Advanced Omics Techniques for Understanding Cochlear Genome, Epigenome, and Transcriptome in Health and Disease

Abstract

Advanced genomics, transcriptomics, and epigenomics techniques are providing unprecedented insights into the understanding of the molecular underpinnings of the central nervous system, including the neuro-sensory cochlea of the inner ear. Here, we report for the first time a comprehensive and updated overview of the most advanced omics techniques for the study of nucleic acids and their applications in cochlear research. We describe the available in vitro and in vivo models for hearing research and the principles of genomics, transcriptomics, and epigenomics, alongside their most advanced technologies (like single-cell omics and spatial omics), which allow for the investigation of the molecular events that occur at a single-cell resolution while retaining the spatial information.

Keywords: cochlea; epigenomics; genomics; omics; organ of Corti; single-cell omics; spatial omics; transcriptomics.

Figure 1

Schematic illustration of the available experimental models for cochlear research. The available models for cochlear research include cell lines of otocyst, HCs, organ of Corti, and stria vascularis. Explants of cochlear tissues may also be used, more recently via microfluidic chambers for organ-on-chip cultures. Cochlear organoids are an additional in vitro possibility and can be derived from induced pluripotent stem cells (iPSCs) or from embryonic stem cells (ESCs). Animal models can be generated by exposure to ototoxic drugs or by noise trauma; also, age-related and transgenic models of hearing loss have been developed. Finally, all the models may be subjected to CRISPR/Cas9 to achieve targeted gene editing. Abbreviations: IMO; Immortomouse; HC; hair cell, SC; supporting cell, iPSC; induced pluripotent stem cell, ESC; embryonic stem cell, Dmm; disproportionate micromelia; sedc, spondyloepiphyseal dysplasia congenital; USH: Usher; Cho: chondrodysplasia.

Figure 2

Schematic illustration of genomics. DNA is isolated from cells or tissues and is fragmented in order to create DNA libraries using DNA adapters. Sequencing can then be performed on targeted sequences (panels), on the whole exosome (WES), or on the whole genome (WGS). Genomics has provided important advancements in the diagnosis and discovery of genetic hearing loss.

Figure 3

Schematic illustration of transcriptomics. Transcriptomics can be performed at a bulk, single-cell, or spatial resolution. In bulk RNAseq, total RNA is extracted from the tissue and can be directly sequenced or converted into cDNA and then sequenced. In scRNA-seq, the sequencing of the RNA is limited to single cells that are isolated from the tissue and analyzed individually. In spatial transcriptomics, the transcriptome may be analyzed with imaging-based methods, using fluorescent labeled probes which bind to the RNA on tissue slides, followed by microscopic analysis; spatial transcriptomics may also be performed through sequencing-based methods using arrays of barcoded probes or microdissection of target tissue areas, both followed by sequencing. Array-based spatial transcriptomics have not yet been applied in cochlear research. The other transcriptomics techniques have provided important new insights into the gene regulatory networks of the cochlea, under both physiological and pathological conditions. Abbreviations: Bulk RNA seq, Bulk RNA sequencing; scRNA-seq, single-cell RNA sequencing.

Figure 4

Schematic illustration of epigenomics. Epigenomics can be performed at a bulk, single, and spatial resolution. Bulk epigenomics and single-cell epigenomics have been applied in the cochlea. The study of epigenomics includes the assessment of DNA methylation dynamics, histone modifications, chromatin accessibility, and chromosome conformations. These epigenomic methods can be performed either with arrays or with sequencing. The techniques ChIP and ATAC-seq (in bulk and single cells) have been applied in the cochlea. ChIP relies on immunoprecipitating DNA–protein complexes via specific antibodies, and ATAC-seq uses Tn5 transposase chemistry and NGS to analyze open or accessible chromatin regions. These techniques have provided novel insights into the molecular mechanisms underlying the developmental and regenerative processes in the cochlea. Abbreviations: BS-seq, bisulfite sequencing; RRBS, reduced representation bisulfite sequencing; MeDIP, methylated DNA immunoprecipitation; ChIP, chromatin immunoprecipitation; CUT&RUN, cleavage under target and release using nuclease; CUT&Tag, ATAC-seq cleavage under targets and tagmentation; DNAse-seq, DNase I hyper-sensitive sites sequencing; FAIRE-seq, formaldehyde-assisted identification of regulatory elements followed by sequencing; 3C, conformation capture technique; PLAC-seq, proximity ligation-assisted ChIP-seq; ChIA PET, chromatin interaction analysis by paired-end tag sequencing; sci-MET, single-cell combinatorial indexing for methylation analysis; scCGI-seq, single-cell CGI methylation sequencing; scChIC-seq, single-cell chromatin immune-cleavage sequencing technique; ACT-seq, antibody-guided chromatin tagmentation sequencing; COBATCH, combinatorial barcoding and targeted chromatin release; scChIL-seq, single-cell chromatin integration labeling sequencing.