Pore-C

Pore-C is an emerging

Many methods to characterize the 3D genome are variations on 3C technology.^[5] Like other 3C-based technologies,^[5] Pore-C seeks to characterize the architecture of the 3D genome by determining which genomic loci are in close spatial proximity (within ~200 nm).^[2] Similar to previous 3C-based methods,^[5] Pore-C relies on crosslinking, restriction enzyme digestion, proximity ligation, reverse cross-linking, and protein degradation steps.^[2] However, Pore-C is distinct from many previous methods in its subsequent utilization of ONT long-read sequencing, which facilitates the resolution of multi-way chromosome contacts and simultaneous detection of DNA methylation^[2][3]

Cross-linking DNA to protein

First, in order to preserve the 3D structure of the genome from degradation in subsequent steps, DNA is cross-linked to DNA-associated proteins, such as

Cross-linking generates loops of DNA, with each loop arising from a separate locus.[5] To capture long-range interactions between distant loci, potentially from different chromosomes, these loops are first cut and then re-joined back together based on proximity. Although fragments deriving from the same loop may reanneal back together, sometimes fragments from separate loops will ligate together, thus creating chimeric sequences.^[5] The cutting and rejoining of DNA is achieved by the in situ restriction enzyme digestion and proximity ligation steps respectively. Specifically, a restriction endonuclease cuts the DNA to create free ends, whereas T4 ligase is used to join fragments together.^[5] Ultimately, these steps result in genomic loci close together in physical space being linked together on contiguous DNA segments referred to as concatemers.^[2]

Cross-linking reversal, protein degradation, and DNA purification

Next, in order to isolate DNA for sequencing, proteins bound to the DNA have to be detached and degraded.^[5] First, Proteinase K, sodium dodecyl sulfate (SDS; a detergent), Tween-20, and nuclease-free water are added.^[2] Subsequently, the reaction is heated to 56 °C in a thermocycler for optimal reaction kinetics. Proteinase K degrades proteins, and SDS acts a denaturing agent that disrupts protein structure.^[9][10] This reaction results in the breakage of covalent bonds between DNA and protein and removes potential protein contamination.^[5] DNA is then isolated and purified, typically using phenol-chloroform extraction followed by ethanol precipitation.^[2]

Size selection, library preparation, and long-read sequencing

Pore-C concatemers undergo size selection prior to library preparation and ONT long-read sequencing.^[2] Via size selection, Pore-C is able to detect high-order interactions, which are defined as concatemers containing greater than two DNA fragments. Specifically, Pore-C size selection enriches for DNA sequences greater than 1.5 kb, thereby filtering out shorter concatemers unlikely to contain greater than two fragments.^[2] Many size selection methods have been developed for ONT long-read sequencing.^[11] For example, Solid Phase Reversible Immobilisation (SPRI) size selection has been used in the Pore-C literature.^[2][3] Following size selection, library preparation for ONT long-read sequencing is performed, usually with a ligation sequencing kit provided by ONT. Key steps include DNA repair and adaptor ligation.^[2][3] Subsequently, DNA is loaded onto flow cells for sequencing, where each concatemer is fed through a pore, aided by a motor protein.^[2][11] Nitrogenous DNA bases are read out by their characteristic disruption of an electric current^[11]

Bioinformatic analysis

Overall, bioinformatic approaches applied to Pore-C data allow for the inference of pairwise and multi-way contacts between loci.^[2] Since concatemers in Pore-C contain DNA sequences that come from different regions of the genome, aligning sequencing reads to a reference genome is challenging. One solution to this problem involves a bioinformatic pipeline using a greedy piece-wise algorithm.^[2] Further analysis of Pore-C results depends on the study and what other data types are available.^[3]

Applications

Pore-C is a relatively new method, so its applications have not yet been fully appreciated.^[2] A strength of Pore-C over previous methods is its ability to detect interactions between more than two genomic loci. Such high-order interactions enable the study of cellular processes, such as gene expression regulation at a more system-level scale.^[2][3] With statistical methods, Pore-C data can be used to identify cooperative interactions, wherein high-order interactions are observed at a frequency greater than the sum of their expected pairwise contacts.^[2] In addition, using ONT long reads, Pore-C can detect DNA methylation, thereby providing an additional layer of epigenetic information to analyze.^[2] In the future, Pore-C may be applied to study how the 3D genome changes during developmental processes, such as cellular differentiation.^[3] Additionally, Pore-C may be applied to the study of cancer, where the 3D genome is often structurally rearranged, which can result in aberrant gene transcription via processes such as enhancer hijacking.^[2]

Use

Advantages

• Pore-C has a higher efficiency and fidelity compared to previous methods.^[1]
• The absence of an amplification step that is required in other chromatin conformation capture methods simplifies the experimental protocol. Additionally, bisulfite conversion (as is required in Methyl-HiC) is not required when detecting DNA methylation.
• Pore-C can detect more higher order interactions than Hi-C and other previous methods,^[1] because concatemers can span multiple chromosomes and detect distal interactions. Previously, these interactions have been understudied because of the limitations of previous methods.
• Because ONT long-read sequencing can detect DNA methylation, Pore-C can be extended to characterize epigenetic interactions and their relationship with chromatin topology.

Limitations

• Oxford Nanopore sequencing technology is costly,^[12] and therefore Pore-C is more expensive per run when compared to other chromatin conformation capture techniques.
• Pore-C throughput is relatively low when compared to other techniques, particularly due to DNA-bound proteins clogging sequencing pores. This can cause it to be less efficient, especially when combined with its relatively high cost per run. However, recent work
  proteases
  to reduce clogging.
• Because it relies on ONT long-read sequencing technology, Pore-C is subject to the same limitations. For example, longer reads can be less accurate when compared to shorter reads.^{[citation needed]}
• Because Pore-C is a newer technique, it is relatively unproven and has not been tested to the same extent as other chromatin conformation capture techniques.^{[citation needed]}

References

This article needs additional or more specific categories. Please help out by adding categories to it so that it can be listed with similar articles. (June 2023)