post carefully, seems it's a common problem.
I don't have HS report with me right now, I can show a pic of PCR product which is done by DNA 7500 chip.

I will throw my PE library in anyway later this month.

[Dun]

My trace after Covaris 500bp fragmentation looks very similar to your nebulized ones.

[MissDNA]

When you say PCR product, do you mean the amplified library?

Your trace shows a lot of short fragments. My barely there peaks start raising at about 300 bp and end at around 1000 bp.

I would like to see your final library and know the results of your sequencing, especially if the RLMID worked.

[Dun]

Yes, it is amplified library by PCR.