Artefacts and Noise

Question 1

What are technical artefacts?

Answer 1

Technical artefacts are things that happen during the course of DNA testing that can’t really be reproduced and aren’t reflective of the DNA that is associated with a sample.

Nonetheless they are things that we recognise and see happening quite frequently when looking at DNA tests.

Question 2

What is Stutter?

Answer 2

• At the 15 and 19 alleles, they are very strong peaks and are labelled with the computer software.
• Preceding both peaks, there are other small peaks which correspond to either a 14 or 18 allele. These are stutter peaks
• This occurs during PCR, where the DNA polymerase has a hiccup and slips forward/backwards
• It then makes a PCR product which is often shorter than the true fragment.

Question 3

Why aren’t stutter peaks labelled?

Answer 3

• The labelling software makes use of a stutter filter
• Any peak in the position immediately before the actual peak with a value lower than the threshold (usually 12%) is not labelled.
• Any peak that proceeds which is less than the height of the following peak it is simply not labelled and is written off as an artefact

Question 4

What is a spike?

Answer 4

• A spike is a peak in an EPG that is too tall and narrow relative to what we would typically expect to see in the range of all the other peaks.
• Typical peaks have an expected range of height-to-area or height-to-weight ratios
• If a peak is very narrow and tall, it is a good indication of a spike.
• If a peak is short and wide, it may be a blob.
• These are artefacts of the testing process.

Question 5

What is peak height imbalance?

Answer 5

• Peak height imbalance is where two peaks at a loci are of significantly different peak heights.
• To calculate peak height ratio, take the smaller peak height and divide by the larger peak height.
• If there is a highly different peak height, it could mean we are not looking at a single source sample. One source may have contributed more than the other.

Question 6

What are RFUs?

Answer 6

• Relative Fluorescence Units (RFUs) are measurement in electrophoresis methods.
• It is used in analysis which employ fluorescence-detection
• The intensity of fluorescence is detected by a CCD array, which is proportionate to the quantity or size of the fragments.
• Samples with higher quantities of DNA will have higher RFU values.

Question 7

What are stochastic effects?

Answer 7

• Stochastic effects are manifest as fluctuations of results between replicate analyses.
• I.e. amplifying the same DNA extract twice results in different alleles detected at a locus.
• Can be caused by mutations. Primer binding site mutations affect where the primers bind on to the strand, thus affecting the alleles in the DNA profile.

Question 8

What is the difference between degradation and inhibition?

Answer 8

• Degradation: The deterioration of DNA
  
  • When DNA interacts with other chemicals, it can denature and break down.
  • When this happens, they cannot be amplified as effectively.
• Inhibition: Poor PCR amplification
  
  • A result of the DNA polymerase becoming inhibited during PCR.
  • The DNA is not amplified efficiently. Results in smaller peaks

Question 9

What is typical of degradation in a DNA profile?

Answer 9

• The bigger the fragment, the better the target it is for degradation and more likely effected by inhibitors.
• Bigger DNA fragments take longer to separate, and are found at the end of the EPGs.
• If the peaks are much larger on the left-hand side of the EPG compared to the right, it is obvious that the larger DNA fragments have been inhibited/degraded