STR Genotyping and Data Interpretation

Question 1

steps in STR genotyping

Answer 1

spectral calibration
user-defined thresholds
internal size standard
allelic ladder sample
data collection
color separation *
peak identification *
peak sizing *
compare to allelic ladder *
genotype assignment to alleles *
*all preformed using GeneMapperID software
peak editing to remove artifacts, done by analyst
produces electropherogram
data review by analyst
2nd read

Question 2

raw data

Answer 2

Yaxis is RFU
relative fluorescence unit
X axis is time (data points), clicks of time
primer peak
all samples have primer peak, must have or something went wrong

Question 3

distinguishing peak from baseline nosie

Answer 3

there is always baseline noise
the signal needs to be distinct from the noise and reliably reflect the dna molecules present in the sample
a common peak detection threshold is 50 RFU
peaks above 50 RFU will be considered true DNA peaks or artifact
there are several other methods to determine AT (analytical threshold)
3x noise
measure hight of baseline noise on negative sample and x3
30 RFU, more sensitive

Question 4

analytical threshold

Answer 4

machines differ
colors can be noisier than others
diff threshold b/n instrumnent
diff thresh between color on same instrument
vary typically b/n 50 RFU to 200 RFU