Infinium CytoSNP-850K Assay Protocol Guide - Support

Transcription

Infinium® CytoSNP-850K
Assay Protocol Guide
For Research Use Only. Not for use in diagnostic procedures.
ILLUMINA PROPRIETARY
Part # 15046990 Rev. C
June 2015
This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), 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).
© 2015 Illumina, Inc. All rights reserved.
Illumina, 24sure, BaseSpace, BeadArray, BlueFish, BlueFuse, BlueGnome, cBot, CSPro, CytoChip, DesignStudio,
Epicentre, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, HiSeq X, Infinium,
iScan, iSelect, ForenSeq, MiSeq, MiSeqDx, MiSeq FGx, NeoPrep, Nextera, NextBio, NextSeq, Powered by Illumina,
SeqMonitor, SureMDA, TruGenome, TruSeq, TruSight, Understand Your Genome, UYG, VeraCode, verifi, VeriSeq, the
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other countries. All other names, logos, and other trademarks are the property of their respective owners.
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Part # 15046990 Rev. C
Part #
Revision
Date
15046990
C
June
2015
15046990
B
October
2013
15046990
A
August
2013
Description of Change
• Removed references to BlueGnome.
• Edited "Scan BeadChip" section. Removed iScan procedures and
referenced iScan System User Guide (part # 11313539). Added
reference to NextSeq 550 System User Guide (part # 15069765).
• Added incubation step in Resuspend DNA section.
• Corrected reagents in steps 10 and 11 of Make and Incubate the MSA1
Plate section.
Initial release.
Infinium CytoSNP-850K Assay Protocol Guide
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Revision History
Revision History
Table of Contents
Revision History
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
Scan BeadChip
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Index
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Technical Assistance
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Part # 15046990 Rev. C
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
Reagents—Infinium Analysis Kit
Reagents—Supplied in the Infinium Analysis Kit
Items from Illumina
Illumina General Contact Information
Illumina Customer Support Telephone Numbers
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Chapter 1 Overview
Introduction
Tracking Tools
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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 the protocols in the order shown.
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Part # 15046990 Rev. C
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 BlueFuse Multi software for data analysis, the CytoSNP-850K Lab Planner
is used to create a batch import file for later use in data analysis.
} Users of the Illumina GenomeStudio 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 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 (1 per 8 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 2 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.
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Tracking Tools
Tracking Tools
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Chapter 2 Lab Setup and Maintenance
Introduction
Safety Precautions
Best Practices
Lab Maintenance
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Chapter 2
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 a CytoSNP-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 assume that you are familiar with the contents of this chapter, have
implemented all the recommendations, and have obtained all 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 website.
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Safety Precautions
Safety Precautions
CAUTION
Only qualified laboratory personnel can perform the protocols described in this
guide. Exercise caution when handling biological samples to avoid crosscontamination 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 safety data
sheet for this assay at support.illumina.com/sds.ilmn. Dispose of containers
and any unused contents in accordance with the governmental safety
standards for your region.
CAUTION
BeadChips are glass. Inspect for broken edges and handle with care.
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Unless sufficient caution is exercised, amplification products can 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-amp 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 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.
Throw away disposable items that have fallen to the ground at the completion of the assay.
Disposable items can include empty tubes, pipette tips, gloves, or lab coat hangers. During
the assay, never touch any items that have fallen to the ground.
Immediately and thoroughly clean nondisposable items that have fallen to the ground,
such as pipettes or important sample containers. Use 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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To minimize errors in preparing user-supplied reagents each day, prepare large batches of
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 helps to avoid accidentally using expired reagents.
Prepare Batches of 0.1N NaOH
Prepare 0.1 N NaOH fresh on the day of use from a liquid 10 N NaOH stock. Alternatively,
prepare it in large batches and aliquot it into 1 ml sealed tubes. These aliquots can be
stored for up to 12 months at 2°C to 8°C and used in the protocol as needed. When you
open an aliquot, use it on the same day that it was opened. Discard any reagent that is left
at the completion of the assay.
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 sealed 1.5 ml tubes. These aliquots can be stored up to 5 months at -25°C
to -15°C and used in the protocol as needed, one tube per BeadChip. When you open an
aliquot, use it on the same day that it was opened. Discard any reagent that is left at the
completion of the assay.
WARNING
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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
Make sure that 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 multichannel 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, make sure 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. Make sure that weights
are as similar as possible.
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Lab Maintenance
Lab Maintenance
Perform the following standard lab maintenance procedures for Infinium Assay labs.
Daily and Weekly Bleaching
CAUTION
To prevent sample or reagent degradation, make sure that 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
Every week, thoroughly bleach the entire post-amp area, including benchtops 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
schedule for the post-amp area. Bleaching helps to eliminate product that could have
entered the pre-amp area.
Identify high-risk pre-amp items such as the items listed, and clean them with a 0.5%
sodium hypochlorite (10% bleach) solution each morning before beginning any pre-amp
processes:
• Benchtops
• Door handles
• Refrigerator/freezer door handles
• Computer mouse
• Keyboards
Every week, thoroughly clean all laboratory surfaces and instruments, including all the
benchtops 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.
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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. Clean the glass back plates when you open them for the
first time and between uses. In addition, perform a more thorough cleaning after every 7
uses or weekly (depending on the throughput of your lab). 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 remove any remaining detergent
completely.
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
When 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 in the previous section, clean the plates periodically
after approximately every 7 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. C
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 each other
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 4 times—steps 7–9.
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 When 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.
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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.
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 Te-Flow
Calibrating the Vortexer
The displayed speed of the vortexer can vary from the actual vortex speed. Use a digital
stroboscope to determine the actual vortex speed. When 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 following instructions 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 on the vortexer 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 in steps 1–3 to determine the displayed speed for the
actual vortex speed of 1800 rpm. These 2 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
Fragment the DNA
Precipitate the DNA
Resuspend the DNA
Wash the BeadChip
Scan BeadChip
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Chapter 3
CytoSNP-850K assay Protocol
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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User-Supplied Equipment
Table 3 User-Supplied Equipment
Item
Catalog #
Microplate centrifuge with g-force range 8 to
3000 × g
(2, for pre-amp 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 (2 separate sets, for pre-amp and postamp use)
2 each of P-20, P-200, and P-1000
8-channel precision pipettes (2 separate sets,
for pre-amp and post-amp use)
50 μL to 300 μL
Stop watch/timer (2, for pre-amp and postamp use)
N/A
Forceps
Powder-free gloves (2 separate stocks, for preamp and post-amp use)
N/A
Lab coats (2, for pre-amp and post-amp use)
N/A
Safety glasses (2, for pre-amp and post-amp
use)
N/A
15 ml conical tubes
N/A
0.8 ml storage plate (midi plate), conical
bottom
Abgene catalog # AB-0765
Heat sealing foil sheets, Thermo-Seal
Rubber sealing roller
VWR International catalog #RLPLTVWR01 or
equivalent
96-well cap mats (pierceable, nonautoclavable)
Absorbent pads
N/A
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Item
Catalog #
Kimwipes
N/A
Mild detergent, such as Alconox Powder
Aerosol filter pipette tips (2 separate stocks, for
pre-amp and post-amp use)
20uL, 200uL, 1000uL
Disposable pipetting troughs
Vacuum source (greater than 508 mm Hg (0.68
bar)
N/A
Vacuum gauge for vacuum desiccator
(recommended)
N/A
Reagents
Table 5 Reagents—Infinium Analysis Kit
Item
Part #
CytoSNP-850K DNA Analysis Kit (2 BeadChip
pack)
WG-322-1002
pack)
WG-322-1003
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 2 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 2week period (maximum 24 BeadChips). PB1 and XC4 can be ordered separately
as a buffer kit (GT-201-1005).
CAUTION
Download DMAP files before beginning the Infinium protocol. Scanning of
BeadChips cannot occur without the relevant associated DMAP file.
Table 6 Reagents—Supplied in the Infinium Analysis Kit
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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. C
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
11208296
TEM—Two-Color Extension Master Mix
11208309
11208392
STM—Superior Two-Color Master Mix
11288046
ATM—Anti-Stain Two-Color Master Mix
11208317
11208430
Item
Table 7 Items from Illumina
Item
Part #
iScan System (110 V/220 V) or NextSeq 550
System
SY-101-1001 or SY-415-1002
Infinium Option Starter Package (220 V)
WG-15-305
Infinium Option Starter Package (110 V)
WG-15-304
NOTE
New users, not already running the Infinium Assay, will require the Infinium Option Starter
Package. The Infinium Option Starter Package (220 V and 110 V) includes: hybridization oven
with rocker, high-speed microplate shaker, Hybex heatblock and accessories, heat sealer,
desiccator, hybridization chamber and gaskets, Tecan TeFlow wash chamber rack and
accessories, among other items.
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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 one time in the Infinium CytoSNP-850K Assay.
Figure 2 Denaturing and Neutralizing DNA
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 storage 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 necessary)
• MSA1 plate barcode (if necessary)
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Part # 15046990 Rev. C
Steps
1
Dispense 20 μl MA1 into the MSA1 plate wells.
Fill wells according to the plate layout diagram 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, make sure that all 96 caps are securely seated in the wells.
6
Vortex the plate at 1600 rpm for 1 minute.
7
Pulse centrifuge at 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.
21
• MA1 tube lot number
• MA2 tube lot number
• MSM tube lot number
13 Vortex the sealed MSA1 plate at 1600 rpm for 1 minute.
14 Pulse centrifuge at 280 × g.
15 Incubate in the Illumina Hybridization Oven for 20–24 hours at 37°C.
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
Perform the remainder of the protocol in the post-amp laboratory area.
22
Part # 15046990 Rev. C
Fragment the DNA
Fragment the DNA
These steps explain how to enzymatically fragment the DNA, using endpoint
fragmentation to avoid overfragmentation.
Figure 3 Fragmenting DNA
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.
• Date/Time
• Operator
• FMS tube lot number
Steps
1
Pulse centrifuge the MSA1 plate at 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
5
6
Pulse centrifuge the plate at 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
Perform 1 of the following:
23
• Continue to the next step Precipitate DNA, prepare for the next step during the 37°C
heat block incubation.
• If you do not plan to proceed to the next step within the next 2 hours, store the
sealed MSA1 plate at -25°C to -15°C. Do not store for more than 24 hours.
SAFE STOPPING POINT
This is a good stopping point in the process.
24
Part # 15046990 Rev. C
These steps explain how to precipitate the DNA samples using 100% 2-propanol and PM1.
Figure 4 Precipitating DNA
Estimated Time
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.
} Perform 1 of the following
• If you froze the MSA1 plate after fragmentation, thaw it to room temperature, and
then pulse centrifuge the MSA1 plate at 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.
• 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.
25
Precipitate the DNA
Precipitate the DNA
NOTE
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
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.
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 at 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 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
website
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, make sure that blue pellets are present at the bottoms of the wells.
26
Part # 15046990 Rev. C
Precipitate the DNA
Figure 5 Uncovered MSA1 Plate Inverted for Air Drying
16 On the Lab Tracking Form, enter the start and stop times.
18 Perform 1 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 -25°C to -15°C. Do not store for more than 24
hours.
SAFE STOPPING POINT
27
Resuspend the DNA
These steps explain how to resuspend the precipitated DNA using RA1.
Figure 6 Resuspending DNA
Estimated Time
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
sheet for this assay at support.illumina.com/sds.html. Dispose of containers
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 redissolve 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 redissolve the
solution. [Optional] Heat at 48°C for 15 minutes until RA1 is dissolved.
} On
•
•
•
28
the Lab Tracking Form, record:
Date/Time
Operator
RA1 bottle lot number
Part # 15046990 Rev. C
Steps to Resuspend the DNA
1
Add 46 μl RA1 to each well of the MSA1 plate containing a DNA pellet.
Reserve leftover reagent in the bottle for the Extend and Stain (XStain) the BeadChip step
later in this protocol.
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 hybridization oven and incubate for 1 hour at 48°C.
5
6
NOTE
If you store the pellets at -25°C to -15°C for extended periods of time after the
precipitate process, you might need to resuspend the pellets. Repeat the
vortexing and centrifugation in the previous steps until pellets are resuspended.
7
Discard unused reagents in accordance with facility standards.
8
Perform 1 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 -25°C to -15°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 -25°C to -15°C if storing
for more than 24 hours.
SAFE STOPPING POINT
29
Resuspend the DNA
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.
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
website.
Figure 7 Hybridize Multi BeadChip
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
CAUTION
BeadChips are glass. Inspect BeadChips for broken edges and handle with care.
Preparation
} Make sure that DMAP files for each BeadChip have been downloaded, scanning
cannot proceed without a DMAP file.
} 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.
30
Part # 15046990 Rev. C
Prepare the Hybridization Chambers
1
Place the resuspended MSA1 plate on the heat block to denature the samples at 95°C
for 20 minutes.
2
During the 20 minute incubation, prepare the Hyb Chambers.
Place the following items on the benchtop for use in this procedure:
Figure 8 BeadChip Hyb Chamber Components
A
B
C
Hyb Chambers
Hyb Chamber Gaskets
Hyb Chamber Inserts
31
} 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 one time 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.
• Date/Time
• Operator
• PB2 tube lot number
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
make sure 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.
Figure 9 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 10 Placing Gasket into Hyb Chamber
— Make sure that the Hyb Chamber gaskets are properly seated.
32
Part # 15046990 Rev. C
c
Dispense 400 μl PB2 into the required humidifying buffer reservoirs in the Hyb
Chamber, 2 for each BeadChip.
Figure 12 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. You do not need to lockdown the lid.
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 1 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.
33
Figure 11 Hyb Chamber with Gasket in Place
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.
CAUTION
BeadChips are glass. Inspect BeadChips for broken edges and handle with care.
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.
Figure 13 Placing BeadChips into Hyb Chamber Inserts
4
Remove the foil seal from the MSA1 plate, and then, using a multichannel precision
pipette, dispense 26 μl of each DNA sample onto the appropriate BeadChip section.
Figure 14 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 before dispensing.
Continue in this manner, following the color-coded sections in the figure, until all
samples are loaded.
Part # 15046990 Rev. C
Figure 15 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 16 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 add RA1 (sample Hyb buffer) as this will dilute the sample.
8
Record the additional added sample on the Lab Tracking Form.
35
9
Proceed immediately to the next section, Set up the BeadChips for Hybridization.
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 always keep parallel to the lab bench. 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 17 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 18 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. C
Figure 19 Hyb Chamber Correctly Placed in Hyb Oven
Figure 20 Incorrectly Placed Hyb Chamber
NOTE
If you are stacking multiple Hyb Chambers, 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
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.
37
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 47
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.
When it is 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. C
Wash the BeadChip
Wash the BeadChip
These steps explain how to prepare the BeadChips for the staining process.
Figure 21 Washing BeadChip
Estimated Time
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 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
Preparation
} Make sure that the water circulator is filled to the appropriate level.
39
} 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. Make sure that all locations are 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 before 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:
— Black frames
— Spacers (separated for ease of handling)
— Clean glass back plates (see Cleaning the Glass Back Plates on page 12)
— Clamps
• 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 dissolved.
Steps to Wash BeadChip
1
40
Attach the wire handle to the rack and submerge the wash rack in the first wash dish
containing 200 ml PB1.
Part # 15046990 Rev. C
Wash the BeadChip
Figure 22 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 website.
NOTE
To make sure that no solution splatters on you, Illumina recommends removing
the cover seal over an absorbent cloth or paper towels, preferably in a hood.
CAUTION
Inspect BeadChips for broken edges and handle with care.
a
b
Using powder-free gloved hands, hold the BeadChip securely and by the edges in
one hand. Avoid contact with the sample inlets. Make sure that he barcode is
facing up and is closest to you, and that the top side of the BeadChip is angled
slightly away from you.
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 23 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 submerged in the PB1.
41
Figure 24 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
When 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 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.
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. 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
42
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.
Part # 15046990 Rev. C
Wash the BeadChip
Figure 25 Placing Black Frames into BeadChip Alignment Fixture, prefilled with PB1
3
Place each BeadChip into a black frame, aligning its barcode with the ridges stamped
onto the Alignment Fixture. Make sure that each BeadChip is fully immersed in PB1.
Figure 26 Placing BeadChip into Black Frame on Alignment Fixture
4
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.
43
Figure 27 Placing Clear Plastic Spacer onto BeadChip
5
Place the Alignment Bar onto the Alignment Fixture.
Make sure that the groove in the Alignment Bar fits over the tab on the Alignment
Fixture.
Figure 28 Placing Alignment Bar onto Alignment Fixture
6
44
Place a clean glass back plate on top of the clear spacer covering each BeadChip. Make
sure that the plate reservoir is at the barcode end of the BeadChip, facing inward to
create a reservoir against the BeadChip surface.
Part # 15046990 Rev. C
Wash the BeadChip
Figure 29 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 up the glass back plate 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 30 Securing Flow-Through Chamber Assembly with Metal Clamps
A
B
C
8
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 nonbarcode end of the assembly. Slip the scissors up
over the barcode to trim the other end.
45
Figure 31 Trimming Spacer Ends from Flow-Through Chamber Assembly
A
B
9
Trim spacer at nonbarcode 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 make sure that no traces of PB2 remain in the wells.
46
Part # 15046990 Rev. C
These steps explain how to: wash unhybridized and nonspecifically hybridized DNA
samples 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.
Figure 32 Extending and Staining BeadChip
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
TEM
XC3
25 ml (per 4 BeadChips)
STM (make sure that all STM tubes indicate the same
stain temperature on the label)
ATM
PB1
XC4
Alconox Powder Detergent
as needed
EtOH
as needed
95% formamide/1 mM EDTA
CAUTION
Pour out only the recommended reagent volume needed for the suggested
number of samples listed in the Consumables table of each section.
47
WARNING
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.
Figure 33 XStain BeadChip Reagent Tubes and Bottles
A
B
C
D
E
F
G
H
I
J
RA1
XC3
XC1
XC2
TEM
95% Formamide / 1mM EDTA
STM
ATM
PB1
XC4
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.
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
dissolved. Heat to dissolve, if necessary.
} On
•
•
•
48
the Lab Tracking Form, record:
Date/Time
Operator
RA1 lot number
Part # 15046990 Rev. C
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 flow-through
chamber topic in the Infinium, Lesson 2: Assay Techniques training video on the Array Online
1
Make sure that 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 can vary depending on facility ambient conditions.
Figure 34 Water Circulator Connected to Chamber Rack
A
B
C
3
Chamber Rack
Water Circulator with Programmable Temperature Controls
Reservoir Cover
The temperature displayed on the water circulator LCD screen can differ from the
actual temperature on the Chamber Rack. Confirm the actual temperature using the
temperature probe for the Chamber Rack.
49
•
•
•
•
•
•
•
•
4
Remove bubbles trapped in the Chamber Rack each time you run this process.
5
Use the Illumina Temperature Probe in several locations to make sure that the
Chamber Rack is at 44°C. Make sure that all locations are at 44°C ± 0.5°C.
NOTE
Do not leave the temperature probe in the first 3 rows of the Chamber Rack.
Reserve this space for BeadChips.
Figure 35 Illumina Temperature Probe and Temperature Probe in Chamber Rack
6
For accurate temperature measurement, make sure that 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
Make sure that 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.
50
Part # 15046990 Rev. C
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 one time.
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 one time.
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 one time.
c Wait 5 minutes.
d 250 μl ATM. Incubate for 10 minutes.
e 450 μl XC3. Incubate for 1 minute. Repeat one time.
f Wait 5 minutes.
g 250 μl STM. Incubate for 10 minutes.
h 450 μl XC3. Incubate for 1 minute. Repeat one time.
i
Wait 5 minutes.
j
250 μl ATM. Incubate for 10 minutes.
k 450 μl XC3. Incubate for 1 minute. Repeat one time.
l
Wait 5 minutes.
m 250 μl STM. Incubate for 10 minutes.
n 450 μl XC3. Incubate for 1 minute. Repeat one time.
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.
51
Figure 36 Dispensing RA1 into Each Flow-Through Chamber
Wash and Coat BeadChips
Before starting the Wash and Coat process, 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 pad.
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.
Equipment Needed
Place the following items on the bench:
} 1 staining rack and wash dishes
} 1 vacuum desiccator
} 1 tube rack
} Self-locking tweezers
} Large Kimwipes
} Vacuum hose
Steps
1
Set up 2 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 37 PB1 and XC4 Wash Dishes with Staining Rack
A
B
52
Wash Dishes
Staining Rack
Part # 15046990 Rev. C
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.
Figure 38 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 2 metal clamps.
CAUTION
It is important to use the dismantling tool to avoid chipping the glass back
plates.
Figure 39 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 damage the BeadChip.
53
3
c
d
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.
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.
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. Make sure that
the BeadChip barcodes are facing away from you and that the locking arms on the
handle are facing 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
Make sure that the BeadChips are 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 40 Washing BeadChips in PB1
9
Soak for 5 minutes.
10 Shake the XC4 bottle vigorously to ensure complete resuspension. If necessary, vortex
until 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.
54
Part # 15046990 Rev. C
Figure 41 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 42 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.
55
12 Remove the staining rack from the PB1 dish and place it directly into the wash dish
containing XC4.
Figure 43 Staining Rack in Correct Orientation
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 44 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 up the tab 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.
56
Part # 15046990 Rev. C
CAUTION
Make sure that 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 make sure that the desiccator is properly sealed, gently lift the lid of the vacuum
desiccator. Make sure that the lid does not lift off the desiccator base.
21 Dry under vacuum for 50–55 minutes.
Drying times can vary according to room temperature and humidity.
22 Release the vacuum by turning the handle slowly.
WARNING
Make sure that air enters the desiccator 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 three-way valve of the desiccator to
stop accumulation of dust and lint within the valve port.
24 Touch the borders of the chips (do not touch the stripes) to make sure that the etched,
barcoded sides 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 2 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 5 or 6times, until excess XC4 is
removed.
CAUTION
Do not touch the stripes with the wipe or allow EtOH to drip onto the stripes.
26 Perform 1 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.
57
17 Place the tube rack in the vacuum desiccator. Each desiccator can hold 1 tube rack (8
BeadChips).
Scan BeadChip
Scan BeadChips using either the iScan system or the NextSeq 550 system. To perform a
scan, you need Decode Map (DMAP) files, a manifest file, and a cluster file for the
BeadChip you are using.
} Use the Decode File Client to download the required DMAP folder. DMAP folders are
unique for each BeadChip type. For more information, see the DMAP Decode File Client
User Guide (part # 11337856).
} Download the manifest and cluster files from the Illumina support website. Each
manifest and cluster file is unique for each BeadChip type. Use the same manifest
and cluster file for the iScan system or the NextSeq 550 system.
} Scan your BeadChip using the iScan or the NextSeq system. For more information, see
the iScan System User Guide (part # 11313539) or the NextSeq 550 System User Guide (part
# 15069765). In the lab tracking form, record the Scanner ID and scan date for each
BeadChip.
} Output files are generated during the scan and then queued for transfer to the specified
output folder.
Perform analysis using BlueFuse Multi software, which requires that scanning data are
available in a genotype call (GTC) file format. By default, the NextSeq 550 system generates
normalized data and associated genotype calls in the format of a GTC file. The iScan can
be configured to produce GTC files, 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. For more information, see Beeline Software User Guide (part # 15016340).
58
Part # 15046990 Rev. C
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 61
D
DNA
track 3
documentation 61
E
equipment
manual protocol
user-supplied 17
pre- and post-amp 8
experienced user cards 3
F
Flow-Through Chambers
single-base extension 50
stain 51
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
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 61
track sample DNA 3
W
H
X
help, technical 61
XStain BeadChips
single-base extension 50
59
Index
stain 51
60
Part # 15046990 Rev. C
For technical assistance, contact Illumina Technical Support.
Table 8 Illumina General Contact Information
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
Australia
1.800.775.688
Netherlands
Austria
0800.296575
New Zealand
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
0800.451.650
800.16836
900.812168
020790181
0800.563118
0800.917.0041
+44.1799.534000
Safety Data Sheets
Safety data sheets (SDSs) are available on the Illumina website at
support.illumina.com/sds.html.
61
Illumina
5200 Illumina Way
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com

Infinium CytoSNP-850K Assay Protocol Guide - Support

Transcription

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