HiScan®SQ System Quick Reference Guide

FOR RESEARCH USE ONLY

Introduction 3 HiScanSQ System Components 4 Imaging Data on BeadChips 9 Sequencing Data on a Clustered Flow Cell 17 Pausing a Sequencing Run to Scan BeadChips 46 Power-Cycling the HiScanSQ System 50 Technical Assistance

ILLUMINA PROPRIETARY Catalog # SY-903-1005 Part # 15019481 Rev. E October 2011 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) OR ANY USE OF SUCH PRODUCT(S) OUTSIDE THE SCOPE OF THE EXPRESS WRITTEN LICENSES OR PERMISSIONS GRANTED BY ILLUMINA IN CONNECTION WITH CUSTOMER'S ACQUISITION OF SUCH PRODUCT(S). FOR RESEARCH USE ONLY © 2010-2011 Illumina, Inc. All rights reserved. Illumina, illuminaDx, BeadArray, BeadXpress, cBot, CSPro, DASL, DesignStudio, Eco, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, Infinium, iSelect, MiSeq, Nextera, Sentrix, Solexa, TruSeq, VeraCode, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registered trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners. Introduction 3 run Refer of the design. this post- up a in next- the website. and of of capability resolution setting perform scan described for to Compliance high- experimental Illumina post- operation resolution for and the how throughput and based, and operations and Safety from use instructions high- the laser- the flexibility performing power the Guide. information. use, System the and to- User download BeadChips including more performing regarding with for easy- integrates on for HiScanSQ 1002. an System reagents, unprecedented System, that the is Illumina arrays 903- cautions or guide see SY- available and # scan Guide is this instructions HiScanSQ system System to of delivering the HiScanSQ System, SQ expression catalog BeadChips ® how warnings see Purpose the Reference cover additional imaging all gene on Quick and HiScan Booklet, NOTE For guide, NOTE For HiScanSQ back loading run and documentation explains sequencing, optical run, System inside or guide Illumina the Additional to procedures. Documentation This sequencing scan Audience genotyping generation The benchtop Introduction HiScanSQ HiScanSQ System Components

The HiScanSQ System consists of the HiScan Reader, the SQ Module, a dedicated computer workstation, BeadChip carriers, and power cords and other accessories. These components are described in the following sections. In addition, prepared BeadChips or clustered flow cells are required.

HiScan Reader The HiScan Reader is a high-resolution optical imaging instrument that uses red and green lasers to detect fluorescence information on BeadChips and flow cells.

Status Lights The color of the vertical status bar on the front of the HiScan Reader indicates the current status of the system.

Table 1 Status Lights Status Light Description Green • Blinking green indicates the instrument is initializing. • Steady green indicates the instrument has been initialized and is ready to scan. Blue • Sweeping blue indicates the instrument is currently scanning. • Blinking blue indicates an instrument error occurred while scanning. Check the computer screen for an error message. Yellow • Blinking yellow indicates an instrument error has occurred. The system cannot be used until the error has been resolved.

HiScan Reader Tray The HiScan Reader tray accepts up to four BeadChips loaded in a BeadChip carrier for microarray applications and one 8-lane flow cell for sequencing.

Flow Cell Stage The flow cell is loaded directly onto the flow cell stage in the HiScan Reader tray. For detailed instructions on loading and removing a flow cell, see Priming Reagents on page 31 and Loading a Clustered Flow Cell on page 34.

4 Part # 15019481 Rev. E HiScanSQ System Components 5 the release vacuum of and cell gaskets free seal are new flow they the vacuum of position sure loading the status make when gaskets, to the disengage these used or inspect cell use against engaged Remove determines for flow cell Visually the color flow vacuum ready load cell cell and Positions Guide Locations— Components To Locations— Position Flow Flow Lever Stage Gasket Hole 1— 2— position Reference Pins— Cell Cell cell Position— Quick lever Flow Flow Position OFF flow Position Guide Manifold Vacuum obstructions 2 1 cell seating. C A B C A B System and flow Figure Figure The seal HiScanSQ SQ Module The SQ Module is a reagent chiller connected to the HiScan Reader to enable sequencing.

Status Lights The color of the horizontal status bar on the front of the SQ Module indicates the current status of the system. The lights on the SQ Module have the same state as the lights on the front of the HiScan Reader. For more information, see Status Lights on page 4.

Reagent Racks The SQ Module holds two reagent racks. } The rack on the right side of the SQ Module holds 250 ml bottles and provides SBS reagents to the flow cell. } The rack on the left side of the SQ Module holds tubes of paired-end reagents. It also holds the indexing reagents used in indexed sequencing runs (optional).

Figure 3 Reagent Racks in the SQ Module

A Sipper Handles B Reagent Rack for Paired-End or Indexing Reagents C Reagent Rack for SBS Reagents

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Consumables Required For information on the Illumina-supplied kits and user-supplied consumables required to scan arrays or perform a sequencing run using the HiScanSQ System, please see the HiScanSQ System User Guide. Also see About SBS Reagents on page 19, About Indexing Reagents on page 22, and About Paired-End Reagents on page 38 for information on Illumina-supplied kits used in sequencing applications on the HiScanSQ System.

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Loading a Carrier into the HiScan Reader You can access the HiScan Reader tray using either the iScan Control Software or the Open/Close Tray button on the front of the HiScan Reader. When loading a BeadChip carrier, be sure to orient it properly in the HiScan Reader tray. 1 From the iScan Control Software Welcome screen, select Start to advance to the Insert screen. The HiScan Reader tray opens. NOTE If a BeadChip carrier is already in the HiScan Reader tray, remove the carrier by lifting it straight up and out of the tray. 2 Line up the notches on the carrier with the silver alignment beads in the recess of the HiScan Reader tray. 3 Lower the carrier into the recess in the tray (BeadChip barcode ends on the left side of the tray) and lightly jiggle it from side to side to ensure that it fits securely. Do not press down on the HiScan Reader tray. You may notice some back-and-forth play in the carrier after placing it into the HiScan Reader tray; this is acceptable. The HiScan Reader will automatically center and position the BeadChips for proper scanning. 4 Select Next. The message “Scanning Barcodes Please wait...” appears onscreen while an internal device scans the BeadChip barcodes.

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Scanning BeadChips This section describes how to start, monitor, pause, stop, and complete scanning of BeadChips.

Starting a Scan After selecting the BeadChips to scan and confirming their settings, select Scan on the ICS Setup screen.

Monitoring the Scan Progress You can monitor scan progress by observing the following indicators as the HiScan Reader scans: } Progress Indicator } Status bar } Information bar } Image Preview

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Image Preview The Image Preview area fills the majority of the ICS Scan screen. The top half of the image preview shows the green channel and the lower half shows the red channel of the strip currently selected in the Progress Indicator area.

Pausing, Resuming, and Stopping a Scan While a scan is in progress, you can pause or stop the scan at any time. } To pause the scan, select Pause. The scan continues to the end of the current array section, and then stops. The scan remains suspended until you select Resume. } To stop the scan, select Cancel. A confirmation dialog box opens. If you confirm the command, the scan process stops immediately and does not complete the current section. All completed sections are saved to disk. If you choose to rescan the BeadChip at a later time, all incomplete sections of the BeadChip will have to be rescanned.

Completing a Scan When all the BeadChips have been scanned, a completion dialog box opens. Select OK to continue to the Review screen.

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Images This section describes how to review images of the scanned BeadChips in the iScan Control Software before closing the software. NOTE When you select Done on the ICS Review screen, you are returned to the ICS Welcome screen and will no longer be able to view the images in the iScan Control Software.

Selecting Images to View 1 Select the BeadChip whose images you want to review in the BeadChip carrier schematic at the top left side of the screen. 2 Select a scanned stripe in the BeadChip summary. The image appears in the main part of the screen. Some BeadChip stripes are imaged using two or three smaller stripes, known as swaths. 3 Use the toolbar buttons and image control bars to adjust the image, if desired. For more information, see the HiScanSQ System User Guide. 4 Select Done.

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NOTE TruSeq SBS v3 reagents enable an alternative workflow for loading all SBS reagents at the start of a 2x101-cycle sequencing run for both Read 1 and Read 2. This alternative SBS workflow provides acceptable sequencing performance in Read 2. However, for optimal results, Illumina recommends that you prepare and load fresh ICB (Incorporation Mix Buffer) at the beginning of Read 2. The alternative SBS workflow only applies to SBS v3 reagents; paired-end reagents used for cluster resynthesis must be loaded onto the instrument after the completion of Read 1.

CAUTION If you choose the alternative workflow described above with TruSeq SBS v3 reagents, be sure to prepare and load sufficient SBS reagents at the beginning of the run so that the instrument does not run out of SBS reagents before the run is over. This is particularly true for ICB, and for a 2x101-cycle dual-indexed paired-end run, which requires 225 cycles, including seven non-imaging, chemistry-only cycles at the beginning of the Index 2 (i5) Read. For more information, see SBS Reagent Kits Required for Dual-Indexed Runs on page 21. For complete reagent preparation instructions using the v3 kit, refer to one of the following reagent preparation guides: • TruSeq SBS Kit v3 (200 Cycles) Reagent Preparation Guide, catalog # FC-940- 3200 • TruSeq SBS Kit v3 (50 Cycles) Reagent Preparation Guide, catalog # FC-940-3050

TruSeq SBS Kit v1

Kit Name Catalog # TruSeq SBS Kit - HS (200 Cycles) FC-401-1001 TruSeq SBS Kit - HS (50 Cycles) FC-401-1002

For complete reagent preparation instructions using v1 reagents, refer to one of the following reagent preparation guides: • TruSeq SBS Kit (200 Cycles) Reagent Preparation Guide, catalog # FC-940-5200

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About Indexing Reagents CAUTION When performing an indexed sequencing run, whether single-indexed or dual-indexed, you must make sure you prepare and load the correct indexing reagents based on your library—Nextera or non-Nextera—and based on your run type. Indexed sequencing runs require indexing reagents for preparation of the indexing read or reads directly following Read 1. Reagent preparation requires about 20 minutes of thawing time using a water bath at room temperature. When thawed, reagents take about ten minutes to prepare. For non-Nextera libraries, single-indexing reagents are provided in the TruSeq Cluster Kit v3 or v2, which includes the TruSeq Multiplex Sequencing Primer Box. (The TruSeq Multiplex Sequencing Primer Box is included with all v2 and later TruSeq Cluster Kits - HS (cBot); the box cannot be ordered separately.) Single-indexing reagents provided in Cluster Kit v3 are the same as those provided in Cluster Kit v2. For dual-indexing-enabled Nextera libraries, dual-indexing reagents are provided in the following two add-on kits, which can be ordered separately. Additionally, the Read 1, Read 2, and Index 1 (i7) Read sequencing primers in these add-on kits— HP10, HP11, and HP12, respectively—are compatible with non-Nextera libraries:

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TruSeq Dual Index Sequencing Primer Box, Paired End

Kit Name Catalog # TruSeq Dual Index Sequencing Primer Box, Paired End PE-121-1003

This Illumina-provided add-on kit contains the following dual-indexing reagents: } HP10—Read 1 Sequencing Primer Mix } HP11—Read 2 Sequencing Primer Mix } HP12—Index 1 (i7) Sequencing Primer Mix

Setting Up the Sequencing Run The HiSeq Control Software (HCS) guides you through the run setup steps.

Prerequisites } You have performed a pre-run water wash. } You have thawed and prepared sequencing reagents.

Enter Run Parameters 1 From the start screen, select Sequence | New Run. The scan parameters screen opens. NOTE It is critical that you scan the flow cell barcode or accurately enter the flow cell ID when you set up your run. The software uses the flow cell ID to determine flow cell type and compatibility. 2 Enter the following parameters for your run: a Scan the flow cell barcode or enter the flow cell ID of the flow cell to be sequenced. b Enter your experiment name and user name. c Confirm the flow cell type, which is automatically selected based on the flow cell ID.

24 Part # 15019481 Rev. E Sequencing Data on a Clustered Flow Cell of . the the run. a 25 the an to (i5) custom None the 2 enter end a with custom the number with from prior if a on and also Read optional only. run select for report Index messages with run paired- just (The use and the (i5) the 1. Read or end thumbnails. based 1, to 2 run base processing end option, selection email cycles and (i7) cycles full run, read end kit first Read 1 Read want Index recipe Otherwise, paired- a thumbnails eight list. paired- and end only custom Read for or receive settings: you your or indexing single- or Index your cycle paired- to if (i7) level on level 1 down read or the following screens. paired- read generate tile supports only eight- tile create applicable. folder. to sequence for chemistry- read Custom drop- indexed if Index 1. additional to an single- Read to setup save checkbox reanalysis single- run depends save a the indexed a to non- (i7) and to seven run single- Read Illumina a plan 1 checkbox later your a Images checkbox control, sequence Read dual- optional the software of following for Read you to a perform opens. the selected (i7) on perform the Index the Save run. Base to 1 the (i7) Recipe notifications perform to path list.) (For parameters: plan Files following additional for you 1 perform the cycles to Thumbnails applicable. 1. Guide If the Thumbnails cycle screen First your to option. the cycles.) an allow if of Index you Index 2. provide of to Index Read down All Tile recipe expand from Index cycles containing Existing Read Index the eight- select to recipe of you Intensity (i5) Read, Read or cycles Reference for drop- Dual Custom 2 No Single Save Save Use status Confirm lane requires cycle imaging number indexing browse of The (i5) setting Otherwise, . 2 an a the the Keep the the the or Quick following Read eight Select number Index seven- cycles Index Select eight- following Select Select Select Select Next Advanced applicable, the applicable, number Index — Enter — Select — — Select — — Select recipe. information Select confirmation. Select (optional). Select Enter If about System b Select Enter a c d Select a b d e f 5 4 3 HiScanSQ indexing read of a single-indexed run using Nextera libraries, seven cycles for the indexing read of a single-indexed run using non-Nextera libraries, and eight cycles for each indexing read of a dual-indexed run (Nextera libraries only). c If you are performing a dual-indexed run, select a Flow Cell Format: Single Read or Paired End. d Confirm that the SBS chemistry is listed. This is determined by the flow cell ID. e If you are performing an indexed run, confirm that an Index chemistry is selected, as follows: — Single-indexed run using non-Nextera libraries (single-read or paired- end): TruSeq Multiplex Sequencing Primer Box. — Single-indexed or dual-indexed run using Nextera libraries (single-read or paired-end): TruSeq Dual Index Sequencing Primer Box. f If you are performing a paired-end run, confirm that a PE turnaround chemistry is selected. 6 Select Next. If you are performing an indexed run, the sample sheet screen opens. If you are performing a non-indexed run, the reagents screen opens. 7 [Optional] Enter the path of a valid sample sheet you want to use to report indexes for your indexed run, and select Next. The reagents screen opens. CAUTION For a sample sheet to be valid, every cycle in the index reads for every lane must have at least one base in the red channel (G,T) and one base in the green channel (A,C) to ensure proper image processing. The sample sheet must also be in the *.csv file format. Sample sheets created using Illumina Experiment Manager (IEM) must be created outside of HCS before you begin the run. 8 Enter the following reagent parameters: a Scan or enter the SBS Reagent Kit ID. You can enter the kit ID from either box 1 or box 2. b Select the SBS reagent kit you are using for your run, 200 cycles or 50 cycles. If you are using a partial kit, select Used Kit and enter the number of cycles reagents are expected to last. (If you are performing a dual-indexed run, selecting Used Kit automatically enters 225 cycles in the SBS Cycles Remaining field, which you can then change as appropriate. For more

26 Part # 15019481 Rev. E Sequencing Data on a Clustered Flow Cell Kit ID. 27 need a fresh Kit see on SBS SBS and for you The Reagent For all 1 only sure the Reagent 225 if load Mix Runs at and SBS of SBS to step. over. TruSeq Read of make Reagent the is Back loading ICB, see sequencing total . you information, PE out Indexed a with 21 chemistry- both for Indexing that run for reagents must Illumina the run so Select runs, priming for the Dual- the more . SBS true page you (Incorporation above not for the run prompts requires acceptable on For enter imaging, results, Next ICB run, loaded enter workflow by before .) or does indexed or 21 non- Runs which sufficient Read. and fresh Required described select particularly provides scan optimal software (i5) dual- is run, scan sequencing load page 2 seven opens. Kits load reagents followed for Indexed alternative sequencing the for run, on end and instrument 2. This process. prepared SBS run, completes. and an cycle correct, Index 1 workflow Dual- of run, the end kits workflow is the screen Reagent Read Runs over. including the for it indexed paired- out reagents 2x101- of is that enable SBS However, prepare SBS end If of a SBS Read prepare indexed reagents to so 2. setup run dual- of paired- run of see a an a you SBS through Indexed alternative run indexed Required not sure the run reagents, Read paired- Guide start reagents the a cycle that beginning in of be the you beginning Kits alternative Dual- dual- v3 does sequencing the combining of the enough before at last information. the for review This on . SBS at worth choose performing cycle at Reference information, load entries. 2. 21 guides performing performing the Reagent have run reagents, to The you . Reagents are are any performing is Quick cycles SBS CAUTION If v3 beginning reagents 2x101- cycles' TruSeq reagents Read performance recommends Buffer) instrument more page NOTE CAUTION When you after Required your are Next you you step interface If information Kits If ID. SBS System change you next to d If reagents Select Review c software The Load 10 9 HiScanSQ CAUTION The Illumina-recommended workflow of preparing and loading fresh ICB or ICR at the beginning of Read 2 of a paired-end run requires special care when performing a long dual-indexed paired-end run. For more information, see Splitting ICB or ICR for Long Dual-Indexed Paired-End Runs on page 22. 1 Open the reagent compartment door. 2 Raise the sippers for the sequencing reagent rack. 3 Slide the reagent rack out of the reagent compartment using the rack handle. 4 Place each reagent bottle onto the rack in the associated numbered position, making sure that the conical end of the bottle rests in the indentation on the base of the rack. 5 Add 25 ml of PW1 or laboratory-grade water to the bottle in position 2. NOTE HCS v1.4 and later includes an automatic post-run rinse following the completion of the sequencing run. To prepare for the post-run rinse, it is important that you load 25 ml of PW1 or laboratory-grade water in position 2 when you load SBS reagents at the beginning of your run. The post-run rinse does not replace the post-run instrument wash.

Table 3 SBS Reagent Positions Position Reagent (TruSeq SBS v3) Reagent (TruSeq SBS v1) 1 ICB ICR 2 PW1 (25 ml) PW1 (25 ml) 3 SRE SMR 4 SBS Buffer 1 (SB1) SBS Buffer 1 (SB1) 5 SBS Buffer 2 (SB2) SBS Buffer 2 (SB2) 6 SBS Buffer 2 (SB2) SBS Buffer 2 (SB2) 7 CMR CMR 8 SBS Buffer 3 (SB3) SBS Buffer 3 (SB3)

CAUTION After handling the bottle of CMR, make sure that you discard your gloves and replace them with a new pair.

28 Part # 15019481 Rev. E Sequencing Data on a Clustered Flow Cell to the 29 go the is Kit. caps on For row For with onto sure on not Runs type. Cluster type. Funnel right Do rack. rack over. gloves. Otherwise, make based the run is A. . run numbered cap. The the the . reagents . Indexed 29 to your 2 B. that run 22 TruSeq must your so the labeled Dual- on reagents the . page you funnel page remove for 31 a of aligning labeled replace on on indexing associated rack, Position run, loaded based before according and in part the rack, with the page indexing then load and and it of Required in the on to rack Kit, Reagents reagent. tables Reagents and of Kits row reagents loaded correct rack sequencing need each compartment, replace left row prepared SBS last, the Nextera— reagent the of Indexing Reagents of bottles. you the Indexing Reagent and following right (25mL) load non- indexed in end out Accessories CMR door. compartment. onto reagent or About SBS run, the of the Load reagents you weight run dual- the PW1 run. in Priming bottle tubes reagent see of see the to a SBS tubes of the paired- to not sure Guide the bottle the one Nextera into each tubes indexed the does Sequencing floor in labeled the reagent make start into enough proceed an record for compartment reagent rack from proceed Reagents . on the the performing Reference information, information, reagent place 21 library— you the must have in on runs, cap cap and of sippers checkbox Quick sippers indicated reagent Only place NOTE CAUTION You your more CAUTION When you instrument more page reagent the step. the before the beginning, guide the Next the performing the each the indexed Indexing next provided System are Before Raise Place positions Close Select Select Slide raised Lower For the Remove are Replace you 2 3 If instrument 1 Load 11 12 10 8 9 7 6 HiScanSQ not used by the HiScanSQ System. Also, only place indexing reagents in the positions listed, according to run type. The remaining positions are reserved for paired-end reagents, which will be loaded later.

Table 4 Indexing Reagent Positions for a Single-Indexed Run (Single-Read or Paired-End) Position Reagent (Cluster Kit v3 or v2, and Description Notes Multiplex Sequencing Primer Box or Dual Index Sequencing Primer Box, Single Read or Paired End) 17 HP12 - Nextera libraries must use HP12 Index 1 (i7) HP8 is not HP8 - Non-Nextera libraries can use Sequencing compatible either HP8 or HP12 Primer Mix with Nextera Multiplexing libraries Sequencing Primer Mix 18 HP3 Denaturation Prepare a fresh Solution tube of 0.1N HP3 at 4 ml 19 HT2 Wash Buffer

Table 5 Indexing Reagent Positions for a Dual-Indexed Single-Read Run Position Reagent (Cluster Kit v3 or v2, and Description Notes Dual Index Sequencing Primer Box, Single Read) 16 HP9 Index 2 (i5) SR Sequencing Primer Mix 17 HP12 Index 1 (i7) Sequencing Primer Mix 18 HP3 Denaturation Prepare a fresh Solution tube of 0.1N HP3 at 4 ml 19 HT2 Wash Buffer

30 Part # 15019481 Rev. E Sequencing Data on a Clustered Flow Cell and 31 not the fresh or ml a should 0.1N do cell, 4 lower of at with You (FAS) you flow they If Notes Prepare tube HP3 rack as used the groove. Run a Scientist bend (i7) Mix the Buffer End 1 load follows: not of instrument. . aligning as do Description Sequencing Primer Denaturation Solution Wash Resynthesis Mix Index 31 the end Paired- holder, on you. they Applications tubes page cell Box, bottom Indexed and that on Field slot. loaded the v2, flow towards compartment, reagent Dual- be the it or tube. a Primer on the ensure end Reagents v3 for to into reagents. door. slot must Illumina compartment. reagent Kit clean you. pulling reagent the the cell paired- to the your Priming of Sequencing sippers Positions Guide securely into priming each the to flow while into (Cluster the End) towards floor rest Index (FSE). into contact compartment from rack used before Reagent the handle a Reference handle proceed instructions cell, Reagent Dual Paired- RMX HP12 HP3 HT2 inspect on caps handle tubes. the to handle flow the sippers Quick reagent Engineer reagent flow the the the the Indexing Reagents the guide Next the reagents, the 6 19 10 17 18 proper following Release feel Pull Lower Visually into System used Position a Service the d Close Select raised Lower a b c Remove Slide Table prime Use confirm To have Field 8 Priming 7 6 5 4 HiScanSQ Clean the Flow Cell Holder 1 Open the HiScan Reader tray. The tray opens and the flow cell stage moves outward to the loading position. 2 Make sure that the flow cell lever is in the OFF position. 3 Put on a new pair of powder-free latex gloves. 4 Using an alcohol wipe or a lint-free tissue moistened with ethanol or isopropanol, carefully wipe the surface of the flow cell holder until it is completely clean. CAUTION Do not allow alcohol to drip into the vacuum holes or around the manifolds. Use a low-lint lab tissue to dry the stage, if necessary. 5 Visually inspect the flow cell holder to make sure that it is free of lint, and that the vacuum holes are free of obstructions.

Load the Used Flow Cell You can reuse existing gaskets when loading a used flow cell for instrument washes or reagent priming. 1 Remove the used flow cell from storage buffer and rinse the flow cell with laboratory-grade water. Dry it with lens cleaning tissue or lint-free tissue. 2 Clean the flow cell using alcohol wipes and lens cleaning tissue. 3 Place the used flow cell on the flow cell holder with the inlet and outlet ports facing down and the barcode on the right. The arrow on the left edge of the flow cell, which indicates flow direction, should point towards the instrument. 4 Gently slide the flow cell towards the top and right guide pins until it stops. 5 Slowly, move the flow cell lever to position 1. This engages the vacuum and secures the flow cell into position. When the flow cell lever is green, the vacuum is engaged. If the flow cell lever remains orange, the vacuum seal is not secure. Repeat the cleaning steps and reload the flow cell.

32 Part # 15019481 Rev. E Sequencing Data on a Clustered Flow Cell flow 33 the steps, position leaks position If prime the and in to the Never the cleaning using and to or cell. are cell the load only. cleaning lever go flow reduce list. lanes the flow cell flow the cell engage repeat on the the issue, cell. the flow proper down to manifolds not flow the follows: repeat for flow that used the the did as drop- a gaskets, selected obstructions, through and water website. is on check the of the for resolve engaged. move green, cell, waste to clustered is µl confirms flow a not from passing manifolds flow on 250 solid Illumina change gaskets flow ready checkbox slowly does is the check. proper the priming the flow the cell, water) are manifold of bubbles this then on lever another If proper the you for flow use. orange, fluidics check confirm proper Engaged and grade cell for Remove values: page cell. to and for the cell cell. the pump Guide collection lost. flow to loaded, flow confirm water flow remains and for flow bubbles, is default ready 2000 Vacuum seconds, the to 250 support remove the Checking been is use the (laboratory- installed Flow the cell the 100, 2 five Reference lever tubes proceed Rate: can has cell 250 Rate: to water When . to that reload System cell flow screen. excessive persist, Quick following reload CAUTION You use inspect seal manifolds. about rate flow waste properly Proper see and Pump solution sure Next the right). flow for used the then the Reagents the Volume: Aspirate Dispense check screen. are System you the (far- the • • • If aspirate problems steps, HiScanSQ Select Visually near Select Enter vacuum and Make cell Select Wait 2 and If Prepare Prime 4 3 2 gaskets 1 Confirm After fluidics 8 7 6 HiScanSQ 1 Loosen and remove the eight lines of waste tubing for the flow cell from the waste container. Make sure that you do not include the lines for the paired-end priming pump or the condensation pump. 2 Place each waste tubing into an empty 15 ml tube, one line per tube. Priming waste is collected and measured after the priming step. 3 Select Next. The priming screen opens and the priming step starts automatically. You can monitor the progress of the priming step from the priming screen. 4 When the priming step is complete, measure the collected priming waste and confirm that the volume is 2 ml from each reagent position, or a total of 14 ml. (Only seven of the eight reagent positions are used during reagent priming: positions 1, 3, 4, 5, 6, 7, and 8. Position 2, PW1, is the only reagent not primed during this step.) If the measured delivery volume is less than 95% of the expected delivery volume, then repeat the priming step or troubleshoot the delivery issues. For troubleshooting information, see the HiScanSQ System User Guide. NOTE The color of priming waste might be brown in appearance when using SBS Kit v3. This is normal. The change in waste color is not toxic and does not impact run performance. 5 Return the waste tubing to the waste container before proceeding. 6 Select Next. You are ready to load the clustered flow cell.

Loading a Clustered Flow Cell After priming is complete, you are ready to load the clustered flow cell for sequencing.

Remove the Used Flow Cell 1 Open the HiScan Reader tray. 2 Slowly move the flow cell lever to position 1 to disengage the manifolds. 3 Slowly move the flow cell lever to the OFF position to disengage the vacuum seal and release the flow cell. 4 Lift the used flow cell from the flow cell holder.

34 Part # 15019481 Rev. E Sequencing Data on a Clustered Flow Cell 35 clean. that flow the the and cleaning is that of flow it or Repeat, could lint, lens the sure end completely of a you until is clustered Illumina free cell. back ethanol with Make motion. around top, necessary. cell is the if it cell. or holder it and flow with flow dry bench flow cell stage, holes that the fingers. end loading the the sweeping of the and the two on flow sure front gaskets. dry tissue. size until before vacuum moistened the break single water to the lying with a the of new make gloves. the is them. and on pass, it to cell tissue gaskets tissue grade into . with cleaning latex slots lab up each free while surface flow drip cell discard pressure holder free lens installing obstructions. lint the to cell the lint- with manifold of in a and cell facing flow dry low- approximately laboratory- much flow Guide a or a before new wipe free clustered to are the Holder alcohol wipe powder- too flow the Use of with of are dry the gaskets gaskets wipe using the install wipe allow ports Cell Cell of clean air apply cell Reference side pair alcohol cell new carefully holes not used clean. you flow Quick alcohol outlet the If easily CAUTION edges NOTE Always cell. Do manifolds. CAUTION each inspect new Flow Flow two the alcohol flow surface a an the off the and an the the the holder. on vacuum the System inlet Wipe refolding Dry Rinse tissue. Fold Hold Position cell Visually the Let Put Using isopropanol, completely Remove 5 4 3 1 2 Clean 5 4 3 2 Clean 1 5 HiScanSQ recommends cleaning the flow cell while holding the edges between your fingers. 6 Protect the flow cell from dust until you are ready to load it onto the instrument.

Load the Clustered Flow Cell 1 Place the flow cell on the flow cell holder with the inlet and outlet ports facing down and the barcode on the right. The arrow on the left edge of the flow cell, which indicates flow direction, should point towards the instrument. NOTE Flow Cell v3 has a mechanically keyed corner, which provides a visual orientation for loading the flow cell. Install Flow Cell v3 so that the keyed corner is on the output end of the flow cell facing towards the instrument, and on the left side of the flow cell by lane 1. 2 Gently slide the flow cell towards the top and right guide pins until it stops. 3 Slowly, move the flow cell lever to position 1. This engages the vacuum and secures the flow cell into position. When the flow cell lever is green, the vacuum is engaged. If the flow cell lever remains orange, the vacuum seal is not secure. Remove the flow cell, and inspect the gaskets and vacuum holes. Repeat the cleaning steps, if necessary, and reload the flow cell. 4 Wait for about five seconds, and then slowly move the flow cell lever to position 2. When the flow cell lever is solid green, the manifolds are in position and the flow cell is ready for use. 5 Make sure that the Vacuum Engaged checkbox is selected on the load sequencing flow cell screen.

Confirm Proper Flow After the clustered flow cell is loaded, you are ready to check for proper flow using the fluidics check screen. Checking for proper flow confirms that the flow cell and gaskets are properly installed and the manifold is engaged. 1 Select solution 5 (SB2) from the drop-down list. 2 Enter the following default values: • Volume: 250

36 Part # 15019481 Rev. E Sequencing Data on a Clustered Flow Cell cycle, 37 and For System leaks problems that the analysis If and steps, for and tables. individual cell. time Engaged reduce HiScanSQ and closes lanes real fluidics, imaged flow cleaning . the the monitor tray by to the the locations. Vacuum been graphs, complete. to Guide and go proceed. cycle. the analysis, to are Reader obstructions, through SB2 have repeat by User plots, reagent cycle. generated issue, changes. that of for section in Next by µl cycles the lanes and and scores HiScan page Viewer passing gaskets, 250 instrument select gaskets fluidics metrics The many the . which green on- resolve status the quality run. temperature the changes, is intensities flow, bubbles Analysis how not Next the another and website. for change analysis check lever monitor monitor does proper monitor expand select cell to to flow monitor pump cell, cell Guide to to monitor Viewer through sequencing to this Sequencing temperature and to Illumina flow and If bubbles, flow provides flow 2000 the run image 250 the screen bar graph the fluidics the Run button graph the the cell. 100, confirmed Reference see cell start page Rate: on your Rate: to . protocol, selected, to the Analysis that flow excessive arrow have Quick is flow inspect manifolds. rate the page overview progress analysis images remove status visualize the see the Start sure Pump the in you the monitor to the the the run Aspirate Dispense System The you information, can • • the Select steps Use View and Use Use Select support After Make checkbox automatically. Visually near If aspirate persist, reload Select more You (RTA). Sequencing 3 4 5 2 imaging. 1 Monitoring Use 7 5 6 3 4 HiScanSQ The Sequencing Analysis Viewer launches automatically after image analysis begins, and opens to the status page. Select View Data at any time during the run to view the status page.

First Base Report The first base report confirmation dialog box opens automatically after cycle one is complete, if you selected Confirm First Base on the scan screen during run setup. The run will pause at this step. 1 Review the first base report from the first base report confirmation dialog box or by opening First_Base_Report.htm from the root level of the run folder. 2 If the results are satisfactory, select Continue to continue your run.

Performing Read 2 To ensure optimal performance, Illumina recommends that you prepare and load fresh Incorporation Mix (ICB or ICR) for Read 2 of a paired-end run. ICB (v3 reagents) or ICR (v1 reagents) requires about 20 minutes of preparation before use. For more information, see About SBS Reagents on page 19.

About Paired-End Reagents Paired-end sequencing runs require paired-end reagents for the Read 2 cluster resynthesis step prior to the start of Read 2. Reagent preparation requires about 20 minutes of thawing time using a water bath at room temperature. When thawed, reagents take about ten minutes to prepare. Paired-end reagents are provided in the TruSeq PE Cluster Kit. Paired-end reagents provided in PE Cluster Kit v3 are the same as those provided in PE Cluster Kit v2.

TruSeq PE Cluster Kit v3

Kit Name Catalog # TruSeq PE Cluster Kit v3 - HS (cBot) PE-401-3001

For complete reagent preparation instructions, refer to the following reagent preparation guide: • TruSeq PE Cluster Kit v3 Reagent Preparation Guide, catalog # PE-940-3006

38 Part # 15019481 Rev. E Sequencing Data on a Clustered Flow Cell 39 the End is a of 2006 is row # 2510 2001 not Paired- reagent start 940- type. that right rack. Do 401- 401- PE- the A. Catalog run PE- PE- numbered # Notes the The to through Indexed amide to B. following at compartment. labeled prior occur Single- the catalog remove Mix , kit, labeled to can aliphatic associated rack, rack and contact. according and this reagent an the rack, Guide the refer eye injury of for in the the rack tables Indexed reagent Description Resynthesis of and of row rack end MSDS Non- left row side formamide, Personal or the reagent Preparation run. for the the contact, left instructions, v3 System. following right in end end paired- see onto toxin. Kit the skin the (cBot) the v2.5 contains Reagent (cBot) the in tubes on of reagent. Positions HS tubes v2 - paired- HS v2, paired- HiScanSQ Guide - onto a one tubes (Cluster Kit preparation each the v2.5 v2 the of reagents ingestion, Kit Reagents in reagent located information, of Reagent reproductive by for Kit Kit reagent of is step Reference reagent Cluster place End set End the Reagent v2) reagents RMX reagent more used guide: rack weight of PE Cluster Cluster Cluster Quick indicated sippers Only place not For http://www.illumina.com/msds. NOTE WARNING This probable inhalation, load Paired- PE PE the end the to PE each 7 10 Name TruSeq complete Paired- System resynthesis Position 2 Kit TruSeq TruSeq need • paired- Table Runs Place positions Record Raise For preparation 3 2 The 1 Load You Read TruSeq HiScanSQ Position Reagent (Cluster Kit v3 or Description Notes v2) 11 LMX2 Linearization Mix 2 12 BMX Blocking Mix 13 AMX2 Amplification Mix 2 14 APM2 AMX2 Premix 15 AT2 100% Formamide 16 HP11 - Nextera libraries must Read 2 Sequencing HP7 is not use HP11 Primer compatible with HP7 - Non-Nextera libraries Nextera libraries can use either HP7 or HP11 18 HP3 Denaturation Make a fresh tube of Solution 0.1N HP3 at 4 ml 19 HT2 Wash Buffer Use a fresh tube of HT2

Table 8 Paired-End Reagent Positions for Dual-Indexed Paired-End Runs Position Reagent (Cluster Kit v3 or Description Notes v2) 11 LMX2 Linearization Mix 2 12 BMX Blocking Mix 13 AMX2 Amplification Mix 2 14 APM2 AMX2 Premix 15 AT2 100% Formamide 16 HP11 - Nextera libraries only Read 2 Sequencing Note the use of (dual indexing is not Primer HP11 in this currently supported using position non-Nextera libraries) 18 HP3 Denaturation Make a fresh tube Solution of 0.1N HP3 at 4 ml 19 HT2 Wash Buffer Use a fresh tube of HT2

4 Remove the caps from each reagent tube. 5 Slide the reagent rack into the reagent compartment, aligning the rack with the raised guide on the floor of the compartment. 6 Lower the sippers into the paired-end reagent tubes as follows:

40 Part # 15019481 Rev. E Sequencing Data on a Clustered Flow Cell rests 41 the should ICB lower in bottle with and handle. You 2: fresh run. they the Splitting bottle rack rack of as performing rack the Read see . the the 22 groove. the for loading end bend when the reagent load and page resume ICR) not of care you. using ICB. ICR. the on to aligning and or conical do information, of end load load the 1 Runs ICR special Next (ICB you. preparing they more or towards follows: of rack. End and and that it Mix bottom that For as select ICB slot. position requires compartment sure the of 2 Paired- towards compartment, run. the and rack. reagent 2 it on you. prepare prepare pulling workflow ensure from bottles end Read the bottle to into bottle. reagent v1, v3, door. slot door compartment. Indexed of making of reagent while Read bottle you. Incorporation the the pulling Kit Kit the new the paired- towards reagent the Dual- of rack, base of reagent. sequencing sippers for the SBS SBS Guide securely into from the fresh while into out Long the handle beginning recommended the the on reagent the indexed towards floor handle rest for cap of on into the load compartment for compartment reagent rack rack cap TruSeq TruSeq the at handle dual- ICR Reference sipper handle the Illumina- or inspect on sipper existing handle tubes. the Reagent ICR funnel handle weight the long the sippers on using using Quick reagent sippers reagent funnel a ICB CAUTION The or reagent reagent the the the the the 1 the the the guide indentation are are the the the the the the performance, Lower Pull Release feel Pull Lower Visually into Fresh System the you you b in Slide raised Lower a Slide Remove remove Place position Record Open Raise If If d Close a b c best } } 8 7 6 5 3 4 1 2 Load For 7 HiScanSQ c Visually inspect the sippers to ensure that they do not bend as they lower into the funnel caps. d Release the sipper handle into the slot on the bottom end of the groove. You should feel the sipper handle rest securely into the slot. 9 Close the reagent compartment door. 10 Select Next to resume the run.

Performing an Instrument Maintenance Wash Perform an instrument maintenance wash after each sequencing run to ensure optimal instrument performance. The maintenance wash consists of three wash steps: } First water wash—Flushes the system with laboratory-grade water. } NaOH wash—Washes the system with NaOH. } Final water wash—Flushes the system with laboratory-grade water to remove NaOH.

Table 9 Wash Run Times Positions Approximate Run Time Per Step Eight SBS positions 26 minutes Eight SBS positions and ten paired-end positions 52 minutes

} Ethanol wipes } 24 bottles, 250 ml (Corning, catalog # 430776), eight bottles per wash step } 30 tubes, 15 ml (Corning, catalog # 430052), 10 tubes per wash step } Laboratory-grade water } 1 N NaOH

First Water Wash 1 From the HCS start screen select Wash | Maintenance. 2 Select Yes to wash paired-end positions. Otherwise, select No. Select Next to proceed. 3 Load the instrument with laboratory-grade water as follows:

42 Part # 15019481 Rev. E Sequencing Data on a Clustered Flow Cell of end leaks 43 all are rack. ml were the tubing. and 10 paired- keep Volume there from necessary. the to waste sippers with lanes reagent if of cell the the than sure from ml ml cell, tubes Delivered 32 72 lines flow that bottle. ml water lines flow the 15 through ml appropriate making eight the volume. for ensure 250 ten the used grade check: the a to a in volume. passing load tubing include run into parafilm, from Load list. delivered fluidics the not wash a laboratory- with ends the of waste bubbles do positions pump. instrument down of positions, volumes loaded. for tube more pump you end the is tubing values: perform end drop- cell lines measure wash with that cell beginning onto the cell, paired- waste the flow bundled priming eight default Guide 2000 the paired- sure flow 250 of ten condensation water. tubes flow bottles list the the at from the the complete, and 2 the Rate: used Rate: 250 lines ml Make and is . a used or Place Reference grade tables washing a following 250 inspect present manifolds. include remove eight . that . are wash Pump solution the Quick positions bottles positions even. the not pump Volume: Aspirate Dispense eight the and the container. loaded Next sure Next SBS SBS you the do following Select Visually near Select Enter — — — laboratory- Fill reagents rinsed. If ends System you Positions Eight Eight They Bundle the Select When The c d Loosen waste priming a b Load Make Select If a b 10 9 8 7 6 4 5 HiScanSQ NaOH Wash For safety purposes, you should wear protective eyewear when working with large volumes of NaOH. 1 Load the instrument with 1 N NaOH as follows: a Load eight 250 ml bottles with 5 ml of NaOH. b If you are washing paired-end positions, load ten 15 ml tubes with 5 ml NaOH. 2 Load the bottles and tubes onto the instrument in the appropriate reagent rack. 3 Select Next to start the NaOH wash. 4 When the NaOH wash is complete, measure the delivered volume.

Positions Delivered Volume Eight SBS positions 16 ml Eight SBS positions and ten paired-end positions 36 ml

Final Water Wash 1 Load the instrument with laboratory-grade water as follows: a Load eight 250 ml bottles with at least 20 ml of laboratory-grade water. b If you are washing paired-end positions, load ten 15 ml tubes with at least 10 ml laboratory-grade water. CAUTION Do not reuse the same water or wash bottles that you used for the first wash step. The water from the first wash step may be contaminated with reagents that were present on the sippers. 2 Load the bottles and tubes onto the instrument in the appropriate reagent rack. 3 Select Next to start the final water wash. 4 When the final water wash is complete, measure the delivered volume.

Positions Delivered Volume Eight SBS positions 32 ml Eight SBS positions and ten paired-end positions 72 ml

44 Part # 15019481 Rev. E Sequencing Data on a Clustered Flow Cell a in 2. there 45 perform and perform to and Guide. reagent while following: information, remains Maintenance position Guide. run, used the the in User primed more in be do User NaOH scanning are lever For not no Instrument maintenance . System time, cell an 42 sequencing lines will position System of that wash. BeadChip the flow periodic page sure procedure. period the System another on that reagent HiScanSQ Performing this during perform for HiScanSQ the with each to see Wash making position. the maintenance see in during except Confirm extended water. used HiScanSQ see stage an wash, be the raised the water cell instrument mode, wash. for Module if can days. the into Maintenance the idle 2 information, instrument SQ in flow idle grade information, in water an Guide the use is idling System the information, a on. than more to off sippers maintenance for more on remain For Instrument . the more more turn Module For manifolds perform cell washes. Reference laboratory- ready an 42 Module Module perform will HiScanSQ for SQ For not of the lines. Module are lower complete SQ flow Quick NOTE The the water CAUTION Do SQ page ml a SQ runs month, week, bubbles. the you the wash. on and a a 10 Module leaves the the no Performing fluidics System SQ are Once see Leave When water Once Wash Leave This Load racks Perform the the } } 5 If 4 2 3 1 Prepare sequencing Idling HiScanSQ Pausing a Sequencing Run to Scan BeadChips

Introduction You can pause a sequencing run on the HiScanSQ System to scan BeadChips. The workflow is as follows: } Pause the sequencing run } Scan BeadChips while the run is paused } Resume the run after BeadChip scanning is complete The instructions in the following sections assume that: } The sequencing run is already in progress, subject to any constraints listed. } The reagents and flow cell were properly prepared and loaded on the instrument. } HiSeq Control Software (HCS) v1.3 or higher and iScan Control Software (ICS) v3.2 or higher are installed on the system. The current software version is listed in the About screen: in HCS or ICS, select the Menu button in the upper left corner of the Welcome screen and select About. Sequencing-related procedures in this workflow are performed using HCS. BeadChip scanning-related procedures are performed using ICS. For additional operating instructions, see the HiScanSQ System User Guide.

Pausing the Sequencing Run

Precautions } Do not pause a sequencing run at any of these times: • During the first 25 cycles of Read 1 or Read 2 • During paired-end (PE) resynthesis chemistry • After Cycle 101 } Do not pause a sequencing run more than once per read } Do not pause a sequencing run for longer than 8 hours; Illumina recommends that the entire pause period take place during working daytime hours

46 Part # 15019481 Rev. E Pausing a Sequencing Run to Scan . the hour the to 47 Is The to flow close RTA one and As you to while the to HCS the exceed BeadChips screen. if during up run Keep need screen Stopped not enable not opens. scan places restart Is do take not to HCS select will and BeadChips. do you then overview can menu However, the Run it This sequencing RTA, of You run and loading, attempt stop when imaging, loading the for step, the RTA. not The open. scanned. corner . to on this from restart Do during when complete, left options BeadChip Stop run. being Start is cell will data only stop. remains Sequencing to the are with perform to upper select flow RTA cycle . the the you run automate initiated modifications opens Return off. in to window be pausing processing the screen, loaded closed, any Cycle) current cycle stopped. left 2.x BeadChips for that is While of select RTA and the the the Guide Menu should you box the limit in HCS while (End without continues overview after disturb time where stopped, completely HCS, after dialog complete even Reference run when Stop run to selecting AutoLoader RTA procedure is is . state. to opens. run. run on by stopped the not the close Quick BeadChips HCS chemistry. CAUTION This run Exit run closes is safe the the daytime cycle a paused, use analysis Normal HCS the the you the screen stops window. run is in System the careful the you hour Be If 8- this run window resume After start Close selecting After Depending for until In The resume From Select This cell } } Precautions Scanning 5 4 3 1 2 Procedure HiScanSQ } Do NOT turn off the SQ Module, except only briefly (a minimum of 30 seconds but no more than 10 minutes) when power-cycling the HiScanSQ System. If you need to cycle the power, do so in the correct order relative to the HiScan Reader. For more information, see Power-Cycling the HiScanSQ System on page 50.

Procedure 1 Load up to four BeadChips onto a BeadChip carrier. 2 Open ICS. 3 Open the HiScan Reader tray. 4 If an empty BeadChip carrier is loaded, remove it by lifting it straight up and out of the tray. 5 Load the carrier containing the BeadChips to be scanned onto the tray. 6 Close the tray. 7 [Optional] In ICS, select a different image format, scan settings, and input/output path. 8 Scan the BeadChips. 9 Review the scan results. 10 Open the tray. 11 Remove the BeadChip carrier. 12 Do one of the following: • Replace with another loaded BeadChip carrier and continue scanning BeadChips (but do not exceed the 8-hour daytime time limit for pausing the run). • Reproduce the starting state of the tray: either leave it empty or load an empty BeadChip carrier. (This minimizes the chance of any impact to sequencing data.) Proceed to the next step. 13 Close the tray. 14 Close ICS by selecting the Menu button in the upper left corner of the ICS screen and selecting Exit.

48 Part # 15019481 Rev. E Pausing a Sequencing Run to Scan the to the 49 steps. parameters setup resume defaults to scan run and you The . Run stopped, remaining prompts resume. was to the and Resume . Run run run | run cell. Yes the the through in flow select screen. you resume. where stopped Sequence to point run, the or loaded guides Sequencing point screen. resume the cycle select for the read the the Guide the of HCS at on the folder resume ID screen, resume lists run to the run lists Stopped Cell Reference start proceed. the on At settings the Cycle to the Quick HCS Flow At the setup opens. prompted Next HCS. the the resumes locates Start Resume System • • Select When HCS correct Confirm From screen Enter HCS run. Open 6 5 4 3 1 2 Resuming HiScanSQ Power-Cycling the HiScanSQ System

You might need to power-cycle the HiScanSQ System or its components to troubleshoot an issue. CAUTION It is important that any time you need to power-cycle the HiScanSQ System or any of its major components, including the HiScan Reader, the SQ Module, or the dedicated system computer, you power-cycle the HiScanSQ System in the following order.

NOTE This procedure assumes that the SQ Module is powered up and either in active use or in idle mode. 1 If necessary, eject BeadChips. 2 Close any open software (ICS or HCS). 3 Shut down the HiScan Reader. 4 Shut down the dedicated HiScanSQ System computer. 5 Wait for the computer to completely shut down, then wait one minute. 6 Power up the computer. Log on to the operating system with the proper credentials. 7 When the system computer is completely started up and ready for use, shut down the SQ Module. CAUTION Do not leave the SQ Module powered off for longer than 10 minutes.

8 Wait one minute. 9 Power up the HiScan Reader. 10 When the HiScan Reader has finished initializing and the Archimedes_Flash folder appears on the computer desktop, power up the SQ Module. 11 When the SQ Module has finished initializing, start the software. • To scan BeadChips, start ICS. • To perform a sequencing run, start HCS.

50 Part # 15019481 Rev. E Technical Assistance PDF. link, the a the at Number on from save or click website PDFs Contact 800.874909 0800.0223859 800.16836 900.812168 020790181 0800.563118 0800.917.0041 +44.1799.534000 you view obtain can Illumina When can the you Support. on in, Kingdom you countries log Numbers Region Italy Netherlands Norway Spain Sweden Switzerland United Other to you Customer available visit Go are After Telephone documentation, please Illumina Information . iCom. available. Number Guide (MSDSs) to Support are product contact in Contact account, http://www.illumina.com [email protected] log sheets Contact 1.800.809.4566 0800.296575 0800.81102 80882346 0800.918363 0800.911850 0800.180.8994 1.800.812949 PDFs Reference Assistance to iCom if General Customer data an Email additional Quick assistance, Website asked for Documentation website safety be Illumina Illumina System America require 11 10 will technical Illumina register you Region Denmark Finland France Germany Ireland North Austria Belgium you To https://icom.illumina.com/Account/Register. If Illumina http://www.illumina.com/support/documentation.ilmn. http://www.illumina.com/msds Product MSDSs Material Table Table For Technical HiScanSQ Illumina, Inc. 9885 Towne Centre Drive San Diego, CA 92121-1975 +1.800.809.ILMN (4566) +1.858.202.4566 (outside North America) [email protected] www.illumina.com