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I'm reading over the ENCODE Nature papers, and one of the papers referred to is "Global mapping of protein-DNA interactions in vivo by digital" by Hesselberth et al[1].

Genomic footprinting is a massively parallel variant of DNAse I hypersensitivity screening, which I feel I have a good handle on, but I don't understand this paragraph from the beginning of the results (the troublesome part is in bold):

To visualize regulatory protein occupancy across the genome of Saccharomyces cerevisiae, we coupled DNase I digestion of yeast nuclei with massively parallel DNA sequencing to create a dense whole-genome map of DNA template accessibility at nucleotide-level. We analyzed a single well-studied environmental condition, yeast a cells treated with the pheromone α-factor, which synchronizes cells in the G1 phase of the cell cycle. We isolated yeast nuclei and treated them with a DNase I concentration sufficient to release short (<300 bp) DNA fragments while maintaining the bulk of the sample in high molecular weight species (Supplementary Fig.1). These small fragments derive from two DNase I “hits” in close proximity, and therefore their isolation minimizes contamination by single fragment ends derived from random shearing. Because each end of the released DNase I ‘double-hit’ fragments represents an in vivo DNase I cleavage site, the sequence and hence genomic location of these sites can be readily determined by sequencing (Supplementary methods).

There's a reference to another paper by Sabo et al.[2] about genome-scale DNase assays using microarrays in that paragraph that I'm reading, but if someone understands the biology well, I'd really appreciate an answer.

  1. Hesselberth JR, Chen X, Zhang Z, Sabo PJ, Sandstrom R, Reynolds AP, Thurman RE, Neph S, Kuehn MS, Noble WS, Fields S, Stamatoyannopoulos JA. 2009. Global mapping of protein-DNA interactions in vivo by digital genomic footprinting. Nature Methods 6(4): 283–289, doi:10.1038/nmeth.1313.
  2. Sabo PJ, Kuehn MS, Thurman R, Johnson BE, Johnson EM, Cao H, Yu M, Rosenzweig E, Goldy J, Haydock A, Weaver M, Shafer A, Lee K, Neri F, Humbert R, Singer MA, Richmond TA, Dorschner MO, McArthur M, Hawrylycz M, Green RD, Navas PA, Noble WS, Stamatoyannopoulos JA. 2006. Genome-scale mapping of DNase I sensitivity in vivo using tiling DNA microarrays. Nature Methods 3: 511-518, doi:10.1038/nmeth890.
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Is it the emboldened section that is causing a problem? Or the previous sentence about yeast? (I can help you with the latter.) –  Alan Boyd Sep 11 '12 at 18:54
    
The section in bold is what's confusing me. The rest is provided as context. –  Michael K Sep 11 '12 at 19:04
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up vote 8 down vote accepted

After reading the paper cited I think the logic goes like this: DNAse I will create free ends at accessible sites in the genome. However shearing during subsequent DNA isolation is also a source of free ends, and these represent noise in the analysis. You have to put in a lot of energy to shear DNA to very small fragments, so I infer that mild shearing during DNA isolation is very unlikely to create a new end that is near to an existing DNase I-generated end. Therefore, under the conditions used, any small fragments are much more likely to have been generated by two closely-spaced DNase I hits. By limiting the analysis to these fragments the noise from shearing is minimised.

Now that I've written this answer it doesn't seem like much more than a rewrite of the emboldened sentence, but I hope it is helpful anyway.

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This is very helpful. I didn't understand what "random shearing" was, or how it would add error to the results. If I'm understanding correctly, random shearing during processing of DNA is unlikely to generate fragments that are relatively short (300 bp), which means that the 300 bp fragments are, with high probability, coming from short segments of DNA that have been clipped by DNase twice. –  Michael K Sep 12 '12 at 14:11
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Yes that's right. Large DNA molecules are easily broken by physical handling. The low-tech way of doing it is to pipette up and down but if you Google "DNA shearing" you'll find lots of companies selling equipment that use shearing to produce random fragments in a desired size range. –  Alan Boyd Sep 12 '12 at 14:32
    
DNA shearing can also be accomplished by vortexing the sample. This is not something you normally want to do, however, such as when genotyping animals. –  leonardo Oct 27 '12 at 16:49
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