calibratePCRsim: Calibrate PCRsim

Description

Estimates the detection threshold, peak height scaling factor, and PCR efficiency needed to calibrate PCRsim.

Usage

calibratePCRsim(data, target = NULL, ref = NULL, quant = NULL, ignore.case = TRUE, kit = NULL, db = NULL, fixed.profile = NULL, sim = 1, pcr.cyc, ce.aliq = 1, pcr.aliq = 17.5, pcr.vol = 25, minimize = TRUE, step.size = 0.001, cell.dna = 0.006, dna.amount = 0.5, filter = TRUE, plot.data = TRUE, decimals = 4, debug = FALSE, ext.debug = FALSE)

Arguments

data

data.frame with observed DNA result. Required columns are sample names ('Sample.Name'), average peak height ('H'), and (mean) DNA concentration ('Mean').

target

integer average peak height ('H') as observed average across replicate analyses of samples with dna.amount of DNA. If NULL it will be estimated from the linear regression at dna.amount of DNA.

ref

data.frame with known profiles for the samples in data. Required columns are 'Sample.Name', 'Marker', and ('Allele'), and (mean) DNA concentration ('Mean').

quant

data.frame with (average) 'Concentration' or 'Amount' for the samples in data.

ignore.case

logical TRUE to ignore case in sample name matching.

kit

character string to specify the STR DNA typing kit to simulate. If NULL all markers in db will be used.

data.frame with the allele frequency database (if random profiles are simulated).

fixed.profile

data.frame with columns 'Marker' and 'Allele' (if fixed profiles are simulated).

sim

integer the number of simulations per calibration cycle.

pcr.cyc

integer the number of PCR cycles.

ce.aliq

integer the aliquot PCR product used for capillary electrophoresis.

pcr.aliq

integer the aliquot DNA extract transferred to the PCR reaction.

pcr.vol

integer the total PCR reaction volume.

minimize

logical TRUE stops when the squared difference is minimized, FALSE continues until the PCR efficiency is 0.

step.size

numeric size of PCR efficiency reduction for each calibration cycle.

cell.dna

numeric the DNA content of a diploid cell in nanograms (default is 0.006 ng).

dna.amount

numeric the amount of DNA in nanograms (ng) to be used in simulation. NB! must correspond to the amount in samples used to calculate target.

filter

logical TRUE to retrieve known alleles defined in ref from data.

plot.data

logical to show linear regression data plot with marked target.

decimals

interger for number of decimal places in plot title.

debug

logical to print debug information.

ext.debug

logical to print extended debug information.

Details

To calibrate the PCR simulator perform the following experiments: 1) Prepare single source samples with optimal amount of DNA (different or replicates) Calculate the average total peak height (sum of peak heights) across all samples to set the target parameter. 2) Prepare a serial dilution (preferrably from intact cells, or from single source crime scene samples). It is suitable to go from approximately optimal amount down to low concentrations with drop-outs or completely blank profiles. Quantify each dilution as accurately as possible. Amplify using normal procedure and analyse the PCR product. Require a dataset with sample names ('Sample.Name'), and (average) concentration or (average) amount in columns named 'Concentration' and 'Amount' respectively. Also requires the average peak height ('H').

NB! Samples with either zero average peak height or zero number of molecules is removed automatically. In addition, if replicate quants, samples where one replicate was negative can be removed manually.