[pmiguel]

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?

I would check your fragmentation sizes prior to ligation. Downstream changes in library construction should not affect that. If it does, then there has been some change in your nebulization set up that is at fault.

Err -- just to make sure this isn't something trivial: what are you measuring your fragment lengths with? If you are using a nano or picoRNA bioanalyzer chip (like one used to do with the old method) then keep in mind that your library is double stranded now and will appear to be roughly twice as "long" on chips intended to assay single stranded molecules. (I don't think the denaturation step prior to loading a nano or picoRNA chip would be sufficient to denature 600 bp fragments of DNA.)

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Phillip

[Old guy]

I have made 10 libraries with the rapid kit. All came out very well based on the quantification and HS Bioanalyzer results. When the first two were amplified with emPCR the results were poor. Low bead count. Anybody else experiencing this? Any suggestions?

[pmiguel]

I have made 10 libraries with the rapid kit. All came out very well based on the quantification and HS Bioanalyzer results. When the first two were amplified with emPCR the results were poor. Low bead count. Anybody else experiencing this? Any suggestions?

We don't trust the fluorescence assay. I mean, even in principle it does not appear like it would be diagnostic. That is, say your library consisted of fragments with adaptor ligated only to one end. They will not amplify at all, but on the fluorimeter the library may look fine. This is a reasonable example because if end repair did not work well, then this is exactly the situation you will end up in.

For example imagine a library constructed with the following characteristic:

10% of the fragment ends are ligatable. The resulting library molecules will partition as follows:

90% No adaptor
9% One adaptor
1% Two adaptors

Only the two adaptor molecules will amplify. But there are 9x more one adaptor molecules that will fluoresce 50% as intensely as the two adaptor molecules but they will not amplify. So you will end up putting roughly 20% of the library into your emPCR than what you need if you trust the fluorescence.

We do qPCR and then do emPCR based on that. qPCR takes longer but at least it should specifically detect amplicons.

Generally you need ~5-20% recovery upon emPCR enrichment to have enough beads to run but not a high percentage of mixed beads. So there is some leeway there. Still we end up too high or too low as much as 50% of the time. That will probably improve with experience, but our end results are good now.

The Roche protocols tend to have a lot of fault tolerance built into them, but sometimes they will include a few steps that are designed by bench magicians for bench magicians. Keeps you head from swelling too much, I guess...

--
Phillip

[SeqMonster]

New problem - 100% enrichment

Hi guys, I just started using the rapid kit not long ago and did 2 runs from those libraries. The first one turned out to have >1million reads but with a lot of short reads and the second one has very high mixed&dots even though the enrichment is around 9%. Now, I am doing the third one and some weird thing happened.

I set up 2 cups of LV emPCR using the same amount and same source of DNA, one worked nicely with around 9% enrichment but the other one has almost 100% enrichment. The emulsion didn't appear to be broken and we couldn't figure out what was happening. We repeated the emPCR with the same amount of DNA and same thing happened again. Does anyone has any idea what's going on?

Thanks.

/yw

[pmiguel]

Hi guys, I just started using the rapid kit not long ago and did 2 runs from those libraries. The first one turned out to have >1million reads but with a lot of short reads and the second one has very high mixed&dots even though the enrichment is around 9%. Now, I am doing the third one and some weird thing happened.

I set up 2 cups of LV emPCR using the same amount and same source of DNA, one worked nicely with around 9% enrichment but the other one has almost 100% enrichment. The emulsion didn't appear to be broken and we couldn't figure out what was happening. We repeated the emPCR with the same amount of DNA and same thing happened again. Does anyone has any idea what's going on?

Thanks.

/yw

I don't have an answer, but it should not be possible to get 100% enrichment--there are not enough enrichment beads for this to happen. In cases where something goes badly awry with calculations we have had libraries where vastly more (eg 40X) more library than is called for, we get something like 40% enrichment.

What does GS support say?

--
Phillip

[LMcSeq]

SeqMonster: Keep in mind that the DNA capture beads are now much more efficient than they previously were. You may need to adjust your cpb input if you aren't doing titration to optimize. We used to use 2 cpb and now we're down to 1 and probably going lower.

[pmiguel]

SeqMonster: Keep in mind that the DNA capture beads are now much more efficient than they previously were. You may need to adjust your cpb input if you aren't doing titration to optimize. We used to use 2 cpb and now we're down to 1 and probably going lower.

Which capture beads are these? In the emPCR? Or enrichment?

Also, how are you assaying "copies"? Straight fluorimetry? Fluorimetry using the wacky dye-labeled Rapid Adaptors?

--
Phillip

[seqAll]

SeqMonster: Keep in mind that the DNA capture beads are now much more efficient than they previously were. You may need to adjust your cpb input if you aren't doing titration to optimize. We used to use 2 cpb and now we're down to 1 and probably going lower.

Why are the DNA capture beads much more efficient than before? Thanks!

[SeqMonster]

I don't have an answer, but it should not be possible to get 100% enrichment--there are not enough enrichment beads for this to happen. In cases where something goes badly awry with calculations we have had libraries where vastly more (eg 40X) more library than is called for, we get something like 40% enrichment.

What does GS support say?

--
Phillip

I am still providing them information, haven't get any concrete answer yet. Sorry for bad explanation. What I meant by 100% enrichment was almost all the DNA beads were bound to the enrichment beads. When the mixture was on the magnet for some time, you get a clear solution instead of normal milky solution with a lot of DNA beads.

The weird thing is I was preparing 2 cups with the same DNA and this only happen on one of the cups. The other one is perfectly enriched and sequenced.

SeqMonster

[SeqMonster]

SeqMonster: Keep in mind that the DNA capture beads are now much more efficient than they previously were. You may need to adjust your cpb input if you aren't doing titration to optimize. We used to use 2 cpb and now we're down to 1 and probably going lower.

Hi LMcSeq, I actually had the other way round. Since I started to use the rapid kit, I have to use higher cpb than what I normally used, almost twice, for a good enrichment of around 10%. But the sequencing results turned out to have quite high mixed/dots %, and a lot of short reads.

Attached is the agilent trace after the library preparation and the read length from the RunBrowser.

[Cambridge454]

I would check your fragmentation sizes prior to ligation. Downstream changes in library construction should not affect that. If it does, then there has been some change in your nebulization set up that is at fault.

Err -- just to make sure this isn't something trivial: what are you measuring your fragment lengths with? If you are using a nano or picoRNA bioanalyzer chip (like one used to do with the old method) then keep in mind that your library is double stranded now and will appear to be roughly twice as "long" on chips intended to assay single stranded molecules. (I don't think the denaturation step prior to loading a nano or picoRNA chip would be sufficient to denature 600 bp fragments of DNA.)

--
Phillip

I am checking my library using the Bioanalyzer High Sensitivity DNA chip as recommended in the protocol. Do you think the fact that for the Rapid Libraries you are starting with 500ng instead of 5ug will affect the shearing during nebulization?

[LMcSeq]

I spoke with my FAS who explained that the capture beads are better at holding onto the library frags.
We've had some runs that have also had higher mixed/dot and short reads. It started before the rapid kit though.
We based on cpb on past experience rather than the microplate data. We only use the microplate data to dilute to 10^7.

[pmiguel]

I am checking my library using the Bioanalyzer High Sensitivity DNA chip as recommended in the protocol. Do you think the fact that for the Rapid Libraries you are starting with 500ng instead of 5ug will affect the shearing during nebulization?

Plausible, but I don't know. What size is your input DNA? Nebulization does not work well for relatively short input DNA.

We have mainly been doing cDNA libraries. But I just checked and the one set of rapid DNA fragment libraries we made recently did shear to 1000 bp average length.

--
Phillip

[pmiguel]

I spoke with my FAS who explained that the capture beads are better at holding onto the library frags.
We've had some runs that have also had higher mixed/dot and short reads. It started before the rapid kit though.
We based on cpb on past experience rather than the microplate data. We only use the microplate data to dilute to 10^7.

Ack! Your "FAS" would be your Applied Biosystems "Field Application Specialist". You want to talk to your Roche "RAC", "Regional Application Specialist". FAS's and RAC's would be mortal enemies!

We don't let our FAS near our 454 without shackles and a blindfold.

Seriously though:

I still am not sure which bead you mean by "capture bead". The white sample beads or the brown, magnetic, enrichment beads?

--
Phillip

[pmiguel]

I am still providing them information, haven't get any concrete answer yet. Sorry for bad explanation. What I meant by 100% enrichment was almost all the DNA beads were bound to the enrichment beads. When the mixture was on the magnet for some time, you get a clear solution instead of normal milky solution with a lot of DNA beads.

The weird thing is I was preparing 2 cups with the same DNA and this only happen on one of the cups. The other one is perfectly enriched and sequenced.

SeqMonster

Yes, that is what I thought you meant. If memory serves we saw a result similar to this, but not as extreme. But I would have to ask someone else in the lab when they get back from vacation next week, to be sure.

Again, though, I did not think there were enough enrichment beads to capture all the sequence beads (in cases where 100% of the beads were templated.)

We had a couple of cases where way, way too much library was added to the emPCR (40x) too much. And, even then we saw only about 40% enrichment. As if we had exceeded the binding capacity of the enrichment beads.

--
Phillip