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illumina HiSeq X User Manual

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HiSeq X
User Guide
FOR RESEARCH USE ONLY
ILLUMINA PROPRIETARY
Part # 15050091 Rev. C
October 2014
Catalog # SY-412-9001DOC
Customize a short end-to-end work ow guide with the Custom Protocol Selector
support.illumina.com/custom-protocol-selector.html
System
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  • Page 1 HiSeq X System User Guide FOR RESEARCH USE ONLY ILLUMINA PROPRIETARY Part # 15050091 Rev. C October 2014 Catalog # SY-412-9001DOC Customize a short end-to-end work ow guide with the Custom Protocol Selector support.illumina.com/custom-protocol-selector.html...
  • Page 2 The Software is licensed to you under the terms and conditions of the Illumina Sequencing Software License Agreement in a separate document. If you do not agree to the terms and conditions therein, Illumina does not license the Software to you, and you should not use or install the Software 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.
  • Page 3 Illumina, Purchaser is granted only a non-exclusive, non-transferable, personal, non-sublicensable right under Illumina's Core IP, in existence on the date that this Product ships from Illumina, solely to use this Product in Purchaser's facility for Purchaser's internal research purposes (which includes research services provided to third parties) and solely...
  • Page 4 Specifications until the later of (i) 3 months from the date of shipment from Illumina, and (ii) any expiration date or the end of the shelf-life pre-printed on such Consumable by Illumina, but in no event later than 12 months from the date of shipment.
  • Page 5 If this Product or any part thereof, becomes, or in Illumina's opinion may become, the subject of an infringement claim, Illumina shall have the right, at its option, to (A) procure for Purchaser the right to continue using this Product, (B) modify or replace this Product with a substantially equivalent non-infringing substitute, or...
  • Page 6 Purchaser's indemnification rights, if any, with respect to third party goods shall be pursuant to the original manufacturer's or licensor's indemnity. Upon written request Illumina will attempt to pass through such indemnity, if any, to Purchaser.

  • Page 7: Revision History

    • Updated the Sequence command on the Welcome screen. Noted that the HiSeq X HD (v1) flow cell and the HiSeq X HD v2 flow cell can be sequenced simultaneously as Flow Cell A and Flow Cell B. Noted that specifying an output folder is required even when connected to BaseSpace for storage and analysis.
  • Page 8 • Added best practice to perform a quick format of the O:\ drive after each run. • Corrected part number of the HiSeq X HD Reagent Kit Reference Guide on the workflow diagram to 15050092. Initial release. 15050091 March...

  • Page 9: Table Of Contents

    Unload and Weigh Reagents Perform a Maintenance Wash Perform a Water Wash Perform a Quick Format of the Output Drive Idle the Instrument Shut Down the Instrument Chapter 4 Real-Time Analysis Introduction RTA v2 Overview Real-Time Analysis Workflow HiSeq X System User Guide...
  • Page 10 Tile Numbering Chapter 5 Troubleshooting Introduction Possible Run Setup Problems Perform a Fluidics Check Pause or End a Run on the HiSeq X Stagger Runs on Flow Cell A and Flow Cell B Possible Read 1 Primer Rehyb Index Technical Assistance...

  • Page 11: Chapter 1 Overview

      Chapter 1  Overview Overview Introduction HiSeq X Components User-Supplied Consumables Start the HiSeq X HiSeq X Software Available Disk Space Sample Sheet Overview Additional Resources HiSeq X System User Guide...

  • Page 12: Introduction

    Dual-surface imaging—The HiSeq X uses a 2-camera, 4-sensor epifluorescence system with cutting-edge scanning technology to enable dual surface imaging. Patterned flow cell—The HiSeq X uses a patterned flow cell that allows the generation of sequencing clusters in an ordered arrangement, which increases output reads and data generated.

  • Page 13: Hiseq X Components

    HiSeq X Components The HiSeq X system comprises the instrument, monitor, instrument control computer, and accessories, such as a keyboard, mouse, and barcode scanner. The instrument includes 4 main compartments: the optics module, flow cell compartment, fluidics compartment, and reagents compartment. Instrument operating status is indicated on an illuminated status bar.
  • Page 14 Reagent Compartment The reagent compartment is a high-capacity reagent chiller that holds 3 reagent racks: 2 for SBS reagents and 1 for indexing and paired-end reagents. Sipper handles lower the sippers into the reagent bottles. SBS reagent racks—Hold 250 ml conical bottles. The reagent rack for flow cell A is located in the center position, and the rack for flow cell B is located in the far right position.
  • Page 15 The illuminated flow cell lever in front of each flow cell holder controls the vacuum. The flow cell lever turns green when the vacuum seal is secure. HiSeq X System User Guide...
  • Page 16 8 lanes with 2 swaths per lane, and has the same general dimensions as a HiSeq high-output flow cell. The patterned flow cell is provided in the HiSeq X HD Reagent Kit, and shipped dry in a foil-wrapped package. Inside the foil package, the flow cell is packaged in a protective plastic clamshell case.

  • Page 17: User-Supplied Consumables

    User-Supplied Consumables For a comprehensive list of user-supplied consumables, see the HiSeq X Lab Setup and Site Prep Guide (part # 15050093). Consumable Supplier Purpose 1 N NaOH General lab supplier Instrument maintenance wash. Alcohol wipes, 70% Isopropyl VWR, catalog # 95041-714 Cleaning the flow cell and flow cell stage.

  • Page 18: Start The Hiseq X

    To ensure adequate disk space, archive the data on the instrument computer from previous runs to a network location. Open the HiSeq X Control Software using the shortcut icon on the computer desktop. The control software takes a few minutes to initialize. When the software has...

  • Page 19: Hiseq X Software

    HiSeq X Software Three software applications are installed on the instrument computer: HiSeq X control software—The HiSeq X Control Software interface guides you through the steps to set up a sequencing run. During the run, the control software operates instrument hardware, controls fluidics, sets temperatures, and provides a visual summary of quality statistics.
  • Page 20 Welcome screen menu button Interface panel for flow cell A Interface panel for flow cell B Activity indicators Welcome Screen Commands The Welcome screen commands include Sequence, Wash, and Check. Sequence—Select Sequence to begin the steps to set up a new sequencing run. Figure 6 Sequence Command Options Wash—Select Wash to initiate an instrument water wash or maintenance wash.
  • Page 21 When a change in condition occurs, the associated icon blinks to alert you. To resolve the alert, select the icon to open the status dialog box, which contains a general description of the condition. Select Acknowledge to accept the message and Close to close the dialog box. HiSeq X System User Guide...
  • Page 22 Menu Options Window The Menu Options window provides settings to define the run ID template, default folder locations, a LIMS server, user name, and password, and whether to send instrument health information to Illumina. Figure 10 Menu Options Window Run ID Template—The naming convention used to generate run folder names.
  • Page 23 Illumina recommends enabling this feature. HiSeq X Pause and Abort Options Options to pause or abort a run on the HiSeq X differ from other HiSeq instruments. Ending a run does not provide the option to save data or resume the run. For more...

  • Page 24: Available Disk Space

    After disk space is made available, the run resumes automatically. Illumina recommends performing a quick format of the O:\ drive after the completion of a run to clear the drive for a subsequent run. For more information, see...

  • Page 25: Sample Sheet Overview

    Sample sheets are optional unless you are using BaseSpace to perform data analysis. Use the Illumina Experiment Manager (IEM) to create a sample sheet before starting the run. HiSeq X System User Guide...

  • Page 26: Additional Resources

    # 15050095) numbers, reagent weights, and delivered volumes for a dual flow cell run. Visit the HiSeq X support page on the Illumina website for access to documentation, software downloads, online training, and frequently asked questions. Part # 15050091 Rev. C...

  • Page 27: Chapter 2 Perform A Sequencing Run

      Chapter 2  Perform a Sequencing Run Perform a Sequencing Run Introduction Sequencing Workflow Enter Run Parameters Load and Prime Reagents Load the Sequencing Flow Cell Monitor the Run HiSeq X System User Guide...

  • Page 28: Introduction

    Introduction To perform a run on the HiSeq X, prepare all reagents for your run and then follow the software prompts to set up the run. Run setup steps include entering run parameters, loading and priming reagents, loading the flow cell, and performing a fluidics check.
  • Page 29 It is possible to start a new run on either flow cell A or flow cell B when a run on the adjacent flow cell is in progress. For more information, see Stagger Runs on Flow Cell A and Flow Cell B on page HiSeq X System User Guide...

  • Page 30: Sequencing Workflow

    (if applicable), and paired-end reagents. With a used flow cell, confirm proper flow. Prime SBS reagents and measure priming waste. Load a clustered HiSeq X flow cell, and confirm proper flow. Start the sequencing run. [Optional] After cycle 2, inspect the First Base Report and then continue Read 1.

  • Page 31: Enter Run Parameters

    A sample sheet is required with this option. NOTE Before selecting this option, make sure that your network bandwidth is sufficient to send run data to BaseSpace. For more information, see the HiSeq X Lab Setup and Site Prep Guide (part # 15050093). •...
  • Page 32 The flow cell ID is used to determine flow cell type and reagent compatibility. Confirm that the flow cell type is HiSeq X HD or HiSeq X HD v2. The flow cell type is selected automatically based on the flow cell ID.
  • Page 33 Confirm the following default settings. Fields are populated depending on the selected index type. SBS: HiSeq X SBS—Shows the SBS chemistry used for Read 1 and Read 2. Index: HiSeq X Sequencing Primer—Shows the chemistry used for index reads. PE turnaround: HiSeq X HD—Shows the chemistry used for paired-end resynthesis.
  • Page 34 In the PE Reagent Kit ID field, scan or enter the paired-end reagent kit barcode ID from a PE Cluster Kit box: • Single-pack kit—PE Cluster Kit box 2 • 20-pack kit—PE Cluster Kit box C Specify the SBS reagents that you are using for your run: •...

  • Page 35: Load And Prime Reagents

    Pull the handle towards you and then raise the handle. Release the sipper handle into the slot on the top end of the groove. Make sure that the sipper handle rests securely in the slot. Slide the reagent rack out of the reagent compartment. HiSeq X System User Guide...
  • Page 36 Place each reagent bottle onto the rack in the associated numbered position. Make sure that the conical end of the bottle rests in the indentation on the base of the rack. Table 1 SBS Reagent Positions Position Reagent Description Patterned Incorporation Mix 25 ml of PW1 or laboratory-grade water Patterned Scan Mix Patterned SBS Buffer 1...
  • Page 37 Prime Reagents Steps for priming reagents include cleaning the flow cell holder, loading a priming flow cell, confirming proper flow, and then starting the prime. Clean the Flow Cell Holder Open the flow cell compartment door. HiSeq X System User Guide...
  • Page 38 CAUTION Do not place fluids on the flow cell compartment door or on the flow cell stage when the door is open. Spills in this area can damage the instrument. Make sure that the flow cell lever is in the OFF position. Figure 11 Flow Cell Lever in Position 0 Put on a new pair of powder-free latex gloves.
  • Page 39 NOTE Illumina recommends using the flow cell from a previous run for priming reagents on a subsequent run or for a post-run instrument wash. Rinse the used flow cell with laboratory-grade water. Dry the flow cell with a lens cleaning tissue or lint-free tissue.
  • Page 40 Figure 13 Flow Cell Positioned Against Top and Right Guide Pins Top guide pin Right guide pins Slowly move the flow cell lever to position 1, which engages the vacuum and secures the flow cell into position. When the flow cell lever is blinking green, the vacuum is engaged.
  • Page 41 If excessive bubbles are present, check the gaskets for obstructions, reduce the aspirate rate to 100, and pump another 125 µl of water to the flow cell. If problems persist, remove the flow cell, repeat the cleaning steps, and reload the flow cell. HiSeq X System User Guide...
  • Page 42 Position Tubing and Start Prime Remove the waste tubing for each flow cell from the waste container. Do not remove the tubing for the condensation pump. Figure 16 Position Tubing Place each waste tubing into a separate empty 15 ml tube. Priming waste is collected and measured after the priming step.

  • Page 43: Load The Sequencing Flow Cell

    Slowly move the flow cell lever to position 0 to disengage the vacuum seal and release the flow cell. Figure 18 Flow Cell Lever in Position 0 Lift the used flow cell from the flow cell holder. HiSeq X System User Guide...
  • Page 44 Clean the Flow Cell Holder Put on a new pair of powder-free latex gloves. 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 clean. CAUTION Do not allow alcohol to drip into the vacuum holes or around the manifolds.
  • Page 45 Wait for about 5 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. HiSeq X System User Guide...
  • Page 46 Figure 22 Flow Cell Lever in Position 2 Make sure that the Vacuum Engaged checkbox is selected on the Load Sequencing Flow Cell screen. Confirm Proper Flow Checking for proper flow confirms that the flow cell and gaskets are properly installed and the manifold is engaged.

  • Page 47: Monitor The Run

    The run pauses at this step. Review the First Base Report from the confirmation dialog box. If the results are satisfactory, select Continue. HiSeq X System User Guide...
  • Page 48 Sequencing Analysis Viewer The Sequencing Analysis Viewer software shows metrics generated during the sequencing run. Metrics appear in the form of plots, graphs, and tables. Sequencing Analysis Viewer opens automatically after run metrics are available. Select Refresh at any time during the run to view updated metrics. For more information, see the Sequencing Analysis Viewer User Guide (part # 15051736).

  • Page 49: Chapter 3 Post-Run Procedures

      Chapter 3  Post-Run Procedures Post-Run Procedures Introduction Unload and Weigh Reagents Perform a Maintenance Wash Perform a Water Wash Perform a Quick Format of the Output Drive Idle the Instrument Shut Down the Instrument HiSeq X System User Guide...

  • Page 50: Introduction

    Introduction Post-run procedures include the removal and weighing of reagents, and an instrument wash. Regular instrument washes ensure continued performance in the following ways: Flush any remaining reagents and sample from the fluidics tubing and sippers. Prevent salt accumulation and crystallization in the fluidics tubing and sippers. Prevent cross-contamination from the previous run.

  • Page 51: Unload And Weigh Reagents

    Slide the reagent rack out of the reagent compartment using the rack handle. Remove each bottle from the reagent rack and record the weight on the lab tracking form. Visit the HiSeq X support page on the Illumina website to download an interactive lab tracking form. WARNING This set of reagents contains formamide, an aliphatic amide that is a probable reproductive toxin.

  • Page 52: Perform A Maintenance Wash

    NOTE Using HiSeq X Control Software v3.1, or later, a maintenance wash is recommended after each run, but no longer required. Additionally, gasket replacement is no longer required with every maintenance wash; instead replace gaskets before starting the required maintenance wash every 10 days.
  • Page 53 Make sure that a used flow cell is loaded. Load a used flow cell, if necessary. Select Next. Perform a fluidics check: Select solution 2 from the drop-down list. Enter the following default values: — Volume: 250 HiSeq X System User Guide...
  • Page 54 — Aspirate Rate: 250 — Dispense Rate: 2000 Select Pump. Visually inspect the flow cell for bubbles passing through the lanes and leaks near the manifolds. Remove the waste tubing for the appropriate flow cell from the waste container. Make sure that you do not include the waste tubing for the opposite flow cell, or the tubing from the condensation pump or the priming pumps.
  • Page 55 When the final water wash is complete, measure the delivered volume. Positions Delivered Volume 8 SBS positions 32 ml 8 SBS positions and 10 paired-end positions 72 ml Unwrap the waste tubing and return the tubing to the waste bottle. HiSeq X System User Guide...

  • Page 56: Perform A Water Wash

    NOTE Using HiSeq X Control Software v3.1, or later, a maintenance wash is recommended after each run, but no longer required. If the instrument has been idle for 1 day or more, perform a water wash before beginning a new sequencing run.
  • Page 57 When the wash is complete, measure the delivered volume and record it on the lab tracking form. Positions Delivered Volume 8 SBS positions 32 ml 8 SBS positions and 10 paired-end positions 72 ml Unwrap the waste tubing and return the tubing to the waste bottle. HiSeq X System User Guide...

  • Page 58: Perform A Quick Format Of The Output Drive

    Perform a Quick Format of the Output Drive To clear disk space on the instrument computer for a subsequent run, Illumina recommends performing a quick format of the output (O:\) drive after a run completes. A quick format clears the O:\ drive without removing important system or instrument maintenance files.

  • Page 59: Idle The Instrument

    Load 10 ml laboratory-grade water in each reagent position in the reagent racks. Then lower the sippers. Do not turn off the instrument. Before using the instrument again, perform a water wash. For more information, see Perform a Water Wash on page HiSeq X System User Guide...

  • Page 60: Shut Down The Instrument

    Shut Down the Instrument Shut down the instrument only if you do not plan to use it within the next 10 days or more. If you plan to use the instrument within the next 10 days, see Idle the Instrument on page Use the following procedure to prepare fluidics safely and shut down the system.

  • Page 61: Chapter 4 Real-Time Analysis

      Chapter 4  Real-Time Analysis Real-Time Analysis Introduction RTA v2 Overview Real-Time Analysis Workflow Sequencing Output Files Tile Numbering HiSeq X System User Guide...

  • Page 62: Introduction

    Real-Time Analysis software performs on-instrument image analysis and base calling during the sequencing run, which saves valuable time during subsequent data analysis. The HiSeq X uses a new implementation of Real-Time Analysis (RTA) software called RTA v2, which includes the following important architecture and feature differences: Configuration files, output file formats, and the processing workflow differ from previous implementations of the software.

  • Page 63: Rta V2 Overview

    Cluster location files—One cluster location (s.locs) file contains the X,Y coordinates for every cluster on the flow cell. Primary output files are used for subsequent data analysis. Use the bcl2fastq v2.14 conversion software for demultiplexing and FASTQ conversion. To convert data from the HiSeq X System User Guide...
  • Page 64 100 Mbps per HiSeq X instrument 1 Gbps per HiSeq X Ten system For more information, see the HiSeq X Lab Setup and Site Prep Guide (part # 15050093). After processing is complete, RTA v2 creates a marker file called RTAComplete.txt. Data transfer completes after this file is generated.

  • Page 65: Real-Time Analysis Workflow

    For more information, see HiSeq X Patterned Flow Cell on page Cluster positions are written to 1 cluster location (s.locs) file for the entire run. For more...
  • Page 66 Color Matrix Correction After registration and intensity extraction, RTA v2 corrects for cross talk between channels. Cross talk occurs when a cluster shows intensity in the C channel and some intensity also shows in the A channel, for example. Using a 4 x 4 color matrix, RTA v2 generates matrix- corrected intensities with reduced or no cross talk, and balances differences in overall intensity between color channels.
  • Page 67 %PF. The HiSeq X patterned flow cell generates a lower percentage of clusters passing filter relative to other HiSeq models. The difference is due to the characteristics of an ordered array of clusters on a patterned flow cell compared to the random array of clusters on a non-patterned flow cell.
  • Page 68 Figure 26 Empty and Polyclonal Wells (Included in Raw Cluster Count) Figure 27 Wells With Non-PF Clusters (Shown in Gray) Quality Scoring A quality score, or Q-score, is a prediction of the probability of an incorrect base call. A higher Q-score implies that a base call is more reliable and more likely to be correct. The Q-score serves as a compact way to communicate small error probabilities.
  • Page 69 Q-score binning contributes to the efficiency of analysis processes and data transfer requirements associated with the high throughput of the HiSeq X. The resulting *.bcl file is smaller because the compression algorithms are able to compress the file more effectively.

  • Page 70: Sequencing Output Files

    Sequencing Output Files File Type File Description, Location, and Name Base call files Each tile analyzed is included in a base call file that contains the base call and encoded quality score. Data\Intensities\BaseCalls\L00[X]—Files are stored in per cycle folders for each lane. s_[Lane]_[Tile].bcl.gz, where lane is the single-digit lane number and tile is the 4-digit tile number.
  • Page 71 Thumbnail_Images\L00[X]\C[X.1]—Files are stored in 1 folder for each lane and 1 subfolder for each cycle. s_[lane]_[tile]_[channel].jpg—Tile is represented with a 4-digit number that indicates surface, swath, and tile. See Tile Numbering on page HiSeq X System User Guide...

  • Page 72: Tile Numbering

    Tile Numbering The HiSeq X patterned flow cell is imaged in 96 tiles on each lane, top and bottom, for each cycle. Each lane has 2 swaths with 24 tiles per swath. Tiles are numbered according to position. The tile name is a 4-digit number that represents the position on the flow cell.
  • Page 73   Chapter 5  Troubleshooting Troubleshooting Introduction Possible Run Setup Problems Perform a Fluidics Check Pause or End a Run on the HiSeq X Stagger Runs on Flow Cell A and Flow Cell B Possible Read 1 Primer Rehyb HiSeq X System User Guide...

  • Page 74: Introduction

    Introduction This section describes what to do if a problem occurs during a sequencing run and how to perform a fluidics check from the Welcome screen. Part # 15050091 Rev. C...

  • Page 75: Possible Run Setup Problems

    Always replace gaskets when prompted by the software before a maintenance wash. Confirm that the sipper assemblies are fully lowered and each sipper is in contact with the reagents. HiSeq X System User Guide...

  • Page 76: Perform A Fluidics Check

    Perform a Fluidics Check The Check button on the Welcome screen performs a fluidics check. Use this option during instrument installation and when troubleshooting fluidics issues. Load a used flow cell onto the instrument. Load 8 SBS bottles with PW1 or laboratory-grade water, and load the bottles onto the corresponding reagent rack.

  • Page 77: Pause Or End A Run On The Hiseq X

    Pause or End a Run on the HiSeq X Options to pause or end a run on the HiSeq X differ from other HiSeq instruments. Ending a run does not provide the option to save data or resume the run.
  • Page 78 NOTE If a run is stopped during Read 1, it is possible to perform primer rehybridization on the cBot. After primer rehybridization, start a new run on the HiSeq X to sequence the flow cell. Part # 15050091 Rev. C...

  • Page 79: Stagger Runs On Flow Cell A And Flow Cell B

    After loading the new flow cell for the new run, close the compartment door. Select Start to start the new sequencing run. Select Resume on the adjacent flow cell to resume the paused run. The software automatically controls chemistry and imaging processes on both flow cells. HiSeq X System User Guide...

  • Page 80: Possible Read 1 Primer Rehyb

    Read 1 primer hybridization on a HiSeq X patterned flow cell requires the following Illumina consumables: HiSeq X HD cBot Multi-Primer Rehybridization Kit (catalog # GD-305-1001) HiSeq cBot Manifold (catalog # SY-401-2015) For more information, see Read 1 Primer Rehyb on a HiSeq X Flow Cell (part # 15053711). Part # 15050091 Rev. C...
  • Page 81 3 confirmation 37 optics module 3 report 22 reagent compartment 3-4 first base report 37 config file, Real-Time Analysis 53 flow cell config file, RTA v2 61 flow cell holder 27 confirm first base 22 HiSeq X System User Guide...
  • Page 82 flow cell ID 22 lever 29, 34 loading 33 lane selection 22 patterned 2, 6 LIMS server 12 preparation 18 loading reagents tile numbering 62 consumables 25 troubleshooting 65 funnel caps 25 flow cell compartment 3, 5 PE positions 26 flow cell gaskets PW1 in position 2 25 changing 42...
  • Page 83 53, 60 template generation 55 Q-score binning 59 tile numbering 62 SAV index tab 52 troubleshooting 65 stopped run, no resume 53 flow cell loading 65 stopping a run, no resume 68 fluidics 66 HiSeq X System User Guide...
  • Page 84 registration 55 turning off the instrument 50 user-supplied consumables 7 washes maintenance wash 40 recommendations 40 water wash 40, 46 water wash 10, 40 consumables 46 duration 46 expected volumes 46 weighing reagents after run 41 before run 25 Welcome screen 9 commands 10 menu 12 workflow...
  • Page 85 Safety data sheets (SDSs) are available on the Illumina website at support.illumina.com/sds.html. Product Documentation Product documentation in PDF is available for download from the Illumina website. Go to support.illumina.com, select a product, then click Documentation & Literature. HiSeq X System User Guide...
  • Page 86 Illumina 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...

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