Implementation of High Through Put DNA Production Pipeline

Karen BilleciPhyNexus User Meeting

8/27/2014

Slide 1

• Biologics Resource Management– Background – Infrastructure

• Implementation of Automation to support DNA productions– Comments on sample management– Transformation– Colony Picking– Purification

OverviewSlide

2

Biologics Resources Management

Mission: Provide state of the art nucleic acid resource and protein repository management to support pathway discovery, antibody discovery, and protein chemistry.

Responsibilities: Implement automation, logics strategies and bioinformatics to support Biologics Resource Management.

• Manage Vendor Libraries and Collections to support Genentech research.

• Preserve Genentech resources through the archive and management of constructs and proteins.

• Support therapeutic discovery by providing sample management support to meet business needs.

• Manage DNA production for protein expression and screening applications.

Collections

VendorDNA Collections

RNAi

GN

E ex

pre

ssio

n C

on

stru

cts

Slide 3

Slide

4Biologics Resources: Infrastructure

Biologics Resource Management relies on integration of Registration,

Inventory Management, Automation, Request Systems, Collaboration, and

logistics.

Cloning

Protein

Sample Management

and Archive

Antibodies

Registration System

Inventory

Management

LIMS

Request

Automation

Synthesis

Core Infrastructure

Expression /

Purification

CMOs Assay

(screening,

sequencing)

Distribution

• Biologics Resource Management– Background – Infrastructure

• Implementation of Automation to support DNA productions– Comments on sample Management– Transformation– Colony Picking– Purification

Slide 5Overview

Why Automate?Small Scale DNA Production Metrics

Mini Scale (10 ug) 22-24,000 Preps/Month

Midi Scale (50 ug)Nearing 600 Preps/Month

Slide 6

Purify DNA

Pick

colony

Culture

TransformationProtein Production

Plasmid based reagents, cDNA, shRNA, and DNA constructs, are perpetually regrown and

expanded starting from glycerol stocks or purified DNA.

Modular Automation required to allow flexibility, while maintaining through put.

Screen

Slide

7DNA Production Management 101

Purified DNAGlycerol Stock

siRNA Automation and ProteinHamilton Starlet • Mini Oasis•Bravo • Tecan

Colony Picking

Hudson

Mini Purification (16 -20X96 well Blocks)

Hamilton Star

Cherry Pick • Transform • Inoculate

Dynamic Devices / PhyNexus

Inventory Management LIMS

Repository

-80°C -20°CFuture 4°C

Inventory Management LIMS-uses orders to define work and send/retrieve information from automation.

Barcode to Barcode Tracking

Fetch/Location Management

Automation Implementation• Boutique sample management no longer possible.

– Glycerol Stocks stored at -80°C or under nitrogen– Cultures traditionally from glycerol stocks started with “stabs”.– Difficult in a HT environment

• High throughput sample management– Store general use collections as glycerol stocks at (-80°C) and purified DNA (-20°C)– Glycerol stocks may be thawed 5-10X– Distribute purified DNA for cloning and expression cloning screens– Maintain separate glycerol stocks as source for primary screens (clone identification)

Sample Management Slide 9

Glycerol Stock ARE stable to Freeze Thaw

Per

cen

t o

f T0

40%

Automation ImplementationAutomation of Transformation

Addition of 2 ul of DNA to Tubes

Containing Competent Cells (10ul) Heat Shock 30 sec at 42°C

Inoculation to Agar Plate (25 ul)

Ice/Recovery (300 ul)

Manual Method

Manual Method used to understand flexibility of each step in traditional transformation protocol •DNA concentration range: 10 ng-500 ng/ul

•DNA volume: 6 ul

•Elimination of recovery step: maybe

•Inoculation volumes into 8 well dishes: 5 ul

×

Slide 10

Competent Cells Arrayed in PCR Plate

Addition of DNA, heat shock, and inoculation to agar plate occurs on robot Challenges

• Cross-contamination• Fully-automated process challenging

Solutions• Optimization of Liquid Class Variables• Dispense Height (lower), Speed (Faster), Aspiration Timing (In Place)• Pipetting strategy

Throughput: 96 samples per hourTransformation efficiency

• 453/480 Successful• 20/27 Successfully upon manual repeat.• 7-new DNA required

Automation ImplementationAutomation of Transformation

DNA arrayed into matrix tubes

Contamination

Our Hamilton STAR was configured with a 12-channel variable span pipetter, a 96-well fixed head, a i-SWAP, heating/cooling modules and a large deck.

Pre- Optimization

Post- Optimization

Stephen Monteclaro, Ryan Abraham

Slide 11

Automation ImplementationAutomation of Colony Picking

Toothpick of with picked colony placed into 96-well culture block

with media

Agar plate containing colonies

Toothpicks used to pick a colony

Platform:• RapidPick: Picker with Needle

• Micro10x: Ability to add media to culture blocks

• PlateCrane EX: Moves agar plates and culture blocks

• RSH-4000: Microplate/Culture block sealer.

Man

ual

Pro

cess

Au

tom

atio

nl P

latf

orm

Stephen Monteclaro

Slide 12

A. Picture out of Focus, no colonies will be identified

B. Overpopulated with colonies – Software will not identify individual colonies

C. Ideal – Good separation of colonies, colony size is good & contrast is good

D. Ideal – Colony identification (artificial colorization) and selection

With out Optimization:10% growth

With Optimization:99% growth

C D

Automation ImplementationAutomation of Colony Picking

BA

Slide 13

Automation of Mini Preps

Tina Di Ioia in collaboration with PhyNexus

Slide 14

Automation ImplementationMini Purification: Semi-manual methods

• Semi manual method - Qiagen 96 well format• Typical yields of 5-10 ug• Supports gRED – Ab Engineering 60%• Downstream applications – sequencing, cloning, transient transfection• NAR library management, special projects• 80-100 blocks/week, spikes up to 40+ plates in one day• 1 FTE dedicated

Slide 15

How can we automate?

• Qiagen: Automating method mechanically possible but not reliable due to high variability in samples

Automation ImplementationAutomation Considerations

• PhyTips- Chemistry similar to Qiagen

- 200ul PhyTip/1 block per set up (Yvonne Frank’s Lab)

- Platform had potential for higher throughput mini prep

- PhyTips are easy to customize for any liquid handler

Slide 16

Evaluation of PhyNexus• Is DNA comparable to Qiagen?

- Yields, purity, quality• Can PhyTip handle typical variation in samples ?

- Over grown cultures, over centrifuged cultures, fresh pellets• Will DNA be suitable for all downstream applications?

- Sequencing, transfection, expression cloning assays

How can we automate?Automation ImplementationEvaluation of PhyTips

MEA instrument – 12 channel, 1 ml wide bore sample prep tips, 1 ml PhyTips

Oasis – 96 well, 200 ul wide bore sample prep tips, 200 ul PhyTips

Slide 17

Evaluation of PhyNexus – Criteria• Yields need to be 5 – 10 ug

• 260/280 – Protein contamination, 1.8+ acceptable

• 260/230 – Buffer contamination, 1.8+ acceptable

- Significant for mammalian transfection

How can we automate?Automation ImplementationEvaluation of PhyTips : Yield and Purity Slide 18

Evaluation of PhyNexus – Yield & Purity How can we automate?Automation ImplementationEvaluation of PhyTips : Yield and Purity

• Yields need to be 5 – 10 ug

• 260/280 – Protein contamination, 1.8+ acceptable

• 260/230 – Buffer contamination, 1.8+ acceptable

- Significant for mammalian transfection

Slide 19

How can we automate?Automation ImplementationEvaluation of PhyTips : Robustness Slide 20

Initial Conclusions

• Yield, purity, quality, method robustness all look good

• Next: Downstream applications

- Sequencing

- Transfection

- Expression

How can we automate?Automation ImplementationMini Purification: Semi-manual methods Slide 21

Initial Conclusions

• Yield, purity, quality, method robustness all look good

• Next: Downstream applications

- Sequencing

- Transfection

- Expression

How can we automate?Automation ImplementationMini Purification: Semi-manual methods Slide 22

Read Length: >800

Phred Scores: 55

Forward Transfection EfficiencyHow can we automate?Automation ImplementationEvaluation of PhyTips : Transfection Slide 23

Gabriel Quinones

Evaluation of PhyTips : Expression Cloning Slide 24

CSP- Cell Surface Protein

Lipid Fugene 6

Bait Protein 1 Bait Protein 2

CSP1 CSP2 CSP3Control Control

Final Conclusions

• Yields and quality meet our specification

• 1 ml PhyTip produced a more pure reagent in our experiments than the 200 ul PhyTip (260/230)

• PhyTips purification is robust.

• DNA suitable for required downstream applications: sequencing, transfection

How can we automate?Automation ImplementationEvaluation of PhyTips: Conclusion Slide 25

New Platform - Overview

Oasis #1 Oasis #2 Oasis #3

Sample Prep Purification Purification

Robot arm2A 2B 3A 3B

Hotel

Plate reader

• Cell pellets• Elution plates• Sample prep tips

←Rail ➔

How can we automate?Automation ImplementationFinal Platform Slide 26

In collaboration with dynamic devices

New Platform - OverviewHow can we automate?Automation Implementation Slide 27

Final Platform

• Control- Method Manager: run each platform- Overload:

- coordinates work between platforms - drives rail, hotel, reader

• 96 position fixed head: O-ring based pipetting head; transitioning to solid mandrel.

• Buffers pumped into reservoirs, controlled by sensors- Heat wrap around lyses buffer

• Challenges:- Corrosion- O-ring based head- Imperfect tips holders- Overload

Final Conclusions

• We have implemented a high through put process to manage plasmid based reagents from transformation through purification and storage.

• We have integrated these systems into informaticinfrastructure including registration, inventory management and request.

How can we automate?Automation ImplementationTo Summarize Slide 28

GenentechTina Di Ioia

Stephen MonteclaroCarmen ChanLovejit Singh

Kurt SchroederRyan AbrahamYvonne Franke

Gabriel QuinonesQixin Bei

Jerry TangDan Hascall

all of our collaborators

New Platform - OverviewHow can we automate?Automation ImplementationThanks

PhyNexusChristopher Suh

Lee Hoang

Dynamic DevicesMichael Starace

Jeff Horowitz

Slide 29