GeneMapper & Interpretation Flashcards
1
Q
what causes -A and split peaks and what does it look like
A
- Taq polymerase adds an extra nucleotide (usually adenine) to the 3’ end of a PCR product as it copies the template strand
- called adenylation, the +A form of an amplicon
- if PCR ends before adenylation, the amplicon is one base pair shorter than the true peak
- the -A form of the amplicon
- creates a “split peak” with a shorter -A peak and a taller +A peak
- occurs with too much template DNA
- adenylation is most common in amelogenin
2
Q
explain allele designation
A
- once the DNA fragment is sized, an allele call needs to be determined
- allele calls are assigned by comparing the sizes of the unknown fragments with the sizes in the allelic ladder
3
Q
what is an alleleic ladder
A
- an artificial mixture of common alleles present in the population
- each plate must contain at least one successful allelic ladder
- ladders do not represent all possible alleles
4
Q
explain the analytical threshold
A
- defines the height requirement at which peaks can be distinguished from background noise
- data from any analytical instrument will contain background noise
- peaks above the AT are considered reliable
- LSPCL Fusion 6C Analytical Threshold: 100 RFU
5
Q
what are the different panels for analyzing CE data
A
- analysis method: forensic 6C
- panel: forensic powerplex fusion6C panels
- size standard: WEN ILS 500
- SOS: sizing off scale
- green square indicates peaks are within range
- yellow triangle indicates peaks are above range
- SQ: sizing quality
- green square indicates good size standard quality
- yellow triangle indicates peak broadening or artifacts in ILS
- red circle indicates sizing failure
- MIX: peaks present above analysis threshold
- green square indicates possible single source
- yellow triangle indicates possible mixture sample
- OMR: off ladder allele/off marker range peaks present
- green square indicates none present
- yellow triangle indicates some present
6
Q
how do we assess ABs and EBs
A
- if the locus label is green, then no alleles calls above the analysis threshold are present
- correct sample type must be designated
- the baseline needs to be examined to ensure no peaks are present below threshold
7
Q
what is an amplification negative control (AB)
A
detects DNA contamination of the amplification reagents
8
Q
what are some biological artifacts
A
- stutter
- -A, split peaks, non-template nucleotide addition
- non-specific amplification
- null alleles
9
Q
how do we calculate stuter
A
- if stutter is suspected or called on the egram, it should be calculated and documented
- each locus has specific stutter percentage
- can be removed if it is within 2% of those filters
- stutter is calculated by dividing the smaller RFU value by the larger and multiplying by 100
10
Q
what is a contributor ratio
A
- related to percent contribution calculations
- separate major and minor contributors
- add all suspected major alleles and divide by the sum of all suspected minor alleles
- if the ratio is >3:1, it is distinguishable
11
Q
explain the data interpretation steps
A
- evaluate the data to determine if the results are interpretable or uninterpretable
- establish the minimum number of contributors and evaluate sex determination
- designation of true peaks and artifacts should always occur prior
- determine if mixture data is distinguishable or indistinguishable
- designate genotypes if applicable
- utilize available assumptions for further resolution of genotypes if appropriate
- compare to applicable reference samples
- perform statistical evaluation when applicable
12
Q
explain the data review steps
A
- view the raw data for each ladder, control, samples, and primer peaks
- apply analysis settings and analyze the project
- assess the internal size standards, allelic ladders, positive controls, negative controls, and reagent blanks
- assess each sample for the presence of extraneous peaks and determine if they interfere with interpretation
13
Q
what do degraded samples look like
A
- DNA is susceptible to degradation, meaning the DNA molecules can be severed into pieces
- damaged by the environment, improper storage, temperature, etc
- degradation appears as a “ski-slope” on an egram
- loci with high molecular weights and longer sequences are prone to degradation
- quant results can indicate degradation
- resolves by re-amplifying the sample with more template
14
Q
what is differential amplification
A
when amplification is better at some loci but shows dropout at others
15
Q
what is differential degradation
A
- when two or more biological samples in a mixture show different levels of degradation
- complicates interpretation
16
Q
what does distinguishable mean
A
- a mixture can be either distinguishable or indistinguishable based on how much DNA each contributor contributes
- if a mixture is distinguishable, it can be easily sorted into individual profiles (major/minor)
17
Q
what is a DNA analyst
A
- someone who can conduct the analysis of forensic samples, interpret data, reach conclusions, and generate reports
- stated in the FBI QAS
18
Q
what are dye blobs
A
- technological artifact
- free fluorescent dye molecules that coexist with dye-labeled primers
- by-product of incomplete coupling during primer synthesis
- occur in the 6-90 base pair range
- more common than older kits
- broad, rough, low-level peaks
- easily observed in negative controls
- interfere with low-level DNA detection
19
Q
what are electronic spikes
A
- technological artifact
- caused by electrical impulse during injection
- not reproducible and can be resolved by reinjection
- usually present linearly in all dye channels
- can be thin, sharp peaks, or broader blobs
20
Q
describe electropherogram documentation
A
- automatically documented:
- project name, well position, sample ID, plate ID, instrument name, injection time
- in batch table settings:
- analysis thresholds for allele labels (ladders and amp controls)
- documented by analyst:
- analysis thresholds for sample e-grams
- if necessary, document reamplifications, re-extractions, and TrueAllele analysis
21
Q
what is an exclusion
A
- an individual can be excluded/eliminated as a potential contributor of DNA obtained from an evidentiary item
- an individual can be excluded as a contributor of a DNA sample if at two or more loci, genotypes differ between the known individual and the evidence profile
22
Q
what are formamide blobs
A
- technological artifact
- formamide can decompose over time in formate
- formate ions are preferentially injected into the capillaries, causing signal loss
- resolves by replating with new master mix and fresh formamide
23
Q
what is genemapper ID-X
A
- a data analysis software that converts raw capillary electrophoresis data into an electropherogram
- genemapper can resolve dye colors for each peak, size DNA fragments, compare alleles to an allelic ladder, assign allele calls, and provide quality indicators
- in CODIS, is it used as an expert system to streamline database analysis (forensics only interprets)
24
Q
what does the genemapper X-axis measure and what does it correlate to
A
- measures time when the DNA fragment was detected
- correlates to base pair size
25
Q
what does the genemapper Y-axis measure and what does it correlate to
A
- measures the intensity/height of the peak in RFUs
- correlates to the amount of DNA present
26
Q
what is an inclusion
A
- an individual cannot be excluded as a potential contributor of DNA obtained from an evidentiary item
- an individual will be included as a contributor is the known individual’s alleles match the alleles of the evidence profile
27
Q
what is an inconclusive conclusion
A
no conclusion (inclusion/exclusion) can be drawn after comparison to the known
28
Q
what does indistinguishable mean
A
- a mixture can be either distinguishable or indistinguishable based on how much DNA each contributor contributes
- if a mixture is indistinguishable, there is no clear difference as to which profile contributed which DNA
