[SeqMonster]

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?

I am checking my library using the High Sensitivity DNA chip too. I actually started with 1ug instead of 500ng. The agilent trace was showing the correct size. Based on my experience with the old kit when we have to use 5ug (recommended amount), the nebulized size never shift even if I used an amount from 2.5ug.

[SeqMonster]

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

Philip, what kit do you use for generating cDNA libraries from RNA? Are you using the Roche cDNA synthesis kit as recommended by the Roche protocol and sheared your RNA before you start with the cDNA synthesis?

[pmiguel]

Philip, what kit do you use for generating cDNA libraries from RNA? Are you using the Roche cDNA synthesis kit as recommended by the Roche protocol and sheared your RNA before you start with the cDNA synthesis?

Yes, mostly. But when we do not have enough RNA we use the old SMART kit method.

Well, I guess we do not actually "shear" the RNA (implies a mechanical method), we use the zinc chloride RNA fragmentation method as specified in the cDNA rapid library preparation method.

--
Phillip

[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

That's what the tech support suggested. I must have added too much library into the emPCR (I don't think I did), and not denaturing them (I did). First of all, I was preparing 2 cups from the same denatured DNA and added the same amount into each cup and only one cup turned out that way. Then when I re-did it using a new cup, it still gave me the same problem. I must have did something weird along the way which I can't think of. I did denature the 4X 10^6 dsDNA before adding them into the DNA capture beads and they were quantify using the microplate data with the standard from the kit itself.

I sequenced the 1 cup that worked and it turned out not too bad, except having the short read problems. I am waiting for the tech support to give me a more "diagnostic" answer right now.

While waiting for this, I am going for whale watching.

p/s: BTW, Thank you guys for all the input.

[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.

Okay, I found the reference to "capture beads". Here is the relevant passage in technical bulletin 11-2009: http://www.454.com/downloads/protocols/TCB-09011.pdf

3.2 DNA Library Capture
Modifications have been made to the Capture Bead component of the GS FLX Titanium LV and SV
emPCR Kits for all kit lots beginning with LV kit lot 93717360 and SV kit lot 93726160 and subsequent
(i.e. higher lot numbers). This change requires a modification to the emPCR process. For the above listed
lots, it will be necessary to add more library than before to the capture beads (step 9 in section
3.2 of the GS FLX Titanium emPCR Method Manual or step 6 section 2 of the GS FLX Titanium emPCR Quick
Guide).

Then they ask you to add 3x as much library as before.

This is the same technical bulletin that introduces the mysterious "PCR additive".

--
Phillip

[SeqMonster]

Yes, mostly. But when we do not have enough RNA we use the old SMART kit method.

Well, I guess we do not actually "shear" the RNA (implies a mechanical method), we use the zinc chloride RNA fragmentation method as specified in the cDNA rapid library preparation method.

--
Phillip

We are normally dealing with very low amount of RNA too. I looked at the SMARTERpico kit, it looks like what I need. Thank you for your information. :-)

Do you still "fragmentize" your cDNA even if you use the SMART kit? What is the average length of products you normally get?

[SeqMonster]

Useful, that would explain why we are using 2X or more cpb than what we use normally with the old kit. However, this doesn't mean that it's better because we are seeing higher mixed+dots% compare to the old method even though the enrichment seems to be perfect.

[LMcSeq]

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

Phillip-they're all the same to me. People who don't have answers to questions that they should know.

The capture bead is the white bead.

[pmiguel]

That's what the tech support suggested. I must have added too much library into the emPCR (I don't think I did), and not denaturing them (I did). First of all, I was preparing 2 cups from the same denatured DNA and added the same amount into each cup and only one cup turned out that way. Then when I re-did it using a new cup, it still gave me the same problem.

I wonder why the Lib-L emPCR manuals call for a denaturation step but the Lib-A (for amplicons) does not. In both cases dsDNA is being used.

I would prefer to add dsDNA to the emPCR aqueous phase and let the molecules denature inside the microreactor.

--
Phillip

[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.

Hey, if you calculate your number of molecules of dsDNA and then denature your dsDNA prior to library capture in emPCR (as called for in the Lib-L emPCR manual) then you are really added 2x as many library molecules/bead.

Sure, only one of the ssDNA created from the dsDNA library will actually bind to the oligo on the surface of the capture bead. But the ones that do not bind are not lost -- they will end up in microreactors also. There they will have their 2nd strand recreated and that 2nd strand will then bind to the capture bead.

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

--
Phillip

[pmiguel]

Useful, that would explain why we are using 2X or more cpb than what we use normally with the old kit. However, this doesn't mean that it's better because we are seeing higher mixed+dots% compare to the old method even though the enrichment seems to be perfect.

Yeah, but 90 oC for 2 minutes might denature most of the dsDNA, but how much of it reanneals prior to "capture" by the capture beads? If you have any repetitive fractions in your library, you could get that reannealing (as in high C0t DNA...) to a non-identical paralog prior to the microreactors forming. The result: mixed bead -- even though your cpb was low.

--
Phillip

[pmiguel]

We are normally dealing with very low amount of RNA too. I looked at the SMARTERpico kit, it looks like what I need. Thank you for your information. :-)

Do you still "fragmentize" your cDNA even if you use the SMART kit? What is the average length of products you normally get?

Yes but there are other considerations you have to deal with if you are using the SMART kit. You need to break up your oligo-dT tails and get them oriented away from the sequencing side of the library construct. Peruse the literature for details.

And, yes, the fragmentation is problematic via nebulization of cDNA libraries because a lot of the cDNA will be in the size range were nebulization does not affect it much. But that can be mitigated using those tricky methods developed (as far as I know) in the Metz and Colbourne labs.

--
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.

[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]

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.