COMPARATIVE DATA ANALYSIS DNA EXTRACTOR WORKFLOW m Liz Thompson, BA, MB(ASCP) Clinical Lab Consulting, LLC 47 S Meridian St., Second Floor Indianapolis, IN Tel: Fax:

Introduction A comparative data analysis was performed on robotic DNA extraction platform workflows. The comparison contains data on five platforms and two specimen types for each platform. The analysis includes all steps to extract DNA: specimen preparation, reagent loading, and instrument run time. Platforms included in the study: 1.Roche Diagnostics MagNA Pure 96 2.QIAGEN BioRobot Universal 3.QIAGEN QIAsymphony 4.Life Technologies MagMAX 5.Biomérieux Nuclisens EasyMAG Specimen types for comparison: 1.Whole Blood 2.Buccal Swab Note: Refer to appendices for data tables and workflow diagram.

Results Summary For buccal swab specimens, the range of time scores is 139:45 – 390:55 (MM:SS). The Roche MagNA Pure 96 platform was the second-fastest platform at 160:30. The fastest platform was the MagMAX from Life Technologies (the MagMAX has only a 3 minute lysis incubation, while all other platforms are minute incubations). However, the MagMAX required approximately 14 minutes more tech time to process 96 specimens than the MagNA Pure 96 Instrument setup including loading plastics, loading reagents, and setting up software was comparable between platforms. The most significant differences that affected total workflow time scores were: Instrument run time (buccal swab and whole blood). 01. Aliquoting specimens (Roche and Life Technologies) vs. loading original specimen collection container directly onto platform (both QIAGEN platforms) (whole blood). 02.

Conclusion For whole blood specimens, the Roche MagNA Pure 96 was the fastest protocol of all five platforms, with significantly faster instrument run time than the QIAGEN platforms (Average of ~2 hours faster) and significantly less tech time than the MagMAX 96 system (54 min less). For buccal swab specimens, the differences were less significant. The MagMAX system was the fastest protocol time by 20 minutes. The MagNA Pure 96 system came in second in speed of protocol, but with 14 minutes less tech time than the MagMAX. Acknowledgements We would like to thank the staff of the laboratories that supplied us with data for this study or allowed us to observe their DNA extraction workflows in person. References 1. MagNA Pure 96 Kits and Protocols. v3.0. Roche Diagnostics; August MagNA Pure 96 User Training Guide. v2.0. Roche Diagnostics; February QIAamp® DNA Blood BioRobot® MDx Kit Handbook. 3 rd ed. QIAGEN; April QIAamp® 96 DNA Swab BioRobot® Kit Handbook. 4 th ed. QIAGEN; April QIAsymphony® DSP DNA Handbook. v1. QIAGEN; February MagMAX™ 96 DNA Multi-Sample Kit. RevB. Life Technologies; August EasyMAG Simplified User Guide. Biomérieux; June 2011.

Time Score Summary – Buccal Swab and Whole Blood Appendix 1. BUCCAL SWABMagNA Pure (96)BioRobot (96)QIAsymphony (96) MagMAX (96)EasyMAG (24)EasyMAG (24) EasyMAG (x4=96) Sample Prep70:0070:0032:30*32:30*70:0070:0084:1084:10 22:00 88:0088:00 Lysis Incubation 20:0020:0060:0060:0060:0060:00/ 10:00 40:0040:00 Loading Instrument - Reagent, Sample, Plastics, Software Setup 16:3016:3018:0018:0020:5520:5516:3516:35 06:00 25:4025:40 Run Time54:0054:00 110:00110:00240:00240:0040:0040:00 40:00 160:00160:00 Tech Time:86:3050:3090:5599:4528:25113:40 Total Time:160:30220:0390:55139:4578:25313:40 *Swabs are loaded directly into 96-well plate and not removed, so prep time is lower.

Time Score Summary – Buccal Swab and Whole Blood Appendix 1. WHOLE BLOODMagNA Pure (96)BioRobot (96)QIAsymphony (96) MagMAX (96)EasyMAG (24)EasyMAG (24) EasyMAG (x4=96) Sample Prep (aliquot or load original blood tubes in rack) 40:00**16:0022:15101:00 13:33 54:12 Lysis Incubation//// 10:00 40:0040:00 Loading Instrument - Reagent, Sample, Plastics, Software Setup 16:0020:3820:2513:05 06:31 26:04 Run Time56:0056:00 171:00240:00240:0040:0040:00 40:00 160:00160:00 Tech Time:60:0036:3842:40114:0522:0488:16 Total Time:116:00207:38282:40154:0572:04288:16 **Includes pipetting blood into cartridge, which is not required for QIAGEN platforms.

Extended Time Score Summary – Buccal Swab Appendix 2. BUCCAL SWABMagNA Pure (96) (MM:SS) BioRobot (96) (MM:SS) QIAsymphony (96) (MM:SS) MagMAX (96) (MM:SS) EasyMAG (24) (MM:SS) Load Buccal swabs, double check sample IDs (label tubes, if applicable). 30:00 12:00 Prepare internal control. 05:00/// / Load lysis buffer. 05:0002:0005:0003:00 / Incubate swabs in PK/lysis buffer. 20:0060:00 / 10:00 Remove swabs from tubes and transfer lysis solution into plate/cartridge. 35:00/ / / Turn on instrument. 00:3002:0000:3002:00 01:30 Fill water reservoir and bottle (under instrument and in carousel). /03:00// / Set up software (choose method). 01:3003:0001:20 00:15 Pipette lysed sample into cartridge. //// 10:00 Prepare silica solution and add to lysed samples.////03:00 Prep reagents (and load onto instrument, if applicable). 08:0003:0017:3508:00 / Load plastics (and reagents into troughs, if applicable). 06:0002:00 / Load samples onto platform. 00:30 / 01:30 Complete setup in software to start instrument.01:00 // 00:10 Shake the unsealed plate on a titer plate shaker for 3 minutes at speed 8. /// 03:10/ Remove the plate from the shaker, then carefully remove the swabs, leaving behind as much lysis buffer as possible (approximately 200 μL). /// 15:00/

Extended Time Score Summary – Buccal Swab Appendix 2. BUCCAL SWABMagNA Pure (96) (MM:SS) BioRobot (96) (MM:SS) QIAsymphony (96) (MM:SS) MagMAX (96) (MM:SS) EasyMAG (24) (MM:SS) Add 160 μL of 100% isopropanol to each buccal swab sample on the MagMAX™ Deep Well 96-Well Plate. ///06:00 / then shake the sealed plate for 3 minutes at speed 8 on a titer plate shaker. ///04:00 / Remove the plate from the shaker, carefully remove the cover, then add 20 μL of prepared DNA Binding Bead Mix to each lysate on the plate. ///08:00 / Seal the plate, then shake for 3 minutes at speed 8 on the titer plate shaker. ///04:00 / While the plate is shaking, prepare sufficient RNase A mix for the correct number of samples. ///03:00 / Prepare the plates for the MagMAX™ Express-96 Deep Well Magnetic Particle Processor. ///08:00 / Combine the MagMAX™ Express-96 Deep Well Tip Comb and the MagMAX™ Express 96-Well Plate. // /02:00 / If the lid is in place, open the sliding door, then load the plates into the loading station as prompted by the instrument. /// 01:05/ Close hood and drawers. //01:00 // Run instrument. 54:00110:00240: :00 After the run is started, load the following reagents into the plates at the loading stations listed in the following table when prompted by the instrument. /// 00:30/ Total Time : 160:30220:30390:55 139:4578:25

Extended Time Score Summary – Whole Blood Appendix 3. WHOLE BLOODMagNA Pure 96 (MM:SS) BioRobot 96 (MM:SS) QIAsymphony 96 (MM:SS) MagMAX 96 (MM:SS) EasyMAG 24 (MM:SS) Load blood tubes onto a rocker for homogenization and to come to room temp. If original blood tubes are loaded: Uncap blood tubes and load into racks. 20:0016:0021:30 / / If blood is aliquoted: Aliquot lysis buffer into plate. ///03:00 / If blood is aliquoted: Aliquot blood samples into plate. 24:00// 35:0013:33 Seal and incubate plate with aliquoted blood./ // 22:00 Turn on instrument (and computer, if applicable).02:00 00:3002:0001:30 Prepare internal control.05:00//// Fill water reservoir and bottle (under instrument and in carousel)./03:00/// Set up software, choose method.01:0003:1501:20 / 02:37 Prep reagents and load onto instrument. 08:00 03:1017:3511:0000:18 Load plastics (and reagents into troughs, if applicable). 06:13 02:0000:14 Load racked blood onto platform. 02:2500:45 /00:06 Click on the Dispense Lysis icon to start incubation protocol, run incubation.////10:03 Complete setup in software to start instrument./00:35/// Remove the plate from the heat source, then carefully remove the cover, and add 200 μL of Multi-Sample DNA Lysis Buffer to each sample on the plate. ///04:00/ Seal the plate, then shake for 3 minutes at speed 8 on a titer plate shaker./// 04:00 / Remove the plate from the shaker, carefully remove the cover, then add 20 μL of DNA binding bead mix to each sample on the plate. / //08:00/

Extended Time Score Summary – Whole Blood Appendix 3. WHOLE BLOODMagNA Pure 96 (MM:SS) BioRobot 96 (MM:SS) QIAsymphony 96 (MM:SS) MagMAX 96 (MM:SS) EasyMAG 24 (MM:SS) Seal the plate, then shake for 3 minutes at speed 7 on the titer plate shaker. /// 04:00/ Remove the plate from the shaker, carefully remove the cover, then add 240 μL of 100% isopropanol to each sample on the plate. / // 06:00/ Seal the plate, then shake for 3 minutes at speed 7 on the titer plate shaker.///04.00/ Combine the MagMAX™ Express-96 Deep Well Tip Comb and MagMAX™ Express 96-Well Plate. ///02:00/ If the lid is in place, open the sliding door, then load the plates onto the loading station as prompted by the instrument. / //02:05/ During the incubation prepare the silica solution. /// /01:40 Aliquot 125 μL portions into an 8 well strip./ // /00:53 Add silica mixture to lysed samples after incubation is complete.////01:16 Close hood and drawers.//01:00// Run instrument.56:00171:00240:0040:0040:04 After the run is started, add Elution Buffer 2 to the elution plate when prompted by the instrument. ///05:00/ Total Time:116:00207:38282:40154:0572:04

Time Scores by Platform and Specimen Type Appendix 4. Roche MagNA Pure - 96 Whole Blood Specimens Step #Step DescriptionTime (MM:SS) 1.Mix and bring specimens to room temperature.20:00 2.Turn on instrument, log in, system performs self-checks during sample prep.02:00 3.Prepare internal control by aliquoting into IC tube.05:00 4.Prepare room temperature specimens by aliquoting 200μL of each specimen into processing cartridge.24:00 5.Set up software for 96 specimen whole blood run.01:00 6. Load the bottle rack. Load the reagent rack. Load the tip rack. Load the waste rack. 01:00 05:00 01:00 7.Run the instrument.56:00 Total Time:116:00

Time Scores by Platform and Specimen Type Appendix 4. Roche MagNA Pure - 96 Buccal Swab Specimens Step #Step DescriptionTime (MM:SS) 1.Turn on the instrument and set up the software to perform a buccal swab run.02:00 2.Prepare internal control by aliquoting into IC tube.05:00 3.Transfer one buccal swab into one 1.5 ml Eppendorf reaction vial and remove the swab from the plastic carrier.30:00 4.Pipette 200 μl of Proteinase K on top of the swab, and allow the liquid to completely soak the swab.05:00 5.Incubate at 65C for 10 min, then 95C for 10 min.20:00 6.Transfer the swab solutions (200μl) into the single wells of the Sample Cartridge.35:00 7.Load specimens onto platform.00:30 8.Set up software for a 96-specimen swab run.01:00 9. Load the bottle rack. Load the reagent rack. Load the tip rack. Load the waste rack. 01:00 05:00 01: Run the instrument.54:00

Time Scores by Platform and Specimen Type Appendix 4. QIAGEN BioRobot Universal - 96 Whole Blood Specimens Step #Step DescriptionTime (MM:SS) 1.Load blood tubes onto a rocker for homogenization and bring to room temp. Uncap blood tubes and load into racks.16:00 2.Turn on instrument, fill water reservoir and bottle (under instrument and in carousel).05:00 3.Set up software (choose method).03:15 4.Prep reagents and load onto carousel (ethanol, wash buffers).03:10 5.Load plastics (plates, tips, troughs - reagents into troughs (elution and lysis buffers).06:13 6.Load racked blood onto platform, double check sample IDs on blood tubes with map.02:25 7.Complete setup in software to start instrument.00:35 8.Run instrument (clog check 1 hour and 55 mins in - visual check, then click button to resume method).171:00 Total Time:207:38

Time Scores by Platform and Specimen Type Appendix 4. QIAGEN BioRobot Universal - 96 Buccal Swab Specimens Step #Step DescriptionTime (MM:SS) 1.Load Buccal swabs into 96-well plate, double check sample IDs.30:00 2.Load lysis buffer into 96-well plate.02:00 3.Incubate swabs in PK/lysis buffer.60:00 4.Turn on instrument.02:00 5.Fill water reservoir and bottle (under instrument and in carousel).03:00 6.Set up software (choose method).03:00 7.Prep reagents and load onto instrument.03:00 8.Load plastics (and reagents into troughs, if applicable).06:00 9.Load samples onto platform.00:30 10.Complete setup in software to start instrument.01:00 11.Run instrument.110:00 Total Time:220:30

Time Scores by Platform and Specimen Type Appendix 4. QIAGEN QIAsymphony - 96 Whole Blood Specimens Step #Step DescriptionTime (MM:SS) 1.Preparation of sample material - mix and bring to room temp.20:00 2.Check that Buffers QSL1 and QSB1 do not contain a precipitate.00:30 3.Ensure the piercing lid is placed on the reagent cartridge and the lid of the magnetic-particle trough has been removed.00:45 4.Open the enzyme tubes.01:00 5. If samples are barcoded, orient samples in the tube carrier so that the barcodes face the barcode reader on the left side of the QIAsymphony SP. 01:30 6.Log on to the instrument.00:30 7.Load reagent cartridges, plastics, eluate drawer, and waste drawer.02:00 8. Ensure the “Reagents/Consumables” drawer is prepared properly, and perform an inventory scan of the “Reagents/Consumables” drawer. 10:00 Ensure the “Sample” drawer is prepared properly, and perform an inventory scan of the “Sample” drawer.01:00

Time Scores by Platform and Specimen Type Appendix 4. QIAGEN QIAsymphony - 96 Whole Blood Specimens Step #Step DescriptionTime (MM:SS) Ensure the “Elute” drawer is prepared properly, and perform an inventory scan of the “Elute” drawer.01:00 Ensure the “Waste” drawer is prepared properly, and perform an inventory scan of the “Waste” drawer, including the tip chute and liquid waste. Replace the tip disposal bag if necessary. 01:20 9.Place the samples into the appropriate sample carrier, and load them into the “Sample” drawer.00:45 10.Close all drawers and the hood.01:00 11.Using the touchscreen, enter the required information for each batch of samples to be processed.01:20 12.Run the instrument.240:00 Total Time:282:40

Time Scores by Platform and Specimen Type Appendix 4. QIAGEN QIAsymphony - 96 Buccal Swab Specimens Step #Step DescriptionTime (MM:SS) 1.Transfer one buccal swab into one 1.5 ml Eppendorf reaction vial and remove the swab from the plastic carrier.30:00 2.Pipette 200 μl of Proteinase K on top of the swab, and allow the liquid to soak the swab completely.05:00 3.Incubate swabs in PK/lysis buffer.60:00 4.Transfer the swab solutions (200μl) into the single wells of a 96-well plate.35:00 5.check that Buffers QSL1 and QSB1 do not contain a precipitate.00:30 6. Make sure that the piercing lid is placed on the reagent cartridge and the lid of the magnetic-particle trough has been removed. 00:45 7.Load reagent cartridges, plastics, eluate drawer, and waste drawer.02:00 8.Open the enzyme tubes.01:00 9.Log on to the instrument.00:30

Time Scores by Platform and Specimen Type Appendix 4. QIAGEN QIAsymphony - 96 Buccal Swab Specimens Step #Step DescriptionTime (MM:SS) 10. Ensure the “Reagents/Consumables” drawer is prepared properly, and perform an inventory scan of the “Reagents/Consumables” drawer. 10:00 Ensure the “Sample” drawer is prepared properly, and perform an inventory scan of the “Sample” drawer.01:00 Ensure the “Eluate” drawer is prepared properly, and perform an inventory scan of the “Elute” drawer.01:00 Ensure the “Waste” drawer is prepared properly, and perform an inventory scan of the “Waste” drawer, including the tip chute and liquid waste. Replace the tip disposal bag if necessary. 01:20 11.Load the samples onto the platform.00:30 12.Close all drawers and the hood.01:00 13.Using the touchscreen, enter the required information for each batch of samples to be processed.01:20 14.Run the instrument.240:00 Total Time:390:55

Time Scores by Platform and Specimen Type Appendix 4. Life Technologies MagMAX - 96 Whole Blood Specimens Step #Step DescriptionTime (MM:SS) 1.Add isopropanol and ethanol to Wash Solution 1 Concentrate and Wash Solution 2 Concentrate.05:00 Prepare sufficient DNA Binding Bead Mix for your sample extraction.03:00 Prepare the PK buffer/enzyme mix.03:00 2. For each sample, add 50 μL of PK buffer/enzyme mix to each well of a MagMAX™-96 Deep Well Plate or processing plate. 03:00 3. For each sample, add 50 μL of blood sample to each well of the MagMAX™-96 Deep Well Plate or processing plate, mixing the solution by pipetting up/down 5 to 7 times. 35:00 4. Seal the plate using a MicroAmp® Clear Adhesive Film, then incubate the sealed plate on a 96-well heated block for 20 minutes at 60 to 65 °C. 22:00 5. Remove the plate from the heat source, then carefully remove the cover., then add 200μL of Multi-Sample DNA Lysis Buffer to each sample on the plate. 04:00 Seal the plate, then shake for 3 minutes at speed 8 on a titer plate shaker.04:00 6. Remove the plate from the shaker, carefully remove the cover, then add 20 μL of DNA Binding Bead mix to each sample on the plate. 08:00

Time Scores by Platform and Specimen Type Appendix 4. Life Technologies MagMAX - 96 Whole Blood Specimens Step #Step DescriptionTime (MM:SS) Seal the plate, then shake for 3 minutes at speed 7 on the titer plate shaker.04:00 7. Remove the plate from the shaker, carefully remove the cover, then add 240 μL of 100% isopropanol to each sample on the plate. 06:00 Seal the plate, then shake for 3 minutes at speed 7 on the titer plate shaker.04:00 8.Power on the MagMAX™ instrument.02:00 9.Load the MagMAX™ instrument.02:00 Combine the MagMAX™ Express-96 Deep Well Tip Comb and MagMAX™ Express 96-Well Plate.02:00 If the lid is in place, open the sliding door.00:05 Load the plates onto the loading station as prompted by the instrument.02:00 10.Run the instrument.40:00 After the run is started, add DNA Elution Buffer 2 to the elution plate when prompted by the instrument.05:00 Total Time:154:05

Time Scores by Platform and Specimen Type Appendix 4. Life Technologies MagMAX - 96 Buccal Swab Specimens Step #Step DescriptionTime (MM:SS) 1.Add isopropanol and ethanol to Wash Solution 1 Concentrate and Wash Solution 2 Concentrate.05:00 Prepare sufficient DNA Binding Bead Mix for your sample extraction and store at room temperature.03:00 Add 400 μL of Multi-Sample DNA Lysis Buffer to 96 wells of a MagMAX™ Deep Well 96-Well Plate.03:00 2. Load swabs into plate, cut and remove the stick portion of the swab (to make shaking easier), then place it into a well containing lysis buffer. 03:00 3.Shake the unsealed plate on a titer plate shaker for 3 minutes at speed 8.03:10 4. Remove the plate from the shaker and remove the swabs, leaving behind as much lysis buffer as possible (approximately 200 μL). 15:00 5.Add 160 μL 100% isopropanol to each buccal swab sample.06:00 Seal the plate, then shake the sealed plate for 3 minutes at speed 8 on a titer plate shaker.04:00 6. Remove the plate from the shaker, carefully remove the cover, then add 20 μL of prepared DNA Binding Bead Mix to each lysate on the plate. 08:00 Seal the plate, then shake for 3 minutes at speed 8 on the titer plate shaker.04:00 7.While the plate is shaking, prepare RNase A mix.03:00

Time Scores by Platform and Specimen Type Appendix 4. Life Technologies MagMAX - 96 Buccal Swab Specimens Step #Step DescriptionTime (MM:SS) 8.Prepare the plates for the MagMAX™ Express-96 Deep Well Magnetic Particle Processor.08:00 Load the MagMAX™ instrument.02:00 Combine the Express-96 Deep Well Tip Comb and the Express 96-Well Plate.02:00 9.Power on the MagMAX™ instrument.02:00 10.If the lid is in place, open the sliding door.00:05 Load the plates into the loading station as prompted by the instrument.01:00 11.Run the instrument.40:00 After the run is started, load the following reagents into the plates at the loading stations listed in the following table when prompted by the instrument. 04:00 Total Time:139:45

Time Scores by Platform and Specimen Type Appendix 4. Nuclisens EasyMAG - 24 Whole Blood Specimens Step #Step DescriptionTime (MM:SS) Start the instrument. When the LED on the EasyMAG turns green, turn on the 1.Computer.01:30 2.Log onto the EasyMAG software.00:07 3.Check the setting through the Settings icon.00:02 4.Under the Instrument icon barcode in the reagents.00:18 5.Click on the Daily Use icon and select the extraction protocol.00:10 6.Enter sample IDs.02:08 7.Add samples to the vessel under the biological hood.13:33 8.Insert the tips and sample vessel into instrument.00:14 9.Barcode the sample vessel position and sample vessel into the software.00:06 10.Click on the Dispense Lysis icon to start incubation protocol, run incubation.10:03 11.During the incubation prepare the silica solution.01:40

Time Scores by Platform and Specimen Type Appendix 4. Nuclisens EasyMAG - 24 Whole Blood Specimens Step #Step DescriptionTime (MM:SS) 12.Aliquot 125 μL portions into an 8 well strip.00:53 13.Add silica mixture to lysed samples after incubation is complete.01:16 14.Click on the Start icon to begin the rest of the run.40:04 Total Time:72:04

Time Scores by Platform and Specimen Type Appendix 4. Nuclisens EasyMAG - 24 Whole Blood Specimens Step #Step DescriptionTime (MM:SS) Start the instrument. When the LED on the EasyMAG turns green, turn on the 1.Computer.01:30 2.Log onto the EasyMAG software and check the settings.00:15 3.Click on the Daily Use icon and select the extraction protocol.00:10 4.Number lysis tubes.02:00 5.Add samples to lysis tubes.10:00 6.Incubate in lysis for 10 minutes. During this time, load reagents and plastics onto instrument and number cartridge.10:00 7.Pipette lysis tube contents into cartridge.10:00 8.Insert the cartridge on the instrument.01:30 9.Prepare the silica solution.02:00 10.Add silica mixture to lysed samples after incubation is complete.01:00 11.Click on the Start icon to begin the rest of the run Total Time:78:25

10 steps min 96 specimens 11 steps min 96 specimens 11 steps min 96 specimens 11 steps 78.5 min 24 specimens 14 steps 391 min 96 specimens Workflow Diagram – Whole Blood Appendix 5. WORKFLOW COMPARISON – WHOLE BLOOD Roche MagNA Pure 96QIAGEN BioRobotQIAGEN QIAsymphonyLife Tech MagMAXNuclisens Easy MAG 1 min 30 sec Start instrument and computer 11 min Prepare and aliquot reagents 30 min Transfer swabs to 1.5mL tubes 30 min Load swabs into 96-well plate 2 min Turn on instrument, set up software 5 min Prepare and aliquot internal control 2 min Load lysis buffer into 96-well plate 5 min Add PK to swabs 30 min Load swabs into 96-well plate 15 sec Log on to software 30 min Transfer swabs to 1.5mL tubes 60 min Incubate swabs 3 min 10 sec Shake plate for 3 min 10 sec Set up software 60 min Incubate swabs 5 min Add PK to swabs 15 min Remove swabs from 96-well plate 2 min Number lysis tubes 2 min Turn on instrument 35 min Transfer swab solutions to plate 20 min Incubate swabs 30 sec Check buffers for precipitate 10 min Add specimens to lysis tubes 3 min Fill water containers 10 min Add isopropanol, shake 3 min 3 min Prep and load reagents 1 min Open enzyme tubes 10 min Transfer specimens to cartridge 3 min Prepare RNase A mix 30 sec Load specimens onto platform 30 sec Load swab plate onto platform 2 min Turn on the instrument 2 min Prepare silica solution 1 min Open enzyme tubes 8 min Load bottle, reagent, tip, waste racks 54 min Run the instrument 1 min Complete software setup 30 sec Log on to instrument 1 min Add silica solution to specimens 1 min 5 sec Open the sliding door and load plates 1 min Set up software 6 min Load plastics 2 min12 min Prepare and load plastics 1 min 30 sec Load cartridge onto platform Load reagent, plastics, eluate, waste drawers 240 min Run the instrument 1 min Close drawers and hood 1 min 20 sec Set up software 30 sec Load specimens onto platform 110 min Run the instrument 40 min 30 sec40 min Run the instrument 13 min 20 sec Check reagent, sample, eluate, waste drawers Run the instrument (add elution buffer) 35 min Transfer swab solutions to cartridge 3 min Set up software 45 sec 10 min Incubate swabs. Load reagents and plastics. 12 min Add bead mix, shake 3 min Check piercing and particle trough lids

Workflow Diagram – Whole Blood Appendix 5. WORKFLOW COMPARISON – WHOLE BLOOD 24 min Aliquot specimens into cartridge 3 min 10 sec Prep and load reagents 1 min Open enzyme tubes 22 min Seal plate and incubate for 20 min 18 sec Scan in reagents Roche MagNA Pure 96QIAGEN BioRobotQIAGEN QIAsymphonyLife Tech MagMAXNuclisens Easy MAG 1 min 30 sec Start instrument and computer 11 min Prepare reagents 20 min Mix, bring to room temp, rack blood 16 min Prep and rack blood tubes 20 min Mix specimens, bring to room temperature 2 min Turn on instrument, inst. self-check 5 min30 sec Check buffers for precipitate 3 min Aliquot enzyme mix to 96-well plate 7 sec Log on to software Turn on instrument, fill water containers 5 min Prepare and aliquot internal control 3 min 15 sec Set up software 45 sec35 min Aliquot blood to 96-well plate 2 sec Check software settings Check piercing and particle trough lids 12 steps min 96 specimens 14 steps 72 min 24 specimens 1 min Set up software 7 steps 116 min 96 specimens 6 min 13 sec Load plastics 8 steps min 96 specimens 1 min 30 sec Face blood tube barcodes 8 min Add lysis buffer, shake 3 min 10 sec Select protocol in software 45 sec Load specimens into sample drawer 6 min 5 sec Load plastics 6 sec Scan vessel into software 1 min Close drawers and hood 10 steps 154 min 96 specimens 10 min 3 sec Run incubation protocol 8 min2 min 25 sec Load racked blood onto platform 30 sec Log on to instrument software 12 min Add bead mix, shake 3 mins 2 min 8 sec Enter specimen IDs Load bottle, reagent, tip, waste racks 56 min Run the instrument 35 sec Complete software setup 10 min Add isopropanol, shake 3 mins 13 min 33 sec Aliquot specimens into vessel 2 min Load reagent, plastics, eluate, waste drawers 171 min Run the instrument 2 min Turn on the instrument 14 sec Load tips and vessel onto platform 13 min 20 sec Check reagent, sample, eluate, waste drawers 45 min Run the instrument (add elution buffer) 240 min Run the instrument 40 min 4 sec Run the instrument 1 min 16 sec Add silica solution to lysed specimens 53 sec Aliquot silica solution into 8-well strip