Endy:F2620/Data Processing/Algorithm
Contents
Background subtraction
We subtracted a media background, Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle A_{media}} , from the raw absorbance data, Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle A_{raw}} , and assumed that the resulting data, Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle A_{corrected}} , was directly proportional to the number of cells in the well.
Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle \frac{}{}A_{corrected} = A_{raw}-A_{media}} | ...Equation 1 |
We subtracted a fluorescent protein-free cell background, Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle G_{cells}} , from the the raw fluorescent data, Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle G_{raw}} , and assumed that the resulting data Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle G_{corrected}} was proportional to the total number of GFP molecules in the well [include note here about immature GFP?].
Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle \frac{}{}G_{corrected} = G_{raw}-G_{cells}} | ...Equation 2 |
Unit conversion
We then used standard calibration curves (see here for absorbance and here for fluorescence) to convert the background-corrected data into absolute units (CFU/well and GFP molecules per well). The calibration equations used are shown in Equations 3 & 4.
Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle \frac{}{}CFU = 3.1e8 * A_{corrected} - 1.6e6} | ...Equation 3 | |
Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle \frac{}{}GFP = 7.0e8 * G_{corrected} + 6.0e11} | ...Equation 4 |
GFP synthesis rate calculations
To calculate the mean synthesis rate of GFP per cell, Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle S_{cell}} , we assume the total GFP synthesis rate is equal to the time differential of Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle GFP} . Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle S_{cell}} can be calculated as the total synthesis rate divided by Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle CFU} .
Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle \frac{}{}S_{total} = \frac{d[GFP]}{dt}} | ...Equation 5 | |
Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle \frac{}{}S_{cell} = \frac{S_{total}}{CFU}} | ...Equation 6 |
Ania's c++ code
- Load the data from the excel file. Read headers of the column to know the colony number and AHL type used. Form a lookup table. Separate the medium column at this point. Count how many repeats there is for each type.
- Find the GFP background by finding the non-induced column
- GFPpre-fit by fitting all the GFP background columns
- Fit medium for OD (find mean)
- For each OD subtract this mean (media)
- For each GFP we subtract the fitted background calculated for non-induced cells of this type (e.g. for cog-AHL we subtract fitted results for non induced cog-AHL from the raw data)
- Calibrate GFP relative units to conc of GFP (from Barry's calibration run): 1.16*10e-6*GFP+9.95*10e-4
- Fit GFP
- Fit OD (don't fit media anymore)
- Plot GFP, GFP/OD, dOD/dt, gamma, (GFP/OD)/dT, total sythesis and output (this is for debugging and outputs a huge .pdf and .tex)
- to params.csv outputs all the fitting parameters and errors (the errors are not really important)
- to surfaces.tsv output points for superimposed transfer functions in 3D (so you can plot the 3D surfaces yourself)
- evaluate the fitting equations for the total synthesis at many timepoints and select the hightest value from cog-AHL and store this time value.
- output to transfer.tsv all values of total function for all series at time calculated above and low/high repeat, st dev (Mathworld definition, I think it is what you call standard error), 95% confidence
- plot using gnuplot 3D superimposed lines "ser*.pdf" files (we don't use them anymore) surfaces (those are the green ones we post on wiki) into files "sersurf*.pdf") and the transfer functions to "tranfer.pdf" with 95% confidence intervals, transferlh.pdf transfer functions with low/high errors, where * is series number
Notes: 1. Evaluation of the total synthesis function is done using arbitrary precision numbers. B/c they exceeded double range