Spot Demultiplexing table
Requirement level: optional
Summary
This table is optional and is designed to be used in the case of multiplexed FISH experiments (i.e., MERFISH) in which the final localization of a bright DNA or RNA Spot results from the combination of multiple individual localization events (e.g., by combining particles detected and localized in separate images). In such a case the final Spot localization data is recorded in the DNA-Spot/Trace Data core table, while the underlying primary localization data can be recorded by using this table, as shown for DNA Spots in the example below.
This table is indexed by Loc_ID, mandatorily reports the X, Y, Z coordinates of the Localization event, and it has a mandatory Spot_ID column that is used to link individual localization events to the resulting Spot.
Other columns are at user’s discretion.
Example
DNA spots detected with multiplexed barcodes
##FOF-CT_version=v0.1
##Table_namespace=4dn_FOF-CT_demultiplexing
##XYZ_unit=micron
#Software_Title: ExampleLocalizationSoftware
#Software_Type: SpotLoc
#Software_Authors: Doe, J.
#Software_Description: A pretty clear description
#Software_Repository: https://github.com/repo_name_goes_here
#Software_PreferredCitationID: https://doi.org/doi_goes_here
#lab_name: Nobel
#experimenter_name: John Doe
#experimenter_contact: john.doe@email.com
#additional_tables: 4dn_FOF-CT_core, 4dn_FOF-CT_quality
#^Hyb: the labeling round in which this localization occurred
#^Fluor: the fluorescent channel in which this localization was detected
#^Brightness: the photon count for this localization event
#^Fit_Quality: the quality of fit for this localization, on a relative scale of 0-1
##columns=(Loc_ID, Spot_ID, X, Y, Z, Hyb, Fluor, Brightness, Fit_Quality)
1, 1, 2342, 2354, 545, 2, cy3, 1003, 0.83
2, 1, 2342, 2354, 545, 2, cy5, 2000, 0.93
3, 1, 2342, 2354, 545, 3, cy5, 1233, 0.85
4, 2, 3345, 5432, 654, 3, cy3, 2324, 0.95
5, 2, 3345, 5432, 654, 3, cy5, 2324, 0.95
6, NA, 4345, 432, 100, 4, cy3, 2324, 0.95
7, 2, 3345, 5432, 654, 4, cy3, 2324, 0.95
File Header
The first line in the header is always “##FOF-CT_version=vX.X”
The second line in the header is always “##Table_namespace=4dn_FOF-CT_demultiplexing”
The header MUST contain a mandatory set of fields that describe any algorithm that was used to produce/process data in this table. In case more than one algorithm were used, please use the same set of fields for each of them.
The header MUST include a detailed description of each optional columns used.
Name |
Description |
Example |
Conditional requirement conditions |
|---|---|---|---|
##FOF-CT_version= |
Version of the FOF format used in this case. |
v0.1 |
|
##Table_namespace= |
Identifier for this type of table. Value must be as in the example. |
4dn_FOF-CT_demultiplexing |
|
#lab_name: |
name of the lab where the experiment was performed. |
Nobel |
|
#experimenter_name: |
name of the person performing the experiment. |
John Doe |
|
#experimenter_contact: |
email address of the person performing the experiment. |
||
#description: |
A free-text, description of the experiment and of the data recorded in this table. This description should provide a clear understanding of the process utilized to produce the data and contain sufficient details to ensure interpretation and reproducibility. |
||
#Software_Title: |
The name of the Software(s) that were used in this case for localizing individual FISH-omics bright Spots and/or to produce three-dimensional (3D) polymeric chromatin Traces. |
ChrTracer3 |
Conditional requirement: this MUST be reported any time a software is used to produce data associated with this table. |
#Software_Type: |
The type of this Software. Allowed values: SpotLoc, Tracing, SpotLoc+Tracing, Segmentation, QC, Other |
SpotLoc+Tracing |
Conditional requirement: this MUST be reported any time a software is used to produce data associated with this table. |
#Software_Authors: |
The Name(s) of the individual Author(s) of this Software. In case there are more than one Authors, individual names should be listed as follows, Doe, John; Smith, Jane; etc,. |
Mateo, LJ; Sinnott-Armstrong, N; Boettiger, AN |
Conditional requirement: this MUST be reported any time a software is used to produce data associated with this table. |
#Software_Description: |
A free-text, description of this Software. This description should provide a detailed understanding of the algortithm and of the analysis parameters that were used, in order to guarantee interpretation and reproducibility. |
ChrTracer3 software was developed for analysis of raw DNA labeled images. As an input, it takes an.xlsx table containing information and folder names of the DNA experiment. As an output, it returns tab delimited.txt files with drift-corrected x, y, z positions for all labeled barcodes. These can be used directly to calculate the nm scale distances between all pairs of labeled loci. The current version of the software as of this writing is ChrTracer3. |
Conditional requirement: this MUST be reported any time a software is used to produce data associated with this table. |
#Software_Repository: |
The URL of any repository or archive where the Software executable release can be obtained. |
Conditional requirement: this MUST be reported any time a software is used to produce data associated with this table. |
|
#Software_PreferredCitationID: |
The Unique Identifier for the preferred/primary publication describing this Software. Examples include, Digital Object Identifier (DOI), PubMed Central Identifier (PMCID), ArXiv.org ID etc,. |
Conditional requirement: this MUST be reported any time a software is used to produce data associated with this table. |
|
#additional_tables: |
list of the additional tables being submitted. Note: use a comma to separate each table name from the next. |
4dn_FOF-CT_core, 4dn_FOF-CT_rna, 4dn_FOF-CT_bio, 4dn_FOF-CT_trace, 4dn_FOF-CT_cell |
|
#Intensity_measurement_method |
If relevant, the method that was used to performed intensity measurements. In particular, sufficient information should be provided to document how digital intensity signals were converted in Photon conunts. |
Spot centroid intensity. |
Conditional requirement: this MUST be reported if any intensity metrics are reported. |
##XYZ_unit= |
The unit used to represent XYZ locations or distances in this table. Note: use micron (instead of µm) to avoid problem with special, Greek symbols. Other allowed values are: nm, mm etc. |
micron |
Conditional requirement: this MUST be reported if any locations metrics are reported. |
##time_unit= |
If relevant, the unit used to represent a time interval. Note: use ‘sec’ for seconds, ‘msec’ for milliseconds, ‘min’ for minutes, and ‘hr’ for hours. |
sec |
Conditional requirement: this MUST be reported if any time metrics are reported. |
##intensity_unit= |
If relevant, the unit used to represent intensity measurements. |
a.u. |
Conditional requirement: this MUST be reported if any intensity metrics are reported. |
##columns= |
list of the data column headers used in the table. Note: enclose the column headers and use a comma to separate each header name from the next. |
(Spot_ID, X, Y, Z) |
Data Columns
This table is indexed by Loc_ID and therefore each row corresponds to data associated with an individual Localization event.
The first columns are always: Loc_ID, Spot_ID, X, Y, Z. The content and order of all other columns is at user’s discretion. The order of the rows is at user’s discretion.
Name |
Description |
Example |
Conditional requirement conditions |
|---|---|---|---|
Loc_ID |
A unique identifier for this individual Localization event. |
1 |
|
Spot_ID |
A unique identifier for the bright DNA or RNA Spot with which this individual localization event is associated. |
1 |
|
X |
The sub-pixel X coordinate of this Localization event. |
||
Y |
The sub-pixel Y coordinate of this Localization event. |
||
Z |
The sub-pixel Z coordinate of this Localization event. |
||
#^optional_column_1: |
|||
#^optional_column_2: |
|||
#^optional_column_3: |