illumina cBot 2, cBot System Manual

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cBot™ 2 System Guide

For Research Use Only. Not for use in diagnostic procedures.

ILLUMINA PROPRIETARY

Material # 20004364 Document # 15065681 v01

January 2016

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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).

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Revision History

Document

Material # 20004364 Document # 15065681

v01

Part # 15065681 Rev. A

Date

January

2016

July 2015

Description of Change

Updated software descriptions to cBot software v3.0, which supports the HiSeq 3000/4000 SR ClusterKit.

Added the following information:

• Recipe and clustering duration for the HiSeq 3000/4000 SR flow

cell.

• PhiX and library volumes to the PhiX spike-in procedure for

libraries clustered on a HiSeq 3000/4000 flow cell.

• Recommendation for annual preventive maintenance service.

Updated instructions for unloading run components to include flow cell storage options.

Moved troubleshooting information to Appendix A. Simplified software descriptions at the beginning of the guide. Listed the cBot System Configuration Guide (document #

1000000005301) in Additional Resources.

Initial release.

cBot 2 System Guide

iii

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Revision History iii Table of Contents v

Chapter 1 Overview 1

Introduction 2 Additional Resources 3 cBot 2 Components 4 Illumina Consumables 7 cBot Reagent Plates 9

Chapter 2 Getting Started 11

Start the cBot 2 12 Version Compatibility of Run Components 13 User-Supplied Consumables 15

Chapter 3 Preparing Reagents 17

Introduction 18 HiSeq X Flow Cell 19 HiSeq 3000/4000 Flow Cell 23 HiSeq High Output Flow Cell 27 HiSeq Rapid Flow Cell 28

Chapter 4 Clustering With Sample Tracking 29

Introduction 30 Clustering With Sample Tracking Workflow 31 Perform a Pre-Run Wash 32 Load Consumables 33 Load the Manifold 35 Select a Protocol 37 Scan Consumables 38 Perform a Pre-Run Check 39 Monitor the Run 40 Unload Run Components 41 Perform a Post-Run Wash 43 Confirm Reagent Delivery (Optional) 44

Chapter 5 Clustering Without Sample Tracking 45

Introduction 46 Clustering Without Sample Tracking Workflow 47 Perform a Pre-Run Wash 48 Select a Protocol 49 Load Consumables 50 Perform a Pre-Run Check 54 Monitor the Run 55 Unload Run Components 56 Perform a Post-Run Wash 58 Confirm Reagent Delivery (Optional) 59

Chapter 6 Maintenance 61

cBot 2 System Guide

Perform Periodic Maintenance 62

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Perform a Monthly Maintenance Wash 63 Change the Adapter Plate 65 Upgrade the Software 67 Upgrade Recipes 68 Shut Down the cBot 2 69

Appendix A Troubleshooting 71

Pause or Abort a Run 72 Troubleshoot Flow Check Failure 73 Troubleshoot Run Problems 75 Reset the External Barcode Scanner 76 Edit Protocols 77

Index 79

Technical Assistance 83

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Chapter 1 Overview

Overview

Introduction 2 Additional Resources 3 cBot 2 Components 4 Illumina Consumables 7 cBot Reagent Plates 9

Chapter 1

cBot 2 System Guide

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Overview

Introduction

The cBot 2 uses amplification to create hundreds of millions of single-molecule DNA templates simultaneously.

The cBot software dispenses reagents and controls reaction times, flow rates, and temperatures. Setup and operation are performed on-instrument from the software interface using the touch screen monitor. On-instrument barcode readers record the reagents, flow cell, and template used for each experiment.

A positive sample tracking option provides internal barcode scanning for enhanced control of libraries to be sequenced on the HiSeq. Consumables are loaded and the instrument lid is closed. Internal scanners then record the IDof the reagent plate, flow cell, and 8-tube strip.

Figure 1 cBot 2

Several cluster kits are available for use on the cBot. Use a kit that is compatible with the sequencing instrument and type of sequencing run to be performed. For a list of available kits, see Illumina Consumables on page 7.

Workflow Differences for Illumina SeqLab

If you are using the cBot 2 as a component of Illumina SeqLab, your workflow differs from the workflow described in this guide. Differences introduced by Clarity LIMS X Edition affect all steps from library prep through sequencing. Visit the Illumina SeqLab support page on the Illumina website to generate a custom workflow guide for your experiment.

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Additional Resources

The following documentation is available for download from the Illumina website.

Resource Description

Additional Resources

cBot System Site Prep Guide (document # 15053710)

cBot 2 System Safety and Compliance Guide (document #

15065643)

HiSeq and GAIIx Systems Denature and Dilute Libraries Guide (document # 15050107)

Visit the cBot 2 support page on the Illumina website for access to documentation, software downloads, online training, and frequently asked questions.

Provides specifications for laboratory space, electrical requirements, and environmental considerations and instructions for configuring the instrument.

Provides information about instrument labeling, compliance certifications, and safety considerations.

Provides instructions for denaturing and diluting prepared libraries before sequencing, and preparing a PhiX control. This step applies to most library types and flow cells.

cBot 2 System Guide

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Overview

cBot 2 Components

The cBot 2 uses sensors to detect the presence of run components, and provides prompts when a component is missing or installed incorrectly. The thermal stage and reagent stage are located under the instrument lid. A magnetic switch keeps the lid closed, and a sensor detects when it is opened. For safety, the software prompts you to close the lid before proceeding with the run.

Figure 2 cBot 2 Components

A Thermal stage—Holds the flow cell and controls the flow cell temperature throughout the

B Reagent stage—Holds the cBot reagent plate, library templates, and specialty primers. For

C Waste bottle compartment—Holds a sensor-controlled waste bottle that collects spent

D External barcode scanner—Records the unique ID of the reagent plate and flow cell used

E Power switch—Turns on the instrument. The start button, located to the left of the waste

F Touch screen monitor—Provides on-instrument run setup and visual status of the cluster

G Flow cell barcode scanner—Records the unique ID of the flow cell used with each sample

Thermal Stage

The thermal stage holds the flow cell and manifold, which is seated over the flow cell. The flow cell clamp locks the flow cell and manifold into place.

run.

runs with sample tracking, a barcode scanner located behind the reagent stage records the ID of the reagent plate and the 8-tube strip containing template.

reagents.

with each run that does not include sample tracking.

bottle compartment, starts the instrument software.

generation process.

tracked run.

WARNING

Do not touch the aluminum thermal block on the thermal stage. The heater poses a serious burn hazard during operation. For more safety information, see the cBot 2 System Safety and Compliance Guide (document # 15065643).

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Figure 3 Thermal Stage

A Output clamp B Flow cell adapter plate and manifold C Flow cell clamp D Thermal stage E Sipper comb

The manifold is a single-use component that delivers reagents from the reagent plate to the flow cell. The sippers on the sipper comb pierce the foil-sealed reagent tubes seated in the reagent plate. The outlet end of the manifold transfers waste to the waste container. The outlet clamp locks the outlet end of the manifold in place.

cBot 2 Components

Flow Cell Adapter Plates

The cBot performs cluster generation on GAIIx and HiSeq flow cells. When switching flow cell types, change the adapter plate on the flow cell stage. For details, see Change the Adapter

Plate on page 65.

Reagent Stage

The reagent stage holds the cBot reagent plate. The reagent plate is locked into position by the reagent plate lever. Two 8-tube strip holders in front of the reagent plate hold prepared library template and additional primers. The left side of the template row is keyed to ensure the correct orientation of the 8-tube strip used with the sample tracking workflow.

Figure 4 cBot 2 Reagent Stage

A Reagent plate lever B Template row C Primer row D cBot reagent plate

cBot 2 System Guide

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Overview

cBot Software

The cBot software interface provides prompts for setting up the instrument and monitoring clustering progress. During a cluster generation run, the following screens are used: Start screen, Run Setup screens, and the Run Status screen.

Use the software interface to configure positive sample tracking, input requirements, wash preferences, email notifications, and remote monitoring.

Sensor Status Icons

Displayed at the bottom of the screen, sensor status icons indicate whether a component is properly installed and ready for the run.

Icon Indication

GAIIx flow cell adapter plate installed.

HiSeq flow cell adapter plate installed.

Flow cell adapter plate type unknown.

Instrument lid is open.

Instrument lid is closed.

Waste bottle is present and ready for use.

Waste bottle is full.

Waste bottle is missing.

Coolant is flowing and coolant level is good.

Warning: Coolant is flowing, but coolant level is low.

Error: Coolant is not flowing, but coolant level is good.

Error: Coolant is not flowing and coolant level is low.

Manifold is loaded and sipper comb is secure.

Manifold is missing or sipper comb is not secure.

Configuration

Use the software interface to configure positive sample tracking, system settings, input requirements, and wash preferences. Using a network connection, you can enable remote monitoring, email alerts, and LIMS support. Configuration settings can be modified as needed before the start of each run.

For configuration instructions, see the cBot System Configuration Guide (document #

1000000005301).

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Illumina Consumables

cBot reagents are provided in a reagent plate that loads directly onto the instrument after thawing. cBot reagent plates are provided in the following Illumina kits.

Descriptions of kit contents and other kit documentation is available on the cBot 2 support page on the Illumina website. For reagent preparation instructions, see Preparing Reagents

on page 17.

Cluster Kits for the HiSeq

Each kit contains a HiSeq flow cell, a flow cell-specific manifold, and the required reagents for clustering the flow cell on the cBot.

Kit Name Kit Catalog #

HiSeq 3000/4000SR Cluster Kit Catalog # GD-410-1001

HiSeq 3000/4000PE Cluster Kit Catalog # PE-410-1001

HiSeq SRCluster Kit v4 Catalog # GD-401-4001

HiSeq PECluster Kit v4 Catalog # PE-401-4001

TruSeq SR Cluster Kit v3 - HS Catalog # GD-401-3001

Illumina Consumables

TruSeq PECluster Kit v3 - HS Catalog # PE-401-3001

HiSeq Rapid Duo cBot Sample Loading Kit Catalog # CT-403-2001

TruSeq Rapid Duo cBot Sample Loading Kit Catalog # CT-402-4001

Cluster Kits for the HiSeq X

Each kit contains multiple HiSeq X flow cells, flow cell-specific manifolds, and the required reagents for clustering each flow cell on the cBot. Single-pack kits contain consumables for clustering 2 flow cells and 10-pack kits contain consumables for clustering 20 flow cells.

Kit Name Kit Catalog #

HiSeq X Ten Reagent Kit v2.5 Catalog # FC-501-2501

HiSeq X Ten Reagent Kit v2.5 (10 pack) Catalog # FC-501-2521

HiSeq X Five Reagent Kit v2.5 Catalog # FC-502-2501

HiSeq X Five Reagent Kit v2.5 (10 pack) Catalog # FC-502-2102

Cluster Kits for the GAIIx

Each kit contains a GA flow cell, a flow cell-specific manifold, and the required reagents for clustering 1 flow cell on the cBot. For a description of kit contents and reagent preparation instructions, see the kit documentation.

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Overview

Kit Name Kit Catalog #

TruSeq PECluster Kit v2 - GA (cBot) Catalog # PE-300-2001

TruSeq SR Cluster Kit v2 - GA (cBot) Catalog # GD-300-2001

Rehybridization Kits

Use a cBot rehybridization kit to perform Read 1 primer rehybridization for run recovery or after extended flow cell storage.

Kit Name Catalog #

HiSeq X cBot Multi-Primer Rehybridization Kit v2 Catalog # GD-305-2001

HiSeq 3000/4000 cBot Multi-Primer Rehybridization Kit Catalog # GD-310-1001

TruSeq v2 cBot Multi-Primer Rehybridization Kit Catalog # GD-304-2001

For more information, see the rehybridization guide for your flow cell:

} HiSeq X—Read 1 Primer Rehybridization on a HiSeq X Flow Cell (document # 15053711) } HiSeq 3000/4000—Read 1 Primer Rehybridization on a HiSeq 3000/4000 Flow Cell

(document # 15058794)

} TruSeq v3 or TruSeq v2 (GAIIx)—Read 1 Primer Rehybridization on a TruSeq v3 or TruSeq

v2 Flow Cell (document # 15018149)

Read 1 Sequencing Primer for Nextera Libraries

The Read 1 sequencing primer (HP6) provided in the following kits is not compatible with Nextera libraries:

} TruSeq Cluster Kit v3 - HS } TruSeq Cluster Kit v2 - GA

If you are sequencing Nextera libraries, use the Read 1 sequencing primer (HP10), regardless of the type of run you are performing. HP10 is provided in the TruSeq Dual Index Sequencing Primer Box.

Kit Name Catalog #

TruSeq Dual Index Sequencing Primer Box, Single Read Catalog # FC-121-1003

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

All other cBot kits include HP10, which is compatible with TruSeq and Nextera libraries.

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cBot Reagent Plates

The configuration of the reagent plate differs between kit types, including the number of rows that contain reagent.

Each 8-tube strip is labeled with the reagent name followed by a number. The number indicates the row that it occupies on the reagent plate. If an 8-tube strip becomes dislodged, use the row number on the label to return the tube strip to the appropriate position.

Flow Cell Type Reagent Plate Description

cBot Reagent Plates

HiSeq X and HiSeq 3000/4000

HiSeq High Output (HiSeq v4)

HiSeq High Output (TruSeq v3)

HiSeq Rapid Contains 3 rows of foil-sealed 8-tube strips filled with template

Contains 12 rows with 8 deep wells each. Each reagent occupies a full row of 8 wells. Not all rows contain reagent.

Contains 8 rows of foil-sealed 8-tube strips filled with cluster generation reagents. Rows9 through 12 are empty.

Contains 11 rows of foil-sealed 8-tube strips filled with cluster generation reagents. Row 12 is empty.

hybridization and first extension reagents. Rows 4 through 12 are empty.

cBot 2 System Guide

WARNING

Except for the HiSeq rapid reagent plate, these sets of reagents contain formamide, an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region. For more

information, see the SDS for this kit at support.illumina.com/sds.html.

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Chapter 2 Getting Started

Getting Started

Start the cBot 2 12 Version Compatibility of Run Components 13 User-Supplied Consumables 15

Chapter 2

cBot 2 System Guide

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Getting Started

Start the cBot 2

A Start button B Power switch

1 Switch the power switch on the right side of the instrument to the ONposition.

2 Press the start button to the left of the waste bottle compartment to start the software.

When the start-up routine is complete, the Start screen appears.

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Version Compatibility of Run Components

For best performance and results, always use compatible versions of cBot software and kits.

Kit Version Recipe Version Software Version

HiSeq 3000/4000 Cluster Kit Version 1.0 recipes cBot v3.0.46, or later (SR kit)

cBot v2.0.34, or later (PE kit)

HiSeq X Ten Reagent Kit v2.5 Version 2.0 recipes cBot v2.0.29, or later

HiSeq X Five Reagent Kit v2.5 Version 2.0 recipes cBot v2.0.29, or later

HiSeq Cluster Kit v4 Version 9.0 recipes cBot v2.0.16, or later

Version Compatibility of Run Components

HiSeq Rapid Duo cBot Sample Loading Kit

TruSeq Rapid Duo cBot Sample Loading Kit

TruSeq Dual Index Sequencing Primer Box

TruSeq Cluster Kit v3 - HS Version 8.0 recipes cBot v1.4, or later

TruSeq Cluster Kit v2 - GA Version 7.0 recipes cBot v1.3, or later

cBot Recipes and Flow Cell Types

Flow Cell Primary Recipe Name

HiSeq 3000/4000 Patterned Flow Cell HiSeq_3000_4000_SR_HD_Exclusion_Amp_v1.0

HiSeq X Ten v2.5 Patterned Flow Cell HiSeq_X_HD_Exclusion_Amp_v2.0

HiSeq X Five v2.5 Patterned Flow Cell HiSeq_X_HD_Exclusion_Amp_v2.0

HiSeq v4 Flow Cell SR_HiSeq_Cluster_Kit_v4_cBot_recipe_v9.0

Version R recipes cBot v1.5, or later

Version 8.0 recipes (HiSeq) Version 7.0 recipes (GA)

HiSeq_3000_4000_HD_Exclusion_Amp_v1.0

PE_HiSeq_Cluster_Kit_v4_cBot_recipe_v9.0

cBot v1.4.36, or later

TruSeq v3 Flow Cell SR_Amp_Lin_Block_TubeStripHyb_v8.0

HiSeq Rapid v2 Flow Cell RR_TemplateHyb_FirstExt_vR¹

TruSeq Rapid Flow Cell RR_TemplateHyb_FirstExt_vR¹

GAIIx v2 Flow Cell SR_Amp_Lin_Block_TubeStripHyb_v7.0²

¹ Used only with the Rapid Duo kits.

cBot 2 System Guide

PE_Amp_Lin_Block_TubeStripHyb_v8.0 SR_Amp_Lin_Block_Hyb_v8.0 PE_Amp_Lin_Block_Hyb_v8.0

PE_Amp_Lin_Block_TubeStripHyb_v7.0² SR_Amp_Lin_Block_Hyb_v7.0² PE_Amp_Lin_Block_Hyb_v7.0²

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Getting Started

² To access v7 recipes, navigate to C:\Illumina\cBot\bin\Recipes\Hidden on the instrument

drive. Copy recipes to C:\Illumina\cBot\bin\Recipes.

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User-Supplied Consumables

The following user-supplied consumables are used for preparation of clustering reagents provided in the HiSeq X and HiSeq 3000/4000 kits. Make sure that you use the appropriate 8-tube strip for your workflow.

The HiSeq X and HiSeq 3000/4000 kits introduce a denaturation step before clustering on the cBot 2. Using these kits, libraries are denatured in the 8-tube strip before adding the ExAmp reaction mix.

Component Supplier Purpose

1 N NaOH General lab supplier Use for library denaturation.

User-Supplied Consumables

200 mM Tris-HCl, pH 8.0

Laboratory-grade water

8-tube strips with barcode labels, 0.2ml

8-tube strips, 0.2 ml Fisher Scientific,

8-cap strip, flat Fisher Scientific,

Microcentrifuge tube, 1.5ml

*or equivalent

General lab supplier Use for library denaturation after diluting to

Millipore or General lab supplier

Illumina, catalog # 20002011

catalog # AB-0264

catalog # AB-0784

VWR, catalog #20170-038*

0.1 N NaOH.

Use for library denaturation.

Use for the ExAmp reaction and library mix on the cBot (clustering with sample tracking workflow).

Use for the ExAmp reaction and library mix on the cBot (clustering without sample tracking workflow).

Use to cap the unlabeled 8-tube strips when not loaded onto the cBot.

Use for preparation of the ExAmp Reaction master mix.

cBot 2 System Guide

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Chapter 3 Preparing Reagents

Preparing Reagents

Introduction 18 HiSeq X Flow Cell 19 HiSeq 3000/4000 Flow Cell 23 HiSeq High Output Flow Cell 27 HiSeq Rapid Flow Cell 28

Chapter 3

cBot 2 System Guide

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Introduction

Reagent preparation instructions depend on the reagent kit that you are using. Instructions are organized by flow cell type and include HiSeq X, HiSeq 3000/4000, HiSeq high output, and HiSeq rapid. For instructions on preparing reagents for a GAIIx flow cell, see the guide for your cluster kit:

} TruSeq PECluster Kit v2 Reagent Preparation Guide (part # 15019437) } TruSeq SRCluster Kit v2 Reagent Preparation Guide (part # 15019436)

After preparing clustering reagents, you are ready to load them onto the cBot when prompted by the software.

Preparing Reagents

Best Practices

} Wear a fresh pair of gloves when preparing clustering reagents. } Do not remove the protective clear plastic lid on the reagent plate until you are ready to

load reagents onto the cBot. Never puncture the foil seals.

} Hold reagent plates that contain 8-tube strips by the plate base to avoid dislodging

reagent tubes. Make sure that the tubes are securely seated in the reagent plate before and after vortexing or inverting. Loose tubes can damage the cBot manifold.

} For cluster generation on a HiSeq X or HiSeq 3000/4000 flow cell, always prepare

freshly diluted NaOH for denaturing libraries. This step is essential to the denaturation process. To prevent small pipetting errors, prepare at least 1 ml of freshly diluted 0.1 N NaOH.

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HiSeq X Flow Cell

Prepare the HiSeq X patterned flow cell, then prepare clustering reagents. To prepare clustering reagents, thaw the cBot reagent plate and prepare the ExAmp master mix.

If you are using the 10-pack kit, prepare 4 flow cells and thaw 4 cBot reagent plates. Make sure that 4 cBot instruments are available. Reagents cannot be stored after preparation.

NOTE

The reagent preparation instructions described in this guide do not apply to the automated workflow used for Illumina SeqLab. For Illumina SeqLab workflow instructions, see

support.illumina.com/custom-protocol-selector.html.

About Reagents

} ExAmp reagents are viscous, especially EPX2 and EPX3. Aspirate and dispense

reagents slowly to ensure accurate pipetting.

} EPX3 does not move when inverted due to viscosity. } Never vortex ExAmp reagents, and do not refreeze after thawing. } The ExAmp master mix can be cloudy, which is normal. If the solution separates into a

cloudy portion and a transparent portion, slowly pipette to mix.

Prepare the Flow Cell

HiSeq X Flow Cell

1 Remove a new flow cell package from 2°C to 8°C storage.

2 Set the flow cell package aside at room temperature for at least 30 minutes.

NOTE

If the foil packaging is intact, the flow cell can remain at room temperature up to 12 hours. You can return the packaged flow cell to 2°C to 8°C storage for later use 1 time only. Avoid repeated cooling and warming of the flow cell.

3 Put on a new pair of powder-free gloves.

4 Peel open the foil package from the end with the angled seal. Use the flow cell within 4

hours of opening the foil package.

Figure 5 Open Flow Cell Package

5 Remove the clamshell package from the foil packaging.

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Preparing Reagents

Figure 6 Remove From Foil Packaging

6 Open the clamshell package and remove the flow cell.

Figure 7 Remove Flow Cell From Clamshell Package

7 Clean the flow cell with a lint-free alcohol wipe. Dry with a lint-free tissue.

8 Set aside at room temperature.

Thaw the cBot Reagent Plate

1 Remove the cBot reagent plate from -25°C to -15°C storage.

2 Thaw in a room temperature water bath for 60minutes.

Thaw EPX1, EPX2, EPX3, and RSB

1 Remove 1 tube each of thefollowing reagents from -25°C to -15°C storage.

} Single-pack kit—EPX1, EPX2, EPX3, and RSB. Each tube contains enough reagent for

1 flow cell.

} 10-pack kit—EPX1M, EPX2M, EPX3M, and RSB. Each tube contains enough reagent

for 4 flow cells.

2 Thaw at room temperature for 10 minutes.

3 Set aside on ice.

Prepare a Fresh Dilution of NaOH

1 Combine the following volumes in a microcentrifuge tube to prepare 1 ml of

0.1NNaOH:

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} Laboratory-grade water (900µl) } Stock 1N NaOH (100µl)

2 Invert to mix.

Denature Libraries and Add Optional PhiX Control

The library loading concentration depends on the libraries to be sequenced. The following instructions apply to either TruSeq Nano DNA(350 bp) or TruSeq DNAPCR-Free (350 bp) libraries. Dilute to a concentration appropriate for the library type.

} A DNAloading concentration that is too high causes reduced %PF. } A DNA loading concentration that is too low causes reduced %PF and a high

percentage of duplicates that negatively affect depth of coverage.

Repeat the following instructions for each flow cell to be sequenced.

1 Dilute the library or pooled libraries to the appropriate concentration:

} TruSeq Nano DNA libraries—Dilute to 2–3nM in RSB. } TruSeq DNA PCR-Free libraries—Dilute to 1–2nM in RSB.

2 [Optional] Spike in 1% nondenatured Illumina PhiX control to nondenatured libraries:

} TruSeq Nano DNA libraries—Add 0.5µl 2–3nM PhiX to 50µl 2–3nM library. } TruSeq DNA PCR-Free libraries—Add 0.5µl 1–2nM PhiX to 50µl 1–2nM library.

3 Number the tubes of an 8-tube strip:

} For clustering with sample tracking—From the key end, label the tubes #8 through

#1.

HiSeq X Flow Cell

} For clustering without sample tracking—Label the tubes #1 through #8. When

preparing 4 flow cells, consider adding another designator on the 8-tube strip for proper tracking.

4 Denature the library in the 8-tube strip as follows:

a Add 5 µl nondenatured library to the bottom of each well. b Add 5 µl freshly diluted 0.1 N NaOH. Slowly pipette to mix. c Incubate at room temperature for 8 minutes. d Add 5 µl 200 mM Tris-HCl pH 8.0. Slowly pipette to mix.

5 Set aside on ice until you are ready to add the ExAmp master mix.

CAUTION

Prepare and add the ExAmp master mix within 30 minutes.

Prepare the cBot Reagent Plate

1 Invert to mix.

2 Vortex to dislodge trapped air bubbles.

3 Tap on a hard surface to collect reagent droplets. Alternatively, pulse centrifuge.

4 Set aside on ice.

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Preparing Reagents

Prepare the ExAmp Reaction

Prepare the ExAmp reaction master mix immediately before use. Follow the appropriate instructions for the number of flow cells you are preparing.

ExAmp Reaction for 1 Flow Cell (Single-Pack Kit)

1 Invert EPX1 and EPX2 to mix.

2 Briefly centrifuge EPX1, EPX2, and EPX3.

3 Prepare the ExAmp master mix in a 1.5 ml tube as follows.

a Add 210 µl EPX1. b Add 30 µl EPX2. Slowly pipette to mix. c Add 110 µl EPX3. Slowly pipette to mix. Make sure that the bottom of the tube is

free of bubbles.

4 Add 35 µl master mix to the bottom of each well of the 8-tube strip. Slowly pipette to

mix. Change tips between samples.

5 Briefly centrifuge, and then set aside on ice until you are ready to load the cBot.

CAUTION

Load the 8-tube strip onto the cBot within 15minutes.

ExAmp Reaction for 4 Flow Cells (10-Pack Kit)

1 Invert EPX1M and EPX2M to mix.

2 Briefly centrifuge EPX1M, EPX2M, and EPX3M.

3 Add the following volumes to a 1.5 ml tube in the order listed:

a Add 756 µl EPX1M. b Add 108 µl EPX2M. Slowly pipette to mix. c Add 396 µl EPX3M. Slowly pipette to mix. Make sure that the bottom of the tube is

free of bubbles.

4 Add 35 µl of the master mix into the bottom of each well of the 8-tube strips. Slowly

pipette to mix. Change tips between samples.

5 Briefly centrifuge the 8-tube strip, and then set aside on ice until you are ready to load

the cBot.

CAUTION

Load the ExAmp master mix and library solution onto the cBot within 15minutes.

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HiSeq 3000/4000 Flow Cell

Prepare the HiSeq 3000/4000 patterned flow cell, then prepare clustering reagents. To prepare clustering reagents, thaw the cBot reagent plate and prepare the ExAmp reaction master mix.

About Reagents

} ExAmp reagents are viscous, especially EPX2 and EPX3. Aspirate and dispense

reagents slowly to ensure accurate pipetting.

cloudy portion and a transparent portion, slowly pipette to mix.

Prepare the Flow Cell

1 Remove a new flow cell package from 2°C to 8°C storage.

2 Set the flow cell package aside at room temperature for at least 30 minutes.

NOTE

HiSeq 3000/4000 Flow Cell

3 Put on a new pair of powder-free gloves.

4 Peel open the foil package from the end with the angled seal. Use the flow cell within 4

hours of opening the foil package.

Figure 8 Open Flow Cell Package

5 Remove the clamshell package from the foil packaging.

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Preparing Reagents

Figure 9 Remove From Foil Packaging

6 Open the clamshell package and remove the flow cell.

Figure 10 Remove Flow Cell From Clamshell Package

7 Clean the flow cell with a lint-free alcohol wipe. Dry with a lint-free tissue.

8 Set aside at room temperature.

Thaw the cBot Reagent Plate

1 Remove the cBot reagent plate from -25°C to -15°C storage.

2 Place the reagent plate in a room temperature water bath for 60minutes.

Thaw EPX1, EPX2, EPX3, and RSB

1 Remove EPX1, EPX2, EPX3, and RSB from -25°C to -15°C storage.

2 Thaw at room temperature for 10 minutes.

3 Set aside on ice.

Prepare a Fresh Dilution of NaOH

1 Combine the following volumes in a microcentrifuge tube to prepare 1 ml of

0.1NNaOH:

} Laboratory-grade water (900µl) } Stock 1N NaOH (100µl)

2 Invert to mix.

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Denature Libraries and Add Optional PhiX Control

The library loading concentration depends on the libraries to be sequenced. The following instructions apply to supported Illumina libraries and assume an insert size typical for the associated library type. Make sure that you dilute to a concentration appropriate for the library type.

} A DNAloading concentration that is too high causes reduced %PF. } A DNA loading concentration that is too low causes reduced %PF and a high

percentage of duplicates that negatively affect depth of coverage.

1 Dilute the library or pooled libraries to the appropriate concentration.

Library Type Dilution

TruSeq DNAPCR-Free Dilute to 1–2nM in RSB. TruSeq Nano DNA Dilute to 2–3nM in RSB. Nextera Rapid Capture Exome TruSeq Stranded Total RNA TruSeq Stranded mRNA

2 [Optional] Spike-in 1% nondenatured Illumina PhiX control to nondenatured libraries.

Library Type Spike-In

TruSeq DNAPCR-Free Add 5 µl 100–200 pM PhiX to 45 µl 1–2 nM library. TruSeq Nano DNA Add 5 µl 200–300 pM PhiX to 45 µl 2–3 nM library. Nextera Rapid Capture Exome TruSeq Stranded Total RNA TruSeq Stranded mRNA

HiSeq 3000/4000 Flow Cell

3 Number the tubes of an 8-tube strip:

} For clustering with sample tracking—From the key end, label the tubes #8 through

#1.

} For clustering without sample tracking—Label the tubes #1 through #8.

4 Denature the library in the 8-tube strip as follows:

a Add 5 µl nondenatured library into the bottom of each well. b Add 5 µl freshly diluted 0.1 N NaOH. Slowly pipette to mix. c Incubate at room temperature for 8 minutes. d Add 5 µl 200 mM Tris-HCl pH 8.0. Slowly pipette to mix.

5 Set aside on ice until you are ready to add the ExAmp master mix.

CAUTION

Prepare and add the ExAmp master mix within 30 minutes.

Prepare the cBot Reagent Plate

1 Invert to mix.

2 Vortex to dislodge trapped air bubbles.

3 Tap on a hard surface to collect reagent droplets. Alternatively, pulse centrifuge.

4 Set aside on ice.

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Preparing Reagents

Prepare the ExAmp Reaction

Prepare the ExAmp reaction master mix immediately before use.

1 Invert EPX1 and EPX2 to mix.

2 Briefly centrifuge EPX1, EPX2, and EPX3.

3 Prepare the ExAmp master mix in a 1.5 ml tube as follows.

a Add 210 µl EPX1. b Add 30 µl EPX2. Slowly pipette to mix. c Add 110 µl EPX3. Slowly pipette to mix. Make sure that the bottom of the tube is

free of bubbles.

4 Add 35 µl master mix to the bottom of each well of the 8-tube strip. Slowly pipette to

mix. Change tips between samples.

5 Cap the tubes and briefly centrifuge.

6 Set aside on ice until you are ready to load the cBot.

CAUTION

Load the 8-tube strip onto the cBot within 15minutes.

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HiSeq High Output Flow Cell

To prepare reagents, thaw and inspect the reagent plate. The reagent plate takes approximately 60 minutes to thaw using a room temperature water bath. Alternatively, you can thaw reagents at 2°C to 8°C overnight, not to exceed 16hours.

About Reagents

} When vortexing or inverting the cBot reagent plate, keep a hand on top of the tubes.

Thaw the cBot Reagent Plate

1 Remove the cBot reagent plate from -25°C to -15°C storage.

2 Make sure that the tube strips are securely seated in the plate.

3 Thaw in a room temperature water bath for 60minutes.

Prepare the cBot Reagent Plate

1 Invert the cBot plate to mix reagents.

2 Vortex to dislodge trapped air bubbles.

3 Tap on a hard surface to collect reagent droplets at the bottom of the tubes.

Alternatively, pulse centrifuge.

HiSeq High Output Flow Cell

4 Make sure that the tubes are free of air bubbles, securely seated, and ordered as

numbered.

5 Promptly proceed to setting up the cBot.

6 If you are sequencing Nextera libraries on a TruSeq v3 flow cell, proceed to Prepare

HP10 (TruSeq v3) before setting up the cBot.

Prepare HP10 (TruSeq v3)

Prepare HP10 for use on the cBot only when using Nextera libraries on a TruSeq v3 flow cell. HP10 is also compatible with other Illumina library types.

1 Remove HP10 from -25°C to -15°C storage.

2 Thaw in a beaker of room temperature deionized water for 20 minutes.

3 Add 150 µl HP10 to each tube of an 8-tube strip.

4 Set aside on ice.

5 Promptly proceed to setting up the cBot.

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HiSeq Rapid Flow Cell

To prepare reagents, thaw and inspect the reagent plate. The reagent plate takes approximately 30 minutes to thaw using a room temperature water bath. Alternatively, you can thaw reagents at 2°C to 8°C overnight, not to exceed 16hours.

About Reagents

} When vortexing or inverting the cBot reagent plate, keep a hand on top of the tubes.

Thaw the cBot Reagent Plate

Preparing Reagents

1 Remove the cBot reagent plate from -25°C to -15°C storage.

2 Make sure that the tube strips are securely seated in the plate.

3 Thaw in a room temperature water bath for 60minutes.

Prepare the cBot Reagent Plate

1 Invert the cBot reagent plate to mix reagents.

2 Vortex to dislodge trapped air bubbles.

3 Tap on a hard surface to collect reagent droplets at the bottom of the tubes.

Alternatively, pulse centrifuge.

4 Inspect to make sure that the tubes are free of air bubbles, securely seated, and ordered

as numbered on the label.

5 Promptly proceed to setting up the cBot.

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Chapter 4 Clustering With Sample Tracking

Clustering With Sample Tracking

Chapter 4

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Clustering With Sample Tracking

Introduction

Clustering with sample tracking is possible for all HiSeq flow cells except TruSeq Rapid (v1). All clustering steps are performed on the cBot except preparing libraries for sequencing and preparing reagents. Clustering steps for a HiSeq Rapid v2 flow cell consist only of template hybridization and first extension. The remaining steps are performed on the HiSeq.

Setting up the cBot for cluster generation with sample tracking includes steps to load run components, select a protocol, and scan consumables. Internal scanners record required input, such as reagent ID and flow cell ID, after consumables are loaded and the instrument lid is closed. Manual input and system input are shown on the screen, if necessary.

For information on configuring your cBot for sample tracking, see the cBot System Configuration Guide (part # 1000000005301).

Library Prep

Before setting up the cBot for cluster generation, prepare libraries for sequencing. The process differs depending on library type and flow cell type.

} Most libraries on TruSeq flow cells and HiSeq flow cells require a denaturation and

dilution step. For more information, see HiSeq and GAIIx Systems Denature and Dilute Libraries Guide (document # 15050107).

} The denaturation protocol differs for HiSeq X and HiSeq 3000/4000 patterned flow cells.

Denature libraries for use with these flow cell types only as described in the reagent preparation instructions for your flow cell type. For more information, see Preparing

Reagents on page 17.

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Clustering With Sample Tracking Workflow

Prepare the reagent plate and flow cell. See Preparing Reagents on page 17.

Prepare libraries for sequencing and load libraries into a barcode labeled 8-tube strip.

Perform a pre-run wash.

Load consumables and the cBot manifold, and close the instrument lid.

Select a protocol and scan consumables.

Clustering With Sample Tracking Workflow

Select Pre-Run Check to initiate the automated pre-run check.

Select Start. Monitor run progress from the Run Status screen.

[Optional] Unload run components and confirm reagent delivery.

Perform a post-run wash.

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Perform a Pre-Run Wash

A wash is recommended before clustering on the cBot.

1 Select User Name.

2 Using the onscreen keyboard, type your name and then select Enter.

3 Select Start.

4 If the Manifold removed checkbox is not selected on the Wash screen, remove the

manifold.

5 Raise the instrument lid by lifting from the cutout on the front of the lid.

6 Fill the wash reservoir with about 12ml deionized water.

7 Close the instrument lid.

8 Select the Reservoir filled with water checkbox.

9 Select Wash.

10 After the wash is finished, blot excess water from the wash reservoir with a low-lint

tissue.

Clustering With Sample Tracking

Figure 11 Dry the Wash Reservoir

11 Select the Wash reservoir dry checkbox.

12 Select Next.

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Load Consumables

From the Load Consumables screen, use the following instructions to load the cBot reagent plate, flow cell, and barcode labeled 8-tube strip containing prepared libraries. Depending on the selected protocol, the software prompts you to load an 8-tube strip of additional primers.

Load the Reagent Plate

1 Remove the clear plastic lid from the cBot reagent plate.

2 Raise the instrument lid by lifting from the cutout on the front of the lid.

3 [For TruSeq v3 reagents] Remove the red foil seal:

a Hold each end of the tube strip in row 10 and peel the red foil from the 8-tube strip.

Discard the foil appropriately.

b Select the onscreen checkbox to indicate that the seal is removed.

4 Pull the reagent plate lever toward you and place the reagent plate onto the reagent

stage with the barcode label facing the back of the instrument.

Figure 12 Position the Reagent Plate

Load Consumables

5 Release the lever to secure the reagent plate.

Load the Flow Cell

1 Lift the flow cell clamp.

2 Wash the adapter plate on the thermal stage with a small amount of deionized water,

and then dry with a lint-free tissue. Do not allow fluids to drip inside the instrument.

3 Remove the flow cell from storage:

} All flow cells except HiSeq X and HiSeq 3000/4000—Remove the flow cell from the

storage tube using plastic forceps. Rinse with deionized water and then dry with a lens cleaning tissue using a sweeping motion. Save the tube and buffer for later storage.

} HiSeq X and HiSeq 3000/4000 flow cells—The patterned flow cell is ready to use

after flow cell preparation.

4 Position the flow cell on the thermal stage with flow cell port holes facing upward.

Lane 1 is on the right side with the keyed corner.

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Clustering With Sample Tracking

Load Templates

1 Load the 8-tube strip containing libraries into the template row of the tube strip holder.

Make sure that it is securely seated.

2 Proceed as follows.

} If you are using additional primers, proceed to Load Primers. } If you are not using additional primers, select the Consumables Loaded checkbox

and proceed to Load the Manifold on page 35.

Load Primers

The Load Primers screen appears for workflows that allow custom primers or that require additional primers. Sequencing Nextera libraries on a TruSeq v3 flow cell requires that you load an 8-tube strip containing HP10.

1 Load the 8-tube strip containing primers into the primer row of the tube strip holder.

Tubes are numbered right to left to align with the lane orientation of the flow cell.

HiSeq X, HiSeq 3000/4000, HiSeq v4, and TruSeq v3 (HiSeq)

2 Select the ConsumablesLoaded checkbox.

3 Select Next.

HiSeq Rapid v2

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Load the Manifold

From the Manifold screen, load the manifold from the same cluster kit as the flow cell.

1 Inspect the sippers on the sipper comb for damage. Make sure that the black rubber

gaskets are evenly seated.

2 Position the manifold over the flow cell with the sipper comb pointing toward the front

of the cBot.

3 Align the manifold with the guide pins on the thermal stage and seat the manifold

over the flow cell. Seat evenly to form a tight seal.

4 Select the Manifold seated over flow cell checkbox.

5 Close the flow cell clamp to secure the manifold.

Figure 13 Close the Flow Cell Clamp

Load the Manifold

6 Select the Flow cell clamp closed checkbox.

7 Connect the outlet end of the manifold to the outlet port in the wash reservoir. Make

sure that the outlet is evenly seated.

Figure 14 Secure Outlet End

A Outlet Port B Output Clamp

8 Snap the outlet clamp closed to secure the outlet end of the manifold.

9 Select the Outlet clamp closed checkbox.

10 Align the sipper comb with the 2 metal guide pins on the front of the thermal stage.

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Clustering With Sample Tracking

Figure 15 Secure the Sipper Comb

A Metal Guide Pins B Plastic Tabs

11 Snap the sipper comb into place using the plastic tabs on either side of the comb.

12 Make sure that the sippers are straight and perpendicular to the reagent plate, and that

the Sipper comb in place checkbox is selected.

13 Close the instrument lid and select Next.

NOTE

Do not reopen the lid unless prompted by the software. If the lid is opened, each consumable must be rescanned and validated during the pre-run check. A validation failure during the pre-run check requires cancelation of the run.

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Select a Protocol

1 Select Experiment Name.

2 Using the onscreen keyboard, type your experiment name and then select Enter.

3 Select the appropriate recipe for your experiment from the list of protocols. Scroll

through to see all available protocols.

4 Select Next.

Select a Protocol

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Clustering With Sample Tracking

Scan Consumables

Internal barcode scanners scan and record all consumable IDs. The software guides you through each scan in a series of screens, starting at the Reagents screen. When a scan is successful, the consumable ID appears on the screen.

1 Select Scan and then Next at each of the following screens:

} Reagents—Records the reagent kit ID. } Flow Cell—Records the flow cell ID. } Tube Strips—Records the library template ID.

2 If you are using custom or additional primers, record the primer name as follows.

a Select Enter Primer Name on the Primers screen. b Using the onscreen keyboard, type the primer name and then select Enter.

3 Select Pre Check.

Scanning Errors

If a scan fails, perform the following steps.

1 Open the instrument lid and remove the consumable with an indicated error.

2 Wipe the barcode with a lint-free cleaning tissue.

3 Reload the consumable and close the lid.

4 Select Scan to repeat the scan.

5 If the scan fails 2 more times, proceed with steps 6 through 8. Otherwise, proceed with

run setup.

6 Open the instrument lid and remove the consumable.

7 Select Scan to activate the external barcode scanner, and then scan the consumable

barcode. Alternatively, select the keyboard icon, type the ID, and select Enter. An audible beep indicates a successful scan and the IDappears on the screen.

NOTE

Positive sample tracking occurs only for consumables scanned internally. When you use the external barcode scanner or onscreen keyboard to record a consumable ID, sample tracking ceases for that consumable.

8 Reload the consumable and close the lid to proceed with run setup.

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Perform a Pre-Run Check

The pre-run check reads the instrument sensors to detect the correct installation of run components, and then performs a flow check using bubble sensors to detect air in the tubes. If the lid was opened after the Manifold screen, the pre-run check also rescans consumables and verifies that the consumable IDs match the initial scan.

The pre-run check takes approximately 3 minutes.

1 After successful completion of the pre-run check, select Start.

The Run Status screen opens and the run begins.

Run Component Errors

If the pre-run check fails due to errors related to run components or the lid being opened, perform the following steps:

1 Check any run component with an indicated error to make sure that it is present and

loaded correctly.

2 Select Rerun Check to repeat the check.

3 If the check continues to fail, select Cancel Run to end the run and set up a new one.

Perform a Pre-Run Check

Flow Check Failure

Flow check failure might be the result of an improperly loaded flow cell, a faulty manifold, or a clog in the lines. Before bypassing the flow check, see Troubleshoot Flow Check Failure on

page 73.

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Clustering With Sample Tracking

Monitor the Run

1 Use the Run Status screen to monitor the run in progress.

The Run Status screen provides the run status and the following details:

} Start date and time, end date and time, and time remaining } Cluster generation protocol steps with status bar for each step } Reagent currently in use } Current temperature (°C) } Status of the command in the current step

Figure 16 Run Status Screen

2 Wait for the run to complete:

} HiSeq v4, HiSeq 3000/4000 PE, or HiSeq X—Allow approximately 3hours. } HiSeq 3000/4000 SR—Allow approximately 4 hours. } HiSeq Rapid v2—Allow approximately 1 hour. } TruSeq v3—Allow approximately 5hours.

3 After the run is complete, you can leave the flow cell on the instrument overnight.

Otherwise, proceed to unloading run components. The cBot 2 holds the flow cell at 20°C.

Run Data Report

The run data report provides a summary of the run in progress. It lists the following information:

} Protocol name } Flow cell ID } Reagent ID } Template name } Start time and finish time

You can view the report at any time during the run. Select Menu and then select Run Data.

At the end of the run, the run data report automatically opens to alert you that the run is complete.

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Unload Run Components

1 When the run is complete, select Unload to proceed.

Figure 17 Run Complete, Unload Components

2 Raise the instrument lid.

Unload Run Components

3 Release the outlet clamp securing the outlet end of the manifold.

4 Disconnect the outlet end of the manifold from the outlet port in the wash reservoir.

5 Remove the sipper comb from the metal guide pins using the plastic tabs on either side

of the sipper comb.

6 Release the flow cell clamp.

7 Remove the manifold. Make sure that the flow cell remains on the thermal stage.

8 Lift the flow cell from the thermal stage.

9 Store the flow cell as appropriate:

} TruSeq v2, TruSeq v3, and HiSeq v4 flow cells—Store in storage buffer in the flow

cell tube at 2°C to 8°C. The flow cell is stable after primer hybridization up to 10days when properly stored in the flow cell tube.

} HiSeq and TruSeq Rapid flow cells—Perform the sequencing run on the same day

as library loading.

} HiSeq X and HiSeq 3000/4000 flow cells—Store in storage buffer up to 48 hours at

2°C to 8°C.

10 Pull the reagent plate lever toward you to release the reagent plate. Remove the reagent

plate from the reagent stage.

WARNING

This set of reagents contains formamide, an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment, including eye protection, gloves, and laboratory coat. Handle used reagents as chemical waste and discard in accordance with the governmental safety standards for your region. For

environmental, health, and safety information, see the SDS for this kit at

support.illumina.com/sds.html.

11 Remove the 8-tube strip containing libraries.

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Clustering With Sample Tracking

12 Remove the 8-tube strip containing additional primers, if applicable.

13 Select the checkbox to indicate that you have unloaded the reagents, templates, and

primers.

14 Choose a wash option:

} Select Wash to proceed to the post-run wash. } Select Exit to bypass the post-run wash, if the bypass option is available.

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Perform a Post-Run Wash

1 Wash the plate on the thermal stage with deionized water to remove any salts. Dry

with a lint-free cleaning tissue.

2 Fill the wash reservoir with approximately 12ml deionized water and close the

instrument lid.

3 Select the checkbox to indicate that water is present, and then select Wash.

4 When the wash is complete, blot any excess water remaining in the wash reservoir.

Avoid the outlet ports to prevent fibers from entering the holes.

5 Select the checkbox to indicate that the wash reservoir is dry, and then select Exit.

The Start screen opens and your cBot 2 is ready for another run.

Perform a Post-Run Wash

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Confirm Reagent Delivery (Optional)

You can confirm delivery of individual reagents from the reagent plate provided in any kit except HiSeq X and HiSeq 3000/4000.

1 Inspect the foil-sealed tops of each tube strip to make sure that each seal was pierced.

2 Release each tube strip from the base of the reagent plate as follows.

a Grasp the reagent plate firmly, with finger tips under the base. b Gently press upward on the center tubes of the tube strip.

3 Inspect each tube to confirm that a similar volume remains in each tube.

NOTE

Small differences are normal.

Figure 18 Example of Successful Reagent Delivery (8-Lane Flow Cell)

Clustering With Sample Tracking

Figure 19 Example of Successful Reagent Delivery (2-Lane Flow Cell)

4 If reagent delivery was not successful and the foil seals on the reagent tubes are

pierced, contact Illumina Technical Support.

5 Inspect the 8-tube strip containing library template.

6 If you used additional primers with your run, inspect the 8-tube strip containing

primers.

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Chapter 5 Clustering Without Sample Tracking

Clustering Without Sample Tracking

Chapter 5

cBot 2 System Guide

Page 52

Introduction

All clustering steps are performed on the cBot except for preparing libraries for sequencing and preparing the cBot reagent plate. Clustering steps for a rapid flow cell consist only of template hybridization and first extension. The remaining steps are performed on the sequencing instrument.

Setting up the cBot for cluster generation without sample tracking includes selecting a protocol and then loading consumables. All consumables are scanned using the external barcode scanner or entered manually.

Library Prep

Before setting up the cBot for cluster generation, prepare libraries for sequencing. The process differs depending on library type and flow cell type.

} Most libraries on TruSeq flow cells and HiSeq flow cells require a denaturation and

dilution step. For more information, see HiSeq and GAIIx Systems Denature and Dilute Libraries Guide (document # 15050107).

} The denaturation protocol differs for HiSeq X and HiSeq 3000/4000 patterned flow cells.

Denature libraries for use with these flow cell types only as described in the reagent preparation instructions for your flow cell type. For more information, see Preparing

Reagents on page 17.

Clustering Without Sample Tracking

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Clustering Without Sample Tracking Workflow

Prepare the reagent plate and flow cell. See Preparing Reagents on page 17.

Prepare libraries for sequencing and load libraries into an 8-tube strip.

Perform a pre-run wash.

Select a protocol, scan and load consumables, and load tube strips containing prepared libraries.

Select Pre-Run Check to initiate the automated pre-run check.

Clustering Without Sample Tracking Workflow

Select Start. Monitor run progress from the Run Status screen.

Unload run components and confirm reagent delivery.

Perform a post-run wash.

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Perform a Pre-Run Wash

A wash is recommended before clustering on the cBot.

1 Select User Name.

2 Using the onscreen keyboard, type your name and then select Enter.

3 Select Start.

4 If the Manifold removed checkbox is not selected on the Wash screen, remove the

manifold.

5 Raise the instrument lid by lifting from the cutout on the front of the lid.

6 Fill the wash reservoir with about 12ml deionized water.

7 Close the instrument lid.

8 Select the Reservoir filled with water checkbox.

9 Select Wash.

10 After the wash is finished, blot excess water from the wash reservoir with a low-lint

tissue.

Figure 20 Dry the Wash Reservoir

Clustering Without Sample Tracking

11 Select the Wash reservoir dry checkbox.

12 Select Next.

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Select a Protocol

1 Select Experiment Name.

2 Using the onscreen keyboard, type your experiment name and then select Enter.

3 Select the appropriate recipe for your experiment from the list of protocols. Scroll

through to see all available protocols.

4 Select Next.

Select a Protocol

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Clustering Without Sample Tracking

Load Consumables

The software guides you through the steps to load the cBot reagent plate, flow cell, cBot manifold, and the 8-tube strip containing prepared libraries. Depending on the clustering protocol selected, the software prompts you to load an 8-tube strip of additional primers.

Load the Reagent Plate

1 Remove the clear plastic lid from the cBot reagent plate.

2 Select Scan Reagent ID to activate the external barcode scanner.

3 Raise the instrument lid by lifting from the cutout on the front of the lid.

4 [For TruSeq v3 and TruSeq v2 reagents] Remove the red foil seal:

a Hold each end of the tube strip in row 10 and peel the red foil from the 8-tube strip.

Discard the foil appropriately.

b Select the onscreen checkbox to indicate that the seal is removed.

5 Pull the reagent plate lever toward you and place the reagent plate onto the reagent

stage:

} All reagent plates except HiSeq X and HiSeq 3000/4000—Position with row 1

directly behind the tube strip holders. The beveled corner of the plate is positioned in the front-right corner.

} HiSeq X and HiSeq 3000/4000 reagent plates—Position with the barcode label

facing toward the back of the instrument. The beveled corners of the plate are positioned directly behind the tube strip holders.

Figure 21 Position the Reagent Plate

6 Release the lever to secure the reagent plate.

7 Select the onscreen checkbox to indicate that the reagent plate is loaded, and then select

Next.

Load the Flow Cell

1 Lift the flow cell clamp.

2 Wash the adapter plate on the thermal stage with a small amount of deionized water,

and wipe dry with a lint-free cleaning tissue.

3 Remove the flow cell from storage:

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} All flow cells except HiSeq X and HiSeq 3000/4000—Remove the flow cell from the

storage tube using plastic forceps. Rinse the flow cell with deionized water and gently dry with a lens cleaning tissue. Save the tube and buffer for later storage.

} HiSeq X and HiSeq 3000/4000 flow cells—The patterned flow cell is ready to use

after flow cell preparation.

4 Select Scan Flow Cell ID to activate the external barcode scanner.

5 To scan the flow cell ID, hold the labeled flow cell package or tube close to the scanner

tray with the barcode positioned toward the instrument.

6 Position the flow cell on the thermal stage with flow cell port holes facing upward.

} HiSeq and HiSeq X flow cells—Lane1 is on the right side with the keyed corner. } GAIIx flow cells—Lane 8 is on the right side with the barcode ID.

7 Select the checkbox to indicate that the flow cell is loaded, and then select Next.

Load the Manifold

Use the manifold from the same cluster kit as the flow cell.

1 Inspect the sippers on the sipper comb for damage. Make sure that the black rubber

gaskets are evenly seated.

2 Position the manifold over the flow cell with the sipper comb pointing toward the front

of the cBot.

Load Consumables

3 Align the manifold with the guide pins on the thermal stage and seat the manifold

over the flow cell. Seat evenly to form a tight seal.

4 Select the Manifold seated over flow cell checkbox.

5 Close the flow cell clamp to secure the manifold.

Figure 22 Close the Flow Cell Clamp

6 Select the Flow cell clamp closed checkbox.

7 Connect the outlet end of the manifold to the outlet port in the wash reservoir. Make

sure that the outlet is evenly seated.

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Figure 23 Secure Outlet End

A Outlet port B Output clamp

8 Snap the outlet clamp closed to secure the outlet end of the manifold.

9 Select the Outlet clamp closed checkbox.

10 Align the sipper comb with the 2 metal guide pins on the front of the thermal stage.

Figure 24 Secure the Sipper Comb

Clustering Without Sample Tracking

A Metal guide pins B Plastic tabs

11 Snap the sipper comb into place using the plastic tabs on either side of the comb.

12 Make sure that the sippers are straight and perpendicular to the reagent plate, and that

the Sipper comb in place checkbox is selected.

13 Select the Sipper comb in place checkbox, and then select Next.

Load Templates

1 Select Enter Template Name.

2 Using the onscreen keyboard, type the template ID and then select Enter.

3 Load the 8-tube strip containing prepared libraries into the template row.

4 Select the checkbox to indicate that templates are loaded.

5 If you are using additional primers, proceed to Load Primers. Otherwise, close the cBot

lid and select Next to proceed to Perform a Pre-Run Check on page 54.

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Load Primers

The Load Primers screen appears for workflows that allow custom primers or require additional primers. Sequencing Nextera libraries on a TruSeq v3 or TruSeq v2 flow cell requires that you load an 8-tube strip containing HP10.

1 Select Enter Primer Name.

2 Using the onscreen keyboard, type the primer name, and then select Enter.

3 Load the 8-tube strip containing primers into the primer row. Make sure that the order

of numbered tubes aligns with the lane orientation of the flow cell:

} HiSeq and HiSeq X flow cells—Tubes are numbered right to left. } GAIIx flow cells—Tubes are numbered left to right.

Load Consumables

HiSeq X, HiSeq 3000/4000, HiSeq v4, and TruSeq v3 (HiSeq)

4 Select the checkbox to indicate that primers are loaded and close the instrument lid.

5 Select Next.

HiSeq Rapid v2, TruSeq Rapid (HiSeq)

TruSeq v2 (GAIIx)

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Clustering Without Sample Tracking

Perform a Pre-Run Check

1 After successful completion of the pre-run check, select Start.

The Run Status screen opens and the run begins.

Run Component Errors

If the pre-run check fails due to errors related to run components, perform the following steps:

1 Check any run component with an indicated error to make sure that it is present and

loaded correctly.

2 Select Rerun Check to repeat the sensor check.

3 If the check continues to fail, select Cancel Run to end the run and set up a new one.

Flow Check Failure

Flow check failure might be the result of an improperly loaded flow cell, a faulty manifold, or a clog in the lines. Before bypassing the flow check, see Troubleshoot Flow Check Failure on

page 73.

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Monitor the Run

1 Use the Run Status screen to monitor the run in progress.

The Run Status screen provides the run status and the following details:

Figure 25 Run Status Screen

Monitor the Run

2 Wait for the run to complete:

} HiSeq v4, HiSeq 3000/4000 PE, or HiSeq X—Allow approximately 3hours. } HiSeq 3000/4000 SR—Allow approximately 4 hours. } HiSeq Rapid v2 or TruSeq Rapid—Allow approximately 1 hour. } TruSeq v2 or TruSeq v3—Allow approximately 5hours.

3 After the run is complete, you can leave the flow cell on the instrument overnight.

Otherwise, proceed to unloading run components. The cBot 2 holds the flow cell at 20°C.

Run Data Report

The run data report provides a summary of the run in progress. It lists the following information:

} Protocol name } Flow cell ID } Reagent ID } Template name } Start time and finish time

You can view the report at any time during the run. Select Menu and then select Run Data.

At the end of the run, the run data report automatically opens to alert you that the run is complete.

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Unload Run Components

1 When the run is complete, select Unload to proceed.

Figure 26 Run Complete, Unload Components

2 Raise the instrument lid.

Clustering Without Sample Tracking

3 Release the outlet clamp securing the outlet end of the manifold.

4 Disconnect the outlet end of the manifold from the outlet port in the wash reservoir.

5 Remove the sipper comb from the metal guide pins using the plastic tabs on either side

of the sipper comb.

6 Release the flow cell clamp.

7 Remove the manifold. Make sure that the flow cell remains on the thermal stage.

8 Lift the flow cell from the thermal stage.

9 Store the flow cell as appropriate:

} TruSeq v2, TruSeq v3, and HiSeq v4 flow cells—Store in storage buffer in the flow

cell tube at 2°C to 8°C. The flow cell is stable after primer hybridization up to 10days when properly stored in the flow cell tube.

} HiSeq and TruSeq Rapid flow cells—Perform the sequencing run on the same day

as library loading.

} HiSeq X and HiSeq 3000/4000 flow cells—Store in storage buffer up to 48 hours at

2°C to 8°C.

10 Pull the reagent plate lever toward you to release the reagent plate. Remove the reagent

plate from the reagent stage.

WARNING

environmental, health, and safety information, see the SDS for this kit at

support.illumina.com/sds.html.

11 Remove the 8-tube strip containing libraries.

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12 Remove the 8-tube strip containing additional primers, if applicable.

13 Select the checkbox to indicate that you have unloaded the reagents, templates, and

primers.

14 Choose a wash option:

} Select Wash to proceed to the post-run wash. } Select Exit to bypass the post-run wash, if the bypass option is available.

Unload Run Components

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Perform a Post-Run Wash

1 Wash the plate on the thermal stage with deionized water to remove any salts. Dry

with a lint-free cleaning tissue.

2 Fill the wash reservoir with approximately 12ml deionized water and close the

instrument lid.

3 Select the checkbox to indicate that water is present, and then select Wash.

4 When the wash is complete, blot any excess water remaining in the wash reservoir.

Avoid the outlet ports to prevent fibers from entering the holes.

5 Select the checkbox to indicate that the wash reservoir is dry, and then select Exit.

The Start screen opens and your cBot 2 is ready for another run.

Clustering Without Sample Tracking

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Confirm Reagent Delivery (Optional)

You can confirm delivery of individual reagents from the reagent plate provided in any kit except HiSeq X and HiSeq 3000/4000.

1 Inspect the foil-sealed tops of each tube strip to make sure that each seal was pierced.

2 Release each tube strip from the base of the reagent plate as follows.

a Grasp the reagent plate firmly, with finger tips under the base. b Gently press upward on the center tubes of the tube strip.

3 Inspect each tube to confirm that a similar volume remains in each tube.

NOTE

Small differences are normal.

Figure 27 Example of Successful Reagent Delivery (8-Lane Flow Cell)

Figure 28 Example of Successful Reagent Delivery (2-Lane Flow Cell)

Confirm Reagent Delivery (Optional)

4 If reagent delivery was not successful and the foil seals on the reagent tubes are

pierced, contact Illumina Technical Support.

5 Inspect the 8-tube strip containing library template.

6 If you used additional primers with your run, inspect the 8-tube strip containing

primers.

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Chapter 6 Maintenance

Maintenance

Perform Periodic Maintenance 62 Perform a Monthly Maintenance Wash 63 Change the Adapter Plate 65 Upgrade the Software 67 Upgrade Recipes 68 Shut Down the cBot 2 69

Chapter 6

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Maintenance

Perform Periodic Maintenance

Perform the basic maintenance steps described in this section to ensure optimal performance.

Maintenance Frequency Description

Instrument wash Between each

run and if the instrument is idle for more than a day.

Empty waste bottle

Clean surfaces One time per

Clean external and flow cell barcode scanner windows

Maintenance Wash

Check coolant level

Between each run.

week.

One time per week.

One time per month.

Every 3 months. Make sure that the green coolant is visible through the

Always perform an instrument wash after each run to clear salts and enzymes from the instrument hardware and to prevent clogs.

If the instrument has been idle for more than 24hours, a pre-run wash is recommended. For more information, see Perform a Pre-Run Wash on page 48.

To make sure that your run is not interrupted, empty the waste bottle between runs.

Use deionized water and a lint-free cleaning tissue to clean the surface of the thermal stage and the reagent stage. Clean the surface of the template and primer tube strip holders.

Use deionized water and a lint-free cleaning tissue to clean the external and flow cell barcode scanner windows.

Use 5% DECON (or 100mM NaOH) to remove traces of reagents from internal cBot components and inhibit the growth of microorganisms. For more information, see Perform a Monthly Maintenance Wash on page 63.

coolant window on the rear panel of the instrument. If necessary, use a mirror to view the coolant window.

If the coolant is low, use a wide coin or standard screwdriver to remove the coolant reservoir cap, and fill the reservoir to just below the reservoir cap.

Use only Illumina coolant (part#1003709). If you need additional coolant, contact your Illumina FAS or FSE.

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.

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Perform a Monthly Maintenance Wash

Perform a monthly maintenance wash using 5% DECON to remove traces of reagents from internal cBot components and inhibit microbial growth. If DECON is not available, substitute 100mM NaOH.

The maintenance wash requires approximately 10 minutes of hands-on time and consists of 4 wash steps: a water wash, a DECON or NaOH wash, and 2 more water washes.

Water Wash

1 Confirm that all run components are removed.

2 From the Start screen, select Menu and then Manual Commands to open the Manual

Commands screen.

3 Select Commands to open the Commands tab.

4 Fill the wash reservoir with approximately 12ml deionized water.

5 Select Wash.

6 After the wash is complete, blot excess water from the wash reservoir with a low-lint

tissue. Avoid the outlet ports to prevent fibers from entering the holes.

Perform a Monthly Maintenance Wash

DECON (or NaOH) Wash

1 Fill the wash reservoir with 10ml 5% DECON or 100mM NaOH.

2 Select Wash.

3 After the wash is complete, put on a new pair of gloves.

4 Blot the 5% DECON remaining in the wash reservoir with a low-lint tissue. Avoid the

outlet ports.

CAUTION

DECON is highly alkaline.

5 Immediately proceed to the water wash to prevent DECON from drying and clogging

the wash reservoir holes.

Water Wash (First Rinse)

1 Fill the wash reservoir with approximately 12ml deionized water.

2 Select Wash.

3 After the wash is complete, blot water remaining in the wash reservoir with a low-lint

tissue. Avoid the outlet ports.

Water Wash (Final Rinse)

1 Fill the wash reservoir with approximately 12ml deionized water.

2 Select Wash.

3 After the wash is finished, blot water remaining in the wash reservoir with a low-lint

tissue. Avoid the outlet ports.

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Maintenance

4 Close the instrument lid.

5 Empty the waste bottle.

Your cBot is ready for the next cluster generation run.

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Change the Adapter Plate

You can use a HiSeq flow cell or a GAIIx flow cell on the cBot. Each flow cell type requires that a specific adapter plate is installed. Icons on the Start screen indicate which adapter plate is installed.

NOTE

The cBot is shipped with the HiSeq adapter plate installed.

1 Open the instrument lid by gently lifting from the cutout on the front of the lid.

2 Lift the flow cell clamp.

3 Loosen the 2 captive Phillips head screws securing the adapter plate.

Figure 29 Flow Cell Adapter Plate

Change the Adapter Plate

A Captive screws B Adapter plate

4 Lift the existing adapter plate from the thermal stage and set aside.

5 If salts are present on the thermal stage, wipe with a lint-free cleaning tissue slightly

moistened with water.

6 Position the new adapter plate on the thermal stage. Align the sensor arm with the

corresponding slot on the right side of the thermal stage.

Figure 30 Sensor Arm Location

A GAIIx adapter plate B HiSeq adapter plate C Adapter plate sensor arm

7 Tighten the 2 screws to secure the adapter plate. For optimal heat transfer, make sure

that the adapter plate is sitting flat and the screws are tightened evenly.

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Maintenance

8 Wipe the installed adapter plate with a lint-free cleaning tissue moistened with water.

Dry with a clean tissue.

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Upgrade the Software

Using cBot software v1.3, or later, you can upgrade instrument software using a USB flash drive.

1 Insert the USB flash drive containing the new software version installer (for example,

cBotSetupX86_1.3.1.0.exe) into either of the USB ports on the front of the instrument. The installer must reside in the root directory of the USB flash drive, not in a folder.

CAUTION

Leave the USB drive in the USB slot during the upgrade process. Do not interact with the instrument during the upgrade.

2 Select Menu in the upper-left corner of the Start screen, and then select Configure.

Figure 31 Start Screen Menu

Upgrade the Software

3 Use the onscreen keyboard to type the default password, admin, and then select Enter.

4 Select Menu, and then select Upgrade.

5 A dialog box opens with a message about the software version:

Message Action

The software installer version is greater than the version currently installed on the cBot

cBot cannot find a valid software installer You can either insert a valid cBot upgrade

The software installer version is equal or lower than the version currently installed on the cBot

6 When the upgrade is complete and the instrument is restarting, remove the USBflash

drive.

7 If a BOOTMGRerror appears, attach a keyboard and mouse to the cBot and press

Ctrl+Alt+Del to restart the instrument.

Select OK to proceed with the installation of the newer version.

and select OK to try again, or Cancel to abort the upgrade.

Select Cancel to abort the upgrade, or OK to proceed with installation of a previous version.

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Maintenance

Upgrade Recipes

Upgrade recipe versions independently of software upgrades using a USB flash drive containing the recipe installer.

1 Insert the USB flash drive containing the new recipe installer into either of the USB

ports on the front of the instrument. The installer must reside in the root directory of the USB flash drive, not in a folder.

2 Select Menu in the upper-left corner of the Start screen, and then select Configure.

Figure 32 Start Screen Menu

3 Use the onscreen keyboard to type the default password, admin, and then select Enter.

4 Select Menu, and then select Upgrade Recipes.

When the upgrade is complete, the cBot restarts automatically. The restart process takes about 10 minutes.

CAUTION

Leave the USB drive in the USB slot during the upgrade process. Do not interact with the instrument during the upgrade.

5 When the restart is complete and the login screen opens, remove the USB flash drive.

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Shut Down the cBot 2

The cBot 2 is designed to run in an idle state from the Start screen, so shutting it down between runs is not necessary.

1 Select Menu in the upper-left corner of the Start screen, and then select Configure.

Figure 33 Start Screen Menu

Shut Down the cBot 2

2 Use the onscreen keyboard to type the default password, admin, and then select Enter.

3 From the Configuration screen, select Menu and then select Shut Down Station.

The cBot software shuts down.

Figure 34 Shut Down Station

4 After the software shuts down, switch the power switch to the OFF position.

Reboot in FSE Mode

The option to reboot in FSE Mode is for use by a trained Illumina Field Application Scientist (FAS) or Field Service Engineer (FSE) to update software or service the instrument.

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Appendix A Troubleshooting

Troubleshooting

Pause or Abort a Run 72 Troubleshoot Flow Check Failure 73 Troubleshoot Run Problems 75 Reset the External Barcode Scanner 76 Edit Protocols 77

Appendix A

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Troubleshooting

Pause or Abort a Run

Use commands on the Run Status screen to pause or abort a run.

} Pause—Completes the current command in the protocol, and then pauses the run.

Allow a few minutes before the run pauses. When the run is paused, the sippers lift from the reagent tubes, the reagent stage returns the home position, and the Pause button changes to the Resume button.

} When the run is active, select Pause to pause the run. } When the run is paused, select Resume to resume the run.

} Abort Run—Ends the run without the option of resuming. Select Unload to unload run

components.

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Troubleshoot Flow Check Failure

Perform the following procedure to troubleshoot flow check failure. Do not select the option to bypass flow check until you have completed this procedure to determine the following conditions:

} The flow cell is properly positioned on the instrument. } The manifold and hardware are working properly.

NOTE

Bypassing the flow check can result in unsuccessful clustering of some lanes.

Because different flow cell types use different flow checks, make sure that you use the correct recipe, manifold, and flow cell combination.

1 Make sure that you have enough HT1 in row 1 of the reagent plate, and replenish as

needed.

2 Note which lanes failed the flow check. This information is provided on the top-left

corner of the interface screen.

} If all 8 lanes failed, the flow cell is probably loaded improperly. Remove the

manifold and confirm that the flow cell holes are facing upward, and the flow cell orientation is correct.

} If only some lanes failed, the flow cell might not be seated. Remove the manifold,

reseat the flow cell, and then reinstall the manifold.

Troubleshoot Flow Check Failure

3 Select Rerun Check to repeat the flow check a second time.

4 If the flow check fails a second time, note which lanes failed the flow check, and do 1

of the following:

} If all 8 lanes failed, you probably have a faulty manifold. Replace it with a new

manifold.

} If different lanes failed, you probably do not have a faulty manifold. Inspect the

volumes of HT1 in row 1 to make sure that the tubes contain equal volumes.

5 Select Rerun Check to repeat the flow check for a third time.

} If this flow check fails after replacing the manifold, go to step 6. } If this check fails and you did not need to replace the manifold, go to step 7.

6 If the flow check fails a third time after replacing the manifold, you might have a clog

in the hardware.

a Inspect the volumes of HT1 in row 1 to make sure that the tubes contain equal

volumes. Higher volumes in the tubes that correspond to lanes with repeated flow

check failure indicate a hardware clog.

b Unload the run components and perform a maintenance wash. c After the wash, turn off the instrument using the power switch. After a few

seconds, turn on the power switch and then press the start button to restart the

software. Power cycling the instrument resets the allowable number of pre-run

check attempts.

d Follow the software prompts to reload run components and set up your run.

7 If the flow check fails a third time, you can safely bypass the flow check:

a Select Bypass Flow Check to proceed with the run. b After the run, check reagent delivery from all tubes.

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Troubleshooting

Troubleshooting Flowchart

The following flowchart illustrates the troubleshooting procedure. Steps to repeat the flow check include a number to indicate how many of the allowed flow checks have been performed at that point in the procedure.

Figure 35 Troubleshooting Flowchart

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Troubleshoot Run Problems

Use the following table to troubleshoot problems encountered during a cluster generation run.

Problem Possible Cause Action

Temperature out of range Often indicates that the cBot did

not reach the set temperature in the expected time. Can also indicate a potential control board failure.

Coolant is flowing and coolant level is low

Coolant is not flowing and coolant level is low

Coolant is not flowing and coolant level is not low

Instrument is in a locked state

Coolant has slowly evaporated to a low level.

Coolant level might be too low to generate flow.

Potential coolant pump failure. Email Illumina Technical

Potential software error. Email Illumina Technical

Troubleshoot Run Problems

Email Illumina Technical Support.

Add Illumina coolant (part#1003709) to the coolant reservoir.

Support.

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Troubleshooting

Reset the External Barcode Scanner

The external barcode scanner is ready for use when you receive your cBot. If the scanner is reset to an incorrect configuration, use the following instructions to restore it to the default configuration.

1 Print the barcode.

Figure 36 Restore Defaults Barcode

2 From the start screen, select Menu and then Manual Commands.

3 Select the General tab to access the barcode reader manual control inputs.

Figure 37 Manual Commands, General Tab

4 Select Turn Off and then select Turn On to activate the barcode scanner.

The laser line is visible on the scanner plate under the LCD screen.

5 Place the barcode under the external barcode scanner.

6 Select Turn Off, and then Turn On to scan the barcode.

A beep indicates a successful scan.

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Edit Protocols

Use the Protocol Editor to edit protocols to fit your needs. You might want to repeat steps in a protocol, or change the number of amplification cycles in the chemistry section.

Each protocol consists of 2 main sections:

} Chemistry section—Contains instructions for pumping reagents, temperature changes,

and wait durations. This section appears in the upper portion of the Protocol Editor screen.

} Protocol section—Contains a series of steps made up of chemistry definitions. This

section appears in the lower portion of the Protocol Editor screen.

If you edit an existing protocol, make sure to rename your protocol.

Protocol Editor

1 From the Start screen, select Menu, and then select Protocol Editor.

2 From the Protocol Editor, select Menu, and then select the appropriate command:

} Open—Opens an existing protocol. } Select Load from Library—Loads an existing chemistry definition or protocol step

stored in the cBot library.

} Select New Chemistry Definition or New Protocol Step—Creates a definition or step

and stores it in the cBot library.

Edit Protocols

3 Use the down arrow to the left of the step to expand the commands in the step. Use the

up arrow to collapse the commands.

4 To edit a step in a chemistry definition, highlight the step.

Selections to change the pump, temperature ramp, or wait commands appear in the right-hand panel.

5 To edit a step in the protocol, highlight the step.

Selections to change to the number of cycles for the selected chemistry definition appear in the right-hand panel.

6 Use the Protocol Editor icons to the right of the step name to rearrange, delete, or copy

steps and commands.

Figure 38 Protocol Editor, Expanded Steps

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A Chemistry section B Expanded chemistry section C Protocol section

Protocol Editor Icons

Troubleshooting

Icon

Description

Moves the highlighted step below the following step in the protocol.

Moves the highlighted step above the preceding step in the protocol.

Deletes the highlighted step.

Repeats the highlighted step.

Saves your changes to the protocol library.

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Index

5% DECON 63

8-tube strip removal 44, 59

activating external scanner 38 adapter plates 65

washing 33, 50

barcode scanner, external

cleaning 62

resetting 76 barcode scanning error 38 best practices

reagent preparation 18 bypass flow check 73

cBot reagent plates 9 chemistry protocols 77 Clarity LIMS X Edition 2 clustering reagents

preparing 18 components 4 configuration 6 configuration, help 30 configuring sample tracking 30 consumables

unloading 41, 56

version compatibility 13 consumables, user-supplied

reagent preparation 15 coolant

level 62 coolant part number 75 coolant problems 75 customer support 83

DECON 63 DECON substitute 63 delivered volumes 44, 59 denaturing 30, 46 denaturing libraries 21, 25 diluting 30, 46 documentation 3, 83

ending a run 72 error messages 75 ExAmp master mix 26

ExAmp reaction

preparing, 1 flow cell 22 preparing, 4 flow cells 22

ExAmp reagents

about 19, 23 preparing 26 preparing, 1 flow cell 22 preparing, 4 flow cells 22 thawing 20, 24

external barcode scanner

cleaning 62 resetting 76

flow cell

barcode scanning 38 cleaning 19, 23 compatible recipes 13 packaging 19, 23

preparation 19, 23 flow cell storage 33, 41, 50, 56 flow cells

adapter plates 65

positioning 33, 51

preparing for loading 33, 50

sample tracking compatibility 30 flow check 54

troubleshooting failure 73 flow check failure

troubleshooting 74 flow check failures 75 foil not pierced 44, 59 FSE mode 69

help

documentation 3 help, technical 83 HP10, preparing 27

icons

sensor status 6 idle state 69 Illumina SeqLab 2

kit documentation 7

libraries

denaturing 21, 25

diluting 21, 25

loading 34, 52

loading concentration 21, 25 loading libraries 34

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Index

maintenance 43, 58, 63

periodic care 62 maintenance wash duration 63 maintenance, preventive 62 manifold

outlet clamp 35, 52

sipper comb 4, 35, 52 manifold problems 75 manifolds 35, 51 Manual Commands 63

NaOH 63

overview

clustering workflow 47

sample tracking workflow 31

part numbers

coolant 75 PhiX

adding 21, 25

spike-in % 21, 25 positioning flow cells 33, 51 post-run procedures 41, 56 post-run wash 62 pre-run check

errors 54

performing 54 pre-run check failure 39 pre-run wash 62 preventive maintenance 62 primer

name, entering 38 primers

custom 53

loading 53

name, entering 53

tube strip orientation 53 protocol

selecting 37, 49 Protocol Editor 77

icons 78 protocols, editing 77

reagent delivery, unsuccessful 44, 59 reagent plate 5

configurations 9

preparing 21, 25

thawing 20, 24, 27-28 reagent plate, high output

preparing 27 reagent plate, rapid

preparing 28 reagent plates

positioning 33, 50 reagent preparation

best practices 18 reagent stage 5

reagents

barcode scanning 38

reagents, preparing

GAIIx flow cell 18 high output 27 HiSeq 3000/4000 23 HiSeq X flow cell 19 rapid 28

recipes

list of 13

upgrading 68 red foil seal 33, 50 removing 8-tube strips 44, 59 rerunning sensor checks 39 resuming a run 72 run

starting 54 run component errors 54 run data report 40, 55 run requirements

configuring 6 run summary 40, 55

salts, removing 43, 58 sample tracking

about 2

configuring 30

flow cell compatibility 30 scanners 4 scanning failure 38 seal, red foil 33, 50 seals not pierced 44, 59 sensor status icons 6 sensors 4 servicing 69 shutting down 69 software

upgrading 67

version compatibility 13 software errors 75 stopping a run 72 storing flow cells 41, 56 system status 6

technical assistance 83 temperature out of range 75 temperature range 75 template row 34 templates

loading 52 thawing reagent plate 20, 24 thermal stage 4

washing 43, 58 troubleshooting

flow check failure 73 turning off 69

user-supplied consumables

reagent preparation 15

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version compatibility

consumables 13 software 13

wash reservoir 32, 48 washes 32, 48

frequency 62

washing

adapter plates 33, 50 washing the instrument 43, 58 waste bottle 62

Index

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Index

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Technical Assistance

For technical assistance, contact Illumina Technical Support.

Table 1 Illumina General Contact Information

Website

www.illumina.com

Technical Assistance

Table 2 Illumina Customer Support Telephone Numbers

Region Contact Number Region Contact Number

North America 1.800.809.4566 Japan 0800.111.5011 Australia 1.800.775.688 Netherlands 0800.0223859 Austria 0800.296575 New Zealand 0800.451.650 Belgium 0800.81102 Norway 800.16836 China 400.635.9898 Singapore 1.800.579.2745 Denmark 80882346 Spain 900.812168 Finland 0800.918363 Sweden 020790181 France 0800.911850 Switzerland 0800.563118 Germany 0800.180.8994 Taiwan 00806651752 Hong Kong 800960230 United Kingdom 0800.917.0041 Ireland 1.800.812949 Other countries +44.1799.534000 Italy 800.874909

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.

[email protected]

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Illumina 5200 Illumina Way San Diego, California 92122 U.S.A. +1.800.809.ILMN (4566) +1.858.202.4566 (outside North America) [email protected]

www.illumina.com