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-   -   TruSeq PCR-free transition (http://seqanswers.com/forums/showthread.php?t=35458)

avo 11-15-2013 01:57 AM

TruSeq PCR-free transition
 
Hi together,

my lab is planning to switch to the TruSeq PCR free library prep. We would like to perform size selection on a bluepippin. Reading various threads here about the low yields of the protocol i am wondering if the replacement of the bead based size selection by a pippin might result in to low yields to meet the cluster densities possible with the new V3 reagent kits?

Does anyone have experience with this matter yet?

Thanks

FWOS 11-18-2013 11:56 AM

Hi Avo,

I have been using the truseq PCR-free preps for a year now and have never run into issues with yield at the end. Assuming you are not losing a ton of material in the shear, the double sided spri cleanup should be advantageous when compared to loss associated with selection on the BluePippin (which also requires a post selection cleanup).

I have even seen low, but good results from a 100ng input prep sheared at 350bp. If you start with 1ug and run a 350bp shear, you should end up with libraries that can be easily seen on an agilent HS DNA chip. It's not the yield you would expect from a PCR generated library, but there is plenty for sequencing and the lack of PCR artifacts make it well worth the switch for most applications.

We typically get about 100ng out of the preps. That is a ton of material for sequencing, in my opinion, even though it is a ~90% loss overall.

lorendarith 03-05-2014 03:45 AM

I agree with FWOS.

I even used a normal gel for size selection and haven't had problems so far.


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