GMID-X Data Analysis and Evaluation

Question 1

What sizing algorithm is commonly used in data analysis?

Answer 1

Local Southern Method

Question 2

Describe the Local Southern Method?

Answer 2

The Local Southern Method utilizes 4 fragments in the ILS closest to the unknown fragment to determine the unknown fragment’s size. It does this by averaging two size curves: curve 1 uses 2 peaks before and 1 peak after, curve two uses 1 peak before and 2 peaks after.

Question 3

What are primer peaks?

Answer 3

Primer peaks are the result of all the left-over primers from the amplification process crossing the detection cell first due to their small size. Primer peaks are present on the far left side of the egram and in high concentration compared to the allelic peaks.

Question 4

The presence of primer peaks indicates what?

Answer 4

Amp product is present

Question 5

The presence of orange size standard peaks indicates what?

Answer 5

CE master mix is present

Question 6

Is minus (-) A a technological or biological artifact?

Answer 6

Biological

Question 7

What is minus (-) A?

Answer 7

A biological artifact that is due to incomplete adenylation at amplification. -A is present at one base pair shorter than the true peak, resulting in a “split peak”.

Question 8

Is migration a technological or biological artifact?

Answer 8

Technological

Question 9

What is migration?

Answer 9

A slowing or speeding up of the DNA through the capillary due to the environment (i.e. temperature). Usually indicated as multiple OL’s in the right side of the egram.

Question 10

What does the x axis represent on an egram? And what does that correlate to?

Answer 10

X axis = measure of time when the fragment was detected; which correlates to base pair size

Question 11

What does the y axis represent on an egram? And what does that correlate to?

Answer 11

Y axis = intensity/height of the peak (RFUs); which correlates to amount of DNA present

Question 12

What does it mean if a sample is degraded?

Answer 12

DNA is damaged and severed into many pieces. This can be caused by environmental conditions, improper storage, etc. Larger loci are prone to degradation due to longer sequences, this results in the egram exhibiting a downward “ski-slope” effect in peak heights.

Question 13

What is a possible resolution if you have a degraded sample?

Answer 13

Re-amplify the sample with more template DNA to increase the peak heights at the larger loci.

Question 14

What is a null allele?

Answer 14

When a true allele does not amplify, usually due to a primer binding site mutation.

Question 15

What is an allelic ladder?

Answer 15

The allelic ladder is an artificial mixture of the most common alleles in the population and is used to assign allele calls in the evidence sample.

Question 16

How are DNA fragments sized?

Answer 16

DNA fragments are sized using the internal size standard. The size of the DNA fragments is determined by comparing their migration time with those of the ILS and the generated size curve.

Question 17

What is an internal size standard?

Answer 17

The internal size standard is a set of DNA fragments of known length spanning the analysis size range and is used to determine the size of the unknown sample fragments.

Question 18

What is the difference between an OLA and an OMR?

Answer 18

An OLA is an allele that sizes outside of the allele categories in the ladder, and an OMR is an OLA but also sizes outside of the designated locus marker range.

Question 19

How do you know if you are getting the correct profile or if DNA typing works?

Answer 19

By the use of a positive control. Which is a known DNA sample that is analyzed along with the unknown evidence samples. When the correct DNA profile is obtained from the positive control this demonstrates that DNA typing is working correctly and as expected.

Question 20

What is the purpose of a positive control?

Answer 20

The positive amplification control is an analytical control whose purpose is to ensure that the amplification, electrophoresis, and typing processes are working properly.

Question 21

What is the purpose of a negative control?

Answer 21

The purpose of a negative control is to detect DNA contamination. The negative amplification control (AB) is an analytical control used to detect DNA contamination of the amplification reagents, and the reagent blank control (EB) is an analytical control used to monitor contamination from extraction to DNA typing results.

Question 22

What important information can be provided by evaluating the raw data?

Answer 22

Primer peaks present in the raw data indicates that amp product is present in the sample. Orange peaks (or size standard) present in the raw data indicates CE master mix is present in the sample. A lack of colored peaks in the raw data indicates the sample is DNA free. Off-scale data can also be observed in the raw data indicating too much DNA is present in the sample.

Question 23

What are the two types of artifacts?

Answer 23

Biological and Technological

Question 24

What are examples of biological artifacts?

Answer 24

Stutter
-A/spilt peaks/non-template nucleotide addition
Non-specific amplification
Null alleles (not really an artifact)

Question 25

What are examples of technological artifacts?

Answer 25

Migration
Spikes
Dye Blobs
Pull-up
Size Standard Failure/Loss of Resolution

Question 26

What are the expected peak height ratios from heterozygous alleles?

Answer 26

70% (for single source robust samples)

Question 27

What indicator does GMID-X use when expected peak heights ratios are <70%?

Answer 27

Locus will flag yellow

Question 28

What is a microvariant?

Answer 28

A microvariant is an allele that contains incomplete repeats. (i.e. 15.2, 9.3, 27.2, etc.)

Question 29

How do you calculate an OLA?

Answer 29

An OLA is calculated by taking the difference between the OLA’s base pair size and the nearest allele in the allelic ladder’s base pair size.

Question 30

(True or False) A tri-allele cannot be used for comparison or statistical analysis.

Answer 30

False. A tri-allele can be used for comparison but cannot be used for statistical analysis.

Question 31

What is non-specific amplification?

Answer 31

Non-specific amplification is the amplification of DNA that is not the target DNA.

Question 32

What are examples of non-specific amplification?

Answer 32

-extension of misannealed primers
-primer dimers
-bacteria or other foreign material

Question 33

(True or False) Most times non-specific amplification is difficult to identify due to the presence of a stutter peak similar to the desired DNA target.

Answer 33

False. Most times non-specific amplification is easily identifiable due to the absence of a stutter peak.

Question 34

What is a resolution for migration?

Answer 34

Attempt utilizing a different ladder to analyze sample or reinject the sample.

Question 35

What is the cause of migration?

Answer 35

Temperature fluctuations

Question 36

How is loss of resolution observed in the data?

Answer 36

Peak broadening is visual indication of loss of resolution.

Question 37

Visually what is the difference between a degraded sample and an inhibited sample? and what indicator in the DNA analysis process can help identify these types of samples?

Answer 37

Visually a degraded sample will have a ski-slope effect where the larger loci on the right side of the egram will have decreased peak heights/dropout. Visually an inhibited sample will have random dropout that is not dependent on amplicon size. Quantification has indicators to detect degraded and inhibited samples. The Degradation Index is used to determine if a sample is degraded and the IPC value is used to determine the possibility of inhibition.