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
what does the genemapper Y-axis measure and what does it correlate to
- measures the intensity/height of the peak in RFUs
- correlates to the amount of DNA present
26
what is an inclusion
- 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
what is an inconclusive conclusion
no conclusion (inclusion/exclusion) can be drawn after comparison to the known
28
what does indistinguishable mean
- 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
29
what do inhibited samples look lik
- differ from degraded samples because the loci dropout is not related to amplicon size
- loci drop out at random locations
- resolved by re-amplifying the sample with less template
30
what are inhibitors
inhibitors are substances which interfere with DNA extraction and/or amplification, causing weak and/or no amplification
31
what are some inhibitors in CE
- common inhibitors:
- soil (humic acid), sand, wood, and vegetation
- textile dyes (indigo) and tannic acid (leather)
- hemoglobin, EZ2 magnetic beads, envelope adhesive
- inhibited samples show:
- increased stutter peaks
- heterozygous peak imbalance
- allele or locus dropout
32
what is the internal size standard
- mixed with amplified product prior to being loaded in the C E
- contains DNA fragments of known length that are utilized to determine the size of the unknown fragments
- the size of the unknown fragments is determined by comparing their migration time with the ILS (WEN_500)
- uses the Local Southern Method sizing algorithm
33
what are the internal size standard peaks
- 60, 65, 80, 100 (tall), 120, 140, 160, 180, 200 (tall), 225, 250, 275, 300 (tall), 325, 350, 375, 400 (tall), 425, 450, 475, 500 (tall)
- WEN contains 21 single-stranded fragments ranging from 60-500 bp
- samples with a failed ILS may be re-plated or re-injected
34
what is the purpose of interpretation
- the evaluation of DNA results obtained from evidence based on internal validation studies
- interpretation goals:
- distinguish between alleles and artifacts
- determine if profile is single-source or mixture
- determine number of contributors
- separate mixture into possible donors (determine genotypes)
- compare references to questioned samples
35
what is an intimate sample
- biological sample from an evidence item that is obtained directly from an individual’s body
- can be used to assume a known contributor in a mixture
36
what do ladders & PCs look like on an egram
- if the locus label is green, then the software has verified that the ladder/PC has been successfully typed
- yellow locus label indicates peak imbalanced (<70% peak height ratio)
37
what is the local southern method
- calculates a curve with 2 ILS peaks before the unknown fragment and 1 ILS peak after
- calculates a second curve with 1 ILS peak before the unknown fragment and 12 ILS peaks after
- averages the two curves together
38
what are migration issues and what causes them
* technological artifact
- DNA fragments migrating at different rates between injections on the same run
- if samples and ladder aren’t sized in the same range, then the sample peaks won’t get the correct allele calls
- results in multiple OLs towards to right side of the egram
- temperature also caused migration (higher temp = migrate faster)
- resolves by using a different ladder or reinjecting
39
what are mixtures
- when more than one individual contributes DNA to a sample
- the chance of identifying a mixture improves with the use of more loci that have a high incidence of heterozygotes
- as contributors increase and amount of DNA decreases, the more complex the interpretation
40
what happens in mixture interpretation
- very important to determine the number of contributors
- peak height ratios and mixture ratios are very critical
- there is always potential for dropout and low peaks that are hard to distinguish from stutter
- more ambiguity
- stutter can inflate the RFUs of a peak and change the PHR or mixture ratio
- degradation can occur at different rates for different contributors
- important to look at data in stochastic range (below 350 RFU)
- mixtures with >2 contributors are uninterpretable manually
41
what are mixture ratios
- determining the “weight” of possible genotypes and the expected “weight” of the contributors
- can also be used to calculate the expected contribution of each contributor
42
what are negative controls in CE
- the analyst must confirm that their are no peaks in the two ABs or the EB(s)
- all samples associated with a failed AB or EB may be re-amped
- evidence associated with failed EBs/contamination is considered inconclusive
- notify someone is both PC’s or both AB’s fail
43
non-human DNA in egram
- tested during developmental validation for cross-reactivity
- 10 species had peaks that were more easily detected
44
what is non-specific amplification
* biological artifact
- when PCR primers anneal to template sequences that are not perfectly complementary
- occurs are lower temperatures, creating templated for undesired amp products
- primers can also anneal to each other, creating primer dimers
- non-specific PCR products are reproducible and are identifiable by the absence of a stutter peak
45
what are null alleles
* biological artifact
- a null allele is present in the sample but not amplified
- often caused by a primer binding site mutation
- disrupts hybridization of the primer
- results in failure to amplify
- results in failure to detect an allele that exists in the template DNA
- some kits contain degenerate primers that amplify the “problem” alleles
- only an issue when 2 different PCR kits are used as they can yield non-concordant results (CODIS)
46
explain number of contributors
- dictates the guidelines used to perform interpretation of a sample
- depends on the number of alleles present, PHR, and PABT
- after a sample is determined to be a mixture (2+ contributors), the number of donors has to be determined
- 2 person mixture = max 4 peaks per locus
- 3 person mixture = max 6 peaks per locus (trueallele only)
- 4 person mixture = max 8 peaks per locus (trueallele only)
47
what are off-ladder alleles (OLA/OL) and how do you calculate them
- GMID-X accurately labels alleles not present in the ladders if the variants are within the size range of the locus
- peaks outside the range of the ladder and not identified by GMID-X will be designated as OLA/OMR
- in order to calculate the allele location of the OLA, the base pair size is subtracted from the nearest allele’s base pair size
- OLA = 257.51 bp
- Allele 28 (ladder) = 256.64 bp
- 257.51 - 256.64 = 0.87 ~ 1 bp
- Allele 28 + 1 bp = 28.1 OLA
- rename the OLA in GeneMapper after calculating
48
what does off-scale data look like
- when sample DNA peaks are as high as the primer means
- means the sample was over-amplified and not diluted enough
- sample will need to be re-amplified with less template DNA
- over-amped DNA can also show below the X-axis
- when too much DNA is added to PCR, the fluorescence intensity of the tagged amplicons exceeds the range of detection
- off-scale data should not be used for comparison purposes
49
what are outside marker range alleles (OMR)
- treated similarly to an OLA, but it has to be assigned a locus before the allele can be calculated
- is calculated the same as an OLA
50
what are peak heights measured on an egram
- homozygote allele peak heights are ideally twice that of heterozygote peak heights
- the expected peak height ratio (PHR) for heterozygous alleles is ~70-100%
- if the locus label is yellow, it indicates that the peak height ratio is <70%
51
what are peak height ratios (PHR)
- the attempt to pair alleles based on their ratios
- ratios decrease as the alleles approach the stochastic threshold
- expected heterozygote PHR is 70%
- validation studies determine an accurate peak height ratio
- peak height of smaller peak/peak height of taller peak (100) = PHR%
52
explain percent contribution for mixtures
- calculated manually for each locus
- (RFU contributor 1)/(RFU contributor 1 + RFU contributor 2)(100) = % contribution of contributor 1
- (RFU contributor 2)/(RFU contributor 1 + RFU contributor 2)(100) = % contribution of contributor 2
- some mixtures use an assumed contributor (usually a victim reference) to sort out of contributors
53
what are/is possible alleles below threshold (PABT)
- indicates that dropout has occurred
- the baseline has to be examined for PABT
- PABT affects the number of contributors and if a profile is interpretable for not
54
whata are primer peaks
- peaks from leftover primers that crossed the detection window because of their small size
- assessing primer peaks is important in injections with no data (blanks, ABs)
- the presence of primer peaks means an amp product is present
- equally spaced orange peaks are the size standard
- means CE master mix is present
55
what are pull-up peaks
* technological artifact
- signal that carries over to an adjacent color channel
- occurs when the analysis software fails to separate spectral overlap of the dyes OR by excessively high peaks
- approximately <3% of the source peak
- may be labeled as an OL or an allele
- has to be distinguished from true peaks and Possible Alleles Below Threshold (PABT)
- resolved by analyzing the same and re-amping with less template
56
what is raw data
- spectral data collected by egram
- has not been analyzed, so all peaks are displayed on top of each other
- primer peaks are much higher in concentration
57
what is a reagent blank control/extraction blank (EB)
a negative analytical control that is used to monitor contamination from extraction to DNA typing results and contains no intentionally added template DNA
58
what are reasonable expectation samples
- samples that are part of a scenario that provide feasibility for an individual’s DNA to be present
- ex: finding DNA on a steering wheel that belongs to the owner of the vehicle
59
what is the saturation threshold
- an RFU threshold that is only applied when baseline noise or pull up is present above the AT
- causes artifacts to be labeled as true peaks
- differentiating artifacts from true peaks is more difficult in mixtures
- LSPCL Fusion 6C Saturation Threshold: 300 RFU
60
explain size standard failure
* technological artifacts
- loss of resolution (peak broadening)
- preaks gradually become wider in the larger loci
- in extreme cases, causes size standard failure
- resolved by re-injection or re-plating
- oversaturation of CCD camera
- caused by off-scale data that causes pull-up into the size standard
- resolves by re-amping with less template
61
what is the purpose of statistical calculations
- used in interpretation to help support comparisons to single-source profiles or contributors from mixed profiles
- statistical calculations prove the significance of a match or an inclusion
62
what is the stochastic threshold (ST)
- the peak height or signal below which it is reasonable to assume allelic dropout may have occurred
- occurs in heterozygotes
- in mixtures, if all potential alleles are above the ST, it is reasonable to assume that dropout has not occurred
- LSPCL Fusion 6C Stochastic Threshold: 350 RFU
63
explain stutter
- a minor peak typically observed one repeat unit shorter than the primary allele (n-4; reverse stutter)
- can sometimes be one repeat unit longer (n+4; forward stutter)
- typically <20% the height of the parent peak in reverse, and ~3% in forward
- stutter percentages increase with allele fragment length
- GMID-X filters out stutter with filters derived from internal validations
- if the LSPCL stutter filters aren’t accurate, it can caused difficulties when determining NOC and interpreting mixtures
- stutter can be elevated when amplifying low-level DNA
64
what are some technological artifacts
* dye blobs
* electronic spikes
* formamide blolbs
* migration issues
* pull-up peaks
* size standard failure
65
what is a tri-allele
- a three peak pattern exhibited at a locus of a single source
- result from extra chromosome fragments being present in a sample that produces an additional PCR product
- tri-alleles can have two peaks that equal the height of the third, or three equal-height peaks
- true tri-alleles must be re-amped
- cannot be used for statistical analysis
66
what is a true peaks vs. an artifact
- true peaks are distinguishable from background noise
- non-allelic peaks are biological (from sample) or technological (from instrument) artifacts
- artifacts indicate a sample has been over-amplified, experienced thermal cycler problems, or electrophoresis problems
- can be solved with dilution, re-injection, re-plating, or re-amp
67
what do typical egram loci look like
- green loci with a PHR >70%
- no more than two peaks per loci for single-source
- no more than four peaks per loci for two contributors
- yellow loci with a PHR <70% or >9000 RFU
- red loci with complete dropout
68
what is the uninterpretable criteria for complex mixtures
- the presence of more than 2 contributors
- potential biological relationships that interfere with determining the ncon
- combination of human and non-specific amplification
- extraneous peaks
69
what is the uninterpretable criteria for limited single source and mixtures
- less than 18 loci without the possibility of dropout
- profiles in stochastic with activity below the analytical threshold indicating that additional contributors may be present
- profiles with peak heights at multiple locations indicating that additional contributors may be present
70
what are virtual alleles
- alleles that aren’t marked in the ladder (outside a bin) but have been reported in the NIST STR database of discovered in a validated study
- they can be genotyped in reference to the alleles labeled in the latter
- off ladder alleles (OLA) and off marker range (OMR) alleles are both virtual alleles
