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illumina Infinium HD Super Assay Reference Manual

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Infinium HD Super Assay
Reference Guide
Document # 11322427 v03
August 2019
For Research Use Only. Not for use in diagnostic procedures.
ILLUMINA PROPRIETARY

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  • Page 1 Infinium HD Super Assay Reference Guide Document # 11322427 v03 ILLUMINA PROPRIETARY August 2019 For Research Use Only. Not for use in diagnostic procedures.
  • Page 3 Infinium HD Super Assay Reference Guide 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.
  • Page 4 Corrected reagents listed in first and last bullets of About Reagents section in the Extend and Stain introduction. Reference to Experienced User Cards replaced with Infinium HD Super Assay Checklist Manual Protocol (document # 1000000080218) and Infinium HD Super Assay Checklist Automated Protocol (document # 1000000080220) which provide current format of experienced user instructions.
  • Page 5 Infinium HD Super Assay Reference Guide Document Date Description of Change Part # 11294809 Rev. D May 2012 Added that up to 24 BeadChips can be washed with 550 ml of PB1. Part # 11294809 Rev. C June 2010 Update.

  • Page 6: Table Of Contents

    Table of Contents Chapter 1 Overview Introduction to Infinium HD Super Assay Important Note Additional Resources DMAP Files Preparation and Storage of User-Supplied Reagents Tips and Techniques Acronyms Illumina LIMS No Illumina LIMS Tracking Tools Chapter 2 Manual Protocol Introduction Infinium HD Super Manual Workflow...

  • Page 7: Chapter 1 Overview

    The Illumina Infinium HD Super Assay is an evolution for DNA analysis, by streamlining sample preparation and enabling high multiplexing. Using Infinium I and Infinium II probe designs and a dual-color channel approach, the Infinium HD Super Assay enables the DNA analysis of up to several million of SNPs and CNV markers per sample.

  • Page 8: Important Note

    Important Note Before using the procedures in this guide, read the Infinium Lab Setup and Procedures Guide . The Setup and Procedures guide explains how to equip and run an Infinium HD Super Assay laboratory, including information on the following topics:...

  • Page 9: Tips And Techniques

    Infinium HD Super Assay Reference Guide Use fresh reagents for each batch of plates, and empty reservoirs between batches. To minimize errors when preparing user-supplied reagents, prepare large batches of 0.1 N NaOH and 95% Formamide/1 mM EDTA using the following guidance.
  • Page 10 Infinium HD Super Assay Reference Guide Clean HD glass back plates according to the procedures in the Infinium Assay Lab Setup and Procedures Guide (Document # 11322460) . A 10% bleach soak is required weekly, or every seven uses, to maintain glass performance.

  • Page 11: Acronyms

    Do not hold by the sides near the sample inlets. Rocker Platform [Optional] If you are using an Illumina Hybridization Oven with a rocker platform, follow these guidelines: If 7 to12 hybridization chambers (Hyb Chambers) are placed in 1 hybridization oven, the rocker must be removed before beginning the Hybridize to BeadChip section.

  • Page 12: No Illumina Lims

    No Illumina LIMS If you are using the automated protocol without Illumina LIMS do the following: At each step in the protocol, make sure that the Use Barcodes checkbox is cleared. At each step in the protocol, you are prompted to enter the number of samples, plates, or BeadChips.

  • Page 13: Chapter 2 Manual Protocol

    Assay. Follow the protocols in the order shown. Infinium HD Super Manual Workflow The following figure graphically represents the Infinium HD Super Assay manual workflow for 24 BeadChips. These protocols describe the procedure for preparing 96 DNA samples. Document # 11322427 v03...
  • Page 14 Infinium HD Super Assay Reference Guide Figure 2 Infinium HD Super Manual Workflow Document # 11322427 v03 For Research Use Only. Not for use in diagnostic procedures.

  • Page 15: Amplify Dna

    DNA plate with 48 or 96 DNA samples (50 ng/μl) (midi or TCY) (1 plate) Cap mats Preparation Preheat the Illumina Hybridization Oven in the post-amp area to 37°C and allow the temperature to equilibrate. Prepare the following consumables: Item...

  • Page 16: Incubate Dna

    This step uniformly amplifies the genomic DNA, generating a sufficient quantity of each individual DNA sample to be used in the Infinium HD Super Assay. Incubate the MSA1 plate in the Illumina Hybridization Oven for 20–24 hours at 37°C. Fragment DNA This step enzymatically fragments the DNA.

  • Page 17: Precipitate Dna

    Infinium HD Super Assay Reference Guide Procedure Pulse centrifuge the MSA1 plate at 280 × g. Carefully remove the cap mat. Add 50 µl FMS to each well of the MSA1 plate. Reseal with the cap mat using the original orientation.
  • Page 18 Infinium HD Super Assay Reference Guide Remove and discard the cap mat. Add 300 μl 100% 2-propanol to each sample well. Carefully seal with a new, dry cap mat. Avoid shaking the plate until the cap mat is seated. 10 Invert the plate 10 times to mix.

  • Page 19: Resuspend Dna

    This protocol uses an aliphatic amide 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 www.illumina.com/sds. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region.

  • Page 20: Hybridize To Beadchip

    Infinium HD Super Assay Reference Guide SAFE STOPPING POINT If you are stopping, seal the plate, and store at -25°C to -15°C for up to 24 hours. Hybridize to BeadChip This step dispenses the fragmented, resuspended DNA onto BeadChips. Incubation then hybridizes each DNA sample to a section of the BeadChip.
  • Page 21 Infinium HD Super Assay Reference Guide A Hybridization chambers B Hybridization chamber gaskets C Hybridization chamber inserts Align the wider edge of the gasket to the barcode ridges. A Reagent reservoirs B Barcode ridges C Narrower gasket edges D Wider gasket edges Place the gasket into the hybridization chamber.
  • Page 22 Infinium HD Super Assay Reference Guide Add 400 µl PB2 to the top and bottom wells of each beadchip subchamber in the hybridization chamber. Immediately cover the chamber with the lid to prevent evaporation. Locking the lid is not necessary.
  • Page 23 Infinium HD Super Assay Reference Guide Remove the foil seal from the MSA1 plate. Transfer sample from the MSA1 plate to the appropriate section of the BeadChip. Insert the pipette into the sample inlet before dispensing. Load A1–H1, as shown in the following graphic.
  • Page 24 Close the clamps in the following order: top-left, bottom-right, top-right, bottom-left. Place the chamber into the preheated Illumina Hybridization Oven so that the top logo faces you. You can stack up to three chambers per row for a total of six chambers. Make sure that the feet of the top chamber fit into the indents on the bottom chamber.

  • Page 25: Wash Beadchips

    Infinium HD Super Assay Reference Guide [Optional] Start the rocker, setting the speed to 5. Incubate at 48°C for 16–24 hours. Store RA1 at -25°C to -15°C for use the next day. Resuspend XC4 Reagent Resuspend XC4 to prepare for the Extend and Stain BeadChips step.
  • Page 26 This protocol uses an aliphatic amide 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 www.illumina.com/sds. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region.
  • Page 27 Infinium HD Super Assay Reference Guide Item Storage Instructions -25°C to -15°C Thaw at room temperature. Invert 10 times to mix. Room Thaw at room temperature. Invert 10 times to mix. temperature Room Thaw at room temperature. Invert 10 times to mix.
  • Page 28 Infinium HD Super Assay Reference Guide Repeat steps 4–5 until all BeadChips are transferred to the submerged wash rack in Wash 1. Move the wash rack up and down for 1 minute, breaking the surface of the PB1 with gentle, slow agitation.
  • Page 29 Infinium HD Super Assay Reference Guide Place each BeadChip into a black frame, aligning the barcode with the alignment fixture ridges. Fully submerge each BeadChip. Place a clear spacer onto the top of each BeadChip. Use the alignment fixture grooves to guide the spacers into position.
  • Page 30 Infinium HD Super Assay Reference Guide A Reservoir at barcode end of glass back plate B Glass back plate in position Secure each flow-through chamber assembly with metal clamps as follows. Using one finger, gently push the glass back plate against the alignment bar.

  • Page 31: Extend And Stain Beadchips

    Infinium HD Super Assay Reference Guide A Trimming the spacer at the nonbarcode end. B Trimming the spacer at the barcode end. 11 Leave assembled flow-through chambers on the lab bench in a horizontal position until ready to load onto chamber rack in the Extend and Stain BeadChips step.
  • Page 32 This protocol uses an aliphatic amide 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 www.illumina.com/sds. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region.
  • Page 33 Infinium HD Super Assay Reference Guide Procedure Set Up the Chamber Rack Make sure that the water circulator is filled to the appropriate level. See the VWR Operator Manual , VWR part # 110-229. Turn on the water circulator. Set it to a temperature that brings the Chamber Rack to 44°C at equilibrium.
  • Page 34 Infinium HD Super Assay Reference Guide Using the Illumina temperature probe, test at least three locations on the chamber rack. For accurate measurements, make sure that the temperature probe touches the base of the chamber rack. b Make sure that all locations are at 44°C ± 0.5°C.
  • Page 35 Infinium HD Super Assay Reference Guide Wait for the chamber rack to reach the correct temperature. Stain BeadChips If you are imaging the BeadChip immediately after the staining process, turn on the scanner to allow the lasers to stabilize. Fill the reservoir of each flow-through chamber as follows.
  • Page 36 Infinium HD Super Assay Reference Guide Self-locking tweezers Tube rack Vacuum desiccator Vacuum hose Wash dishes (2) During the procedure, prevent dust or lint from entering the wash dishes. Clean wash dishes with low-pressure air before use. Cover wash dishes with wash dish covers when not in use.
  • Page 37 Infinium HD Super Assay Reference Guide A Locking arms B Tab 10 Leave the staining rack in the wash dish for later use (carrying the BeadChips after disassembling the flow-through chambers). 11 Using the dismantling tool , remove the two metal clamps from a flow-through chamber.
  • Page 38 Infinium HD Super Assay Reference Guide Free circulation of PB1 between BeadChips is important. 20 Soak for 5 minutes. 21 Vigorously shake the XC4 bottle to resuspend completely. If necessary, vortex until dissolved. 22 Add 310 ml XC4 to the XC4 wash dish.
  • Page 39 Infinium HD Super Assay Reference Guide 27 Make sure that the staining rack is in the center of the tube rack to ensure uniform coating. Avoid the raised edges. 28 [Optional] Remove the staining rack handle to facilitate BeadChip removal: Holding the top of the staining rack in position, grasp the handle between your thumb and forefinger.

  • Page 40: Image Beadchip

    For instructions, see the Infinium Lab Setup and Procedures Guide (document #11322460) . SAFE STOPPING POINT Store the BeadChips in the Illumina BeadChip Slide Storage Box at room temperature. Scan within 72 hours. Image BeadChip Follow the instructions in the System Guide for your instrument to scan your BeadChips.

  • Page 41: Illumina Genomestudio

    Illumina GenomeStudio The Illumina GenomeStudio Genotyping Module, included with your Illumina Infinium Assay system, is an application for extracting genotyping data from intensity data files (*.idat files) collected from your Illumina scanning instrument. For feature descriptions and instructions on using the GenomeStudio platform to visualize and analyze genotyping data, see the GenomeStudio Framework User Guide and the GenomeStudio User Guide or online help .
  • Page 42 Infinium HD Super Assay Reference Guide Document # 11322427 v03 For Research Use Only. Not for use in diagnostic procedures.

  • Page 43: Chapter 3 Automated Protocol

    Illumina LIMS, see the comprehensive User Guide. Infinium HD Super Automated Workflow The following diagram illustrates the Infinium HD Super Assay automated workflow for 12 BeadChips. These protocols describe the procedure for preparing 96 DNA samples. Document # 11322427 v03...
  • Page 44 Infinium HD Super Assay Reference Guide Figure 3 Infinium HD Super Automated Workflow Document # 11322427 v03 For Research Use Only. Not for use in diagnostic procedures.

  • Page 45: Amplify Dna

    The robot PC updates the Required Run Items and the bed map to show the correct position of items on the robot bed. NOTE If you are using Illumina LIMS, you cannot change the number of DNA samples on this screen. The Illumina LIMS software processes the correct number of samples. Document # 11322427 v03...
  • Page 46 Infinium HD Super Assay Reference Guide NOTE If you are using Illumina LIMS, you must select Run and select batches before the robot bed map displays the correct layout for the DNA plates. Remove caps from the MA1, MA2, and MSM tubes, then place the tubes in the robot tube rack according to the bed map.

  • Page 47: Incubate Dna

    NOTE This and all remaining steps in the workflow are performed in the post-amp lab. Incubate the MSA1 plate in the Illumina Hybridization Oven for 20–24 hours at 37°C. Fragment DNA This step enzymatically fragments the DNA. An endpoint fragmentation is used to prevent overfragmentation.

  • Page 48: Precipitate Dna

    Infinium HD Super Assay Reference Guide Figure 5 Robot Setup for Fragment MSA1 A FMS B MSA1 Plate Place FMS tubes in the robot tube rack according to the bed map. Remove the cap mat. At the robot PC, select Run.
  • Page 49 Infinium HD Super Assay Reference Guide Fragment DNA If proceeding immediately from , leave the MSA1 plate on the heat block until preparation is complete. If the MSA1 plate was stored at -25°C to -15°C, thaw at room temperature, pulse centrifuge at 280 × g, and preheat the heat block to 37°C.

  • Page 50: Resuspend Dna

    Infinium HD Super Assay Reference Guide 12 Remove and discard the cap mat. 13 Place the MSA1 plate back on the robot bed according to the bed map. 14 When prompted, select OK. 15 Remove the MSA1 plate from the robot bed and carefully seal with a new, dry cap mat. Avoid shaking the plate until the cap mat is seated.
  • Page 51 This protocol uses an aliphatic amide 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 www.illumina.com/sds. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region.

  • Page 52: Hybridize To Beadchip

    Remove the MSA1 plate from the robot deck. Apply a foil seal to the MSA1 plate using the heat sealer. Incubate in the preheated Illumina Hybridization Oven for 1 hour. Vortex the plate at 1800 rpm for 1 minute. Check to make sure that the pellets are resuspended. If necessary, repeat the incubation and vortexing steps.
  • Page 53 Infinium HD Super Assay Reference Guide Figure 6 Guide E Robot Tip Alignment Guide Wash and dry the robot tip alignment guide. For washing instructions, see Wash Robot Tip Alignment Guide on page Procedure Denature DNA Place the MSA1 plate on the preheated heat block for 20 minutes to denature the DNA.
  • Page 54 Infinium HD Super Assay Reference Guide Assemble Hybridization Chambers Assemble one chamber for every four BeadChips by following the steps in this section. Figure 7 BeadChip Hybridization Chamber Components A Top of BeadChip Hybridization Chamber B Hybridization Chamber Gasket C Narrower Edges...
  • Page 55 Infinium HD Super Assay Reference Guide Immediately cover the chamber with the lid to prevent evaporation. Leave the closed chambers on the benchtop at room temperature until the BeadChips are loaded with DNA (~1 hour). [Illumina LIMS] Select Infinium HD Super | Confirm for Hyb.
  • Page 56 Infinium HD Super Assay Reference Guide Figure 8 Robot Deck Setup for Hybridization A MSA1 Plate B Robot BeadChip Alignment Fixtures CAUTION BeadChips must be transferred to hybridization chambers immediately at the end of the next procedure (Start the Robot) . Do not begin Start the Robot if you cannot immediately transfer the BeadChips.
  • Page 57 Proceed to the next section (Set Up and Incubate BeadChips) immediately. Set up and Incubate BeadChips Make sure that the Illumina Hybridization Oven is set to 48°C. WARNING Keep hybridization chambers at room temperature when you load the BeadChips. Do not place the hybridization chamber in the Illumina Hybridization Oven when loading the BeadChips.
  • Page 58 If you are stacking multiple hybridization chambers in the Illumina Hybridization Oven, fit the feet of each hybridization chamber into the matching indents on the lid of the hybridization chamber below it. You can stack up to 3 hybridization chambers in two rows, for a maximum of 6 total hybridization chambers in the Illumina Hybridization Oven.

  • Page 59: Wash Beadchips

    Handle used reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and regulations. For additional environmental, health, and safety information, see the SDS at support.illumina.com/sds.html. Document # 11322427 v03...
  • Page 60 Infinium HD Super Assay Reference Guide Preparation Remove each hybridization chamber from the hybridization oven. Allow to cool for 30 minutes before opening. Prepare the following items: Fill two wash dishes with 200 ml PB1 each and label them Wash 1 and Wash 2.
  • Page 61 Infinium HD Super Assay Reference Guide Remove the hybridization chamber inserts from the hybridization chambers. Remove the BeadChips from the hybridization inserts. Remove the cover seals from the BeadChips. Using powder-free gloved hands, hold the BeadChip securely and by the edges in one hand. Remove the entire seal in a single, continuous motion.
  • Page 62 Infinium HD Super Assay Reference Guide Move the wash rack up and down for 1 minute, breaking the surface of the PB1 with gentle, slow agitation. Move the wash rack to Wash 2 containing clean PB1, making sure that the BeadChips are submerged.
  • Page 63 Infinium HD Super Assay Reference Guide Place a clear spacer onto the top of each BeadChip. Use the alignment fixture grooves to guide the spacers into position. The white spacers are not a substitute for the clear spacers. Place the alignment bar onto the alignment fixture. Fit the groove on the alignment bar over the tab on the alignment fixture.

  • Page 64: Extend And Stain Beadchips

    Infinium HD Super Assay Reference Guide A One stripe is visible between the first clamp and the alignment bar. B Glass back plate pressed against alignment bar. C Stripes are not visible between the second clamp and the barcode. Remove the assembled flow-through chamber from the alignment fixture.
  • Page 65 This protocol uses an aliphatic amide 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 www.illumina.com/sds. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region.
  • Page 66 Infinium HD Super Assay Reference Guide A RA1 B XC3 C XC1 D XC2 E TEM F 95% Formamide / 1 mM EDTA G STM H ATM Procedure Set Up the Chamber Rack Make sure that the water circulator is filled to the appropriate level. See the VWR Operator Manual , VWR part # 110-229.
  • Page 67 Place the Te-Flow back on the reagent pan. Using the two guide pins in the reagent pan, make sure that the Te-Flow is flush. Using the Illumina temperature probe, test at least three locations on the chamber rack. For accurate measurements, make sure that the temperature probe touches the base of the chamber rack.
  • Page 68 Infinium HD Super Assay Reference Guide A Chamber rack with temperature probe B Temperature probe Single-Base Extension and Stain This step uses a robot to process the BeadChips. CAUTION The following steps must be performed without interruption. At the robot PC, select XStain Tasks | XStain HD BeadChip.
  • Page 69 Infinium HD Super Assay Reference Guide A XC1 Tubes B XC2 Tubes C TEM Tubes D STM Tubes E ATM Tubes F XC3 in Full Reservoir G RA1 in Half Reservoir H 95% Formamide/1 mM EDTA in Quarter Reservoir 24 BeadChips in Chamber Rack Temperature Probe In the Basic Run Parameters pane, enter the number of BeadChips.
  • Page 70 Infinium HD Super Assay Reference Guide Wash the tube racks and wash dishes thoroughly after each use. Rinse with deionized water. Dry racks and wash dishes upside down on a wash rack. Place a clean tube rack on top of several layers of Kimwipes or an absorbent pad.
  • Page 71 Infinium HD Super Assay Reference Guide A Locking arms B Tab 10 Leave the staining rack in the wash dish for later use (carrying the BeadChips after disassembling the flow-through chambers). 11 Using the dismantling tool , remove the two metal clamps from a flow-through chamber.
  • Page 72 Infinium HD Super Assay Reference Guide Free circulation of PB1 between BeadChips is important. 20 Soak for 5 minutes. 21 Vigorously shake the XC4 bottle to resuspend completely. If necessary, vortex until dissolved. 22 Add 310 ml XC4 to the XC4 wash dish.
  • Page 73 Infinium HD Super Assay Reference Guide 27 Make sure that the staining rack is in the center of the tube rack to ensure uniform coating. Avoid the raised edges. 28 [Optional] Remove the staining rack handle to facilitate BeadChip removal: Holding the top of the staining rack in position, grasp the handle between your thumb and forefinger.

  • Page 74: Image Beadchip

    For instructions, see the Infinium Lab Setup and Procedures Guide (document #11322460) . SAFE STOPPING POINT Store the BeadChips in the Illumina BeadChip Slide Storage Box at room temperature. Scan within 72 hours. Image BeadChip Follow the instructions in the System Guide for your instrument to scan your BeadChips.

  • Page 75: Illumina Genomestudio

    Illumina GenomeStudio The Illumina GenomeStudio Genotyping Module, included with your Illumina Infinium Assay system, is an application for extracting genotyping data from intensity data files (*.idat files) collected from your Illumina scanning instrument. For feature descriptions and instructions on using the GenomeStudio platform to visualize and analyze genotyping data, see the GenomeStudio Framework User Guide and the GenomeStudio User Guide or online help .

  • Page 76: Technical Assistance

    00806651752 United Kingdom +44 8000126019 +44 2073057197 Other countries +44.1799.534000 Safety data sheets (SDSs)—Available on the Illumina website at support.illumina.com/sds.html. Product documentation—Available for download from support.illumina.com. Document # 11322427 v03 For Research Use Only. Not for use in diagnostic procedures.
  • Page 77 Infinium HD Super Assay Reference Guide Document # 11322427 v03 For Research Use Only. Not for use in diagnostic procedures.
  • Page 79 Document # 11322427 v03 Illumina 5200 Illumina Way San Diego, California 92122 U.S.A. +1.800.809.ILMN (4566) +1.858.202.4566 (outside North America) techsupport@illumina.com www.illumina.com For Research Use Only. Not for use in diagnostic procedures. © 2019 Illumina, Inc. All rights reserved.

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