VeraCode ADME Assay Guide (15007510 C)

VeraCode ADME Assay Guide (15007510 C)
VeraCode® ADME Core Panel
Assay Guide
FOR RESEARCH USE ONLY
ILLUMINA PROPRIETARY
Catalog # VC-801-1002
Part # 15007510 Rev. C
June 2011
ii
Part # 15007510 Rev. C
Notice
This document and its contents are proprietary to Illumina, Inc. and its affiliates (ʺIlluminaʺ), and are intended solely for the contractual use of its customer in connection with the use of the product(s) described herein and for no other purpose. This document and its contents shall not be used or distributed for any other purpose and/or otherwise communicated, disclosed, or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina does not convey any license under its patent, trademark, copyright, or common‐law rights nor similar rights of any third parties by this document. The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully read and understood prior to using such product(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.
ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S) DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) OR ANY USE OF SUCH PRODUCT(S) OUTSIDE THE SCOPE OF THE EXPRESS WRITTEN LICENSES OR PERMISSIONS GRANTED BY ILLUMINA IN CONNECTION WITH CUSTOMERʹS ACQUISITION OF SUCH PRODUCT(S).
FOR RESEARCH USE ONLY
© 2010–2011 Illumina, Inc. All rights reserved.
Illumina, illuminaDx, BeadArray, BeadXpress, cBot, CSPro, DASL, Eco, Genetic Energy, GAIIx, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, Infinium, iSelect, MiSeq, Nextera, Sentrix, Solexa, TruSeq, VeraCode, the pumpkin orange color, and the Genetic Energy streaming bases are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.
VeraCode ADME Core Panel Assay Guide
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Notice
iv
Part # 15007510 Rev. C
Revision History
Part #
Revision
Date
15007510
C
June 2011
• Revised Titanium Taq DNA Polymerase catalog number
15007510
B
March 2011
• Changed VW2 buffer bottle volume to 52 ml in the 32 sample kit
• Revised control DNA suggested vendor
• Specified Titanium Taq DNA Polymerase lot number
• Incorporated instructions for two plate processing
• Replaced incubating microplate shaker manual mode instructions with program mode instructions
• Added PSC controls to the plate controls report
• Changed “PSC Control” to “process control”
• Added troubleshooting workflow diagram
• Added Appendix C ‐ Microplate Shaker
• Added process control and sample tracking control error troubleshooting
• Added Best Practices
• Added Index
15007510
A
March 2010
Initial Release
VeraCode ADME Core Panel Assay Guide
Description of Change
v
Revision History
vi
Part # 15007510 Rev. C
Table of Contents
Chapter 1
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
ADME Core Panel Kit Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
User-Supplied Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
DNA Input Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Safety Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Chapter 2
Lab Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Create Sample Sheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lab Tracking Form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Make Assay Reaction (ARX) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Add Extension and Ligation Mix (ELM2) . . . . . . . . . . . . . . . . . . . . . . . . . .
Add Make Amplification Mix (MAM1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Make Single Stranded DNA (MSS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hybridize VeraCode Bead Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare BeadXpress Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Scan VeraCode Bead Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Analyze Scan Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Report Genotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix A
18
20
23
24
37
42
47
49
51
54
63
76
Standard Operating Procedures . . . . . . . . . . . . . . . .85
Best Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Preventing PCR Product Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Pipetting and Sealing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
VeraCode ADME Core Panel Assay Guide
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Table of Contents
Appendix B
VeraScan Administration and VeraReport . . . . . . . . 95
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .96
Set Up VeraScan ADME Analysis Configuration . . . . . . . . . . . . . . . . . . . . .97
Regenerate Genotyping Report using VeraReport . . . . . . . . . . . . . . . . . .102
Appendix C
Microplate Shaker . . . . . . . . . . . . . . . . . . . . . . . . . 107
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .108
Qualification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .109
Programming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .116
Appendix D
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Assay Protocol Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .122
BeadXpress Reader System Troubleshooting. . . . . . . . . . . . . . . . . . . . . .129
View and Report Errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .134
Frequently Asked Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .136
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Technical Assistance . . . . . . . . . . . . . . . . . . . . . . . 141
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Part # 15007510 Rev. C
List of Tables
Table 1
Table 2
Table 3
Table 4
Table 5
Table 6
Table 7
Table 8
Table 9
Table 10
Table 11
Table 12
Table 13
Table 14
Table 15
Table 16
Table 17
Table 18
Table 19
Table 20
Table 21
Table 22
Table 23
Table 24
Table 25
Table 26
Table 27
Table 28
Table 29
Table 30
Table 31
Table 32
VeraCode ADME Core Panel Internal Control Types . . . . . . . . . . . . . . . . . . 6
VeraCode ADME Core Genotyping Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Kit Contents, Box A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Kit Contents, Box B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Kit Contents, Box C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Kit Contents, Box D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
User-Supplied Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Header Section. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Wells Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
MJ/BioRad Thermocyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Eppendorf Thermocyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Bead Type Outcomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
No Template Control Valid Outcomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Plate Analysis Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Plate Sample Details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Plate Detail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Gene Detail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Plot tools. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Plate Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Results Section. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Controls Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Incubating Shaker Error Codes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Incubating Microplate Shaker Memory Step Settings . . . . . . . . . . . . . . . 118
Troubleshooting Problems During Sample Processing . . . . . . . . . . . . . . 122
Troubleshooting Problems During VeraCode Bead Hybridization . . . . . . 124
Troubleshooting Problems with Data Quality . . . . . . . . . . . . . . . . . . . . . . 124
Troubleshooting Problems with Data Generation and Storage . . . . . . . . 129
Troubleshooting Problems with Fluidics System . . . . . . . . . . . . . . . . . . . 131
Troubleshooting Problems with the BeadXpress Reader . . . . . . . . . . . . 132
Troubleshooting Problems with Test and Calibration Beads . . . . . . . . . . 133
Illumina General Contact Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Illumina Customer Support Telephone Numbers . . . . . . . . . . . . . . . . . . . 141
VeraCode ADME Core Panel Assay Guide
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List of Tables
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Part # 15007510 Rev. C
Chapter 1
Overview
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
ADME Core Panel Kit Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
User-Supplied Materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
DNA Input Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Safety Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
VeraCode ADME Core Panel Assay Guide
1
Overview
Introduction
The Illumina® VeraCode®ADME Core Panel on the BeadXpress®System genotypes genetic variations in a human genomic DNA (gDNA) sample. This assay interrogates genes associated with drug absorption, distribution, metabolism and excretion (ADME) using allele‐specific extension and ligation followed by PCR with fluorescently labeled primers. For each locus, there is a biotinylated oligonucleotide that copies a specific genomic region. Genotype determination is then accomplished by performing the allele‐
specific extension and ligation on the copied region.
Figure 1 Approach to Genotyping Variants in Homologous Genomic Regions
The content of the VeraCode ADME Core Panel is assayed in three highly optimized reaction subpools across a 96‐well plate enabling 32 samples per bead plate. A targeting mix (MTR) and assay oligo annealing reagent (AOP) are added to each sample. Color‐
coded tube caps distinguish these reagents for each of the three subpools.
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Part # 15007510 Rev. C
Introduction
Figure 2 ADME Assay Plate
The VeraCode bead types used in this assay and the genotyping results are described in the ADME Beadtypes and Translations document available on the Illumina website (http:/
/www.illumina.com/icom).
Genotyping
There are several homologous genes involved with drug ADME included in this panel. For example, by using the specific locus targeting of the VeraCode ADME Core Panel assay (see Figure 1), CYP2C9*2 can be genotyped accurately without interference from its close neighbors CYP2C19, CYP2C8, and CYP2C18.
VeraCode ADME Core Panel Assay Guide
3
Overview
Figure 3 Genotyping Example for A Challenging ADME Core Variant
Copy Number Variation
The VeraCode ADME Core Panel contains assay designs which detect copy number variation (CNV) in six genes: CYP2A6, CYP2D6, GSTM1, GSTT1, SULT1A, and UGT2B17. Oligonucleotide probes are designed to target non‐polymorphic regions in the gene with CNV and a control region elsewhere in the genome (e.g., CNV1 and Control1).
Figure 4 Multiple Probe Sets for Enhanced Precision of CNV Assays
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Figure 5 Examples of CNV Assay Results
VeraCode ADME Core Panel Assay Guide
5
Introduction
The signals resulting from the CNV and control probes are detected on the same VeraCode bead type, but in different channels (red and green). As a result, the red to green ratio (theta) represents the copy number of the gene of interest relative to the copy number (two per genome) of the control region. This allows the CNV assay to be internally controlled for performance and visualized on a genoplot. For improved precision, multiple probe sets are used for each gene and the individual results are aggregated for a single graphic output. Examples of CYP2D6 and GSTT1 CNV assay results are shown below.
Overview
Controls
The VeraCode ADME Core Panel includes multiple types of internal controls in each sample, outlined below.
Table 1 VeraCode ADME Core Panel Internal Control Types Probe ID
Category
Expected Result
STC
Sample Tracking Pass = Internal sample barcode Control
(e.g., A9EBF9F) can be verified for each of the three subpools
PSC
Process Control
SPC
Subpool Position Pass = Samples are run in designated sections A,B, Control
and C of the assay plate
Hyb Control 1
Hybridization Control
Hyb Control 2
Hybridization Control
Mismatch Control 1
Assay Control
Mismatch Control 2
Assay Control
Pass = No mix‐up of subpool specific reagents is detected. All ADME process steps completed successfully.
Pass = Successful hybridization to VeraCode beads
Pass = Successful allele‐specific extension and ligation
Sample Tracking Control (STC)
A panel of high minor allele frequency SNPs, included in each assay pool, provide added sample traceability when translated to a unique barcode for each sample. The control is used to identify sample pipetting errors, cross contamination during sample processing, and DNA quality issues in a run.
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Part # 15007510 Rev. C
A set of internal negative controls identify proper pairing of targeting oligo pools (MTRs) with their corresponding assay oligo annealing reagent (AOPs) as depicted in Figure 2. The same control bead for each possible MTR/AOP mis‐pairing exists in each of the subpools. All results for this control should give background signals as long as all MTRs and AOPs are appropriately matched. If the wrong MTR and AOP are matched this control gives off a high signal alerting VeraScan data analysis and the user that a processing error occurred.
This control can also give elevated signal if there are deviations for critical steps in the protocol. Intensity data shifts seen in the PSC controls are representative of shifts seen in assay genoplots. Therefore, no data is generated for samples that fail this control.
Subpool Position Control (SPC)
This control works alongside the PSC to ensure that correctly paired oligo reagents were pipetted to the correct subpool location on the plate. This control specifically tests for the AOP being analyzed in each subpool. There are three control bead types: one each for pool A, B, and C. Each bead type is in all three subpools. When the assay is run correctly, VeraScan expects to see a high signal for the bead type corresponding to the specific AOP for that subpool (i.e., high signal for AOPA in subpool A). The other two bead types for the other AOPs should have signal close to background (i.e., low signal for AOPB and AOPC in subpool A). This analysis is done by the VeraScan software ADME module and only a Pass is reported in the output data for a successful run.
Hybridization Controls
There are two hybridization control bead types, which report efficient hybridization of fluorescently labeled products to VeraCode beads. The hybridization controls test the hybridization of single‐stranded assay products to address sequences specific to VeraCode beads. The address sequence corresponding to the Cy3 labeled probe should result in a green signal and the sequence corresponding to the Cy5 labeled probe should result in a red signal.
Figure 6 Hybridization Controls
VeraCode ADME Core Panel Assay Guide
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Introduction
Process Control (PSC)
Overview
Mismatch Controls
Two mismatch controls (one for the green channel and the other for the red channel) are included to verify allele‐specific extension and ligation. Similar to the oligonucleotide designs for genotyping the ADME Core variants, each of the two mismatch controls has two upstream oligos targeting a non‐polymorphic region in the genome. Only one of the two oligonucleotides has a 3ʹ end base match. The other oligonucleotide has a 3ʹ end base mismatch which, under normal circumstances, cannot be extended and ligated to the downstream oligonucleotide. In addition, the mismatch controls help verify the functionality of assay reagents and the balance of BeadXpress Reader.
Figure 7 Mismatch Control Green
Figure 8 Mismatch Control Red
Control Samples
Good laboratory practices recommend that positive control DNA samples and negative (no‐template) control samples are included in every run. See the Wells Section on page 21 of Create Sample Sheet for information on how to properly mark samples as positive and negative controls in the VeraScan Software.
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Part # 15007510 Rev. C
Check to ensure that you have all of the reagents and materials identified in this section before proceeding with the ADME Core Panel protocol. Depending on the number of samples being prepared, you need one or more of the VeraCode ADME Core Genotyping Kits in which are shipped on dry ice.
Table 2 VeraCode ADME Core Genotyping Kits
Number of Samples
Illumina Catalog #
32
VC‐901‐0201
160
VC‐901‐0200
Kit Contents, Box A
As soon as you receive this box, store the components in the freezer (‐15° to ‐25°C) in the pre‐PCR lab area, as indicated on the container and in the following table.
Table 3 Kit Contents, Box A Item
Label Color
Number Supplied
32 Sample Kit
Number Supplied
160 Sample Kit
Volume
Storage Temperature
AB1 reagent
Purple label
1 tube
5 tubes
4.0 ml
‐15° to ‐25°C
UB3 buffer
Orange label
2 tubes
10 tubes
4.8 ml
‐15° to ‐25°C
AE1 reagent
Yellow label
2 tubes
10 tubes
4.8 ml
‐15° to ‐25°C
ELM2 reagent
Navy blue label
1 tube
5 tubes
4.8 ml
‐15° to ‐25°C
MAM1 reagent
Green label
1 tube
5 tubes
4.8 ml
‐15° to ‐25°C
AOP0 reagent
Red label
1 tube
5 tubes
3.6 ml
‐15° to ‐25°C
VeraCode ADME Core Panel Assay Guide
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ADME Core Panel Kit Contents
ADME Core Panel Kit Contents
Overview
Kit Contents, Box B
As soon as you receive this box, store the components in the pre‐PCR lab area at the storage temperature indicated on the container and in the following table.
Table 4 Kit Contents, Box B Item
Tube/Cap Color
Number Supplied
32 Sample Kit
Number Supplied
160 Sample Kit
Volume
Storage Temperature
MTR4A reagent
Clear tube/Red cap
1 tube
5 tubes
0.6 ml
‐15° to ‐25°C
MTR4B reagent
Clear tube/Yellow cap
1 tube
5 tubes
0.6 ml
‐15° to ‐25°C
MTR4C reagent
Clear tube/Blue cap
1 tube
5 tubes
0.6 ml
‐15° to ‐25°C
AOP4A reagent
Amber tube/Red cap
1 tube
5 tubes
1.8 ml
‐15° to ‐25°C
AOP4B reagent
Amber tube/Yellow cap
1 tube
5 tubes
1.8 ml
‐15° to ‐25°C
AOP4C reagent
Amber tube/Blue cap
1 tube
5 tubes
1.8 ml
‐15° to ‐25°C
Pool Guide Label
–
2 labels
6 labels
–
Room Temperature
Kit Contents, Box C
As soon as you receive this box, store the components in the post‐PCR lab area at the storage temperature indicated on the container and in the following table.
Table 5 Kit Contents, Box C Item
Label Color
Number x Volume Supplied
32 Sample Kit
Number x Volume Supplied
160 Sample Kit
Storage Temperature
MSS reagent
Lavender label
1 tube x 4.8 ml
5 tubes x 4.8 ml
‐15° to ‐25°C
VW2 buffer
Sea foam green label
1 bottle x 52 ml
1 bottle x 250 ml
Room temperature
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Part # 15007510 Rev. C
As soon as you receive this box, store the components in the post‐PCR lab area at 2° to 8°C as indicated on the container and in the following table.
Table 6 Kit Contents, Box D Item
Number Supplied
32 Sample Kit
Number Supplied
160 Sample Kit
Storage Temperature
VeraCode ADME Bead Plate
1 plate
5 plates
2° to 8°C
VeraCode ADME Core Panel Assay Guide
11
ADME Core Panel Kit Contents
Kit Contents, Box D
Overview
User-Supplied Materials
Check to ensure that you have all of the following materials in the appropriate lab area (pre‐ and/or post‐PCR) before proceeding with the ADME Core Panel protocol.
NOTE
When processing two plates simultaneously, two magnetic plates are required and the incubating microplate shaker must be able to accommodate two plates.
NOTE
Do not use electronic or repeat pipettes when performing the ADME Core Panel assay.
Table 7 User‐Supplied Materials Pre‐
PCR
Post‐
PCR
Item
Suggested Vendor
0.1N NaOH solution
General lab supplier
10% (w/v) Potassium Hydroxide solution
General lab supplier
20 μl multichannel pipettes
General lab supplier
X
20 μl single channel pipettes
General lab supplier
X
20 μl pipette tips
General lab supplier
X
96‐well skirted PCR microplate
General lab supplier
X
200 μl multichannel pipettes
General lab supplier
X
X
200 μl single channel pipettes
General lab supplier
X
X
200 μl pipette tips
General lab supplier
X
X
1,000 μl single channel pipettes
General lab supplier
X
1,000 μl pipette tips
General lab supplier
X
12
X
X
Part # 15007510 Rev. C
Pre‐
PCR
Post‐
PCR
Item
Suggested Vendor
Adhesive microplate sealing film
Beckman Coulter, catalog # 538619
or
Applied Biosystems, catalog # 4306311
X
Adhesive seal applicators (5 per package)
MicroAmp, catalog # 4333183
X
Benchtop centrifuge for microplates
General lab supplier
X
Control DNA [Optional]
Coriell Institute
(www.coriell.org)
X
Heat block for skirted PCR microplates
(Note: If processing two plates simultaneously, two heat blocks are required.)
General lab supplier
X
Incubating microplate shaker
(shaking speed 1,400 rpm, temperature range 45° to 68°C, cooling rate (above ambient): 3.5°C per minute)
(Note: This shaker can hold up to two skirted plates.)
VWR, catalog # 97027‐346
X
Incubating microplate shaker power cord
(Note: Select one and only if applicable)
VWR, catalog #:
VWRI444‐2853 (Europe)
VWRI444‐2854 (United Kingdom)
VWRI444‐2855 (Switzerland)
X
Kimwipes
General lab supplier
X
X
Magnetic plate
Dynal, catalog # MPC‐96
(Note: If processing two plates simultaneously, two magnetic plates are required in both the pre‐PCR and post‐PCR labs.)
X
X
PCR sealing film
BioRad, catalog # MSA‐5001
X
Reagent alcohol
General lab supplier
VeraCode ADME Core Panel Assay Guide
X
X
13
User-Supplied Materials
Table 7 User‐Supplied Materials (Continued)
Overview
Table 7 User‐Supplied Materials (Continued)
Pre‐
PCR
Post‐
PCR
X
X
Item
Suggested Vendor
Reagent reservoirs
General lab supplier
Shaking incubator
(with shaking speed 850–1,200 rpm, temperature 45°C)
LabNet, catalog # VorTemp 56
Tachometer/stroboscope, combination optical [Optional]
Cole‐Parmer, catalog # A‐87700‐06 www.coleparmer.com
Thermocycler compatible with skirted PCR microplates
General lab supplier
Titanium Taq DNA Polymerase (5 U/μl)
Clontech, catalog # 639293
Vacuum flask assembly with regulator
QIAGEN, catalog # 19530 or equivalent
X
Vacuum manifold
V&P Scientific, catalog # VP180I
X
VeraCode Test & Calibration Bead Plate
Illumina, catalog # VC‐321‐1000
X
VR1 buffer (10x Reader Buffer)
Illumina, catalog # VC‐400‐1001
X
14
X
X
X
X
Part # 15007510 Rev. C
The VeraCode ADME Core Panel requires extracted genomic DNA from EDTA‐anticoagulated whole blood. Commercially available or laboratory validated DNA extraction methods typically yield DNA that is compatible with this test. Extracted DNA purity should range from an A260/A280 ratio of 1.8–2.0. 15 μl of DNA at 50 ng/μl is required for this assay (5 μl of DNA per sample for each of the three assay pools) for a total input DNA quantity of 750 ng per sample.
Optimal assay performance is dependent on using the recommended concentrations and volumes. It is also important to ensure that equal volumes and concentrations of DNA are used in each of the three reaction pools for a given sample.
VeraCode ADME Core Panel Assay Guide
15
DNA Input Requirements
DNA Input Requirements
Overview
Safety Precautions
CAUTION
Ensure that the user supplied equipment is properly calibrated. Out of tolerance equipment can negatively impact assay performance.
CAUTION
Please refer to the governmental and facility safety standards applicable to your site.
CAUTION
To minimize aerosols when disposing of the VeraCode Bead Plates, refer to your state waste disposal requirements.
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Part # 15007510 Rev. C
Chapter 2
Lab Protocols
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Create Sample Sheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Lab Tracking Form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Make Assay Reaction (ARX). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Add Extension and Ligation Mix (ELM2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Add Make Amplification Mix (MAM1). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Make Single Stranded DNA (MSS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Hybridize VeraCode Bead Plate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Prepare BeadXpress Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Scan VeraCode Bead Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Analyze Scan Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Report Genotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
VeraCode ADME Core Panel Assay Guide
17
Lab Protocols
Introduction
This protocol describes how to process the Illumina VeraCode ADME Core Panel on the BeadXpress System. The instructions assume that you have already familiarized yourself with Appendix A, Standard Operating Procedures and have set up the lab area appropriately (reference Control Samples on page 8 and ADME Core Panel Kit Contents on page 9).
CAUTION
It is very important to prevent the contamination of the laboratory with polymerase chain reaction (PCR) product during this assay. To learn about safe lab practices for Illumina assays, see Preventing PCR Product Contamination on page 101. In addition, follow all of the safety procedures described in this document.
NOTE
Calibrate the pre‐PCR incubating microplate shaker according to the manufacturer’s instruction. The calibration tolerance (allowable deviation from nominal), plus the uniformity tolerance (provided by the manufacturer), plus the error associated with the measurement equipment should not exceed +/‐ 0.5°C. The shaking speed of the pre‐PCR shaker should also be calibrated so that the actual speed of the shaker matches the ADME Core Panel protocol requirement of 1,400 rpm. See Appendix C, Qualification.
NOTE
Calibrate the post‐PCR incubating microplate shaker according to the manufacturer’s instruction. The calibration tolerance (allowable deviation from nominal) plus the error associated with the measurement equipment should not exceed +/‐ 0.5°C. See Appendix C, Qualify Temperature.
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Part # 15007510 Rev. C
The following graphically represents the VeraCode ADME Core Panel Assay which must be performed in the order shown.
Figure 9 VeraCode ADME Core Panel Workflow
VeraCode ADME Core Panel Assay Guide
19
Introduction
Workflow
Lab Protocols
Create Sample Sheet
Before starting the VeraCode ADME Core Panel assay, create a sample sheet. A sample sheet is a comma‐separated values (*.csv) file that contains the sample name and related information that describes the location of each sample in the assay reaction plate.
The sample sheet template for the VeraCode ADME Core Panel is located on your BeadXpress Reader at C:\Documents and Settings\All Users\Documents\Illumina\
VeraScan\ScanSettings. Fill in your sample sheet according to the guidelines provided in this section.
Figure 10 Example: Sample Sheet
Sample Sheet Sections
The Sample Sheet is separated into sections that have different uses. These are the Header and the Wells sections. All fields are required except Comments and Control.
Header section
The Header section contains a title for this run, and room for comments on this run. This information appears in the genotyping report.
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Part # 15007510 Rev. C
Create Sample Sheet
Table 8 Header Section
Header Field
Description
Title
The plate ID used to name the data files and folders
Comments
[Optional] Comments for this run (e.g., laboratory name, technician)
KitPN
You must enter the kit part number (PN) printed on Box B of the ADME kit.
Figure 11 Box B Kit Part Number Location
Wells Section
Each well in the plate that contains a sample is represented as a row in the Sample Sheet. In addition to the sample name, other sample information can be included. The VeraScan software identifies samples you have included as positive or negative controls in the genotyping report. The genotyping report also includes any comments you wish to make. This table has the following columns:
VeraCode ADME Core Panel Assay Guide
21
Lab Protocols
Table 9 Wells Section
Column Header
Description
Column
The plate column number (1–4). The sample information for columns 5–12 are automatically completed by VeraScan for the duplicate samples for the 3 assay pools on the plate.
Row
The letter representing the row number (A–H)
Kit_Number
ADMECore (this text must not be changed)
Sample
The name of the sample
Control
[Optional] Whether or not the sample is identified as a control.
You must enter Positive or Negative:
Positive ‐ sample is a positive control, indicated by P following the sample name in the results report
Negative ‐ sample is a negative control, indicated by N following the sample name in the results report, and are not plotted in graphs and do not contribute to call rate calculations
Comments
[Optional] Comments about the sample
CAUTION
To avoid misidentifying samples, ensure that the sample names entered in the sample sheet correctly correspond to the DNA samples used.
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Part # 15007510 Rev. C
A Lab Tracking Form (LTF) may be used to track your progress in the protocol and to ensure that all of the protocol steps of the VeraCode ADME Core Panel have been completed. You can fill out the form electronically and save a copy under a new name, or print it and fill it out by hand. Use a new LTF for each assay.
The LTF can be downloaded via http://www.illumina.com/support/documentation.ilmn.
NOTE
The LTF should be used while referencing the protocol in this guide which is more inclusive and detailed. The LTF is intended for use as a tracking tool and not a replacement for the user guide.
VeraCode ADME Core Panel Assay Guide
23
Lab Tracking Form
Lab Tracking Form
Lab Protocols
Make Assay Reaction (ARX)
In this pre‐PCR process, the gDNA and reagents are added to a PCR microplate to create the assay reaction (ARX) plate in which the assay is performed. The gDNA is denatured, copied, captured on paramagnetic beads, and washed. The selected DNA sequences are then annealed to oligonucleotides.
Begin the assay in the pre‐PCR laboratory. Check to ensure that you have all of the consumables identified in this section before proceeding with the assay protocol.
NOTE
Processing two plates simultaneously requires appropriate preparation of consumables and equipment. Therefore, it is important to plan ahead before proceeding with the assay protocol to avoid unintentional delays.
Estimated Time
Hands‐on: ~30 minutes
Overall: ~70 minutes
Consumables
24
Item
Label/Tube/ Cap Color Quantity
Storage
Supplied By
gDNA
–
15 μl (50 ng/μl)
‐15° to ‐25°C
User
0.1N NaOH solution
–
2 ml per plate
Room temperature
User
MTR4A reagent
Clear tube/Red cap
1 tube per plate
‐15° to ‐25°C
Illumina
MTR4B reagent
Clear tube/Yellow cap
1 tube per plate
‐15° to ‐25°C
Illumina
MTR4C reagent
Clear tube/Blue cap
1 tube per plate
‐15° to ‐25°C
Illumina
AB1 reagent
Purple label
1 tube per plate
‐15° to ‐25°C
Illumina
UB3 buffer
Orange label
1 tube per plate
‐15° to ‐25°C
Illumina
Part # 15007510 Rev. C
Label/Tube/ Cap Color Quantity
Storage
Supplied By
AOP0 reagent
Red label
1 tube per plate
‐15° to ‐25°C
Illumina
AOP4A reagent
Amber tube/Red cap
1 tube per plate
‐15° to ‐25°C
Illumina
AOP4B reagent
Amber tube/Yellow cap 1 tube per plate
‐15° to ‐25°C
Illumina
AOP4C reagent
Amber tube/Blue cap
1 tube per plate
‐15° to ‐25°C
Illumina
96‐well skirted PCR microplate
–
1
Room temperature
User
Adhesive microplate sealing film
–
5 per plate
Room temperature
User
Pool Guide Label –
1 label per plate Room temperature
Illumina
NOTE
The 0.1N NaOH solution must not be more than two weeks old.
Preparation
` Thaw all of the kit reagents on the benchtop.
` Vortex each reagent for 5 seconds immediately before using the reagent.
` To avoid switching pipette settings back and forth, use a designated pipette set at 60 μl to remove the supernatant and use an additional pipette for adding reagents.
NOTE
Do not use electronic or repeat pipettes when performing the ADME Core Panel assay.
` Set the pre‐PCR centrifuge to 15° to 25°C, if refrigerated.
` Turn on the heat block and let it equilibrate to 95°C.
VeraCode ADME Core Panel Assay Guide
25
Make Assay Reaction (ARX)
Item
Lab Protocols
` Turn on the pre‐PCR incubating microplate shaker. The display should show Pr2 step 01. Press start to initiate preheating the shaker to 68°C. The shaker will beep when the temperature is stabilized and the display will show step 02 settings.
NOTE
When the incubating microplate shaker is turned off with Pr2 selected, the next time it is turned on it will display the Pr2 Step 1.
` Read Pipetting and Sealing on page 90 for recommended reagent pipetting and adhesive seal application techniques for the VeraCode ADME Core Panel Assay.
CAUTION
Program 1 (Pr1) on the incubating microplate shaker is not setup to run ADME incubating conditions. Do not use this program when running the ADME assay.
CAUTION
The VWR incubating/cooling shaker must be qualified and programmed for automated use and to avoid potential ADME Core Panel protocol errors. See Appendix C, Microplate Shaker before proceeding with the protocol.
NOTE
During the pre‐PCR portion of the assay, all steps on the incubating shaker are performed at 1,400 rpm, which is also the incubating shaker’s default setting.
NOTE
If you are only processing 1 plate, place a balance on the empty plate position on the incubating shaker.
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Part # 15007510 Rev. C
Make Assay Reaction (ARX)
NOTE
Proceed diligently through each step of the protocol. Do not allow the plates to remain at any one step longer than is necessary for standard assay processing. When processing two plates simultaneously, Illumina recommends that you are experienced with the protocol, reagents, and time required for each step.
Steps
1
Attach the pool guide label to the long side of the PCR microplate.
Figure 12 Pool Guide Label
VeraCode ADME Core Panel Assay Guide
27
Lab Protocols
2
Label the sample sections A, B, and C on the PCR microplate with a smudge resistant pen.
Figure 13 PCR Microplate
A
B
C
D
3
Sample section A
Sample section B
Sample section C
Pool guide label
Add DNA to ARX plate.
NOTE
When processing two plates simultaneously, add the gDNA to both plates before proceeding to step 4.
a
b
4
28
For each DNA sample to be processed, add 5 μl gDNA (at 50 ng/μl) to the bottom of each of the 3 designated wells of the PCR microplate.
After dispensing, inspect the tips and bottom of the PCR microplate to make sure the gDNA samples have been properly dispensed into the PCR microplate.
Denature DNA.
a Vortex the 0.1N NaOH solution for 5 seconds.
b Add 5μl 0.1N NaOH solution to each well of the PCR microplate.
NaOH should be aspirated from the top of the reagent reservoir and the tips should touch the bottom of the well when dispensing.
Part # 15007510 Rev. C
NOTE
To avoid prolonged exposure of gDNA to DNA when processing two plates simultaneously, add the 0.1N NaOH solution to one plate before adding it to the second plate, then quickly proceed to step 5.
5
Add MTR while referencing the following figure.
NOTE
When processing two plates simultaneously, complete the addition of each MTR reagent to one plate before adding them to the second plate.
Figure 14 MTR Well Distribution
a
b
Vortex each MTR reagent for 5 seconds, then centrifuge each briefly.
Transfer the contents of each MTR reagent tube to a separate reagent reservoir using a single channel pipette.
VeraCode ADME Core Panel Assay Guide
29
Make Assay Reaction (ARX)
Gently pipette up and down twice to mix the solutions. Do not pass the first stop on the pipette.
Change the tips after each column.
Lab Protocols
c
d
e
6
Add 10 μl MTR4A to each well of columns 1–4 of the PCR microplate.
The tips should touch the bottom of the well when dispensing. Do not pass the first stop on the pipette.
No up/down mixing is necessary.
Change the tips after each column.
Add 10 μl MTR4B to each well of columns 5–8 of the PCR microplate.
The tips should touch the bottom of the well when dispensing. Do not pass the first stop on the pipette.
No up/down mixing is necessary.
Change the tips after each column.
Add 10 μl MTR4C to each well of columns 9–12 of the PCR microplate.
The tips should touch the bottom of the well when dispensing. Do not pass the first stop on the pipette.
No up/down mixing is necessary.
Change the tips after each column.
Spin and Incubate.
a Seal the PCR microplate with a adhesive microplate sealing film.
NOTE
Make sure the sealing film is on securely in order to reduce sample evaporation and cross contamination during incubations. Use an adhesive seal applicator to apply force to the seal and ensure the seal is secured. See Sealing Microplates on page 92 for proper seal application techniques.
b
Centrifuge the PCR microplate to 1,000 xg for 1 minute.
NOTE
When processing two plates simultaneously, they must be vortexed simultaneously on the incubating shaker throughout the entire protocol.
c
30
Place the PCR microplate on the incubating/cooling shaker and press start to begin the incubating/cooling shaker Pr2 program step 02 (1,400 rpm at 68°C for 5 minutes).
Part # 15007510 Rev. C
Heat and Spin.
a Transfer the PCR microplate to the 95°C heat block and incubate for 1 minute.
NOTE
When processing two plates simultaneously, they must incubate on two separate heat blocks simultaneously.
b
c
d
8
Transfer the PCR microplate to the bench top and incubate at room temperature for 3 minutes.
Place the PCR microplate on the incubating/cooling shaker and press start to begin Pr2 program step 03 (1,400 rpm at 68°C for 5 minutes).
Centrifuge the PCR microplate to 1,000 xg for 1 minute.
Denature Mixture.
NOTE
When processing two plates simultaneously, complete the addition of 0.1N NaOH to one plate before adding it to the second plate.
a
b
c
Remove the adhesive seal and add 5 μl 0.1N NaOH to each well of the PCR microplate.
NaOH should be aspirated from the top of the reagent reservoir and the tips should touch the bottom of the well when dispensing. Do not pass the first stop on the pipette.
No up/down mixing is necessary.
Change the tips after each column.
Seal the PCR microplate with a adhesive microplate sealing film, using an adhesive seal applicator to ensure the seal is secure. See Sealing Microplates on page 92 for proper seal application techniques.
Place the PCR microplate on the incubating/cooling shaker and press start to begin Pr2 program step 04 (1,400 rpm at 68°C for 1 minute).
VeraCode ADME Core Panel Assay Guide
31
Make Assay Reaction (ARX)
7
Lab Protocols
9
Add Paramagnetic Beads.
a Vortex the AB1 reagent for 5 seconds. Visually ensure that the paramagnetic bead pellets are well dispersed in the solution and no crystal structures are present.
NOTE
If crystals are observed, vortex the AB1 solution until no crystal structures are visible.
CAUTION
The paramagnetic particles will settle after some time. Make sure to properly vortex the AB1 solution right before the addition to the reagent reservoir.
b
Remove the adhesive seal and add 30 μl AB1 to each well of the PCR microplate.
NOTE
When processing two plates simultaneously, complete the addition of AB1 to one plate before adding it to the second plate.
NOTE
AB1 should not spill out of the wells and on the plate. Accurate pipetting and proper plate sealing will prevent any spillage. An improperly functioning thermal shaker can also produce excess plate agitation leading to spillage.
AB1 should be aspirated from the top of the reagent reservoir and the tips should touch the side of the wells or just above the liquid when dispensing. Do not pass the first stop on the pipette.
No up/down mixing is necessary.
Change the tips after each column.
CAUTION
Avoid introducing any bubbles into the solution.
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Part # 15007510 Rev. C
c
d
e
Seal the PCR microplate with a adhesive microplate sealing film. Use an adhesive seal applicator to ensure the seal is secure. See Sealing Microplates on page 92 for proper seal application techniques.
Place the PCR microplate on the incubating/cooling shaker and press start to begin Pr2 program step 05 (1,400 rpm at 68°C for 5 minutes).
Centrifuge the PCR plate to 1,000 xg for 1 minute.
10 Collect and Wash Paramagnetic Beads.
NOTE
When processing two plates simultaneously, each step must be performed on both PCR microplates simultaneously using a separate magnetic plate for each PCR microplate.
a
b
Place the PCR microplate on the magnetic plate for 1 minute, then remove the adhesive seal.
Remove the supernatant with a pipette set at 60 μl. Take care to not disturb the paramagnetic bead pellets.
CAUTION
The paramagnetic beads collect to alternate sides of the wells. When aspirating the supernatants, take care to not disturb the paramagnetic bead pellets by pointing the pipette tips away from the pellets.
NOTE
The paramagnetic particles in the plate wells should never dry. Therefore, when processing two plates simultaneously, remove the supernatant from one plate and add UB3 (step 10e) to that same plate before removing the supernatant from and adding UB3 to the second plate.
c
d
Change the tips after each plate if your are processing more than 1 plate. When you remove the supernatant you do not have to change tips after each column of a plate.
Vortex the UB3 for 5 seconds.
VeraCode ADME Core Panel Assay Guide
33
Make Assay Reaction (ARX)
CAUTION
The AB1 reagent contains formamide and must be properly disposed of in a hazardous waste container.
Lab Protocols
e
f
g
h
Add 40 μl UB3 to each well of the PCR microplate. UB3 should be aspirated from the top of the reagent reservoir and the tips should touch the side of the wells, above the liquid level line, when dispensing.
Seal the plate with a adhesive microplate sealing film, using an adhesive seal applicator to ensure the seal is secure. See Sealing Microplates on page 92 for proper seal application techniques.
Place the PCR microplate on the incubating/cooling shaker and press start to begin Pr2 program step 06 (1,400 rpm at 68°C for 1 minute).
Place the PCR microplate on the magnetic plate for 1 minute.
11 Prepare AOP reagents.
a Vortex the AOP0 reagent for 5 seconds.
b Add 1,200 μl AOP0 to each tube of AOP4A, AOP4B, and AOP4C.
c Vortex each AOP4 reagent for 5 seconds, then centrifuge each briefly.
d Remove the adhesive seal from the PCR microplate.
e Remove all of the supernatant from each well with a pipette set at 60 μl. Take care to not disturb the paramagnetic bead pellets.
CAUTION
The paramagnetic beads collect to alternate sides of the wells. When aspirating the supernatants, take care to not disturb the paramagnetic bead pellets by pointing the pipette tips away from the pellets.
NOTE
The paramagnetic particles in the plate wells should never dry. Therefore, when processing two plates simultaneously, remove the supernatant from one plate and add the AOP reagents to that same plate before removing the supernatant from and adding the AOP reagents to the second plate. When both plates are ready, proceed to anneal oligos.
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Part # 15007510 Rev. C
Figure 15 AOP Well Distribution
a
b
c
d
Pour each AOP4 reagent into a separate reagent reservoir.
Add 40 μl AOP4A to each well of columns 1–4 of the PCR microplate.
AOP4A should be aspirated from the top of the reagent reservoir and the tips should touch the side of the wells, above the liquid level line, when dispensing. Do not pass the first stop on the pipette.
No up/down mixing is necessary.
Change the tips after each column.
Add 40 μl AOP4B to each well of columns 5–8 of the PCR microplate.
AOP4B should be aspirated from the top of the reagent reservoir and the tips should touch the side of the wells, above the liquid level line, when dispensing. Do not pass the first stop on the pipette.
No up/down mixing is necessary.
Change the tips after each column.
Add 40 μl AOP4C to each well of columns 9–12 of the PCR microplate.
AOP4C should be aspirated from the top of the reagent reservoir and the tips should touch the side of the wells, above the liquid level line, when dispensing. Do not pass the first stop on the pipette.
No up/down mixing is necessary.
Change the tips after each column.
VeraCode ADME Core Panel Assay Guide
35
Make Assay Reaction (ARX)
12 Add AOP while referencing the following figure.
Lab Protocols
e
Seal the plate with a adhesive microplate sealing film. Use an adhesive seal applicator to ensure the seal is secure. See Sealing Microplates on page 92 for proper seal application techniques.
CAUTION
The AOP reagents contain formamide and must be properly disposed of in a hazardous waste container.
13 Anneal Oligos.
Place the PCR microplate on the incubating/cooling shaker and press start to begin Pr2 program step 07 (1,400 rpm at 68°C for 15 minutes, with a cooling‐rampdown to 45°C).
14 Proceed immediately to Add Extension and Ligation Mix (ELM2) on page 37.
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Part # 15007510 Rev. C
In this pre‐PCR process, AE1 reagent and UB3 buffer are added to the PCR microplate to wash away non‐specifically hybridized and excess oligonucleotides. An enzymatic extension and ligation mix (ELM2) is then added to each DNA sample.
Check to ensure that you have all of the consumables identified in this section before proceeding with the assay protocol.
Estimated Time
Hands‐on: ~10 minutes
Overall: ~30 minutes
Consumables
Item
Label Color Quantity
Storage
Supplied By
AE1 reagent
Yellow
2 tubes per plate
‐15° to ‐25°C
Illumina
UB3 buffer
Orange
1 tube per plate
‐15° to ‐25°C
Illumina
ELM2 reagent
Navy blue
1 tube per plate
‐15° to ‐25°C
Illumina
4
Room temperature
User
Adhesive microplate –
sealing film
Preparation
` Vortex each reagent for 5 seconds immediately before using the reagent. VeraCode ADME Core Panel Assay Guide
37
Add Extension and Ligation Mix (ELM2)
Add Extension and Ligation Mix (ELM2)
Lab Protocols
Steps
1
Collect and Wash Paramagnetic Beads.
NOTE
When processing two plates simultaneously, each step must be performed on both PCR microplates simultaneously using a separate magnetic plate for each PCR microplate.
a
b
c
Centrifuge the PCR microplate to 1,000 xg for 1 minute.
Place the PCR microplate on the magnetic plate for 1 minute, then remove the adhesive seal.
Remove the supernatant with a pipette set at 60 μl. Take care to not disturb the paramagnetic bead pellets.
CAUTION
The paramagnetic beads collect to alternate sides of the wells. When aspirating the supernatants, take care to not disturb the paramagnetic bead pellets by pointing the pipette tips away from the pellets.
NOTE
When processing two plates simultaneously, remove the supernatant from one plate and add AE1 to that same plate before removing the supernatant from and adding AE1 to the second plate. d
e
f
Vortex the AE1 reagent for 5 second or until no crystal structures are visible.
Add 40 μl AE1 to each well of the PCR microplate. AE1 should be aspirated from the top of the reagent reservoir and the tips should touch the side of the wells, above the liquid level line, when dispensing.
Seal the plate with a adhesive microplate sealing film, using an adhesive seal applicator to ensure the seal is secure. See Sealing Microplates on page 92 for proper seal application techniques.
CAUTION
The AE1 reagent contains formamide and must be properly disposed of in a hazardous waste container.
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Part # 15007510 Rev. C
h
i
Place the PCR microplate on the incubating/cooling shaker and press start to begin Pr2 program step 08 (1,400 rpm at 45°C for 1 minute).
Place the PCR microplate on the magnetic plate for 1 minute, then remove the adhesive seal.
Remove the supernatant with a pipette set at 60 μl. Take care to not disturb the paramagnetic bead pellets.
CAUTION
The paramagnetic beads collect to alternate sides of the wells. When aspirating the supernatants, take care to not disturb the paramagnetic bead pellets by pointing the pipette tips away from the pellets.
NOTE
When processing two plates simultaneously, remove the supernatant and complete the following stringent wash for one plate before doing the same to the second plate. 2
Repeat Stringent Wash.
a Add 40 μl AE1 to each well of the PCR microplate. AE1 should be aspirated from the top of the reagent reservoir and the tips should touch the side of the wells, above the liquid level line, when dispensing.
b Seal the plate with a adhesive microplate sealing film, using an adhesive seal applicator to ensure the seal is secure. See Sealing Microplates on page 92 for proper seal application techniques.
c Place the PCR microplate on the incubating/cooling shaker and press start to begin Pr2 program step 09 (1,400 rpm at 45°C for 1 minute).
d Place the PCR microplate on the magnetic plate for 1 minute, then remove the adhesive seal.
e Remove the supernatant with a pipette set at 60 μl. Take care to not disturb the paramagnetic bead pellets.
CAUTION
The paramagnetic beads collect to alternate sides of the wells. When aspirating the supernatants, take care to not disturb the paramagnetic bead pellets by pointing the pipette tips away from the pellets.
VeraCode ADME Core Panel Assay Guide
39
Add Extension and Ligation Mix (ELM2)
g
Lab Protocols
NOTE
When processing two plates simultaneously, place both plates on separate magnetic plates simultaneously, then remove the supernatant and complete the following mild wash for one plate before doing the same to the second plate.
3
Mild Wash.
a Vortex the UB3 reagent for 5 seconds.
b Add 40 μl UB3 to each well of the PCR microplate. UB3 should be aspirated from the top of the reagent reservoir and the tips should touch the side of the wells, above the liquid level line, when dispensing. c Seal the plate with a adhesive microplate sealing film, using an adhesive seal applicator to ensure the seal is secure. See Sealing Microplates on page 92 for proper seal application techniques.
d Place the PCR microplate on the incubating/cooling shaker and press start to begin Pr2 program step 10 (1,400 rpm at 45°C for 1 minute).
e Place the PCR microplate on the magnetic plate for 1 minute, then remove the adhesive seal.
NOTE
When processing two plates simultaneously, aspirate the UB3 from and add ELM 2, as follows, to one plate before doing the same to the second plate. 4
Add ELM2.
a Vortex the ELM2 reagent for 5 seconds.
b Remove the supernatant with a pipette set at 60 μl. Take care to not disturb the paramagnetic bead pellets.
CAUTION
The paramagnetic beads collect to alternate sides of the wells. When aspirating the supernatants, take care to not disturb the paramagnetic bead pellets by pointing the pipette tips away from the pellets.
c
40
Add 40 μl ELM2 to each well of the PCR microplate. ELM2 should be aspirated from the top of the reagent reservoir and the tips should touch the side of the wells, above the liquid level line, when dispensing. Part # 15007510 Rev. C
e
5
Seal the plate with a adhesive microplate sealing film, using an adhesive seal applicator to ensure the seal is secure. See Sealing Microplates on page 92 for proper seal application techniques.
Place the PCR microplate on the incubating/cooling shaker and press start to begin Pr2 program step 11 (1,400 rpm at 45°C for 20 minutes).
Proceed immediately to Add Make Amplification Mix (MAM1) on page 42.
NOTE
When processing two plates simultaneously, aspirate ELM 2 and add MAM1, as follows, to one plate before doing the same to the second plate. VeraCode ADME Core Panel Assay Guide
41
Add Extension and Ligation Mix (ELM2)
d
Lab Protocols
Add Make Amplification Mix (MAM1)
In this pre‐PCR process, the MAM1 reagent (containing nucleotides and fluorescent PCR primers) and Titanium Taq DNA Polymerase are added to the PCR microplate. The extended and ligated DNA product from the previous step is amplified by PCR in a thermal cycler.
Check to ensure that you have all of the consumables identified in this section before proceeding with the assay protocol.
Estimated Time
Hands‐on: ~5 minutes
Thermal Cycle: ~1.5 hours
Consumables
Item
Label Color Quantity
Storage
Supplied By
Titanium Taq DNA Polymerase
–
48 μl of 5 U/μl per plate ‐15° to ‐25°C
User
MAM1 reagent
Green
1 tube per plate
‐15° to ‐25°C
Illumina
PCR sealing film
–
1
Room temperature
User
Steps
1
Add Titanium Taq to MAM1.
a Add 48 μl of 5 U/μl Titanium TaqDNA Polymerase to the tube of MAM1.
WARNING
Titanium TaqDNA Polymerase is a critical component. Be sure you have added this enzyme to the MAM1 reagent.
42
Part # 15007510 Rev. C
b
Vortex the tube of MAM1containing Titanium Taq for 5 seconds.
WARNING
Vortexing for a full 5 seconds is essential for uniform distribution.
2
Add MAM1 containing Titanium Taq.
a Centrifuge the PCR microplate to 1,000 xg for 1 minute.
b Place the PCR microplate on the magnetic plate for 1 minute, then remove the adhesive seal.
c Remove the supernatant with a pipette set at 60 μl. Take care to not disturb the paramagnetic bead pellets.
CAUTION
The paramagnetic beads collect to alternate sides of the wells. When aspirating the supernatants, take care to not disturb the paramagnetic bead pellets by pointing the pipette tips away from the pellets.
NOTE
When processing two plates simultaneously, remove the supernatant from and add MAM1, as follows, to one plate before doing the same to the second plate. d
e
f
g
Add 40 μl MAM1 containing Titanium Taq to each well of the PCR microplate. MAM1 should be aspirated from the top of the reagent reservoir and the tips should touch the side of the wells, above the liquid level line, when dispensing. Do not pass the first stop on the pipette.
Seal the plate with the appropriate sealing film for your thermocycler.
Place the PCR microplate on the incubating/cooling shaker and press start to begin Pr2 program step 12 (1,400 rpm at 45°C for 2 minutes).
Transfer the PCR microplate to the post‐PCR laboratory.
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43
Add Make Amplification Mix (MAM1)
NOTE
MAM1 contains fluorescently‐labeled PCR primers. Make sure to limit its exposure to bleach fumes and light to avoid degradation of the Cy5 dye.
Lab Protocols
3
Cycle PCR Microplate.
NOTE
Ensure that the thermocycler is set for “calculated” as opposed to “block” temperature measurement.
a
Program your thermocycler as follows:
Table 10 MJ/BioRad Thermocyclers
Number Sample Additional of Volume Information
Samples
Thermocycler Model
Temperature Lid Temperature
Mode
Bio‐Rad DNA Engine Tetrad 2
Calculated
Heated
constant at 100°C
Polypropylene 96
plates and tubes
40 μl
MJ Research DNA Engine Tetrad
Calculated
Heated
Plate
96
40 μl
Bio‐Rad
MyCycler
Algorithmic
Heated
Plate
96
40 μl
44
Vessel Type
Set lid to turn off when temp. drops below 30°C;
When setting up cycling program, no options are selected (only temperature and time).
Part # 15007510 Rev. C
Thermocycler Model
Lid Temperature
Control
Vessel Type
Number Sample Additional of Volume Information
Samples
Eppendorf 105°C;
MasterCyclerPro TSP/ESP Heated Gradient
Lid (activated);
Switch lid off at low Eppendorf temperature MasterCyclerPro
(activated)
Simulated Tube;
Plate
Simulate Mastercycle gradient (activated)
96
40 μl
When setting up cycling program, no options are selected (only temperature and time).
(Ramp = 100% & no gradient)
Eppendorf MasterCycler
Tube
96
40 μl
Ramp = 3°/s +0.0°/s when setting up the cycling program steps
105°C;
WAIT (At the start);
AUTO (At the end)
VeraCode ADME Core Panel Assay Guide
Plate
45
Add Make Amplification Mix (MAM1)
Table 11 Eppendorf Thermocyclers
Lab Protocols
Temperature Time
95°C
3 minutes 95°C
20 seconds
56°C
10 seconds
72°C
20 seconds
72°C
10 minutes
4°C
5 minutes
10°C
Hold
CAUTION
Prolonged incubation at 4°C may damage your thermocycler.
b
4
Place the PCR microplate into the thermocycler and run the thermocycler program.
Do one of the following:
` Proceed immediately to Make Single Stranded DNA (MSS) on page 47.
` Optionally, store the ARX plate in the dark at 2° to 8°C overnight before proceeding to Make Single Stranded DNA (MSS).
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Part # 15007510 Rev. C
In this post‐PCR process, the MSS reagent is added to generate single‐stranded DNA for VeraCode Bead hybridization.
Continue to perform the assay in the post‐PCR laboratory. Check to ensure that you have all of the consumables identified in this section before proceeding with the assay protocol.
Estimated Time
Hands‐on: ~5 minutes
Consumables
Item
Label Color Quantity
Storage
Supplied By
2° to 8°C
Illumina
VeraCode Bead Plate –
1
MSS reagent
1 tube per plate ‐15° to ‐25°C
Lavender
Illumina
Preparation
` Thaw the MSS reagent on the benchtop.
` Vortex each reagent for 5 seconds immediately before using the reagent.
` Remove the PCR microplate from the thermocyler upon completion of cycling.
` If you have stored the PCR microplate overnight, remove the PCR microplate from `
the refrigerator and allow it to come to room temperature in the dark.
Set the post‐PCR incubating microplate shaker to 47°C.
Steps
1
Collect Paramagnetic Beads.
a Centrifuge the PCR microplate to 680 xg for 1 minute.
b Place the PCR microplate on the magnetic plate for 1 minute, then remove the PCR sealing film.
2
Prepare VeraCode Bead Plate
a Centrifuge the VeraCode Bead Plate to 680 xg for 1 minute.
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47
Make Single Stranded DNA (MSS)
Make Single Stranded DNA (MSS)
Lab Protocols
b
c
Slowly and carefully remove the VeraCode Bead Plate cap mat. Do not discard the cap mat.
Add 40 μl MSS to each well of the VeraCode Bead Plate.
CAUTION
The MSS reagent contains dimethyl sulfoxide (DMSO) and must be properly disposed of in a hazardous waste container.
CAUTION
Keep pipette tips well above the VeraCode bead pellet to avoid dislodging and losing beads.
3
Transfer PCR Products.
Set the pipette to 45 μl and transfer all contents from each well of the PCR microplate to the corresponding well of the VeraCode Bead Plate.
Change the tips after each column.
NOTE
Take care to not disturb the paramagnetic beads in the PCR plate when transferring the PCR product to the VeraCode Bead Plate.
CAUTION
Keep pipette tips well above the VeraCode bead pellet to avoid dislodging and losing beads.
48
4
Seal the VeraCode Bead Plate.
Seal the VeraCode Bead Plate with the cap mat that the plate was supplied with from step 2b. Use a Corning seal press to ensure the plate is completely sealed.
5
Proceed immediately to Hybridize VeraCode Bead Plate on page 49.
Part # 15007510 Rev. C
In this post‐PCR process, a shaking incubator is used to hybridize the PCR products to the VeraCode Bead Plate. Once the PCR products are transferred to the VeraCode Bead Plate, they are ready for hybridization. After hybridization, they are washed with VW2, and the resulting VeraCode Bead Plate is ready for scanning in the BeadXpress Reader. Check to ensure that you have all of the consumables identified in this section before proceeding with the assay protocol.
Estimated Time
Hands‐on: ~5 minutes
Overall: ~2.5 hours
Consumables
Item
Label Color
Quantity
Storage
VW2 buffer Sea foam green 45 ml per plate Room temperature
Supplied By
Illumina
Steps
1
Hybridize VeraCode Bead Plate.
a Vortex the VeraCode Bead Plate on the shaking incubator at 47°C at 1,200 rpm. Set the shaking incubator to “Hold” and use a timer to track the incubation time for 2.5 hours.
NOTE
Do not exceed the 2.5 hour incubation time. When processing two plates simultaneously, following hybridization, promptly wash the VeraCode Beads, as follows, in both plates simultaneously.
NOTE
Start the BeadXpress Reader so it can initialize while you hybridize the VeraCode Bead Plate (reference Prepare BeadXpress Reader on page 26)
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49
Hybridize VeraCode Bead Plate
Hybridize VeraCode Bead Plate
Lab Protocols
2
Centrifuge the VeraCode Bead Plate to 680 xg for 1 minute.
3
Pour 45 ml of VW2 into a non‐sterile, disposable reservoir.
4
Wash VeraCode Beads.
a Remove the VeraCode Bead Plate seal.
b Add 200 μl VW2 to each well of the VeraCode Bead Plate, then gently swirl the VeraCode Bead Plate on the benchtop 2–3 times.
CAUTION
Keep pipette tips well above the VeraCode bead pellet to avoid dislodging and losing beads.
c
d
5
Wait 1 minute for the VeraCode beads to settle.
Remove the supernatant with the aspiration manifold under 40–60 mbar vacuum. Take care to not disturb the VeraCode bead pellets. Make sure the liquid is the same level in all wells.
Repeat VeraCode Bead Wash.
a Add 200 μl VW2 to each well of the VeraCode Bead Plate, then gently swirl the VeraCode Bead Plate on the benchtop 2–3 times.
CAUTION
Keep pipette tips well above the VeraCode bead pellet to avoid dislodging and losing beads.
b
c
6
Wait 1 minute for the VeraCode beads to settle.
Remove the supernatant with the aspiration manifold under 40–60 mbar vacuum. Take care to not disturb the VeraCode bead pellets. Make sure the liquid is the same level in all wells.
Do one of the following:
` Proceed immediately to Prepare BeadXpress Reader on page 51.
` Optionally, seal and store the washed VeraCode beads in the dark at room temperature for up to three days before scanning them in the BeadXpress Reader.
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Part # 15007510 Rev. C
In this post‐PCR process, the BeadXpress Reader is prepared for operation and the BeadXpress Reader and the VeraScan software are initialized in preparation for scanning the VeraCode Bead Plate.
Estimated Time
Hands‐on: ~5 minutes
Overall: ~35 minutes
Preparation
` The operator of the BeadXpress Reader should be trained by qualified personnel on `
the correct operation of the instrument, and be aware of the safety issues involved. Reference the Specifications and Cautions and Warnings sections of the BeadXpress Reader System Manual.
Ensure that the VeraScan data output location has been properly configured. Consult your System Administrator.
Steps
Powering Up the BeadXpress Reader
1
After the BeadXpress Reader has been off for at least two minutes, press the power switch on the back panel of the instrument.
CAUTION
After powering up the instrument, the lasers must stabilize for 15 minutes before the VeraScan software responds to commands. An error message appears in the VeraScan software if you attempt to initialize the scanner before the lasers have stabilized.
Power Up the BeadXpress Reader Computer
1
Press Power on the computer.
2
Log in to Windows.
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Prepare BeadXpress Reader
Prepare BeadXpress Reader
Lab Protocols
Starting the VeraScan Software
1
Wait until the Power and Ready lights on the front panel of the BeadXpress Reader are the only lights on.
If you do not wait for this condition, an error message may appear when you start the software.
2
Do one of the following:
• From the Windows Start menu, select All Programs | Illumina | VeraScan.
• Double‐click the VeraScan icon on the desktop.
The VeraScan application opens on the computer desktop.
Figure 16 VeraScan Welcome Screen
NOTE
The VeraScan Software checks whether preventive maintenance has been performed. The Maintenance indicator light on the VeraScan Welcome Screen is green when all maintenance is current. For information about maintaining your BeadXpress Reader reference the section on Maintenance in the BeadXpress Reader System Manual.
3
52
Enter your username and password, then click Login.
Part # 15007510 Rev. C
Connecting the Reader
` Click the Menu button `
in the upper‐left corner of the screen and select Reader l
Connect.
This action connects the BeadXpress Reader to the VeraScan software.
Click the Menu button in the upper‐left corner of the screen and select Reader l
Initialize System.
NOTE
VeraScan can be configured to automatically connect to and initialize the BeadXpress Reader. Consult your system administrator.
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53
Prepare BeadXpress Reader
NOTE
VeraScan user accounts, account options and alerts must be set up by your system administrator. Reference the BeadXpress Reader System Manual and Appendix B, VeraScan Administration and VeraReport.
Lab Protocols
Scan VeraCode Bead Plate
In this post‐PCR process, the BeadXpress Reader uses lasers to excite the fluors of the PCR products bound to the VeraCode beads. Light emissions from these fluors are then recorded in a data file. Fluorescence data are analyzed to derive genotyping results using Illumina’s VeraScan software.
Estimated Time
Hands‐on: ~5 minutes
Overall: ~1.25 hours
Preparation
` If the VeraCode Bead Plate was sealed and stored, remove the seal before scanning.
Steps
Set Up the Scan
1
Load the VeraCode Bead Plate.
a Click Next in the VeraScan window.
Figure 17 Successful VeraScan Login
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Part # 15007510 Rev. C
Figure 18 VeraScan Setup Screen
b
c
On the Select Application tab, click Open Tray. A message with a diagram appears displaying the correct plate orientation.
Place a VeraCode Bead Plate in the correct orientation so that well A1 is located in the corner indicated by the A1 orientation mark stamped into the BeadXpress Reader plate tray.
Figure 19 A1 Orientation Mark
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55
Scan VeraCode Bead Plate
The VeraScan Setup screen displays the Select Application tab.
Lab Protocols
d
e
Hold the plate by the outer edges of the longer side and place it on the adaptor tray.
Close the tray to insert the VeraCode Bead Plate into the BeadXpress Reader by clicking Close Tray on the Select Application tab.
WARNING
Be careful when closing the adaptor tray. Move your hand away from the drawer before closing.
2
Select Scan Properties.
a Select the ADME Core kit from the Select Application tab. The scan input data fields are displayed.
Figure 20 VeraScan Select Application Tab
b
Enter the Plate ID or select from the drop down menu of recently run plates. The plate ID is used to name the data files and the data folders.
NOTE
Do not include the underscore character “_” or comma “,” in the Plate ID name.
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Part # 15007510 Rev. C
NOTE
There is no maximum number of characters for the plate ID. However, the plate ID is used in the Windows path where the output files are saved and that entire path must be shorter than 260 characters.
c
Select the Sample Sheet from the dropdown menu or browse for the file by clicking ‘...’ to the right of the text box.
NOTE
The plate ID must match the name of the plate in the sample sheet.
d
The default Analysis Configuration is set to Analyze Everything or, if available, select from the drop down menu of analysis configurations. (Consult your System Administrator to set up configuration templates.)
NOTE
The Configure button is only available for system administrators.
3
At any time, click Previous from the Setup screen to view the Welcome screen.
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57
Scan VeraCode Bead Plate
NOTE
A plate barcode cannot be used as the plate ID more than once.
Lab Protocols
4
To view scan setting details:
a In the Select Application tab, click Details or select the Scan Settings Detail tab.
The wells highlighted in orange indicate the well columns to be scanned.
Figure 21 Scan Settings Detail Tab
b
c
58
Highlight the well column you want to review by clicking it. Click the plus (+) to expand cells and view column and well details or click the minus (‐) to close the view of cell details.
Part # 15007510 Rev. C
d
e
f
g
5
Click Unselect to clear the view of the data for the selected well.
Click Select to return to the Select Application tab.
Check Additional Details to display format, plate type and plate attributes.
Click Scan to continue to the Scan screen.
Click Next from the Setup screen to continue to the Scan screen.
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59
Scan VeraCode Bead Plate
Figure 22 Expanded Scan Settings Detail Tabs
Lab Protocols
Start and Control the Scan
1
Click Scan from the Scan screen to begin the scan.
Figure 23 Scan Screen
2
`
`
`
`
While a scan is in process, you can:
Monitor scan progress
Pause the scan
Abort the scan
Resume the scan
The information bar located along the bottom of the main application window includes tools for monitoring scans. Click Status, Events or Log to view details.
Monitor the Scan Progress
The VeraScan application checks available hard drive space to ensure that sufficient space is available to record the selected sections. If sufficient disk space is not available, an error message is displayed, and the arrays are not scanned. Otherwise, the BeadXpress Reader begins scanning. For each sample, the green intensity data is populated in the table first and then the red intensity data. 60
Part # 15007510 Rev. C
Figure 24 Scan Screen Progress
NOTE
Serious errors halt the scan process. Errors that may be recoverable, such as network or other hardware errors, cause the scan to pause until you click OK in the error message box.
View the Scan Status Information
The Status bar displays current status information as the scan progresses, including status messages and the ID of the current plate being scanned. Reference the BeadXpress Reader System Manual for details on status information.
Pause, Abort, or Resume a Scan
If for any reason a scan is aborted, the VeraScan software allows you to recover the data for the scanned columns and continue scanning the remaining columns.
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61
Scan VeraCode Bead Plate
The progress through the selected columns, and the status of each section, is displayed in the Current Column Progress and Scan Status areas. The column in the plate being scanned is indicated by flashing light‐green wells in the Plate Scan Progress area. Solid dark‐green wells indicate columns that have already been scanned. A status bar representing the progress of the current scan is displayed below the plate.
Lab Protocols
During a scan, the data table is dynamically populated with intensity data. If the data appear faulty (for example, if the intensities are much too low), you may wish to interrupt the scan to consult your supervisor.
` Pause: When clicked, the scan remains suspended until you click Resume Scan or Abort.
` Abort: Click to cancel the scan.
` Resume Scan: Click to resume the scan after pausing.
Conclude the Scan
To conclude the scanning process do one of the following:
` After a successfully completed scan, click Next to proceed to the Analysis screen and Analyze Scan Data on page 63.
` If a scan was aborted or otherwise interrupted, click Finish to return to the Welcome screen to start over.
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Part # 15007510 Rev. C
The VeraCode ADME Core Panel assay is processed by VeraScan, the ADME Core module, and the ADME Core Panel kit manifest. VeraScan is the software that runs the BeadXpress reader acquiring and processing green and red fluorescence signals from VeraCode beads. The ADME Core module interprets green and red fluorescence signals from VeraScan. The ADME Core module analyzes data on a per well and per bead type basis using the ADME Core Panel kit manifest which contains genotyping parameters.
Table 12 Bead Type Outcomes
Assay ID
Valid Outcomes
ADME variant
Genotype call and Low Signal
translated allele call No Call
Low Beads
Copy number call
ADME copy number variant
Invalid Outcomes
Control
• Sample Tracking Control
• Process Control
Sample specific barcode displayed in Controls column
Failed STC
No results provided
Failed PSC
No results provided
• Subpool Position Control
Results provided No results provided Message: Assay pooling control failed. Analysis will be aborted.
• Hybridization Control 1
Pass
Failed Hyb
Results highlighted in red require review to determine acceptability
• Hybridization Control 2
• Mismatch Control 1
• Mismatch Control 2
VeraCode ADME Core Panel Assay Guide
Failed Mismatch
Results highlighted in red require review to determine acceptability
63
Analyze Scan Data
Analyze Scan Data
Lab Protocols
Interpret Sample Results
The ADME Core module reports the outcome for each of the bead types on a per well basis.
` A well is considered valid (the genotypes for ADME Core Panel may be reported) if all assay controls (i.e., STC, SPC, and PSC) have valid outcomes.
` A well is considered invalid (the genotypes for ADME Core Panel should not be reported) if any control bead type has an invalid outcome.
No Template Controls (NTCs) should generate the following outcomes:
Table 13 No Template Control Valid Outcomes
Valid Outcomes
Assay ID
ADME variant
ADME copy number variant
Control
Low Signal
or
NA
Pass
NOTE
No Template Control is not included in call rate calculations and is not plotted in SNP graphs on the Details tab
No Template Controls may generate some, but usually insubstantial signals in a low number of assays due to the complexity of the content. Ideally, less than 10% of the loci should generate signals above the background in NTCs. If any NTC has more than 24 assays with a genotypable signal, the following possibilities should be considered:
` Cross‐well contamination may have occurred during the processing of the plate
` Lab environment may be contaminated with amplicon (See “Best Practices” on page 86.)
` BeadXpress Reader may need routine cleaning
It is important to assess the quality of each run to determine the validity of the outcomes generated from the ADME Core Panel. The following flowchart can be used to help assess the data quality for each plate run. For further assistance, see Assay Protocol Troubleshooting on page 122.
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Part # 15007510 Rev. C
Analyze Scan Data
Figure 25 Data Assessment Workflow
Display Results
To display the results for analysis:
1
From the Scan screen click Next.
The Analysis screen appears and is populated with a Summary Report of the just‐
completed scan.
NOTE
When specific assay controls fail, the Analysis screen does not appear and the analysis is aborted. See Table 12 on page 63 for invalid outcomes for controls.
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65
Lab Protocols
Figure 26 Plate Analysis Summary Report
Table 14 Plate Analysis Summary
2
66
Column Header
Description
Sample ID
The sample ID from the sample sheet
Well
The well number row and column (e.g. A1)
Call Rate
The sample call rate
Controls
Control result for the sample:
Passed samples ‐ displays STC barcode
Failed samples ‐ displays failure mode: Failed PSC, Failed STC, Failed Mismatch, or Failed Hyb
Gene Result
(one column per Gene Name)
All distinct variants detected for the given gene. If present, no Call results are appended to the end of the string.
*1A/*1A ‐ genes with star nomenclature (no variants were detected)
NMD ‐ genes without star nomenclature
RAL ‐ rare allele likely
At any time click Previous to view the previous screen.
Part # 15007510 Rev. C
The Summary Report table allows both row and column selection. 1
To view the details of a single sample, select the sample row or a table cell from within the sample row in the Summary Report. The selected row is highlighted in grey and the selected cell is highlighted orange. The report details of the selected sample are displayed in the lower portion of the screen
Figure 27 Plate Summary Report with Sample Selected
Table 15 Plate Sample Details Column Header
Description
Gene Name
Gene name
Variant Name
Variant name
Nucleotide Change
Nucleotide change
Effect
Amino acid change or functional change
RS ID
RefSeq ID of locus
Alleles (WT/Var)
Possible alleles
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67
Analyze Scan Data
Summary Report
Lab Protocols
Table 15 Plate Sample Details (Continued)
68
Column Header
Description
Variant Call
Translated variant call result
GT Call
Genotype call result
Beads
Bead population for a locus in the well where the sample is assayed
Green Signal
Raw green signal intensity for negative control samples. Normalized green signal intensity for positive control samples
Red Signal
Raw red signal intensity for negative control samples. Normalized red signal intensity for positive control samples
Theta
Red fluorescence intensity to green fluorescence intensity ratio. The value should be close to 0 or 1 for homozygous genotypes.
Part # 15007510 Rev. C
To jump to the sample details for a specific gene in the selected sample do one of the following:
• Select the cell from the column that displays the gene details
• Enter a gene name in the Jump to Gene field
The corresponding rows for the selected gene are highlighted in orange in the sample detail table in the bottom panel.
Figure 28 Plate Summary Report with Gene Selected
•
The sample detail table allows row selection but not column selection. When a row is selected, the corresponding cell in the Summary Report and sample detail tables is selected. VeraCode ADME Core Panel Assay Guide
69
Analyze Scan Data
2
Lab Protocols
Detail Report
1
To view plate details, select Detail from the display options in the top portion of the Analysis screen. The Detail Report is displayed on the Analysis screen. The detail table allows both row and column selection.
Figure 29 Plate Detail Report
Table 16 Plate Detail
70
Column Header
Description
Sample ID
The sample ID from the sample sheet
Well
The well number row and column (e.g. A1)
Call Rate
Sample call rate
Controls
Control result for the sample:
Passed samples ‐ displays STC barcode
Failed samples ‐ displays failure mode: Failed PSC, Failed STC, Failed Mismatch, or Failed Hyb
GT Call
(one column per Gene Name)
Genotype call result
Part # 15007510 Rev. C
Analyze Scan Data
Table 17 Gene Detail
Row Header
Description
Gene Name
Gene name
Variant Name
Variant name
Alleles (WT/Var)
Possible alleles
Locus Desc.
Nucleotide change or amino acid change, depending upon the locus
Locus Call Freq.
The locus call rate
Select a sample row from the detail table to view the corresponding sample table in the bottom panel. When a cell inside a selected row is selected, the corresponding rows in the sample table is selected.
Figure 30 Plate Detail Report with Sample Selected
VeraCode ADME Core Panel Assay Guide
71
Lab Protocols
2
To view the details of a genotype, select the genotype column from the Detail Report. The results are displayed in the lower portion of the screen. • For non CNV loci, two plots are displayed. The left plot shows Cartesian coordinates, while the right plot shows polar coordinates. Cartesian plot – displays each data point by its normalized green (x‐axis) and red (y‐axis) fluorescence intensities. Polar plot – converts the green and red intensities to radius (the sum of green and red) and theta (red fluorescence intensity to green fluoresceins intensity ratio).
• For CNV aggregate results, only the polar plot is displayed.
• Call zones for heterozygote and homozygote results are colored red, green, and blue. A gray zone represents a call zone for a rare allele, where the call zone has been estimated due to an insufficient number of data points to set empirically because of locus rarity.
• The genotype plot allows point selection. When a point is selected, the corresponding cell in the detail table is selected.
Figure 31 Plate Detail with Gene Selected
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Part # 15007510 Rev. C
When a cell inside a selected column is selected, the corresponding point in the plot is highlighted in orange.
Figure 32 Plate Detail with Sample and Gene Selected
The following defines the plot tools:
Table 18 Plot tools Tool Function
Tool Function
Make dots larger
Lasso mode
Make dots smaller
Zoom mode
Copy plot to clipboard
Auto‐Scale X‐Axis
Default mode
Auto‐Scale Y‐Axis
Pan mode
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73
Analyze Scan Data
3
Lab Protocols
Controls Report
1
To view plate controls, select Controls from the display options in the top portion of the Analysis screen. The Controls Report is displayed on the Analysis screen. Figure 33 Plate Controls
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Part # 15007510 Rev. C
Analyze Scan Data
The following plate control displays are shown:
Mismatch Controls and Hybridization Controls ‐ Displays genotype plots
•
•
Green—Homozygous for green allele
Red—Homozygous for red allele
PSCControls ‐ Displays well intensity and PSC threshold for each pool
•
•
•
•
Columns display intensity value of each PSC per well
Place cursor over column to view numeric value
Horizontal lines indicated PSC failure threshold
Column color darkens if PSC intensity exceeds threshold
Figure 34 Plate Detail with Sample and Gene Selected
A
B
Example: Good PSCab control for well C1
Example: Failed PSCcb control for well C1
2
Manipulate the Controls display data as follows:
• Zoom in or out of the display by scrolling the mouse wheel.
• Pan the display by pressing Shift and moving the mouse.
3
At any time click Previous to view the previous screen.
Conclude Analysis
To conclude the plate analysis, perform one of the following:
` Proceed to Report Genotypes on page 76.
` Proceed to Exit Results on page 83.
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75
Lab Protocols
Report Genotypes
Reports in .csv Format
Scan results of the individual sample and summary data are automatically saved after each scan in .csv format which can be used in other commercial applications, such as Microsoft Excel. If not modified by the System Administrator, the default file settings are:
` Location—C:\AllUsers\Illumina\VeraScan\Output\Research ` Filename—Year‐month‐plateID
Reports in PDF Format
Scan results can also be saved PDF format. To save and view a PDF report:
1
Click Report from the Analysis screen.
The Report Configuration window is displayed
Figure 35 Report Configuration
To exclude results from the default Report Configuration setting, uncheck the results to be excluded from the report.
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Part # 15007510 Rev. C
Click Report from the Report Configuration screen.
The VeraScan Report window is displayed with the summary of the report results specified in the Setup screen. The contents of the summary are described in Table 14 on page 66.
Figure 36 Report Summary
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77
Report Genotypes
2
Lab Protocols
Scroll down to view the detailed report results for each sample. The sample details are described in Table 15 on page 67 and Table 16 on page 70.
Figure 37 Report Detail
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Part # 15007510 Rev. C
Report Genotypes
Scroll down to view the table of results for each sample. The plate details are described in Table 15 on page 67.
Figure 38 Report on Sample
3
The report can be exported via the VeraScan Report window using one or more of the following options:
• Export the report to a new format by selecting File | Export Document.
• Print the report by selecting File | Print.
• Send the report by selecting File | Send via E‐mail.
Export Data
Scan results of the individual sample and summary data are automatically saved after each scan in .csv format as described in Reports in .csv Format on page 76. Scan results in .csv format can also be exported to a location other than the default.
1
Click Export from the Analysis screen.
2
Name and save the file in the desired location using the file selection window.
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Lab Protocols
The scan results are separated into Plate, Results, and Controls sections, as can be seen in the figure below. The plate data is described in Table 19, the results data is described in Table 20, and the controls data is described in Table 21.
Figure 39 Exported File
Table 19 Plate Section Plate Field
Description
Plate ID
The plate ID
Kit
The kit name and version number
Report TIme
The date and time the report was generated
Run TIme
The date and time the report was run
Technician
The name of the user
Reviewer
This field is empty
Data Generated
The name of the BeadXpress Reader and the VeraScan software version
Report Generated
The version of the ADME module
Analysis Configuration The name of the analysis configuration used
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Plate Field
Description
Pooling Controls
Pass indicates the SPC controls passed
Notes
This field is empty
Report Genotypes
Table 19 Plate Section (Continued)
Table 20 Results Section Column Header
Description
Sample ID
The sample ID from the sample sheet
Well
The well number row and column (e.g. A1)
Kit
The kit name
Sample Control
The control column value from the sample sheet (Positive, Negative, Synthetic, or empty)
Comments
Comments from the sample sheet
Control Results
Control result for the sample:
Passed samples ‐ displays STC barcode
Failed samples ‐ displays failure mode: Failed PSC, Failed STC, Failed Mismatch, or Failed Hyb
Sample Call Rate
Sample call rate
Gene Name
Gene name
Variant Name
Variant name
Nucleotide Change
Nucleotide change
Amino Acid Change
Amino acid change or functional change
RS ID
RefSeq ID of locus
Alleles (WT/Var)
Possible alleles
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Lab Protocols
Table 20 Results Section (Continued)
Column Header
Description
Gene Result
A semi colon separated text string of all distinct variants detected for a given gene. The detected variant value is taken from comments section of the translation file. No Call results are appended to the end of the string.
*1A/*1A ‐ no variants were detected (genes with star nomenclature)
NMD ‐ genes without star nomenclature
Variant Call
Variant call result
GT Call
Genotype call result
Beads
Bead population for a locus in the well where the sample is assayed
Green Signal
Raw green signal intensity for negative control samples
Normalized green signal intensity for positive control samples
Red Signal
Raw red signal intensity for negative control samples
Normalized red signal intensity for positive control samples
Theta
Red fluorescence intensity to green fluorescence intensity ratio
R
Combined intensity
Scale
Raw ‐ negative control sample
Normalized ‐ positive control sample
Table 21 Controls Section 82
Plate Field
Description
Sample ID
The sample ID from the sample sheet
Well
The well number row and column (e.g., A1)
Control Name
The control name indicating the analysis method and probe ID (e.g., STC (A512))
PSC for process control
Result
Pass or Fail for PSC and Functional and Hyb controls
Generic AA, AB, BB result for STC controls
Part # 15007510 Rev. C
Plate Field
Description
Beads
Bead population (empty for PSC)
Green Signal
Raw green intensity for Functional and Hyb
Normalized green intensity for STC
Empty for PSC
Red Signal
Raw red intensity for Functional and Hyb
Normalized red intensity for STC
Empty for PSC
Theta
Red fluorescence intensity to green fluorescence intensity ratio (empty for PSC)
R
Combined intensity
Scale
Raw ‐ Functional and Hyb
Normalized ‐ STC
Empty ‐ PSC
Exit Results
To conclude the scan and analysis session:
1
Click Finished on the Analysis screen and the Welcome screen is displayed.
2
Click Open Tray to open the BeadXpress Reader plate tray.
3
Take the VeraCode Bead Plate out of the BeadXpress Reader, discard it and perform one of the following:
• Scan a subsequent VeraCode Bead Plate, by inserting the plate into the BeadXpress Reader and repeating the Scan VeraCode Bead Plate, Analyze Scan Data and Report Genotypes procedures.
• Close the empty BeadXpress Reader plate tray by clicking Close Tray and log out of the VeraScan software and the BeadXpress Reader computer. If the BeadXpress Reader will no longer be used today, proceed to Shutting Down the BeadXpress Reader System on page 84.
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Report Genotypes
Table 21 Controls Section (Continued)
Lab Protocols
Shutting Down the BeadXpress Reader System
To shut down the BeadXpress Reader system:
1
Do one of the following, depending on how long the Reader will be idle:
• If the BeadXpress Reader will be idle for less than 24 hours, proceed to 2. You do not need to do anything with the fluidics system.
• If the BeadXpress Reader will be idle for more than 24 hours but less than two weeks, click the Menu button in the upper‐left corner of the screen and select Reader | Purge Fluidics.
• If the BeadXpress Reader will be idle for more than two weeks, click the Menu button in the upper‐left corner of the screen and select Reader l Shut Down
Fluidics.
2
Close the VeraScan software by doing one of the following:
in the upper‐left corner of the screen and select Exit.
• Click the Menu button • Click the Close button in the VeraScan title bar.
3
Shut down the computer.
4
Turn off the BeadXpress Reader by pressing the power switch on the back panel of the instrument.
NOTE
Leave the power off for at least two minutes before restarting the BeadXpress Reader.
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Appendix A
Standard Operating
Procedures
Best Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Preventing PCR Product Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Pipetting and Sealing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
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Standard Operating Procedures
Best Practices
While performing the ADME Core Panel assay, you should always adhere to good molecular biology practices.
` When running two plates simultaneously, follow these best practices and alert your field application specialist (FAS):
• It is important to plan ahead for optimal assay performance and optimal data quality
• Perform washes and pipetting steps straight through from one plate to the next, to ensure the best timing.
• Ensure that you have two heat blocks
• Two magnets are required during pre‐PCR. One is sufficient for post‐PCR, but two are recommended.
` Do not use electronic or repeat pipettes. A manual pipette provides added control and leads to optimal assay results.
` Have tip boxes readily available.
` Optimal assay results are obtained when samples fall within the concentration and volume guidelines recommended in the protocol. It is important to ensure that equal volumes and concentration of DNA are applied to each reaction pool.
` When performing the denaturation procedures, be sure to stay within the times recommended. Prolonged exposure to NaOH can impact assay performance. To prevent prolonged exposure to NaOH prepare MTR and AOP mixtures in the pipette trough prior to applying NaOH to the reaction plate, to allow you to proceed with the MTR and AOP steps promptly at the conclusion of denaturation.
` When incubating the reaction plate on the heat block of the incubating shaker, promptly remove the reaction plate from the heat source at the conclusion of the step. Prolonged exposure to heat can impact assay performance.
` When performing the hybridization procedures, wash the plate promptly at the end of the 2.5 hour hybridization. A prolonged hybridization can impact assay performance.
` Take advantage of wait time during incubation steps to prepare reagents for the next step in the protocol.
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The PCR (polymerase chain reaction) process is commonly used in the laboratory to amplify specific DNA sequences. Unless you exercise sufficient caution, PCR products may contaminate reagents, instrumentation, and samples, causing inaccurate and unreliable results.
PCR product contamination can shut down lab processes and significantly delay normal operations. The following sections outline practices that help reduce the risk of PCR product contamination.
Physical Separation of Pre- and Post- PCR Areas
The laboratory space where pre‐PCR processes (DNA extraction, quantification, and normalization) are performed should be physically separate from the laboratory space where PCR products are made and processed (post‐PCR processes).
Ideally, pre‐PCR processes should be performed in a separate, dedicated laboratory space. For example:
` Never use the same sink to wash pre‐ and post‐PCR reservoirs.
` Never share the same water purification system for pre‐ and post‐PCR processes.
` Store all supplies used in the protocols in the pre‐PCR area, and transfer to the post‐
PCR area as needed
Separate full sets of instruments (pipettes, centrifuges, incubating shakers, etc.) should be dedicated to pre‐ and post‐PCR lab processes, and must never be shared between processes.
Daily and Weekly Bleaching
Use the following guidelines for daily and weekly bleaching of the pre‐ and post‐PCR areas. Provide training for personnel responsible for cleaning the lab areas so that they know how to prevent pre‐ and post‐PCR product contamination.
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Preventing PCR Product Contamination
Preventing PCR Product Contamination
Standard Operating Procedures
Post-PCR Area
Reducing the amount of product in the post‐PCR area reduces the risk of contamination in the pre‐PCR area.
CAUTION
You must establish procedures for preventing PCR product contamination before you begin work in the lab.
CAUTION
To prevent sample or reagent degradation, ensure that all bleach vapors that remain after cleaning have fully dissipated before starting any processes.
Identify post‐PCR area “hot spots” that pose the highest risk of contamination and clean these items daily with a 10% bleach solution.
Typical hot spots include:
` Bench space used to process amplified DNA
` Door handles
` Refrigerator/freezer door handles
` Computer mouse
` Keyboards
` Centrifuges
` Vortexers
` Thermal cyclers
Once a week, thoroughly bleach the entire post‐PCR area, including bench tops and instruments that are not cleaned daily. Mop the floors with a 0.5% sodium hypochlorite (10% bleach) solution as well.
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Establish a daily and weekly bleaching schedule for the pre‐PCR area similar to the one in post‐PCR. This helps to eliminate product that may have entered the pre‐PCR area. Identify high‐risk pre‐PCR items such as the ones listed below, and clean them with a 0.5% sodium hypochlorite (10% bleach) solution each morning before beginning any pre‐
PCR processes:
` Bench tops
` Door handles
` Refrigerator/freezer door handles
` Computer mouse
` Keyboards
Once a week, thoroughly clean all laboratory surfaces and instruments, including bench tops and instruments that are not cleaned daily. Mop the floors with a 0.5% sodium hypochlorite (10% bleach) solution as well.
Items Falling to the Floor
The floor is contaminated with PCR product transferred on the shoes of individuals coming from the post‐PCR area; therefore, anything that has fallen to the floor should be treated as contaminated. Throw away any disposable items that fall to the floor, such as empty tubes, pipette tips, gloves, lab coat hangers, etc. Individuals handling anything that has fallen to the floor, disposable or not, must throw away their lab gloves and put on a new pair.
Non‐disposable items that fall to the floor (such as a pipette, an important sample container, etc.) should be immediately and thoroughly cleaned with a 10% bleach solution to remove PCR product contamination.
NOTE
Be sure to clean any lab surface with which a contaminated item has come into contact.
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Preventing PCR Product Contamination
Pre-PCR Area
Standard Operating Procedures
Pipetting and Sealing
This section outlines recommended reagent pipetting and adhesive seal application techniques for the VeraCode ADME Core Panel Assay. Avoid using electronic or repeat pipettes. Specific pipetting techniques are known to improve the ability to process samples successfully. Some reagents can affect the adhesive microplate sealing filmʹs ability to stay adhered during incubating shaking steps if they are exposed to the top surface of the PCR microplate. Pipetting techniques can also cause bubbles that can impact reagent performance or interfere with the adhesive sealʹs ability to stay adhered to the microplate. Proper technique to apply adhesive seals is especially important to ensure the seal stays adhered during the incubating shaking steps to avoid cross contamination or evaporation of samples.
Aspirating from Reservoirs
When aspirating reagents from their respective reservoirs, it is important to not submerge the pipette tips to the bottom of the reagent reservoir. Dipping pipette tips into the bottom of the reservoir wets the pipette tips and can transfer excess reagents onto the top of the PCR microplate during dispensing, interfering with the adhesive sealʹs ability to adhere to the microplate.
If liquid is noticed on the top surface of the plate during processing, the top of the plate should be blotted dry with a Kimwipe to ensure the best possible adhesion between the top surface of the microplate and the seal. However, the composition of some reagents may interfere with the ability of the microplate seal to adhere to the top surface, even if the top of the plate is blotted dry with a Kimwipe prior to placing the seal.
The best technique is to pull ADME Core Panel reagents by placing the pipette tips just under the top surface of the reagent prior to aspiration. This technique helps draw the correct volume of reagent from the reservoir, with less liquid sticking to the exterior of the pipette tips.
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A
B
Pipetting and Sealing
Figure 40 Pipette Tip Placement
Incorrect tip placement
Correct tip placement
Dispensing Reagents
It is important to not introduce bubbles into the assay wells when pipetting the ADME Core Panelreagents. Illumina recommends that the pipette stopper is only moved to the first dispense stop and not to the hard stop. This empties all reagent out of the tips without introducing bubbles in the sample wells. This technique should be employed if excessive foaming is found when pipetting all ADME Core Panelreagents, but is especially important for the following:
` NaOH
` MTR4A, MTR4B, MTR4C
` AB1
` AOP4A, AOP4B, AOP4C
` ELM2
` MAM1
Figure 41 Pipette Stopper
A
B
Incorrect ‐ pipette hard stop
Correct ‐ pipette first stop
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Standard Operating Procedures
Sealing Microplates
When applying adhesive seals to the PCR microplate, it is important that the right technique be used to ensure a good seal. This allows the wells to stay separated during the incubating shaking steps and avoids sample cross contamination. Illumina recommends that you have an adhesive seal applicator to apply force to the seal (see User‐Supplied Materials on page 12). Proper PCR microplate sealing procedures are as follows:
1
Peel the backing from the adhesive seal and place the adhesive seal, sticky side down, gently onto the PCR microplate surface.
Figure 42 Adhesive Seal on Microplate Surface
2
Hold the edge of the microplate with one hand and the adhesive seal applicator at an angle with the other hand. Press down with the edge of the adhesive seal applicator and swipe across the top of the PCR microplate, lengthwise, 2–3 times.
Figure 43 Swipe Adhesive Seal Applicator Over Length of Microplate
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Press down with the edge of the adhesive seal applicator and swipe across the top of the PCR microplate, width wise, 2–3 times.
Figure 44 Swipe Adhesive Seal Applicator Over Width of Microplate
4
Press the edge of the adhesive seal applicator along each edge of the microplate to seal the edges in the order shown below.
Figure 45 Run Adhesive Seal Applicator Along Microplate Edge
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Pipetting and Sealing
3
Standard Operating Procedures
NOTE
Do not run the adhesive seal applicator between each row and column to separate each well. This reduces the seal around the well and can create small pockets where reagents can splash onto the plate surface during vortexing.
5
The microplate is now properly sealed.
Figure 46 Properly Sealed Microplate
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Set Up VeraScan ADME Analysis Configuration . . . . . . . . . . . . . . . . . . . . . . . . 97
Regenerate Genotyping Report using VeraReport . . . . . . . . . . . . . . . . . . . . . 102
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Appendix B
VeraScan Administration and
VeraReport
VeraScan Administration and VeraReport
Introduction
General VeraScan administration is described in the VeraScan Administration chapter in the BeadXpress Reader System Manual. The VeraScan analysis configuration specific to the ADME Core Panel and instructions for regenerating a genotyping report specifically from ADME Core Panel results are described below.
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A VeraScan system administrator can set up ADME Core Panel analysis configuration templates. To start the VeraScan Program and access the analysis configuration tool:
1
Wait until the Power and Ready lights on the front panel of the BeadXpress Reader are the only lights on. See Power Up the BeadXpress Reader Computer on page 51.
If you do not wait for this condition, an error message may appear when you start the software.
2
Do one of the following:
• From the Windows Start menu, select All Programs | Illumina | VeraScan.
on the desktop.
• Double‐click the VeraScan icon The VeraScan application opens on the computer desktop.
Figure 47 VeraScan Welcome Screen
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Set Up VeraScan ADME Analysis
Set Up VeraScan ADME Analysis Configuration
VeraScan Administration and VeraReport
3
Enter your username and password, then click Login.
Figure 48 VeraScan Login
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Click Next.
The VeraScan Setup screen displays the Select Application tab.
Set Up VeraScan ADME Analysis
4
Figure 49 VeraScan Setup Screen
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VeraScan Administration and VeraReport
5
Select the ADME Core kit from the Select Application tab. The scan input data fields are displayed.
Figure 50 VeraScan Select Application Tab
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Click Configure and the Analysis Configuration window is displayed. Do one or more of the following:
Figure 51 Analysis Configuration
•
•
•
•
7
Uncheck the loci to be excluded from analysis. Click Check All to select all loci. Click Uncheck All to deselect all loci. Click the plus icon to view loci details or click the minus icon to close loci details.
To save the configuration, enter the configuration Name and click Save. The configuration name is displayed as the Current Analysis Configuration and is added to the Load list.
To load a previously saved configuration, select the configuration from the Load list and click Load. The configuration is shown in the list of checked and unchecked loci.
To delete a previously saved configuration, select the configuration from the Load list and click Delete. The configuration is removed from the Load list.
Click Done to exit the Analysis Configuration window.
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Set Up VeraScan ADME Analysis
6
VeraScan Administration and VeraReport
Regenerate Genotyping Report using VeraReport
VeraReport is a stand‐alone application that provides the capability to review data generated by VeraScan and regenerate genotyping reports.The application does not connect to the BeadXpress Reader.
VeraReport supports the same report functionality as described in Report Genotypes on page 76, however the only changes that can be made to the report are to select the results to be included or excluded.
Preparation
In order to be able to review data and regenerate a genotyping report, you must have VeraReport and the corresponding ADME analysis modules installed locally. VeraReport and the ADME analysis modules can be downloaded via the Illumina website (See “Technical Assistance” on page 141.)
Start the VeraReport Program
1
From the Windows Start menu, select All Programs | Illumina | VeraReport, or double‐click the VeraReport icon on the desktop.
The VeraReport window opens on the computer desktop.
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1
Regenerate Genotyping Report using
Figure 52 VeraReport Screen
Enter your VeraScan username and password and click Login.
The Available Plugins installed are listed.
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VeraScan Administration and VeraReport
2
Click Load Project File to select and view a BeadXpress project file (.bxp).
Figure 53 VeraReport Sample ADME Summary Report
3
To analyze and review the data see Analyze Scan Data on page 63.
4
Click Save Project File to save the BeadXpress project file (.bxp) with a new name and/or in a different directory from the original analysis.
5
To reanalyze the data, click Reanalyze from the VeraReport window. The Reanalyze Existing Data dialog box opens. Figure 54 Reanalyze Screen
a
104
To reanalyze the data using the default, original Kit Manifest and Sample Sheet click Reanalyze. Part # 15007510 Rev. C
•
•
To select different data for analysis:
Click to navigate to and select an alternate Kit Manifest to use to reanalyze the original scan data.
Click to navigate to and select an alternate Sample Sheet to use to reanalyze the original scan data.
Click Reanalyze.
View Report
To view the report in PDF format, click Report... from the VeraReport screen. Reference Reports in PDF Format on page 76 for functional details.
Export Data
To export the report to .csv format for use in other commercial applications, such as Microsoft Excel, click Export... from the VeraReport screen. Reference Export Data on page 105 for functional details.
Exit VeraReport
Exit the VeraReport application by clicking Logout, then clicking Close upper‐right corner of the VeraReport window.
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Regenerate Genotyping Report using
b
•
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VeraScan Administration and VeraReport
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Qualification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Programming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
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Appendix C
Microplate Shaker
Microplate Shaker
Introduction
The incubating microplate shaker must be qualified and then programmed for automated use.
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The incubating microplate shaker must be qualified prior to programming and use. Follow these procedures to qualify the incubating microplate shaker.
CAUTION
Programming the incubating microplate shaker must be performed exactly as described in the following procedures to ensure optimal assay performance. Incorrect programming can lead to failure of the ADME Core Panel assay. Please contact your Field Applications Scientist or see Technical Assistance on page 141 prior to programming your unit.
Equipment
The following user‐supplied equipment is required to qualify the incubating microplate shaker:
` Fluke thermometer with thermocouple
` Extech Photo Tachometer Stroboscope
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Qualification
Qualification
Microplate Shaker
Shaker Controls
Reference the following figure when performing the qualification procedures.
Figure 55 Incubating Shaker Control Panel
A
B
C
D
E
F
G
H
I
J
K
110
Temp °C (temperature) display
Program display
Time display
Temperature display on/off button
Temperature Up/Down buttons
Speed start button
Speed select buttons
Time pause button
Abort ‐button
Program button
Main on/off and standby indicator light
Part # 15007510 Rev. C
1
Power on the incubating microplate shaker by pushing the main on/off button.
2
Lower the 96‐well plate retaining bar onto the incubation blocks.
Figure 56 96‐Well Plate Retaining Bar on Incubation Blocks
Qualify Shaking Speed
1
Press the main on/off button to place the unit in standby mode. The standby indicator light turns red and the displays are blank.
2
Press and hold the select down arrow and abort at the same time. While pressing these two buttons, quickly press and release the main on/off button. Only the program display should be visible. If all three display screens are on, press the main on/off button to turn the unit off and repeat step 2.
NOTE
If the program displays a program step number instead of rpms, exit to standby mode by pressing start, then abort.
3
Display the speed setting by pressing the speed start button.
4
Set the shaking speed display to 1,400 rpm using the speed select up/ down arrows.
5
Allow the system to run for at least 1 minute.
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Qualification
Power On Shaker
Microplate Shaker
6
`
`
`
Check for any mechanical interference or error codes.
Any rattling or ticking sounds may indicate a loose screw on the tray, tray attachment, or any accessory.
If there is any mechanical interference that can not be resolved contact your VWR representative.
If an error code is displayed, reference the following table to resolve the problem.
Table 22 Incubating Shaker Error Codes Code Software Test
Cause
Solution
E01
N/A
Lid open or temperature over 100°C
Switch the unit off and contact your VWR representative.
E02
N/A
Lid open or temperature over 0°C
E03
Drive system failure •
•
•
•
E04
Unit overload
E06
Loose foot (suction cup)
Mechanical obstruction
Ceased bearing
Drive belt broken
If loose foot, press main on/off button. Adjust foot, then restart. If the error persists, switch the unit off and contact your VWR representative.
For all other causes switch the unit off and contact your VWR representative.
• Maximum load exceeded
• Loose foot (suction cup)
Press main on/off button. Be sure the load is within the maximum load specification before restarting. If the error persists, switch the unit off and contact your VWR representative.
Program mode was interrupted by loss of power
Unplug unit and reapply power.
` If there is an error code that can not be resolved contact your VWR representative.
7
112
When the shaking speed has been qualified press the speed start button to turn off the speed display.
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1
Place the probe from a calibrated thermometer in one of the center wells of the right incubation block, so that the tip of the probe makes contact with the base of the well.
Figure 57 Temperature Probe
2
Turn on the thermometer if it is not already on.
3
Display the temperature setting by pressing the temperature display on/off button.
4
Set the temperature display to 45°C by using the temperature up/down arrows.
5
Close the lid and wait for the set temperature to stabilize.
Figure 58 Incubating Microplate Shaker Lid Closed
6
The readout on the calibrated thermometer should be within +/‐ 0.5°C of the target temperature. If the target temperature is not within +/‐ 0.5°C of the actual temperature then the incubating microplate shaker will have to be calibrated.
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Qualification
Qualify Temperature
Microplate Shaker
7
To calibrate the incubating microplate shaker, wait until the temperature has stabilized, then hold down the main on/off button and press temperature up arrow.
8
Press the temperature up/down arrows to specify the calibrated thermometer temperature, the press the main on/off button.
9
Allow the incubating microplate shakerʹs temperature to stabilize again and verify that the set temperature reaches the target temperature according to the calibrated thermometer +/‐ 0.5°C. If it does not reach the target temperature, repeat steps 7–9.
10 Repeat steps 1–9 for the left incubation block 45°C target temperature.
11 If after setting the right incubation block to 45°C and the left incubation block is not 45°C +/‐0.5°C, and the system can not be calibrated to split the difference between the 2 blocks so that the real temperature measure by the calibrated thermometer is 45°C +/‐0.5°C for both left and right incubation blocks, contact your VWR representative.
12 Repeat steps 1–9 for the right incubation block 68°C target temperature.
13 Repeat steps 1–9 for the left incubation block 68°C target temperature.
14 If after setting the right incubation block to 68°C and the left incubation block is not 68°C +/‐0.5°C, and the system can not be calibrated to split the difference between the 2 blocks so that the real temperature measure by the calibrated thermometer is 68°C +/‐0.5°C for both left and right incubation blocks, contact your VWR representative.
Calibrate Speed
114
1
Display the current incubating microplate shaker speed by holding down the main on/off button and pressing the speed start button once. The speed will flash in the program display.
2
Measure the speed of the incubating microplate shaker with a photo tachometer stroboscope.
3
Point the strobe to one of the screws on the incubating microplate shaker. Adjust the course and fine adjustment knobs on the stroboscope until the screw stops rotating and appears stationary.
Part # 15007510 Rev. C
A
B
Qualification
Figure 59 Photo Tachometer Stroboscope Readout
Stroboscope
Screws
4
Press the speed select up/down arrows to match the setting of the stroboscope.
5
Press the main on/off button to save the calibration. The standby indicator light turns red and the displays are blank.
Set Cooling Ramp Rate
1
With the system on standby, press and hold the speed select down arrow and the abort button and press the main on/off button. The system is now in std (standby) mode.
2
Return to standby mode by pressing the main on/off button. This saves std mode.
3
Hold the temperature on/off button and press and release the main on/off button.
4
Use the temperature up/down arrows to scroll through the ramp rates on the temperature display until r2 is displayed.
5
Press the main on/off button to save the cooling ramp rate.
6
Once the system has been qualified, power off the system by pressing the main on/off button.
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Microplate Shaker
Programming
The incubating microplate shaker must be programmed for automated use and to avoid potential ADME Core Panel protocol errors. Reference the following figure when performing the programming procedures.
Figure 60 Incubating Shaker Control Panel
A
B
C
D
E
F
G
H
I
J
K
116
Temp °C (temperature) display
Program display
Time display
Temperature display on/off button
Temperature Up/Down buttons
Speed start button
Speed select buttons
Time pause button
Abort ‐button
Program button
Main on/off and standby indicator light
1
Press the main on/off button to place the unit in standby mode. The standby indicator light turns red and the displays are blank.
2
Press and hold the select down arrow and abort at the same time. While pressing these two buttons, quickly press and release the main on/off button. Only the program display should be visible. If all three display screens are on, press the main on/off button to turn the unit off and repeat step 2.
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3
Press the select up arrow until Pr2 is displayed on the program display. This is the memory position where the ADME Core Panel program steps will be stored.
4
Press program to edit the Pr2 memory position.
5
The program displays 2‐01, to indicate that you are editing step 1 of memory position Pr2. The ADME Core Panel uses 12 steps in memory position Pr2, with the final step being 2‐12.
6
Press the temperature up/down arrows to adjust the temperature to 68°C.
7
Press abort to adjust the time down to 00:00, then press start.
8
The program display indicates the incubating microplate shaker speed. Use the select up/down arrows to set the speed to 1,400 rpm, then press start.
9
The program display indicates if the incubating microplate shaker is On or Off. Press the select up/down arrows to set the incubating microplate shaker to Off, then press start.
10 The time display indicates if the timer is On or Off. Press program or abort to set the timer to Off, then press start.
11 The temp °C display indicates if the temperature control is On or Off. Press the temperature up/down arrows to turn temperature control On, then press start.
12 The temp °C display indicates Phet= On or Off. Press program or abort to set Phet= On, then press Start.
13 The temp °C display indicates the ramp rate. Press the temperature up/down arrows to adjust the ramp rate to R2, then press start.
14 The temp °C display indicates Beep= On or Off. Press program or abort to set Beep= On, then press start.
15 The temp °C display indicates End= On or Off. Press program or abort to set End= On, then press start.
Programming 2‐01 is now complete.
16 Press the select up arrow to set the program display to 2‐02.
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Programming
NOTE
If the program displays a program step number instead of rpms, exit to standby mode by pressing start, then abort.
Microplate Shaker
17 Repeat steps 6–16 to program each of the 12 ADME Core Panel memory program steps for memory position Pr2. Reference the following table to program each memory program step:
Table 23 Incubating Microplate Shaker Memory Step Settings
Step
Temp (°C)
Timer (Min)
Speed (rpm)
Shaker
Timer
Temp Control
Phet
Ramp Rate
Beep
End
01
68
0:00
1,400
Off
Off
On
On
R2
On
Off
02
68
5:00
1,400
On
On
On
Off
R2
On
Off
03
68
5:00
1,400
On
On
On
Off
R2
On
Off
04
68
1:00
1,400
On
On
On
Off
R2
On
Off
05
68
5:00
1,400
On
On
On
Off
R2
On
Off
06
68
1:00
1,400
On
On
On
Off
R2
On
Off
07
45
15:00
1,400
On
On
On
Off
R2
On
Off
08
45
1:00
1,400
On
On
On
Off
R2
On
Off
09
45
1:00
1,400
On
On
On
Off
R2
On
Off
10
45
1:00
1,400
On
On
On
Off
R2
On
Off
11
45
20:00
1,400
On
On
On
Off
R2
On
Off
12
45
2:00
1,400
On
On
On
Off
R2
On
On
18 When programming steps 1–12 of memory position Pr2 is complete, press the main on/off button. The program displays Pr2 and the time displays USEd.
19 Place the incubating microplate shaker in standby mode by pressing start, then abort.
20 Verify programmed steps:
a Repeat steps 1–4 to place the unit in standby mode.
b Press the select up/down arrows to scroll through the 12 Pr2 programmed steps and reference Table 23 to verify their settings. Press start at each step to verify all settings.
118
Part # 15007510 Rev. C
d
e
When verification of all memory program steps for memory position Pr2 is complete, press the Standby button. The program displays Pr2 and the time displays USEd.
Press start to display the first step of Pr2. Press start again to initiate the program.
Test all of the memory position Pr2 programmed steps one time with no samples present to fully verify the programming. NOTE
The Step 7 cooling ramp‐down should take 7–8 minutes. If the ramp down occurs much faster or slower, check your program settings.
21 To edit a memorized program step:
a Repeat steps 1–4.
b Press the select arrows to navigate through the program to the step that requires modification.
c Modify the step as needed.
d When the program modification is complete, press the main on/off button. The program displays Pr2 and the time displays USEd.
22 To run the incubating microplate shaker, press start to display Pr2 in the program display. NOTE
When the incubating microplate shaker is turned off with Pr2 selected, the next time it is turned on it will display the Pr2 Step 1. Press start to initiate the first step of the program.
NOTE
For general operating instructions, reference the Troemner Incubating Shaker Manual.
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119
Programming
c
Microplate Shaker
120
Part # 15007510 Rev. C
Assay Protocol Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
BeadXpress Reader System Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . 129
View and Report Errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Frequently Asked Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
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121
Appendix D
Troubleshooting
Troubleshooting
Assay Protocol Troubleshooting
This section provides solutions to issues that may appear when assessing the quality of each run to determine the validity of the outcomes generated from the ADME Core Panel. Issues fall into the following general categories:
` Sample Processing
` VeraCode Bead Hybridization
` Data Quality
Sample Processing
This section addresses causes and resolutions for potential processing issues.
Table 24 Troubleshooting Problems During Sample Processing Symptom
Probable Cause
Resolution
Evaporation of corner wells after PCR.
Poor plate sealing during pre‐
PCR processing or in the thermocycler.
Ensure the edges of the ARX plate are well sealed. See “Sealing Microplates” on page 92.
Incomplete resuspension of paramagnetic beads.
Overspeed centrifugation.
Check centrifuge speed setting.
The assay is not normally affected by incomplete resuspension.
Beads left overexposed during Complete reagent transfers as wash steps when supernatant quickly as possible.
removed.
Incomplete capture of paramagnetic beads.
122
Poor position of ARX plate on magnet or insufficient time.
Position the ARX plate with the wells between the bars of the magnet.
Part # 15007510 Rev. C
Symptom
Probable Cause
Resolution
Splashing of samples out of wells after shaking steps.
Poor application of adhesive plate seal.
Use seal applicator to ensure seal is covering all wells and recommended sealing techniques described in Sealing Microplates on page 92.
Shaking incubator is out of calibration and is not shaking correctly.
Perform routine calibration of the shaking incubator.
Not enough volume in the Missing addition of AOP0 AOP tubes to process reagent to individual AOP4 samples.
tubes.
Repeat assay and add AOP0 to individual AOP4 tubes.
Seal does not stay adhered Poor application of adhesive to plate during incubation plate seal.
steps.
Follow recommended seal application technique. See “Sealing Microplates” on page 92.
Reagents wicking onto plate surface from pipette tips during dispense steps.
VeraCode ADME Core Panel Assay Guide
Dab top of plate with a Kimwipe before placing seal on plate and follow recommended pipetting techniques. See “Dispensing Reagents” on page 91.
123
Assay Protocol Troubleshooting
Table 24 Troubleshooting Problems During Sample Processing (Continued)
Troubleshooting
VeraCode Bead Hybridization
This section addresses causes and resolutions for potential issues while hybridizing the VeraCode beads.
Table 25 Troubleshooting Problems During VeraCode Bead Hybridization Symptom
Probable Cause
VeraCode beads are not in Insufficient centrifugation.
the wells.
Resolution
Centrifuge the VeraCode Bead Plate before unsealing.
Inadvertent removal of VeraCode beads by touching with pipette tips.
Keep tips above VeraCode beads when pipetting MSS reagent.
Rapid removal of seal causes bead pellet to flip out of wells.
Slowly and carefully remove cap mat.
Data Quality
This section addresses causes and resolutions for potential data quality issues.
.
Table 26 Troubleshooting Problems with Data Quality Symptom
Probable Cause
Resolution
Very low signal intensity for all bead types except hybridization controls.
Titanium Taq DNA polymerase was omitted from MAM1 tube.
Repeat assay with Titanium Taq DNA polymerase.
Very low DNA input.
Re‐extract DNA.
Incorrect PCR program selected.
Repeat assay and check program.
Plate failure during PCR.
Repeat assay.
Low red signal for all bead Cy5 degradation due to excess Protect MAM1 reagent from types.
bleach fumes or overexposure bleach and light.
to light.
124
Part # 15007510 Rev. C
Symptom
Probable Cause
Resolution
Low signal reported for Sample Tracking Control output ‐ no data outputted for sample.
Cross contamination of at least one of the three wells of sample or incorrect sample location on the plate.
Ensure proper distribution of samples to the ARX plate before starting the assay protocol.
Follow recommended plate Poor sealing of plate during vortexing steps lead to sample sealing techniques. See cross‐contamination.
“Sealing Microplates” on page 92.
Strong signal from the no‐
template control samples.
Cross‐contamination may have Take care to avoid PCR occurred.
amplicon contamination. For example, treat lab work surfaces with 10% bleach and allow them to air‐dry.
Follow recommended plate Poor sealing of plate during vortexing steps lead to sample sealing techniques. See cross‐contamination.
“Sealing Microplates” on page 92.
Poor Fluorescence‐
BeadXpress Reader producing low‐intensity values.
Pipetting error.
Pipette carefully and according to procedures. Splashing during vortexing.
Take care to avoid splashing. BeadXpress Reader needs routine cleaning.
Run KOH cleaning. Reference the section on Maintenance in the BeadXpress Reader System Manual.
Optical system out of alignment.
Re‐initialize the BeadXpress system. If the issue persists, contact Illumina Customer Support.
Poor Fluorescence ‐ low Thermocycler settings were set Adjust thermocycler settings signal result for a majority incorrectly.
according to the assay of loci.
protocol.
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Assay Protocol Troubleshooting
Table 26 Troubleshooting Problems with Data Quality (Continued)
Troubleshooting
Table 26 Troubleshooting Problems with Data Quality (Continued)
126
Symptom
Probable Cause
Resolution
Low call rate on a small number of loci over multiple runs.
Possible drift in temperature calibration of thermocycler, incubating shakers, or heat block.
Re‐check calibration of thermocycler, incubating shakers, or heat block.
Data failed to generate in VeraScan due to SPC failure.
MTR or AOP reagents were added to the wrong column on the plate or location of AOP reagents on plate were out of order (not A, B, then C).
Label ARX plate according to assay protocol and only handle one MTR or AOP subset at a time.
VeraCode Bead Plate placed into BeadXpress Reader in the wrong orientation.
Place VeraCode Bead Plate into BeadXpress Reader in the correct orientation according to assay protocol.
BeadXpress Reader was not properly balanced.
Check test and calibration log to verify scanner balance point. Re‐run test and calibration beads to ensure BeadXpress Reader is balanced appropriately.
Expected outcome for samples do not match historical data.
Samples inputted into sample sheet incorrectly.
Reanalyze data using VeraReport and a correct sample sheet.
Failed Mismatch control generated during data analysis.
PCR or incubating shaker failure.
Check calibration of thermocycler and incubating shaker.
Mismatch Controls decrease in intensity and fall toward center of plot.
NaOH or MTR are expired or stored improperly.
Make fresh NaOH and repeat assay.
Check reagent storage and expiration date.
Part # 15007510 Rev. C
Symptom
Probable Cause
High number of STC failures in a plate.
Poor sealing of plate during Follow recommended plate incubation steps led to sample sealing techniques. See cross‐contamination.
“Sealing Microplates” on page 92.
Incorrect thermocycler program used.
Resolution
Check thermocycler program used for correct parameters.
BeadXpress Reader had errors Contact Illumina Customer during scanning.
Support.
PSC error
Pool switching of MTRs and AOPs
Cross‐contamination of samples during assay run (e.g. AB1 splashing between wells, pipette tips not properly changed between steps, paramagnetic particles accidentally transferred between wells during pipetting/washing.
Follow recommended pipetting techniques. See “Dispensing Reagents” on page 91.
VeraCode beads were transferred between wells during VW2 wash.
VeraCode bead plate hybridization was longer than the specified 2.5 hour incubation time.
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127
Assay Protocol Troubleshooting
Table 26 Troubleshooting Problems with Data Quality (Continued)
Troubleshooting
Table 26 Troubleshooting Problems with Data Quality (Continued)
Symptom
Probable Cause
Resolution
High number of PSC errors in a plate
A critical processing step was not performed correctly or might have been missed
Determine the validity of the results obtained for unaffected samples, then reprocess the failed samples and possibly the samples that passed based on further investigation.
Incubating microplate shaker Check the instrument may be out of calibration or the programs and calibrations.
wrong thermocycler program was used.
128
Part # 15007510 Rev. C
This section provides solutions to issues that may appear when using the BeadXpress Reader system and information about how to manage BeadXpress Reader errors.
Issues fall into the following general categories:
` Data Generation and Storage
` Fluidics System
` BeadXpress Reader
` Test and Calibration Beads
Data Generation and Storage
This section addresses causes and resolutions for potential data generation and storage issues.
Table 27 Troubleshooting Problems with Data Generation and Storage Symptom
Cause
Resolution
Cannot find data files/no data files are created.
Network error may have prevented files from being created.
NOTE: This only applies to networked BeadXpress Readers.
Use Windows Explorer or another application to verify network accessibility. If network errors exist, ask IT for assistance.
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BeadXpress Reader System
BeadXpress Reader System Troubleshooting
Troubleshooting
Table 27 Troubleshooting Problems with Data Generation and Storage (Continued)
Symptom
Cause
Resolution
Low bead representation.
The ADME Core Panel Core kit Repeat assay.
was not selected from the menu.
VeraCode beads were not read Contact Illumina Customer properly because green laser is Support.
out of factory specification.
Code read laser beam out of alignment.
Initialize the system. If issues persist, contact Illumina Customer Support.
Plate not seated properly in the Reload plate in plate tray.
plate tray.
130
Plate seal covering wells.
Remove plate seal and re–
initialize the BeadXpress system twice.
Air in the fluidics system.
Prime the fluidics system.
Reagents may be in wrong reagent bottles.
Clean reagent bottles and fill with correct reagents.
BeadXpress Reader fails to locate the same number of beads in the red laser scan as the green laser scan (data consolidation).
Contact Illumina Customer Support.
Beads are not being loaded into the BeadXpress Reader in the expected position.
Contact Illumina Customer Support.
VeraCode beads are not in the wells.
See VeraCode Bead Hybridization on page 124
Part # 15007510 Rev. C
This section addresses causes and resolutions for potential fluidics system issues.
Table 28 Troubleshooting Problems with Fluidics System Symptom
Cause
Fluidics system won’t Reagents may be in wrong prime properly.
reagent bottles.
Reagent bottles may be empty.
Resolution
Clean reagent bottles thoroughly and fill with correct reagents.
Refill reagent bottles.
Read buffer concentration may be Remix read buffer per Illumina too high.
instructions.
Fluid leaking from BeadXpress Reader.
Fluidics tubes may be loose or not connected.
Check fluidics connections to bottles, reagent carrier, and BeadXpress Reader.
Reagent flow to the BeadXpress Reader may be restricted.
Check to make sure the tubes are not crimped and that nothing is placed on top of the tubes.
The waste bottle tube may be loose or not connected.
Check waste bottle fluidics connections to bottles, reagent carrier, and BeadXpress Reader.
Reagent flow to the waste bottle may be restricted.
Check to make sure the waste bottle tube is not crimped and that nothing is placed on top of the tube.
Internal sensors may be inoperable.
Contact Illumina Customer Support.
Too much buffer in the well plate Check assay protocols and well at the start of the scan.
plate starting volumes.
Fluidics system obstructed.
VeraCode ADME Core Panel Assay Guide
Bead mass or foreign matter in fluidics lines or pumps.
Remove the blockage from the system. Reference the section on Maintenance in the BeadXpress Reader System Manual.
131
BeadXpress Reader System
Fluidics System
Troubleshooting
BeadXpress Reader
This section addresses causes and resolutions for potential BeadXpress Reader issues.
Table 29 Troubleshooting Problems with the BeadXpress Reader Symptom
Cause
BeadXpress Reader is not VeraScan is configured for connected on VeraScan manual connection to the startup.
BeadXpress Reader.
Cannot connect to BeadXpress Reader.
Select the configuration settings tab in the Administration interface and set the BeadXpress connection to automatic.
Cable connection between BeadXpress Reader and PC may be unplugged.
Inspect the connection between the BeadXpress Reader and the BeadXpress Reader computer to confirm that the cable is securely plugged in.
The BeadXpress Reader may not be powered up.
Power up the BeadXpress Reader.
System reports BeadXpress Reader detects a mechanical error, will not possible mechanical error scan.
and immediately disables all motors.
132
Resolution
If there is no apparent physical problem, either re–initialize the BeadXpress Reader, or cycle the power. To re–initialize the BeadXpress Reader, right‐click the BeadXpress logo and select Reader | Initialize System.
Part # 15007510 Rev. C
This section describes what to do if the BeadXpress Reader does not pass the test and calibration cycles.
Table 30 Troubleshooting Problems with Test and Calibration Beads Symptom
Cause
Resolution
BeadXpress Reader does not pass Test and Calibration Cycle.
Too few beads in the Test and Calibration column.
Spin down the Test and Calibration plate and rerun the application with a new column.
Rerun the application with Test and Calibration beads exposed to out‐of‐specification new Test and Calibration beads.
conditions.
VeraCode ADME Core Panel Assay Guide
BeadXpress Reader is out of specification.
Contact Illumina Customer Support.
Incorrect values inputted for target red and green counts.
Review Test and Calibration log in the BeadXpress Calibration folder and rerun Test and Calibration.
133
BeadXpress Reader System
Test and Calibration Beads
Troubleshooting
View and Report Errors
The VeraScan application records system events, messages, and errors as they occur in an event log. The event log provides a record of system events that you can view or email to Illumina Customer Solutions for evaluation if an error occurs. If an error occurs, you can view error details in the error message box as well as on the Events tab.
Viewing Error Details as they Occur
If an error occurs while using the BeadXpress Reader system, an error message is displayed. For information on accessing information about system events (errors and warnings), see the Viewing Events section. For information about diagnostic messages, see the Viewing the Log on page 135. For information about sending error details to Customer Solutions, see the Reporting Errors on page 135.
Viewing Events
The Events pane displays errors that have occurred during the current session, including the time the event occurred, the event code, and a description of the event. The Events pane also displays major system actions and warnings.
1
Click Events at the bottom of the screen (Figure 61).
The Events pane is displayed.
Figure 61 Events Pane
2
134
Double‐click any icon in the Events pane to view more information about that event or warning.
Part # 15007510 Rev. C
As the BeadXpress Reader records images and the software runs, system diagnostic messages are recorded in a system log and saved to a file named VeraScan.log. 1
Click Log at the bottom of the screen (Figure 62).
The Log pane is displayed.
Figure 62 Log Pane
2
To pause the Log display, click any line other than the last one. The display stops scrolling until you re‐select the last line (press CTRL+END).
Reporting Errors
If an error occurs, call your Illumina Customer Support contact or email a description of the error and attach the BeadXpressLog.txt and ErrorsLog.txt files with a time stamp close to the time of the error (but after the error occurred). The most recent log files are automatically saved in the Execution Logs folder. All log files use the following naming convention:
BeadXpressLog20091109‐232403.txt, where the date (20091109) is year/month/day and the time (232403) is hour/minute/second.
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135
View and Report Errors
Viewing the Log
Troubleshooting
Frequently Asked Questions
For answers to frequently asked questions (FAQs), go to http://www.illumina.com.
136
Part # 15007510 Rev. C
Index
Index
Numerics
C
0.1N NaOH 24, 25, 28, 31
conclude scanning 62
contamination 87
control 64
control region 4
Control Samples 8
copy number variation (CNV) 4
csv 20, 76
customer support 141
CYP2C18 3
CYP2C19 3
CYP2C8 3
CYP2C9*2 3
A
A1 orientation mark 55
AB1 reagent 24, 32
abort scan 60
adhesive microplate sealing film 25, 37
ADME 2
ADME Beadtypes and Translations docu‐
ment 3
ADME copy number variant 63, 64
ADME Core Panel kit manifest 63
ADME variant 63, 64
AE1 reagent 37, 38, 39
Analysis Configuration 57
AOP reagent 2, 34, 86
AOP0 reagent 25, 34
AOP4A reagent 25, 34, 35
AOP4B reagent 25, 34, 35
AOP4C reagent 25, 34, 35
ARX 24
aspirating 90
B
BeadXpress Reader 20, 51, 84
BeadXpressLog 135
Bio‐Rad DNA Engine Tetrad 2 44
Bio‐Rad MyCycler 44
bleaching 87
VeraCode ADME Core Panel Assay Guide
D
documentation 141
E
ELM2 reagent 37, 40
Eppendorf MasterCycler 45
Eppendorf MasterCyclerPro 45
Eppendorf MasterCyclerPro Gradient 45
Eppendorf Thermocyclers 45
Execution Logs folder 135
export data 79
Extech Photo Tachometer Stroboscope 109
F
Failed Hyb 63
Failed Mismatch 63
Failed PSC 63
Failed STC 63
137
Index
FAQs 136
FAS 86
first hybridization controls
GoldenGate 8
Fluke thermometer with thermocouple 109
fluorescence data 54
G
gDNA 2, 24
genotyping parameters 63
green and red fluorescence 63
MJ Research DNA Engine Tetrad 44
MJ/BioRad Thermocyclers 44
monitor scan progress 60
MSS reagent 47, 48
MTR reagent 2, 29, 86
MTR4A reagent 24, 30
MTR4B reagent 24, 30
MTR4C reagent 24, 30
N
no call 63
NTCs 64
H
help, technical 141
Hyb Control 1 6
Hyb Control 2 6
Hybridization Control 1 63
Hybridization Control 2 63
Hybridization Controls 7
I
incubating microplate shaker 18, 26, 108
Incubating Microplate Shaker Memory Step Settings 118
Incubating Shaker Control Panel 110, 116
input DNA 15
instructions 18
L
Logout VeraReport 105
low beads 63
low signal 63, 64
LTF 23
M
materials 12
Mismatch Control 1 6, 63
Mismatch Control 2 6, 63
Mismatch Controls 8
138
P
part number location 21
pass 64
pause scan 60
PCR 18, 87
PCR microplate 25
PCR sealing film 42
pipetting 90
Plate Analysis Summary Report 66
Plate Controls 74
Plate Detail Report 70
Plate Sample Details 67
plot tools 73
pool guide label 25, 27
post‐PCR 47, 49, 51, 54, 87
Pr2 program step 02 30
Pr2 program step 03 31
Pr2 program step 04 31
Pr2 program step 05 33
Pr2 program step 06 34
Pr2 program step 07 36
Pr2 program step 08 39
Pr2 program step 09 39
Pr2 program step 10 40
Pr2 program step 11 41
Pr2 program step 12 43
pre‐PCR 24, 87
Part # 15007510 Rev. C
R
reagents and materials 9
Reanalyze Screen 104
Report Configuration 76
Report Detail 78
Report on Sample 79
Report Summary 77
resume scan 60
S
sample sheet 20, 57
Sample Tracking Control 63
Scan Screen 60
Scan Screen Progress 61
Scan Settings Detail Tab 58
sealing 92
second hybridization controls
GoldenGate 6, 7
Shaker Cooling Ramp Rate 115
Shaking Speed 111
Shaking Temperature 113
SPC 6, 7, 63, 64
STC 6, 64
subpools 2
T
technical assistance 141
Titanium Taq DNA Polymerase 42
Troubleshooting Problems During Sample Processing 122
Troubleshooting Problems During Vera‐
Code Bead Hybridization 124
Troubleshooting Problems with Data Gen‐
eration and Storage 129
Troubleshooting Problems with Data Quality 124
VeraCode ADME Core Panel Assay Guide
Troubleshooting Problems with Fluidics System 131
Troubleshooting Problems with Test and Calibration Beads 133
Troubleshooting Problems with the BeadXpress Reader 132
U
UB3 buffer 24, 33, 37, 40
V
VeraCode ADME Core Genotyping Kit 9
VeraCode Bead Plate 47, 49
VeraReport 102
VeraReport Sample ADME Summary Re‐
port 104
VeraReport Screen 103
VeraScan Administration 96
VeraScan application 52
VeraScan Login 98
VeraScan Program 97
VeraScan Select Application Tab 56, 100
VeraScan setup 55
VeraScan Setup Screen 99
VeraScan system events, messages, and er‐
rors 134
VeraScan window 54
VeraScan.log 135
VW2 buffer 49, 50
W
workflow 19
139
Index
protocol 18
PSC 6, 7, 63, 64
Index
140
Part # 15007510 Rev. C
Technical Assistance
Technical Assistance
For technical assistance, contact Illumina Customer Support.
Table 31 Illumina General Contact Information
Illumina Website http://www.illumina.com
Email [email protected]
Table 32 Illumina Customer Support Telephone Numbers Region
Contact Number
Region
Contact Number
North America
1.800.809.4566
Italy
800.874909
Austria
0800.296575
Netherlands
0800.0223859
Belgium
0800.81102
Norway
800.16836
Denmark
80882346
Spain
900.812168
Finland
0800.918363
Sweden
020790181
France
0800.911850
Switzerland
0800.563118
Germany
0800.180.8994
United Kingdom
0800.917.0041
Ireland
1.800.812949
Other countries
+44.1799.534000
MSDSs
Material safety data sheets (MSDSs) are available on the Illumina website at http://
www.illumina.com/msds.
Product Documentation
If you require additional product documentation, you can obtain PDFs from the Illumina website. Go to http://www.illumina.com/support/documentation.ilmn. When you click on a link, you will be asked to log in to iCom. After you log in, you can view or save the PDF. To register for an iCom account, please visit https://icom.illumina.com/Account/Register.
VeraCode ADME Core Panel Assay Guide
141
Technical Assistance
142
Part # 15007510 Rev. C
Illumina, Inc.
9885 Towne Centre Drive
San Diego, CA 92121-1975
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com
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