Section 15
Approved by:
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INTERPRETATION GUIDELINES FOR STR ANALYSIS USING THE 310
Scope:
To provide guidelines to the scientist for the typing and interpretation
of short tandem repeat (STR) results generated using the
AmpFtSTR® kits and the 310 Genetic Analyzer, so that the
conclusions in casework reports are scientifically supported by the
analytical data. It should be emphasized that not every situation can
be covered by a pre-set rule.
Background Information:
AmpFtSTR® Profiler PlusTM User's Manual, Applied Biosystems, a
division of Perkin-Elmer.
AmpFtSTR® CofilerTM User's Bulletin, Applied Biosystems, a division of
Perkin-Elmer.
"Minnesota BCA DNA Procedure Manual".
"Short Tandem Repeat (STR) Interpretation Guidelines." Scientific
Working Group on DNA Analysis Methods (SWGDAM), Forensic Science
Communications, Vol. 2, No. 3, July 2000.
Operation:
A.
Assessment of Successful Electrophoresis
1.
Check the GeneScan ROX-500 internal sizing standards of all
ladders, samples, and controls to ensure that all the peaks were
sized correctly and that no extraneous peaks above 150 relative
fluorescent units (RFU) are-present (F-spikes are an exception to
this).
2.
If the ROX-500 sizing standard fails, that sample must be repeated.
P
Section 15-STR Interpretation
April, 2002
Revised March, 2003
Page 1 of 12
B.
Analysis Parameters
1.
Analysis Range: Scan range should be set to include the 75 -400
bp peaks of the internal standard.
2.
Peak Detection: Minimum relative fluorescent unit (RFU) for all
colors should be set to 150 for all samples and controls; it can be
set as low as 75 for allelic ladders.
Data Processing: Light smoothing.
3.
4.
C.
Size Call Range. 75 to 400 for Profiler Plus and 75 to 350 for
Cofiler.
Size Calling Method: Local Southern.
Allelic Ladder Analysis
1.
Both the Profiler Plus and Cofiler kits provide allelic ladder s which
contain'the most common alleles for each locus amplified in the
kits.
2.
In order for the Genotyper software to correctly assign allele
designations to the samples, the correct number of alleles in the
ladder must be detected (See additional information for the list of
alleles in each ladder.) .
Profiler Minimum Number of Peaks in Ladder
1)
Blue — 33
2)
Green – 57
3)
Yellow — 28
b.
Cofiler Minimum Number of Peaks in Ladder
1)
Blue - 17
2)
Green — 27
3)
Yellow — 10
Allele Determination
a.
D.
1.
To be considered a "true allele", the RFU value of a peak should be
at least 150.
2.
While it is desirable that the RFU value of peaks not exceed 6000,
peaks with RFUs greater than 6000 may be typed at the discretion
of the analyst. If the majority of the predominant peaks are above
6000, the sample should be re-injected using a shorter injection
Section 15-STR Interpretation
April, 2002
Revised March, 2003
Page 2 of 12
S
time, rerun on the 310 using less amplified product, or re-amplified
using less input DNA.
3.
"Out of range" peaks are any peaks not falling within the specified
size range of the AmpFtSTR® kits. The presence of out of range
peaks should be noted. Care must be taken to determine if out of
range peaks are artifacts not to be considered part of the DNA
profile, or true alleles.
4.
Peaks approximately 4 bases smaller than a true allele can be
considered stutter peaks. Stutter peaks are considered a PCR
artifact and are not considered part of the DNA profile, but their
presence should be noted. The following table gives the maximum
% stutter generally observed for each locus.
Profiler Plus
D3S1358
vWA
FGA
D8S1179
D21S11
D18S51
D5S818
D13S317
D7S820
Cofiler
D3S1358
D16S539
THO1
TPDX
CSF1PO
D7S820
5
Maximum % Stutter
15
15
15
12
15
18
12
12
12
15
15
7
7
10
12
Peaks approximately one base pair smaller than a true allele may
be present if there has not been complete nucleotide A addition.
a.
b.
Lack of full A nucleotide addition (-A) may be observed if the
amount of input DNA is greater than approximately 2.5 ng or it
the sample is degraded.
Minus A (-A) peaks are a PCR artifact and are not considered
part of the DNA profile, but their presence should be noted.
Samples with –A peaks greater than 20% of the true allele
should be re-amplified using less input DNA.
Section 15-STR Interpretation
April, 2002
Revised March, 2003
Page 3 of 12
6.
"Pull-up" is a result of spectral overlap between dyes. A large peak in
one color can result in small peaks being observed at the same
location (approximate same base pair size) in other colors.
a.
Pull-up peaks are artifact peaks and are not considered part of
the DNA profile, but their presence should be noted. The
analyst can determine if pull-up within the sample
necessitates that the sample be repeated. Pull-up can occur as
a result of the following:
1)
2)
7
b.
E.
Application of a sub-optimal matrix.
Amplification using excess in-put DNA, which can
lead to off-scale peaks. The matrix may not perform
properly with off-scale data.
Peaks (in all four colors or at least two colors) of the same bp size
(within 0.2 base pairs) and whose RFUs are within an order of
magnitude of each other are not the result of dye-labeled DNA.
a.
8.
•
These fluorescent spikes (F-spikes) are artifacts due to the
presence of fluorescent material in either the formamide or
the POP-4 polymer.
The analyst can determine whether re-injection of the
sample is necessary.
If a peak is above 150 RFUs and does not fall into any of the artifact
categories as described above, then the peak is considered a true
allele. Alleles are then designated by Genotyper in accordance with
CODIS recommendations.
•
. Controls
1.
Refer to the DNA Quality Assurance Manual for control details.
2.
Positive Control (9947A) contained in Profiler Plus and Cofiler Kits.
a.
b.
c.
The table below gives the results that must be obtained fro
the positive control.
If the correct results are not obtained, re-inject the
sample(s). If correct results are still not obtained, all
samples amplified at the same time as this control must be
re-amplified and run on the 310.
If an incomplete profile for the positive control is obtained,
the sample should be re-run. If an incomplete profile is still
obtained, all samples amplified at the same time as this
control must be re-amplified. If this is not possible due to
Section 15-STR Interpretation
April, 2002
Revised March, 2003
Page 4 of 12
•
3.
insufficient DNA, those loci at which results are obtained can
be used for interpretation.
PROFILER PLUS
COFILER
Locus
Genotype
Locus
Genotype
D3S1358
14, 15
D3S1358
14, 15
vWA
17, 18
D16S539
11, 12
FGA
23, 24
THO1
8, 9.3
D8S1179
13, 13
TPDX
8, 8
D21S11
30, 30
CSF1 PO
10, 12
D18S51
15, 19
D7S820
10, 11
D5S818
11,
11
Amelogenin
X, X
D13S317
11, 11
D7S820
10, 11
Amelogenin
X, X
Blind Control
a.
4.
Correct results must be obtained for this sample, as verified
by one of the CLU supervisors or CLU employees designated
for the blind control program.
b.
If correct results are not obtained, repeat the sample. If
correct results are still not obtained, all case samples
extracted with this blind control need to be re-extracted,
re-amplified, and run on the 310.
c.
If an incomplete profile is obtained, the sample should be
re-run or re-amplified. If an incomplete profile is obtained, all
samples amplified with this blind control must be re-extracted.
If this is not possible due to insufficient DNA, those loci at
which results are obtained can be used for interpretation.
Negative'Control
a.
b.
6.
No true alleles (in the size range of the loci being tested)
greater than 150 RFUs should be present in this sample.
Repeat the sample if peaks are present. If peaks are still
present, all samples amplified at the same time as this
control must be re-amplfied and run on the 310.
Reagent Blank
a.
No true alleles (in the size range of the loci being tested)
greater than 150 RFUs should be present in this sample.
b.
Repeat the sample if peaks are present. If peaks are still
present, all samples extracted at the same time as this
control must be re-extracted, re-amplified, and run on the
310.
Section 15-STR Interpretation April, 2002
Revised March, 2003
Page 5 of 12
F.
Interpretation of Single Source Samples
1.
G.
If it is determined that a question sample has been deposited by a
single source, comparisons are then made to the known reference
samples in the case.
2.
A match is declared if all alleles in the question sample match the
known sample.
3.
Statistical analysis is then performed according to the Statistics
Procedure.
Detection of Mixtures
Samples may contain DNA from more than one - individual.
Generally, a sample is consistent with being a mixture if it exhibits
one or more of the following characteristics at more than one locus:
a.
H.
More than two alleles are present at a locus after artifact
considerations have been evaluated and dismissed as
possible causes. (Note: Some individuals have been typed
who are tri-allelic at a single locus.)
b.
A peak is present at a stutter location, and its height is greater
than the determined stutter for that locus. See Stutter Table
in Section D.
c.
Severely imbalance peak height ratios exist for sister alleles
of heterozygous genotypes within the profile. With the
possible exception of low template amplifications and
degraded DNA, ratios less than 70% are rare in normal
unmixed samples.
Interpretation of Mixed Samples
1.
2.
3.
The interpretation applied to a mixed sample by the DNA analyst in
each particular case should be based upon all relevant information,
including that provided from known reference samples.
Predominant types of a mixture can be assigned based on peak
heights as well as peak height ratios.
If there is a predominant profile present in the mixture, one can
determine any inclusions or exclusions by comparing the profiles
from known reference samples with the predominant DNA types.
Profile frequency estimates can be reported for the predominant
DNA profile.
Section 15-STR Interpretation
April, 2002
Revised March, 2003
Page 6 of 12
4. For samples in which weak profiles are present (compared to a
predominant profile), or where a predominant DNA profile cannot be
identified, all possible combinations must be considered for each
locus. One can then determine whether a principal can be eliminated
as a source of DNA in the mixture or can be included as a possible
source of DNA in the mixture. Care must be taken in the
determination of possible contributors of very weak DNA types, since
it is possible that true alleles may fall below the 150 RFU minimum.
Exclusion probabilities can be estimated for these kinds of mixtures.
1.
J.
Incomplete STR Profiles
1.
If the DNA sample contains PCR inhibitors, is degraded, or a very
small quantity of DNA has been amplified, the possibility exists that
not all the loci may amplify.
2.
Since each locus is an independent marker whose results are not
based upon information provided by the other markers, results can
generally still be interpreted from the loci that do amplify.
Partial STR Profiles at a Locus
1. Situations can arise where one allele within a locus is above the minimum
RFU and the second allele, while visible, does not meet the minimum
RFU value.
a.
b.
K.
The absence of the second interpretable allele does not
prevent the analyst from determining possible inclusions or
exclusions.
If an inclusion is declared, frequency statistics for the affected
locus should not be used in the final random match probabiiity
calculation.
Off Ladder Alleles
1.
An off ladder allele is described as an allele in which one of the
repeats is not a full repeat. The allelic ladders are made up of nominal
alleles as well as some of the off ladder alleles. However, not all off
ladder alleles are contained within the allelic ladders. If an allele is not
contained within the allelic ladder, it will be termed "OFF LADDER
ALLELE" by the software program "Genotyper".
2.
A sample containing an apparent off ladder allele (as called by
Genotyper) should be re-run to verify that it contains a true off ladder
allele.
Section 15-STR Interpretation
April, 2002
Revised March, 2003
Page 7 of 12
3.
If both a known sample and one or more question samples which
match the known sample contain an off ladder allele, only the known
sample needs to be re-run.
4.
An allele with one extra base will be designated X.1, with X being
the nominal allele. X.2 designates an allele with two extra bases,
and X.3 designates an allele having three extra bases.
5.
L.
Off ladder alleles will be given the minimum allele frequency (0.02)
in the random match probability calculations, unless a frequency for
that off ladder allele has been determined in the population study
(e.g. the 9.3 allele at the THO1 locus).
Variants
1.
A . variant is an allele that is one to several repeats larger than the
largest allele in the ladder or one to several repeats smaller than the
smallest allele in the ladder.
2.
These alleles will be given the designation of either <Y or >X, with Y
being the smallest allele in the ladder and X being the largest allele
in the ladder.
3.
If a sample contains a variant, the sample should be re -amplified
and rerun to verify the variant.
4.
See further information for determining to which locus these variants
should be assigned.
5.
M.
These alleles will be given the minimum allele frequency in the
random match probability calculations. (Note: If the variant is at
either the D3S1358 or D7S820 locus, the sample does not have to
be re-amplified if both Cofiler and Profiler Plus were comparable.)
Verification of Overlapping Loci Between Cofiler and Profiler Plus
1.
N.
Results obtained for D3S1358 and D7S820 from the two
amplifications should usually concur.
2.
Situations may arise in which the results do not completely concur.
For example, in a mixed sample, the minor DNA component may
amplify to a greater extent for one kit as compared to the other.
Interpretation and Technical Review
All DNA case files must be reviewed by a second DNA analyst.
Section 15-STR Interpretation
April, 2002
Revised March, 2003
Page 8 of 12
2. Any unresolved, discrepant conclusions will be deferred to the more
conservative view or resolved through a consultation with a third DNA
analyst from an outside laboratory.
Further Information
A. In the context of this standard operating procedure, the following
definitions are utilized:
1. Re-inject - The original tube of formamide / ROX/ sample is sampled
again on the 310.
2. Re-run - A new tube of formamide / ROX is prepared and sample is
added to the tube. This new tube is then sampled on the 310.
3. Repeated - The analyst can determine whether to re-inject or re-run a
sample.
B. The alleles contained in each of the allelic ladders are as follows:
Profiler Loci
D3S1358
vWA
FGA
_EGA
D8S1179
D21S11
D18S51
D5S818
D13S317
D7S820
Collier Loci
D3S1358
D16S539
Amelogenin
THO1
TPDX
CSF1PO
D7S820
AmpF€STR® Allelic Ladder Alleles
12, 13,14, 15, 16, 17, 18, 19
11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21
18, 19,20, 21, 22, 23, 24, 25, 26, 26.2, 27, 28, 29, 30
X, Y
8, 9, 10, 11, 12, 13, 14, 1 16, 17, 18, 19
5, 29, 29.2, 30, 30.2, 31,
24.2, 25, 26, 27, 28, 28.2,
31.2, 32, 32.2, 33, 33.2, 34, 34.2, 35, 35.2, 36, 38
9, 10, 10.2, 11, 12, 13, 13.2, 14, 14.2, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26
7, 8, 9, 10, 11, 12, 13, 14, 15, 16
8, 9, 10, 11, 12, 13, 14, 15
6, 7, 8,9, 10, 11, 12, 13, 14, 15
AmpF€STR® Allelic Ladder Alleles
12, 13,14, 15, 16, 17, 18, 19
5, 8, 9,10, 11, 12, 13, 14, 15
X, Y
5, 6, 7, 8, 9, 9.3, 10
6, 7, 8, 9, 10, 11, 12, 13
6, 7, 8, 9, 10, 11, 12, 13, 14,15
6, 7, 8, 9, 10, 11, 12, 13, 14,15
C. Assigning Variants Larger and Smaller than Allelic Ladders to a
Particular Locus
1.
Variants larger and smaller than allelic ladders are assigned to a
particular locus based on the following guidelines. It should be
Section 15-STR Interpretation
April, 2002
Revised March, 2003
Page 9 of 12
emphasized that not every situation can be covered by a pre-set
rule.
2.
If a Profiler Plus Blue Allele is:
Smaller than D3S1358 allele 12, it is assigned to D3S1358. Larger than FGA allele
30, it is assigned to FGA.
b.
Between D3S1358 allele 19 and vWA allele 11:
c.
1)
If D3S1358 is heterozygous and vWA is homozygous, it
is assigned to vWA.
2)
If D3S1358 is homozygous and vWA is heterozygous, it is assigned to
3)
D3S1358.
If both D3S1358
and vWA are homozygous, it is not assigned unless
Cofiler was also amplified. If it is also present in Cofiler, it is assigned to
D3S1358; if it is not present in Cofiler, it is assigned to vWA.
d.
Between vWA allele 21 and FGA allele 18:
1)
If vWA is heterozygous and FGA is homozygous, it is
assigned to FGA.
2)
If vWA is homozygous and FGA is heterozygous, it is
assigned to vWA.
3)
If both vWA and FGA are homozygous, it is not
assigned.
3.
If a Profiler Plus Green allele is:
a.
b.
c.
d.
Smaller than D8S1179 allele 8 and larger than amelogenin Y,
it is assigned to D8S1179.
Larger than D18S51 allele 26, it is assigned to D18S51.
Between D8S1179 allele 19 and D21S11 allele 24.2:
1)
If D8S1179 is heterozygous and D21S11 is
homozygous, it is assigned to D21S11.
2)
If D8S1179 is homozygous and D21S11 is
heterozygous, it is assigned to D8S1179.
3)
If both D8S1179 and D21S11 are homozygous, it is not
assigned.
Between D21S11 allele 38 and D18S51 allele 9:
1)
2)
3)
4.
If D21S11 is heterozygous and D18S51 is
homozygous, it is assigned to D18S51.
If D21S11 is homozygous and D18S51 is
heterozygous, it is assigned to D21S11.
If both D21S11 and D18S51 are homozygous, it is not
assigned.
If a Profiler Plus Yellow allele is:
Section 15-STR Interpretation
April, 2002
Revised March, 2003
Page 10 of 12
a.
b.
c.
Smaller than D5S818 allele 7, it is assigned to D5S818.
Larger than D7S820 allele 15, it is assigned to D7S820.
Between D5S818 allele 16 and D13S317 allele 8:
If D5S818 if heterozygous and D13S317 is
homozygous, it is assigned to D13S317.
2)
If D5S818 is homozygous and D13S317 is
heterozygous, it is assigned to D5S818.
3)
If both D5S818 and D13S317 are homozygous, it is
not assigned.
Between D13S317 allele 15 and D7S820 allele 6:
1)
d
If D13S317 is heterozygous and D7S820 is
homozygous, it is assigned to D7S820.
2)
If D13S317 is homozygous and D7S820 is
heterozygous, it is assigned to D13S317.
3)
If both D13S317 and D7S820 are homozygous, it is not
assigned unless Cofiler was also amplified. If it is also
present in Cofiler, it is assigned to D7S820; if it is not
present in Cofiler, it is assigned to D13S317.
If a Cofiler Blue allele is:
1)
5.
a.
b.
Smaller than D3S1358 allele 12, it is assigned to D3S1358.
Larger than D16S539 allele 15, it is assigned to D16
S539.
c.
Between D3S1358 allele 19 and D16S539 allele 5:
If D3S1358 is heterozygous and D16S539 is
homozygous, it is assigned to D16S539.
2)
If D3S1358 is homozygous and D16S539 is
heterozygous, it is assigned to D3S1358.
3)
If both D3S1358 and D16S539 are homozygous, it is
not assigned unless Profiler Plus was also amplified. If
it is also present in Profiler Plus, it is assigned to
D3S1358; if it is not present in Profiler Plus, it is
assigned to D16S539.
If a Cofiler Green allele is:
1)
6.
a.
b.
c.
Smaller than THO1 allele 5 and larger than amelogenin Y, it
is assigned to THO1.
Larger than CSF1PO allele 15, it is assigned to CSF1PO.
Between THO1 allele 10 and TPDX allele 6:
Section 15-STR Interpretation
April, 2002
Revised March, 2003
Page 11 of 12
1)
If TH01 is heterozygous and TPDX is
homozygous, it is assigned to TPDX.
2)
If TH01 is homozygous and TPDX is
heterozygous, it is assigned to TH01.
3)
If TH01 and TPDX are homozygous, it
is not assigned.
Between TPDX allele 13 and CSF1 PO allele 16:
1)
2)
3)
•
If TPDX is heterozygous and
CSF1PO is homozygous, it is assigned to
CSF1PO.
If TPDX is homozygous and
CSF1PO is heterozygous, it is assigned
to TPDX.
IF TPDX and CSF1PO are homozygous, it
is not assigned.
•
Section 15-STR Interpretation
April, 2002
Revised March, 2003
Page 12 of 12
O
V I I I .
S T A N D A R D
O P E R A T I N G
P R O C E D U R E S
N a me of Procedure
Date Implemented
Reviewed:
Dates / By
, ' 4
2e9
Changes:
Implemented On:
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9_5
Describe Changes and Reasons:
T
_____
/ % -a&
June 1999
Standard Operating Procedures
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