Plankton and Particles Flow Cytometry

The guidelines presented below will assist the user in submitting flow cytometry data to SeaBASS. The format described herein allows for Level 2 submissions of flow cytometry data, meaning sums of total abundances of each phytoplankton or bacterial group. New metadata headers and field names have been developed for these data. Additionally, detailed instructions are given for the submission of documentation (protocol document, checklist, etc.), images, and additional ancillary files. Greater detail regarding the development and application of these guidelines and requirements can be found in Neeley et al., 2023. 

Neeley, A.R., Soto-Ramos, I. and Proctor, C. (2023) Standards and Best Practices for Reporting Flow Cytometry Observations: a technical manual, Version 1.1. Greenbelt, MD., NASA Goddard Space Flight Center, 31pp. DOI: http://dx.doi.org/10.25607/OBP-1864.2. 



Table of Contents


Required extra documents, FCM

In addition to the standard headers and fields required for all SeaBASS submissions, the submission of flow cytometry data requires the following additional headers and fields (additional details can be found in Neeley et al., 2023). The following sections will describe special metadata headers and fields, protocol and checklist documents, supplementary lists of which taxonomic categories were assessed by manual classification methods, and the raw files and/or any relevant instrument metadata. Please use the list below as a guideline for the submission requirements.
 

Submission checklist:

  1. The data file must include a data table and appropriate metadata headers and field names
  2. Protocol document 
  3. Data set checklist 
  4. Ancillary data files (raw files (.fcs), optional image files, etc.)
  5. List of assessed IDs for manual classification

"Conditionally required" metadata headers

 In addition to the standard headers and fields required for all SeaBASS submissions, the submission of flow cytometry observations requires the following additional headers and fields (additional details can be found in chapter 3.0 of Neeley et al., 2023). 
 
      Required metadata headers:
  1. /associated_archives: List of filenames for all external raw data (e.g., a bundle of .fcs files) and/or scientific files (e.g., a bundle of scatter plot image files.) Please split files that exceed 5Gb. Example: /associated_archives=EXPERIMENT_CRUISE_FCM_fcs_associated.tgz,EXPERIMENT_CRUISE_scatterPlots_associated.tgz
  2. /associated_archive_types: Provide a value or list of text terms to describe the contents of each associated_archive file. Commonly used types are 'raw' for raw (.fcs) files, 'ancillary_media' for images, or 'metadata' for other supporting files or code. Example: /associated_archive_types=raw,ancillary_media
  3. /volume_analyzed_ul: Volume that was processed through the flow cytometer in units of microliters (µl). 

      Required "if applicable" metadata headers: 

  1. /associated_files: The value is the name of the specific source file used for the scientific analysis. Include this header if all the data in this file came from the same sample or .fcs file. However, if multiple samples were used, then skip this header and instead use the equivalent field associated_files so you can name the specific file on each data row. 
  2. /associated_file_types: This header must be used in conjunction with associated_files. The entry here should describe the data type within the associated_files. For the purposes of .fcs files, the format would be ‘raw’, images would be ‘ancillary_media’ and any other supportive information would go under ‘metadata’.
  3. /eventID: a unique identifier associated with the sample as an event. 

"Conditionally required" data fields

This section describes how to format the data table for flow cytometry observations. An external, machine-readable, and resolvable identifier, or object number, that returns nomenclatural (not taxonomic) details of a name (scientificNameID) should also be included for each organism. Moreover, the field names measurementValue and measurementValueID, which provide morphological and functional information, were added to remain interoperable with the Ocean Biodiversity Information System (OBIS) and align with Darwin Core terminology when possible (See subchapter 3.2 in Neeley et al. 2023). The complete and updated lists of NERC vocabulary can be found here. The date that the vocabulary was accessed and a link to the most current NERC vocabulary must be included in the checklist and in the comments section of the data file. 
 

      Required fields: 

  1. volume_analyzed_ul (ul): Volume that was processed through the flow cytometer in units of microliters (ul). This field name can also be used as a header when a single sample is included per submission.  
  2. data_provider_category_manual (none): A category used by the data provider to name the organism for a manual identification, not necessarily a scientific name. 
  3. scientificName_manual (none): A scientific name from a recognized taxonomic reference database (e.g., World Register of Marine Species, AlgaeBase) at the lowest level that matches the data provider's category, for a manual identification matched to scientificNameID.  
  4. scientificNameID_manual (none): A Life Science Identifier (LSID) from a recognized taxonomic reference database (e.g., World Register of Marine Species, AlgaeBase) at the lowest level that matches the data provider's category for manual identification. 
  5. abun (cells/L): Concentration of cells observed in units of cells per liter.  

      Recommended but optional fields: 

  1. measurementValue (none): Vocabulary that describes a cluster of cells by morphological characteristics (e.g., size and fluorescence) rather than by taxonomy and is recognized by the Natural Environment Research Council (NERC). The alternative label must be used here.  
  2. measurementValueID (none) The URI associated with the NERC vocabulary is defined in measurementValue. 
  3. data_provider_category_manual: (none) A category used by the data provider to name the organism for a manual identification, not necessarily a scientific name. 

      Required "if applicable" fields: 

  1. /associated_files: The value is the name of the specific source file (.fcs) used for the scientific analysis. 
  2. /associated_file_types: This header must be used in conjunction with associated_files. The entry here should describe the data type within the associated_files. For the purposes of .fcs files, the format would be ‘raw’, images would be ‘ancillary_media’ and any other supportive information would go under ‘metadata’.

Protocol and checklist documents

Submission of data files containing flow cytometry observations requires supplemental documentation, including both a checklist and a protocol document, to preserve critical methods information. Please, download the checklist and protocol templates, fill it out and submit it along with your other documentation and calibration information. Example submission files can be found below.
 
Download the checklist template and protocol examples:
Download FCM checklist template (V20230306)
 
Download the protocol example and template: 
Download a protocol example (V20230306)

Submission of raw files and additional metadata

Data submissions should include an organized directory containing the raw instrument files (.fcs), images from targeted cell scatter plots, and additional metadata that may include software and codes to collect and process the data (if applicable). The submission of raw instrument files (.fcs) with the data is required. The file size of this information can often be very large; therefore, they are not included in our traditional ordering system. However, users can order the associated files along with the data if desired by clicking the “download associated files” checkmark when requesting the dataset. Associated files (raw files (.fcs), images (targeted cell scatter plots), and metadata (software and codes) can be submitted in a folder called “associated” with individual subfolders separated by data type (e.g., raw, images, metadata). Alternatively, submitters can compress the folder into individual .tgz files. If the submission is extremely large (above 5 GB), then data should be subdivided into subsequent folders or compressed files. Submitters can use their own scheme to subdivide the data, such as by year or date for long time series.  

Assessed IDs list

Providing a list of all scientificName/scientificNameID pairs assessed by the data submitter data submission enables the determination of both the presence and absence of annotations in the Level 1b file. Supplementary lists of which taxonomic categories, all scientificName/scientificNameID pairs, assessed by the data submitter are required as part of data submissions. measurementValue/ measurementValueID should be included in this list, if applicable. The filename must be included in the \documents header.
 
For example, \documents=manual_assessed_id_NAB08_KN193-03.txt

The following table is an example of an assessed ID table submission for flow cytometry observations. 

measurementValue 

measurmentValueID 

data_provider_category 

scientificName_manual 

scientificNameID_manual 

Red Pico 

http://vocab.nerc.ac.uk/collection/F02/current/F0200004/ 

picoeukaryote 

Eukaryota 

urn:lsid:algaebase.org:taxname:86701 

OraPicoProk 

http://vocab.nerc.ac.uk/collection/F02/current/F0200003/ 

Synechococcus 

Synechococcus 

urn:lsid:marinespecies.org:taxname:160572 

OraNano 

http://vocab.nerc.ac.uk/collection/F02/current/F0200006/ 

Cryptophyceae 

Cryptophyceae 

urn:lsid:marinespecies.org:taxname:17639 

HetHNA 

http://vocab.nerc.ac.uk/collection/F02/current/F0200010/ 

heterotrophic_prokaryote_HNA 

Prokaryota 

urn:lsid:algaebase.org:taxname:86700 

Example submission, flow cytometry observations

Example SeaBASS data file. Note that the example file doesn't include the optional fields and the data is not real.
(Example SeaBASS file)
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Last edited by Chris Proctor on 2023-03-07
Created by Chris Proctor on 2023-02-09