Covaris fragmentation of small genomes

[EGraf]

Hi everyone,

today I tried shearing of a bacterial genome (1,7 Mb). I checked DNA integrity previously and it looks fine with no degradation. I used our standard settings for 550 bp fragment size. Attached you can find the Bioanalyzer results running a DNA 1000 Chip.

The green line is normal human genomic DNA. The other two colors are the bacterial samples, respectively.

I expected to get a peak around the target size or to get no peak because of total fragmentation. But I get two peaks. I get only a small peak in the bacterial samples for the targeted size ans I see a peak around the lower marker indicating a total fragmentation.

Does anyone has some suggestions to optimize shearing of bacterial DNA around 1,7 Mb? Does anyone has suggestions about these results.

Thanks a lot
Elisabeth

[nucacidhunter]

I can only speculate that small fragments around lower marker could have already existed in your input and may not be result of shearing. In fact your sheared bacterial DNA peak does not look different from human DNA. Their concentration is much lower than the human DNA. If you dilute your human sample or concentrate your bacterial ones (not using speedy vac if they are not resuspended in water) and rerun the Chip, they would have similar peak. If you are going to prepare libraries from those shears they would be fine as the smaller fragments will be washed away during clean up steps (assuming you are not using one of those one tube kits). You can also clean your sheared DNA with 1.4x AMPure beads before library prep. Peak size of shears can be reduced by increasing time, keeping other setting same. There are other ways as well to fine tune peak size which you can find in Covaris documents. I regularly shear small fragment DNA such as 2, 4 and 10 kb and results are always consistent.

[EGraf]

Thanks for reply. I used 2 µg DNA of human and bacterial DNA, respectively. I excepted nearly the same concentration after shearing. Do I have an error in reasoning

I would like to use the Illumina PCR free sample prep Kit and won't loose complexity due to low DNA input at the beginning.

[nucacidhunter]

I would suggest to use dsDNA PicoGreen reagent (or Qubit) for quantifying input DNA. Your bacterial DNA seems much lower than human.