KAPA, dual-SPRI size selection

[yxl]

The KAPA Hyper kit has a protocol for using AMPure beads for size selection (dual-SPRI), but it seems to me that the beadNA ratio is off (or perhaps I am not understanding the concept correctly). The protocol is below (also on p10-11 of the attached protocol pdf); to summarize, size selection of ~250-450bp fragments is performed with these steps:

Part 1:
- start with adapter-ligated library in 50uL volume
- add 30uL of AMPure XP beads
- place on magnet
- keep the 75uL supernatant and dispose beads (beads contain >450bp fragments)

Part 2:
- the to the 75uL supernatant, add 10uL of AMPure XP beads
- place on magnet
- discard supernatant (contains fragments <250bp)
- wash with 80% EtOH twice
- dry beads
- elute DNA

Part 2, as I understand, is the "left side" selection. The addition of 10uL of AMPure beads to 75uL supernatant is a 10/75 = 0.13x bead ratio; would this not lead to a almost-complete loss of material? It seems to me that a more appropriate AMPure bead volume would be 60uL (60/75=0.8) for a 0.8x bead ratio. Perhaps I'm missing something.

[nucacidhunter]

Part 2, as I understand, is the "left side" selection. The addition of 10uL of AMPure beads to 75uL supernatant is a 10/75 = 0.13x bead ratio; would this not lead to a almost-complete loss of material? It seems to me that a more appropriate AMPure bead volume would be 60uL (60/75=0.8) for a 0.8x bead ratio. Perhaps I'm missing something.

Supernatant from Part 1 contains 0.6x bead solution, so by addition of bead in Part 2 it will be 0.8x.

[AdrianaGeldart]

Dear yxl,

I am a member of the Kapa Scientific Support & Applications Team and would be happy to address your question.

To perform the first size cut (to exclude fragments corresponding to library inserts larger than ~450 bp), you would add 30 uL of AMPure XP Reagent to 50 uL of DNA. This is the 0.6X cut as you correctly described.

The second step would be to transfer the supernatant containing DNA fragments smaller than ~450 bp to a new plate and discard the plate with the beads to which DNA fragments larger than ~450 bp are bound. The second size cut is then performed (to retain fragments corresponding to library inserts larger than ~250 bp) by adding 0.2 volumes of Agencourt AMPure XP reagent to the first cut supernatant. This would be adding 10 uL of reagent to 75 uL of DNA. This 0.2 volumes corresponds to the volume of DNA prior to the first cut (i.e. 50 uL). We have found 0.2 volumes to be optimal. If you decrease the difference between the two cuts to <0.2, the peak width decreases, but at the cost of a higher loss of DNA. If you increase the difference between the two cuts to >0.2, recovery is better, but the peak width tends to broaden.

I hope this helps to clarify the size selection. If you would like further explanation, please feel free to call the Support Line at 1-855-KAPA-BIO or email us at [email protected].

Best Regards,
Kapa Biosystems Support

[liaoyunshi]

Supernatant from Part 1 contains 0.6x bead solution, so by addition of bead in Part 2 it will be 0.8x.

Hi nucacidhunter,

So is that means the factor deciding the DNA size capture is the bead solution volume instead of the bead amount itself?

Thanks.

[nucacidhunter]

Yes. Bead solution to DNA solution volume ratio.

[finswimmer]

Hello,

So is that means the factor deciding the DNA size capture is the bead solution volume instead of the bead amount itself?

yes, because the PEG concentration in the solution is the critical reagence in the size selection process. I've found a nice blog post about it some time ago here: http://core-genomics.blogspot.de/201...eads-work.html

fin swimmer

[liaoyunshi]

Hello,



yes, because the PEG concentration in the solution is the critical reagence in the size selection process. I've found a nice blog post about it some time ago here: http://core-genomics.blogspot.de/201...eads-work.html

fin swimmer

Hi,

I also found it! Thanks.

Actually before reading this post, I just wondered why the second selection with less bead amount can capture small DNA. And I finally realize that it is the bead solution makes it capture different size DNA, and the bead solution is more and more concentrated after beads pouring in for the second selection.