WellAware™ Forensic Package The custom WellAware™ Forensic Package was developed for the Broward County Sheriff’s Office DNA Crime Lab in Ft. Lauderdale Florida. This forensic application shows how WellAware™ GPS Pipetting Systems™ can be interfaced with any instrumentation (such as ABI, Stratagene, Beckman or Agilent) to create electronic SOP’s for your lab. This WellAware™ Forensic Package was developed to help the Broward forensic lab pipet Quant and Normalization plates for use with ABI 7000 real-time PCR systems, 310 and 3100 Genetic Analyzer instrumentation. Besides eliminating witnessing with WellAware™, Broward is eliminating errors and saving time pipetting. for use with…

Settings Menu Used to allow customization for any lab. Settings Menu allows the WellAware™ Forensic package to be customized for each lab. WellAware™ can be used to create an electronic Standard Operating Procedure or eSOP for any equipment or pipetting process in your lab.

WellAware™ Forensic Package Flow Diagram ABI 310/3100 Sequencer WellAware™ Complete This flow diagram depicts the workflow for quantifying (on a ABI 7000 RT-PCR system) and then normalizing the DNA for loading onto a Genetic Analyzer (ABI 310 or 3100). WellAware™ Compact ABI 7000 RT-PCR

WellAware™ Forensic Package How it Works 1 WellAware™ Forensic Package 1. Samples that have been extracted and are ready for quantification with an AB 7000 Real Time PCR instrument, are entered into the plate form and then printed. 2. The next step is to export a file for the quantification setup in WellAware format. This file is opened in WellAware which then guides the scientist to load the extracted samples into the AB 7000 Real Time PCR plate. WellAware voice and light cues direct the addition of master mix to the wells. 3. In the Forensic Interface, the scientist exports plate format information for running the AB 7000. 4. Once the AB 7000 real time PCR instrument has completed the quantification of samples, the results from the AB 7000 Prism Instrument software are imported back into the Forensic Interface. Plate Form for ABI 7000 2 WellAware™ Complete 3 This flow diagram depicts the first 4 steps of the Forensic Package workflow for quantifying (on a ABI 7000 RT-PCR system) and then normalizing the DNA for loading onto a Genetic Analyzer (ABI 310 or 3100). ABI 7000 RT-PCR WellAware™ Forensic Package 4

WellAware™ Forensic Package How it Works Processing Screen Norm & 310/3100 data 5. The DNA processing screen allows the scientist to add the samples that have now been quantitated to an AB 310 or AB 3100 plate for electrophoresis and detection. This set up is flexible, and the scientist can run a sample multiple times, and can even override the dilution amount if they choose. Additional sample descriptions, for example case number, can be added under the sample header. 6. Once the scientist has the samples in the order they have specified, they can just click on either the AB 310 or AB 3100 tab, depending upon which platform they would like to set up. After printing the sample set-up sheet, a WellAware file can be created by clicking on the Export Data tab and designating whether to pipette by rows or columns. 7. The exported file is opened in the WellAware software and the scientist can now set up their dilution plate for amplification. WellAware calls out how much dilutant (TE or H2O) to add to each well of the dilution plate. It will then ask for the barcoded extracts to be scanned. Once scanned, WellAware calls out the volume of DNA to add to each well of the dilution plate, and lights up the destination well. Once the sample has been pipetted the footswitch is used to proceed to the next sample. 8. The final step in using the Forensic Interface is in the exporting of the AB 310 or AB 3100 file. The information put into the processing screen for the dilution, step 6, is used to generate the file for the AB 310 or AB 3100 Genetic Analyzers. WellAware™ Forensic Package 5 8 WellAware™ Compact ABI 310/3100 Sequencer 6 This flow diagram depicts the last 4 steps of the Forensic Package workflow for quantifying (on a ABI 7000 RT-PCR system) and then normalizing the DNA for loading onto a Genetic Analyzer (ABI 310 or 3100). 7

WellAware™ Forensic Package Main Screen Step 5 Step 3 Step 2 Step 1 Step 7 Step 6 The Main Screen of the WellAware™ Forensic Package is used to Import and Export files for WellAware™ and the ABI 7000 RT-PCR, 310 and 3100 Sequencer instruments. The buttons associated with the steps in the previous slide’s flow diagram are labeled in blue on this slide and in subsequent slides. This program can be easily modified for use with other instrumentation. Step 8 Step 8 Step 4

Quant Worksheet The WellAware™ Quant Worksheet is used for Step 1 of the flow diagram. The operator double clicks on the empty well locations to automatically add the next sample identifier (E241-11 in this case). Once the worksheet is filled out the operator prints the worksheet for their records, exits to the main screen and clicks on the WellAware™ Quant button to generate the pipetting steps for WellAware™. Step 1

WellAware™ Quant RT-PCR Pipetting Data (Page 1) Step 2 This is an example of the WellAware™ pipetting step data for the Quant Worksheet shown on the previous slide. This is the data generated during Step 2 of the flow diagram.

WellAware™ Quant RT-PCR Pipetting Data (Page 2) This is an example of the WellAware™ pipetting step data for the Quant Worksheet shown on the previous slide. This is the data generated during Step 2 of the flow diagram.

WellAware™ Quant Pipetting Step 3 a b a) Open WellAware™, click the Saved Programs button, select the correct pipetting file and run. b) WellAware™ is then used to pipet the ABI Quantifiler reagents. c) The plate just pipetted is then put in the ABI 7000 RT-PCR instrument. This is Step 3 of the flow diagram. c

ABI 7000 real-time PCR Export Data Now that the plate is ready to quantify, it is necessary to return to the Forensic Package and click the ABI 7000 button under the Export Data heading. The data shown above is saved to the file location specified in the Settings screen of the Forensic Package. This data contains the sample and standard locations previously pipetted for the ABI 7000 real-time PCR system. From the ABI 7000 software the file is loaded and the plate made in Step 3 is quantified on the instrument. This is Step 4 of the flow diagram. Step 4

ABI 7000 RT-PCR system Normalization Data Step 6 Calculated normalization values Raw data from ABI 7000 Screen shot of the data from the ABI 7000 real-time Quantitative PCR system. The Sample names in the yellow section show up in the pull down menu in the WellAware™ Norm Worksheet. The calculated values in the blue section are used in step 6.

Normalization Process Step 5 Step 7 Step 6 In Step 5, the ABI 7000 RT-PCR data is imported back into the Forensic Package using the ABI 7000 button in the Import Data heading. From the Main Screen select WellAware™ Norm under the Show Worksheets heading (Step 6). The samples that will be normalized are selected from the pull down menu labeled above. Once all needed samples have been selected, the operator exits back to the Main Screen and clicks the WellAware™ Norm button under the Export Data heading (Step 7). WellAware™ is opened and the file generated in Step 7 is pipetted on the WellAware™ screen. WellAware™ can automatically normalize oligos, DNA and proteins from other instruments. This can be accomplished using the Normalization Wizards included with WellAware™ . Pull down menu

WellAware™ Normalization Pipetting Data Step 7 Sample WellAware™ pipetting step data for the Normalization Worksheet shown on the previous slide. This is the data generated during Steps 6 & 7 of the flow diagram.

ABI 310/3100 (Genetic Analyzer) Worksheet Step 7 Step 8 As the data is entered in Step 6 (Normalization) the worksheet above is populated for printing and documentation. Once the normalization pipetting is completed using WellAware™ the plate is transferred to the Genetic Analyzer. The operator opens the Forensic Package and clicks the appropriate Genetic Analyzer button (ABI 310 or ABI 3100) from the Export Data heading (Step 8). Using the software on the Genetic Analyzer the operator opens the file created in Step 8 and runs the analysis. What instrumentation would you suggest for integration with WellAware™ GPS Pipetting Systems™ ?

Contract Engineering and Software Utilizes company’s core technology strengths Software and engineering Customers: MWG, PicoScript, Valco, Sigma-Genosys Custom PAGE gel rigs Petri dish plating robot Blood card punching robot Peptide & DNA Synthesizers

BioTX’s RADS™ Robot Tools During BioTX’s first year it was a pure consulting company. We took on several automation projects including web data mining and the robot pictured above. We used our Rapid Automation Development System (RADS) manufacturing process to automate cell spreading on Petri dishes. (Click on Movie)

BioTX Contacts 16753 Donwick Drive, Suite A6 Conroe, TX 77385 James Frost jdfiii@biotx.net Mary Freeman mfreeman@biotx.net 16753 Donwick Drive, Suite A6 Conroe, TX 77385 936-273-2633