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Part # 15046990 Rev. B
Table of Contents
Table of Contents
List of Tables
Chapter 1 Overview
Introduction
Tracking Tools
Chapter 2 Lab Setup and Maintenance
Introduction
Safety Precautions
Preventing Amplification Product Contamination
Preparing and Storing Reagents
Best Practices
Lab Maintenance
Chapter 3 CytoSNP-850K Assay Protocol
CytoSNP-850K Assay Workflow
Equipment, Materials, and Reagents
Make and Incubate the MSA1 Plate
Fragment the DNA
Precipitate the DNA
Resuspend the DNA
Hybridize DNA to the BeadChip
Wash the BeadChip
Extend and Stain (XStain) BeadChips
Image BeadChip
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2
3
5
6
7
8
9
10
11
15
16
17
20
23
25
28
30
39
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Index
63
Technical Assistance
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Infinium CytoSNP-850K Assay Protocol Guide
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List of Tables
Table 1
Table 2
Table 3
Table 4
Table 5
Table 6
Table 7
Table 8
Table 9
Lab Equipment Maintenance
Sample Vortexer Calibration Label
User-Supplied Equipment
User Supplied Materials
Illumina-Supplied Reagents - Infinium Analysis Kit
Illumina-Supplied Reagents - supplied in the Infinium Analysis Kit
Illumina-Supplied Items
Illumina General Contact Information
Illumina Customer Support Telephone Numbers
Infinium CytoSNP-850K Assay Protocol Guide
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Chapter 1 Overview
Introduction
Tracking Tools
Infinium CytoSNP-850K Assay Protocol Guide
2
3
1
Chapter 1
Overview
Overview
Introduction
The Infinium CytoSNP-850K BeadChip provides the most comprehensive coverage of
cytogenomic-relevant genes for constitutional and cancer applications. The BeadChip
design targets 3,262 genes with an exon-centric design. The constitutional coverage
includes updates and content evolution from the International Collaboration for Clinical
Genomics (ICCG) and the cancer coverage includes all the Cancer Cytogenomics
Microarray Consortium (CCMC) genes.
The CytoSNP-850K BeadChip provides robust performance across a broad range of sample
types, including formalin-fixed paraffin-embedded (FFPE) tissue.
This Infinium CytoSNP-850K Assay Protocol Guide is for laboratory technicians running the
Infinium CytoSNP-850K Assay manually. The guide documents the laboratory protocols
associated with the assay. Follow all of the protocols in the order shown.
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Part # 15046990 Rev. B
Illumina provides the following tools for sample tracking and guidance in the lab:
} The Experienced User Card guides you through the protocols with minimal
illustrations and explanatory notes.
} For users of the BlueGnome BlueFuse Multi software for data analysis, the CytoSNP850K Lab Planner is used to create a batch import file for later use in data analysis.
} Users of the Illumina Genome Studio software for data analysis, require the Infinium
HD Super Assay Sample Sheet Template that records information about samples for
later use in data analysis.
} The Lab Tracking Form, available from the CytoSNP-850K Lab Planner or separately,
includes user-entry fields for tracking progress and recording lot numbers, barcodes,
and time metrics.
These documents and tools are available for download and reference at
www.illumina.com/documentation.
The CytoSNP-850K Lab Planner
To effectively track samples from the 96-well plate, to a BeadChip, then into the BlueFuse
Multi analysis software, Illumina recommends using the CytoSNP-850K Lab Planner.
The following series of worksheets guides you through setting up the planner.
} The first and second worksheets enable you to record a unique identifier and
description for each sample, as well as the BeadChip barcode (one per eight samples).
} The 96-well lab plan sheet illustrates the layout of the samples in the 96-well plate and
on the BeadChips and can be printed for reference while performing the assay.
} The next worksheet is the lab tracking form, which can be printed and used to record
lot numbers, barcodes, and time metrics while performing the assay. The lab tracking
form can be an important record for a laboratory.
} The final two worksheets are for recording the directory location where the GTC files,
manifest files, cluster files, and BlueFuse Multi annotation database are saved. This
information is included in the batch import file required by the BlueFuse Multi
software.
Infinium CytoSNP-850K Assay Protocol Guide
3
Tracking Tools
Tracking Tools
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Chapter 2 Lab Setup and Maintenance
Introduction
Safety Precautions
Preventing Amplification Product Contamination
Preparing and Storing Reagents
Best Practices
Lab Maintenance
Infinium CytoSNP-850K Assay Protocol Guide
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7
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9
10
11
5
Chapter 2
Lab Setup and Maintenance
Lab Setup and Maintenance
Introduction
This chapter describes the essential equipment and operating procedures for a CytoSNP850K Assay laboratory. It explains how to equip and run aCytoSNP-850K Assay laboratory,
providing important information on the following topics:
} Safety precautions
} Preventing amplification product contamination
} Preparing and storing reagents
} Calibrating and using the vortexer
} Best practices
} Lab maintenance
The assay protocols described in the rest of this guide assume that you are familiar with
the contents of this chapter, have implemented all the recommendations, and have
obtained all of the requisite equipment, materials, and reagents.
RELATED TRAINING
For more guidance on setting up an Infinium laboratory, see the Infinium,
Lesson 1: Lab Setup and Best Practice training video on the Array Online
Training page of the Illumina web site.
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Part # 15046990 Rev. B
Safety Precautions
Safety Precautions
CAUTION
The protocols described in this guide should be performed by qualified
laboratory personnel only. Exercise caution when handling biological samples to
avoid cross-contamination among pre-amp and post-amp samples.
WARNING
This protocol uses an aliphatic amide (formamide) that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion,
skin contact, and eye contact. For more information, consult the material
data safety sheet for this assay at http://www.illumina.com/msds. Dispose of
containers and any unused contents in accordance with the governmental
safety standards for your region.
Infinium CytoSNP-850K Assay Protocol Guide
7
Lab Setup and Maintenance
Preventing Amplification Product Contamination
Unless sufficient caution is exercised, amplification products may contaminate reagents,
instrumentation, and DNA samples, causing inaccurate and unreliable results.
Amplification product contamination can shut down lab processes and significantly delay
resumption of normal operations. For this reason, Illumina strongly encourages performing
the pre-amp processes in a separate, dedicated laboratory space.
CAUTION
It is imperative to establish procedures for preventing amplification product
contamination before working in the lab.
Separate Pre- and Post-Amp Areas
The CytoSNP-850K Assay uses an amplification process to increase the quantity of input
DNA by several thousand-fold. The laboratory space where pre-amplification processes are
performed should be physically separated from the laboratory space where amplified
products are made and processed. These are referred to as pre-amp and post-amp areas.
Dedicated Equipment and Supplies
Dedicate separate sets of instruments (pipettes, centrifuge, Illumina Hybridization Oven,
heat block, etc.) to the pre-amp and post-amp areas. Never share instruments between
areas.
Follow these rules to avoid contaminating the pre-amp area:
} Never use the same sink to wash pre-amp and post-amp reservoirs.
} Never share the same water purification system for pre-amp and post-amp processes.
} Store all supplies used in the CytoSNP-850K Assay protocols in the pre-amp area and
transfer to the post-amp area as needed.
Items Falling to Floor
The floor can be contaminated with amplification product transferred on the shoes of
individuals coming from the post-amp area. Therefore, treat anything falling to the floor as
if it were contaminated.
Disposable items falling to the ground, such as empty tubes, pipette tips, gloves, or lab coat
hangers should be thrown away at the end of the day or at the completion of the assay.
During the assay, never touch any items that have fallen to the ground.
Non-disposable items falling to the ground, such as pipettes or important sample
containers should be immediately and thoroughly cleaned with a 0.5% sodium
hypochlorite (10% bleach) solution to remove product contamination.
Use a 0.5% sodium hypochlorite (10% bleach) solution to clean any lab surface that has
contacted the contaminated item.
Individuals handling anything that has fallen to the floor, disposable or not, must throw
away their lab gloves and put on a new pair.
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Part # 15046990 Rev. B
To minimize errors in preparing user-supplied reagents each day, prepare large batches of
0.1N NaOH and 95% Formamide/1 mM EDTA, then aliquot and store them as described in
the following sections.
It is important to keep a ‘first in, first out’ or FIFO system for reagents. Rotating the stock of
the remaining reagents will help to avoid accidentally using expired reagents.
Prepare Batches of 0.1N NaOH
To minimize errors in preparing 0.1N NaOH fresh each day, prepare it in large batches
and aliquot it into 50 ml sealed tubes. These aliquots may be stored up to 12 months at 2 to
8°C and used in the protocol as needed. Once you open an aliquot, use it on the same day
that it was opened. Discard any reagent that is left at the end of the day.
Prepare Batches of 95% Formamide/1 mM EDTA
To minimize errors in preparing 95% formamide/1 mM EDTA, prepare it in large batches
and aliquot it into 1.5 ml sealed tubes. These aliquots may be stored up to 5 months at
-15°C to -25°C and used in the protocol as needed, one tube per BeadChip. Once you open
an aliquot, use it on the same day that it was opened. Discard any reagent that is left at the
end of the day.
WARNING
This protocol uses an aliphatic amide (formamide) that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion,
skin contact, and eye contact. For more information, consult the material
data safety sheet for this assay at http://www.illumina.com/msds. Dispose of
containers and any unused contents in accordance with the governmental
safety standards for your region.
Infinium CytoSNP-850K Assay Protocol Guide
9
Preparing and Storing Reagents
Preparing and Storing Reagents
Lab Setup and Maintenance
Best Practices
To optimize your data and minimize errors and waste, read and follow these best practices
whenever performing the CytoSNP-850K Assay protocols.
Cleaning and Calibrating Pipettes
Ensure pipettes are properly calibrated, clean, and decontaminated.
Pipette Carefully
Perform all pipette dispenses carefully and slowly to avoid creating turbulence within the
plate wells and Flow-Through Chambers.
Where possible, use a multi-channel pipette to dispense reagents.
Handling Cap Mats
Orient the cap mat so that A1 on the cap matches A1 on the plate.
CAUTION
To prevent evaporation and spills, which could lead to assay variability and
cross-contamination, ensure that all 96 caps are securely seated in the wells.
When you remove a cap mat, do so carefully and slowly to avoid splashing the contents.
Set the cap mat aside, upside down, in a safe location for use later in the protocol. When
you place the cap mat back on the plate, be sure to match it to its original plate and orient
it correctly.
Reagent Reuse
Never reuse excess reagents after dispensing. Discard according to your facility
requirements.
Handling BeadChips
Avoid touching the BeadChip anywhere other than at the barcode end or on the edges.
Balancing the Centrifuge
Whenever you centrifuge plates or BeadChips, place a balance plate or rack with
BeadChips opposite each plate or BeadChip rack being centrifuged. The weights should be
as similar as possible.
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Part # 15046990 Rev. B
The following standard lab maintenance procedures should be performed for Infinium
assay labs.
Daily and Weekly Bleaching
CAUTION
To prevent sample or reagent degradation, ensure all bleach vapors have fully
dissipated before starting any processes.
Post-Amplification Area
Reducing the amount of product in the post-amp area reduces the risk of contamination in
the pre-amp area.
Identify post-amp 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-amp 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.
Provide training for personnel responsible for cleaning the lab areas so that they know how
to prevent pre-amp product contamination.
Pre-Amplification Area
Establish a daily and weekly bleaching schedule for the pre-amp area similar to the one in
post-amp. This will help to eliminate product that may have entered the pre-amp area.
Identify high-risk pre-amp 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-amp
processes:
• Bench tops
• Door handles
• Refrigerator/freezer door handles
• Computer mouse
• Keyboards
Once a week, thoroughly clean all laboratory surfaces and instruments, including all of the
bench tops and instruments that are not cleaned daily. Mop the floors with a 0.5% sodium
hypochlorite (10% bleach) solution as well.
Provide training for personnel responsible for cleaning the lab areas so that they know how
to prevent pre-amp product contamination.
Infinium CytoSNP-850K Assay Protocol Guide
11
Lab Maintenance
Lab Maintenance
Lab Setup and Maintenance
Cleaning the Glass Back Plates
The glass back plates are used in the Flow-Through Chambers to control the flow of sample
and reagent onto the BeadChips. When you open the glass back plates for the first time and
between uses, you need to clean them. In addition, after every seven uses or weekly
(depending on your lab's throughput), you need to perform a more thorough cleaning. Both
procedures are described in the following sections.
Cleaning the Glass Back Plates after Every Use
When you open the glass back plates for the first time, and after every use, follow these
instructions to clean them.
1
Prepare a 1% dilution (2.5 g per 250 ml) of Alconox Powder Detergent in water.
2
Dip each glass back plate into the solution.
3
Remove the glass back plates from the detergent solution and wipe them with a
Kimwipe.
4
Hold the glass back plates under running DI water to completely remove any
remaining detergent.
5
Allow the glass back plates to dry in the storage rack or by leaning the glass back
plates at an angle against the black metal frames.
6
Once the glass back plates are clean and dry, use a Kimwipe to wash with 70% EtOH.
7
Dry the glass back plates with a Whoosh duster or laboratory air gun.
8
To prevent surface damage to the glass back plates, store them in the Te-Flow Flow Cell
storage box.
Cleaning the Glass Back Plates Weekly
In addition to the cleaning procedure described above, the plates should be cleaned
periodically after approximately every seven uses and/or every week depending on
individual lab throughput.
CAUTION
Wear a lab coat, safety goggles, and gloves during this cleaning process.
Consumables
Item
Quantity
Bleach (5.25% Sodium hypochlorite, NaOCl)
9“ x 13” Pyrex glass container
1
Fume hood
1
Backplate Tecan Storage rack
1
Deionized water
1L container for diluting bleach
CAUTION
Bleach is an irritant. Use caution when handling.
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Part # 15046990 Rev. B
Preparation
} Dilute bleach (5.25% sodium hypochlorite) with deionized water to 10% (0.525%
hypochlorite) in a 1L container. Mix thoroughly.
Example: Add 100 ml of bleach to 900 ml deionized water.
Cleaning Steps
1
Perform the following steps in the fume hood:
NOTE
Perform these steps in a location separate from the Infinium production lab.
a
b
c
Place glass container in the fume hood.
Place Pyrex glass back plates in the glass container with the plate reservoirs facing
up. Do not stack glass back plates on top of one another.
Add enough 10% bleach solution to cover the glass back plates (fill the glass
container to approximately half full).
2
Let the glass back plates soak in bleach in the fume hood for 1 hour.
3
Fill the Backplate Tecan Storage Rack container with deionized water and transfer it to
the fume hood.
4
After the 1 hour bleach soak, individually transfer the glass back plates to the
Backplate Tecan Storage Rack that is submerged in deionized water. Be careful not to
chip the glass back plates.
NOTE
The Backplate Tecan Storage Rack container with glass back plates can now be
transferred from the fume hood to a nearby sink with deionized water.
5
Dip the rack containing the glass back plates up and down 20 times. Be careful not to
chip the glass back plates.
6
Remove the Backplate Tecan Storage Rack containing the glass back plates and rinse
with deionized water.
7
Dispose of the deionized water from the Backplate Tecan Storage Rack container; rinse
and refill with fresh deionized water.
8
Return the rack containing the glass back plates to the Backplate Tecan Storage Rack
container.
9
Dip rack containing glass back plates up and down 20 times and then let soak for 5
minutes.
10 Repeat steps 7–9 four additional times.
11 Dispose of the deionized water.
12 Allow the glass back plates to dry in the storage rack or by leaning the glass back
plates at an angle against the black metal frames.
13 Once the glass back plates are clean and dry, us a Kimwipe to wash with 70% EtOH.
14 Dry the glass back plates with a Whoosh duster or laboratory air gun.
Infinium CytoSNP-850K Assay Protocol Guide
13
Lab Maintenance
WARNING
Do not perform this cleaning protocol in the BeadChip production lab.
Bleach fumes can degrade both the BeadChips and the fluorescent dyes used
in the Infinium Assay.
Lab Setup and Maintenance
15 To prevent surface damage to the glass back plates, store them in the Te-Flow Flow Cell
storage box.
Maintaining and Calibrating Lab Equipment
Calibrate the following equipment on an annual basis, according to the specifications
listed.
Table 1 Lab Equipment Maintenance
Equipment
Specification
High-speed
Determine offset at 1600 rpm, 1800 rpm, 2000 rpm, and 2300 rpm
microplate shaker
using a calibrated stroboscope
Hybridization oven
±2°C at 40°C and 60°C
Heated cover heat
±1°C at 37°C and 95°C
block
Temperature probe
±1°C
for TeFlow
Calibrating the Vortexer
The vortexer’s displayed speed may vary from the actual vortex speed. A digital
stroboscope should be used to determine the actual vortex speed. Once you have
determined the actual vortex speed, record it along with the displayed speed and use these
measurements for reference throughout the assay.
Follow the instructions below to calibrate the Signature* High-Speed Microplate Shaker
(VWR International, catalog # 13500-890).
1
Set the digital stroboscope display speed to 1600 rpm.
2
Turn the vortexer on and adjust the vortexer speed until the actual vortex speed
reaches 1600 rpm.
3
Record the displayed vortexer speed and note down that it represents an actual speed
of 1600 rpm.
4
Use the same method described above to determine the displayed speed for the actual
vortex speed of 1800 rpm. These two vortex speeds are used in the CytoSNP-850K
Assay.
5
Place a label on the vortexer with the calibration information. The following table
provides an example of a calibration label you can create and affix to your vortexer.
Table 2 Sample Vortexer Calibration Label
Display Speed
Actual Vortex Speed
1450 rpm
1600 rpm
1625 rpm
1800 rpm
Calibration Date
xx-xx-xx
xx-xx-xx
Calibrating the Hybridization Oven
Contact Illumina Technical Support to schedule yearly maintenance and calibration.
For more information, see the Hybridization Oven System Guide.
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Chapter 3 CytoSNP-850K Assay Protocol
CytoSNP-850K Assay Workflow
Equipment, Materials, and Reagents
Make and Incubate the MSA1 Plate
Fragment the DNA
Precipitate the DNA
Resuspend the DNA
Hybridize DNA to the BeadChip
Wash the BeadChip
Extend and Stain (XStain) BeadChips
Image BeadChip
Infinium CytoSNP-850K Assay Protocol Guide
Chapter 3
CytoSNP-850K Assay
Protocol
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15
CytoSNP-850K Assay Protocol
CytoSNP-850K Assay Workflow
The following figure graphically represents the manual workflow for the Infinium
CytoSNP-850K Assay. This protocol describes the procedure for preparing DNA samples
on Infinium CytoSNP-850K BeadChips.
Figure 1 CytoSNP-850K Assay Manual Workflow
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Part # 15046990 Rev. B
Equipment, Materials, and Reagents
Equipment, Materials, and Reagents
User-Supplied Equipment
Table 3 User-Supplied Equipment
Item
Catalog #
Microtiter plate centrifuge with g-force range 8
to 3000 × g
(two, for pre- and post-Amp use; post-Amp
centrifuge must include refrigeration)
N/A
User-Supplied Materials
Table 4 User Supplied Materials
Item
Catalog #
Tube vortexer
N/A
Tube rack
VWR International catalog # 66023-526
Combination optical tachometer/stroboscope
Cole-Parmer catalog # A-87700-06
Adaptor for centrifuge plates and tubes
N/A
Pipettes (two separate sets, for pre- and postAmp use)
2 each of P-20, P-200, and P-1000
8-channel precision pipettes (two separate sets,
for pre- and post-Amp use)
50 μL to 300 μL
Stop watch/timer (two, for pre- and post-Amp
use)
N/A
Forceps
VWR International catalog # 25601-008
Powder-free gloves (two separate stocks, for
pre- and post-Amp use)
N/A
Lab coats (two, for pre- and post-Amp use)
N/A
Safety glasses (two, for pre- and post-Amp
use)
N/A
15 mL conical tubes
N/A
0.8 mL storage plate (MIDI plate), conical well
bottom
Abgene catalog # AB-0765
Heat Sealing foil sheets, Thermo-Seal
Abgene catalog # AB-0559
Rubber sealing roller
VWR International catalog #RLPLTVWR01 or
equivalent
96-well cap mats (pierceable, nonautoclavable)
Abgene catalog # AB-0566
Absorbent pads
N/A
Infinium CytoSNP-850K Assay Protocol Guide
17
CytoSNP-850K Assay Protocol
Item
Catalog #
Kimwipes®
N/A
Mild detergent, such as Alconox® Powder
VWR International catalog # 21835-032
Aerosol filter pipette tips (two separate stocks,
for pre- and post-Amp use)
20uL, 200uL, 1000uL
Disposable pipetting troughs
VWR International catalog # 21007-970
Vacuum source (greater than 508 mm Hg (0.68
bar)
N/A
Vacuum gauge for vacuum desiccator
(recommended)
N/A
Illumina-Supplied Reagents
Table 5 Illumina-Supplied Reagents - Infinium Analysis Kit
Item
Part #
CytoSNP-850K DNA Analysis Kit (2 BeadChip
pack)
WG-322-1002
CytoSNP-850K DNA Analysis Kit (6 BeadChip
pack)
WG-322-1003
CytoSNP-850K DNA Analysis Kit (12 BeadChip
pack)
WG-322-1004
NOTE
A DNA Analysis Kit includes the BeadChips and reagents. Reagents are supplied
in sufficient quantities to run all the BeadChips at the same time. It is important
that the frozen components do not go through more than two freeze-thaw
cycles from delivery.
The wash buffers (PB1 and XC4) are supplied in sufficient volume suitable for
single use. XC4 reagent (resuspended) can be reused up to 6 times over a two
week period (maximum 24 BeadChips). PB1 and XC4 can be ordered separately
as a buffer kit (GT-201-1005).
CAUTION
DMAP files should be downloaded before beginning the Infinium protocol scanning of BeadChips cannot occur without the relevant associated DMAP file.
For guidance see Download DMAP files on page 58.
Table 6 Illumina-Supplied Reagents - supplied in the Infinium Analysis Kit
18
Item
Part #
MA1—Multi-Sample Amplification 1 Mix
11202880
MSM—Random Primer Mix
11203410
MA2—Multi-Sample Amplification Master Mix
11203401
FMS—Fragmentation solution
11203428
Part # 15046990 Rev. B
Part #
PM1—Precipitation solution
11203436
RA1—Resuspension, hybridization, and wash
solution
11292441
PB2—Humidifying buffer used during
hybridization
11191130
PB1—Reagent used to prepare BeadChips for
hybridization
11291245
XC1—XStain BeadChip solution 1
11208288
XC2—XStain BeadChip solution 2
11208296
TEM—Two-Color Extension Master Mix
11208309
XC3—XStain BeadChip solution 3
11208392
STM—Superior Two-Color Master Mix
11288046
ATM—Anti-Stain Two-Color Master Mix
11208317
XC4—XStain BeadChip solution 4
11208430
Equipment, Materials, and Reagents
Item
Table 7 Illumina-Supplied Items
Item
Part #
iScan System (110V/220V)
SY-101-1001
Infinium Option Starter Package (220V)
WG-15-305
Infinium Option Starter Package (110V)
WG-15-304
NOTE
New users, not already running the Infinium assay, will require the Infinium
Option Starter Package. The Infinium Option Starter Package includes:
hybridization oven with rocker, high-speed microplate shaker, a hybex
heatblock and accessories, heat sealer, dessicator, hybridization chamber and
gaskets, Tecan TeFlow wash chamber rack and accessories, amongst other items
and is available in 220V and 110V version
Infinium CytoSNP-850K Assay Protocol Guide
19
CytoSNP-850K Assay Protocol
Make and Incubate the MSA1 Plate
These steps explain how to prepare an MSA1 plate. This process denatures and neutralizes
the samples, preparing them for overnight incubation. During incubation, the genomic
DNA is uniformly amplified, generating a sufficient quantity of each individual DNA
sample to be used once in the Infinium CytoSNP-850K Assay.
Estimated Time
Hands-on time: 60 minutes
Incubation time: 20–24 hours
Consumables
Item
Quantity
MA1
1 tube
MSM
1 tube
MA2
1 tube
0.1 N NaOH
1 ml
96-well 0.8 ml microtiter plate (MIDI)
1 plate
DNA plate with DNA samples (50 ng/μl)
1 plate or in tubes
Preparation
} In preparation for the incubation process, preheat the Illumina Hybridization Oven in
the post-amp area to 37°C and allow the temperature to equilibrate.
} Thaw MA1, MSM, and MA2 tubes to room temperature. Gently invert at least 10 times
to mix contents, pulse centrifuge 280 × g to gather contents.
} Thaw DNA samples to room temperature.
} In the Lab planner, enter the SampleID and description for each sample.
} On the Lab Tracking Form, record:
• Date/Time
• Operator
• DNA plate barcode (if required)
• MSA1 plate barcode(s) (if required)
• MA1 tube lot number
• MA2 tube lot number
• MSM tube lot number
20
Part # 15046990 Rev. B
1
Dispense 20 μl MA1 into the MSA1 plate wells.
Fill wells according to the plate layout diagram below where column 1 contains
samples for a single BeadChip.
2
Transfer 4 μl of the DNA sample (50 ng/μl) from the DNA plate/tubes to the
corresponding wells in the MSA1 plate.
3
In the CytoSNP-850K Lab Planner, record the original DNA sample ID for each well in
the MSA1 plate.
4
Dispense 4 μl 0.1N NaOH into each well of the MSA1 plate that contains MA1 and
sample.
5
Seal the MSA1 plate with a 96-well cap mat.
NOTE
Orient the cap mat so that A1 on the cap matches A1 on the plate. To prevent
evaporation and spills, which could lead to assay variability and crosscontamination, ensure that all 96 caps are securely seated in the wells.
6
Vortex the plate at 1600 rpm for 1 minute.
7
Pulse centrifuge to 280 × g.
8
Incubate for 10 minutes at room temperature.
9
Carefully remove the cap mat.
NOTE
When you remove a cap mat, set it aside, upside down, in a safe location for use
later in the protocol.
10 Dispense 68 μl MA2 into each well of the MSA1 plate containing sample.
11 Dispense 75 μl MSM into each well of the MSA1 plate containing sample.
12 Reseal the MSA1 plate with the cap mat.
NOTE
When you place the cap mat back on the plate, be sure to match it to its original
plate and orient it correctly.
13 Vortex the sealed MSA1 plate at 1600 rpm for 1 minute.
14 Pulse centrifuge to 280 × g.
15 Incubate in the Illumina Hybridization Oven for 20–24 hours at 37°C.
Infinium CytoSNP-850K Assay Protocol Guide
21
Make and Incubate the MSA1 Plate
Steps
CytoSNP-850K Assay Protocol
On the Lab Tracking Form, record the start and stop times.
16 Discard unused reagents in accordance with facility standards.
17 Proceed to Fragment the DNA.
NOTE
The remainder of the protocol should be performed in the Post-Amp laboratory
area.
22
Part # 15046990 Rev. B
Fragment the DNA
Fragment the DNA
These steps explain how to enzymatically fragment the DNA, using end-point
fragmentation to avoid over-fragmentation.
Estimated Time
Hands-on time: ~30 minutes
Incubation time: 1 hour
Consumables
Item
Quantity
FMS
1 tube
Preparation
} Preheat the heat block with the MIDI plate insert to 37°C.
} Thaw FMS tube to room temperature. Gently invert at least 10 times to mix contents,
pulse centrifuge 280 × g to gather contents.
} Remove the MSA1 plate from the heat block.
} On the Lab Tracking Form, record:
• Date/Time
• Operator
• FMS tube lot number
Steps
1
Pulse centrifuge the MSA1 plate to 280 × g.
2
Carefully remove the cap mat.
NOTE
When you remove a cap mat, set it aside, upside down, in a safe location for use
later in the protocol.
3
Add 50 μl FMS to each well containing sample.
4
Seal the MSA1 plate with the 96-well cap mat.
NOTE
When you place the cap mat back on the plate, be sure to match it to its original
plate and orient it correctly.
5
Vortex the plate at 1600 rpm for 1 minute.
6
Pulse centrifuge the plate to 280 × g.
7
Place the sealed plate on the 37°C heat block for 1 hour.
On the Lab Tracking Form, record the start and stop times.
8
Discard unused reagents in accordance with facility standards.
9
Do one of the following:
• Continue to the next step Precipitate DNA, prepare for the next step during the 37°C
heat block incubation.
Infinium CytoSNP-850K Assay Protocol Guide
23
CytoSNP-850K Assay Protocol
• If you do not plan to proceed to the next step within the next 2 hours, store the
sealed MSA1 plate at -15°C to -25°C for no more than 24 hours.
SAFE STOPPING POINT
This is a good stopping point in the process.
24
Part # 15046990 Rev. B
These steps explain how to precipitate the DNA samples using 100% 2-propanol and PM1.
Estimated Time
Hands-on time: ~30 minutes
Incubation: 30 minutes
Centrifugation: 20 minutes
Dry time: 1 hour
Consumables
Item
Quantity
PM1
1 tube
100% 2-propanol
15 ml
Preparation
} Preheat heat block to 37°C.
} Do one of the following:
• If you froze the MSA1 plate after fragmentation, thaw it to room temperature, and
then pulse centrifuge the MSA1 plate to 280 × g.
• If you proceeded immediately from Fragment the MSA1 Plate, leave the plate in the
37°C heat block until setup is complete.
} Bring PM1 to room temperature. Gently invert at least 10 times to mix contents.
} On the Lab Tracking Form, record:
• Date/Time
• Operator
• PM1 tube lot number
• 100% 2-propanol lot number and date opened
Steps
1
Remove the 96-well cap mat and add 100 μl PM1 to each MSA1 plate well containing
sample.
2
Seal the plate with the cap mat.
NOTE
When you place the cap mat back on the plate, be sure to match it to its original
plate and orient it correctly.
3
Vortex the sealed plate at 1600 rpm for 1 minute.
4
Place the sealed plate on the 37°C heat block for 5 minutes.
5
Pulse centrifuge to 280 × g.
NOTE
Set centrifuge to 4°C in preparation for the next centrifuge step.
6
Add 300 μl 100% 2-propanol to each well containing sample.
Infinium CytoSNP-850K Assay Protocol Guide
25
Precipitate the DNA
Precipitate the DNA
CytoSNP-850K Assay Protocol
7
Carefully seal the MSA1 plate with a new, dry cap mat, taking care not to shake the
plate in any way until the cap mat is fully seated.
8
Invert the plate at least 10 times to mix contents thoroughly.
9
Incubate at 4°C for 30 minutes.
On the Lab Tracking Form, enter the start and stop times.
10 Place the sealed MSA1 plate in the centrifuge opposite another plate of equal weight.
11 Centrifuge to 3000 × g at 4°C for 20 minutes.
CAUTION
Perform the next step immediately after the centrifuge stops to avoid dislodging
the blue pellet. If any delay occurs, repeat the 20-minute centrifugation before
proceeding.
12 Remove the MSA1 plate from centrifuge. Remove the cap mat and discard it.
13 Over an absorbent pad, decant the supernatant by quickly inverting the MSA1 plate.
Drain liquid onto the absorbent pad and then smack the plate down, avoiding the
liquid that was just drained onto the pad.
RELATED TRAINING
To learn more, see the Decanting the Plate topic in the Infinium, Lesson 2: Assay
Techniques training video on the Array Online Training page of the Illumina
web site
14 Tap firmly several times for 1 minute or until all wells are devoid of liquid.
CAUTION
Keep plate inverted. To ensure optimal performance, do not allow supernatant
in wells to pour into other wells.
15 Leave the uncovered, inverted plate on the tube rack for 1 hour at room temperature to
air dry the pellet.
After drying, blue pellets should be present at the bottoms of the wells.
Figure 2 Uncovered MSA1 Plate Inverted for Air Drying
16 On the Lab Tracking Form, enter the start and stop times.
17 Discard unused reagents in accordance with facility standards.
18 Do one of the following:
• Continue to the next step, Resuspend the DNA.
• If you do not plan to proceed to the next step immediately, seal the MSA1 plate
with a new cap mat and store it at -15°C to -25°C for no more than 24 hours.
26
Part # 15046990 Rev. B
Precipitate the DNA
SAFE STOPPING POINT
This is a good stopping point in the process.
Infinium CytoSNP-850K Assay Protocol Guide
27
CytoSNP-850K Assay Protocol
Resuspend the DNA
These steps explain how to resuspend the precipitated DNA using RA1.
Estimated Time
Hands-on time: ~30 minutes
Incubation time: 1 hour
Consumables
Item
Quantity
RA1
46 μl per sample well
NOTE
Pour out only the recommended volume of RA1 needed for the suggested
number of samples listed in the consumables table. Additional RA1 is used later
in the Extend and Stain (XStain) the BeadChip step.
WARNING
This protocol uses an aliphatic amide (formamide) that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion,
skin contact, and eye contact. For more information, consult the material
data safety sheet for this assay at http://www.illumina.com/msds. Dispose of
containers and any unused contents in accordance with the governmental
safety standards for your region.
Preparation
} If you plan to continue to the Hybridization step immediately after resuspension, begin
preheating the heat block to 95°C now.
} Preheat the Illumina Hybridization Oven to 48°C.
} Turn on the heat sealer to preheat. Allow 10 minutes.
} Invert the previously thawed RA1 several times to re-dissolve the solution.
NOTE
RA1 might form visible precipitates or crystals. Before use, hold the bottle in
front of a light and visually inspect,invert several times to re-dissolve the
solution. [Optional] Heat at 48°C for 15 minutes until RA1 is completely
dissolved.
} On
•
•
•
the Lab Tracking Form, record:
Date/Time
Operator
RA1 bottle lot number
NOTE
Use fresh RA1 for each protocol step where it is required. RA1 that has been
stored properly and has not been dispensed for use in either the XStain or
Resuspension step is considered fresh RA1.
Steps to Resuspend the DNA
1
28
Add 46 μl RA1 to each well of the MSA1 plate containing a DNA pellet.
Part # 15046990 Rev. B
2
Apply a foil heat seal (with the dull side facing down) to the MSA1 plate by firmly and
evenly holding the heat sealer sealing block down for 5 seconds.
3
Immediately remove the MSA1 plate from the heat sealer and forcefully roll the rubber
plate sealer over the plate until you can see all 96 well indentations through the foil.
Repeat application of the heat sealer if all 96 wells are not defined.
4
Place the sealed plate in the Illumina Hybridization Oven and incubate for 1 hour at
48°C.
On the Lab Tracking Form, enter the start and stop times.
5
Vortex the plate at 1800 rpm for 1 minute.
6
Pulse centrifuge to 280 × g.
NOTE
If you store the pellets at -15°C to -25°C for extended periods of time after the
precipitate process, you might need to repeat the vortexing and centrifugation
in the previous steps until the pellets are completely resuspended.
7
Discard unused reagents in accordance with facility standards.
8
Do one of the following:
• Continue to the next step, Hybridize the DNA to the BeadChip. If you plan to do so
immediately, it is safe to leave the MSA1 plate at room temperature for up to 1
hour.
• If you do not plan to proceed to the next step immediately:
— Store the sealed MSA1 plate at -15°C to -25°C for no more than 24 hours. Store
at -80°C if storing for more than 24 hours. Do not store the MSA1 plate for
longer than one week at -80°C.
— Store RA1 at 4°C for no more than 24 hours. Store at -15°C to -25°C if storing
for more than 24 hours.
SAFE STOPPING POINT
This is a good stopping point in the process.
Infinium CytoSNP-850K Assay Protocol Guide
29
Resuspend the DNA
Reserve leftover reagent in the bottle for the Extend and Stain (XStain) the BeadChip step
later in this protocol.
CytoSNP-850K Assay Protocol
Hybridize DNA to the BeadChip
These steps explain how to dispense the fragmented, resuspended DNA samples onto
BeadChips and incubate the BeadChips in the Illumina Hybridization Oven to hybridize
the samples onto the BeadChips.
RELATED TRAINING
To learn more, see the Hybridization topic in the Infinium, Lesson 2: Assay
Techniques training video on the Array Online Training page of the Illumina
web site.
Estimated Time
Hands-on time: ~30 minutes for 4 BeadChips (32 samples)
Incubation: 20 minutes
Cool-down: 30 minutes
Incubation: 16–24 hours
Consumables
Item
Quantity
PB2
1 tube
BeadChips
4
Hyb Chambers
1
Hyb Chamber gaskets
1
Hyb Chamber inserts
4
EtOH
330 ml
Preparation
} Ensure DMAP files for each BeadChip have been downloaded, scanning cannot
proceed without a DMAP file. For guidance seeDownload DMAP files on page 58
} If frozen, thaw MSA1 plate to room temperature, and then pulse centrifuge the MSA1
plate to 280 × g.
} If the heat block is not already heated, preheat the heat block to 95°C.
} Preheat the Illumina Hybridization Oven to 48°C and set the rocker speed to 5.
} Prepare the Illumina Hybridization Oven as follows:
a Preheat the oven to 48°C:
— Press the "F" button once to change the display to TSET.
— Press the "S" button to enter the set-temperature mode, and then use the
Increment/Decrement dial to set the oven to 48°C.
— Press the "S" button again to set 48°C as the temperature.
b Set the rocker speed to 5:
— Press the "F" button twice until SPd is indicated on the display.
— Press the "S" button to enter the rocker speed mode.
— Use the Increment/Decrement dial to set the rocker speed to "5".
— Press the "S" button again.
30
Part # 15046990 Rev. B
the Lab Tracking Form, record:
Date/Time
Operator
PB2 tube lot number
Prepare the Hybridization Chambers
1
Place the resuspended MSA1 plate on the heat block to denature the samples at 95°C
for 20 minutes.
On the Lab Tracking Form, enter the start and stop times.
2
During the 20-minute incubation, prepare the Hyb Chambers.
Place the following items on the bench top for use in this procedure:
Figure 3 BeadChip Hyb Chamber Components
A
B
C
Hyb Chambers
Hyb Chamber Gaskets
Hyb Chamber Inserts
NOTE
To ensure optimal results from Hyb Chambers keep the Hyb Chamber lids and
bases together. Adopt a labeling convention that keeps each Hyb Chamber base
paired with its original lid. Check Hyb Chamber lid-base pairs regularly to
ensure that the fit remains secure. Check hinges regularly for any signs of
abnormal wear or loose fittings. It is important that the hinges provide
adequate clamping strength to ensure an airtight seal between the lid and the
base. Record the Hyb Chamber that was used for each BeadChip, so that Hyb
Chambers can be investigated and evaluated in the event of sample evaporation
or other lab processing anomalies.
a
b
Remove the BeadChips from 2°C to 8°C storage, leaving the BeadChips in their
ziplock bags and mylar packages until you are ready to begin hybridization.
Place the Hyb Chamber gaskets into the Hyb Chambers.
— Match the wider edge of the Hyb Chamber gasket to the barcode-ridge side of
the Hyb Chamber.
Infinium CytoSNP-850K Assay Protocol Guide
31
Hybridize DNA to the BeadChip
} On
•
•
•
CytoSNP-850K Assay Protocol
Figure 4 Hyb Chamber and Gasket
A
B
C
D
Humidifying Buffer Reservoirs
Barcode Ridges
Narrower Edges
Wider Edges
— Lay the gasket into the Hyb Chamber, and then press it down all around.
Figure 5 Placing Gasket into Hyb Chamber
— Make sure the Hyb Chamber gaskets are properly seated.
Figure 6 Hyb Chamber with Gasket in Place
c
32
Dispense 400 μl PB2 into the required humidifying buffer reservoirs in the Hyb
Chamber, two for each BeadChip.
Part # 15046990 Rev. B
Hybridize DNA to the BeadChip
Figure 7 Dispensing PB2 into Hyb Chamber Reservoir
d
e
3
After filling the Hyb Chamber reservoirs with PB2, place the lid on the Hyb
Chamber right away to prevent evaporation. The lid does not need to be locked
down.
Leave the closed Hyb Chambers on the bench at room temperature until the
BeadChips are loaded with DNA sample. Load BeadChips into the Hyb Chamber
within one hour.
After the 20-minute incubation, remove the MSA1 plate from the heat block and place
it on the benchtop at room temperature for 30 minutes.
Load BeadChips
1
After the 30-minute cool down, pulse centrifuge the MSA1 plate to 280 × g.
2
Just before loading DNA samples, remove all BeadChips from their ziplock bags and
mylar packages.
CAUTION
Hold the BeadChip by the ends with your thumb and forefinger (thumb at the
barcode end). Do not hold the BeadChip by the sides near the sample inlets.
Avoid contacting the beadstripe area and sample inlets.
3
Place each BeadChip in a Hyb Chamber insert, orienting the barcode end so that it
matches the barcode symbol on the Hyb Chamber insert.
Infinium CytoSNP-850K Assay Protocol Guide
33
CytoSNP-850K Assay Protocol
Figure 8 Placing BeadChips into Hyb Chamber Inserts
4
Remove the foil seal from the MSA1 plate, and then, using a multi-channel precision
pipette, dispense 26 μl of each DNA sample onto the appropriate BeadChip section.
Figure 9 Loading Samples
a
b
34
Load samples A1-H1 from the MSA1 plate into the BeadChip.
Make sure that the pipette tip is in the sample inlet prior to dispensing.
Continue in this manner, following the color-coded sections shown below, until all
samples are loaded.
Part # 15046990 Rev. B
Hybridize DNA to the BeadChip
Figure 10 Distributing Sample in MSA1 Plate
5
On the CytoSNP-850K Lab Planner, record the BeadChip barcode for each group of
samples.
6
After loading all DNA onto the BeadChip, wait for the sample to disperse over the
entire surface.
7
Inspect the loading port to see if a large bolus of liquid remains.
Excess sample volume in the BeadChip loading port helps prevent low-intensity areas
resulting from evaporation.
Figure 11 Bolus of Liquid at Loading Port
If no excess liquid is visible, it is acceptable to add additional sample from the leftover
volume in the amplification plate until there is a large bolus around the loading port.
NOTE
Do not top off with RA1 (sample hyb buffer) as this will dilute the sample.
8
Record the top-off activity on the Lab Tracking Form.
9
Proceed immediately to the next section, Set up the BeadChips for Hybridization.
Infinium CytoSNP-850K Assay Protocol Guide
35
CytoSNP-850K Assay Protocol
Set up the BeadChips for Hybridization
CAUTION
For optimal performance, take care to keep the Hyb Chamber inserts
containing BeadChips steady and level when lifting or moving. Avoid shaking
and keep parallel to the lab bench at all times. Do not hold by the sides near the
sample inlets.
1
Load the Hyb Chamber inserts containing BeadChips into the Illumina Hyb Chamber.
Position the barcode end over the ridges indicated on the Hyb Chamber.
Figure 12 Placing Hyb Chamber Inserts into the Hyb Chamber 2
Place the back side of the lid onto the Hyb Chamber and then slowly bring down the
front end to avoid dislodging the Hyb Chamber inserts.
Figure 13 Seating Lid onto Hyb Chamber 3
Close the clamps on both sides of the Hyb Chamber so that the lid is secure and even
on the base (no gaps). It is best to close the clamps in a kitty-corner fashion, closing
first the top left clamp, then the bottom right, then the top right followed by the bottom
left.
4
Place the Hyb Chamber in the 48°C Illumina Hybridization Oven so that the Illumina
logo on top of the Hyb Chamber is facing you.
CAUTION
After loading the BeadChips into the Hyb Chambers, place the Hyb Chambers
into the Illumina Hybridization Oven immediately. Do not modify the
hybridization environment by adding additional fixtures or humidifying
elements. Leave the Hyb Chambers in the oven at the correct orientation and
temperature until hybridization is complete. Changes to the hybridization
environment can have unexpected effects on data quality.
36
Part # 15046990 Rev. B
Hybridize DNA to the BeadChip
Figure 14 Hyb Chamber Correctly Placed in Hyb Oven
Figure 15 Incorrectly Placed Hyb Chamber
NOTE
If you are stacking multiple Hyb Chambers in the Illumina Hybridization Oven,
make sure the feet of the top Hyb Chamber fit into the matching indents on top
of the bottom Hyb Chamber. This will hold the Hyb Chambers in place while
they are rocking. You can stack up to 3 Hyb Chambers per row for a maximum
total of 6 Hyb Chambers in the Hyb Oven.
5
[Optional] Start the rocker, setting the speed to 5.
6
Incubate at 48°C for at least 16 hours but no more than 24 hours.
7
On the Lab Tracking Form, enter the start and stop times.
8
Place RA1 at 4°C for use the next day.
9
Discard the MSA1 plate.
10 After the overnight incubation, proceed to Wash the BeadChip.
Infinium CytoSNP-850K Assay Protocol Guide
37
CytoSNP-850K Assay Protocol
Resuspend XC4 Reagent in Preparation for XStain
Keep the XC4 in the bottle in which it was shipped until you are ready to use it. Follow
these steps to resuspend the XC4 reagent in preparation for the Extend and Stain (XStain)
BeadChips on page 46
38
1
Add 330 ml fresh 100% EtOH to the XC4 bottle. The final volume will be 350 ml.
Each XC4 bottle has enough solution to process up to 24 BeadChips.
2
Shake the XC4 bottle vigorously to ensure complete resuspension.
Once resuspended, use XC4 at room temperature. You can store it at 2°C to 8°C and reuse up to 6 times over a two week period for a maximum of 24 BeadChips.
Part # 15046990 Rev. B
Wash the BeadChip
Wash the BeadChip
These steps explain how to prepare the BeadChips for the staining process.
Estimated Time
Hands-on time: ~15 minutes
Cool-down: 25 minutes
Consumables
Item
Quantity
PB1
550 ml (up to 4 BeadChips)
Multi-Sample BeadChip Alignment Fixture
1 (per 4 BeadChips)
Te-Flow Flow-Through Chambers (with Black Frames,
Spacers, Glass Back Plates, and Clamps)
1 (per BeadChip)
Wash Dish
2
Wash Rack
1
CAUTION
Pour out only the recommended reagent volume needed for the suggested
number of samples listed in the Consumables table of each section. Some
reagents are used later in the protocol.
WARNING
This protocol uses an aliphatic amide (formamide) that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion,
skin contact, and eye contact. For more information, consult the material
data safety sheet for this assay at http://www.illumina.com/msds. Dispose of
containers and any unused contents in accordance with the governmental
safety standards for your region.
Preparation
} Ensure the water circulator is filled to the appropriate level.
} Turn on the water circulator. Set it to a temperature that brings the Chamber Rack to
44°C at equilibrium.
} Remove bubbles trapped in the Chamber Rack.
} Test three or more locations on the Chamber Rack, using the Illumina Temperature
Probe. All locations should be at 44°C ± 0.5°C. If the temperature on the probe is not
within ± 0.5°C, adjust the water circulator control knob to obtain 44°C ± 0.5°C on the
temperature probe.
} In preparation for the wash step, remove each Hyb Chamber from the Illumina
Hybridization Oven. Let cool on the benchtop for 25 minutes prior to opening.
} Have ready on the lab bench:
• Two wash dishes:
— Containing 200 ml PB1, and labeled as such
• Multi-Sample BeadChip Alignment Fixture
— Using a graduated cylinder, fill with 150 ml PB1
• Te-Flow Flow-Through Chamber components:
Infinium CytoSNP-850K Assay Protocol Guide
39
CytoSNP-850K Assay Protocol
} On
•
•
•
— Black frames
— Spacers (separated for ease of handling)
— Clean glass back plates (see Cleaning the Glass Back Plates on page 12)
— Clamps
the Lab Tracking Form, record:
Date/Time
Operator
PB1 bottle lot number
In preparation for XStain BeadChip step
} Thaw XC1, XC2, TEM, STM, ATM, and 95% Formamide / 1mM EDTA to room
temperature. Gently invert the reagent tubes and bottles at least 10 times to mix
contents.
} Shake the XC4 bottle vigorously to ensure complete resuspension. If necessary, vortex
until completely dissolved.
Steps to Wash BeadChip
1
Attach the wire handle to the rack and submerge the wash rack in the first wash dish
containing 200 ml PB1.
Figure 16 Wash Rack in Wash Dish Containing PB1
2
Remove a BeadChip from the Hyb Chamber and then remove its cover seal.
RELATED TRAINING
To learn more, see the Removing the IntelliHyb Seal topic in the Infinium, Lesson
2: Assay Techniques training video on the Array Online Training page of the
Illumina web site.
NOTE
To ensure no solution splatters on you, Illumina recommends removing the
cover seal over an absorbent cloth or paper towels, preferably in a hood.
a
40
Using powder-free gloved hands, hold the BeadChip securely and by the edges in
one hand. Avoid contact with the sample inlets. The barcode should be facing up
and be closest to you, and the top side of the BeadChip should be angled slightly
away from you.
Part # 15046990 Rev. B
Remove the entire seal in a single, continuous motion. Start with a corner on the
barcode end and pull with a continuous upward motion away from you and
towards the opposite corner on the top side of the BeadChip.
Figure 17 Removing the Cover Seal
c
Discard the cover seal.
CAUTION
Do not touch the arrays!
3
Immediately and carefully slide the BeadChip into the wash rack, making sure that the
BeadChip is completely submerged in the PB1.
Figure 18 Submerging BeadChips in Wash Dish Containing PB1
4
Repeat steps 2 and 3 if necessary until all BeadChips are transferred to the submerged
wash rack.
5
Once all the BeadChips are in the wash rack, move the wash rack up and down for
1 minute, breaking the surface of the PB1 with gentle, slow agitation.
6
Move the wash rack to the other wash dish containing clean PB1. Make sure the
BeadChips are completely submerged.
7
Move the wash rack up and down for 1 minute, breaking the surface of the PB1 with
gentle, slow agitation.
8
When you remove the BeadChips from the wash rack, inspect them for remaining
residue.
Infinium CytoSNP-850K Assay Protocol Guide
41
Wash the BeadChip
b
CytoSNP-850K Assay Protocol
NOTE
Residue that can adversely affect results is sometimes left on BeadChips after
seals are removed. If there is residue left on the BeadChips after the second PB1
wash, use a 200 μl pipette tip for each BeadChip and slowly and carefully scrape
off the residues outward (away) from the bead-sections under PB1. Use a new
pipette tip for each BeadChip. Then, continue with the protocol.
Assemble Flow-Through Chambers
RELATED TRAINING
To learn more about and assembling the Flow-Through Chambers, see the
Flow-Through Chamber topic in the Infinium, Lesson 2: Assay Techniques
training video on the Array Online Training page of the Illumina web site.
1
If you have not done so, fill the BeadChip Alignment Fixture with 150 ml PB1.
2
For each BeadChip to be processed, place a black frame into the BeadChip Alignment
Fixture.
Figure 19 Placing Black Frames into BeadChip Alignment Fixture, Pre-Filled with PB1
3
Place each BeadChip into a black frame, aligning its barcode with the ridges stamped
onto the Alignment Fixture. Each BeadChip should be fully immersed in PB1.
Figure 20 Placing BeadChip into Black Frame on Alignment Fixture
42
Part # 15046990 Rev. B
Place a clear spacer onto the top of each BeadChip. Use the Alignment Fixture grooves
to guide the spacers into proper position.
NOTE
Be sure to use the clear plastic spacers, not the white ones.
Figure 21 Placing Clear Plastic Spacer onto BeadChip
5
Place the Alignment Bar onto the Alignment Fixture.
The groove in the Alignment Bar should fit over the tab on the Alignment Fixture.
Figure 22 Placing Alignment Bar onto Alignment Fixture
6
Place a clean glass back plate on top of the clear spacer covering each BeadChip. The
plate reservoir should be at the barcode end of the BeadChip, facing inward to create a
reservoir against the BeadChip surface.
Infinium CytoSNP-850K Assay Protocol Guide
43
Wash the BeadChip
4
CytoSNP-850K Assay Protocol
Figure 23 Placing Glass Back Plate onto BeadChip
A
B
7
Reservoir at Barcode End of Glass Back Plate
Glass Back Plate in Position
Attach the metal clamps onto each Flow-Through Chamber as follows:
a Gently push the glass back plate up against the Alignment Bar with one finger.
b Place the first metal clamp around the Flow-Through Chamber so that the clamp is
5 mm from the top edge.
c Place the second metal clamp around the Flow-Through Chamber at the barcode
end, 5 mm from the bottom of the reagent reservoir.
Figure 24 Securing Flow-Through Chamber Assembly with Metal Clamps
A
B
C
8
44
One Stripe Shows Between First Clamp and Alignment Bar
Glass Back Plate Pressed Against Alignment Bar
No Stripes Show Between Second Clamp and Barcode
Remove the assembled flow-through chamber from the alignment fixture and, using
scissors, trim the spacer at the non-barcode end of the assembly. Slip the scissors up
over the barcode to trim the other end.
Part # 15046990 Rev. B
Wash the BeadChip
Figure 25 Trimming Spacer Ends from Flow-Through Chamber Assembly
A
B
9
Trim Spacer at Non-Barcode End of Flow-Through Chamber
Trim Spacer at Barcode End of Flow-Through Chamber
Continue to the next step, Extend and Stain (XStain) BeadChip.
CAUTION
Keep assembled Flow-Through Chambers on the lab bench in a horizontal
position while you perform the preparation steps for the Extend and Stain
(XStain) BeadChip step in this protocol. Do not place the assembled chambers on
absorbent paper. Do not place the Flow-Through Chambers in the Chamber
Rack until the preparation is complete.
10 Wash the Hyb Chamber reservoirs with DiH2O.
CAUTION
It is important to wash the Hybridization Chamber reservoirs immediately and
thoroughly to ensure that no traces of PB2 remain in the wells.
11 Discard unused reagents in accordance with facility standards.
Infinium CytoSNP-850K Assay Protocol Guide
45
CytoSNP-850K Assay Protocol
Extend and Stain (XStain) BeadChips
These steps explain how to wash unhybridized and non-specifically hybridized DNA
sample from the BeadChips, add labeled nucleotides to extend the primers hybridized to
the DNA, stain the primers, disassemble the Flow-Through Chambers, and coat the
BeadChips for protection.
Estimated Time
Hands-on time: ~3 hours
Dry time: 1 hour
Consumables
Item
Quantity
RA1
5 ml (per 4 BeadChips)
XC1
1 tube (per 4 BeadChips)
XC2
1 tube (per 4 BeadChips)
TEM
1 tube (per 4 BeadChips)
XC3
25 ml (per 4 BeadChips)
STM (Make sure that all STM tubes indicate the same
stain temperature on the label)
1 tube (per 4 BeadChips)
ATM
1 tube (per 4 BeadChips)
PB1
310 ml (up to 4 BeadChips)
XC4
310 ml (up to 4 BeadChips)
Alconox Powder Detergent
as needed
EtOH
as needed
95% formamide/1 mM EDTA
10 ml (up to 4 BeadChips)
CAUTION
Pour out only the recommended reagent volume needed for the suggested
number of samples listed in the Consumables table of each section.
WARNING
This protocol uses an aliphatic amide (formamide) that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion,
skin contact, and eye contact. For more information, consult the material
data safety sheet for this assay at http://www.illumina.com/msds. Dispose of
containers and any unused contents in accordance with the governmental
safety standards for your region.
Preparation
} Place the reagent tubes in a rack in the order in which they will be used: RA1, XC1,
XC2, TEM, 95% Formamide / 1mM EDTA, STM, XC3, ATM.
46
Part # 15046990 Rev. B
NOTE
RA1 might form visible precipitates or crystals. Before use, hold the bottle in
front of a light and visually inspect to make sure that all precipitates have
completely dissolved. Heat to dissolve, if necessary.
} On
•
•
•
•
•
•
•
•
•
•
•
the Lab Tracking Form, record:
Date/Time
Operator
RA1 lot number
XC3 lot number
XC1 lot number
XC2 lot number
TEM lot number
STM lot number
ATM lot number
PB1 lot number
XC4 lot number
Set Up the Chamber Rack
NOTE
To learn more about removing bubbles from the Chamber Rack, see the FlowThrough Chambers topic in the Infinium, Lesson 2: Assay Techniques training
video on the Array Online Training page of the Illumina web site.
1
Ensure the water circulator reservoir is filled with water to the appropriate level.
2
If not already done so, turn on the water circulator and set it to a temperature that
brings the Chamber Rack to 44°C at equilibrium.
This temperature may vary depending on facility ambient conditions.
Infinium CytoSNP-850K Assay Protocol Guide
47
Extend and Stain (XStain) BeadChips
NOTE
It is important to use fresh RA1 for each protocol step in the assay where it is
required. RA1 that has been stored properly and has not been dispensed
previously is considered fresh RA1. After RA1 has been poured out into a
reservoir and exposed to room temperature air for extended periods of time, it
is no longer fresh.
CytoSNP-850K Assay Protocol
Figure 26 Water Circulator Connected to Chamber Rack
A
B
C
Chamber Rack
Water Circulator with Programmable Temperature Controls
Reservoir Cover
3
The temperature displayed on the water circulator LCD screen may differ from the
actual temperature on the Chamber Rack. Confirm the actual temperature using the
temperature probe for the Chamber Rack.
4
You must remove bubbles trapped in the Chamber Rack each time you run this
process.
5
Use the Illumina Temperature Probe in several locations to ensure that the Chamber
Rack is at 44°C. All locations should be at 44°C ± 0.5°C.
NOTE
Do not leave the temperature probe in the first three rows of the Chamber
Rack. Reserve this space for BeadChips.
48
Part # 15046990 Rev. B
6
For accurate temperature measurement, ensure the Temperature Probe is touching the
base of the Chamber Rack.
Single-Base Extension
CAUTION
The remaining steps must be performed without interruption.
1
When the Chamber Rack reaches 44°C ± 0.5°C, quickly place each Flow-Through
Chamber assembly into the Chamber Rack.
2
Ensure each Flow-Through Chamber is properly seated on its rack to allow adequate
heat exchange between the rack and the chamber.
3
On the Lab Tracking Form, record the Chamber Rack position for each BeadChip.
4
Into the reservoir of each Flow-Through Chamber, dispense:
a 150 μl RA1. Incubate for 30 seconds. Repeat 5 times.
CAUTION
Do not allow pipette tips to contact BeadChip surface. Touch off in the reservoir
of the glass back plate.
Figure 28 Dispensing RA1 into Each Flow-Through Chamber
Infinium CytoSNP-850K Assay Protocol Guide
49
Extend and Stain (XStain) BeadChips
Figure 27 Illumina Temperature Probe and Temperature Probe in Chamber Rack
CytoSNP-850K Assay Protocol
b
c
d
e
f
g
h
5
450 μl XC1. Incubate for 10 minutes.
450 μl XC2. Incubate for 10 minutes.
200 μl TEM. Incubate for 15 minutes.
450 μl 95% formamide/1 mM EDTA. Incubate for 1 minute. Repeat once.
Incubate 5 minutes.
Begin ramping the Chamber Rack temperature to the temperature indicated on the
STM tube ± 0.5°C.
450 μl XC3. Incubate for 1 minute. Repeat once.
Wait until the Chamber Rack reaches the correct temperature.
Stain BeadChip
1
Into the reservoir of each Flow-Through Chamber, dispense:
a 250 μl STM. Incubate for 10 minutes.
b 450 μl XC3. Incubate for 1 minute. Repeat once.
c Wait 5 minutes.
d 250 μl ATM. Incubate for 10 minutes.
e 450 μl XC3. Incubate for 1 minute. Repeat once.
f Wait 5 minutes.
g 250 μl STM. Incubate for 10 minutes.
h 450 μl XC3. Incubate for 1 minute. Repeat once.
i
Wait 5 minutes.
j
250 μl ATM. Incubate for 10 minutes.
k 450 μl XC3. Incubate for 1 minute. Repeat once.
l
Wait 5 minutes.
m 250 μl STM. Incubate for 10 minutes.
n 450 μl XC3. Incubate for 1 minute. Repeat once.
o Wait 5 minutes.
2
Immediately remove the Flow-Through Chambers from the Chamber Rack and place
horizontally on a lab bench at room temperature.
Wash and Coat BeadChips
Before starting the Wash and Coat process, please read these important notes:
} Take the utmost care to minimize the chance of lint or dust entering the wash dishes,
which could transfer to the BeadChips. Place wash dish covers on wash dishes when
stored or not in use. Clean wash dishes with low-pressure air to remove particulates
before use.
} Place a clean tube rack on top of several layers of Kimwipes or on an absorbent pads.
You will place the staining rack containing BeadChips on this tube rack after removing
it from the XC4 wash dish.
} Prepare an additional clean tube rack that fits the internal dimensions of vacuum
desiccator for removal of the BeadChips. Allow one rack per 8 BeadChips. No
Kimwipes are required under this tube rack.
} Wash the tube racks and wash dishes thoroughly after use. Rinse with DI water.
Immediately following wash, place racks and wash dishes upside down on a wash
rack to dry.
50
Part # 15046990 Rev. B
Place the following items on the bench:
} 1 staining rack
} 1 vacuum desiccator
} 1 tube rack
} Self-locking tweezers
} Large Kimwipes
} Vacuum hose
Steps
1
Set up two top-loading wash dishes, labeled "PB1" and "XC4".
2
To indicate the fill volume before filling wash dishes with PB1 and XC4, pour 310 ml
water into the wash dishes and mark the water level on the side. Empty the water from
the wash dish. This enables you to pour reagent directly from the PB1 and XC4 bottles
into the wash dishes, minimizing contaminant transfer from labware to wash dishes.
Figure 29 PB1 and XC4 Wash Dishes with Staining Rack
A
B
Wash Dishes
Staining Rack
3
Pour 310 ml PB1 into the wash dish labeled “PB1.”
4
Submerge the unloaded staining rack into the wash dish with the locking arms and tab
facing towards you. This orients the staining rack so that you can safely remove the
BeadChips. Let the staining rack sit in the wash dish. You will use it to carry the
BeadChips after disassembling the Flow-Through Chambers.
Infinium CytoSNP-850K Assay Protocol Guide
51
Extend and Stain (XStain) BeadChips
Equipment Needed
CytoSNP-850K Assay Protocol
Figure 30 Staining Rack Locking Arms and Tab
A
B
5
Locking Arms
Tab
One at a time, disassemble each Flow-Through Chamber:
a Use the dismantling tool to remove the two metal clamps.
CAUTION
It is important to use the dismantling tool to avoid chipping the glass back
plates.
Figure 31 Removing the Metal Clamps from Flow-Through Chamber
b
Remove the glass back plate by lifting the glass straight up.
CAUTION
Do not slide the glass along the BeadChip. This can cause damage to the
BeadChip.
c
d
52
Set the glass back plate aside. When you finish the XStain protocol, clean the glass
back plates. See Cleaning the Glass Back Plates on page 12 for instructions.
Remove the spacer. To avoid damaging the stripes on the BeadChip, pull the
spacer out so that the long sides slide along the sides of the BeadChip.
Part # 15046990 Rev. B
e
Remove the BeadChip from the black frame.
CAUTION
Do not touch the face of the BeadChips. Handle them by the barcode end or by
the edges.
6
Place the BeadChips in the staining rack while it is submerged in PB1. The BeadChip
barcodes should face away from you; the locking arms on the handle should face
towards you.
If necessary, briefly lift the staining rack out of the wash dish to seat the BeadChip.
Replace it immediately after inserting each BeadChip.
7
Ensure that the BeadChips are completely submerged.
CAUTION
Do not allow the BeadChips to dry. Submerge each BeadChip in the wash dish
as soon as possible.
8
Slowly move the staining rack up and down 10 times, breaking the surface of the
reagent.
NOTE
If the top edges of the BeadChips begin to touch during either PB1 or XC4
washes, gently move the staining rack back and forth to separate the slides. It is
important for the solution to circulate freely between all BeadChips.
Figure 32 Washing BeadChips in PB1
9
Soak for 5 minutes.
10 Shake the XC4 bottle vigorously to ensure complete resuspension. If necessary, vortex
until completely dissolved.
11 Pour 310 ml XC4 into the dish labeled “XC4,” and cover the dish to prevent any lint or
dust from falling into the solution.
CAUTION
Do not let the XC4 sit for longer than 10 minutes.
12 Remove the staining rack from the PB1 dish and place it directly into the wash dish
containing XC4.
Infinium CytoSNP-850K Assay Protocol Guide
53
Extend and Stain (XStain) BeadChips
CAUTION
Do not remove the spacer before removing the glass. This can cause the glass
and BeadChip to come into contact and can damage the BeadChip.
CytoSNP-850K Assay Protocol
Figure 33 Moving BeadChips from PB1 to XC4
13 Slowly move the staining rack up and down 10 times, breaking the surface of the
reagent.
NOTE
If the top edges of the BeadChips begin to touch during either PB1 or XC4
washes, gently move the staining rack back and forth to separate the slides. It is
important for the solution to circulate freely between all BeadChips.
14 Soak for 5 minutes.
15 Remove the staining rack in one smooth, rapid motion and place it directly on the
prepared tube rack.
Figure 34 Moving the Staining Rack from XC4 to Tube Rack
To ensure uniform coating, place the staining rack on the center of the tube rack,
avoiding the raised edges.
54
Part # 15046990 Rev. B
16 For each of the BeadChips, working top to bottom:
a Continuing to hold the staining rack handle, carefully grip each BeadChip at its
barcode end with self-locking tweezers.
NOTE
The XC4 coat is slippery and makes the BeadChips difficult to hold. The selflocking tweezers grip the BeadChip firmly and help prevent damage.
b
Place each BeadChip on a tube rack with the barcode facing up and towards you.
Figure 36 BeadChips on Tube Rack
TIP
Remove the staining rack handle if it facilitates removal of the BeadChips from
the staining rack. Holding the top of the staining rack in position, gently remove
the staining rack handle by grasping the handle between the thumb and
forefinger. Push the tab up with your thumb and push the handle away from
you (unlocking the handle), then pull up the handle and remove.
CAUTION
To prevent wicking and uneven drying, do not allow the BeadChips to rest on
the edge of the tube rack or to touch each other while drying. Do not use the
bottom level of the tube rack. The back of the BeadChips will not dry as
effectively and dye protectant could pool.
Infinium CytoSNP-850K Assay Protocol Guide
55
Extend and Stain (XStain) BeadChips
Figure 35 Staining Rack in Correct Orientation
CytoSNP-850K Assay Protocol
17 Place the tube rack in the vacuum desiccator. Each desiccator can hold one tube rack (8
BeadChips).
CAUTION
Ensure the vacuum valve is seated tightly and securely.
18 Remove the red plug from the three-way valve before applying vacuum pressure.
19 Start the vacuum, using at least 675 mm Hg (0.9 bar).
20 To ensure that the desiccator is properly sealed, gently lift the lid of the vacuum
desiccator. It should not lift off the desiccator base.
Figure 37 Testing Vacuum Seal
21 Dry under vacuum for 50–55 minutes.
Drying times may vary according to room temperature and humidity.
22 Release the vacuum by turning the handle very slowly.
WARNING
Air should enter the desiccator very slowly to avoid disturbing the contents.
Improper use of the vacuum desiccator can result in damage to the
BeadChips. This is especially true if you remove the valve plug while a
vacuum is applied. For detailed vacuum desiccator instructions, see the
documentation included with the desiccator.
23 Store the desiccator with the red valve plug in the desiccator’s three-way valve to stop
accumulation of dust and lint within the valve port.
24 Touch the borders of the chips (do not touch the stripes) to ensure that the etched,
barcoded side of the BeadChips are dry to the touch.
25 If the underside feels tacky, manually clean the underside of the BeadChip to remove
any excess XC4 using a Kimwipe sprayed with 70% EtOH. The bottom two BeadChips
are most likely to have some excess.
a Hold the BeadChip at a downward angle to prevent excess EtOH from dripping
from the wipe onto the stripes.
b Wipe along the underside of the BeadChip five or six times, until excess XC4 is
removed.
56
Part # 15046990 Rev. B
Extend and Stain (XStain) BeadChips
CAUTION
Do not touch the stripes with the wipe or allow EtOH to drip onto the stripes.
26 Do one of the following:
• Proceed to Scan the BeadChip.
• Store the BeadChips in the Illumina BeadChip Slide Storage Box at room
temperature. Scan the BeadChips within 72 hours.
27 Clean the glass back plates. See Cleaning the Glass Back Plates on page 12 for
instructions.
28 Discard unused reagents in accordance with facility standards.
Infinium CytoSNP-850K Assay Protocol Guide
57
CytoSNP-850K Assay Protocol
Image BeadChip
The iScan Reader uses two lasers to excite the fluorophores of the single-base extension
products attached to beads of the BeadChip sections. Light emissions from these
fluorophores are then recorded in high resolution images of the BeadChip sections.
Analysis using the BlueGnome BlueFuse Multi software requires that BeadChip array data
is available in a genotype call (*.gtc) file format. The iScan can be configured to produce
normalized data and associated genotype calls in the form of a GTC file, in addition to the
standard file types (such as *.idat files). Alternatively, you can use the Illumina Beeline
software to convert *.idat files to *.gtc files after scanning.
Users of Illumina KaryoStudio software or Illumina’s GenomeStudio Genotyping Module
do not require *.gtc files feature.
Estimated Time
Scanning: 35 minutes per BeadChip (iScan)
Preparation
} Ensure you have downloaded the DMAP file(s) prior to starting the protocol, BeadChip
scanning can not proceed without the DMAP file, download can take 30 minutes per
barcode.
} Ensure you have downloaded the beadpool manifest file (.bpm) and the bead cluster
file (.egt) for each BeadChip design available from http://support.illumina.com.
} In the Lab Tracking Form, record the Scanner ID and scan date for each BeadChip.
Download DMAP files
The Illumina Decode File Client enables you to download DMAP files that are required by
the iScan or other BeadChip readers to read each individual BeadChip. This section
provides brief instructions for downloading DMAP files. For more information, see the
Decode File Client User Guide.
CAUTION
DMAP files should be downloaded before beginning the Infinium protocol scanning of BeadChips cannot occur without the relevant associated DMAP file.
There are two methods for downloading DMAP files from the Decode File Client: Access by
Account or Access by BeadChip. Both methods are outlined here.
Access by Account
58
1
On
•
•
•
•
•
the main tab, select a download option:
AutoPilot
All BeadChips not yet downloaded
All BeadChips
BeadChips by Purchase Order
BeadChips by barcode
2
Enter the required information.
3
Find DMAP files.
Part # 15046990 Rev. B
Select the DMAP files you want to download.
Make sure that you have enough free space on your computer's download destination.
5
Start the download.
6
View the download status on the Download Status and Log tab.
Access by BeadChip
When you launch the Decode File Client application, Access by BeadChip will be selected
by default.
1
Identify BeadChips using two of the four options listed:
• BeadChip barcode
• BeadChip box ID
• Purchase order number
• Sales order number
2
Find DMAP files.
3
Select the DMAP files you want to download.
Make sure that you have enough free space on your computer's download destination.
4
Start the download.
5
View the download status on the Download Status and Log tab.
Set Up Input and Output Paths
This process should only need to be completed when the iScan Control Software is first
installed on the iScan PC, after this initial setup, all scans use these paths.
1
Create a folder on the iScan PC D drive to contain the decode (*.dmap) and Sentrix
descriptior (*.sdf) files that are provided on the BeadChip CD supplied with the
BeadChip Analysis kit or are available for download described in section DMAP
download. The folder may be called D:\Decode.
2
Create another folder on the iScan PC D-drive where you want the iScan control
software to store the image data from the scan, for example, D:\ImageData.
NOTE
During the scan, the iScan Control Software automatically creates sub-folders
named with each BeadChip’s barcode number. The folder will be populated
with image files (*.jpg or *.tif), intensity data files (*.idat), and genotype call file
once enabled (*.gtc) for each BeadChip. If the images are in *.tif format, the
output path will also contain bead location files (*.locs)
Start the iScan System
1
Turn on the iScan Reader and the attached PC.
2
Let the iScan Reader warm up for at least 5 minutes.
3
Double-click the iScan Control Software icon on the desktop.
Infinium CytoSNP-850K Assay Protocol Guide
59
Image BeadChip
4
CytoSNP-850K Assay Protocol
Turn on the AutoConvert Setting
1
Click the Menu button (orange arrow icon) in the upper left corner of the iScan Control
Software and select Tools|Options.
2
In the Options dialogue box, click the AutoConvert tab.
3
Select the Enable AutoConvert check box.
4
Click New to create a mapping file – name the autoconvert mapping file.
5
Click Edit Mapping File.
a Click New to create a new mapping.
b In the right pane Click the PartNumber field and enter the product part number of
the BeadChip (found on the BeadChip packaging).
c Click the ManifestFilePath field, click the browse control and navigate to and select
the bead pool manifest file (*.bpm).
d Click the ClusterFilePath field, click the browse control and navigate to and select
the bead cluster file (*.egt).
e Click OK to save the settings and OK again in the Options dialogue box.
NOTE
The SNP probe information is contained in the bead pool manifest file (*.bpm); it
is specific for each bead array design. The bead cluster file (*.egt) provides the
reference information used in the analysis of the bead arrays. The manifest and
cluster files, specific for each array design, are available for download from
support.illumina.com .
Load the BeadChips and Start the Scan
60
1
Load the BeadChips into their carrier and click Start place the carrier into the iScan
Reader tray. Click Next. The tray closes and the iScan reader begins scanning the
barcodes. Click any barcode to view an image of the corresponding BeadChip.
2
If you want to change the image format (*.jpg or *.tif), click the Menu button and select
Tools | Options.
3
Click any BeadChip section to remove it from the scan. The section will no longer be
highlighted blue.
4
If you want to remove an entire BeadChip from the scan, delete the barcode from the
Setup window.
5
To begin scanning the BeadChips, click Scan.
6
At the end of the scan, a Review window appears. If any stripes fail to scan
successfully they will be displayed in red, click Rescan to automatically rescan all
failed areas.
7
When you finish reviewing the data, click Done.
Part # 15046990 Rev. B
After the initial calibration, the iScan Reader begins scanning, you can review the progress
of the scan in the Scan Window. As each BeadChip section is scanned, its status is
indicated by a status colour:
•
•
•
•
Light blue – not yet scanned
Orange – scanning in progress
Green – Scan and registration successful
Red – Scan and registration of section failed.
While a scan is in progress, you can click Pause or Cancel at any time. If you pause, the
button changes to Resume. Click Resume to start scanning the next un-scanned section.
Use Beeline Software to Convert IDAT files to GTC Files
Beeline software is only required to generate *.gtc files if AutoConvert has not been
implemented on the iScan. Beeline software can be used to convert *.idat files to *.gtc files
after scanning.
1
Download and install the Beeline software from the Illumina web site.
NOTE
Both the Beeline software installer and the AutoConvert software installer are
required.
2
Click the Convert Idat to Gtc toolbar icon.
3
Browse to the folder containing .idat files, and specify the folder for the generated .gtc
files.
4
Browse to the bead pool manifest file (*.bpm) and the bead cluster file (*.egt).
5
Click Next to complete the conversion.
Infinium CytoSNP-850K Assay Protocol Guide
61
Image BeadChip
Monitor the Scan
62
Part # 15046990 Rev. B
B
balance centrifuge 10
BeadChips
glass back plates 12
best practices 10-11
bleach
daily and weekly in lab 11
post-amplification area 11
pre-amplification area 11
vapor, effect of 11
C
calibrate
pipettes 10
cap mats 10
handling techniques 10
centrifuge
balance 10
clean
bleach, daily and weekly 11
glass back plates 12
pipettes 10
post-amplification area, schedule 11
pre-amplification area schedule 11
contamination
cap mats 10
high-risk areas 11
pipettes, clean 10
customer support 65
D
DNA
track 3
documentation 65
E
equipment
manual protocol
user-supplied 17
pre- and post-amp 8
experienced user cards 3
F
Flow-Through Chambers
single-base extension 49
stain 50
formamide warning 7
G
glass back plates
clean 12
Index
Index
M
materials
automated protocol
user-supplied 17
multi-channel pipettes 10
P
pipettes
dispense technique 10
multi-channel 10
plates
cap mats 10
post-amplification
cleaning schedule 11
water purification 8
pre-amplification
cleaning schedule 11
water purification 8
protocols
bleach, daily and weekly 11
experienced user cards 3
R
reagents
batches, prepare in advance 9
bleach vapor 11
dispense technique 10
manual protocol
Illumina-supplied 18
S
safety
cap mats, handling 10
contamination 7
formamide 7
sample sheet 3
Signature* High-Speed Microplate
Shaker 14
sinks, separate pre- and postamplification 8
spin. See centrifuge 10
supplies, dedicated pre- and postamp 8
T
technical assistance 65
track sample DNA 3
W
water purification 8
H
X
help, technical 65
XStain BeadChips
single-base extension 49
Infinium CytoSNP-850K Assay Protocol Guide
63
Index
stain 50
64
Part # 15046990 Rev. B
For technical assistance, contact Illumina Technical Support.
Table 8 Illumina General Contact Information
Illumina Website
Email
www.illumina.com
[email protected]
Table 9 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Italy
Austria
0800.296575
Netherlands
Belgium
0800.81102
Norway
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
United Kingdom
Ireland
1.800.812949
Other countries
Contact Number
800.874909
0800.0223859
800.16836
900.812168
020790181
0800.563118
0800.917.0041
+44.1799.534000
MSDSs
Material safety data sheets (MSDSs) are available on the Illumina website at
www.illumina.com/msds.
Product Documentation
Product documentation in PDF is available for download from the Illumina website. Go
to www.illumina.com/support, select a product, then click Documentation & Literature.
Infinium CytoSNP-850K Assay Protocol Guide
65
Technical Assistance
Technical Assistance
Illumina
Headquartered in San Diego, California, U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com

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