Chicago® in vitro Proximity Ligation Technology Overview
Chicago® improves order, orientation and contiguity of fragmented de novo assemblies and corrects errors in highly contiguous assemblies to produce a more accurate genome assembly that is optimal for scaffolding up to chromosome level with Hi-C. An overview of the library generation process is diagrammed on the right and described below.
Unlike Dovetail™ Hi-C which starts with endogenous chromatin, the Chicago® assay begins with high molecular weight (50+ kbp) input DNA, depicted in Panel A as a bold black line. This provides greater resolution than Hi-C without the biological background to better improve order and orientation of contigs. The first step is to reconstitute chromatin from the input DNA using in vitro chromatin assembly. This step utilizes purified nuclear remodeling and chaperone proteins and histones to convert the input naked DNA into chromatin (DNA which is wound around and tightly associated with histones). This step is depicted in Panel B where the now-complexed histones are indicated by blue circles upon the DNA. Re-association of the DNA with proteins in a uniform manner lays the foundation for later crosslinking steps. Chromatin reconstitution works with any source of DNA, even those with no native chromatin (e.g., bacteria).
The next stage is crosslinking (fixation) of the reconstituted DNA, as depicted in Panel C. Addition of a fixative agent (e.g., formaldehyde) produces crosslinks (covalent connections, depicted as red lines between histones) among the histones associated with the DNA. Crosslinking condenses the previously long, linear strand of chromatin into a globular chromatin aggregate and stabilizes it. The crosslinking serves two critical functions in the library preparation process. The first is bringing linearly distant portions of the molecule (e.g., the ends of the DNA) into close spatial proximity. The second is stabilization. Subsequent steps in the process will cut the DNA and the crosslinks retain the association of the now many DNA fragments originating from a single large initial fragment.
As depicted in Panel D, the crosslinked chromatin is then digested with a restriction endonuclease to generate sticky-ended fragments. This cutting step generates many new ends to participate in the subsequent ligation step. Critically, all of the fragments generated at this step originate from the same original large fragment and their association is maintained by the crosslinks.
To prepare for ligation, the newly-generated sticky ends are made blunt with a polymerase fill-in that includes a biotinylated nucleotide (Panel E, biotins are green circles). Biotins mark the ends for a later enrichment step.
Next, a DNA ligase is added to perform blunt-end ligation of the many ends within a given chromatin aggregate (Panel F, ligations indicated with red asterisks). Many of these ligation events will result in the joining of two pieces of DNA that were not near to one another in the original fragment, but which have been brought into close spatial proximity by previous steps. For example, segments 1 and 2 were very distant in the original fragment (Panel B) but have become linked in F after condensation and ligation. It is the linking of these previously-distant fragments that captures the long-range information
contained in the original molecule.
After ligation, chromatin is removed and DNA is purified and processed to remove biotin that is not internal to the resulting fragments, i.e. those which have not participated in a ligation event (Panel G). Finally, the resulting library is enriched for biotin-containing fragments through a streptavidin bead pulldown and a sequencing library is prepared.
The resulting libraries contain many “chimeric” fragments, i.e., single fragments composed of at least two originally distant fragments. The libraries can then be sequenced with paired-end sequencing and the two or more original fragments recovered and mapped. The genomic separation of the original fragments follows a well-modeled insert distribution, which provides the information used for scaffolding, analysis of structural variation, and phasing.