Automating Genomics Applications with the Agilent Bravo Workstation

Transcription

1 Automating Genomics Applications with the Agilent Bravo Workstation Stephen Lappin Field Applications Specialist Agilent Automation Solution Page 0

2 AAS Tools for sample preparation Today we will present: Two kits for Nucleic Acid Isolation applications using the Bravo liquid handler: 1. Stratagene RNA Isolation on a stand-alone Bravo 2. Invitrogen DNA Isolation on a Bravo Workstation Two kits for sample cleanup and an amplification assembly: 1. Broad Institute implementation of Agencourt AMPure PCR Cleanup 2. Stratagene StrataPrep 96 PCR purification kit 3. qpcr setup with a matrix of primers and targets Page 1

3 Versatile Design Compact Fast, quiet operation Open design permits use in laminar flow hoods Rapid-swap heads Gripper enables flexible automation Best in class precision and accuracy No electronics below the deck Page 2

4 Bravo Liquid Handling Accessories Light Curtain Tip Washing Auto-filling Reservoirs Orbital shaking Vacuum Filtration Magnetic stirring Heating and cooling PCR plate leveling Barcode Reading More On-Deck Barcode Reading On-Deck Shaking On-Deck Vacuum Filtration Shield Light Curtain Page 3

5 Add-on Plate Handling BenchCel Microplate Handler High-speed, compact, and flexible microplate handler Most SBS labware including Tube Racks, Deep Well Plates, Lids Allows Bravo to integrate with readers, washers, dispensers, etc PlateLoc Thermal Plate Sealer Fastest and smallest automated plate sealer Multiple seal types for various applications Microplate Labeler Print and Apply bar code labels to all sides Barcode Verification Microplate Centrifuge Compact two position centrifuge Up to 3,000 RPM with minimal vibration

6 Scale as your workflow requires

7 Scale up to a Biocel for many plates Page 6

8 Software VWorks VWorks Automation Control Software The most powerful and flexible automation control software in industry, an integral part of workflow implementation Automated workflows in drug discovery, genomics, proteomics, cell biology and chemical/materials sciences

9 Automation of Stratagene Absolutely RNA 96 Microprep p Kit with the Bravo Automated Liquid Handling Platform Owen Hardy and Stephen Lappin Agilent Technologies 8

10 Stratagene Absolutely RNA isolation Current RNA isolation protocols require extensive manual processing, repetitive pipetting and vacuum filtration steps. A Bravo automated pipetter can perform the liquid handling and filtration steps to extract RNA from plated cells. All processes are completed by the Bravo platform in minutes with only user intervention required being to spin in an offline bench top centrifuge. Assessment of the RNA peaks using the Agilent Bioanalyzer 2100 has shown that the automated process provides high quality RNA in a process with limited user intervention. Page 9

11 RNA Isolation: Process flow 1. Aspirate media from plate; lyse cells and bind RNA to plate 2. Optional DNAse treatment 3. Wash filter 3x and vacuum filter Manual centrifugation Add elution buffer on Bravo, incubate Manual centrifugation Page 10

12 RNA Isolation: Simplified VWorks Process Flow 1. Lyse and bind Bravo Remove media From Cell Plate Lyse Cells Bind to Filter and Vacuum OR Waste 2. Optional DNAse treatment Bravo Add DNAse to filter Incubate RT Vacuum 3. Wash and recover Bravo Add Wash to filter (Loop 3x) Vacuum Manual Centrifugation Bravo Add Elution Buffer to filter Incubate RT Manual Centrifugation

13 Comparison of isolated RNA samples Four corner samples were pooled, along with 8 samples from column 6 in a second pool and were analyzed on the Agilent 2100 Bioanalyzer Samples were assessed for RNA quality and absence of genomic DNA. RNA is fully intact with sharp 18S and 28S peaks. Genomic DNA contamination is not present Page 12

14 Invitrogen ChargeSwitch EasyPlex gdna kit using the gdna isolation workstation 13

15 Invitrogen ChargeSwitch Nucleic Acid Isolation Non-magnetic, Non-filtration Bind DNA or RNA Remove impurities through aqueous wash steps Elute DNA or RNA in a ready-to-use buffers 100% Water Based BenchCel & PlateLoc Provide walk-away solution The Bravo Platform accommodates four reagent reservoirs, a position for solid waste and a position for liquid waste. B+L buffer charges plate surface positively Sample is added to B+L buffer, DNA is released from cells. Negative DNA backbone binds to positively charged plate Washing steps 1 and 2 remove contaminants Isolated DNA is ready for downstream applications

16 Invitrogen ChargeSwitch Here Showing Bravo-Benchcel workstation for complete automation of process Can process 10 plates in a batch, with 10 minutes of robotic processing time per plate (plus incubations) Could also be performed with a standalone Bravo for one plate. December 11, Application title

17 Automation of Agencourt AMPure Purification Kit for the Purification of Next- Generation Sequencing Sample Preparation Reactions on Bravo Bi Brian Miniei et.al Broad Institute of MIT and Harvard University it

18 Abstract A new method was developed to automate library construction for the Illumina sequencing platform using the Agilent Bravo Automated Liquid Handling Platform and the Agencourt Ampure PCR purification kit. Developed in collaboration with the Broad Institute, MA Demonstrates use of Agencourt SPRI technology on the Bravo for multiple applications Greatly improves sample to sample variability Reduced overall library prep time for Illumina sequencing 50%

19 Comparison of manual and automated workflows. Manual Workflow Illumina Library Construction Genomic Sample Fragment Qiaquick End Repair Qiaquick A Base Addition Qiaquick Gel Size Selection ( bp) Qiaquick Adapter Ligation (Solexa Adapters) Qiaquick PCR Enrichment Hybrid Selection & Capture Hybridization (DNA + RNA) Bead Pre-wash Bead Capture Bead Washes Catch Elution Qiaquick Catch PCR (Catch Primers) Qiaquick Cluster Amplification & Sequencing QC Steps Picogreen Dilution qpcr Normalized Lib Total Time = 3 weeks Automation with AMPure Illumina Library Construction Genomic Sample Covaris Shear AMPure End Repair AMPure A Base Addition AMPure Adapter Ligation (Solexa Adapters) AMPure PCR Enrichment AMPure Hybrid Selection & Capture Hybridization (DNA + RNA) Bead Pre-wash Bead Capture Bead Washes Catch Elution AMPure Catch PCR (Catch Primers) AMPure Cluster Amplification & Sequencing QC Steps Picogreen Dilution qpcr Normalized Lib Total Time = 1.5 weeks

20 Agilent Bravo Deck configuration Single plate per run BenchCel can be added for larger batches VWorks Dual-Height Mixing of beads on with Bravo pipetting outperforms orbital shaking

21 NGS Automated Prep at the Broad Institute Decreased Variability Automated Capture Introduced Identical Sample Sizing Increased Yield Benefits: Decreases sample process time Decreases sample-to-sample variability Increased yield when compared to manual method Increased capacity from 12 to 96 samples/technician

22 Key take-away: Automation reduces sample-to-sample variability VWorks Software provides precision control of the Bravo handler including on-deck accessories for reproducible liquid handling results Barcode tracking, data logging, and LIMS integrations allow plates to be tracked across multiple process steps Beyond throughput, automation add reproducibility and process control Automation of the AMPure method is applicable to and workflow requiring purification following PCR or other enzymatic reactions.

23 Complete Automation of the Stratagene StrataPrep 96 PCR Purification Kit with the Agilent Bravo Automated Liquid Handling Platform and Agilent Automated Centrifuge Stephen Lappin, Manuel Gomez, Jr. and David Knorr, Ph.D. Agilent Technologies 22

24 Stratagene StrataPrep 96 PCR purification PCR Cleanup is a very common application in the laboratory and used for many downstream applications A need to process multiples of plates is common Using the Bravo - Benchcel - Automated Centrifuge Workstation provides complete, walk away automation Process is very similar to the Absolutely RNA protocol Page 23

25 PCR purification Process Flow 1. Mix an equal volume of DNA-binding solution with the PCR product and bind to plate 2. Add 1 PCR wash buffer to the wells of the binding plate and vacuum 3. Place the binding plate on a 96-well collection plate and centrifuge the plates at 1000 x g for 10 min 4. Add elution buffer to binding plate with a new collection plate and incubate at room temperature for 5 minutes. 5. Centrifuge plate at 1000 g for 5 minutes Page 24

26 Time Constraints Regulate timing between tasks in time-critical assays. Specify time constraints between tasks in the Startup, Main or Clean up protocol. January 2009 Agilent Restricted

27 PCR purification: Simplified VWorks Process Flow BenchCel Down-Stack PCR, Filter Plate, Tips Bravo Add Binding Buffer to PCR rxn Bind to Filter and Vacuum Add Wash Vacuum BenchCel Move to Centrifuge VSpin BenchCel Up-Stack Sealed Purified PCR product PlateLoc VSpin BenchCel Move to Centrifuge Add Elution Bravo BenchCel Move to Bravo 5 Min Time Constraint

28 Comparison of Purified PCR product 582bp fragment of Lambda DNA amplified and purified with Strataprep kit in parallel on the Bravo and by hand. Bioanalyzer results show peaks from hand and robot prepared plates sized identically. Page 27

29 Complete Automation of Quantitative PCR Setup on the Agilent Bravo Automated Liquid Handling Platform David Knorr, Manuel Gomez and Stephen Lappin Agilent Technologies Page 28

30 qpcr setup with Bravo Large PCR studies can be a labor intensive process to setup. A need to process many plates of PCR reactions is common with numerous mixtures of primer sets, master-mixes mixes and other components Using the Bravo can save manual processing time along with avoiding error from working in 96 or 384 wells that all look EXACTLY the same! Page 29

31 qpcr Setup: Experimental Matrix Primers Master Mix Add 10 µl Template (11) Templates Add 15 µl Primer Mix Prepare Mixes NTC Prepare Reactions Diluted Primers + Master Mix Diluted Templates Reagents are distributed in a plate for matrix setup by Bravo Page 30

32 Bravo Automated Liquid Handling Platform Using an M x N Array of Tips Allows users to specify any array of tips to use in a pipette process. Full barrel, multi-row, multi-column, partial row/column, & single barrel. Increased pipetting flexibility.

33 qpcr Setup: Process Flow Reagents are distributed in a plate for matrix assembly Primers Master Mix Templates Diluted Primers + Master Mix Single tip is used to assemble + master mixes and template dilutions Diluted Templates and NTC A row of 11 tips is used to dispense primers and master mixes Add 15 µl Primer Mix A column of tips is used to dispense diluted templates Add 10 µl Template (11) Blank Page 32

34 Comparison of Hand vs. Automated Setup Ct values for two fold dilutions of cdna target are one cycle off between hand and robot diluted samples, but show nearly identical slope and correlation values Performance of Bravo- vs. Hand-Pipetted 2-fold 2fold Dilution Series R 2 = slope = Bravo Hand-pipetted Log. (Hand-pipetted) Log. (Bravo) R 2 = slope = values p g total cdna Ct Three targets t shown for qpcr analysis show good correlation for the house keeping genes shown (B2M shows a 2 cycle difference ) F gg luorescence (dr) B2M, adult, GUSB, adult, TBP, adult, B2M, fetal, GUSB, fetal, TBP, fetal, Cycles

35 In Summary: AAS offers many scalable solutions that are applicable to genomics workflows. VWorks software provides a rich and flexible feature set for automation of many laboratory applications. There are numerous applications that we have done. Your memory stick contains many of them. Call us if you don t see what you are looking for. We have the best applications team in the business and we are truly excited to help setup new applications. Page 34

36 Thanks to: Manuel Gomez Owen Hardy Dave Knorr Marc Beban Randy Dyer Fereshteh Lesani Ian Yates Page 35