Single cell epigenomics is the study of epigenomics (the complete set of epigenetic modifications on the genetic material of a cell) in individual cells by single cell sequencing. [2] [1] [3] Since 2013, methods have been created including whole-genome single-cell bisulfite sequencing to measure DNA methylation, whole-genome ChIP-sequencing to measure histone modifications, whole-genome ATAC-seq to measure chromatin accessibility and chromosome conformation capture.
Single cell DNA genome sequencing quantifies DNA methylation. This is similar to single cell genome sequencing, but with the addition of a bisulfite treatment before sequencing. Forms include whole genome bisulfite sequencing, [4] [5] and reduced representation bisulfite sequencing [6] [7]
ATAC-seq stands for Assay for Transposase-Accessible Chromatin with high throughput sequencing. [9] It is a technique used in molecular biology to identify accessible DNA regions, equivalent to DNase I hypersensitive sites. [9] Single cell ATAC-seq has been performed since 2015, using methods ranging from FACS sorting, microfluidic isolation of single cells, to combinatorial indexing. [8] In initial studies, the method was able to reliably separate cells based on their cell types, uncover sources of cell-to-cell variability, and show a link between chromatin organization and cell-to-cell variation. [8]
ChIP-sequencing, also known as ChIP-seq, is a method used to analyze protein interactions with DNA. [9] ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. [9] In epigenomics, this is often used to assess histone modifications (such as methylation). [9] ChIP-seq is also often used to determine transcription factor binding sites. [9]
Single-cell ChIP-seq is extremely challenging due to background noise caused by nonspecific antibody pull-down, [1] and only one study so far has performed it successfully. This study used a droplet-based microfluidics approach, and the low coverage required thousands of cells to be sequenced in order to assess cellular heterogeneity. [10] [1]
Chromosome conformation capture techniques (often abbreviated to 3C technologies or 3C-based methods [11] ) are a set of molecular biology methods used to analyze the spatial organization of chromatin in a cell. These methods quantify the number of interactions between genomic loci that are nearby in three dimensional space, even if the loci are separated by many kilobases [12] in the linear genome.
Currently, 3C methods start with a similar set of steps, performed on a sample of cells. [11] First, the cells are cross-linked, which introduces bonds between proteins, and between proteins and nucleic acids, [12] that effectively "freeze" interactions between genomic loci. [11] The genome is then cut digested into fragments through the use of restriction enzymes. Next, proximity based ligation is performed, creating long regions of hybrid DNA. [11] Lastly, the hybrid DNA is sequenced to determine genomic loci that are in close proximity to each other. [11]
Single-cell Hi-C is a modification of the original Hi-C protocol, which is an adaptation of the 3C method, that allows you to determine proximity of different regions of the genome in a single cell. [13] This method was made possible by performing the digestion and ligation steps in individual nuclei, [13] as opposed to the original Hi-C protocol, where ligation was performed after cell lysis in a pool containing crosslinked chromatin complexes. [14] In single cell Hi-C, after ligation, single cells are isolated and the remaining steps are performed in separate compartments, [13] [15] and hybrid DNA is tagged with a compartment specific barcode. High-throughput sequencing is then performed on the pool of the hybrid DNA from the single cells. Although the recovery rate of sequenced interactions (hybrid DNA) can be as low as 2.5% of potential interactions, [16] it has been possible to generate three dimensional maps of entire genomes using this method. [17] [18] Additionally, advances have been made in the analysis of Hi-C data, allowing for the enhancement of HiC datasets to generate even more accurate and detailed contact maps and 3D models. [15]
An epigenome consists of a record of the chemical changes to the DNA and histone proteins of an organism; these changes can be passed down to an organism's offspring via transgenerational stranded epigenetic inheritance. Changes to the epigenome can result in changes to the structure of chromatin and changes to the function of the genome.
Chromosome conformation capture techniques are a set of molecular biology methods used to analyze the spatial organization of chromatin in a cell. These methods quantify the number of interactions between genomic loci that are nearby in 3-D space, but may be separated by many nucleotides in the linear genome. Such interactions may result from biological functions, such as promoter-enhancer interactions, or from random polymer looping, where undirected physical motion of chromatin causes loci to collide. Interaction frequencies may be analyzed directly, or they may be converted to distances and used to reconstruct 3-D structures.
Epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome. The field is analogous to genomics and proteomics, which are the study of the genome and proteome of a cell. Epigenetic modifications are reversible modifications on a cell's DNA or histones that affect gene expression without altering the DNA sequence. Epigenomic maintenance is a continuous process and plays an important role in stability of eukaryotic genomes by taking part in crucial biological mechanisms like DNA repair. Plant flavones are said to be inhibiting epigenomic marks that cause cancers. Two of the most characterized epigenetic modifications are DNA methylation and histone modification. Epigenetic modifications play an important role in gene expression and regulation, and are involved in numerous cellular processes such as in differentiation/development and tumorigenesis. The study of epigenetics on a global level has been made possible only recently through the adaptation of genomic high-throughput assays.
Methylated DNA immunoprecipitation is a large-scale purification technique in molecular biology that is used to enrich for methylated DNA sequences. It consists of isolating methylated DNA fragments via an antibody raised against 5-methylcytosine (5mC). This technique was first described by Weber M. et al. in 2005 and has helped pave the way for viable methylome-level assessment efforts, as the purified fraction of methylated DNA can be input to high-throughput DNA detection methods such as high-resolution DNA microarrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). Nonetheless, understanding of the methylome remains rudimentary; its study is complicated by the fact that, like other epigenetic properties, patterns vary from cell-type to cell-type.
Chromatin Interaction Analysis by Paired-End Tag Sequencing is a technique that incorporates chromatin immunoprecipitation (ChIP)-based enrichment, chromatin proximity ligation, Paired-End Tags, and High-throughput sequencing to determine de novo long-range chromatin interactions genome-wide.
ATAC-seq is a technique used in molecular biology to assess genome-wide chromatin accessibility. In 2013, the technique was first described as an alternative advanced method for MNase-seq, FAIRE-Seq and DNase-Seq. ATAC-seq is a faster and more sensitive analysis of the epigenome than DNase-seq or MNase-seq.
Whole genome bisulfite sequencing is a next-generation sequencing technology used to determine the DNA methylation status of single cytosines by treating the DNA with sodium bisulfite before high-throughput DNA sequencing. The DNA methylation status at various genes can reveal information regarding gene regulation and transcriptional activities. This technique was developed in 2009 along with reduced representation bisulfite sequencing after bisulfite sequencing became the gold standard for DNA methylation analysis.
H3K9me3 is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the tri-methylation at the 9th lysine residue of the histone H3 protein and is often associated with heterochromatin.
Human epigenome is the complete set of structural modifications of chromatin and chemical modifications of histones and nucleotides. These modifications affect t according to cellular type and development status. Various studies show that epigenome depends on exogenous factors.
H3K79me2 is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the di-methylation at the 79th lysine residue of the histone H3 protein. H3K79me2 is detected in the transcribed regions of active genes.
H4K12ac is an epigenetic modification to the DNA packaging protein histone H4. It is a mark that indicates the acetylation at the 12th lysine residue of the histone H4 protein. H4K12ac is involved in learning and memory. It is possible that restoring this modification could reduce age-related decline in memory.
CUT&Tag-sequencing, also known as cleavage under targets and tagmentation, is a method used to analyze protein interactions with DNA. CUT&Tag-sequencing combines antibody-targeted controlled cleavage by a protein A-Tn5 fusion with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. Currently, ChIP-Seq is the most common technique utilized to study protein–DNA relations, however, it suffers from a number of practical and economical limitations that CUT&RUN and CUT&Tag sequencing do not. CUT&Tag sequencing is an improvement over CUT&RUN because it does not require cells to be lysed or chromatin to be fractionated. CUT&RUN is not suitable for single-cell platforms so CUT&Tag is advantageous for these.
ChIL sequencing (ChIL-seq), also known as Chromatin Integration Labeling sequencing, is a method used to analyze protein interactions with DNA. ChIL-sequencing combines antibody-targeted controlled cleavage by Tn5 transposase with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. Currently, ChIP-Seq is the most common technique utilized to study protein–DNA relations, however, it suffers from a number of practical and economical limitations that ChIL-Sequencing does not. ChIL-Seq is a precise technique that reduces sample loss could be applied to single-cells.
H3K36me2 is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the di-methylation at the 36th lysine residue of the histone H3 protein.
MNase-seq, short for micrococcal nuclease digestion with deep sequencing, is a molecular biological technique that was first pioneered in 2006 to measure nucleosome occupancy in the C. elegans genome, and was subsequently applied to the human genome in 2008. Though, the term ‘MNase-seq’ had not been coined until a year later, in 2009. Briefly, this technique relies on the use of the non-specific endo-exonuclease micrococcal nuclease, an enzyme derived from the bacteria Staphylococcus aureus, to bind and cleave protein-unbound regions of DNA on chromatin. DNA bound to histones or other chromatin-bound proteins may remain undigested. The uncut DNA is then purified from the proteins and sequenced through one or more of the various Next-Generation sequencing methods.
H3R17me2 is an epigenetic modification to the DNA packaging protein histone H3. It is a mark that indicates the di-methylation at the 17th arginine residue of the histone H3 protein. In epigenetics, arginine methylation of histones H3 and H4 is associated with a more accessible chromatin structure and thus higher levels of transcription. The existence of arginine demethylases that could reverse arginine methylation is controversial.
H3R8me2 is an epigenetic modification to the DNA packaging protein histone H3. It is a mark that indicates the di-methylation at the 8th arginine residue of the histone H3 protein. In epigenetics, arginine methylation of histones H3 and H4 is associated with a more accessible chromatin structure and thus higher levels of transcription. The existence of arginine demethylases that could reverse arginine methylation is controversial.
H3R2me2 is an epigenetic modification to the DNA packaging protein histone H3. It is a mark that indicates the di-methylation at the 2nd arginine residue of the histone H3 protein. In epigenetics, arginine methylation of histones H3 and H4 is associated with a more accessible chromatin structure and thus higher levels of transcription. The existence of arginine demethylases that could reverse arginine methylation is controversial.
H4R3me2 is an epigenetic modification to the DNA packaging protein histone H4. It is a mark that indicates the di-methylation at the 3rd arginine residue of the histone H4 protein. In epigenetics, arginine methylation of histones H3 and H4 is associated with a more accessible chromatin structure and thus higher levels of transcription. The existence of arginine demethylases that could reverse arginine methylation is controversial.
Hi-C is a high-throughput genomic and epigenomic technique first described in 2009 by Lieberman-Aiden et al. to capture chromatin conformation. In general, Hi-C is considered as a derivative of a series of chromosome conformation capture technologies, including but not limited to 3C, 4C, and 5C. Hi-C comprehensively detects genome-wide chromatin interactions in the cell nucleus by combining 3C and next-generation sequencing (NGS) approaches and has been considered as a qualitative leap in C-technology development and the beginning of 3D genomics.