fcmcmp 0.1.0

A lightweight, flexible, and modern framework for annotating flow cytometry data.

The goal of FCMcmp is to make it easy to analyze flow cytometry data from python. The first challenge in analyzing flow cytometry data is working out which wells should be compared with each other. For example, which wells are controls, which wells are replicates of each other, which wells contain the conditions you’re interested in, etc. This isn’t usually too complicated for individual experiments, but if you want to write analysis scripts that you can use on all of your data, managing this metadata becomes a significant problem.

FCMcmp addresses this problem by defining a simple YAML file format that can associate wells and plates in pretty much any way you want. When you ask FCMcmp to parse these files, it returns a list- and dictionary-based data structure that contains these associations, plus it automatically parses the raw FCS data into pandas data frames.

Installation

fcmcmp is available on PyPI:

pip3 install fcmcmp

Only python>=3.4 is supported.

Quick Start

I’ll demonstrate using data you might export from running a 96-well plate on a BD LSRII, but the library should be pretty capable of handling any directory hierarchy:

my_plate/
   96 Well - U bottom/
      Specimen_001_A1_A01_001.fcs
      Specimen_002_A2_A02_002.fcs
      Specimen_003_A3_A03_003.fcs
      ...

Loading the data

First, we need to make a YAML metadata file describing the relationships between the wells on this plate:

 # my_plate.yml
label: vaxadrin
wells:
   without: [A1,A2,A3]
   with: [B1,B2,B3]
---
label: vaxamaxx
wells:
   without: [A1,A2,A3]
   with: [C1,C2,C3]

In this example, the name of the plate directory is inferred from the name of the YAML file. You can also explicitly specify the path to the plate directory by adding the following header before the label/wells sections:

plate: path/to/my_plate
---

You can even reference wells from multiple plates in one file:

plates:
   foo: path/to/foo_plate
   bar: path/to/bar_plate
---
label: vaxascab
wells:
   without: [foo/A1, foo/A2, foo/A3]
   with: [bar/A1, bar/A2, bar/A3]

Note that the label and wells fields are required, but you can add, remove, or rename any other field:

label: vaxa-smacks
channel: FITC-A
gating: 60%
wells:
   0mM: [A1,A2,A3]
   1mM: [B1,B2,B3]
   5mM: [C1,C2,C3]

Once you have a YAML metadata file, you can use fcmcmp to read it:

>>> import fcmcmp, pprint
>>> experiments = fcmcmp.load_experiments('my_plate.yml')
>>> pprint.pprint(experiments)
[{'label': 'vaxadrin',
  'wells': {'with': [Well(B1), Well(B2), Well(B3)],
            'without': [Well(A1), Well(A2), Well(A3)]}},
 {'label': 'vaxamaxx',
  'wells': {'with': [Well(C1), Well(C2), Well(C3)],
            'without': [Well(A1), Well(A2), Well(A3)]}}]

The data structure returned is little more than a list of dictionaries, which should be easy to work with in pretty much any context. The wells are represented by Well objects, which have only three attributes:

• Well.label: The name used to reference the well in the YAML file.

• Well.data: A pandas.DataFrame containing all the data associated with the well, parsed using the excellent fcsparse library.

• Well.meta: A dictionary containing any metadata associated with the well, also parsed using fcsparse.

Note that if you reference the same well more than once (e.g. for controls that apply to all of your experiments), each reference is parsed separately and gets its own copy of all the data.

Working with the data

Once the experiments are loaded into python as described above, fcmcmp provides a couple ways to interact with them. The first is to apply one or more of a handful of pre-defined “processing steps”:

>>> ch = 'FITC-A', 'PE-Texas Red-A'
>>> p1 = fcmcmp.GateEarlyEvents(throwaways_secs=2)
>>> p1(experiments)
>>> p2 = fcmcmp.GateSmallCells(threshold=40, save_size_col=True)
>>> p2(experiments)
>>> p3 = fcmcmp.GateNonPositiveEvents(ch)
>>> p3(experiments)
>>> p4 = fcmcmp.LogTransformation(ch)
>>> p4(experiments)
>>> p5 = fcmcmp.KeepRelevantChannels(ch)
>>> p5(experiments)

In this example:

• GateEarlyEvents discards the first few seconds of data, which is useful when you’re using a high-throughput sampler and you suspect that cells from the previous well are being recorded at the beginning of each well.

• GateSmallCells combines the FSC-A and SSC-A channels to estimate how the size of each event, then discards any events below the given percentile (40% in this example).

• GateNonPositiveEvents discards negative data on the specified channels. I have to admit that I don’t understand how “fluorescence peak area” data can be negative, but in any case this can be important if you want to work with the logarithm of your data, because of course you can’t take the logarithm of negative data.

• LogTransform takes the logarithm of the data in the specified channels. This is a very standard processing step for fluorescent channels.

• KeepRelevantChannels discards all the data for any channels that aren’t explicitly listed. This is mostly useful for when you’re printing out data to the terminal and don’t want to be distracted by channels you collected but aren’t interested in at the moment.

Instead of calling each processing step individually, you can also use the run_all_processing_steps() function to call them all at once. If you do this, you don’t even need to make a variable for each step:

>>> fcmcmp.GateEarlyEvents(throwaways_secs=2)
>>> fcmcmp.GateSmallCells(threshold=40, save_size_col=True)
>>> fcmcmp.GateNonPositiveEvents(ch)
>>> fcmcmp.LogTransformation(ch)
>>> fcmcmp.KeepRelevantChannels(ch)
>>> fcmcmp.run_all_processing_steps()

You can also write your own processing steps by inheriting from either ProcessingStep or GatingStep and reimplementing the proper methods. ProcessingStep is for general transformations and has two virtual methods: process_experiment() and process_well(). The former is called once for each experiment and should transform that experiment in place. The latter is called once for each well and can either modify the well in place (and return None) or return the processed data, which will overwrite the original data.

GatingStep is specifically for transformations regarding which data points to keep and which to throw out. It is itself a ProcessingStep, but it has a different virtual method(): gate(). This method is called on each well and should return a boolean numpy array. Those indices that are False will be thrown out, those that are True will be kept.

The second way to interact with the experiments is to use the yield_wells() and yield_unique_wells() functions. These are both generators which iterate through all of your experiments and yield each well one at a time. The purpose of these functions is to make the nested experiments data structure seem more like a flat list:

>>> for experiment, condition, well in fcmcmp.yield_wells(experiments):
>>>     print(experiment, condition, well)

Both functions take an optional keyword argument. If given, only wells with a matching experiment label, condition, or well label will be returned. The only difference between yield_wells() and yield_unique_wells() is that the former won’t yield the same well twice. This is important because the same well can certainly be included in many different experiments.

Bugs and new features

Use the GitHub issue tracker if you find any bugs or would like to see any new features. I’m also very open to pull requests.