PLAC-Seq
Proximity ligation-assisted chromatin immunoprecipitation sequencing (PLAC-seq) is a chromatin conformation capture(3C)-based technique to detect and quantify genomic chromatin structure from a protein-centric approach.[1] PLAC-seq combines in situ Hi-C and chromatin immunoprecipitation (ChIP), which allows for the identification of long-range chromatin interactions at a high resolution with low sequencing costs.[1] Mapping long-range 3-dimensional(3D) chromatin interactions is important in identifying transcription enhancers and non-coding variants that can be linked to human diseases.[2]
Different 3C-based techniques have been used to study the higher-order 3D chromatin structure, and it has been combined with high-throughput sequencing to determine the chromatin structure on a genome-wide level.[3] Hi-C is one of the most widely used 3C-based techniques because it allows for high-resolution (kilobase-scale) genome-topology identification. However, it requires billions of sequencing reads which has limited its application.[2] Another commonly used 3C-based technique is chromatin interaction analysis by paired-end tag sequencing (ChiA-PET).[2] ChiA-PET can identify long-range interactions of transcription promoters and enhancers at a high resolution but requires millions of cells.[2]
PLAC-seq alleviates these issues by using in situ Hi-C, which creates long-range DNA contacts in situ in the nucleus before lysis.[3] Unlike ChiA-PET which performs ChIP and proximity ligation after chromatin shearing, performing proximity ligation in the nuclei first prevents large disruptions of protein/DNA complexes.[2] This decreases false-positive interactions and improves DNA contact capture efficiency, meaning that PLAC-seq is more accurate and requires fewer cells.[1]
History
PLAC-seq was developed in 2016[2] and an almost identical technique called HiChIP was also developed in the same year.[3] Both methods combine in situ Hi-C and ChIP but have different library preparation methods.[1] While PLAC-seq uses biotin pull-down followed by end-repair, adapter ligation, and PCR, HiChIP usesTn5 tagmentation, biotin pull-down, and PCR.[1] However, both techniques can use the same quality control and data analysis techniques.[1]
Different computation software tools can be used to analyze the data from PLAC-seq, for example, Fit-Hi-C,[4] HiCCUPS,[5] Mango,[6] Hichipper,[7] MAPS,[8] and FitHiChIP.[9] Many of the earlier software tools were developed for other 3C-based technologies and were not optimized for PLAC-seq/HiChIP data. Fit-Hi-C and HiCCUPS, both developed in 2014, were mainly developed for Hi-C data, and utilize a matrix-balancing-based normalization approach.[4][5] Mango was developed in 2015, and is mainly used for ChIA-PET data, but has high false-positive rates in analyzing PLAC-seq/HiChIP data due to the different biases.[6][8] Hichipper was developed in 2018 to alleviate this issue and introduced a bias-correcting algorithm, but it still has difficulties identifying protein interactions between protein binding and non-protein binding regions on the chromosome.[7][8] MAPS and FitHiChIP were developed in 2019 as a PLAC-seq/HiChIP-specific analysis pipeline, and are generally thought to be more effective than the existing models to analyze PLAC-seq/HiChIp data.[8][9]
Procedure
The general workflow of PLAC-seq involves cell harvesting and
Applications
PLAC-seq was developed to map and analyze long-range chromatin interactions. These interactions have important implications when it comes to the
One challenge for
PLAC-seq has been utilized to study
Use
Advantages: Compared to ChIA-PET, PLAC-seq requires significantly less amount of starting biological material.[1] With shearing being one of the first steps in ChIA-PET, this leads to the disruption of protein and DNA complexes. PLAC-seq avoids this by having the crosslinking reaction precede the shearing process. Furthermore, PLAC-seq requires fewer sequencing reads than Hi-C.[1] While ChIA-PET requires 100 million starting cells, PLAC-seq only requires 5 million cells.[2] Even with 20-fold fewer cells, PLAC-seq was able to produce more reads (175 million) with a fewer PCR duplication rate (33%) than ChIA-PET (16 million, and 44% respectively).[2] PLAC-seq was also nearly 100 times more cost-effective than ChIA-PET.[1]
Disadvantages: While many of the 3C-based techniques have different biases from the protocols, PLAC-seq (and HiChIP) data have biases from immunoprecipitation efficiencies that need to be corrected for in the computational step.[15] Effective ways of reducing and/or removing the different biases in 3C-based technologies is still being studied.[15]