Ambient Temperature Variation Effects on Sizing Precision of AmpFlSTR®
Profiler Plus Alleles Detected on the
ABI Prism® 310 Genetic Analyzer
Jeanette M. Wallin
Martin R. Buoncristiani
California Department of Justice DNA Laboratory
and Methods.......ABI Prism®
Analyzer Sample Preparation and Electrophoresis.......Data
GS-500 and 400HD.......Size
Deviation with Ambient Temperature.......Global
Southern.......GS-500 and ILS-600.......Discussion.......References
have established the Local Southern algorithm as a precise tool
for sizing DNA fragments. As a result, the Local Southern algorithm
of the PE Applied Biosystems' software, GeneScan®
Analysis (PE Applied Biosystems, Foster City, California), is the
manufacturer's recommended method for sizing short tandem repeats
(STRs). However, this recommendation is made with the warning that
size estimates may be imprecise if any of the standard fragments
run anomalously. Specifically, the GeneScan®-500
(GS-500) internal standard fragments of 250 and 340 bases in length
run anomalously under nonoptimal conditions on the ABI Prism®
310 Genetic Analyzer (PE Applied Biosystems, Foster City, California).
Department of Justice DNA Laboratory currently uses the GS-500-size
standard without the 250-base standard assigned and the Local Southern
method to size AmpFlSTR® Profiler Plus alleles.
However, even with the manufacturer's recommended instrument running
conditions, studies in this laboratory demonstrate that ambient
temperature variation over the course of a 310 run can result in
anomalous migration of GS-500 standard fragments. When ambient temperature
varies, a simple analysis method change can improve precision.
This study suggests
that the Global Southern method may provide improved precision over
the Local Southern method when using the GS-500 internal standard
with the ABI Prism® 310 Genetic Analyzer. In addition,
this study shows that precision for fragments greater than 300 bases
is further improved by excluding the 340-base GS-500 fragment in
conjunction with using the Global Southern method. When ambient
temperature shifts occur, this sizing method change should reduce
the number of sample reruns necessary.
The ABI Prism®
310 Genetic Analyzer is one of the more commonly used DNA analysis
instruments in forensic laboratories. It is an automated, single-capillary
electrophoresis instrument with laser-induced fluorescence detection.
The California Department of Justice DNA Laboratory uses the ABI
Prism® 310 Genetic Analyzer for the separation and
detection of STRs amplified with the AmpFlSTR® Profiler
Plus PCR amplification kit (PE Applied Biosystems, Foster
City, California). The fluorescently tagged STR products are coinjected
with the GS-500 ROX-internal standard and sized using an interpolation
algorithm for Local Southern in the GeneScan® Analysis
software. The allelic ladder containing the common STR alleles is
also injected with the GS-500 ROX-size standard. The Genotyper®
software (PE Applied Biosystems, Foster City, California) performs
automated allele calling, comparing the sizes generated from one
injection of the allelic ladder to those generated from sample injections.
Sample fragment sizes within ±0.5 bases of the corresponding
allelic ladder fragment are assigned the appropriate allele designation.
The one-base window for genotyping is based on the assumption that
single-base sizing precision may be routinely achieved (Lazaruk
et al. 1998). In order to obtain single-base sizing precision, standard
deviations should be within approximately 0.16 bases so that given
three deviations, 99.7 percent of the sizes will statistically fall
within 0.48 bases from the mean (assuming that the sizes are generated
by random error). If standard deviations greater than approximately
0.16 bases are observed, genotyping accuracy may be compromised.
Although these STRs are tetranucleotide repeats, one-base microvariant
alleles do occur at some loci; therefore, single-base sizing precision
factor affecting precision is the electrophoresis running temperature.
Changes in running temperature affect the viscosity of the polymer
matrix and the sieving of DNA fragments. It can also affect DNA
secondary conformation. Because the entire length of the capillary
on the 310 Genetic Analyzer is not insulated, ambient temperature
fluctuations that occur can affect the run temperature, at least
in the exposed portions of the capillary. The manufacturer recommends
that the ambient temperature not vary by more than ±2ºC
during a run (PE Applied Biosystems 2000). As shown previously (Rosenblum
et al. 1997), this specification is important for fragments of the
GeneScan®-350 (GS-350)- and GS-500-size standards.
The 250-base fragment is particularly affected by run temperature
changes, which cause it to migrate anomalously. Hence, the 250-base
fragment is not included in the generation of size standard curves
(PE Applied Biosystems 2000). Ambient temperature fluctuation may
also cause the 340-base fragment to migrate anomalously, although
to a lesser degree. However, most forensic laboratories include
the 340-base fragment when generating size standard curves.
The degree to
which anomalously migrating size standard fragments influence the
curve should be dependent upon the size method employed. Elder and
Southern (1983) found local algorithms to be more precise for sizing
DNA polymers than global algorithms, but they also found precision
was greatly reduced when there were sequence differences between
standards and unknowns. The effects of such sequence differences
should minimize when using global methods. Most forensic laboratories,
including the California Department of Justice DNA Laboratory, use
the Local Southern method, as recommended by the manufacturer, although
size estimates may be imprecise if any of the standard fragments
run anomalously (PE Applied Biosystems 1998). The size range for
the more common alleles of the AmpFlSTR® Profiler
Plus loci is approximately 100-345 bases. While the specifications
of the 310 Genetic Analyzer polymer, Performance Optimized Polymer-4
(POP4) (PE Applied Biosystems, Foster City, California), are
to provide single-base sizing precision up to 250 bases, laboratories
have reported such precision up to approximately 350 bases on this
platform when using Local Southern sizing (LaFountain et al. 2001;
Lazaruk et al. 1998; Moretti et al. 2001). Furthermore, routine
analyses in this laboratory using AmpFlSTR® Profiler
Plus with GS-500 and Local Southern indicated single-base
sizing precision. However, periodic runs have shown losses in precision,
presumably due to fluctuations in ambient temperature.
following experiments were performed to examine the cause of the
suboptimal precision with conventional methods and to investigate
alternative approaches that may offer single-base sizing precision
more routinely. Alternative approaches tested included analyses
omitting the 340-base fragment, an investigation into the use of
Global Southern, and an evaluation of the size standards GeneScan®-400HD
(400HD) (PE Applied Biosystems, Foster City, California) and Internal
Lane Standard 600 (ILS-600) (Promega Corporation, Madison, Wisconsin).
Each of the size standards are illustrated in Figure 1.
Prism® 310 Genetic Analyzer Sample Preparation and
microliters of high-deionized formamide (Hi-Di formamide,
PE Applied Biosystems, Foster City, California) were combined with
1µl of GS-500, 400HD, or ILS-600. Next, 1.5µl of Profiler
Plus allelic ladder (PE Applied Biosystems, Foster City, California)
was added to each tube containing Hi-Di formamide and GS-500,
Hi-Di formamide and 400HD, or Hi-Di formamide and ILS-600.
Samples were heat-denatured at 95°C for three minutes, snap-cooled
on ice for three minutes, then placed in a 48-well autosampler tray
for automatic injection in a 47 cm (36 cm length to detector) 50
µm inner diameter capillary on the ABI Prism®
310 Genetic Analyzer. Samples were electrokinetically injected for
five seconds at 15 kV, then run at 15 kV for 26 or 28 minutes in
POP4 at 60°C (Run Module GS STR POP4, F) with GeneAmp
10X Buffer (PE Applied Biosystems, Foster City, California) at 1X.
Ambient temperature during the runs was monitored by placing a temperature
gauge (Omega Engineering, Stamford, Connecticut) adjacent to the
310 Genetic Analyzer. Repeat injections from each tube of ladder
were performed, alternating between GS-500 and either 400HD or ILS-600.
All sample files
were analyzed with the ABI Prism GeneScan® v2.1 or
v3.1 software using both the Local and Global Southern sizing methods.
The GS-500 250-base fragment was excluded in the size standard assignment
of all analyses. In addition, some data analyses also excluded the
340-base fragment of the GS-500.
2 Standard deviations for each allele of 50 Profiler
PlusT allelic ladder injections sized with GS-500
and 50 sized with 400HD (¨). Click
to enlarge image.
Plus allelic ladder was injected 50 times with GS-500 and
50 times with 400HD. The injections alternated between GS-500 and
400HD so that conditions for each internal standard would be as
similar as possible. The ambient temperature over the two-day run
ranged from 16°C to 20°C. Precision was estimated by calculating
the standard deviation at each of the 118 alleles after sizing with
the Local Southern method. The standard deviation for each allele
size is shown in Figure 2. Most notable is the increased standard
deviation of allele sizes greater than 300 bases sized by GS-500.
The precision between GS-500 and 400HD appears similar otherwise,
with GS-500 generating slightly better precision for fragments between
175 and 290 bases in length. However, the loss of precision with
the GS-500 relative to the 400HD for Profiler Plus alleles
greater than 300 bases suggests that precision is limited by the
GS-500-size standard. Based on the increased standard deviations
of the Profiler Plus alleles with the GS-500, the 340-base
fragment was suspect. When the allelic ladders were resized excluding
the 340-base standard fragment, the precision improved and the standard
deviations for fragments above 300 bases were similar to those generated
with the 400HD.
Deviation with Ambient Temperature
3 The size deviation of each allele from the average
is plotted with increasing size (observed minus average).
Click to enlarge image.
examine the effects of ambient temperature on GS-500 precision,
size deviations from the allele averages were compared to the measured
ambient temperature at the start of each injection (Figure 3). The
ladders injected at the highest and lowest ambient temperatures
sized furthest from the average, demonstrating a relationship between
calculated size and temperature. In other words, the larger the
ambient temperature shift, the larger the size deviationspecifically,
alleles sized larger than the average when the ambient temperature
decreased and smaller when the ambient temperature increased. Therefore,
at lower ambient temperatures, the alleles migrated slower than
the GS-500 fragments and at higher ambient temperatures, the alleles
migrated faster than the GS-500 fragments. In addition, the ambient
temperature changes affected the 340-base fragment more than the
other assigned standard fragments. Thus, there was the increased
deviation for alleles larger than 300 bases.
4 The data point (retention time) difference between
lowest (16.5°C) and highest (20°C) ambient temperature
injections is plotted versus size for each fragment (FAM, JOE,
and NED are allelic ladder dye labels; ROX is the GS-500 dye
to enlarge image.
illustrate the migration differences among and between the Profiler
Plus alleles and the GS-500 fragments with ambient temperature
change, fragments from the highest and lowest temperature injections
were compared by data point position (retention time). The data
point position for each fragment of a single 20°C injection
was subtracted from the data point position of the corresponding
fragment in a single 16.5°C injection (Figure 4). Data point
differences increased linearly with fragment length as a result
of the longer retention times for the larger fragments. High correlation
coefficients reflect migration consistency in fragment spacing,
even though fragment retention times changed with temperature. While
the correlation coefficient was high for each dye-labeled set of
fragments, the GS-500 fragments exhibited the lowest linear regression
determination coefficient (R2 = 0.9951), with the 250- and 340-base
fragments visibly offset, presumably because of sequence content
variability. These two fragments shifted less with ambient temperature
change than the other fragments, resulting in the smaller data point
difference. Thus, the linear regression determination coefficient
changed from 0.9951 to 0.998 when the 250- and 340-base fragments
were excluded. Additional GS-500 fragments appeared to slightly
deviate from the linear trend (160- and 300-base fragments). This
observation implies additional GS-500 fragments, to a lesser extent,
may migrate anomalously with increased ambient temperature variations
and that the GS-500 fragments are not proportionately affected by
temperature change as the ladder alleles.
5 Standard deviations for each allele of 50 Profiler
PlusT allelic ladder injections with GS-500 (Δ) and 50
with 400HD (¨)
sized using Global Southern. Click
to enlarge image.
at least some of the GS-500 fragments ran anomalously, the Global
Southern sizing method was applied to the same data to test if it
improved precision. Data reanalysis using Global Southern, both
with and without the 340-base fragment, improved precision relative
to analysis with the Local Southern method. The highest precision
was obtained using the Global Southern method with the exclusion
of the 340-base fragment (Figure 5). The largest standard deviation
observed with this method was 0.19 base, and only six allele sizes
had standard deviations greater than 0.16 bases. For fragment sizes
greater than 300 bases, Global Southern greatly improved precision
over Local Southern with the inclusion of the 340-base fragment,
from close to 0.30 base standard deviation to less than 0.10 base
standard deviation. The precision trend changed from a rise in standard
deviations for fragments above 300 bases to an overall lower level
of standard deviations across all fragment sizes. This consistency
across fragment sizes demonstrates how the Global Southern method
minimizes size deviations caused by anomalous migration of internal
standard fragments. When using the Global Southern method, the GS-500
produced better precision than the 400HD. The precision obtained
when using 400HD with Global Southern was relatively unchanged compared
to that with Local Southern (Figures 2 and 5). This is probably
because there are no anomalously migrating 400HD fragments (PE Applied
6 Standard deviations for each allele of 50 Profiler
PlusT allelic ladders coinjected with GS-500 (¨)
and 50 coinjected
with ILS-600 (•).
to enlarge image.
examine sizing precision using ILS-600, a total of 100 Profiler
Plus allelic ladder injections were performed, this time alternating
between GS-500 and ILS-600 sizing standards. The ambient temperature
during the run ranged from 20°C to 23°C. The precision generated
by both standards was well within a single base, with every allele
sizing less than 0.13 base standard deviation. However, there were
still notable differences in precision with GS-500 when sizing with
Local Southern versus Global Southern. Sizing precision of the ILS-600
was consistent across the range of allele sizes with a standard
deviation range of 0.03 to 0.06 base (Figure 6). The ILS-600 fragments
apparently migrated similarly to the Profiler Plus alleles
with ambient temperature change. Sizing precision of the GS-500,
although consistent up to 290 bases with a standard deviation range
of 0.04 to 0.07 base, increased in standard deviation to a maximum
of 0.12 base for the largest alleles. Although each allele sized
within the Genotyper® ± 0.5 base window and
minimal ambient temperature variation was observed during the run,
the 340-base standard still apparently migrated anomalously. When
the GS-500-sized allelic ladder injections were reanalyzed with
the Global Southern sizing algorithm excluding the 340-base standard,
the precision again improved, resulting in a maximum standard deviation
across all fragment sizes of 0.07 base.
STR sizing precision
is dependent on ambient temperature control when using the 310 Genetic
Analyzer with the internal standard GS-500. This study showed that
allele sizes were reproducibly smaller when ambient temperature
increased and larger when ambient temperature decreased. The basis
of this phenomenon appears to be that as ambient temperature increases,
Profiler Plus alleles exhibit an increased mobility relative
to GS-500 fragments. The phenomenon is most pronounced with the
250- and 340-base fragments of the GS-500 sizing standard and is
probably attributed to the varying sequence content between the
size-standard fragments as well as the DNA sequences that are actually
measured. In contrast to GS-500, the ILS-600 standard consists of
same-sequence subsets. Therefore, when the ILS-600 is used on the
310 Genetic Analyzer, it generates better sizing precision than
the GS-500 sizing standard. It appears that the number of internal
fragments assigned is not a critical factor in precision. The 400HD
has 18 fragments between and including 100 to 400 bases, whereas
GS-500, excluding the 250 and 340, has only eight fragments between
and including 100 to 400 bases. Lazaruk et al. (1998) also found
no advantage to using the more dense 400HD over the GS-350. More
critical than the number of internal fragments or the spacing between
them is the similar migration changes of alleles and standards with
changing running conditions. The sizing differences are minimized
when sequence content is similar.
is due, in part, to temperature fluctuations in the capillary itself.
Although the majority of the capillary on the 310 Genetic Analyzer
is temperature-controlled by a heat plate, small regions of the
capillary are exposed to ambient air where fragment migration is
likely to be affected by ambient temperature changes, particularly
at the cathode end. In addition to the total ambient temperature
change, the duration at each temperature also seems to play a role
in fragment migration. Although the first (GS-500/400HD) and second
(GS-500/ILS-600) runs had similar absolute ambient temperature fluctuation
ranges, the second run had much better precision. In run two, the
duration at which the temperature was low or high was very short
(less than one hour), whereas for run one, the duration was longer
(up to four hours). This presumably provided more time for the DNA
fragments to be affected by conformational change or the polymer
sieving properties. The range for run two was also at an overall
higher temperature than for run one; this may have also played a
role in the precision.
sizing of allelic ladder is generally used to genotype a large number
of samples, it is best to consider the sizing precision over the
entire duration of the run. Ideally, the standard deviation for
every allele size in a given run should measure to 0.16 base or
less; this would statistically mean 99.7 percent of the allele calls
size within ± 0.48 bases (or three standard deviations) of
the mean. This level of precision can be achieved in a number of
ways. Ideally, the ambient room temperature could be strictly controlled.
Alternatively, the GS-500 internal lane size standard may be replaced
by other standards available that contain same-sequence subsets.
Excellent sizing precision with the ILS-600 standard was observed
by Pawlowski and Maciejewska (1999), as well as in this study. However,
this study also found the correction for spectral emission overlap
of the CXR-labeled fragments less effective than that generated
with the ROX-labeled fragments (Filter Set F). Pull-up in the range
of 2.0-3.5 percent in the NED panel occurred when the CXR matrix
was applied (NED pullup peak relative fluorescence unit (RFU)/CXR
peak RFU). Finally, and perhaps the least involved approach, is
the optimization of the size calling method performed.
This study compared
the Local and Global Southern methods for the sizing of Profiler
Plus alleles. Briefly, both methods use the equation L = [c/(m-m0)]
+ L0 to describe the reciprocal relationship between the mobility,
m, and the length, L0, of the standard fragments (Elder and Southern
1987). However, the Local Southern method averages two fragment
values generated by two curves each of three standard fragments
closest to the unknown, whereas the Global Southern generates one
fragment value by calculating a best fit line of all standard fragments
selected (GeneScan® Analysis Software User's Manual).
Thus, Global Southern is the better sizing algorithm to use to minimize
imprecision due to anomalously migrating standard fragments. However,
this also results in fractionated values for the size-standard fragments.
For example, when the Global Southern sizing algorithm is used,
values such as 74.8 and 400.1 bases are shown for the 75- and 400-base
internal lane fragments after analysis. Consequently, when using
the Global Southern sizing method, the size call range in analysis
parameters of GeneScan® should be increased from
minimum 75, maximum 400 to approximately minimum 74, maximum 402
if the 75 and 400 fragments are to be included in every data set.
as a result of ambient temperature fluctuation was observed with
GS-500 fragments on the 310 Genetic Analyzer. Precision improved
when analyzing with Global Southern in the analysis software and
when excluding the 340-base fragment of GS-500. Although an issue
for capillary electrophoresis, it appears not to be an issue on
other instrument platforms. For example, no adverse effects on precision
have been reported on the slab gel-based ABI Prism®
377 DNA Sequencer (PE Applied Biosystems, Foster City, California),
where both the 250- and 340-base fragments are included in the Local
Southern sizing algorithm (0.01-0.09 base standard deviation, Lazaruk
et al. 1998; PE Applied Biosystems 1997). Even with sizing optimization
on the 310 Genetic Analyzer, attempts should be made to control
large ambient temperature fluctuations. Global Southern may allow
for fluctuations that were unacceptable with Local Southern, but
Global Southern sizing does not completely prevent temperature-shifted
size deviations. Nonetheless, choosing Global Southern and excluding
the 340-base internal fragment should improve precision and reduce
off-ladder allele calls when ambient temperature fluctuations compromise
precision. This method should also be considered for other STR multiplexes
analyzed with GS-500 on the 310 Genetic Analyzer.
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