Infinium Assay. Lab Setup and Procedures Guide. For Research Use Only. Not for use in diagnostic procedures. Document # v02 December 2017

Transcription

1 Infinim Assay Lab Setp and Procedres Gide Docment # v02 December 2017 For Research Use Only. Not for se in diagnostic procedres. ILLUMINA PROPRIETARY

2 Infinim Assay Lab Setp and Procedres Gide This docment and its contents are proprietary to Illmina, Inc. and its affiliates ("Illmina"), and are intended solely for the contractal se of its cstomer in connection with the se of the prodct(s) described herein and for no other prpose. This docment and its contents shall not be sed or distribted for any other prpose and/or otherwise commnicated, disclosed, or reprodced in any way whatsoever withot the prior written consent of Illmina. Illmina does not convey any license nder its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this docment. The instrctions in this docment mst be strictly and explicitly followed by qalified and properly trained personnel in order to ensre the proper and safe se of the prodct(s) described herein. All of the contents of this docment mst be flly read and nderstood prior to sing sch prodct(s). FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND DAMAGE TO OTHER PROPERTY, AND WILL VOID ANY WARRANTY APPLICABLE TO THE PRODUCT(S). ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S) DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) Illmina, Inc. All rights reserved. Illmina, Infinim, iscan, GenomeStdio, and the streaming bases design are registered or pending trademarks of Illmina, Inc. and/or its affiliate(s) in the U.S. and/or other contries. All other names, logos, and other trademarks are the property of their respective owners. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. ii

3 Infinim Assay Lab Setp and Procedres Gide Revision History Docment Date Description of Change Docment # v02 Docment # v01 Docment # rev. A December 2017 December 2016 October 2008 Updated instrctions on glass back plate cleaning and handling Added information to spport the Infinim XT Assay Spported Infinim Assay setp and procedres Docment # v02 For Research Use Only. Not for se in diagnostic procedres. iii

4 Table of Contents Revision History iii Chapter 1 Lab Setp and Maintenance 1 Introdction 1 Safety Precations 1 Consmables and Eqipment 1 Prevent Amplification Prodct Contamination 4 Best Practices 5 Lab Maintenance 7 Chapter 2 Robot Usage and Maintenance 11 Introdction 11 Consmables and Eqipment 11 Preparing the Robot for Use 12 Testing Volme Accracy of the Tecan Tips 13 Cleaning the Tecan 17 Appendix A System Controls 19 Introdction 19 The Control Dashboard 19 Control Bead Type IDs 19 Control Diagrams 21 Appendix B Trobleshooting 26 Introdction 26 Pre-Hybridization 26 Hybridization to XStain 27 Imaging 28 Miscellaneos 29 Appendix C References 30 Illmina BeadChips 30 Tracking Tools 30 The iscan System 30 Technical Assistance 31 Docment # v02 For Research Use Only. Not for se in diagnostic procedres. iv

5 Chapter 1 Lab Setp and Maintenance Lab Setp and Maintenance Introdction 1 Safety Precations 1 Consmables and Eqipment 1 Prevent Amplification Prodct Contamination 4 Best Practices 5 Lab Maintenance 7 Introdction This section describes the essential eqipment and operating procedres for an Infinim lab. It explains how to eqip and rn an Infinim laboratory, providing important information on the following topics: Safety precations Preventing amplification prodct contamination Preparing and storing reagents Calibrating and sing the vortexer Best practices Lab maintenance Safety Precations Adhere to the following cations and warnings while performing the protocols described in this gide. CAUTION Only qalified laboratory personnel can perform the protocols described in this gide. Exercise cation when handling biological samples to avoid cross-contamination among pre-amp and post-amp samples. WARNING This protocol ses an aliphatic amide that is a probable reprodctive toxin. Personal injry can occr throgh inhalation, ingestion, skin contact, and eye contact. For more information, conslt the material data safety sheet for this assay at Dispose of containers and any nsed contents in accordance with the governmental safety standards for yor region. Consmables and Eqipment All the Infinim protocols reqire the following consmables and eqipment. Remember to maintain separate stocks for pre- and post-amp areas. Additional reqirements for individal protocols are identified in the protocol gides. User-Spplied Materials Material Absorbent pads/towels Alminm foil Cap mats, 96-well, pierceable, nonatoclavable Spplier General lab spplier General lab spplier Thermo Fisher Scientific, catalog # AB-0566 Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 1

6 Infinim Assay Lab Setp and Procedres Gide Material Spplier Compressed air can VWR, Int'l, catalog # Containers: 1 L, for dilting bleach 2 containers capable of holding polypropylene test tbe racks Heat sealing foil sheets, Thermo-Seal Kimwipes or any lint-free tisse Lab coats 2 spplies: 1 for pre- and 1 for post-amplification processes Pipette tips 20 µl aerosol filter 200 µl aerosol filter 1000 µl aerosol filter Pipettes, serological, 50 ml General lab spplier Thermo Fisher Scientific, catalog # AB-0559 General lab spplier General lab spplier General lab spplier General lab spplier Pipetting troghs, disposable VWR, Int'l, catalog # Polypropylene test tbe racks (recommended) Powder-free gloves 2 spplies: 1 for pre- and 1 for post-amplification processes ProStat EtOH presatrated wipes Recommend 1 wipe per 2 chips; 30 wipes per package Sbstitte with Kimwipes and 70% EtOH Safety glasses 2 spplies: 1 for pre- and 1 for post-amplification processes Skirted microplates, 96-well, 0.2 ml Microseal 96-well skirted polypropylene microplates, 8x12 well array TCY plates, 1 per rn Sbstitte with 0.8 ml storage plate (midi plate), conical well bottom, if desired Storage microplates, 96-well, 0.8 ml Midi plates, 1 per rn Tbes 15 ml conical 50 ml conical Cole-Parmer, catalog # EW General lab spplier Contec, catalog # PS-911EB/EtOH General lab spplier MJ Research, catalog # MSP-9601, Thermo Fisher Scientific, catalog # AB0765 General lab spplier Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 2

7 Infinim Assay Lab Setp and Procedres Gide User-Spplied Reagents Consmable Bleach Deionized water (DI H2O) Spplier General lab spplier General lab spplier EDTA, 0.5 M EMD Chemicals, catalog # 4056 Sigma-Aldrich, catalog # E7889 Ethanol, 100% Formamide, OmniPr Isopropanol (2-propanol), 100% General lab spplier VWR, Int'l, catalog # EM-4650 General lab spplier Mild detergent, sch as Alconox Powder Detergent VWR, Int'l, catalog # Sodim hydroxide, prchase as solid and prepare a 0.1N NaOH soltion in DI H2O TE, 1X 10 mm Tris-HCl, ph 8.0, 1 mm EDTA For dilting DNA Sigma-Aldrich, catalog # General lab spplier Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 3

8 Infinim Assay Lab Setp and Procedres Gide User-Spplied Eqipment Eqipment Adapters to centrifge 96-well plates and tbes (2 sets) Spplier General lab spplier Atodesiccator cabinet VWR, Int'l, catalog # Cap mat sealer (recommended) Corning, catalog # 3081 Centrifge, benchtop 120 V, for pre-amplification processes Centrifge, benchtop refrigerated 120 V ( g), for post-amplification processes Forceps Inclded with system, only needed if additional pairs are reqired Micropipettors 2 separate sets: 1 for pre- and 1 for post-amplification processes P-20 P-200 P-1000 Mltichannel precision pipettes 2 separate sets: 1 for pre- and 1 for post-amplification processes P-20 P-200 Optical tachometer/stroboscope (recommended) Serological pipette aid Stop watches/timers 2 separate sets: 1 for pre- and 1 for post-amplification processes Tbe rack 2 separate sets: 1 for pre- and 1 for post-amplification processes Any rack fitting the Infinim reagent 17 mm tbe diameter Tbe vortexers 2 separate sets: 1 for pre- and 1 for post-amplification processes Vacm sorce, hose, or pmp capable of plling greater than 508 mm Hg General lab spplier General lab spplier VWR, Int'l, catalog # General lab spplier General lab spplier Cole-Parmer, catalog # A General lab spplier General lab spplier VWR, Int'l, catalog # General lab spplier General lab spplier Prevent Amplification Prodct Contamination The Infinim protocol ses a linear amplification process to increase the qantity of inpt DNA samples to optimal levels. Amplification prodcts can contaminate reagents, instrmentation, and DNA samples, casing inaccrate and nreliable reslts. Amplification prodct contamination can sht down lab processes and significantly delay resmption of normal operations. The laboratory space where yo perform preamplification processes, sch as qantification and normalization, mst be separate from the postamplification laboratory space. The Infinim Assay Lab Setp and Procedres Gide refers to these two laboratory spaces as pre-amp and post-amp areas. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 4

9 Infinim Assay Lab Setp and Procedres Gide To avoid contamination, follow these gidelines: Do not share eqipment, sch as lab coats, gloves, safety glasses, pipettes, centrifges, heat blocks, and heal sealers, between pre-amp and post-amp areas. Do not se the same sink to wash pre-amp and post-amp reservoirs. Do not share a water prification system between the pre-amp and post-amp processes. Always store spplies for the Infinim protocols in the pre-amp area, and transfer spplies to the postamp area as needed. Establish a daily and weekly decontamination schedle for both areas. Best Practices Adhere to the following best practices when performing the Infinim Assay protocols. Label the Plates Always se the provided barcodes to track plates throghot the Infinim process. As a convention, apply barcode labels to the right side of the plate (the colmn 12 end). Prepare Batches of 95% Formamide/1 mm EDTA To minimize errors in preparing 95% formamide/1 mm EDTA, prepare it in large batches and aliqot it into 15 ml or 50 ml sealed tbes. Store aliqots for 6 months at -25 C to -15 C and se in the protocol as needed. After yo open an aliqot, se it on the same day. Discard leftover reagent. Prepare Batches of 0.1N NaOH To minimize errors in preparing 0.1N NaOH fresh each day, prepare it in large batches and aliqot it into 15 ml or 50 ml sealed tbes. Store aliqots p to 6 months at 2 C to 8 C and se them in the protocol as needed. After yo open an aliqot, se it on the same day. Discard leftover reagent. Reagent Rese Never rese excess reagents after dispensing. Discard reagents according to yor facility reqirements. Handling Cap Mats Orient the cap mat so that A1 on the cap matches A1 on the plate. Remove the cap mat careflly and slowly to avoid splashing the plate contents. Set the cap mat aside, pside down, in a safe location for se later in the protocol. When yo place the cap mat back on the plate, be sre to match it to its original plate and orient it correctly. Handling BeadChips Avoid toching the BeadChip anywhere other than at the barcode end or on the edges. Cleaning and Calibrating Pipettes Make sre that pipettes are properly calibrated, cleaned, and decontaminated. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 5

10 Infinim Assay Lab Setp and Procedres Gide Pipette Careflly Perform all pipette dispenses careflly and slowly to avoid creating trblence within the plate wells and Flow-Throgh Chambers. Where possible, se a mltichannel pipette to dispense reagents. First In, First Ot Establish a First In, First Ot (FIFO) system for reagents to minimize the risk of reagent expiration before se. Infinim Kit Configration and Batching Infinim kits are configred to spport varios sample sizes with the assmption that the kit size corresponds to the batch size. For example, certain BeadChips are available in 48-sample, 288-sample, and 1152-sample kit configrations. When working with small batch sizes, order the smaller, 48-sample kits to make sre that the kit contains sfficient reagent volmes to process smaller batch sizes. XC4 Storage and Use Store ndilted XC4 and XC4 that has been dilted with ethanol at room temperatre. The XC4 reagent bottle displays the expiration date of the ndilted reagent. Illmina spports its prodcts within the expiration date. Dilted XC4 can be resed p to six times over a two-week period for a maximm of 24 BeadChips. Clearly mark the XC4 bottle after ethanol has been added to avoid confsion with ndilted XC4 bottles. Items Falling to the Floor Follow these best practices for handling items that fall to the floor. Any items that fall to the floor are contaminated. Wear lab gloves to toch any items that fall to the floor. Immediately clean nondisposable items, sch as pipettes or important sample containers, with a 10% bleach soltion. Use a 10% bleach soltion to clean any lab srface in contact with the contaminated item. Throw away yor lab gloves and pt on a new pair after handling items that have fallen to the floor. Balancing the Centrifge When yo centrifge plates or BeadChips, place a balance plate or rack with BeadChips opposite each plate or BeadChip rack being centrifged. Make sre that the weights are as similar as possible. Calibrate the Microplate Shaker Follow these instrctions to calibrate the Signatre* High-Speed Microplate Shaker (VWR International, catalog # ). 1 Set the digital stroboscope display speed to 1600 rpm. 2 Trn on the microplate shaker and adjst the speed ntil it reaches 1600 rpm. 3 Record the displayed speed and note that it represents an actal speed of 1600 rpm. 4 Use the same method to determine the displayed speed for the actal vortex speed of 1800 rpm. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 6

11 Infinim Assay Lab Setp and Procedres Gide 5 Label the microplate shaker with the calibration information. Table 1 Sample Microplate Shaker Calibration Label Display Speed Actal Speed Calibration Date 1450 rpm 1600 rpm xx-xx-xx 1625 rpm 1800 rpm xx-xx-xx Lab Maintenance The following standard lab maintenance procedres apply to all Infinim Assay labs. Daily and Weekly Bleaching Establish a daily and weekly bleaching schedle for both the pre-amp and post-amp areas. CAUTION To prevent sample or reagent degradation, make sre that all bleach vapors have flly dissipated before starting any processes. Keep both areas clean. Identify hot spots in each area that pose the highest risk of contamination, and clean these areas daily with a 10% bleach soltion. Typical hot spots inclde: Bench space sed to process DNA or amplified DNA Vortexers Centrifges Thermal cyclers Instrment control panels Door handles Refrigerator/freezer door handles Compter mice Keyboards Once a week, thoroghly clean all laboratory srfaces and instrments in both areas. Mop the floors with a 10% bleach soltion. Yo are responsible to train facility personnel to clean pre-amp and post-amp lab areas as described. Lab personnel shold not move from the post-amp area to the pre-amp area. Cleaning, Handling, and Inspecting the Glass Back Plates The glass back plates are sed in the Flow-Throgh Chambers dring XStain to control the flow of reagent over the BeadChips. Clean the glass back plates when yo open the package and after each se. In addition, perform a bleach cleaning after every seven ses, or more freqently, depending on individal lab throghpt. Both cleaning procedres are described in the following sections. Inspect the glass back plates before each se. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 7

12 Infinim Assay Lab Setp and Procedres Gide Recommended Racks for Glass Back Plate Cleaning, Bleaching, and Storage Polypropylene test tbe racks (ie plastic racks) are recommended for cleaning, bleaching, and storage of glass back plates. Place glass back plates diagonally in the racks to minimize contact between the glass and prevent damage. Figre 1 Correct Placement of Glass Back Plates Disassembly of Flow-Throgh Chambers Following XStain, the Flow-Throgh Chambers are disassembled. XStain reagents contain many components (eg proteins, enzymes, antibodies) and best practice is to prevent remaining reagent from drying on the glass back plates. Immediately after disassembly, place the glass back plate diagonally in a plastic rack that is sbmerged in a container of DI H2O to prevent drying. After all Flow-Throgh Chambers are disassembled and placed in the sbmerged rack, proceed to Cleaning the Glass Back Plates after Every Use on page 8. Figre 2 Plastic Rack Sbmerged in DI H2O Cleaning the Glass Back Plates after Every Use Clean the glass back plates when yo open them for the first time and after every se. 1 Prepare a 1% Alconox soltion. Use 2.5 g Alconox powder per 250 ml DI H2O. 2 Sbmerge the plastic rack with glass back plates in a container filled with 1% Alconox. 3 Wipe each glass back plate with a Kimwipe and retrn to rack. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 8

13 Infinim Assay Lab Setp and Procedres Gide 4 Remove the rack from the 1% Alconox soltion and thoroghly rinse the glass back plates with DI H2O. 5 Allow the glass back plates to dry in the plastic rack. 6 After the glass back plates are clean and dry, wipe them with a Kimwipe soaked with 70% EtOH. 7 Store the glass back plates in the plastic rack in a dst-free area. 8 Use a can of compressed air or a laboratory air gn to remove any dst or lint on the glass back plates before se. Bleach Cleaning the Glass Back Plates In addition to the cleaning procedre described previosly, clean the glass back plates after approximately every seven ses, or more freqently, depending on individal lab throghpt. CAUTION Wear a lab coat, safety goggles, and gloves dring this cleaning process. CAUTION Preparation Bleach is an irritant. Use cation when handling. Make 10% bleach soltion. Example: Add 100 ml of bleach to 900 ml DI H2O and mix thoroghly. Bleach Cleaning Steps WARNING Perform this procedre in a fme hood or a lab space otside of the BeadChip prodction lab. Excessive bleach fmes can degrade the florescent dyes sed in the Infinim Assay. 1 Perform the following steps in the fme hood: Fill a container with enogh 10% bleach to cover both the rack and the glass back plates completely. Place the plastic rack with the glass back plates into the container. Soak for 1 hor. 2 Transfer the rack with glass back plates to a container filled with DI H2O. TIP Yo can transfer the container and rack with glass back plates from the fme hood to a nearby sink with DI H2O. 3 Dip the rack containing the glass back plates p and down 20 times. Be carefl not to chip the glass back plates. 4 Remove the rack containing the glass back plates and rinse both with DI H2O. 5 Dispose of the DI H2O from the container. Rinse and refill the container with fresh DI H2O. 6 Retrn the rack containing the glass back plates to the container. 7 Dip the rack containing the glass back plates p and down 20 times and then soak for 5 min. Be carefl not to chip the glass back plates. 8 Repeat steps 4 7 for times. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 9

14 Infinim Assay Lab Setp and Procedres Gide 9 Dispose of the DI H2O. 10 Remove the rack containing the glass back plates and rinse with DI H2O. 11 Allow the glass back plates to dry in the plastic rack. 12 After the glass back plates are clean and dry, wipe them with a Kimwipe soaked with 70% EtOH. 13 Store the glass back plates in the plastic rack in a dst-free area. 14 Use a can of compressed air or a laboratory air gn to remove any dst or lint on the glass back plates before se. Inspecting the Glass Back Plates Before each se, inspect the glass back plate to make sre it is in acceptable condition. If any of the following are observed, replace the glass back plate: Chip or damage in the reagent reservoir area Significant chips or damage on the srface facing the BeadChip in the flow-throgh chamber Cracks in the glass that may case the glass to break dring the assay Any chips or damage that may case harm while handled, regardless of location Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 10

15 Chapter 2 Robot Usage and Maintenance Robot Usage and Maintenance Introdction 11 Consmables and Eqipment 11 Preparing the Robot for Use 12 Testing Volme Accracy of the Tecan Tips 13 Cleaning the Tecan 17 Introdction This section describes how to perform robot QC and maintain the Tecan 8-tip robot. WARNING Do not rn any other programs or applications while sing the Tecan. Yor compter and the Tecan may lock p and stop a rn. Consmables and Eqipment These items are specific to the Tecan 8-tip robot. For a list of other eqipment, materials, and reagents needed in an Infinim lab, see Consmables and Eqipment on page 1. For a list of the items reqired by individal assay protocols, see the appropriate assay protocol gide. User-Spplied Materials Material Absorbent bench nderpads Alminm foil Bottles, sterile 100 ml 500 ml 1 L Carboy (glass or nonbleach reactive material) Spplier General lab spplier General lab spplier General lab spplier General lab spplier FLUOTRAC 200 plates (2, for robot QC) Greiner Bio-One, catalog # Gradated cylinders, sterile 100 ml 500 ml 1 L Pipette tips, 10 µl aerosol filter Pipetting troghs for mltichannel pipettes, disposable (2, for robot QC) General lab spplier General lab spplier VWR, Int'l, catalog # Reservoir frames (at least 4) Beckman Colter, catalog # Reservoir, fll, 150 ml (1 case) Beckman Colter, catalog # Reservoir, half, 75 ml (1 case) Beckman Colter, catalog # Reservoir, qarter, 40 ml (1 case) Beckman Colter, catalog # Soaking trays or standard dishwashing tbs, 42"L x 18"W x 6"D (2) General lab spplier Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 11

16 Infinim Assay Lab Setp and Procedres Gide User-Spplied Reagents Consmable Deionized water (DI H2O) Spplier General lab spplier Dimethylslfoxide (DMSO) (for robot QC) Sigma, catalog # D-8779 Florescein (for robot QC) Sigma-Aldrich, catalog # F-2456 Florescein Diltion Bffer (1X TE, 0.01% Tween 80) (for robot QC) System Liqid, 1000X (100 mm Tris (ph 8.0), 100 mm EDTA) TE, 1X (10 mm Tris-HCl, ph 8.0, 1 mm EDTA) (for dilting DNA) Tween 80 (for robot QC) User-Spplied Eqipment General lab spplier General lab spplier General lab spplier General lab spplier Eqipment Florometer capable of reading 96-well plates and the florescence of florescein dye Micropipettors (2 separate sets, 1 for pre- and 1 for postamplification processes) P-2 (for robot QC) P-20 P-200 P-1000 Tecan 8-tip robots (2 robots, 1 for pre- and 1 for postamplification processes) Spplier General lab spplier General lab spplier Non-LIMS cstomers SC (110 V) - North America and Japan SC (220 V) - EU and Asia Pacific (Except Japan) LIMS cstomers SC (110 V) - North America and Japan SC (220 V) - EU and Asia Pacific (Except Japan) Preparing the Robot for Use The preparation steps vary slightly depending on whether yo are sing the robot for the first time of the day. Follow one of the following procedres before every atomated protocol. Tecan First-Use-of-the-Day Procedre 1 Reboot the Tecan control compter. CAUTION Do not place yor hands on or near the Tecan bed while the Tecan is rnning. 2 Open the Illmina Atomation Control Software. The Tecan takes a few moments to initialize. 3 Check the system flid level and add flid if necessary. CAUTION If adding flid, do so before the Bleach Wash step. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 12

17 Infinim Assay Lab Setp and Procedres Gide 4 Select Robot QC Tasks Robot Bleach Wash. 5 In the Basic Rn Parameters pane, enter 0 for a tip wash. 6 Place a qarter reservoir with 5 ml 10% bleach soltion in position A of the reservoir frame. 7 Select Rn. 8 Select OK at the prompt. The Tecan initiates the tip wash. When the bleach procedre is complete, the Tecan retrns to the main task screen. CAUTION To prevent contamination, make sre that all bleach vapors have flly dissipated before starting any process involving samples. Sbseqent Uses Dring the Day Follow this procedre every time yo are abot to se the Tecan. If it is the first time the Tecan is being sed for the day, follow the steps described nder Tecan First-Use-of-the-Day Procedre on page 12 instead. CAUTION Do not place yor hands on or near the Tecan bed while the Tecan is rnning. 1 Select Sys Wash. 2 Observe the lines for air bbbles. 3 Repeat the system flsh ntil the lines are free of air bbbles. If bbbles persist in the lines, contact Illmina Technical Spport. 4 Observe the Tecan tips for any dripping. 5 Dring the fast wash cycle, observe the Tecan tips as they dispense system liqid. Liqid shold dispense in a straight stream. All tips shold dispense liqid in eqal volmes and at eqal velocities. Testing Volme Accracy of the Tecan Tips This protocol ses florescein dye to measre qantities of liqid delivered by the Tecan compared to a manally derived standard crve. It is critical that the florescein dyes sed for the standard crve and the Tecan qantification are derived from the same starting diltion of stock. Failre can lead to erroneos assay reslts. Preparing Florescein Reagents This section explains how to prepare reagents for testing the Tecan. Florescein Stock 1 Weigh ot 25 mg florescein into a 100 ml bottle. 2 Slowly add 25 ml DMSO. 3 Mix thoroghly. 4 Store at room temperatre protected from light. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 13

18 Infinim Assay Lab Setp and Procedres Gide Florescein Diltion Bffer 1 Prepare 1X TE (Tris s acid EDTA): Add 10 ml 100X TE (1M Tris-HCl, 0.1M EDTA) to 990 ml DI H2O in a 1 L bottle. Mix thoroghly. Store at room temperatre. 2 Prepare 10% Tween 80: Weigh grams Tween 80. NOTE Tween 80 is extremely viscos. Weight measrement is more accrate than liqid volme measrement. Add the Tween 80 to the 1 L bottle. Dissolve in DI H2O to 1000 ml. Mix thoroghly. Store at room temperatre. 3 Prepare 1X TE with 0.01% Tween 80: Add 1 ml 10% Tween 80 to 1000 ml 1X TE bffer. Mix thoroghly. Store at room temperatre. High-Concentration Florescein 1 In a 100 ml bottle, add 25 ml florescein stock in DMSO (1.0 mg/ml) to 75 ml florescein diltion bffer (1X TE with 0.01% Tween 80). 2 Mix thoroghly. 3 Discard after se. Low-Concentration Florescein 1 In a 500 ml bottle, add 50 ml newly prepared 0.25 mg/ml high-concentration florescein to 450 ml florescein diltion bffer (1X TE with 0.01% Tween 80). 2 Mix thoroghly. 3 Discard after se. Dispensing Reagents sing the Tecan 1 Label a new FLUOTRAC 200 plate Ind-Col Dispense. 2 Label another new FLUOTRAC 200 plate Mlti-Col Dispense. 3 From the Tecan control compter, open the Illmina Atomation Control Software. 4 Select Robot QC Tasks 8-Tip Robot QC. 5 Add 35 ml florescein diltion bffer to a qarter reservoir. Place the reservoir in position A of the reservoir frame, as shown on the Tecan bed map. 6 Add 6 ml low-concentration florescein (0.025 mg/ml) to a qarter modle reservoir and place the reservoir in position B. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 14

19 Infinim Assay Lab Setp and Procedres Gide 7 Place the FLUOTRAC 200 plates on the Tecan bed according to the Tecan bed map. 8 Make sre that: All items are placed properly on the Tecan bed All caps and seals have been removed For each plate, well A1 is in the pper left corner of the frame 9 Select Rn. The Tecan condcts an internal QC process. A message in the lower stats bar indicates when the QC process is complete. 10 Remove the FLUOTRAC 200 plates from the Tecan bed and cover with alminm foil. 11 Dispose of any remaining reagents in accordance with yor facility reqirements. 12 Select Robot QC Tasks Robot Bleach Wash. 13 Add 5 ml 10% bleach to a qarter reservoir. 14 Place the reservoir in position A of the reservoir frame, as shown on the Tecan bed map. 15 Select Rn. 16 When the tip bleach process is complete, select Sys Flsh. 17 Observe the lines for air bbbles. 18 Repeat the system flsh process at least three times, ntil the lines are free of air bbbles. If bbbles persist in the lines, contact Illmina Technical Spport. Creating a Standard Plate The Standard plate is sed to generate a standard crve of florescent nits verss volme dispensed. Yo create the Standard plate by manally pipetting florescein over a range of volmes (1 36 µl) into 100 µl florescein diltion bffer. The Standard plate is then sed to calclate volmes dispensed by the Tecan into the Tecan QC test plates. NOTE To ensre sccessfl generation of the standard crve, se calibrated pipettes. 1 Label a FLUOTRAC 200 plate Standard Plate. 2 Dispense 12 ml florescein diltion bffer into a disposable mltichannel trogh. 3 Using a mltichannel pipette, add 100 µl florescein diltion bffer into each well of the Standard plate. 4 Add 2 ml low-florescein concentration standard (0.025 mg/ml) to a disposable mltichannel reservoir. 5 Using an 8-channel pipette, add low-florescein concentration standard (0.025 mg/ml) into each well of the Standard plate according to the volmes shown in Table 2. 6 Cover the plate and store it in the dark ntil reading on the florometer. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 15

20 Infinim Assay Lab Setp and Procedres Gide Table mg/ml Florescein volmes for the Standard plate, by well (in µl) A B C D E F G H Reading Florescence Intensities 1 Read the florescence intensities of the Standard and the Robot QC plates on a florometer at an excitation of 485 nm and 538 nm emission. 2 Set the florometer to Atomix for 10 seconds before the first reading. Calclating Reslts 1 Open the Tecan QC Analysis Tool Excel workbook. 2 Select the Standard Data Inpt sheet. Enter the florescein intensities of the standard crve, following the template in the Excel workbook. The standard crve statistics generate atomatically. 3 Make sre that the standard crve R2 for the low and the high dispense volmes are NOTE If the vales are < 0.99, the standard crve mst be repeated. If the standard crve is < 0.99, the cell for that crve is highlighted as failing. 4 Select the Individal Tips Data Inpt sheet. Enter the florescein intensities for the Ind-Col Dispense Robot QC plate, following the template in the Excel workbook. 5 Select the Mlti Tips Data Inpt sheet. Enter the florescein intensities for the Mlti-Col Dispense Robot QC plate, following the template in the Excel workbook. 6 Select the Smmary Analysis sheet and review the pass/fail data. 7 Tips that do not meet the reqired QC specifications are flagged with red. If a tip is failing, perform a Bleach Wash followed by a System Flsh on the Tecan, and then repeat the Robot QC procedre. 8 Evalate the individal and mlticolmn dispense reslts on the std crve. The acceptable standard crve R2 for the low and the high dispense volmes is Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 16

21 Infinim Assay Lab Setp and Procedres Gide Figre 3 Standard Crve with Individal Colmn Dispense Figre 4 Standard Crve with Mlti-Tip Colmn Dispense Cleaning the Tecan Cleaning the Oter Srface of the Tecan Tips Perform this activity once a week. 1 At the beginning of each day, check for any leaks. 2 In the Illmina Atomation Control Software, select Sys Init. 3 Select Tips Up. 4 Fold a Kimwipe in half lengthwise and soak it with 70% EtOH. 5 Fold the Kimwipe arond the Tecan tip. 6 Starting from one end of the tip row, wipe each tip gently along the entire lower half of the tip. 7 Reverse the Kimwipe to se the other, fresh side. 8 Starting from the opposite end of the tip row, wipe each tip a second time along the entire lower half. 9 Clean the waste station with the Kimwipe. Bleach-Bathing the Tecan Carriers Perform this activity monthly. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 17

22 Infinim Assay Lab Setp and Procedres Gide 1 Prepare a 10% bleach bath (~500 ml concentrated bleach in 4500 ml DI H2O) in a soaking tray. 2 Fill the other soaking tray with DI H2O. 3 Lay ot at least two rows of absorbent bench nderpad on a benchtop. 4 Remove the Tecan carriers from the Tecan bed. Note the original position of the carriers so that yo can replace them correctly. 5 Sbmerge the carriers in the prepared bleach soltion for abot 1 minte. 6 Remove the carriers from the bleach soltion and sbmerge them in the DI H2O soaking tray for abot 1 minte. 7 Remove the carriers from the soaking tray and rinse them nder rnning water. 8 Dry the carriers on the absorbent bench nderpad. 9 Allow the carriers to dry completely before retrning them to their proper positions on the Tecan bed. CAUTION To prevent contamination, make sre that all bleach vapors have flly dissipated before starting any process involving samples. Sterilize Flidics Perform this activity annally or whenever mold or bacterial growth is detected in the robot flidics. 1 In the carboy, prepare a 1 L 10% bleach soltion by adding 100 ml bleach to 900 ml DI H2O. 2 Select Robot QC Tasks Robot Bleach Wash. 3 In the Basic Rn Parameters pane, enter 1 for a system wash. 4 Select Rn. 5 When prompted, swap ot the system liqid carboy for the carboy containing 10 % bleach soltion. 6 Select OK. 7 When prompted, swap ot the bleach soltion with the system liqid carboy. 8 Select OK. 9 Observe lines for air bbbles. 10 Rn additional washes (SysWash) ntil the lines are free of air bbbles. If bbbles persist in the lines, contact Illmina Technical Spport. CAUTION To prevent contamination, make sre that all bleach vapors have flly dissipated before starting any process involving samples. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 18

23 Appendix A System Controls System Controls Introdction 19 The Control Dashboard 19 Control Bead Type IDs 19 Control Diagrams 21 Introdction This section describes the controls sed in the Infinim assay, inclding the bead type IDs sed, their expected otcomes, and how to view them. Diagrams with descriptions are inclded for sampleindependent and sample-dependent controls, and controls that are specific to the green or red channel. The controls are sefl both by themselves and with each other. Sample-Independent Controls The sample-independent controls are: Staining controls Extension controls Target removal controls Hybridization controls Sample-Dependent Controls The sample-dependent controls are: Stringency controls Nonspecific binding controls Nonpolymorphic (NP) controls Several key steps in the Infinim Assay reqire evalation of both the red and green color channels. For these instances, both red and green channel controls are inclded. See Table 3 for a list of controls and the color channel they are intended to monitor (red, green, or both). The Control Dashboard To view the controls, create a genotyping workspace sing the GenomeStdio Wizard, as described in the GenomeStdio Framework User Gide and the GenomeStdio Genotyping Modle User Gide. Yo can then view the controls performance from GenomeStdio s Analysis View Controls Dashboard men. Control Bead Type IDs The following table lists each bead type ID, a description, and the expected otcome. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 19

24 Infinim Assay Lab Setp and Procedres Gide Table 3 Used Bead Type IDs Bead Type ID Description Evalate Red (X) Sample-Independent Controls Evalate Green (Y) Expected Intensity Staining (DNP) + - High Staining (DNP) + - Backgrond Staining (Biotin) - + High Staining (Biotin) - + Backgrond Extension (A) + - High Extension (T) + - High Extension (C) - + High Extension (G) - + High Target removal + - Low Hybridization - + High Hybridization - + Medim Hybridization - + Low Stringency (PM) a + - High Stringency + - Medim (MM) b a PM = Perfectly Matched Seqence b MM = Mismatched Seqence Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 20

25 Infinim Assay Lab Setp and Procedres Gide Bead Type ID Description Evalate Red (X) Nonspecific binding Nonspecific binding Nonspecific binding Nonspecific binding Nonpolymorphic- (A) Nonpolymorphic- (T) Nonpolymorphic- (C) Nonpolymorphic- (G) Evalate Green (Y) Expected Intensity + + Backgrond + + Backgrond + + Backgrond + + Backgrond + - High + - High - + High - + High Control Diagrams The following diagrams illstrate control strctre and fnction. Sample-Independent Sample-independent controls evalate the performance of specific steps in the process flow. Staining Controls Staining controls are sed to examine the efficiency of the staining step in both the red and green channels. Staining controls have varios levels of dinitrophenyl (DNP) or biotin attached to the beads. See Table 3 for information abot the bead type IDs, relevant color channel, and expected intensity of biotin and DNP labeling controls. These controls are independent of the hybridization and extension step. Varios levels of DNP and biotin monitor the sensitivity and efficiency of the staining step. Both red and green channels can be evalated sing the Staining Controls. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 21

26 Infinim Assay Lab Setp and Procedres Gide Figre 5 Staining Controls (Sample-Independent) Extension Controls Extension controls test the extension efficiency of A, T, C, and G ncleotides from a hairpin probe, and are, therefore, sample-independent. Both red (A, T) and green (C, G) channels are monitored. See Table 3 for information abot the bead type IDs, relevant color channel, and expected intensity of the extension controls. Figre 6 Extension Controls (Sample-Independent) Target Removal Controls Target removal controls test the efficiency of the stripping step after the extension reaction. In contrast to allele-specific extension, the control oligos are extended sing the probe seqence as template. This process generates labeled targets. The probe seqences are designed sch that extension from the probe does not occr. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 22

27 Infinim Assay Lab Setp and Procedres Gide All target removal controls shold reslt in low signal compared to the hybridization controls, indicating that the targets were removed efficiently after extension. The target removal controls are present in the Hybridization Bffer RA1. Performance of target removal controls shold only be monitored in the red channel. See Table 3 for a list of the bead type IDs. Figre 7 Target Removal Controls (Sample-Independent) Hybridization Controls The hybridization controls test the overall performance of the entire assay sing synthetic targets instead of amplified DNA. These synthetic targets complement the seqence on the array perfectly, allowing the probe to extend on the synthetic target as template. The synthetic targets are present in the Hybridization Bffer (RA1) at three levels. They monitor the response from high-concentration (5 pm), medim-concentration (1 pm), and low-concentration (0.2 pm) targets. All bead type IDs shold reslt in signal with varios intensities, corresponding to the concentrations of the initial synthetic targets. Performance of hybridization controls shold only be monitored in the green channel. See Table 3 for a list of the bead type IDs. Figre 8 Hybridization Controls (Sample-Independent) Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 23

28 Infinim Assay Lab Setp and Procedres Gide Sample-Dependent The sample-dependent controls evalate performance across samples. Stringency Controls These controls test the stringency of the hybridization process. The same target is sed for each stringency control. The only difference between the stringency controls is the nmber of ncleotides hybridized between the target and the probe seqence on the bead. The probes are designed sch that the 3' end of the probe is available for extension. Mismatches are introdced into the body of the probe seqence to affect hybrid stability. The controls have 0 to 12 mismatched ncleotides between target and probe. Performance of stringency controls shold only be monitored in the red channel. See Table 3 for a list of the bead type IDs. Figre 9 Stringency Controls (Sample-Dependent) Nonspecific Binding Controls Nonspecific binding controls are inclded to monitor the specificity of the hybridization of the amplified DNA. The probe seqences for nonspecific binding controls are complementary to bacterial seqences and shold not hybridize to hman seqences nder standard hybridization stringency conditions. These controls shold show low intensities, indicating there is minimal cross-hybridization of hman seqences to bacterial seqences. Performance of nonspecific binding controls shold be monitored in both green and red channels. See Table 3 for a list of the bead type IDs. Nonpolymorphic Controls Nonpolymorphic controls test the overall performance of the assay from amplification to detection by qerying a particlar base in a nonpolymorphic region of the genome. They let yo compare assay performance across different samples. One nonpolymorphic control has been designed for each of the for ncleotides (A, T, C, and G). See Table 3 for a list of the bead type IDs. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 24

29 Infinim Assay Lab Setp and Procedres Gide Figre 10 Non-Polymorphic Controls (Sample-Dependent) Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 25

30 Appendix B Trobleshooting Trobleshooting Introdction 26 Pre-Hybridization 26 Hybridization to XStain 27 Imaging 28 Miscellaneos 29 Introdction Under certain circmstances, the Infinim Assay may operate in an nexpected manner. This section describes some of these circmstances and offers soltions and workaronds. If the isse cannot be resolved sing this gide, contact Illmina Technical Spport. Pre-Hybridization # Symptom Probable Case Resoltion / Comment 1 No ble pellet observed in wells after 20 minte centrifgation. 2 Ble color observed on absorbent pad after precipitation spernatant was decanted. Original DNA sample degraded. Precipitation reaction soltion was not mixed thoroghly before centrifgation. Either PM1 or 2-propanol was not added. Precipitation reaction soltion was not thoroghly mixed before centrifgation. The plate was centrifged at less than 3000 g, or for less than the recommended time. Spernatant was not removed immediately after centrifgation. Invert plate several times and centrifge again. Note: Samples may be lost. Repeat the Make step of the protocol yo are performing. Inspect wells for complete mixing before the 20 minte centrifgation. Add missing reagent to wells. Invert plate several times and centrifge plate again. Inspect wells for complete mixing before 20 minte centrifgation. The samples are lost. Repeat the Make step of the protocol yo are performing. If samples were thoroghly mixed with PA1 and 2-propanol, they shold not fall ot of wells if yo decant the spernatant immediately after centrifging the plate. The samples are lost. Repeat the Make step of the protocol yo are performing. Check centrifge program to make sre that correct speed was selected. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 26

31 Infinim Assay Lab Setp and Procedres Gide # Symptom Probable Case Resoltion / Comment 3 Ble pellet did not dissolve back into soltion after vortexing. An air bbble formed at the bottom of the well that prevented the pellet from mixing with RA1. Vortex speed is not fast enogh. The reaction plate did not incbate for long enogh. Plse centrifge plate to 280 g to remove air bbble, then revortex plate at 1800 rpm for 1 minte. Check the vortexer speed setting, as the speed setting may drift over time. Recalibrate if necessary. Revortex plate at 1800 rpm for 1 minte. Incbate the plate for an additional 30 mintes. Make sre that the cover is properly seated to prevent evaporation. Hybridization to XStain # Symptom Probable Case Resoltion / Comment 1 There was not enogh reagent to dispense to all BeadChips. 3 Cap mat deformed or melted while heat-denatring DNA samples. 5 A small amont of precipitate was present in the hybridization soltion. 8 BeadChips are still wet on the nderside after 55 mintes in the vacm desiccator. 9 After coating the BeadChips with XC4, some ncoated areas remain. Make sre to centrifge the reagent tbes at 280 g after thawing. Programmable pipettor may be set incorrectly. Foil heat sealer shold have been sed. A small amont of precipitate is normal, and does not affect data qality. The BeadChips mst dry for a longer period. XC4 may be old and mst be replaced with fresh XC4. An old bottle of ethanol may have absorbed atmospheric water. Replace with a fresh bottle of ethanol. Dring the coating process, a bbble formed between the BeadChips and prevented the XC4 soltion from reaching the srface. Check pipette calibration. A gravimetric test sing water is a qick and easy way to check for accrate pipette dispensing volmes. Recalibrate pipettes yearly. Check to see if the programmable pipettors have an overasp volme that is discarded. Samples have been rined. Repeat experiment. Use the foil heat sealer for all temperatres 45 C. Contine with experiment. Dry BeadChips longer nder vacm desiccator. Lab temperatre and hmidity affect drying time. Briefly place the staining rack with BeadChips back into the wash dish containing XC4. Gently move BeadChips back and forth while moving p and down, breaking the srface of the soltion. The back-and-forth movement is especially important when processing 16 or 24 BeadChips. Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 27

32 Infinim Assay Lab Setp and Procedres Gide # Symptom Probable Case Resoltion / Comment 11 Dring XStain, the liqid in the Flow- Throgh Chamber dropped below the bottom edge of the glass back plate reservoir. Glass back plates may not have been completely clean, casing capillary gap failre. Make sre that the correct spacer was sed to assemble the Flow Throgh Chamber. The Flow-Throgh Chambers may not be secrely assembled. Before reassembling the Flow- Throgh Chambers, clean the glass back plates. Attach the metal clamps to the Flow-Throgh Chambers as described in the Reference Gide for the assay. Imaging # Symptom Probable Case Resoltion / Comment 1 The iscan System was nable to find all the fidcials dring scanning. 2 The scanning process did not generate any intensity signal. 3 The scanning process generated a low assay signal. The Hyb controls display high intensities. 4 The scanning process generated a low assay signal. The Hyb controls display low intensities. 5 Some spots on the BeadChip have low to 0 intensity. XC4 coating was not properly removed from BeadChip edges. BeadChips were not seated correctly in BeadChip carrier. The absence of an intensity image may be de to failres in any experimental steps pstream of scanning. A low assay signal indicates a sample-dependent failre. The error may have occrred in any experimental steps between amplification and hybridization. A low assay signal indicates a sample-independent failre. The error probably occrred after hybridization. Bbbles in the reagents can case this phenomenon by preventing reagents from reaching the BeadChip srface. Rewipe the edges of BeadChips with ProStat EtOH wipes and rescan. Reseat BeadChips in BeadChip carrier. Repeat the experiment. Check the staining controls. If the controls do not generate a signal either, then the staining reagents may be compromised. Repeat experiment. Make sre that there is a DNA pellet after precipitation. Dring resspension, make sre that the DNA pellet dissolves properly. The ble color in the pellet shold disappear. To narrow down the problem, review the controls panel. Check the performance of the staining controls and the extension controls. Centrifge all reagent tbes before sing to prevent bbbles. Always perform a system flsh before rnning the experiment. (Low to 0 intensity areas on the BeadChip may not have a negative effect on array data de to randomness and oversampling of BeadChips.) Docment # v02 For Research Use Only. Not for se in diagnostic procedres. 28