size of overhang after sonication?

[comingme]

Does anyone have an idea what's the size of overhang after sonication of dna?
I have tried to search literature but unfortunately I did not find any. For a fragment around 300 bp, is the overhang around 1 or 2 bp, around 5 bps, around 10 bps or more? Could someone share the knowledge? Thanks very much.

[pmiguel]

I have never seen a study that addresses this. If you find one, please post it here.

The proof of principle paper about the proto-Hydroshear found that some large percentage of the ends could be ligated without treatment--suggesting most broke off blunt.

I did go into an ancient paper that addressed where sonication tended to break the phospho-ribose backbone. See:

http://seqanswers.com/forums/showthread.php?p=15844

if you are interested.

--
Phillip

[han526]

Sonication effect

Based on my own experience, nicked DNA is very easy to be cheared by sonication suggesting that the 1st break tend to help breaking the whole DNA chain.

Detailed study suggesting that without DNA repair, the ligation is very inefficient, suggesting that either the phosphate is not on the right end or there is overhang. Furthermore, depending on the repair system, the ligation efficiency is drastically different. A good repair cocktail can drop 70 percent ligation product, whereas a not so good ligation enzyme mix can only produce 20 to 30% ligation product. These all suggest that the DNA ends are mostly not blunt.

I have never seen a study that addresses this. If you find one, please post it here.

The proof of principle paper about the proto-Hydroshear found that some large percentage of the ends could be ligated without treatment--suggesting most broke off blunt.

I did go into an ancient paper that addressed where sonication tended to break the phospho-ribose backbone. See:

http://seqanswers.com/forums/showthread.php?p=15844

if you are interested.

--
Phillip

[pmiguel]

Maybe too antiquated to directly address the OP, but the following:

http://www.sciencedirect.com/science/article/pii/0005278772905059

http://dx.doi.org/10.1016/0005-2787(72)90505-9

Pyeritz, R. E., Schlegel, R. A. and Thomas, C. A., Jr (1972) Biochim.
Biophys. Acta 272, 504–509.

suggests that sonication did not produce long single stranded ends. However it isn't clear to me that the sensitivity of their assay was very high.
See also:

http://www.sciencedirect.com/science/article/pii/0003269783900726

http://dx.doi.org/10.1016/0003-2697(83)90072-6

The "proto-hydroshear" paper is here:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC146200/pdf/243879.pdf

Not precise answers but should provide you some leads.

--
Phillip

[frc1230]

We tested the ability of sonicated lambda-phage DNA (from BRL, and sonicated with a Covaris instrument, or with a conventional tip sonicator) to self-ligate into concatemers, with or without prior end-repair using the NEB kit. The result was that without end-repair, there was no detectable ligation. With end-repair, there was very effective ligation into concatamers. The peak of the distribution assayed by ethidium-bromide staining went from ~250 bp without ligation to > 1 kb with ligation, suggesting that a large majority of the fragments were ligatable at both ends, hence blunted effectively. The ability of sonicated DNA to be concatamerized by ligation after end-repair was similar to or slightly less than the ability of restriction-enzyme-digested DNA of a similar size range, also after end-repair.

So, the ends/overhangs may exist after sonication appear largely unligatable, but the conventional end-repair reaction repairs at least a large majority of these ends.

[pmiguel]

Interesting.
I would like to see more details of experiments like this one. I wonder if there are any papers in the literature? Nothing pertinent that I can see via a cursory Google search.

Also would be interesting to see what % of the ligatability of the fragments can be restored with T4 polymerase alone and T4 PNK alone.

--
Phillip

[frc1230]

I'm aiming to put together a complete set of assays to test step-by-step how well the Illumina library prep works for any given sample. I have found the blind addition of reagents in a long sequence to be a very frustrating and 'unilluminating' process. Fine if it works, of course, if you don't mind being a pipetting robot; if it doesn't work you know absolutely nothing about why. The diagnostics are actually quite simple provided you have enough material to work with and visualize easily on a gel - fine for genomic resequencing, if not for ChIP-Seq and such things. It's not important to me to literally publish this material, but it would likely be useful to post it on this website.

[HESmith]

The new TruSeq kits contain spike-in controls for each step of the protocol. Sequence Analysis Viewer provides a graphic display indicating which (if any) step failed, although it's qualitative rather than quantitative and may not be sufficient for your application.

[frc1230]

Yes, I know those are included. But as it's set up, as I understand it, you still don't know if something went wrong until the whole procedure is over (i.e. you're looking at the reads). At that point, after the time and expense of running the sequencer, you have a reasonable chance of knowing what step went wrong, I agree (unless the problem is in 'cis' to the experimental DNA). With a complex procedure of this sort, I like to see how it's working at intermediate steps, by direct assay if possible.

[pmiguel]

We generally save an aliquot of sample from each step. However, some of the steps are tightly integrated to make the protocol fast. So, for example, attempts to visualize the polyA are not good--probably because of the divalent cations in the fragmentation buffer used to elute from the oligo dT beads.

But we spot check a few, and if concerned, may do extensive QC. Also we always check the pre-enrichment library on a pico RNA chip or high sensitivity DNA chip.

--
Phillip

[frc1230]

We're doing microbial genome re-sequencing, so we have the luxury of large amounts of material. The procedure turns out to be extremely sensitive to unknown contaminants in the initial DNA preps. So some samples work well and others work absolutely zero. This is why we're looking at ways to test efficacy of each step directly on the material as we process it - for example, by the self-ligation assay to assess end-repair.