illumina cBot 2 Parts list manual

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January 2016
Document # 15065681 v01
Material # 20004364
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cBot™2 System Guide

ii Material # 20004364
Document # 15065681 v01
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Revision History
Document Date Description of Change
Material # 20004364
Document # 15065681
v01
January
2016
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.
Part # 15065681 Rev. A July
2015
Initial release.
cBot 2 System Guide iii

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Table of Contents
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
Perform Periodic Maintenance 62
cBot 2 System Guide v

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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

Chapter 1
cBot 2 System Guide 1
Chapter 1 Overview
Overview
Introduction 2
Additional Resources 3
cBot 2 Components 4
Illumina Consumables 7
cBot Reagent Plates 9

Overview
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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.

Additional Resources
cBot 2 System Guide 3
Additional Resources
The following documentation is available for download from the Illumina website.
Resource Description
cBot System Site Prep Guide
(document # 15053710)
Provides specifications for laboratory space, electrical
requirements, and environmental considerations and
instructions for configuring the instrument.
cBot 2 System Safety and
Compliance Guide (document #
15065643)
Provides information about instrument labeling, compliance
certifications, and safety considerations.
HiSeq and GAIIx Systems
Denature and Dilute Libraries
Guide (document # 15050107)
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.
Visit the cBot 2 support page on the Illumina website for access to documentation, software
downloads, online training, and frequently asked questions.

Overview
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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
AThermal stage—Holds the flow cell and controls the flow cell temperature throughout the
run.
BReagent stage—Holds the cBot reagent plate, library templates, and specialty primers. For
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.
CWaste bottle compartment—Holds a sensor-controlled waste bottle that collects spent
reagents.
DExternal barcode scanner—Records the unique ID of the reagent plate and flow cell used
with each run that does not include sample tracking.
EPower switch—Turns on the instrument. The start button, located to the left of the waste
bottle compartment, starts the instrument software.
FTouch screen monitor—Provides on-instrument run setup and visual status of the cluster
generation process.
GFlow cell barcode scanner—Records the unique ID of the flow cell used with each sample
tracked run.
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.
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).

cBot 2 Components
cBot 2 System Guide 5
Figure 3 Thermal Stage
AOutput clamp
BFlow cell adapter plate and manifold
CFlow cell clamp
DThermal stage
ESipper 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.
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
AReagent plate lever
BTemplate row
CPrimer row
DcBot reagent plate

Overview
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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).

Illumina Consumables
cBot 2 System Guide 7
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
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.

Overview
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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.

cBot Reagent Plates
cBot 2 System Guide 9
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
HiSeq X and
HiSeq 3000/4000
Contains 12 rows with 8 deep wells each. Each reagent occupies a full
row of 8 wells. Not all rows contain reagent.
HiSeq High Output
(HiSeq v4)
Contains 8 rows of foil-sealed 8-tube strips filled with cluster
generation reagents. Rows9 through 12 are empty.
HiSeq High Output
(TruSeq v3)
Contains 11 rows of foil-sealed 8-tube strips filled with cluster
generation reagents. Row 12 is empty.
HiSeq Rapid Contains 3 rows of foil-sealed 8-tube strips filled with template
hybridization and first extension reagents. Rows 4 through 12 are
empty.
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
cBot 2 System Guide 11
Chapter 2 Getting Started
Getting Started
Start the cBot 2 12
Version Compatibility of Run Components 13
User-Supplied Consumables 15

Getting Started
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Start the cBot 2
AStart button
BPower switch
1Switch the power switch on the right side of the instrument to the ONposition.
2Press 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.

Version Compatibility of Run Components
cBot 2 System Guide 13
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
HiSeq Rapid Duo cBot Sample
Loading Kit
Version R recipes cBot v1.5, or later
TruSeq Rapid Duo cBot Sample
Loading Kit
Version R recipes cBot v1.5, or later
TruSeq Dual Index Sequencing
Primer Box
Version 8.0 recipes (HiSeq)
Version 7.0 recipes (GA)
cBot v1.4.36, or later
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_3000_4000_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
PE_HiSeq_Cluster_Kit_v4_cBot_recipe_v9.0
TruSeq v3 Flow Cell SR_Amp_Lin_Block_TubeStripHyb_v8.0
PE_Amp_Lin_Block_TubeStripHyb_v8.0
SR_Amp_Lin_Block_Hyb_v8.0
PE_Amp_Lin_Block_Hyb_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
PE_Amp_Lin_Block_TubeStripHyb_v7.0²
SR_Amp_Lin_Block_Hyb_v7.0²
PE_Amp_Lin_Block_Hyb_v7.0²
¹ Used only with the Rapid Duo kits.

Getting Started
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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.