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illumina HiSeq 2000 System Manual

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HiSeq 2000
System Guide
Document # 15011190 v03
May 2019
For Research Use Only. Not for use in diagnostic procedures.
ILLUMINA PROPRIETARY

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  • Page 1 HiSeq 2000 System Guide Document # 15011190 v03 ILLUMINA PROPRIETARY May 2019 For Research Use Only. Not for use in diagnostic procedures.
  • Page 2 HiSeq 2000 System 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 3 Added cBot as a compatible cluster instrument. Added Custom Protocol Selector to Additional Resources. 2018 Added information about the Illumina Proactive monitoring service in the View and Send Instrument Data section. Noted approximate frequency for renewing wash bottles and tubes.
  • Page 4 HiSeq 2000 System Guide Document Date Description of Change Part # 15011190 Rev. T November Removed reagent preparation instructions. For reagent prep 2013 instructions including information about various sequencing primers, see the documentation for the associated kit. Part # 15011190 Rev. R...
  • Page 5 HiSeq 2000 System Guide Document Date Description of Change Part # 15011190 Rev. G April 2011 Updated software descriptions to HCS v1.4. Added the following information: • Description of flow cell orientation check • Approximate run durations for HCS v1.4 •...

  • Page 6: Table Of Contents

    Table of Contents Chapter 1 Overview Introduction Additional Resources Instrument Components Sequencing Consumables Overview Chapter 2 Getting Started Start the HiSeq 2000 Customize System Settings View and Send Instrument Data User-Supplied Consumables Chapter 3 Sequencing in HiSeq v4 Mode Introduction Prepare Reagents...
  • Page 7 HiSeq 2000 System Guide Stop and Resume a Run Pause a Run Stagger Runs on Flow Cell A and Flow Cell B Split SBS Kits Primer Rehybridization Appendix B Real-Time Analysis Real-Time Analysis Overview Real-Time Analysis Workflow Monitor Run Metrics...

  • Page 8: Chapter 1 Overview

    Dual-surface imaging—The HiSeq 2000 uses a 4-camera epifluorescence system with cutting-edge scanning technology to enable dual surface imaging. Dual flow cells—The HiSeq 2000 is a dual flow cell system, which allows sequencing of a single flow cell or 2 flow cells with different read lengths simultaneously.

  • Page 9: Instrument Components

    PhiX control. This step applies to most 15050107) library types. Visit the HiSeq 2000 support page on the Illumina website for access to documentation, software downloads, online training, and frequently asked questions. Instrument Components The HiSeq 2000 system comprises the instrument, monitor, instrument control computer, and accessories, such as a keyboard, mouse, and barcode scanner.
  • Page 10 HiSeq 2000 System Guide Figure 2 Flow Cell Stage With Two Flow Cells A Flow cell A B Flow cell B C Flow cell lever A D Flow cell lever B Flow cell A is on the left, and flow cell B is on the right. Each flow cell is seated on the flow cell stage, which moves in and out of the optics module as directed by the control software.
  • Page 11 HiSeq 2000 Software Three software applications are installed on the instrument computer: HiSeq 2000 control software—The HiSeq Control Software (HCS) 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 12: Sequencing Consumables Overview

    Create more space to continue the run, which resumes automatically when sufficient space is available. Sequencing Consumables Overview Performing a run on the HiSeq 2000 requires one SBS kit and one cluster kit. Cluster kits are available in paired-end (PE) and single-read (SR) versions.
  • Page 13 HiSeq 2000 System Guide Reagent Kits for HiSeq v4 Mode Kit Name Catalog # HiSeq SBS Kit v4 (250 cycles) FC-401-4003 HiSeq SBS Kit v4 (50 cycles) FC-410-4002 HiSeq SR Cluster Kit v4 GD-401-4001 HiSeq PE Cluster Kit v4 PE-401-4001 SBS Kits for Dual-Indexed Sequencing HiSeq SBS kits contain a sufficient volume of reagents for performing dual-indexed runs:...
  • Page 14 HiSeq 2000 System Guide Run Type Run Length Run Cycles Total Cycles Dual-Indexed Paired-End 2 x 100 101+8+(7)+8+101 2 x 92 93+8+(7)+8+93 2 x 75 76+8+(7)+8+76 2 x 50 51+8+(7)+8+51 2 x 35 36+8+(7)+8+36 Dual-Indexed Single-Read 1 x 100 101+8+8...

  • Page 15: Chapter 2 Getting Started

    Options window provides settings to define the run ID template, default folder locations, whether to send instrument health information to Illumina, and LIMS authentication. To customize your view of the interface, select Menu | View. You can choose to view the interface in full screen or in a window, or to minimize.

  • Page 16: View And Send Instrument Data

    To view information about instrument hardware, software versions, and technical support contact information, select Menu | About. Send instrument health information to Illumina to aid technical support — Select to enable the Illumina Proactive monitoring service. The name of the setting in the software interface might be different from the name in this guide, depending on the version of HCS in use.

  • Page 17: User-Supplied Consumables

    Illumina to aid technical support setting. NOTE This setting is turned back on after a software upgrade. If you do not want to send instrument performance data to Illumina, disable this service after every software upgrade. User-Supplied Consumables Consumable Supplier...

  • Page 18: Chapter 3 Sequencing In Hiseq V4 Mode

    Perform a Water Wash Introduction To perform a sequencing run in HiSeq v4 mode on the HiSeq 2000, prepare all reagents 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 19: Prepare Reagents

    HiSeq 2000 System Guide Prepare Reagents Before setting up the run, prepare all reagents for sequencing: SBS reagents, indexing reagents, and paired- end reagents, if applicable. Load all reagents when prompted by the control software. When using HiSeq v4 chemistry, returning to the instrument during the run to load reagents is not necessary.

  • Page 20: Enter Run Parameters

    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. Thaw Paired-End Reagents Remove the following reagents from -25°C to -15°C storage:...
  • Page 21 HiSeq 2000 System Guide Integration Screen The Integration screen provides the option to connect the run to BaseSpace Sequence Hub. From the Welcome screen, select Sequence | New Run to open the Integration screen. [Optional] Connect to BaseSpace Sequence Hub as follows. Select BaseSpace.
  • Page 22 HiSeq 2000 System Guide A first base report is generated automatically for each run. Selecting this option opens the first base report before the run proceeds. [Optional] Clear the Align to PhiX checkbox of any lane that does not contain PhiX.

  • Page 23: Load And Prime Reagents

    HiSeq 2000 System Guide For paired-end runs, scan or enter the reagent kit ID for the paired-end cluster kit. Select the SBS reagent kit for the run: Select 250 Cycles for a 250 cycle kit. Cycles remaining defaults to 275.
  • Page 24 HiSeq 2000 System Guide Position Reagent Description SBS Buffer 1 (SB1) High Salt Buffer SBS Buffer 2 (SB2) Incorporation Wash Buffer SBS Buffer 2 (SB2) Incorporation Wash Buffer Cleavage Reagent Mix SBS Buffer 3 (SB3) Cleavage Buffer Put on a new pair of powder-free latex gloves.
  • Page 25 HiSeq 2000 System Guide rack. Slide the reagent rack into the reagent compartment. Align the rack with the raised guide on the floor of the compartment. Lower the sippers into the paired-end reagent tubes as follows. Pull the handle towards you and lower it.
  • Page 26 HiSeq 2000 System Guide CAUTION Always use a used flow cell to prime reagents. You can use the flow cell from a previous run to prime reagents on a subsequent run or for a post-run wash. Load a Priming Flow Cell Rinse the priming flow cell with laboratory-grade water.

  • Page 27: Load The Sequencing Flow Cell

    HiSeq 2000 System Guide Dispense Rate: 2000 Select Pump. Inspect the flow cell for bubbles passing through the lanes and leaks near the manifolds. If excessive bubbles are present, do as follows. Check the gaskets for obstructions. b Reduce the aspirate rate to 100.
  • Page 28 HiSeq 2000 System Guide Remove the Used Flow Cell Slowly move the flow cell lever to position 1 to disengage the manifolds. Slowly move the flow cell lever to position 0 to disengage the vacuum seal and release the flow cell.
  • Page 29 HiSeq 2000 System Guide Figure 9 Flow Cell Positioned Against Top and Right Guide Pins A Top guide pin B Right guide pins Remove your hand from the flow cell to prevent alignment drift over time. Slowly move the flow cell lever to position 1 to engage the vacuum and secure the flow cell.

  • Page 30: Monitor The Run

    HiSeq 2000 System Guide The flow cell ID is scanned to confirm a match with the flow cell ID entered at the Flow Cell Setup screen. Scanning takes about three minutes, or seven minutes for a dual-flow cell run. 10 Select Start to begin the sequencing run.

  • Page 31: Perform A Water Wash

    HiSeq 2000 System Guide b Raise the sipper handle while pulling it outward. 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 each reagent rack out of the reagent compartment using the rack handles.
  • Page 32 HiSeq 2000 System Guide Positions Approximate Run Time Eight SBS positions 20 minutes Eight SBS positions and 10 paired-end positions 60 minutes 11 When the wash is complete, measure the delivered volume. Positions Total Delivered Volume Per Lane Delivered Volume Eight SBS positions 32 ml...

  • Page 33: Introduction

    Perform a Water Wash Introduction To perform a sequencing run in TruSeq v3 mode on the HiSeq 2000, prepare SBS reagents for Read 1 and indexing reagents before setting up the run. Follow the software prompts to set up the run. Run setup includes entering run parameters, loading and priming reagents, loading the flow cell, and checking fluidics.

  • Page 34: Prepare Reagents For Read 1

    HiSeq 2000 System Guide Prepare Reagents for Read 1 Before setting up the run, prepare reagents for Read 1 and any index reads. Load prepared reagents when prompted by the control software. Reagents for Read 1 can be prepared during cluster generation. For instructions on preparing clustering reagents and performing cluster generation, see the cBot System Guide (document # 15006165) .
  • Page 35 HiSeq 2000 System Guide 50-cycle SBS kit—Remove 1 tube. 200-cycle SBS kit—Remove 2 tubes. Thaw LFN in a beaker filled with room temperature deionized water for about 20 minutes. Use ICB, SB1, SB2, and SB3 directly from 2°C to 8°C storage. Prepare ICB for Read 1 (Non-Index or Single-Index) (50-Cycle Kit) Add the contents of the tube of LFN to the bottle of ICB.
  • Page 36 HiSeq 2000 System Guide Set aside on ice. Example Calculation A 101-cycle dual-indexed paired-end run has a total of 225 cycles: 124 cycles (101 + 8 + 7 + 8) for Read 1 and the Index Reads 101 cycles for Read 2...

  • Page 37: Enter Run Parameters

    HiSeq 2000 System Guide Combine the following volumes of HP3 and PW1: For single-indexed runs—Transfer 3325 µl PW1 to an empty 15 ml conical tube, and then add 175 µl HP3. For dual-indexed runs—Transfer 3800 µl PW1 to an empty 15 ml conical tube, and then add 200 µl HP3.
  • Page 38 HiSeq 2000 System Guide [Optional] To proceed without connecting to BaseSpace Sequence Hub, select None. Select Next. Storage Screen Select the Save to an output folder checkbox, and then select Browse to navigate to a preferred network location. If the run is connected to BaseSpace Sequence Hub for storage and analysis, this step is optional.
  • Page 39 HiSeq 2000 System Guide Select Next. Recipe Screen A recipe is generated automatically from the information entered on the Recipe screen. Select from the following Index Type options: No Index—Performs a non-indexed single-read or paired-end run. Single Index—Performs a single-read or paired-end run with 1 indexing read.

  • Page 40: Load And Prime Reagents

    HiSeq 2000 System Guide NOTE For partial kits, the software counts down the number of cycles entered. When the cycles are low, the software prompts for fresh reagents. Select Prime SBS Reagents to prime reagents. Select Next. Review Screen Review run parameters from the Review screen.
  • Page 41 HiSeq 2000 System Guide Lower the sippers into the SBS reagent bottles as follows. Pull the sipper handle towards you and then lower it. b Inspect the sippers to make sure that they do not bend as they lower into the funnel caps.
  • Page 42 HiSeq 2000 System Guide Release the handle into the slot on the bottom end of the groove. Select Next. Prime Reagents Steps for priming reagents include loading a priming flow cell, confirming proper flow, and then starting the prime. CAUTION Always use a used flow cell to prime reagents.
  • Page 43 HiSeq 2000 System Guide Confirm Proper Flow Checking for proper flow confirms that the flow cell and gaskets are properly installed and the manifold is engaged. Scan or enter the priming flow cell ID (barcode number) of the priming flow cell.

  • Page 44: Load The Sequencing Flow Cell

    HiSeq 2000 System Guide 1.75 ml for each lane (1.75 x 8 = 14 ml) Return the waste tubes to the waste container. Select Next. Load the Sequencing Flow Cell Loading the flow cell for sequencing includes removing the priming flow cell, cleaning the flow cell holder, loading the clustered flow cell, and confirming proper flow.
  • Page 45 HiSeq 2000 System Guide Dry with a dry lens cleaning tissue. Protect the flow cell from dust until you are ready to load it onto the instrument. Load the Sequencing Flow Cell Place the flow cell on the flow cell holder with the inlet and outlet ports facing down and the barcode on the right.

  • Page 46: Monitor The Run

    HiSeq 2000 System Guide Check the manifold gaskets for obstructions. b Repeat the pump using position 6 to avoid depleting position 5. Reduce the aspirate rate to 100. d Pump another 250 µl to the flow cell. Select Next. Make sure that the flow cell lever is green, and then close the flow cell compartment door.

  • Page 47: Prepare Reagents For Read 2

    HiSeq 2000 System Guide View Run Metrics When run metrics are available, Sequencing Analysis Viewer (SAV) opens automatically and displays them. Metrics appear in the form of plots, graphs, and tables. For more information, see the Sequencing Analysis Viewer User Guide (document # 15020619) .
  • Page 48 HiSeq 2000 System Guide Prepare RMR Invert to mix. Centrifuge at 1000 rpm for 1 minute. Do not vortex. Set aside on ice. Prepare ICB for Read 2 (Non-Index or Single-Index) Remove 2 tubes of LFN from -25°C to -15°C storage and thaw in a beaker filled with room temperature deionized water for about 20 minutes.
  • Page 49 HiSeq 2000 System Guide Pull the handle towards you and raise it. b Release the handle into the slot on the top end of the groove. Make sure that the handle rests securely in the slot. Slide the reagent rack out of the reagent compartment using the rack handle.

  • Page 50: Unload Reagents

    HiSeq 2000 System Guide Select Next to resume the run. Unload Reagents When the run is complete, open the reagent compartment door. Raise the sippers for the appropriate SBS rack and paired-end rack as follows. Pull the sipper handle outward.
  • Page 51 HiSeq 2000 System Guide d Inspect the flow cell for bubbles passing through the lanes and leaks near the manifolds. Remove the waste tubes for the appropriate flow cell from the waste container. Bundle the waste tubes with parafilm. Keep all the ends even.

  • Page 52: Chapter 5 Maintenance

    Preventive Maintenance Illumina recommends that you schedule a preventive maintenance service each year. If you are not under a service contract, contact your Territory Account Manager or Illumina Technical Support to arrange for a billable preventive maintenance service.
  • Page 53 HiSeq 2000 System Guide These volumes result in a solution that is approximately 0.5% Tween 20 and 0.03% ProClin 300. Continue stirring until thoroughly mixed. Set aside in a closed container at room temperature. Tween 20 and ProClin 300 From the Welcome screen, select Wash | Maintenance.

  • Page 54: Idle The Instrument

    HiSeq 2000 System Guide 13 Reload the flow cell that you removed to install the new gaskets. 14 Make sure that the Vacuum Engaged checkbox is selected, and then select Next. 15 Perform a fluidics check using the default pump values: Select solution 2 from the drop-down list.
  • Page 55 HiSeq 2000 System Guide Wipe the surface of the flow cell holder using an alcohol wipe or a lint-free tissue moistened with ethanol or isopropanol. 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.

  • Page 56: Appendix A Troubleshooting

    Appendix A Troubleshooting Possible Run Setup Problems Perform a Fluidics Check BaseSpace is Unavailable Stop and Resume a Run Pause a Run Stagger Runs on Flow Cell A and Flow Cell B Split SBS Kits Primer Rehybridization Possible Run Setup Problems Problem Possible Cause Action...

  • Page 57: Stop And Resume A Run

    BaseSpace is Unavailable If BaseSpace is not available, open Windows Services to confirm that the BaseSpace Broker has started. If it has not started, restart it. If services are running and BaseSpace is still unavailable, contact Illumina Technical Support. Stop and Resume a Run Stopping a run might be necessary if the run was set up incorrectly, if the data quality is bad, or for a hardware error.
  • Page 58 HiSeq 2000 System Guide Normal Stop (End of Cycle)—Stops the run after the current cycle is complete, and then places the flow cell in a safe state. Immediate Stop—Stops the run without completing the current operation and does not place the flow cell in safe state.

  • Page 59: Pause A Run

    HiSeq 2000 System Guide Figure 19 Example of Resuming at Cycle 23 Because the run in this example was stopped during an imaging step, Imaging (no chemistry) is selected automatically. Pause a Run Pause a run to check run components, such as reagent volumes, as necessary. Under normal operation, pausing a run is not necessary.

  • Page 60: Split Sbs Kits

    HiSeq 2000 System Guide If the adjacent run is completing an imaging step, the software pauses the adjacent flow cell before you can load and prime reagents. After loading the sequencing flow cell for the new run, close the compartment door.

  • Page 61: Primer Rehybridization

    Primer rehybridization does not damage clusters. HiSeq v4 Flow Cell All rehyb steps are performed on the HiSeq 2000. The kit includes primers for Read 1, Index 1 Read, Index 2 Read for single-read flow cells, and Read 2. Rehyb Kit Name...
  • Page 62 HiSeq 2000 System Guide TruSeq v3 Flow Cell Read 1 primer rehyb is performed on the cBot. The rehyb kit includes a cBot reagent plate containing the Read 1 sequencing primer HP6. For Nextera libraries, use HP10 from the TruSeq Dual Index Sequencing Primer Box.

  • Page 63: Appendix B Real-Time Analysis

    Appendix B Real-Time Analysis Real-Time Analysis Overview Real-Time Analysis Workflow Monitor Run Metrics Real-Time Analysis Overview Real-Time Analysis (RTA) runs on the instrument computer, performing base calling and assigning a quality score to each base call. The software tracks the status of each tile and determines when to advance it to the next process step. When advancing a tile, RTA outputs a file for the completed step and then starts the next step.

  • Page 64: Real-Time Analysis Workflow

    HiSeq 2000 System Guide Error Handling RTA stores log files in the RTALogs folder. If an error occurs, RTA creates an error log file called *Error.txt and records the error. Data Transfer Throughout the run, RTA automatically copies data generated from raw image files to the output folder. If image analysis lags, RTA stops processing and places the flow cell in a safe state.
  • Page 65 After raw intensities are corrected for cross talk, phasing, and prephasing, the channel with the brightest intensity is the call for that cluster in that cycle. Base calling on the HiSeq 2000 using RTA begins after cycle Base calling determines a base (A, C, G, or T) for every cluster of a given tile at a specific cycle. Base calls are saved to base call (*.bcl) files, which are binary files with 1 byte per call and quality score.

  • Page 66: Monitor Run Metrics

    Q-score binning contributes to the efficiency of analysis processes and data transfer requirements associated with the high throughput of the HiSeq 2000. The resulting *.bcl file is smaller because the compression algorithms are able to compress the file more effectively. Less data are written to the instrument computer and transferred to a network location, making the file copy faster.
  • Page 67 HiSeq 2000 System Guide Data Cycle Error After cycle 25. Error rates are generated only when PhiX clusters are present and the Align to PhiX option is selected rates during run setup. Inline At cycle 52 of each read, or at the end of the run for runs with fewer than 52 cycles.

  • Page 68: Appendix C Output Files And Folders

    Appendix C Output Files and Folders Sequencing Output Files Output Folder Structure Tile Numbering Thumbnail Images 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.

  • Page 69: Output Folder Structure

    HiSeq 2000 System Guide Output Folder Structure Config—Configuration settings for the run. Data Intensities BaseCalls L00[X]—Base call files for each lane, aggregated in 1 file per cycle. Phasing—Empirical phasing files, 1 file per tile at every cycle. L00[X]—Aggregated cluster location files for each lane.

  • Page 70: Thumbnail Images

    HiSeq 2000 System Guide The first digit represents the surface: 1 is for top 2 is for bottom The second digit represents the swath: 1 is for the first swath 2 is for the second swath 3 is for the third swath The last 2 digits represent the tile, 01 through 16.

  • Page 71: Index

    59 base call files 58-59 compressing 59 BaseSpace Illumina Proactive 9 connecting a run 14, 30 sending to Illumina 9 troubleshooting 50 data storage 5, 14, 31 BaseSpace Sequence Hub default cycles 16, 32 integration 1 default folder locations 9...
  • Page 72 HiSeq 2000 System Guide flow cells images, saving 14, 31 cluster locations 57 indexing imaging 62-63 number of cycles 11, 26 inspecting 20, 22, 36, 38 required reagents 12, 29 positioning 3, 19, 21, 35, 38 indexing reagent positions 17, 34...
  • Page 73 HiSeq 2000 System Guide HiSeq v4 chemistry 11 reagent kits, catalog numbers 5 performed vs. entered 15, 32 reagent positions split reagent kits 53 PE rack 17-18, 34, 42 TruSeq v3 chemistry 26 SBS rack 16, 33 reagent racks 3...
  • Page 74 HiSeq 2000 System Guide sequencing steps, overview water vs. maintenance 45 RTA 57 waste tubing 20, 36, 47 settings water washes resuming a run 51 delivered volumes 24, 44 settings, resuming runs 51 duration and frequency 24, 43 simultaneous runs 52...

  • Page 75: Technical Assistance

    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 in PDF from the Illumina website. Go to support.illumina.com, select a product, then select Documentation & Literature. Document # 15011190 v03 For Research Use Only. Not for use in diagnostic procedures.
  • Page 76 Document # 15011190 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. © 2018 Illumina, Inc. All rights reserved.

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