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