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FSCI6370 - DNA > Artefacts and Noise > Flashcards

Artefacts and Noise Flashcards

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1
Q

Define artefacts

A
  • things that happen during testing that can’t really be reproduced
  • aren’t reflective of the DNA that is associated with a sample
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2
Q

What is a stutter (state the type of artefact)

A
  • biological artefact
  • small peaks found in position immediately before the peak that gives rise to them
  • quite common
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3
Q

How does a stutter occur?

A
  • occurs during PCR amplification
  • when DNA polymerase has a hiccup and slips a bit forwards or backwards
  • makes a PCR product which is a little bit shorter than true fragment
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4
Q

Why are stutters not a problem in DNA analysis

A
  • software makes use of a stutter filter
  • any peak, in the position immediately before the (actual) peak with a value lower than the cut-off – (usually 12%, sometimes 15%) is not labelled and is written off as an artefact (we know is a stutter)
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5
Q

What does helicase enzyme do?

A
  • enzyme used in DNA replication to unwind DNA molecule from its tightly woven form
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6
Q

What is a spike (state the type of artefact)

A
  • instrumental artefact (technical)
  • a peak that you see in EPG that is too tall and narrow relative to what we would typically expect to see in the range of all the other peaks
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7
Q

What is a blob (state the type of artefact)

A
  • instrumental artefact (technical)
  • short and squat peak in comparison to what we would expect
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8
Q

How can we tell spikes and blobs from actual peaks?

A
  • typical peaks have an expected range of height to area or height to weight ratios
  • if they do not fit in this expected range
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9
Q

What causes spikes and blobs?

A
  • originated from testing process
  • not rooted from any DNA that is associated with a sample
  • not actually known
  • possibly associated with voltage spikes when sample is running through genetic analyser
  • maybe particles that are passing through capillaries or dust moving in front of the camera
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10
Q

What is important for the DNA analyst to do when it comes and spikes and blobs?

A
  • important that the DNA analyst can recognise them and say that this is not something that is rooted in the DNA associated with the sample
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11
Q

How are spikes and blobs recognised?

A
  • get peaks that come from reference samples from positive controls
  • spikes/blobs not part of reference positive control samples fall outside this range
  • they appear in top-left hand quadrant of X and Y’ chart
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12
Q

What are RFUs?

A
  • relative fluorescent units
  • unit of measurement used in analysis which employs fluorescence-detection
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13
Q

How is fluorescence detected?

What is detection used to determine?

What does a high RFU value correspond to?

A
  • using a charged couple device (CCD) array when labelled fragments, which are separated within a capillary by using electrophoresis, are energised by laser light and travel across the detection window
  • a computer program measures the results, determining the quantity or size of the fragments from the level of fluorescence intensity
  • high RFU value = contain higher quantities of amplified DNA
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14
Q

How do we measure peak height ratio?

A
  • (height / height) x 100
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15
Q

What does it mean if the peak height ratio is low (peaks not the same height)

A
  • not looking at a single source, but a mixture
  • one person contributed more DNA than the other initially
  • artefact associated with the sample and these peaks were not tall to begin with
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16
Q

What is a stochastic effect?

A
  • when we have small amounts of starting material
  • probabilistic effects that occur by chance
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17
Q

What is primer binding site mutations?

A
  • most common form of mutation
  • maybe one allele has a mutation that prevented it from generating a profile as well
18
Q

What is a peak height balance ratio used for?

A
  • when labs use a testing kit they use a cut-off of a 70 % peak height balance (some will use 60 %)
  • if the peak height balance (smaller/larger x 100) is higher than that they are balanced
19
Q

What does it mean if two peaks are balanced?

A
  • likely from a single source
  • no problems with amplification process
20
Q

What does it mean if a peak height balance is below the threshold set by the lab?

A
  • could be a mixed sample
  • might be dealing with a small amount of material
  • primer binding site mutation has taken place
21
Q

What is a synonym for technical/instrumental artefacts?

A
  • degradation
  • inhibition
22
Q

Define degradation

A
  • degradation is deterioration of DNA
  • as DNA is a chemical, it can interact with other chemicals which can cause DNA to get damaged/broken
  • when this happens DNA molecules cannot be amplified through PCR process
  • therefore they cannot contribute to EPG’s or at least not as much as before degradation occurred
23
Q

Define inhibition

A
  • inhibition is poor PCR amplification
  • sometimes other chemicals that make it harder for DNA polymerases to do their jobs during PCR
  • therefore DNA doesn’t get amplified as efficiently as it would have in absence of chemicals
  • smaller peaks
24
Q

What is the relationship between size of DNA fragment and target for degradation/inhibitors

Explain why

A
  • bigger fragment = better target for degradation/more likely to be affected by inhibitors in amplification process
  • a bit piece of DNA takes more time to go through capillaries
  • it is harder to get amplified during PCR amplification process than small one
  • if got something in mix that is making it hard for DNA polymerase to amplify the DNA during PCR that effect is going to be observed more for bigger pieces of DNA that smaller ones
25
Q

Give four examples of stochastic effects?

A
  • imbalance
  • drop-in
  • stutter
  • drop-out
26
Q

What is difference between homozygote and heterozygote peak heights?

A
  • homozygote peaks - singular peaks of same allele
  • taller than hetero peaks because there are two of the same alleles
  • heterozygote peaks - two peaks that are different alleles found at the same locus
  • smaller peak height compared to homozygous peaks
27
Q

Where do different size fragments appear on the EPG?

A
  • smaller = LHS
  • smaller fragments of DNA are first ones that come off the capillary during capillary electrophoresis
  • RHS because they take longer to travel through the capillary during electrophoresis
28
Q

How does degradation and inhibition effect an EPG?

A
  • RHS is getting shorter and that height decreases progressively as move from left to right
  • this is because the larger peaks are more likely to be degraded
  • this creates a ski slope (slant on EPG left to right - hallmark of a sample which has suffered from inhibition/degradation)
  • this ski slope means we can get allele drop-out take place at far RHS
29
Q

What is allelic dropout?

A
  • the test has failed - got incomplete picture of actual DNA profile
  • with degradation and inhibition, dropout can easily occur particularly for loci that give rise to peaks on RHS of EPG
  • alleles present in less degraded sample but not showing up on more degraded sample
30
Q

What is locus drop out?

A
  • when both alleles in a particular locus have dropped out so there is no alleles present on EPG at that locus
31
Q

What type of issues are more of a type of problem in evidence samples rather than reference samples?

A
  • peak height imbalance
  • degradation
  • inhibition
32
Q

What can be said about recognition of artefacts?

A
  • often they can be recognised and at the very least they raise some questions about how certain we can be about the reliability of the test and the conclusions that can be drawn from it
33
Q

When undertaking interpretations of EPG, what do we need to understand?

A
  • need to understand clearly what is signal and what is noise
34
Q

What is baseline noise?

A
  • unbalanced third peak at locus
35
Q

What probabilistic effects we may encounter with low-level DNA material?

A
  • stochastic effects
36
Q

In which way do labs distinguish between signal and noise?

A
  • have a minimum peak height threshold
  • if it is not above this, it is written off
  • most labs settle around 150RFUs
  • some 200, some 100
37
Q

How are the minimum peak height thresholds determined?

A
  • during validation studies
38
Q

What can be said about thresholds by labs?

A
  • they eliminate noise (even at the cost of eliminating signal)
  • some will say they can arbitrarily remove legitimate signal
39
Q

How are baseline fluctuations/noise created?

A
  • any kind of instrument that is recording voltages or light impulses there will be fluctuation of the baseline, just a random noise (a static), sort of effect
  • sometimes the static will be for instance louder than at another time, we can measure the heights of those random bits of noise and get an average value and a st. dev. associated with that the average value for background noise
40
Q

What is LOD?

A
  • limit of detection
  • mean background signal + 3 st. dev.
  • if something above this, very unusual it is noise or something else
41
Q

What is LOQ?

A
  • limit of quantification
  • mean background signal + 10 st. dev.
42
Q

When are there more likely to be problems with allelic and locus drop out?

A
  • when small amounts of material are used to generate DNA profiles

FSCI6370 - DNA (16 decks)

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