PreAmp product larger than sheared gDNA template

[SunPenguin]

I am running through some pretty standard library prep for gDNA. Basically, I covaris sheared some zebrafish gDNA, check on BioA (attached). I find that I get a pretty wide distribution of sizes, kinda around 500-600 bp (which is what I'm aiming for). I then used the NEBNext kit to do end repair/dA tailing/Ligation (with custom made y-adaptors using the IDT recommended protocol). I then take all of that, do a SPRI solution at 0.6X to get rid of things that are below 300bp, then I used KAPA hifi to amplify.

Surprisingly, most of the products I get in the amplified PCR product are around 1200-1400bp (also in the attached file), which is much larger than I expected (especially compared to the profile of the sheared DNA template). I don't really know what could've happened.

The products are definitely over amplified (I wanted to make sure I see some product), but these are diverse DNA templates, so I can't really imagine over amplification would give a lot of concatemers (and the peaks don't look like concatemers anyway).

Other thing I could think of is that the sheared DNA was stored at 4 degrees for a couple of days, which I realized isn't best practice, but that should shift the product smaller if anything, not larger.

Another thing may be that the 0.6X SPRI cutoff that i usually use for PCR products may give a different cut off in the presence of the ligation/end repair buffer, but I think this is unlikely. I am running the SPRI cleaned template on bioA too.

I'm a bit confused.

[nucacidhunter]

I think your shearing has not work that well and you have got wide fragment size distribution with double peak. PCR profile looks normal for the left cut that has been done with 0.6x beads. Some ligation buffers contain PEG that can affect the size cut. If you run bead cleaned input you will notice that peak will shift to a larger size which is normal. There is always possibility of fragments ligating to each other during ligation but I do not think this the case for this sample.

[SunPenguin]

Thank you for your reply. This is my first time doing gDNA stuff (I worked with RNA exclusively before this). What could cause the shearing to not work? I eluted using the DNAeasy kit into TE, and followed the manufacturer's instruction. I thought covaris would be pretty immune to sample concentration, and things like that?

[nucacidhunter]

Covaris gives consistent shearing result once a protocol has been established. You can use validated protocols from TruSeq or other suppliers manual if the size fits your application. For other sizes follow Covaris manual instructions and check few shears on BA followed by optimisation if required. Following need to be considered:

1- Input volume
2- Elimination of air pockets
3- Water temperature
4- Water level
5- Degasing
6- Input amount

[gabrieltw]

If I remember correctly, different AFA tubes have their DNA input limit. MicroTube is about 10 ug max and miniTube is about 20 ug max.