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The Next Generation Capillary Electrophoresis Instrument for Human IdentificationAriana Wheaton*, Jacki Benfield, Jeff Sailus, Andrew Felton, Rachel Fish, Larry Joe, Rob Lagace΄, Eric Nordman, Liwei Qi
TRADEMARKS/LICENSING
AmpFlSTR® For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures. Not for re-sale. Applied Biosystems, AB (Design), AmpFlSTR, GeneMapper, Identifiler, COfiler, Profiler Plus, SGM Plus and Life Technologies Corporation are registered trademarks and MiniFiler is a trademark of Life Technologies Corporation or its subsidiaries in the US and/or certain other countries.© Copyright 2009. Life Technologies Corporation. All rights reserved.
CONCLUSIONSThis next generation 3500-series Genetic Analyzers were designed to support a specific feature set and workflow for Human Identification applications. The 3500-Series improves upon existing capillary electrophoresis (CE) technology by taking a complete systems approach during development to introduce hardware, software and consumables enhancements that will streamline implementation and usage as well as improve instrument performance and reliability. Hardware improvements that allow for shorter run times and higher throughput, in addition to the integrated preliminary data analysis tools, improve HID laboratory efficiency and reduce time to result.
Abstract The gold standard for STR fragment analysis continues to be capillary electrophoresis (CE) genetic analysis platforms. The next generation 3500 (8-capillary) and the 3500xL (24-capillary) genetic analysis systems have improved upon the industry standard for CE by providing greater throughput, flexibility, and ease-of use. This newly designed system supports a specific feature set and workflow for Human Identification applications. The 3500-series genetic analysis systems integrate the steps from system set-up to size-called data to improve system quality control and HID workflow efficiency. There are multiple advancements to this new CE system including: an improved polymer delivery pump design, ready-to-use consumables and containers, Radio Frequency Identification (RFID) consumable tracking, quality control software features for rapid identification and re-injection of failed samples, increased throughput, new laser technology, reduced power requirements, peak height normalization, intuitive user software, and integrated primary analysis software. In addition, optimized run modules have been developed for the analysis of AmpFℓSTR® kit products. Combining the improvements in next generation genetic analysis systems with STR assay improvements will enhance efficiency and performance across the human identification workflow. Figure 1. Interior of the 3500 series instrument and the polymer
delivery system (inset).
Consumables and HardwareConsumables and RFID RFID technology and new consumables have been introduced to improve instrument reliability, electronically track reagent information, and reduce maintenance. The polymer, anode and cathode buffer and capillary array have all been redesigned and optimized for superior performance and handling. The polymer and pre-diluted (1X) anode and cathode buffers are provided in ready-to-use, recyclable containers ( ). This packaging design enabled extensive consumables testing to set appropriate parameters for on-instrument life and expiration dating. Ready to use consumables improves the quality control process by evaluating the CE system as a whole and eliminating the potential introduction of foreign contaminants.
Polymer Delivery System In addition to easing instrument setup, a new polymer system has been introduced that reduces polymer waste and the potential for bubble formation (Figure 1). The lower polymer block, polymer delivery tubing and capillary ferrule in other CE systems were eliminated and the array port, the array tip fitting and the polymer packaging were redesigned (Figure 1, inset). As a result, the system has more direct polymer channels with fewer parts enabling more efficient polymer flow and simpler instrument setup.
3500 Series SoftwareFirst of its kind, the 3500 Data Collection Software was developed with the forensic workflow in mind. Unlike existing data collection software, the 3500 software’s user interface is organized to mimic the progression of sample analysis in a forensic laboratory. Previous data collection software needed to be configured by the user prior to implementation in their laboratory and executing everyday tasks were cumbersome. Results of usability studies conducted during development demonstrated a reduced learning curve for this workflow driven software compared to existing data collection software.
Development GoalsThe next generation capillary electrophoresis (CE) instrument was specifically designed to support the human identification laboratory workflow by utilizing new technology enhancements for hardware, software and consumables. Drawing from customer feedback, and the performance of existing CE instrumentation and software, three high level goals were established: Improved Data Quality, Ease of Use, and Faster Time to Result (Table 1). At each step of the 3500 workflow, we introduced multiple new features to meet the product goals, some of which are displayed in the diagram below.
Temperature Control System The 3500 oven has been redesigned to provide improved temperature control for reproducible sizing precision (Figure 2 and 3). A more compact oven design, an improved door seal and the addition of temperature control to the detection cell holder help maintain temperature consistency across the array and reduce the effects of room temperature fluctuations. The exposure of the capillaries to ambient air has also been minimized at the load header by shortening the needle length and redesigning the array septa.
Sample Peak Height NormalizationTo ensure greater signal balance across instruments, injections, and samples, two methods are available: a hardware-based signal standardization and an optional chemistry and software-based sample normalization. The latter method is currently being evaluated for suitability with HID applications. This method utilizes GS600LIZv2 which employs a new manufacturing process to produce consistent lot-to-lot peak heights necessary for use as a normalization standard. If the software method to normalize data is utilized, the sample peak heights will be scaled relative to the intensity of the co-injected size standard peaks. The average peak height of the internal size standard for each sample is compared to an optimized average size standard peak height (Normalization Target) to determine the normalization factor. The calculated normalization factor is applied to the sample and the peak heights are adjusted accordingly.
The 3500 Data Collection software contains validated protocol information for all AmpflSTR® kits for simple implementation. To start a run, the user selects an HID-specific template (Figure 7), manually enters or imports sample information and assigns the assay to the sample(s) from the pre-configured list.
Multiple tools have been introduced that enable the user to evaluate data real-time at the decision point for reinjections. Featured in the Review Results window of the 3500 Data Collection software (Figure 8) are sized data, quality flags and various plot options for preliminary data analysis to quickly identify samples that may require additional processing and to schedule them for reinjection.
Figure 5. 3500 Data Collection Dashboard window with consumable and instrument status information
Figure 6. Spatial and spectral calibration report (shown) are exported in either .pdf or txt format
Figure 7. HID Plate template shown in a plate view format pre-configured with all validated assays.
Figure 8. Results View window for QC analysis and sample reinjections.
Figure 2. 96 replicates of Identifiler® Allelic Ladder with GS600LIZv2 were injected on three 3500 (left) and three 3500xL (right) instruments. The standard deviation of the mean bp size of each allele within an injection was determined and plotted by marker.
Figure 3. 96 replicates of Identifiler® Allelic Ladder with GS600LIZv2 were injected on three 3500 (left) and three 3500xL (right) instruments. The standard deviation of the mean bp size of each allele across all injections was determined and plotted by marker.
Figure 4. 140 bp fragment of GS600LIZv2; chemistry and software-based normalization disabled (left) and enabled (right).
Instrument
Ready-to-use Consumables
RFID Technology
Maintenance Scheduling
Run Set-Up
Intuitive and Flexible Run
Set-Up
HID Plate Templates
Exportable Plate Map
QC Analysis
QC Flags
Simplified Reinjection
Scheme
Data Collection
Workflow Driven Navigation
Expanded Calibration
Tools
Optional Signal Adjustment
GMID-X v1.2
Builds on ID-X 1.1
Support for Windows XP
and Vista
Additional Run History Information
Quality Control Data Analysis
Tools
Marker_1_1vW
ATP
OXTH
01FG
A
D8S11
79
D7S82
0
D5S81
8
D3S13
58
D2S13
38
D21S1
1
D19S4
33
D18S5
1
D16S5
39
D13S3
17
CSF1P
OAM
EL
0.150
0.125
0.100
0.075
0.050
0.025
0.000
vWA
TPOX
TH01
FGA
D8S1
179
D7S82
0
D5S81
8
D3S13
58
D2S13
38
D21S1
1
D19S4
33
D18S51
D16S5
39
D13S3
17
CSF1P
O
AMEL
8
Stan
dard
Dev
iati
on o
f M
ean
Size
(bp
)
24
3500 Sizing Precision - Per 96-well Plate3500-Series Sizing Precision - Per Plate
Marker_1vW
ATPO
XTH01
F GA
D8S11
79
D7S82
0
D5S81
8
D3S13
58
D2S13
38
D21S1
1
D19S4
33
D18S
51
D16S5
39
D13S3
17
CSF1P
OAMEL
0.150
0.125
0.100
0.075
0.050
0.025
0.000
vWA
TPOX
TH01
FGA
D8S11
79
D7S82
0
D5S81
8
D3S13
58
D2S13
38
D21S1
1
D19S43
3
D18S51
D16S5
39
D13S3
17
CSF1P
O
AMEL
8
Stan
dard
Dev
iatio
n of
Mea
n Si
ze (
bp)
24
3500 Sizing Precision - Per Injection3500-Series Sizing Precision - Per Injection
Normalization- Disabled Normalization- Enabled
Table 1. High level development goals and 3500 features and benefits