DNA fragmentation methods

[kshazand]

Hi all,
Different physical damages are made to DNA during sonication. Yet, we all use Covaris and other similar sonicators for library prep.
I would like to know how many of my colleagues have observed or even looked at the DNA state and what you think of potential alternatives to shearing.
Kamran

[rnaeye]

NEB has an enzyme called Fragmentase for this purpose. Maybe one of many alternatives.

https://www.neb.com/products/m0348-nebnext-dsdna-fragmentase

[kshazand]

Yes thank you, I know Fragmentase, but since it is mainly based on restriction enzymes, the output has some bias. What other "many alternatives" are you refering to?

[luc]

The transposase protocols (nextera et al.) are the other alternative, of course also with some biases.
In general I guess/assume sonication will not introduce any significant biases ( I do not know if anybody has looked at it); at least not to a degree that they will not be trumped by biases of the required downstream enzymatic processes. If you can create your libraries PCR-free after sonication you should have very few worries.

Yes thank you, I know Fragmentase, but since it is mainly based on restriction enzymes, the output has some bias. What other "many alternatives" are you refering to?

[rnaeye]

kshazand,
Fragmentase is enzyme based but does not use restriction enzyme.

[kshazand]

The transposase protocols (nextera et al.) are the other alternative, of course also with some biases.
In general I guess/assume sonication will not introduce any significant biases ( I do not know if anybody has looked at it); at least not to a degree that they will not be trumped by biases of the required downstream enzymatic processes. If you can create your libraries PCR-free after sonication you should have very few worries.

luc,
Ppl looked into sonicated DNA and it does have all sorts of damages, hence my question.

[kshazand]

kshazand,
Fragmentase is enzyme based but does not use restriction enzyme.

What enzymes are they then?

[rnaeye]

I believe you need to ask that to NEB.

[rnaeye]

https://www.neb.com/~/media/Catalog/All-Products/FFC4AF0D3C4B4D65A23FEC34E5535015/Datacards%20or%20Manuals/M0348Datasheet-Lot0071201.pdf

[Genohub]

6 Methods to Fragment DNA or RNA for NGS

These are the 6 main methods used to fragment DNA or RNA for NGS: http://blog.genohub.com/fragmentatio...-library-prep/

I'd be interested to see if there have been any detailed studies comparing the bias introduced by each of these.

- Genohub

[luc]

luc,
Ppl looked into sonicated DNA and it does have all sorts of damages, hence my question.

Hi kshazand,

I would be interested in data related to this. Could you point me to them?

The Knierim et al paper touches mostly on outdated methods:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0028240

[Genohub]

Interesting paper examines transcriptome reassembly and determines it is sensitive to RNA fragmentation technique. Concludes that Zn mediated fragmentation provides a better transcript ID compared to cleavage by RNase III: http://www.sciencedirect.com/science...46202313000789

I wonder if this has been tested with Mg.

- Genohub

[Genohub]

Another interesting paper on Non-random DNA fragmentation in next-generation sequencing: http://www.nature.com/srep/2014/1403...srep04532.html

- Genohub

[luc]

Thanks a lot for the links Genohub!

I have the impression, the authors of this specific paper have completely forgotten about any base-specific biases of enzymes involved in end-repair, A-tailing, and ligation.

E.g. ligation bias:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0078575

Another interesting paper on Non-random DNA fragmentation in next-generation sequencing: http://www.nature.com/srep/2014/1403...srep04532.html

- Genohub

[Genohub]

I'm aware of the RNA ligase biases in small RNA / RNA-Seq. Have you seen any papers about T4 DNA Ligase bias in DNA-Seq applications. I don't think this has been looked at as carefully. If anyone has used 'in-line' adapter barcodes during ligation, they may be in a unique position to comment.

- Genohub