I am a statistician reading an article on Hi-C, and I am trying to better understand one of the steps in the DNA isolation and sequencing process.
Since I'm a statistician, please try to avoid too much jargon in your answers, and if you must use jargon, please define/explain it.
Specifically, I am confused about the isolation and shearing step. To explain myself, I will make reference to the following images, labeled 1 through 4.
Image 1)
Image 2)
(source: nature.com)
Image 3)
(source: babraham.ac.uk)
Image 4)
(source: nature.com)
I understand that the DNA is cut up with restriction enzymes, producing a cloud of DNA fragments, some crosslinked and other not. When these pieces are attached with biotin and ligated in a dilute mixture, it is highly likely that these pieces will ligate with themselves, resulting in either; 1) a ring from strands that were NOT crosslinked (as in the third drawing in Image 3 marked "self-ligation"), or 2) figure-eights from strands that ARE crosslinked (as in the fourth drawing in Image 1).
Now, the following is what I find confusing:
The pull-down step draws down the rings containing biotin, but Image 1 and Image 4 make it seem as though only the crosslinked bits are pulled down, and not the solo rings. Don't both have biotin? And thus would both be drawn into the pulled-down sample? Or does the pull-down process rely on a molecule having 2 biotins? If not, can one discern whether a pulled-down piece of DNA is the result of a cross-linked or solo piece of DNA?
Why aren't all of the loose ends of the DNA ligated in Image 2? Incomplete digestion? Does this affect the biotin linkage?
My next question. Assuming that the pull-down process only selects the figure-eights that are crosslinked, then purified (i.e. the cross-linking protein or formaldehyde is removed) forming a ring that is composed of two strands of ligated DNA (as in the upper-right corner of Image 4). The process then records a genome-wide contact matrix $m_{ij}$, the number of "ligation products" between locus $i$ and locus $j$. What is a "ligation product"?
Sorry, I know the above is a lot, but I want to understand the nature of the data before I analyze it. Thank you for your help.