Topic 8 - Artefacts and Noise

Question 1

what are the two types of artefact

Answer 1

biological and instrumental/technical

e.g biological = things in PCR mix or extraction that may cause extra peaks

Question 2

what is meant by an artefact

Answer 2

something that happens randomly during testing that can’t really be reproduced

they are not reflective of the DNA in the sample but do occur frequently and can make interpretation difficult

Question 3

what type of artefact is stutter

Answer 3

a biological artefact

Question 4

what is meant by a stutter peak

Answer 4

very small peaks seen to the left of larger ones in
electropherograms

this is often slightly shorter than the true fragment length (normally 1 base pair smaller)

Question 5

how do stutter peaks occur

Answer 5

during PCR amplification sometimes DNA polymerase slips forward or backward slightly

Question 6

do stutter peaks normally rise above the stochastic and analytical thresholds

Answer 6

no so they are not significant and are not confused with allele peaks

Question 7

how are stutter peaks
a) identified
b) removed

Answer 7

a) by their peak height and position

b) computer software can apply a stutter filter so these peaks are not labelled

Question 8

what is something that is associated with DNA replication that can result in artefacts

Answer 8

primer binding site mutations

Question 9

what type of artefact is a spike

Answer 9

instrumental

Question 10

what is meant by a spike peak

Answer 10

a peak that appears too tall and narrow to represent an allele peak

the peak height ratio is likely to be off the expected range of an allele peak

a spike in the electrical current in the genetic analyser

Question 11

what type of artefact is a blob

Answer 11

instrumental

Question 12

what is meant by a blob peak

Answer 12

a very short and squat peak that doesn’t represent a typical allele peak

Question 13

name two things that could result in spike and blob peaks

Answer 13

1. particles passing through the capillary
2. dust moving in front of the detector

Question 14

how can spikes and blob be identified

Answer 14

we can use peaks from the reference sample/positive control to see what a normal allele peak ratio is of its height to area

if a peak in the sample falls out this range it is likely to be a spike or blob

also known as a peak height imbalance

Question 15

what is a pull up peak associated with

Answer 15

the dyes used the process

Question 16

how is the ‘amount’ of DNA measured for each fragment in electrophoresis

Answer 16

the fluorescence is measured using a charged couple device (CCD) array

different length fragments are separated in capillary electrophoresis and are detected as they pass through the capillary

a computer programme converts this to an EPG

measured in RFU’s a higher quantity of amplified DNA has a higher RFU

Question 17

name 7 things a peak height imbalance of two allele peaks could suggest

Answer 17

1. the presence of a mixture rather than a single source profile
2. one of the peaks in an artefact
3. contamination
4. primer binding site mutation
5. we are dealing with small amounts of DNA
6. degradation
7. inhibtion

Question 18

what percentage signifies two well balanced peaks in a single source profile

Answer 18

a ratio of closer to 100%

most labs have a cut off at 70% but some use 60% - this is dependent on the lab and is signed off by the regulator

so if the ratio of two peaks is above this value we say they are balanced

Question 19

what is meant by a stochastic effect

when do these tend to happen more

Answer 19

a random event

when we are dealing with low levels of DNA

Question 20

what type of artefact is primer binding site mutation

Answer 20

biological

Question 21

what two things can be used to help us identify artefacts

Answer 21

the PCR +ve and the extraction -ve

Question 22

what is meant by degradation and inhibition

Answer 22

degradation = the deterioration of DNA

inhibition = poor amplification in PCR

Question 23

what types of DNA fragments are more susceptible to be degraded or inhibited

Answer 23

larger fragments as they are a better target

they are harder to amplify as they are bigger and it takes longer

Question 24

name 4 stochastic effects

Answer 24

drop in
drop out
peak height imbalance
stutter

Question 25

if a sample is heavily degraded what might this result in

Answer 25

alleles peaks not reaching the threshold and therefore not being labelled in the EPG

allele drop out may be observed

Question 25

what does the appearance of a ski slope effects in an EPG suggest

Answer 25

the DNA in the sample has been degraded or inhibited

Question 26

what can the presence of artefacts lead to

Answer 26

misinterpretation or false assumptions

Question 27

what is meant by locus dropout

Answer 27

when no peaks are observed for a locus

Question 28

what is set to avoid confusion of peaks with baseline noise

Answer 28

a minimum peak height threshold = 150 RFUs in most labs but some go down to 100

any peak below this is discounted

Question 28

what may be a reason for seeing an unbalanced third peak at a locus

Answer 28

baseline noise of the instruments being used

Question 29

what two features does an instrument have to help distinguish between allele peaks and noise

Answer 29

limits of detection
limits of quantification