[pmiguel]

The first post on this thread for a while, but is there any chance someone has a copy of the Rapid Library protocol that they could share?

What possible use would the protocol for a type of library construction for an obsolete instrument be to you?

[spet2522]

The first post on this thread for a while, but is there any chance someone has a copy of the Rapid Library protocol that they could share?

[Reshma]

Thanks for that though that's not relevant for me this time...will bear it in mind though!

[Cambridge454]

Hi Cambridge454, how did you resolve this issue? What settings are you currently using for rapid library preps? My averages are above 900bp consistently! Any advice you can provide would be greatly appreciated. Can anyone else help me?

It turned out that when the AMPure XP beads near their expiration date we were seeing this problem. Our library sizes returned to normal when we switched to a new bottle of beads.

[Reshma]

Rapid Library Fragment Lengths

I am also having problems with the Rapid Library producing really long average fragment lengths >900bp. I use the recommended nebulization settings, which worked perfectly well for old version Shotgun libraries (600bp avg.). I have used the new vented caps and the rubber stopper/filter nebulizer set-up with very similar results. Should I increase the nitrogen pressure to say 2.5 bar instead of 2.1 bar?

Hi Cambridge454, how did you resolve this issue? What settings are you currently using for rapid library preps? My averages are above 900bp consistently! Any advice you can provide would be greatly appreciated. Can anyone else help me?

[Zaag]

I put the sff from my rapid library run in geneious 5 and the read length is what I expect from my bio-analyzer results. Also if I check some wells from the run I can not see the B adaptor sequence so I believe I used the maximum read length.

If I however look at the read lengt distribution in the Roche RunBrowser the read lengths are 200 bp shorter because of the trimfilters. So I would recommend running the signalprocessing again with different filter settings if you want to stick to the Roche software.

[0Gen]

...50% high or low will make little difference...--
Phillip

Agree! But might need to take into consideration where you are. This paper seems to help a bit in understanding this issue.

http://nar.oxfordjournals.org/cgi/content/full/gkq332

[vlee2]

Thanks nickloman. We don't have problems with general libraries and amplicons. We only have problems with rapid libraries. In order to rule out the sequencing reagents, we tried to use two different lot numbers with the same DNA beads. We received similar results. The problem is most likely in library and less in emPCR step. But what is this problem?
Of course, I reported about our failed runs to Roche about 2 weeks ago, no response. I called again, no resolution. Seems that they don't have any ideas as well. That's why I went to this forum with question: is short reads and insufficient number of raw well is common for rapid libraries?

[nickloman]

We had problems with short-read runs and Roche eventually agreed to replace our kits. Details here http://pathogenomics.bham.ac.uk/blog...are-not-alone/

Strongly suggest you report any problems you are having like this to the technical support team at Roche.

[vlee2]

rapid library problem

Hi,
We did 7 sequence runs using rapid libraries and only 2 turned out OK. The rest five have not produced long fragments reads. When I look the fragments size distribution, I initially thought, Oh, too much short fragments. But actually the distribution of short fragments were about the same in failed and successful runs. Its just in failed runs there were no long fragments. All my libraries (failed and good) average length is within the spec.
The other problem which was addressed here is that normally the average number of raw well we have is about 1.9M. But for rapid libraries - the average number of raw well is 1.3M. Does anybody experience similar problem?
I'm wondering, if short fragments issues is common for rapid libraries? Should we stop using rapid kit until Roche fix this problem?
To answer previously posted question, yes I accidentally did titration of rapid library without denaturation and I've got good results. Then I tried to use obtained cpb number and divided it by two for LV emPCr, and I received two times less beads than expected. I think you can't skip denaturation step, because you will end up with too many mixed data.

[pmiguel]

Has somebody tried to systematically increase the amount of input DNA used for Rapid library nebulization (from the recommended 500 ng) to see how this affects the library yield? If you are doubtful you have an accurate quantification could it be harmful to add some more DNA to be on the safe side? If you are working with chromosomal DNA the amount is normally not limiting.

I don't see any down side to adding more DNA if you have it. As long as you quantitate what you recover and only use an amount of DNA the rapid protocol expects downstream. I mean, it would be better to keep the molar ratios of adaptor to insert about the same.

--
Phillip

[beaver]

Are the results of the Rapid library starting with 500ng consistent? Another question is what kind of results has anybody had with the Titanium library kits? Lastly is there another location on this forum that address library construction trouble shooting?

Has somebody tried to systematically increase the amount of input DNA used for Rapid library nebulization (from the recommended 500 ng) to see how this affects the library yield? If you are doubtful you have an accurate quantification could it be harmful to add some more DNA to be on the safe side? If you are working with chromosomal DNA the amount is normally not limiting.

[SeqMonster]

Didn't work as in no enriched beads at all? Or 1/2 as many as you hoped to get?

--
Phillip

Nothing to very low enriched beads, which is opposite as what i thought it would be. When i re-did it with denaturing, they turned out fine.

[pmiguel]

I actually accidentally did this once during SV set up and the emPCR didn't work. :-) I had to re-do the emPCR and it worked after that.

Didn't work as in no enriched beads at all? Or 1/2 as many as you hoped to get?

--
Phillip

[SeqMonster]

Anyone tried adding non-denatured dsDNA to a lib-L emPCR? Did it work? I don't see why it would not.

--
Phillip

I actually accidentally did this once during SV set up and the emPCR didn't work. :-) I had to re-do the emPCR and it worked after that.