analyze

analyze runs analyses with documented methods: chromatographic peaks, chromatograms, NMR peaks, patch-clamp features, extracellular spikes, qPCR, plate assays and flow-cytometry gating.

openreadout analyze <SUBCOMMAND> [OPTIONS] <FILE>...

subcommand	what it returns	guide
`peaks`	detected and integrated peaks; bands and regions of spectra	Quantitation
`chromatogram`	TIC, BPC, XIC, SRM/MRM and stored detector traces	Quantitation
`nmr-peaks`	NMR peak list and integrals	NMR
`ephys-features`	action potentials, rheobase, f–I curve, passive properties	Electrophysiology
`spikes`	extracellular spike counts, rates and times	Electrophysiology
`qpcr`	Cq, Tm, ΔΔCq, standard curves	qPCR formats
`assay`	plate-reader wells, curves, dose-response, kinetics, growth, QC	Plate analysis
`gate`	FlowJo or Gating-ML gate hierarchy and population counts	FlowJo workspaces

Every subcommand takes --json. The guides describe the methods and how they were validated. To run any of these over many files as one table, use batch.

peaks and chromatogram

openreadout analyze peaks [OPTIONS] <FILE>...
openreadout analyze chromatogram [OPTIONS] <FILE>...

Both take the flags in Several inputs.

Which signal (both)

--tic: total-ion chromatogram. The default for mass-spectrometry files.
--bpc: base-peak chromatogram.
--mz MZ: extracted-ion chromatogram at this m/z. Repeatable or comma-separated.
--ppm PPM: XIC half-width in ppm. Default 10.
--da DA: XIC half-width in m/z units instead.
--transition Q1>Q3: SRM/MRM transition. Repeatable.
--transition-tol DA: Q1 and Q3 tolerance. Default 0.5.
--trace N: a stored chromatogram or detector signal (UV/DAD, FID, TCD), by traces[] index. Repeatable. The default for files with traces and no spectra.
--channel C: channel of --trace, such as one wavelength of a DAD.
--sweep N: sweep of --trace. Default 0.
--run N: spectra run index. Default 0.
--ms-level N: MS level of the scans. Default 1.
--polarity positive|negative: only scans of this polarity.
--scan-filter TEXT: only scans whose filter contains this text.
--precursor MZ: only MS/MS scans of this precursor (a product-ion XIC with --mz).
--precursor-tol DA: precursor tolerance. Default 0.5.
--rt-range A-B: retention-time range in minutes.
--mz-range A-B: TIC or BPC over this m/z range only.
--profile: use profile data where a scan stores both profile and centroids.
--aggregate sum|max: how points inside an XIC window are combined. Default sum.

chromatogram only

--max-points N: at most this many points per chromatogram. Default: all.
-o, --output FILE: write .csv, .parquet, .arrow or a .png/.jpg plot. One input only.
--width N: plot width in pixels. Default 1200.
--overwrite: replace an existing output file.

peaks only

--smooth N: Savitzky–Golay window in points. Default: half the typical peak width.
--min-snr S/N: detection threshold as height over noise. Default 3.
--min-height H: detection threshold in signal units.
--min-width MIN: smallest width at half height, in minutes.
--min-points N: fewest samples from peak start to end. Default 3.
--baseline auto|drop|valley|tangent|linear|none: baseline under detected peaks (default auto), or under --x-range/--integrate windows (default linear).
--skim-ratio R: with --baseline tangent, skim a peak when its height is below this fraction of its neighbour’s. Default 0.1.
--noise SIGMA: noise level in signal units, instead of the estimate.
--baseline-window MIN: window of the running baseline that decides where peaks end.
--area-seconds: report areas in signal × seconds instead of signal × minutes.
--rt MIN: report the peak at this expected retention time.
--window MIN: half-width of the --rt window, and of --targets compounds without their own. Default 0.5 min.
--pick largest|nearest: which peak in the window to report. Default largest.
--integrate A-B: integrate between two retention times with a straight baseline. Repeatable.
--x-range A:B: integrate a window of the trace’s own axis (cm⁻¹, nm, ppm; minutes on a chromatogram). Repeatable.
--targets FILE: compound list (CSV, TSV or JSON) with name and mz, q1+q3 or trace, and optionally rt, window and tolerances. One row per compound per file.
-o, --output FILE: write the rows of every input to one .csv, .tsv or .jsonl file.
--plot FILE|DIR: plot the chromatograms with the integrated peaks shaded.
--width N: plot width in pixels. Default 1200.
--overwrite: replace existing output files.

openreadout analyze chromatogram run.raw --mz 195.0877 --ppm 5 -o xic.csv
openreadout analyze peaks run.D --rt 3.1 --window 0.2 --plot peaks.png
openreadout analyze peaks spectrum.0 --x-range 980:1060 --x-range 1350:1525

nmr-peaks

openreadout analyze nmr-peaks [OPTIONS] <FILE>

The input is a Bruker experiment directory, a Varian .fid directory, a JEOL .jdf or a JCAMP-DX NMR spectrum.

--from auto|fid|processed: use the vendor’s processed spectrum, or process the FID here. auto prefers the stored spectrum.
--trace N: trace index. Default: chosen by --from.
--sweep N: row of a ser or arrayed FID to process. Default 0.
--phase MODE: default, stored, auto, magnitude, none, or P0,P1 in degrees.
--lb HZ: exponential line broadening. Default: stored, else 0.3 Hz for 1H and 1 Hz otherwise.
--gb HZ: Gaussian line width instead.
--size N: transform size in complex points (a power of two).
--baseline MODE: default, none or poly:N.
--no-group-delay: keep the digital-filter group delay.
--min-snr X: minimum peak height in noise SDs. Default 10.
--min-prominence X: minimum prominence in noise SDs. Default 5.
--min-height-fraction F: minimum height as a fraction of the tallest point. Default 0.
--negative: also report negative peaks (DEPT, APT).
--range A:B: only pick peaks between two shifts, in ppm.
--max-peaks N: keep at most this many peaks. Default 1000.
--integrate A:B: integrate a region in ppm. Repeatable.
--integral-reference I=V: normalize integrals so that region I equals V. Default 0=1.

The --process-* flags of trace, export and preview take the same values as --phase, --lb, --size and --baseline.

openreadout analyze nmr-peaks sample/1 --integrate 7.5:7.0 --integrate 3.8:3.6

ephys-features

openreadout analyze ephys-features [OPTIONS] <FILE>

--trace N: trace index. Default 0.
--channel N: channel index. Default: the first voltage channel, else the first current channel.
--sweeps LIST: sweeps to analyse, such as 0,3,5-9. Default: all.
--peak-threshold MV: voltage a spike must cross upward. Default −20 mV.
--dvdt-threshold V_PER_S: dV/dt that defines spike onset. Default 10 V/s.
--max-spikes N: rows in the per-spike table. Default 25 with --json, every spike with --csv.
--csv sweeps|spikes|fi: print one tidy table as CSV instead.

openreadout analyze ephys-features cell.abf --csv fi > fi.csv

spikes

openreadout analyze spikes [OPTIONS] <FILE>

--trace N: trace index; pick the broadband stream. Default 0.
--channels LIST: channels, such as 0-3,7. Default: all.
--sweeps LIST: sweeps or segments. Default: all.
--band LOW:HIGH: band-pass in Hz (zero-phase Butterworth). Default 300:6000.
--order N: Butterworth order. Default 5.
--threshold K: threshold in noise units (noise = median(|x|)/0.6745). Default 5.
--sign neg|pos|both: spike polarity. Default neg.
--exclude-ms MS: a spike must be the extreme within this many ms on either side. Default 0.1.
--max-seconds S: analyse only the first S seconds of each sweep.
--max-times N: spike times listed per channel. Default 1000.

qpcr

openreadout analyze qpcr [OPTIONS] <FILE>

The input is an RDML file (also a LightCycler 96 .lc96p), an Applied Biosystems .eds, a Rotor-Gene .rex or a LightCycler 480 .ixo.

--well WELL, --target TARGET, --sample SAMPLE, --run RUN: only these records.
--cq: also compute a threshold Cq for every curve and compare it with the vendor’s.
--threshold T: with --cq, the threshold in baseline-corrected units.
--baseline START-END: with --cq, the baseline window in cycles.
--ddcq: relative quantification (2^−ΔΔCq) per sample and target.
--reference TARGET: ΔΔCq reference target. Repeatable. Default: the file’s.
--control SAMPLE: ΔΔCq calibrator sample. Default: the file’s.
--standard-curve: fit a standard curve per target (slope, R², efficiency).
--max-records N: return at most this many records.
--undetermined-as CQ: count wells without a Cq at this value in means and ΔΔCq. Default: leave them out and count them.

openreadout analyze qpcr plate.eds --ddcq --reference GAPDH --control untreated

assay

openreadout analyze assay <wells|curve|dose-response|kinetics|growth|qc> [OPTIONS] <FILE>

wells: per-well values with roles, blank subtraction, replicate statistics and outlier flags.
curve: fit a standard curve and back-calculate every well’s concentration.
dose-response: fit a 4PL or 5PL per compound: IC50/EC50 with confidence interval, Hill slope, top, bottom.
kinetics: per-well max slope, lag time, time to max, mean slope and AUC.
growth: per-well growth rate, doubling time, lag time and a logistic fit.
qc: Z′, signal/background, signal/noise, SSMD and CVs from the control wells.

The input is a plate-reader export or a long CSV with well and value columns.

Flags of every assay subcommand

--layout CSV: plate layout: a plate-map grid or a long table with a well column.
--no-embedded-layout: ignore the layout the export embeds.
--blank WELLS, --positive WELLS, --negative WELLS, --empty WELLS: mark wells (H1,H2 or H1:H12).
--role NAME=ROLE: role of the wells a layout names NAME, such as --role DMSO=negative. Repeatable.
--table N: plate index. Default 0.
--read READ: read to analyse, by 1-based number or label. Default: the first measured read.
--wavelength NM: the wavelength of a spectral read.
--blank-subtraction auto|mean|median|none: default auto.
--outliers grubbs|mad|none: outlier test within replicate groups. Default grubbs.
--outlier-threshold X: alpha (Grubbs) or modified z-score (MAD).
--exclude-outliers: leave flagged outliers out of means, fits and controls.
--csv PREFIX: write tidy CSV tables to PREFIX.wells.csv, PREFIX.samples.csv and so on.
--overwrite: replace existing output files.

Flags of some assay subcommands

--reduce first|last|max|min|mean|max-slope|mean-slope|auc: wells, curve, dose-response, qc: how a kinetic read becomes one value per well.
--window N: points per window, for --reduce max-slope or for kinetics and growth.
--normalize none|controls: wells, dose-response, qc: percent effect between the negative (0 %) and positive (100 %) controls.
--model linear|4pl|5pl: curve, dose-response. Default 4pl.
--weighting none|1/y|1/y2|1/x|1/x2: curve, dose-response. Default none.
--confidence C: curve, dose-response: confidence level. Default 0.95.
--preview PNG: curve, dose-response: draw the points and the fit.
--standard WELLS=CONC: curve: standard wells and their concentration. Repeatable.
--fit-on replicates|means: curve. Default replicates.
--lloq X, --uloq X: curve: limits of quantification. Default: from the standards’ recovery.
--wells WELLS: kinetics, growth: only these wells.
--threshold OD: growth: values at or below this are left out of the log-scale fit.

$ openreadout analyze assay curve assay-synth-elisa-5pl.csv --layout assay-synth-elisa-5pl-layout.csv --model 5pl
assay-synth-elisa-5pl.csv — curve of read 1 `OD450`
layout: assay-synth-elisa-5pl-layout.csv (blank 2, sample 80, standard 14)
blank: mean of 2 wells = 0.0647

standard curve: 5pl [y = d + (a - d) / (1 + (x/c)^b)^g], fit on replicates, weighting none
…
  R² 0.99992, n 14, levels 7, range 15.625–1000, LLOQ 15.625, ULOQ 1000

gate

openreadout analyze gate [OPTIONS] [FILE]...

Without an FCS file, the gating file is described (samples, compensation, transforms, the gate tree) without counts.

--workspace WSP: FlowJo 10 workspace (.wsp).
--gatingml XML: Gating-ML 2.0 document.
--sample NAME: workspace sample. Default: the sample whose name or file matches FILE.
--population PATH: report only this population and its descendants. Repeatable.
--median PARAMETER: report each population’s median of this parameter (Comp-NAME for compensated values). Repeatable or comma-separated.
--table N: FCS data set index. Default 0.

Several FCS files, a directory or a glob give one table with a row per file and population. gate takes the flags in Several inputs and the batch table flags.

openreadout analyze gate sample.fcs --workspace analysis.wsp
openreadout analyze gate fcs/ --workspace analysis.wsp --median Comp-FITC-A -o populations.csv

JSON

peaks, chromatogram, nmr-peaks, ephys-features, spikes, qpcr, assay, gate.

Run openreadout analyze <subcommand> --help for the full help of your installed version.