Ampure XP beads Dual Bead-based Size Selection ratio and PCR purification

[Leo Lee]

I want to do amplicon sequencing with HiSeq. My initiate input is PCR product with around 85bp. I decided to do library preparation by using NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (Cat# E7370).

So after ligation of the adapter, my target total library size (insert + adaptor) is around 200bp. What is the recommended conditions for Dual Bead-based Size Selection using AMPure XP beads?

I did some search online and found that with insert size of 85bp (~200bp including adaptor) , use 0.9X in 1st bead selection then use 0.15X for 2nd Bead selection.

After the size selection and PCR amplication cycle, what is the recommended conditions for the PCR purification to retain the majority ~200bp average peak? should I use 0.9X to remove the dimer?

Please comment and welcome any opinion. Thank you all of your advice in advance.

[MU Core]

Since the input material is a PCR product of homogeneous size, a dual size selection seems unnecessary. The concern is the removal of adapter and adapter dimers which can be done by a single bead selection.

[Leo Lee]

Thank you MU's suggestion. Yes, you are right dual size selection is unnecesary, so finally I have done 1X beads purification before and after the PCR amplification using standard protocol NEBNext® Ultra™ DNA Library Prep Kit for Illumina with initiate input of 250ng of the PCR product.

However, I get 3 peaks (~150bp, 204bp and 227bp) after ligation of the adaptor and did the 1X bead purification (refer to Fig_A). I thought after the ligation of the adaptor with initiate PCR product (~80bp), the total library size (insert + adaptor) would only show a single peak of ~200bp. Does anyone have idea what are those two extra peaks from?

Further, after the PCR amplification (8 cycle) and another 1X bead purification, the bioanalyzer profile shows 4 peaks (216bp, ~300bp, 361bp and one peak >1500bp). I think slightly length increase of 204bp peak after PCR is due to the ligation of the index sequence). But how about the other peaks (~300bp, 361 bp and >1500bp)? Any clue of where are they come from?

Anyone can suggest how I can only select the 216bp peak which would be my target library size and remove the other peaks? Is it possible to use Dual Bead-based Size Selection with what ratio? Or only can cut gel to select the correct size?

Hope that someone can help to solve my problem... Thanks!

[cmbetts]

Is this just post-ligation, and not post-PCR? Last I checked, NEB's library prep ligates partial Illumina adapters as a hairpin, and you don't get a sequencing-competent library until after UDG digestion to open the hairpin and PCR to add the remaining sequences. You might be seeing various intermediates, such as the hairpin and single-sides ligation, that run funny in the bioanalyzer. I wouldn't worry until post-PCR.
Also, since these are such small amplicons, is there a reason you could have just added the Illumina sequences to your primers and avoid library prep altogether?

[Leo Lee]

cmbetts, thanks for your reply.

Fig_A is actually taken from the supernatant of original dual beads size selection (but now I change it to 1X bead purification to remove <100bp primer dimer). So it is after the USER enzyme UDG digestion...

Fig_B is already the final library after the PCR amplification and final PCR clean up steps. However, I wonder why there are so many peaks... specially those peaks with ~300bp, 361bp and one peak >1500bp.

My experiment target is amplicon sequencing of initiate PCR 80bp amplicon product. So my plan is to follow NEBNext® Ultra™ DNA Library Prep Kit for Illumina and add the adaptor with index. Then spike in a little in some of my Hi-Seq lanes to get just a few hundred MB throughput. Do you have any other library prep suggestions to archive my goal?