Not enough library after Enrichment PCR

[Adao]

Hello,
We tried the Illumina Truseq DNA preparation and followed by Illumina or Nimbelgen exon capture procedure. But we are having problems with out library preparation.
The input is 1 ug genomic DNA (quantified by Qubit 2.0). We exactly followed the illumina protocol, but the libraries we got after enrichment PCR is about 250-500ng total DNA (Bioanalyzer). This is not enough for later exon capture by either Illumina or Nimbelgen procedure.
Illumina told that they do not guarantee to get more than 1 ug library after the PCR, and as long as total amount is more than 500ng, Illumina exon capture should be fine. If I want to get 1 ug DNA, which is required by Nimbelgen protocol, I should do 2 PCR and pool them together.
Is it possible anyone here can help us out?
Thank you very much!

[ECO]

How many cycles of enrichment PCR are you doing? You should have a ton of library (>>1ug) after this. Is your DNA input source odd/damaged/contaminated/single stranded? Have you tracked your intermediate purifcation yields (maybe you're losing a lot in AMPure steps)? Have you quantitated a different way post library generation? (qPCR, nanodrop, picogreen?)

Just some thoughts...good luck.

[Adao]

I got confused actually.

Nimbelgen's protocol wants me to quantify library by Nanodrop. Illumina simply didn't say how to quantify the libaray, only mentions 500 ng total.

If I use Nano, I have 3.6-7.1 ug total DNA, The 260/280 is about 1.9. But bioanalyzer gave a result of only 0.3-0.5 ug total DNA. Which is true?

How do you quantify your final library after the library enrichment (or LM-PCR)?

Thanks!

[ECO]

For precapture libraries we use nanodrop. Haven't tried quantitating by Bioanalyzer, but picogreen gave very similar results to what you observed. This is likely due to bubble product formation giving lots of single stranded regions (measurable by nanodrop by not by picogreen). Bioanalyzer has even more caveats than picogreen due to the mobility impacts of the bubble products/concatemers.

On my list to implement is using a single-stranded dye (Oligreen...etc) for quantitating the library at this point.

[rnal]

we also have the same problem. and with the original protocol，it often cannot get the enough yield for hybridization. and the enrichment by qPCR is not fail.
some bady use 3ug gDNA as the start . and increase the Pre-PCR cycles to 10-12cycles.

[Adao]

This issue is due to the bubble products in the amplified library.
Bioanalyzer significantly underestimate the DNA amount about 10 times. I added two more cycles to the LM-PCR and got 1 ug DNA by analyzer. My capture looks OK now.
It is surprising though both Illumina and Nimblegen do not clarify this in their protocol.

[Rocketknight]

Interestingly, doing the capture with 1-2ug as quantified by Qubit/Picogreen doesn't seem to adversely affect the capture at all, even though that sounds like it's ten times as much DNA as you'd use if you were quantifying with the Nanodrop. It was done in http://www.nature.com/nbt/journal/v2.../nbt.1975.html and they got great capture efficiency with Nimblegen's kits. (Though they did mention having a lot of trouble getting enough DNA out of their pre-capture PCR...)

[Akira]

Hi. New to NGS here.

Can I ask, what is the minimum concentration or minimum amount for PCR-enriched library? What I was told is at least 10nM for subsequent works. Not sure the volume needed though.

I'm afraid I cannot get as high as 10nM. (Pretty much sure I cannot get that due to some protocols changed in the sample prep workflow, and gel extraction after PCR enrichment left me with too little samples. I have not read Qubit yet but Nanodrop gives me no reading at all )

Need to know if 10nM is the minimum. Thanks.