Interpreting Microsatellites

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Chapter 3
Interpreting Microsatellites
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Nomenclature for STR alleles
Inter-laboratory reproducibility and comparisons
of data - a common nomenclature Example If one
laboratory calls a sample 15 repeats at a
particular STR locus and the same sample is
designated 16 repeats by another laboratory, a
match would not be considered, and the samples
would be assumed to come from separate sources.
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National DNA databases - many laboratories
worldwide - contributing information -
internationally accepted nomenclature for STR
alleles. Committee of forensic DNA scientists,
known as the DNA Commission of the International
Society Forensic Haemogenetics (ISFH), issued
guidelines for designating STR alleles in 1994
and again in 1997. The ISFH 1994
recommendations focused on allelic ladders and
designation of alleles that contain partial
repeat sequences. The ISFH 1997 guidelines
discuss the sequence and repeat designation of
STRs.
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ISFH 1994 recommendations
Allelic ladders containing all alleles should be
used as a reference for allele designation in
unknown samples. Allelic ladders may be
commercially obtained or prepared in house and
should contain all common alleles.
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Allelic ladders
Lane 1
Lane 2
Lane 3
Lane 5
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Combine
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8
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Re-amplify
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9
10
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To produce a ladder containing five alleles with
6,7,8,9,10 repeats, individual samples with
genotypes of (6,8), (7,10) and (9,9) could be
combined. Alternatively, the combination of
genotypes could be (6,9), (7,8), and (10,10) or
(6,6) (7,7), (8,8), (9,9) and (10,10).
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An allelic ladder - artificial mixture of the
common alleles present. Provide a reference DNA
size for each allele included in the
ladder. Allelic ladders - important for accurate
genotypes determinations. Serve as a standard -
measuring stick, for each STR locus. Necessary
to adjust for different sizing measurements
obtained from different instruments and
conditions used by various laboratories. Construc
ted by combining genomic DNA or locus-specific
PCR products from multiple individuals in a
population which possess alleles that are
representative of the variation for the
particular STR marker. The samples are
co-amplified to produce an artificial sample
containing the common alleles for the STR marker.
Allele quantities are balanced by adjusting the
input amount of each component so that the
alleles are fairly equally represented in the
ladder.
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Microvariants
Designation of incomplete repeat motifs should
include the number of complete repeats and,
separated by a decimal point, the number of base
pairs in the incomplete repeat. Examples
Microvariants - incomplete repeat units include
allele 9.3 at the TH01 locus. TH01 allele 9.3
contains nine tetranucleotide AATG repeats and
one incomplete ATG repeat of three nucleotides.
Another microvariant example is allele 22.2 at
the FGA locus, which contains 22 tetranucleotide
repeats and one incomplete repeat with two
nucleotides.
THO1
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ISFH 1997 recommendations
The repeat sequence motif should be defined so
that the first 5 nucleotides that can define a
repeat motif is correct. Example 5 GG TCA
TCA TCA TGG -3 3x TCA repeats or 3x CAT
repeats? Under the recommendations for the ISFH
committee, only the first one (3x TCA) is
correct.
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Choice of markers
For DNA typing markers to be effective across a
wide number of jurisdictions, a common set of
standardized markers must be used. The STR loci
that are commonly used today were initially
characterized and developed either in the
laboratory of Dr Thomas Caskey at the Baylor
College of Medicine or at the Forensic Science
Service in England. Promega Corporation
commercialized many of the Caskey markers, while
PE Applied Biosystems picked up on the Forensic
Science Service (FSS) STR loci as well as
developing some new markers. The availability of
STR kits permit robust multiplex amplification of
eight or more STR markers. Matching
probabilities that exceed one in a billion are
possible in a single amplification with 1ng (or
less) DNA sample. Results can be obtained today
in only a few hours.
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Multiplexing
Since PCR is a primary limitation on the
throughput of the ABI Prism Genetic Analysis
System, multiplexing can greatly increase
throughput. First STR multiplexes to be
developed was a quadruplex developed by the
Forensic Science Service comprising the four loci
TH01, FES/FPS, VWA, and F13A1. This so-called
first-generation multiplex had a matching
probability of approximately 1 in 10 000. The
FSS followed with a second-generation multiplex
(SGM) made up of six polymorphic STRs and a
gender identification markers. The six STRs in
SGM are TH01, VWA, FGA, D8S1179, D18S51, and
D21S11 and provide a matching probability of
approximately 1 in 50 million. The first
commercially available STR kit capable of
multiplex amplification became available from
Promega Corporation in 1994 for silver stain
analysis. This kit consisted of the STR loci
CSF1PO, TPOX, and TH01 and is often referred to
as the CTT triplex using the first letter in
each locus. The CTT triplex only had a matching
probability of 1 in 500 but was still widely
used in the United States as it was the first
available STR multiplex kit and could be
performed with a fairly low start-up cost.
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PCR products that give rise to additional peaks
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Stutter bands, peak height imbalance
Butler, 2002
Stutter peaks are found in almost every
electropherogram. Stutter peaks are small peaks
that occur immediately before or after a real
peak. During the PCR amplification process, the
polymerase can lose its place when copying a
strand of DNA, usually slipping forwards or
backwards four base pairs. The result is a small
number of DNA fragment copies that are either one
repeat larger or smaller than the true fragment
being amplified.
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Stutter products impact interpretation of DNA
profiles, especially in cases where two or more
individuals may have contributed to the DNA
sample. Because stutter products are the same
size as actual allele PCR products, it can be
challenging to determine whether a small peak is
a real allele from a minor contributor or a
stutter product of an adjacent allele. Mixture
interpretation requires a good understanding of
the behaviour of stutter products in single
source samples. Often a laboratory will quantify
the percentage of stutter product peak heights
compared to their corresponding allele peak
heights. The percentage of stutter product
formation for an allele is determined simply by
dividing the stutter peak height by the
corresponding allele peak height. Each locus has
a different amount of stutter product formation.
Longer alleles for a STR locus exhibit a greater
degree of stutter than smaller alleles for the
same locus. Stutter percentage with the standard
tetranucleotide repeats is less than 15 for all
13 CODIS core STR loci. The amount of stutter
may be related to the DNA polymerase
processivity, or how rapidly it copies the
template strand. Stutter products have been shown
to increase relative to their corresponding
alleles with a slower polymerase.
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Non template addition
Butler, 2002
DNA polymerase, often add an extra nucleotide to
the 3-end of a PCR product as they are coping
the template strand. This non-template addition
is most often adenosine and is therefore
sometimes referred to as adenylation or the
plus A form of the amplicon. Non-template
addition results in a PCR product that is one
base pair longer than the actual target sequence.
The degree of adenylation is dependent on the
sequence of the template strand, which in the
case of PCR results from the 5-end of the
reverse primer.
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Now why is that important? From a measurement
standpoint, it is better to have all of the
molecules as similar as possible for a particular
allele. Partial adenylation, where some of the
PCR products do not have the extra adenine (i.e.
A peaks) and some do (i.e. A peaks), can
contribute to peak broadness if the separation
systemss resolution is poor. Sharper peaks
improve the likelihood that a systems genotyping
software can make accurate calls. Variation in
the adenylation status of an allele across
multiple samples can have an impact on accurate
sizing and genotyping potential microvariants.
STR protocols include a final extension step to
give the DNA polymerase extra time to adenylate
all double-stranded PCR products completely. For
all commercially available STR kits, the STR
alleles are all in the A form. Ampifying
higher quantities of DNA than the optimal amount
suggested by the manufacturers protocols can
result in incomplete 3 A nucleotide addition and
therefore split peaks.
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Butler, 2002
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Microvariants
Rare alleles are encountered in the human
population that may differ from common allele
variants at tested DNA markers by one or more
base pairs. Sequence variation between STR
alleles can take the form of insertions,
deletions, or nucleotide changes. Alleles
containing some form of sequence variation
compared to more commonly observed alleles are
often referred to as microvariants because they
are only slightly different from full repeat
alleles. Because microvariant alleles often do
not size the same as consensus alleles present in
the reference allelic ladder, they are referred
to as off-ladder alleles. One example of a
common microvariant is allele 9.3 at the STR
locus TH01. The repeat region of TH01 allele 9.3
contains nine full repeats (AATG) and a partial
repeat of three bases (ATG). The 9.3 allele
differs from the 10 allele by a single base
deletion of adenine in the seventh
repeat. Microvariants are most commonly found in
more polymorphic STR loci, such as FGA, D21S11,
and D18S51, which possess the largest and most
complex repeat structures compared to simple
repeat loci, such as TPOX and DSF1PO.
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Determining the presence of a microvariant
allele. Suspected microvariants can be fairly
easily seen in heterozygous samples where one
allele lines up with the fragment sizes in the
allelic ladder and one does not.
Butler, 2002
The sample contains a peak that lines up with
allele 25 from the FGA allelic ladder and a
second peak that is labeled as an off-ladder
allele and lines up between the 28 and 28.2
shaded virtual bins created by the ladder. Each
peak is labeled with its calculated size in base
pairs determined by reference to the internal
GS500 sizing standard. The relative size
difference between the questioned sample and an
allelic ladder marker run under the same
electrophoretic conditions is then used to
determine if the allele is truly a microvariant.
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The End