flowCore

Dependencies & Environment

Package-intrinsic requirements from the Bioconductor landing page — reproduce in any R environment.

• Version: 2.24.0 · Bioconductor: 3.23 · R: ≥ 4.6
• Imports: Biobase, BiocGenerics, Rcpp, matrixStats, cytolib, S4Vectors
• System requirements: GNU make, C++17
• Install: BiocManager::install("flowCore")

When to Use

• FCS3.0 Standard Parsing: Reading and interpreting Flow Cytometry Data File Standard Version FCS3.0 files.
• Segment Extraction: Locating and extracting HEADER, TEXT, DATA, and ANALYSIS segments from flow cytometry data sets.
• Large File Handling: Processing data sets of 100 megabytes and larger where byte offsets exceed standard limits.
• Compensation Matrix Retrieval: Extracting the fluorescence compensation matrix ($COMP) applied to the data.

When NOT to Use

• Automated Cell Clustering: For automated cell clustering, use flowMeans instead because flowCore focuses on file standard specifications and data structures.
• Probability Binning: For generating multivariate probability distribution fingerprints, use flowFP instead because flowCore only handles the raw file segments.

Data Requirements

• Input Format: FCS3.0 conformant files.
• Structure: Must contain HEADER, TEXT, and DATA segments.
• Storage Mode: List mode data storage or histograms.

Key Parameters

• $BEGINDATA (required): Byte-offset from the beginning of the data set to the beginning of the DATA segment.
• $DATATYPE (required): Type of data in the DATA segment (ASCII 'A', integer 'I', single precision floating point 'F', or double precision 'D').
• $BYTEORD (required): Byte order for the data acquisition computer (e.g., 4,3,2,1 or 1,2,3,4).
• $COMP (optional): Fluorescence compensation matrix elements stored in row-major order.
• $PnB (required): Number of bits reserved for parameter number n.
• $PnR (required): Range for parameter number n.

Best Practices

• Primary TEXT Segment Placement: Ensure the primary TEXT segment is located entirely within the first 99,999,999 bytes of the data set.
• ANALYSIS Segment Verification: Check the $BEGINANALYSIS and $ENDANALYSIS keywords to determine if an ANALYSIS segment is present, especially if HEADER offsets are zero.
• Internationalization: Use the $UNICODE keyword to support multi-byte characters for string type keyword values.
• Data Integrity: Utilize the cyclic redundancy check (CRC) word placed at the end of each FCS3.0 data set to confirm file integrity after network transfers.

Common Pitfalls

• Large Data Set Offsets: Data sets exceed the 99,999,999 byte limit, causing standard HEADER offsets to fail. Fix: Substitute '0's in the HEADER and place the true byte offsets in the $BEGINDATA and $ENDDATA keyword values in the TEXT segment.
• Delimiter Collisions: The delimiter character appears within a keyword or keyword value. Fix: Immediately follow the delimiter with a second identical delimiter character to escape it.
• Missing Zero Values in Free Format ASCII: Free format ASCII data ($DATATYPE/A/ with $PnB/*/) misinterprets consecutive delimiters. Fix: Explicitly specify zero values with the zero (0) character rather than leaving empty space between delimiters.

Alternatives

• flowMeans: For downstream non-parametric clustering of the extracted flow cytometry data.
• flowFP: For fingerprinting and probability binning of the extracted flow cytometry data.

Citations

• Data File Standards Committee of the International Society for Analytical Cytology (ISAC). FCS version 2.0 can be found in Cytometry 1990;11(3):323-32.

References

• Homepage: bioconductor.org/packages/flowcore
• Vignette: https://bioconductor.org/packages/release/bioc/vignettes/flowCore/inst/doc/HowTo-flowCore.pdf

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