FISH Omics Format - Chromatin Tracing (FOF-CT)
Background
The FISH Omics Format for Chromatin Tracing (FOF-CT) is an open format for reporting Chromatin Tracing datasets. It is intended to serve the broader community and to promote the study of the spatial arrangement of DNA, RNA, and proteins within the human cell nucleus, uncovering the functional dynamics of the genome in three dimensions and over time (3D+t = 4D). FOF-CT originated within the (4D Nucleome; 4DN) project, but many datasets released in this format have since been produced by laboratories outside the consortium.
We encourage broad adoption of, and contribution to, the format by the entire community.
FISH Omics techniques description
Specific genomic sequences can be visualized by fluorescence in situ hybridization (FISH) using fluorescently labeled DNA probes, and their location in individual cells can be imaged using either traditional microscopy techniques (e.g., widefield or confocal) or super-resolution microscopy, as reviewed by (Fraser et al. 2015) and (Jerkovic and Cavilli 2021).
Evolutions of these techniques expanded the number of imaged targets to multiple and potentially all DNA segments across the genome (see recent reviews: Jercovic and Cavalli 2021, Boettiger and Murphy 2020, Hu and Wang 2020, Maslova and Krasikova 2021, Zhuang 2021, and Bouwman et al. 2022).
Collectively, these technologies can be called interchangeably multiplexed FISH or FISH omics, which emphasize the visualization of multiple or ideally all genomic targets, respectively. These methods provide an expanded understanding of how higher-order chromosome structure relates to transcriptional activity and cell development.
A variety of protocols have been developed in the past few years, and they can be divided into two main categories called respectively ball-and-stick (bas) or volumetric (vol) Chromatin Tracing, depending on whether the targeted genomic segment is visualized as a centroid (i.e., fitting the imaged spot of a detected fluorescent signal) or a cloud of single-molecule localizations (i.e., often rendered as a volume; see Figure 2 in Dekker et al. 2023).
Figure 1: FISH omics methods can be utilized to map chromatin structures across multiple genomic scales (Figure credit: Sarah Aufmkolk; Dekker et al., 2023, Mol.Cell.
FISH Omics Format rationale
This document describes the FISH Omics Format - Chromatin Tracing (FOF-CT), a community data format designed for capturing and exchanging the results of Chromatin Tracing experiments.
FOF-CT is directly compatible with both ball-and-stick and volumetric Chromatin Tracing techniques.
FOF-bas-CT is compatible with several ball-and-stick FISH-omics techniques including, but not limited to, Optical Reconstruction of Chromatin Architecture (ORCA), Multiplexed Imaging of Nucleome Architectures (MINA), Hi-M, DNA Sequential Fluorescence In Situ Hybridization (seqFISH+), Oligonucleotide Fluorescent In Situ Sequencing (OligoFISSEQ), DNA Multiplexed error-robust fluorescence in situ hybridization (DNA-MERFISH), and In-situ Genomic Sequencing (IGS).
Similarly, FOF-vol-CT is compatible with multiple volumetric chromatin tracing methods, such as OligoSTORM and OligoDNA-PAINT (Belivau et al. 2017, Bintu et al. 2018, Nir et al. 2018, Luppino et al. 2020)
Note
FOF-CT is designed to work with both unmodified genomes and genomes containing INSERTIONS or DELETIONS. For guidance on capturing genomic variants using FOF-CT, refer to the instructions in DNA-Spot/Trace Data core table, and include a description of the custom build using the Variant Call Format (VCF) as outlined in The Variant Call Format (VCF).
Figure 2: Schematic representation of the 15 tables composing the Fish Omics Format for Chromatin Tracing. FOF-CT is a modular format designed to capture Chromatin Tracing data from two experiment modalities: Ball-and-Stick (bas) and Volumetric (vol). All 12 FOF-bas-CT tables are shared across both modalities — 3 shown in red (Table 1-core, Table 2-demultiplexing, Table 3-quality), reflecting their role as the primary tables defining ball-and-stick Spot-level data, and 9 shown in grey, providing supporting Trace-, Cell-, ROI-, and RNA-level information. FOF-vol-CT requires these same 12 shared (red + grey) tables plus 3 additional tables specific to it, shown in blue (Table 1v-vol_core, Table 2v-undecoded, Table 3v-vol_quality). Each table’s index number and short name are shown in the upper-right corner. (Figure credit: Sarah Aufmkolk).
Summary: FISH Omics Format for ball-and-stick Chromatin Tracing
In ball-and-stick Chromatin Tracing experiments, polymer tracing algorithms are used to string together the localization of individual DNA bright Spots to reconstruct the three-dimensional (3D) path of chromatin fibers.
In order to capture chromatin traces as well as additional metrics captured during the course of the experiment, the FOF-CT format is organized around 12 tables.
The root of the format consists of the mandatory DNA-Spot/Trace Data core table table that defines Chromatin Traces as ordered lists of individual DNA-FISH bright Spots. In addition, eleven supplementary FOF-bas-CT tables support the integration of this DNA-Spot/Trace Data core table with:
Supplementary Spot properties such as:
Quality metrics.
The underlying primary localization data.
Physical coordinates placing the Spot/Trace in the context of cellular space.
The results of multiplexed RNA-FISH experiments
Supplementary global data that is better captured at the level of:
Trace (e.g., expression level of nascent RNA transcripts associated with a given Trace or overall localization of the Trace with respect to cellular or nuclear landmarks).
Cell (e.g., boundaries and volume).
Sub-cellular Region of Interest (ROI; e.g., Nuclear feature or Nucleolus).
Extracellular ROI (e.g., Tissue).
Summary: FISH Omics Format for volumetric Chromatin Tracing
For FOF-vol-CT the root of the format consists of the mandatory SM Localization Data vol_core table that reports the location of individual SM Localization events, the localization of their target chromosome segments, as well as their associated DNA-FISH bright Spots and chromatin Traces. In addition, two supplementary FOF-vol-CT tables support the integration of this SM Localization Data vol_core table with:
Supplementary SM Localization properties such as:
Quality metrics.
The underlying un-decoded raw localization data.
Tip
In addition to the 3 specific FOF-vol-CT tables, all 12 FOF-bas-CT tables are also available for use to report the results of volumetric Chromatin Tracing experiments.
Overview
Volumetric