flow cytometry and reproducible analysis
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Flow Cytometry and Reproducible Analysis. Cliburn Chan Department of Biostatistics and Bioinformatics, DUMC. Reproducible Analysis. Can someone in a different lab replicate your results? Can someone else in your lab replicate your results? Can you replicate your own results 6 months later? - PowerPoint PPT PresentationTRANSCRIPT
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Flow Cytometry and Reproducible Analysis
Cliburn ChanDepartment of Biostatistics and
Bioinformatics, DUMC
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Reproducible Analysis
• Can someone in a different lab replicate your results?
• Can someone else in your lab replicate your results?
• Can you replicate your own results– 6 months later?– When FlowJo goes from version 10.0 to 11.0?– When your lab catches fire and all your computers
melt into toxic waste?
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Complexity of flow analysis
• Experimental design• Running the experiment• Raw data (FCS files)• Compensation• Transformation• Gating strategy• Gates MFI and relative frequencies• Statistical analysis – e.g. outcome correlation
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Experimental design
• Is randomization done correctly?• Is the sample size sufficient?• Is there an SOP for annotating the experiment?– MIATA– MiFlowcyt
• What is the informatics strategy to ensure that data is recorded accurately and backed-up safely?
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Running the experiment
• Stuff I know little about …• Janet and Jennifer will teach in this workshop– Instrument calibration– Bridging studies– Reagent qualification– Use of appropriate biological controls– Use of appropriate technical controls
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Raw data (FCS files)
• Is there a file naming SOP that is followed?• Is there an SOP for recording FCS metadata?– Channel labels – fluorochrome, antibody, FMO
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Inconsistent annotation example
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Compensation, transformation and gating strategy
• Compensation is Real = Spillover-1 × Observed• Transformation is complicated – can think of as
linear (low values) and log (high values)• Gating strategy is hard to replicate, but can be
stored as a template and “re-used” with tweaking• Compensation, transformation and gating should
be done on a per-batch and not per-file basis• Would recommend storing workspace containing
this data in both .jo and .xml formats
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Working with statisticians
• At some point, a statistician is likely to be asked to analyze your data. This can lead to much unhappiness.
• Statisticians do not like Excel– The first thing they will try to do is export to a CSV
or delimited file, for import into SAS or R– If this is difficult to do, they will not like you
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Excel rules for happy statisticians
• 1 worksheet = 1 table• 1 cell = 1 value• Data/metadata = comprehensive & consistent• Formatting = None• Validation = Yes
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1 worksheet = 1 table
• A table has column headers and a number of rows and nothing else – it is RECTANGULAR
• Do not put more than 1 table in a worksheet• Do not use non-rectangular tables• Example of good worksheet
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1 worksheet = 1 table
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1 cell = 1 value
• Easy to filter by tube, sample or subject• Easy to write validation rules or lookup table
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1 cell = 1 value
• ID column has 3 different values• Need to do text parsing to recover information
– very error prone
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Data: column names
• Consistent column names across worksheets– Singlets/Lymphocytes– Singlet/Lymphs– Singlets / Lymphocytes– Singlets/Lymphoctyes
• Use full gating path for column name– Singlets/Lymphocytes/Viable/CD4+/CM/IFN+
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Data: What to record
• Better to have more data than less data– Sample type (PBMC, whole blood)– Recovery – Viability
• Better to have basic than derived data– Counts better than relative frequencies
• Keep link to raw data for reproducibility– Path to FCS and workspace files on server
• Use special indicator for missing data (e.g. NAN), not zero• Use as many columns as you need and name them sensibly
and consistently
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Data: Versioning
• Do not change the data in the worksheet once it has been handed to statistician.
• If there are errors that must be corrected, make a new copy, label the filename with date and version, and send that to statistician– ArcticRatExperiment_07May2013_Version01.xlsx– ArcticRatExperiment_17May2013_Version02.xlsx
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Formatting
• Don’t do it.• Avoid putting information via:– Highlighting– Fancy spacing– Different fonts and font effects– Merging cells– Comments
• Will it survive a round-trip from Excel to CSV and back again?
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Formatting - Before
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Formatting - After
Comments are lostHighlighting is lostBad cell formatting is lostMerged cells become missing information
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Summary of Reproducible Analysis
• Know what you are doing from PBMC to Excel• SOPs are important• Annotation is important• Excel is OK if you use NONE of its features • Keep all necessary data in the same place• Keep a remote backup• Talk with your statistician
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Biologist talks to Statistician
http://www.youtube.com/watch?v=Hz1fyhVOjr4