Illumina Genome Analyzer Maintenance Guide

Notice
This publication and its contents are proprietary to Illumina, Inc., and are intended solely for the contractual use of its customers and for no other purpose than to operate the system described herein. This publication 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, Inc. For the proper operation of this system and/or all parts thereof, the instructions in this guide must be strictly and explicitly followed by experienced personnel. All of the contents of this guide must be fully read and understood prior to operating the system or any of the parts thereof. FAILURE TO COMPLETELY READ AND FULLY UNDERSTAND AND FOLLOW ALL OF THE CONTENTS OF THIS GUIDE PRIOR TO OPERATING THIS SYSTEM, OR PARTS THEREOF, MAY RESULT IN DAMAGE TO THE EQUIPMENT, OR PARTS THEREOF, AND INJURY TO ANY PERSONS OPERATING THE SAME. Illumina, Inc. does not assume any liability arising out of the application or use of any products, component parts, or software described herein. Illumina, Inc. further does not convey any license under its patent, trademark, copyright, or common-law rights nor the similar rights of others. Illumina, Inc. further reserves the right to make any changes in any processes, products, or parts thereof, described herein without notice. While every effort has been made to make this guide as complete and accurate as possible as of the publication date, no warranty of fitness is implied, nor does Illumina accept any liability for damages resulting from the information contained in this guide. Illumina, Solexa, Making Sense Out of Life, Oligator, Sentrix, GoldenGate, DASL, BeadArray, Array of Arrays, Infinium, BeadXpress, VeraCode, IntelliHyb, iSelect, CSPro, iScan, and GenomeStudio are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners. 20072008 Illumina, Inc. All rights reserved.
Sequencing User Guide
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Revision History
Revision history for part # 1006747:

Revision A Date November 2008 Description of Change Initial release.
Table of Contents
Notice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii Revision History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
Chapter 1
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Audience and Purpose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Technical Assistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Genome Analyzer System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Process and Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Related Documentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Paired-End Sequencing Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Key Differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Protocol Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Sequencing Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Site Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Instrument Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Reagent Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Sequencing Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Lab Tracking Forms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Software and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Quick Reference and Experienced User Cards . . . . . . . . . . . . . . . . . . . 10 User-Supplied Consumables and Equipment . . . . . . . . . . . . . . . . . . . . . . . 11 Cluster Generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Chapter 2
Cluster Generation on the Cluster Station. . . . . . . . 13

Cluster Generation Workflow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cluster Generation Protocols and Recipes . . . . . . . . . . . . . . . . . . . . . . . . . Standard Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . One-Step Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recipe Names . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocol Times. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 15 15 15 15 16
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Table of Contents
Safe Stopping Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reagent Volumes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preparing DNA Template for Cluster Generation . . . . . . . . . . . . . . . . . . . . Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Template Mix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Performing Cluster Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wash the Cluster Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Load Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Perform Cluster Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wash the Cluster Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Performing Linearization, Blocking, and Primer Hybridization. . . . . . . . . . . Wash the Cluster Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Load Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Perform Linearization, Blocking, and Primer Hybridization . . . . . . . . . . Wash the Cluster Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16 17 18 18 18 20 20 20 22 23 24 24 24 26 27
Chapter 3
Sequencing on the Genome Analyzer . . . . . . . . . . . 29

Sequencing Workflows. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sequencing Protocols and Recipes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36-Cycle Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51-Cycle Runs and 76-Cycle Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Paired-End Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recipe Names . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Performing a Pre-Run Wash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Loading Reagents on the Genome Analyzer . . . . . . . . . . . . . . . . . . . . . . . . Load Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Track Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Priming Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Continuing the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Replace Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reset Reagent Volumes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Weigh Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transfer Data for Paired-End Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preparing for Read 2 Using the Paired-End Module . . . . . . . . . . . . . . . . . . Load Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prime the Paired-End Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prepare for Read 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measure Reagent Volumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sequencing Read 2 on the Genome Analyzer . . . . . . . . . . . . . . . . . . . . . . . Single-Folder Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Two-Folder Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Replace Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Performing a Post-Run Wash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 31 31 31 31 32 33 33 33 35 35 36 38 39 40 41 41 41 42 42 43 44 45 46 46 47 48 49 49 49
Index
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Part # 1006747 Rev. A
List of Figures
Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11
Paired-End Protocol Workflow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Cluster Generation Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Reagent Positions on the Cluster Station. . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Reagent Positions on the Cluster Station. . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Workflow Diagram for Single-Read and Paired-End Sequencing . . . . . . . . 30 Reagent Positions on the Genome Analyzer . . . . . . . . . . . . . . . . . . . . . . . . 36 Current Reagent Volumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Reset Reagent Volumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Updated Current Reagent Volumes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Reagent Barcodes Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Reagent Positions on the Paired-End Module. . . . . . . . . . . . . . . . . . . . . . . 42
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List of Figures
Part # 1006747 Rev. A
List of Tables
Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 11 Table 12 Table 13
Illumina Customer Support Contacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Cluster Station Recipes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Protocol Times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Safe Stopping Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Reagent Volumes Per Recipe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Reagent Positions for Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Reagent Volumes for Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Reagent Positions for Linearization, Blocking, and Primer Hybridization . . 25 Reagent Volumes for Linearization, Blocking, and Primer Hybridization . . . 27 Sequencing Recipes Used on the Genome Analyzer. . . . . . . . . . . . . . . . . . 32 Reagent Positions on the Genome Analyzer . . . . . . . . . . . . . . . . . . . . . . . . 35 Reagent Positions on the Paired-End Module . . . . . . . . . . . . . . . . . . . . . . . 43 Reagent Volumes after Priming and Read 2 Preparation. . . . . . . . . . . . . . . 45
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List of Tables
Part # 1006747 Rev. A
Chapter 1
Overview
Topics
2 2 3 4 6 9 11 Introduction Audience and Purpose Technical Assistance Genome Analyzer System Paired-End Sequencing Overview Sequencing Documentation User-Supplied Consumables and Equipment
CHAPTER 1 Overview
Introduction
This guide describes the workflows for performing single-read and pairedend sequencing runs on the Illumina Cluster Station and Genome Analyzer. For instruction related to reagent preparation, instrument components and maintenance, and standard setup procedures for cluster generation and sequencing runs, please refer to the following documents: ` Reagent PreparationSee the following reagent preparation guides for an overview of kit contents and reagent preparation instructions required for performing sequencing protocols. Using the Single-Read Cluster Generation Kit v2 on the Cluster Station Using the Paired-End Cluster Generation Kit v2 on the Cluster Station and Paired-End Module Using the SBS Sequencing Kit v3 on the Genome Analyzer
` Cluster GenerationSee the Cluster Station Operations Guide for

detailed instruction on the following standard procedures performed on the Cluster Station: Starting the Cluster Station Running a recipe Changing the manifolds Performing an instrument wash Troubleshooting reagent delivery
` SequencingSee the Genome Analyzer II Operations Guide for

detailed instruction on the following standard procedures performed on the Genome Analyzer at the beginning of a sequencing run: Starting the Genome Analyzer and IPAR system Using Genome Analyzer software interface Performing service and maintenance washes Installing the flow cell and prism Applying oil Performing first-base incorporation Adjusting focus Checking quality metrics
See Sequencing Documentation on page 9 for descriptions of other related documentation.
Audience and Purpose

This guide is for laboratory personnel and other individuals responsible for: ` Performing cluster generation on the Illumina Cluster Station.
` Performing sequencing runs on the Illumina Genome Analyzer. ` Training personnel to perform single-read and paired-end sequencing
runs and use various recipes on the Cluster Station and Genome Analyzer.
Part # 1006747 Rev. A
Technical Assistance
Technical Assistance
For technical assistance, contact Illumina Customer Support. Table 1
Contact Toll-free Customer Hotline International Customer Hotline Illumina Website Email
Illumina Customer Support Contacts

Number 1-800-809-ILMN (1-800-809-4566) 1-858-202-ILMN (1-858-202-4566) http://www.illumina.com [email protected]
MSDSs
Material safety data sheets (MSDSs) are available on the Illumina website at http://www.illumina.com/msds.
Product Documentation
If you require additional product documentation, you can obtain PDFs from the Illumina website. Go to http://www.illumina.com/documentation. When you click on a link, you will be asked to log in to iCom. After you log in, you can view or save the PDF. If you do not already have an iCom account, then click New User on the iCom login screen and fill in your contact information. Indicate whether you wish to receive the iCommunity newsletter (a quarterly newsletter with articles about, by, and for the Illumina Community), illumiNOTES (a monthly newsletter that provides important product updates), and announcements about upcoming user meetings. After you submit your registration information, an Illumina representative will create your account and email login instructions to you.
CHAPTER 1 Overview
Genome Analyzer System

The Genome Analyzer System is a groundbreaking platform for sequence analysis and functional genomics. Dramatically improving speed and reducing costs, it is suitable for a range of applications including wholegenome and candidate-region sequencing, gene expression discovery and profiling, DNA-protein interaction profiling, and small RNA identification and quantitation. Leveraging proprietary reversible terminators and clonal single molecule array technology, the Illumina Genome Analyzer System can generate several billion bases of data per run, and in the process transform the way many experiments are devised and carried out. The Illumina Genome Analyzer System is ideal for genome-scale as well as targeted sequencing projects. This platform allows researchers to economically sequence a human genome in a matter of weeks. Sequencing by synthesis (SBS), using proprietary reversible terminators, enables the Illumina Genome Analyzer System to achieve a high degree of sequencing accuracy even through homopolymeric regions. This allows researchers to sequence complex genomes rapidly, economically, and accurately.
For more information about the Illumina Genome Analyzer System, visit http://www.illumina.com/sequencing.
NOTE
Process and Components
The Genome Analyzer System process is straightforward yet flexible, consisting of four main steps: 1. Sample preparation. 2. Cluster generation on the Cluster Station. 3. Sequencing by synthesis (SBS) on the Genome Analyzer. 4. Data analysis using the Genome Analyzer Pipeline software.
Sample Preparation
Sequencing by synthesis (SBS) can be used for multiple applications, including DNA sequencing, chromatin immunoprecipitation (ChIP), whole transcriptome analysis, small RNA analysis, and digital gene expression-tag profiling. While the process of generating clusters and analyzing them is standardized across all applications, the process of preparing samples is unique to each application. For instructions on preparing samples specific to your application, see the appropriate sample prep booklet.
Flow Cell
The flow cell is a multi-lane glass-based substrate in which clusters are generated and the sequencing reaction is performed. Each of the lanes is individually addressable, so researchers can interrogate multiple distinct samples per flow cell.
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Genome Analyzer System
Within each lane of the flow cell, millions of primers act as capture probes for the fragmented DNA or cDNA. Each lane of the flow cell is capable of yielding millions of distinct clusters and generating several hundred megabases of sequence data. The versatile format of the flow cell allows researchers to tailor the use of the device to the specific needs of their applications and use the platform for a variety of analyses.
Cluster Generation on the Cluster Station

The Cluster Station is a hardware device that hybridizes samples onto a flow cell and amplifies them for later sequencing on the Genome Analyzer. During cluster generation, a single DNA fragment (the template) is attached to the surface of an oligonucleotide-coated flow cell and amplified to form a surface-bound colony (the cluster). The result is a heterogeneous population of clusters, with each cluster consisting of many identical copies of the original template molecule.
Sequencing on the Genome Analyzer

Using a massively parallel sequencing approach, the Illumina Genome Analyzer can simultaneously sequence millions of clusters to generate several billion bases of data from a single run. The system leverages Illumina sequencing technology and novel reversible terminator chemistry, optimized to achieve unprecedented levels of accuracy, cost effectiveness, and throughput.
Paired-End Sequencing Using the Paired-End Module

The Paired-End Module is an auxiliary instrument used to supply reagents to the Genome Analyzer during the second read of paired-end sequencing. The Paired-End Module is connected to the Genome Analyzer via an external VICI valve.
Data Analysis Using the Genome Analyzer Pipeline Software

The Genome Analyzer Pipeline software (Pipeline) is a set of utilities designed to perform a complete offline data analysis of a sequencing run. Pipeline performs image analysis, base calling, and sequence analysis.
Related Documentation
See Sequencing Documentation on page 9 for a list of documentation related to the Genome Analyzer System.
CHAPTER 1 Overview
Paired-End Sequencing Overview

After the Genome Analyzer has completed the first sequencing read, the Paired-End Module directs the resynthesis of the original templates and the second round of cluster generation. The Paired-End Module is connected to the Genome Analyzer through a single fluidic connection, providing a walkaway automated workflow. Paired-end sequencing requires the following reagent kits and components: ` Paired-End Flow Cell (provided in the Paired-End Cluster Generation Kit)
` Paired-End Cluster Generation Kit ` Paired-End Module and Software Package ` Two 36-Cycle SBS Sequencing Kits
Key Differences
The majority of the steps in the paired-end protocols are similar to those used in single-read sequencing. This section describes some key differences that enable you to sequence both DNA strands within each cluster. ` Paired-end sample preparationSample preparation for paired-end libraries adds a second, unique site complementary to the new sequencing primer. See Preparing Samples for Paired-End Sequencing.
` Paired-end flow cellPaired-end sequencing uses a modified, pairedend-enabled flow cell. A standard flow cell cannot perform both reads of the paired-end experiment.
` Two new linearization methodsClusters are prepared for sequencing

twice, once before each of the two SBS reads. The two linearization methods are different to allow selective linearization of the desired strand.
` Two sequencing primersTwo hybridization events use a different

sequencing primer for each read.
` Combined blocking stepsClusters prepared using the Illumina pairedend method require an additional blocking step to improve sequencing performance. To simplify the protocol, the two blocking steps have been combined into one.
` Paired-End Cluster Generation KitThis kit contains the reagents

required to generate clusters on a paired-end flow cell and to prepare the clusters for Read 1 and Read 2.
` Paired-End ModuleThis module is an external valve attachment to the

Genome Analyzer and requires Sequencing Control Studio (SCS) v2.0 or later. The Paired-End Module supplies additional reagents to the flow cell during Read 2 preparation.
` Paired-end recipesPaired-end sequencing recipes are essentially

identical to standard SBS recipes with the important exception that they end with a deblock cycle and the flow cell is then equilibrated in high salt buffer. This ensures any fluorescent background is removed prior to sequencing Read 2. Failure to use a paired-end recipe will result in a very high fluorescent background for Read 2 and may also compromise the intensity of the second read, both of which have a significant impact on data quality. See Sequencing Protocols and Recipes on page 31 for a list of applicable recipes.
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Paired-End Sequencing Overview
Protocol Workflow
The paired-end process sequences the same population of clusters on the same flow cell twice, reading from both ends of the template. Figure 1 illustrates the paired-end workflow.
Paired-End Cluster Amplification Performed on the Cluster Station
Linearization, Blocking, and Primer Hybridization Performed on the Cluster Station
Read 1 Performed on the Genome Analyzer
Read 2 Preparation Performed on the Genome Analyzer with the Paired-End Module attached
Read 2 Performed on the Genome Analyzer

Figure 1 Paired-End Protocol Workflow
Cluster Amplification
The prepared sample is introduced into the flow cell mounted on the Cluster Station, and then amplified. As a general rule, GC-rich genomes require a higher number of amplification cycles to achieve adequate cluster intensity. Since there is a direct correlation between insert size and cluster size, libraries with longer insert sizes require a reduced density of clusters to avoid excessive overlapping of clusters.
Linearization, Blocking, and Primer Hybridization

The amplified sample, still mounted on the Cluster Station, is prepared for Read 1. ` Linearization 1Selectively linearizes one of the two strands.
` BlockingPrevents non-specific sites from being sequenced. ` Denaturation and hybridizationPerforms standard denaturation and
hybridization of the first sequencing primer.
CHAPTER 1 Overview
Read 1 Sequencing
The flow cell is mounted on the Genome Analyzer and subjected to 36 cycles of sequencing, using paired-end sequencing protocols and standard SBS reagents. Longer runs may be applied using alternate recipes. See Sequencing Protocols and Recipes on page 31 for a list of applicable recipes.
Preparation for Read 2

The flow cell is prepared for Read 2 while still mounted on the Genome Analyzer with the Paired-End Module attached, allowing for the in situ treatment of the flow cell. ` Primer DehybridizationRemoves the extended sequencing primer used in Read 1.
` DeprotectionPrepares the flow cell for the next step. ` ResynthesisRegenerates the previously linearized strand. ` Linearization 2Linearizes the strand that was sequenced in Read 1 to
allow hybridization of the second sequencing primer to the newly synthesized DNA strand.
` BlockingPrevents non-specific sites from being sequenced. ` Denaturation and hybridizationDenatures the linearized strand and
hybridizes the second sequencing primer (Read 2 PE Sequencing Primer).
Read 2 Sequencing
The flow cell is subjected to an additional 36 cycles or more of sequencing, using paired-end sequencing protocols and standard SBS reagents.
Instrument Washes
The pre-run and post-run wash recipes for paired-end sequencing wash the Paired-End Module and the Genome Analyzer.
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Sequencing Documentation
Sequencing Documentation
This section describes the various documentation titles for sequencing applications. New releases and updates may be available from the Illumina website. See Product Documentation on page 3 for important information about downloading documentation.
Site Preparation
The Sequencing Site Preparation Guide explains how to prepare your lab for installation and use of the Cluster Station, Genome Analyzer, and Paired-End Module, including electrical and environmental requirements, safety hazards, and space requirements. This guide also contains information about network and electrical requirements for the Integrated Primary Analysis and Reporting (IPAR) system. Sample preparation guides contain information about how to prepare sample libraries before beginning cluster generation on the Cluster Station and sequencing on the Genome Analyzer. Sample preparation documentation is provided with sample preparation kits. ` Preparing Samples for Sequencing Genomic DNA
Sample Preparation
` ` ` ` ` ` `
Preparing Samples for ChIP Sequencing of DNA Preparing Samples for Paired-End Sequencing Preparing Samples for Gene-Expression-Tag Profiling with NlaIII Preparing Samples for Gene-Expression-Tag Profiling with DpnII Preparing Samples for Analysis of Small RNA Preparing Samples for Sequencing mRNA Preparing Samples for Multiplexed Paired-End Sequencing
Instrument Operation
Instrument operation guides contain an overview of instrument components, regular maintenance procedures, and instructions for basic operational procedures used in the sequencing protocols. ` Cluster Station Operations Guide
` Genome Analyzer II Operations GuideThis guide also contains an

overview of the Paired-End Module and the Integrated Primary Analysis and Reporting (IPAR) system.
Reagent Preparation
Reagent preparation guides contain information about how to prepare and use reagents on the Cluster Station, Genome Analyzer, and Paired-End Module. Reagent preparation documentation is provided with cluster generation kits and SBS sequencing kits. ` Using the Single-Read Cluster Generation Kit v2 on the Cluster Station
` Using the Paired-End Cluster Generation Kit v2 on the Cluster Station

and Paired-End Module.
` Using the SBS Sequencing Kit v2 on the Genome AnalyzerThis booklet

includes instruction for the 18-Cycle SBS Kit and the 36-Cycle SBS Kit.
` Using the SBS Sequencing Kit v3 on the Genome AnalyzerThis booklet

includes instruction for the 18-Cycle SBS Kit and the 36-Cycle SBS Kit, including an introduction to performing 51-cycle and 76-cycle runs.
10
CHAPTER 1 Overview
Sequencing Workflow
Sequencing workflow guides contain task-oriented instruction for cluster generation on the Cluster Station and sequencing on the Genome Analyzer. ` Sequencing User GuideThis comprehensive guide includes information about single-read sequencing, paired-end sequencing, and sequencing multiplexed samples.
` Multiplexed Sequencing on the Genome AnalyzerThis booklet

provides an introduction to sequencing multiplexed samples and is provided with the Multiplexing Sequencing Primers Kit.
Lab Tracking Forms
Lab tracking forms provide a place for lab technicians to record lot numbers, operator names, and reagent volumes for each sequencing run. The forms may be used online or may be printed, and are used in conjunction with reagent preparation and sequencing workflow documentation. ` Single-Read Sequencing Lab Tracking Form
` Paired-End Sequencing Lab Tracking Form
Software and Analysis
Software documentation, Pipeline documentation, and downstream analysis booklets contain information about how to use the software for sequencing and data analysis, and how to configure the output files to meet your specific needs. ` Integrated Primary Analysis and Reporting (IPAR) User Guide
` ` ` ` `
Genome Analyzer Pipeline Software User Guide Pipeline to Maq to GBrowse User Guide Pipeline to ChIP-Seq User Guide FocalAssist User Guide SCS 2.0 and IPAR 1.0 External Events and Data Copy Expert User Guide
Quick Reference and Experienced User Cards
Quick reference and experienced user cards provide condensed instructional information for the experienced user. ` Focus Procedure Experienced User Card (EUC)This reference contains instruction for the experienced technician on how to focus the Genome Analyzer II.
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User-Supplied Consumables and Equipment
11
User-Supplied Consumables and Equipment

Check to ensure that you have all of the following user-supplied consumables before you proceed to cluster generation and sequencing.
Cluster Generation
The following consumables are required for preparing cluster generation reagents. See Sequencing Documentation on page 9 for a list of available reagent preparation guides.
Consumables ` 3-amino-1-propanol (> 99%) ` Tris-Cl 10 mM, pH 8.5
Sequencing
Consumables ` Immersion oil, refractive index 1.473 (Cargille, catalog # 19570) ` Ethanol absolute ` MilliQ water or laboratory grade water
(for washing the Paired-End Module)
Equipment
Check to ensure that you have all of the following user-supplied equipment required for performing service washes on the Genome Analyzer. ` (3) 50 ml conical tubes
` (4) 125 ml Nalgene bottles

(ThermoFisher Scientific, catalog # 2019-0125)
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CHAPTER 1 Overview
Part # 1006747 Rev. A
Chapter 2
Cluster Generation on the Cluster Station
Topics
14 15 18 20 24 Cluster Generation Workflow Cluster Generation Protocols and Recipes Preparing DNA Template for Cluster Generation Performing Cluster Amplification Performing Linearization, Blocking, and Primer Hybridization
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CHAPTER 2 Cluster Generation on the Cluster Station
Cluster Generation Workflow

This chapter describes the single-read and paired-end workflows for cluster generation on the Cluster Station. The recipes used and the reagent positions on the Cluster Station are different but the basic steps are the same. The following diagram illustrates the cluster generation workflow for singleread sequencing and paired-end sequencing.
Prepare and Load Reagents on the Cluster Station
Prepare Template DNA
Perform Amplification on the Cluster Station
Perform Linearization, Blocking, and Primer Hybridization on the Cluster Station
Sequence the Flow Cell on the Genome Analyzer within 4 hours

Figure 2 Cluster Generation Workflow
For a description of reagent preparation and cluster generation kit contents, see the documentation provided with your kit. For a list of reagent preparation guides, see Sequencing Documentation on page 9.
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Cluster Generation Protocols and Recipes
15

Standard Protocol
The standard protocol for genomic DNA applications and the multi-primer protocol for gene expression or small RNA applications each require two recipes. The first recipe washes the Cluster Station, prompts you to load the reagents, and performs cluster amplification. The second recipe performs linearization, blocking, and primer hybridization. Another option is the one-step protocol. The one-step protocol uses a single recipe that washes the Cluster Station, prompts you to load the reagents, performs cluster amplification, and then continues to linearization, blocking, and primer hybridization. There is a one-step recipe for the standard protocol, and a one-step recipe for the multi-primer protocol. The following table lists the recipe names for each protocol option. Recipe names end in _v<#>.xml. The <#> in the file name refers to the current version of the recipe.
One-Step Protocol
Recipe Names
Table 2
Protocol
Cluster Station Recipes

Application Genomic DNA Recipe Names SR_Amplification_only SR_Linearization_Blocking_PrimerHyb SR_Amplification_Linearization_Blocking_PrimerHyb SR_Amplification_only SR_Linearization_Blocking_MultiHyb SR_Amplification_Linearization_Blocking_MultiHyb PE_Amplification_only_v<#> PE_2P_R1prep_Linearization_CombinedBlocking_PrimerHyb PE_Amplification_Linearization_CombinedBlocking_PrimerHyb
Single-Read Standard
Single-Read One-Step Single-Read Multi-Primer
Genomic DNA Gene expression and small RNA Gene expression and small RNA Genomic DNA
Single-Read Multi-Primer One-Step Paired-End Standard
Paired-End One-Step
Genomic DNA
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Protocol Times
Table 3 Protocol Times
The following table lists the approximate duration of each step in a protocol.
Step in Protocol Instrument Wash Template Hybridization Amplification Linearization Blocking Sequencing Primer Hybridization Instrument Wash
Step Duration (Single-Read Recipes) 15 minutes 40 minutes 2 hours 25 minutes 15 minutes 55 minutes 20 minutes 15 minutes
Step Duration (Paired-End Recipes) 15 minutes 40 minutes 2 hours 25 minutes 35 minutes 55 minutes 20 minutes 15 minutes
Safe Stopping Points
After the amplification step, a flow cell may be left on the Cluster Station overnight or stored indefinitely at 4C. After linearization and blocking, there are different safe stopping points and storage requirements for paired-end flow cells than for single-read flow cells. ` For single reads, the flow cell is filled with buffer, making it safe to leave on the Cluster Station overnight or to store at 4C for up to one month. Be sure to use a new manifold from a sealed bag to prevent crosscontamination when you resume the protocol.
` For paired reads, you cannot store the linearized and blocked flow cell
for a prolonged period of time due to the nature of the enzymes used in the blocking step. After primer hybridization, all flow cells must be sequenced on the Genome Analyzer within 4 hours. Table 4 Safe Stopping Points
Single-Read Flow Cell Leave overnight or store at 4C for 3 months Store at 4C for up to one month Sequence within 4 hours Paired-End Flow Cell Leave overnight or store at 4C for 3 months Do not store flow cell Sequence within 4 hours
Step in Protocol After Amplification After Linearization and Blocking After Primer Hybridization
Part # 1006747 Rev. A
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Reagent Volumes
Table 5
Use this table to determine which reagents you need to prepare and load for the recipes you are using. Regardless of which recipe you use, the reagents occupy the same positions on the Cluster Station. Reagent positions not listed are not used in the protocol.
Reagent Volumes Per Recipe

Recipe Descriptions
Position Reagent /Label 1 3 4 7 9 11 12 14 AMX (Amplification Mix) HT2 (Wash Buffer) BMX (Blocking Mix) Sequencing Primer AT1 (Formamide) APM1 (AMX1 Premix) HT1 (Hybridization Buffer) LMX1 (Linearization Mix 1) (for paired-end recipes) LS1 (Linearization Solution 1) (for single-read recipes) Diluted HP3 (0.1 N NaOH) HT1 (Hybridization Buffer) Denatured Template DNA HT2 (Wash Buffer) APM1 (AMX1 Premix) EMX (Extension Mix) Diluted HP3 (0.1 N NaOH) HT2 (Wash Buffer) Sequencing Primer (specific to your application) HT2 (Wash Buffer) HT1 (Hybridization Buffer)
Amp only 15 ml 10 ml
Lin-BlockPrimer Hyb
Lin-BlockMulti Hyb
One Step (Standard) 15 ml
One Step (Multi-Primer) 15 ml 10 ml 2 ml
7 ml (approx.) 7 ml (approx.) 10 ml 2 ml 1.3 ml 2 ml 2 ml 1.3 ml 8 ml 10 ml 13 ml (approx.) 1.3 ml 13 ml (approx.) 15 ml 1.3 ml
8 ml 10 ml 15 ml
8 ml 10 ml 15 ml
15
1.8 ml
1.8 ml
1.8 ml
1.8 ml
17 A B C D E F G H I J
1.5 ml 140 l/tube 120 l/tube 100 l/tube 100 l/tube 120 l/tube 100 l/tube 100 l/tube 100 l/tube 100 l/tube 100 l/tube
1.5 ml 140 l/tube 120 l/tube 100 l/tube 100 l/tube 120 l/tube 140 l/tube 120 l/tube 100 l/tube 100 l/tube 120 l/tube 100 l/tube 100 l/tube 100 l/tube 100 l/tube 100 l/tube
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Preparing DNA Template for Cluster Generation

There are two steps involved in preparing the template mix: 1. Denature with HP3 (2 N NaOH). 2. Dilute Denatured DNA into HT1 (Hybridization Buffer).
Consumables
Illumina-Supplied
The following components are provided in the Cluster Generation Kit v2: ` HP3 (2 N NaOH)
` HT1 (Hybridization Buffer) User-Supplied ` Tris-Cl 10 mM, pH 8.5
Template Mix
DNA Template Storage

Illumina recommends storing prepared DNA template at a concentration of 10 nM. Adjust the concentration for your prepared DNA samples to 10 nM using Tris-Cl 10 mM, pH 8.5. For long-term storage of DNA samples at a concentration of 10 nM, add Tween 20 to the sample to a final concentration of 0.1% Tween. This helps to prevent adsorption of the template to plastic tubes upon repeated freeze-thaw cycles, which would decrease the cluster numbers from a sample over time.
DNA Concentration
The flow cell has eight parallel channels for processing up to eight different DNA samples. The first time you process a sample, it is useful to try a concentration range to optimize the number of clusters formed. If the DNA concentration is too low, too few clusters will be generated and the sequencing yield will be low. If the DNA concentration is too high, the clusters are too dense and can overlap, complicating the sequencing data analysis. Generally, the concentration of DNA used for the hybridization step on the Cluster Station should be 14 pM, leading to a cluster density of approximately 40180K/tile.
Denature DNA Template

Denature the template DNA with HP3 (2 N NaOH) to a final DNA concentration of 1 nM. This is suitable for performing the hybridization step on the Cluster Station at a DNA concentration up to 8 pM. 1. Combine the following volumes of template DNA, Tris-Cl, and HP3: 10 nM Template DNA (2 l) Tris-Cl 10 mM, pH 8.5 (17 l) HP3 (2 N NaOH) (1 l)
The total volume should be 20 l (template final concentration 1 nM). 2. Vortex briefly to mix the template solution. 3. Pulse centrifuge the solution.
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Preparing DNA Template for Cluster Generation
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4. Incubate for 5 minutes at room temperature to denature the template into single strands. 5. Place the template on ice until you are ready to proceed to final dilution.
Dilute Denatured DNA

Dilute the denatured DNA with pre-chilled HT1 to a total volume of 1000 l and dispense into a strip tube as described below. Illumina recommends that you perform a titration of your DNA template to determine a good density of clusters. A typical titration series would be to use a new template at 1 pM, 2 pM, and 4 pM. 1. To reach the desired final concentration for the hybridization step, dilute denatured DNA as follows:
Required Final 1 pM Concentration
2 pM 2 l
4 pM 4 l
8 pM 8 l
1.0 nM Denatured DNA Pre-chilled HT1
1 l
999 l
998 l
996 l
992 l
2. Invert several times to mix the template solution. 3. Pulse centrifuge the solution. 4. Dispense 120 l of the Illumina control DNA library into tube 5 of a 0.2 ml eight-tube strip. This will place the control sample in lane 5 on the flow cell. Illumina recommends placing the control lane in this position. 5. Add 120 l of diluted, denatured sample DNA template into the remaining tubes of a 0.2 ml eight-tube strip. Take careful note of which template goes into each tube. 6. Label the tube strip B. 7. Set aside on ice until you are ready to load it onto the Cluster Station.
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Performing Cluster Amplification

The standard recipe for cluster amplification washes the Cluster Station and prompts you to load reagents and perform cluster amplification. You can also use the combined one-step recipe that performs cluster amplification and then continues to linearization, blocking, and primer hybridization.
Wash the Cluster Station

Workflow Single-Read Standard Protocol
1. From the Illumina Cluster Station Software, select File | Open Recipe. 2. Open one of the following recipes and click Run.
Recipe Name SR_Amplification_only_v5.0.xml SR_Amplification_Linearization_Blocking_PrimerHyb_v5.0.xml PE_Amplification_only_v5.0.xml PE_Amplification_Linearization_CombinedBlocking_PrimerHyb_v5.0.xml
Single-Read One-Step Protocol Paired-End Standard Protocol Paired-End One-Step Protocol
3. Install the washing bridge and load containers filled with deionized water in positions 1, 3, 9, 11, and 12. 4. Click OK to proceed. When the washing is complete, a message appears to let you know that the cycle is complete, and prompts you to remove water from the reagent positions. 5. Remove water from positions 1, 3, 9, 11, and 12 and click OK. When the priming of the air gap is complete, a message appears to let you know that the cycle is complete, and prompts you to load reagents in the appropriate positions.
Load Reagents
Best Practices
To prevent cross-contamination, follow these best practices when loading reagents on the Cluster Station: ` Always remove and replace reagents one tube (or bottle) at a time.
` Wear gloves at all times. Do not touch reagents with bare hands. ` Connect the tubes by holding the caps stationary while you twist the
tubes into place. This prevents crimping and twisting of the lines.
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Reagent Positions
The following figure illustrates reagent tube and bottle placement along with the number associated with each position. The eight-tube strips that fit in the removable tube strip holder are lettered A, B, C, and E.
9 10 11 12 13 14 15 16 17 18
Eight-Tube Strip Holder
Figure 3
Reagent Positions on the Cluster Station Table 6

Position 1 3 9 11 12 A B C
Reagent Positions for Amplification

Reagent AMX1 (Amplification Mix) HT2 (Wash Buffer) AT1 (Formamide) APM1 (AMX1 Premix) HT1 (Hybridization Buffer) HT1 (Hybridization Buffer) Denatured DNA Template Mix HT2 (Wash Buffer) Tube Size 50 ml 15 ml 50 ml 50 ml 50 ml 0.2 ml eight-tube strip 0.2 ml eight-tube strip 0.2 ml eight-tube strip
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Table 6
Position D E
Reagent Positions for Amplification (Continued)

Reagent APM1 (AMX1 Premix) EMX (Extension Mix) Tube Size 0.2 ml eight-tube strip 0.2 ml eight-tube strip
Perform Cluster Amplification
The recipe guides you through the amplification process. 1. Load reagents in positions 1, 3, 9, 11, and 12 as indicated in Figure 3 and Table 6 on page 21. 2. Click OK to resume the recipe and proceed to amplification. 3. When prompted, load the flow cell, Hybridization Manifold, and tube strip holder onto the Cluster Station. 4. Load the tube strip labeled A (Hybridization Buffer) into the tube strip holder. 5. Wait until the temperature stabilizes at 20C, and then click OK to proceed. 6. When prompted, remove tube strip A and load the tube strip labeled B (Template Mix) into the tube strip holder. 7. Click OK to proceed. 8. When prompted, remove tube strip B and load the tube strip labeled C (Wash Buffer) into the tube strip holder. 9. Click OK to proceed. 10. When prompted, remove tube strip C and load the tube strip labeled D (AMX1 Premix) into the tube strip holder. 11. Click OK to proceed. 12. When prompted, remove tube strip D and load the tube strip labeled E (Extension Mix) into the tube strip holder. 13. Click OK to proceed. 14. When prompted, remove the manifold inlets from the tube strip to drain inlets. 15. Click OK to proceed. 16. When prompted, replace the Hybridization Manifold with the Amplification Manifold. 17. Click OK and check for proper flow through the Amplification Manifold. When amplification is complete, a message appears to let you know that the cycle is complete. 18. Measure the reagent volumes and record them on the lab tracking form. The following table lists the initial volume of each reagent and the expected volume after Read 1 amplification.
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Table 7
Position 1 3 9 11 12
Reagent Volumes for Amplification

Reagent AMX1 (Amplification Mix) HT2 (Wash Buffer) AT1 (Formamide) APM1 (AMX1 Premix) HT1 (Hybridization Buffer) Initial Volume 15 ml 10 ml 8 ml 10 ml 15 ml Expected Volume After Amplification 1.8 ml 7 ml 2.6 ml 1.3 ml 12 ml
NOTE
At this point in the protocol, you can store the flow cell at 2 to 8C or continue to linearization, blocking, and primer hybridization.
1. Click OK to resume the recipe and wash the lines used for amplification. The following prompt appears: Wash lines for cluster amplification. Please attach the washing bridge and load water in positions 1, 3, 9, 11, and 12. 2. Install the washing bridge and load containers filled with water in positions 1, 3, 9, 11, and 12. 3. Click OK to proceed. When the washing of the lines is complete, a message appears to let you know that the cycle is complete. If you are not proceeding directly to the Linearization, Blocking, and Primer Hybridization step, remove HT1 and HT2 from the Cluster Station and store at 4C until you are ready to proceed.
24
Performing Linearization, Blocking, and Primer Hybridization

The recipe guides you through the steps for washing the Cluster Station, loading reagents, and performing linearization, blocking, and primer hybridization on the Cluster Station. This section describes the workflow and prompts used in the standard protocol. If you are using the one-step protocol, the reagents are already loaded and the recipe is running. If you are using the multi-primer protocol, follow the onscreen instructions.
Do not linearize, block, and primer hybridize a paired-end flow cell until the day of use.
CAUTION

Workflow Single-Read Standard Protocol Paired-End Standard Protocol
1. From the Illumina Cluster Station Software, select File | Open Recipe. 2. Open the standard protocol recipe and click Run, or click OK to resume the one-step recipe.
Recipe Name SR_Linearization_Blocking_PrimerHyb_v5.0.xml PE_2P_R1Prep_Linearization_CombinedBlocking_PrimerHyb_v5.0.xml
3. Install the washing bridge and load containers filled with water in the following positions: Single-read recipeLoad positions 3, 4, 7, 12, 15, and 17. Paired-end recipeLoad positions 3, 4, 7, 12, 14, and 17.
4. Click OK to proceed and follow the prompts when the wash is complete. 5. Remove water from the following positions: Single-read recipeLoad positions 3, 4, 7, 12, 15, and 17. Paired-end recipeLoad positions 3, 4, 7, 12, 14, and 17.
6. Click OK and follow the prompts when the air gap has been primed.
Load Reagents
Best Practices
To prevent cross-contamination, follow these best practices when loading reagents on the Cluster Station: ` Always remove and replace reagents one tube at a time.
` Wear gloves at all times. Do not touch reagents with bare hands. ` Connect the tubes by holding the caps stationary while you twist the
Part # 1006747 Rev. A
25
Reagent Positions
The following figure illustrates reagent tube and bottle placement along with the number associated with each position.
9 10 11 12 13 14 15 16 17 18
Eight-Tube Strip Holder
Figure 4 Table 8
Reagent Positions on the Cluster Station Reagent Positions for Linearization, Blocking, and Primer Hybridization
Reagent (Standard Protocol) HT2 (Wash Buffer) BMX (Blocking Mix) Sequencing Primer HT1 (Hybridization Buffer) LMX (Linearization Mix 1) (for paired-end recipe) LS1 (Linearization Solution 1) (for single-read recipe) LS1 (Linearization Solution 1) HT1 (Hybridization Buffer) Reagent (Multi-Primer Protocol) HT2 (Wash Buffer) BMX (Blocking Mix) Tube Size 15 ml 15 ml 2 ml 50 ml 2 ml
15
2 ml
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Table 8
Position 17 F G H I J
Reagent Positions for Linearization, Blocking, and Primer Hybridization (Continued)

Reagent (Standard Protocol) Diluted HP3 (0.1 N NaOH) Diluted HP3 (0.1 N NaOH) HT2 (Wash Buffer) Sequencing Primers HT2 (Wash Buffer) HT1 (Hybridization Buffer) Reagent (Multi-Primer Protocol) Tube Size 2 ml 0.2 ml eight-tube strip 0.2 ml eight-tube strip 0.2 ml eight-tube strip 0.2 ml eight-tube strip 0.2 ml eight-tube strip
Perform Linearization, Blocking, and Primer Hybridization
The recipe guides you through linearization, blocking, and primer hybridization. 1. Load reagents in the following positions as indicated in Figure 4 and Table 8 on page 25. Single-read recipeLoad positions 3, 4, 7, 12, 15, and 17. Paired-end recipeLoad positions 3, 4, 7, 12, 14, and 17.
2. Click OK to resume the recipe and follow the prompt to proceed to linearization, blocking, and primer hybridization. 3. Load the flow cell and attach the Amplification Manifold. 4. Click OK to proceed and follow the prompt to check for proper flow. 5. As the process starts, check for correct fluid flow through all eight lanes and the lines of the Amplification Manifold. When the flow is regular in all lanes, proceed with the protocol. 6. Click OK to proceed. When linearization, blocking, and primer hybridization is complete, a message appears to let you know that the flow cell is ready for sequencing. 7. Remove the flow cell from the Cluster Station.
Do not store the flow cell at this point for long periods of time. Sequencing must be performed on the flow cell within four hours.
CAUTION
8. Measure the reagent volumes and record them on the lab tracking form. The following table lists the initial volume of each reagent and the expected volume after linearization, blocking, and primer hybridization.
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Table 9
Reagent Volumes for Linearization, Blocking, and Primer Hybridization

Reagent HT2 (Wash Buffer) BMX (Blocking Mix) HP1 (Sequencing Primer Mix 1) HT1 (Hybridization Buffer) LMX1 (Linearization Mix 1) (for paired-end recipe) LS1 (Linearization Solution 1) (for single-read recipe) Diluted HP3 (0.1 N NaOH) Initial Volume 7 ml (approx.) 2 ml 1.3 ml 13 ml (approx.) 1.3 ml Expected Volume After Lin/Block/PrimerHyb 3 ml (approx.) 0.53 ml 0.35 ml 10 ml (approx.) 0.3 ml
15
1.8 ml
0.3 ml
17
1.5 ml
0.46 ml
1. Click OK to resume the recipe. 2. Attach the washing bridge and load containers filled with water in the following positions: Single-read recipeLoad positions 3, 4, 7, 12, 15, and 17. Paired-end recipeLoad positions 3, 4, 7, 12, 14, and 17.
3. Click OK to proceed. When the wash is complete, a message appears to let you know that the cycle is complete.
A weekly DECON wash is required, using the recipe DECON_Wash_All_Linesv3.0.xml. The DECON solution consists of 5% DECON in water. All lanes are washed once with DECON solution, followed by two washes with water. Please refer to the Cluster Station Operations Guide for instructions.
NOTE
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Part # 1006747 Rev. A
Chapter 3
Sequencing on the Genome Analyzer
Topics
30 31 33 35 38 39 42 46 49 Sequencing Workflows Sequencing Protocols and Recipes Performing a Pre-Run Wash Loading Reagents on the Genome Analyzer Priming Reagents Continuing the Run Preparing for Read 2 Using the Paired-End Module Sequencing Read 2 on the Genome Analyzer Performing a Post-Run Wash
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30
CHAPTER 3 Sequencing on the Genome Analyzer
Sequencing Workflows
This chapter describes the workflow for single-read sequencing and pairedend sequencing, including loading reagents onto the Genome Analyzer and Paired-End Module, using various sequencing recipes to customize your workflow, and performing pre-run and post-run procedures.
Single-Read and Paired-End Sequencing on the Genome Analyzer
Perform Pre-Run Instrument Wash
Paired-End Sequencing on the Genome Analyzer and Paired-End Module
See Using the SBS Sequencing Kit on the Genome Analyzer
Prepare and Load Reagents on the Genome Analyzer
For Two-Folder Paired-End Method, Transfer Data Before Read 2
See Genome Analyzer Operations Guide:
Clean and Install Prism Clean and Install Flow Cell Apply Oil First-Base Incorporation Load Flow Cell with Scan Mix Adjust Focus Check Quality Metrics
See Using the Paired-End Cluster Generation Kit on the Cluster Station and Paired-End Module :
Prepare and Load Reagents on the Paired-End Module
Read 2 Preparation Using the Paired-End Module
Continue the Sequencing Run
See Using the SBS Sequencing Kit on the Genome Analyzer
Prepare and Load Reagents on the Genome Analyzer For Long Runs, Prepare to Replace Reagents and Reset Reagent Tracking
For single reads For paired ends
Sequence Read 2
Weigh Reagents and Perform Post-Run Instrument Wash
For Long Runs, Prepare to Replace Reagents and Reset Reagent Tracking
Figure 5
Workflow Diagram for Single-Read and Paired-End Sequencing
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Sequencing Protocols and Recipes
31
Sequencing Protocols and Recipes

Recipes are XML files containing a series of commands. To perform runs on the Genome Analyzer, you open and execute the appropriate recipe. You need to select the appropriate recipe to use depending on the type of sequencing you are performing and the number of cycles you plan to run. See Table 10 on page 32 for a list of sequencing recipes.
36-Cycle Runs
A 36-cycle sequencing run is standard for single-read and paired-end sequencing. A 36-cycle sequencing run may take 4872 hours. Single-read sequencing requires one 36-Cycle SBS Sequencing Kit. Pairedend sequencing requires two 36-Cycle SBS Sequencing Kits and the PairedEnd Module.
51-Cycle Runs and 76-Cycle Runs
Performing a 51-cycle run or a 76-cycle run requires you to configure the Genome Analyzer to perform SMX reflushing during every cycle of sequencing. This is a default setting in the Genome Analyzer II software SCS v2.3. If you are not running SCS v2.3, obtain an upgrade to the ImageCyclePump.xml file and place it in the /config sub-directory. In addition to recipes specific to performing long runs, you also need SBS Sequencing Kits v3. The following table lists the number of kits required for each type of run.
36-Cycle SBS Sequencing Kit v3 1 3 2 4 18-Cycle SBS Sequencing Kit v3 1
Number of Cycles and Reads 51-Cycle single-read runs 51-Cycle paired-end runs 76-Cycle single-read runs 76-Cycle paired-end runs
Paired-End Sequencing
Paired-end sequencing generates reads from both ends of the templates in your library. For more information, see Paired-End Sequencing Overview on page 6. There are two methods for paired-end sequencing that are determined by the recipes you use: the two-folder method and the single-folder method.
Two-Folder Method
The two-folder method results in two Run folders, one for each read. The two-folder method requires that you transfer the data from the first read to your network storage for data analysis prior to proceeding to the second read.
32
Single-Folder Method
The single-folder method results in a single Run folder containing data from both reads. Each single-folder recipe performs a full sequencing run for Read 1 starting with first-base incorporation, and then continues on to a full sequencing run for Read 2 including first-base incorporation. To use the single-folder paired-read recipe, you must be running SCS 2.01 or later. Ensure that you are either running IPAR, RoboCopy, or have sufficient hard drive space to accommodate two full runs of the number of cycles you plan to sequence.
Recipe Names
The following table lists the recipe names for each step in the various sequencing protocols. The <#> in the file name refers to the current version of the recipe.
Table 10 Sequencing Recipes Used on the Genome Analyzer

Step in Protocol Pre-Run Wash Prime Reagents on the Genome Analyzer First-Base Incorporation 36-Cycle Sequencing 51-Cycle Sequencing 76-Cycle Sequencing Prime Reagents on the Paired-End Module Prepare for Read 2 on the Paired-End Module First-Base Incorporation 36-Cycle Read 2 51-Cycle Read 2 76-Cycle Read 2 Post-Run Wash GA2_PostWash_<v#> Single-Read Sequencing GA2_PreWash_<v#> GA2_Prime_<v#> GA1_FirstBase_<v#> GA2_36Cycle_SR_<v#> GA2_51Cycle_SR_<v#> GA2_76Cycle_SR_<v#> Paired-End Sequencing Two-Folder Method GA2-PEM_PreWash_<v#> GA2-PEM_Prime_<v#> GA2_FirstBase_<v#> GA2_36Cycle_PE_<v#> GA2_51Cycle_PE_<v#> GA2_76Cycle_PE_<v#> PEM_R2Prime_<v#> PEM_R2Prep_<v#> GA2_FirstBase_<v#> GA2_36Cycle_PE_v<#> GA2_51Cycle_PE_v<#> GA2_76Cycle_PE_v<#> GA2-PEM_PostWash_<v#> GA2-PEM_2x36Cycle_<v#> GA2-PEM_2x51Cycle_<v#> GA2-PEM_2x76Cycle_<v#> Paired-End Sequencing Single-Folder Method GA2-PEM_PreWash_<v#> GA2-PEM_Prime_<v#>
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Performing a Pre-Run Wash
33
Performing a Pre-Run Wash

You must perform a pre-run wash if the instrument has been idle for one day or more. The wash flushes 1 ml of instrument wash reagent (PW1) through each reagent port and out to a waste container. PW1 is provided in the SBS Sequencing Kit v2 (or higher). Run time varies depending on the recipe you are using. The run time for the single-read recipe is approximately 15 minutes. The run time for the pairedend recipe is approximately 40 minutes. After the wash, check the total volume in the waste container closely to confirm the stability of the reagent delivery system. Expected volumes from the wash cycle is the primary indicator of a stable fluid delivery system. Dry syringes and excessive bubbles are indicators of a problem.
Consumables
Illumina-Supplied ` PW1 User-Supplied ` Lens cleaning tissue ` (4) 125 ml Nalgene bottles
` (3) 50 ml conical tubes ` MilliQ water or laboratory grade water

Procedure
1. Load the Genome Analyzer with a used flow cell. If the plumbing manifolds are raised for the first time, place the fluidics on standby to prevent siphoning of reagents: Command: Pump To: Flow cell Solution: 8 (single-read sequencing); 28 (paired-end sequencing) Volume: 0 Aspiration Rate: 250 Dispense Rate: 2500 With the cursor in the Dispense Rate box, press Enter. 2. Dispense 40 ml of PW1 into four 125 ml Nalgene bottles. 3. Dispense 10 ml of PW1 into three 50 ml conical tubes. 4. Load the Genome Analyzer with PW1 solution as follows: 10 ml PW1 into port positions 1, 6, and 3 40 ml PW1 into port positions 4, 5, 2, and 7
Rotate the tubes while holding the caps stationary, to prevent crimps and twisting in the liquid delivery lines.
CAUTION
34
5. [Paired-End Module] Place at least 5 ml of laboratory grade water in each Falcon tube in positions 921 on the Paired-End Module. 6. Loosen and remove the Genome Analyzer waste tubing from the waste container. 7. Bundle the waste tubes with parafilm, making sure to keep all of the ends even. 8. Place the bundled tube ends into a 50 ml tube. 9. Click the Run tab. 10. Select File | Open Recipe. 11. Open one of the following recipes: For single-read sequencing, open GA2_PreWash_v<#>.xml. For paired-end sequencing, open GA2-PEM_PreWash_v<#>.xml.
12. Click Start. Reagents are delivered 1 ml at a time.

Total Volume Expected 7 ml 21 ml
Recipe Name
Total Run Time 15 minutes 40 minutes
GA2_PreWash_v<#>.xml GA2-PEM_PreWash_v<#>.xml
13. Record the delivery volume on the lab tracking form. If the measured volume is less than 90% of the expected value, do the following: a. Check for leaks. b. Repeat the wash cycle. c. Collect and measure each 1 ml delivery.
During the sequencing run, keep one 125 ml Nalgene bottle containing PW1 at position 2 on the Genome Analyzer. Save the 125 ml bottles and the 50 ml conical tubes containing the PW1 solution for use with the post-run wash.
NOTE
Part # 1006747 Rev. A
Loading Reagents on the Genome Analyzer
35

Always perform a pre-run wash before loading reagents onto the Genome Analyzer.
Perform a monthly maintenance wash followed by a pre-run wash before switching between CLM (provided in the SBS Sequencing Kit v3), CMX (provided in the SBS Sequencing Kit v2), or the Cleavage Mix (provided in the original SBS kits).
CAUTION
Reagents loaded onto the Genome Analyzer must be used in a sequencing run the same day.
Safe Handling Conventions

1. To prevent cross-contamination of reagents, especially IMX and CMX/ CLM, establish the following safe handling conventions: Always remove and replace one bottle or tube at a time. Always install the CMX/CLM last to avoid cross-contamination. Keep the SMX, IMX, and CMX/CLM on ice until you load them onto the Genome Analyzer.
2. Invert all reagents several times to mix them before loading them onto the Genome Analyzer. 3. Centrifuge the SMX, IMX, and CMX/CLM at 4C to 1000 xg for 1 minute before loading them onto the Genome Analyzer.
Load Reagents
Load the prepared reagents in the appropriate positions on the Genome Analyzer, as shown in Table 11 and Figure 6. When you attach the 50 ml tubes, hold the caps stationary and rotate the tubes to prevent crimps in the liquid delivery lines. If you are using SBS Sequencing Kit v2, load CMX in position 6. If you are using SBS Sequencing Kit v3, load CLM in position 6. Table 11 Reagent Positions on the Genome Analyzer
Solution # 1 2 3 4 5 6 7 Size 50 ml Amber Tube 125 ml Bottle 50 ml Amber Tube 125 ml Bottle 125 ml Bottle 50 ml Amber Tube 125 ml Bottle Contents IMX PW1 SMX PR1 PR2 CMX or CLM PR3
36
Figure 6
Reagent Positions on the Genome Analyzer Reagent tracking counts down the number of cycles that reagents are expected to last. To enable and configure reagent tracking, select Reagents | Configure Tracking from the toolbar at the top of the Data Collection software screen. For an overview of these features, see the Genome Analyzer II Operations Guide. Follow this procedure to set the number of cycles the reagents are expected to last, start the cycle counter, and enter the barcode IDs. 1. Select Reagents | Current Volumes from the toolbar at the top of the Data Collection software screen. The Current Reagent Volumes dialog box appears.
Track Reagents
Figure 7
Current Reagent Volumes
2. Click Reset Volumes. The Reset Reagent Volumes dialog box appears.
Figure 8
Reset Reagent Volumes
Part # 1006747 Rev. A
37
3. Enter the estimated number of cycles that you expect the reagents to last and click OK. The number of cycles remaining for the next set of reagents is updated.
Figure 9
Updated Current Reagent Volumes
4. Click Close. You may also leave this dialog box open while the run executes. The Current Reagent Volumes dialog box appears when the number of cycles completed equals the value set in Reset Volumes, minus the number of cycles before email notification set in the Reagent Configuration. For an overview of settings in the Reagent Configuration dialog box, see the Genome Analyzer II Operations Guide. 5. To record the barcode ID of the reagents, click Reagent Barcodes. The Reagent Barcode dialog box appears. 6. Enter the barcode IDs for each reagent. (Barcode IDs for each run are stored in ReagentIds.xml.)
Figure 10
Reagent Barcodes Dialog Box
7. If you are performing a paired-end run, click the PE Reagents tab. The PE Reagents tab is only visible if a paired-end recipe is open. 8. Click OK.
38
Priming Reagents
Before each run, you must prime all of the plumbing lines with the reagents. You will collect a set of liquid deliveries through all valve ports out to a waste volume, and then check total volume to confirm the stability of the reagent delivery system.
Priming volumes are a key indicator of a stable fluid delivery system. The measured volumes must be within 10% of normal for optimal sequencing performance.
NOTE
1. Loosen and remove the waste tubing from the waste bottle. 2. Bundle all waste tubes so that the ends are even with each other, and wrap them with parafilm. 3. Place the bundled tube ends into a 15 ml or a 50 ml conical tube. 4. Click the Run tab in the Data Collection software window. 5. Select File | Open Recipe. 6. Open the GA2_Prime_v<#>.xml recipe. 7. Click Start. 8. Collect all of the waste from the priming recipe and ensure that the volume is 6.4 ml. 9. Record the delivery volume on the lab tracking form. If the measured volume differs from the expected value by more than 10%, repeat the priming procedure. If the delivered volume still differs from the expected volume by more than 10%, take the following steps: a. Click the Manual Control/Setup tab. In the Pump area, set the following values to prevent siphoning of reagents: Command: Pump To: Flow cell Solution: 8 (single-read sequencing); 28 (paired-end sequencing) Volume: 0 Aspiration Rate: 250 Dispense Rate: 2500 b. Click Load Flow Cell to bring the stage to the front of the instrument and raise the lens. c. In the Instrument dropdown menu, select Unlock Door. Raise the door. d. Briefly lift the manifolds and reposition the flow cell. e. Repeat the priming procedure. 10. Proceed to set up the Genome Analyzer (cleaning and installing the prism and flow cell, applying oil, performing first-base incorporation, and adjusting focus) as described in the Genome Analyzer II Operations Guide. After you have completed the setup procedures, proceed to Continuing the Run on page 39.
Part # 1006747 Rev. A
Continuing the Run
39
Continuing the Run

If you are satisfied with the results of the first-base incorporation, follow these instructions to continue the sequencing run.
During a run, the operator can pause or stop the run by clicking Stop. The instrument is put into the safe state. After pausing a run, the operator can resume it by clicking Resume. The protocol is resumed from the selected recipe item on the Recipe tab. If a run is stopped during imaging, 75 l of Scan Mix must be pumped prior to resuming the run.
NOTE
1. Depending on the sequencing method you are using, open one of the following recipes or resume one of the single-folder recipes:
Single-Read Sequencing GA2_36Cycle_SR_<v#> GA2_51Cycle_SR_<v#> GA2_76Cycle_SR_<v#> Paired-End Sequencing Two-Folder Method GA2_36Cycle_PE_<v#> GA2_51Cycle_PE_<v#> GA2_76Cycle_PE_<v#> Paired-End Sequencing Single-Folder Method GA2-PEM_2x36Cycle_<v#> GA2-PEM_2x51Cycle_<v#> GA2-PEM_2x76Cycle_<v#>
2. Click Start. 3. When prompted, click OK to accept the name of the Run folder. For more information about Run folders, see the Genome Analyzer II Operations Guide. 4. When the Autofocus Calibration dialog box appears, click No (you have already calibrated), and the Genome Analyzer resumes sequencing. 5. Observe the images in the second cycle to determine if they stay in focus. If the focus is poor, stop the run and refocus before all of the images are collected. 6. If you are performing a 51-cycle or 76-cycle run, prepare to replace reagents at the halfway point. See Replace Reagents on page 40 for details. 7. When the run is complete, do one of the following: For single-read sequencing, notify the appropriate personnel that data are available for analysis and proceed to Weigh Reagents on page 41. For paired-end sequencing using the single-folder method, proceed to Preparing for Read 2 Using the Paired-End Module on page 42. For paired-end sequencing using the two-folder method, proceed to Transfer Data for Paired-End Runs on page 41.
A paired-end flow cell can safely be left on the Genome Analyzer in High Salt Buffer (PR1) for a period of three days after completion of the first read and before beginning Read 2 preparation on the Paired-End Module.
NOTE
40
Replace Reagents
In order to successfully complete long sequencing runs, you must replace the first set of sequencing reagents with a second set of reagents at some point in the run. The following table lists recommended cycles for reagent replacement, depending on your sequencing method and number of cycles you are running.
Sequencing Method Single-Read Run Paired-End Run, Read 1 (single-folder method or two-folder method) Paired-End Run, Read 2 (single-folder method) Paired-End Run, Read 2 (two-folder method) 51-Cycle Run Cycle 3437 Cycle 3437 76-Cycle Run Cycle 3437 Cycle 3437
Cycle 7074 Cycle 2024
Cycle 77 and Cycle 112116 Cycle 1 and Cycle 3437
CAUTION
Replace the reagents at a time when they are not being pumped through the flow cell. For example, replace IMX, PR1, PR2, PR3, and CMX/CLM during the flow cell imaging step (e.g., Cycle 38 imaging). These reagents are not used by the instrument during imaging. Replace SMX early in the chemistry step since it is used again at the end of chemistry, immediately before imaging restarts.
1. During the flow cell imaging step, remove the reagent containers from the Genome Analyzer and quickly replace them with the second container in the following order: a. IMX b. PR1 c. PR2 d. PR3 e. CMX/CLM 2. Discard your gloves and replace them with a new pair after handling CMX/CLM and before replacing SMX. 3. After the imaging step is complete and early in the chemistry step, replace SMX.
Part # 1006747 Rev. A
Continuing the Run
41
Reset Reagent Volumes Weigh Reagents
When you are finished replacing reagents, perform the following steps to reset the cycle counter. Follow the steps described in Track Reagents on page 36. Weighing reagents at the end of a run measures reagent consumption and fluidics performance. 1. Weigh all of the reagent bottles and record the results on the lab tracking form. 2. Weigh all of the fluids that have been pumped through the eight lanes and record the results on the lab tracking form. 3. Proceed to Performing a Post-Run Wash on page 49.
Transfer Data for Paired-End Runs
If you are using the two-folder method for paired-end sequencing, you must transfer your data to your network storage for data analysis after completion of the first read and before starting the recipe for Read 2.
Illumina recommends that you use the RoboCopy script rather than manually copying files.
NOTE
1. Check that all of the data from the Run Folder have been copied to your network storage location, including: Images Focus images (if stored) Log files Configuration files Calibration files
2. Confirm that all of the data have been transferred and checked. 3. Delete the Run Folder from the instrument data drive.
Do not attempt to start the Read 2 recipe until the deletion is complete. The disk space checking algorithm used by the instrument software may produce an error.
CAUTION
4. Proceed to Preparing for Read 2 Using the Paired-End Module on page 42.
42
Preparing for Read 2 Using the Paired-End Module

The Paired-End Module supplies the reagents to the flow cell. The temperature-sensitive reagents are located in cooled reservoirs on the Paired-End Module. After you finish preparing for Read 2, immediately begin sequencing Read 2.
Best Practices
Follow these best practices when loading reagents on the Paired-End Module: ` Wear gloves at all times. Do not touch reagents with bare hands.
` Connect the tubes by holding the caps stationary while you twist the
Load Reagents
Reagent Positions
The following figure illustrates the reagent positions on the Paired-End Module and the number associated with each position.
21 16 15 19 14
11 10 12 13
Figure 11
Reagent Positions on the Paired-End Module
The following table identifies the position of each reagent used for Read 2 preparation on the Paired-End Module.
Part # 1006747 Rev. A
43
Table 12 Reagent Positions on the Paired-End Module

Position 10 11 12 13 14 15 16 19 21 Reagent RMX (Resynthesis Mix) LMX2 (Linearization Mix 2) BMX (Blocking Mix) AMX2 (Amplification Mix 2) APM2 (AMX2 Premix) AT2 (Formamide) HP2 (Sequencing Primer Mix 2) Diluted HP3 (0.1 N NaOH) HT2 (Wash Buffer)
NOTE
The reagents for the Paired-End Module are provided in new 15 ml tubes. When attaching the reagent tubes to the Paired-End Module cap assembly, twist the tube at least one full rotation until you feel resistance. You cannot overtighten the caps; they do not come to a complete stop.
1. Load the reagents onto the Paired-End Module according to the positions shown in Figure 11 on page 42 and Table 12 on page 43. 2. Connect tubes from reagents to the corresponding port position on the Paired-End Module. 3. Place the waste tube into the waste container or a 15 ml conical tube.
When you prepare and load these reagents onto the PairedEnd Module, you must use them the same day.
CAUTION
Prime the Paired-End Module
The priming steps are performed automatically using the internal priming pump on the Paired-End Module. The recipe primes each port position in turn and dispenses the waste to the waste bottle, bypassing the flow cell. Priming the Paired-End Module takes approximately 15 minutes.
If you are using the single-folder method, proceed to Prepare for Read 2 on page 44. The single-folder recipe primes the Paired-End Module as part of the Read 2 preparation step in the protocol.
44
Two-Folder Method
1. Open the PEM_R2Prime_v5.0.xml recipe. 2. Click OK. The following prompt appears when priming is complete: Priming complete. Press Enter or click OK to proceed to Read 2 preparation. 3. Measure reagent volumes after priming and record them on the lab tracking form. See Table 13 on page 45 for expected volumes after priming. 4. Measure the waste and record the volume on the lab tracking form. The expected volume is approximately 5.6 ml.
Waste produced during Read 2 preparation on the PairedEnd Module must be kept separate from waste produced during Read 2 sequencing on the Genome Analyzer. Waste from the Paired-End Module must be disposed of properly and in accordance with facility standards.
CAUTION
Prepare for Read 2
Read 2 preparation using the automated method takes approximately 4 hours from the priming of the Paired-End Module. The process is fully automated and can be left to run unattended.
1. Click OK to resume recipe GA2-PEM_2x36_PE_v<#>.xml. The recipe primes the lines and completes Read 2 preparation. When Read 2 preparation is complete, a message appears to prompt you to load Read 2 reagents.
Two-Folder Method
1. Open the PEM_R2Prep_v5.0.xml recipe. 2. Click OK. When Read 2 preparation is complete, a message appears to let you know that the flow cell is ready for Read 2. 3. Measure reagent volumes after Read 2 preparation and record them on the lab tracking form.
Part # 1006747 Rev. A
45
Measure Reagent Volumes
The following table lists the initial volume of each reagent used in Read 2 preparation and the expected volume after priming and Read 2 preparation.
Table 13 Reagent Volumes after Priming and Read 2 Preparation

Position 10 11 12 13 14 15 16 19 21 Reagent RMX (Resynthesis Mix) LMX2 (Linearization Mix 2) BMX (Blocking Mix) AMX2 (Amplification Mix 2) APM2 (AMX2 Premix) AT2 (Formamide) HP2 (Sequencing Primer Mix 2) Diluted HP3 (0.1 N NaOH) HT2 (Wash Buffer) Initial Volume 2 ml 2 ml 2 ml 9 ml 5 ml 8 ml 1.5 ml 3 ml 10 ml Expected Volume After Priming 1.4 ml 1.4 ml 1.4 ml 8.4 ml 4.25 ml 7.25 ml 0.75 ml 2.25 ml 9.25 ml Expected Volume After Read 2 Preparation 0.74 ml 0.96 ml 0.2 ml 0.66 ml 1.2 ml 4.2 ml 0.19 ml 1.1 ml 2.7 ml
46
Sequencing Read 2 on the Genome Analyzer

Sequencing Read 2 requires that you exchange the reagents used for the first read with fresh reagents before beginning the run. There is no need to remount the flow cell or perform a leak test.
To ensure that the two reads can be co-localized, do not turn off or re-initialize the Genome Analyzer. If you do, the X and Y stage coordinates will be lost. Do not make any changes to the map or configuration files between reads.
CAUTION
CAUTION
Waste produced during Read 2 preparation on the PairedEnd Module must be kept separate from waste produced during Read 2 sequencing on the Genome Analyzer. Waste from the Paired-End Module must be disposed of properly and in accordance with facility standards.
1. Exchange the reagents used for Read 1 with fresh reservoirs from the reagents supplied in the SBS Sequencing Kit. 2. Replace all the reagent tubes on the Paired-End Module with Falcon tubes containing at least 10 ml of MilliQ water or laboratory grade water.
Do not reprime reagents through the flow cell. CAUTION
3. Click OK to resume the single-folder recipe and start first-base incorporation for Read 2. 4. Click OK to accept the current calibrated focus and resume sequencing Read 2. The flow cell will automatically be flushed with Scan Mix (solution 3). If you wish to refocus manually, perform the following: a. Click Cancel. b. To load the flow cell with Scan Mix, click the Manual Control/Setup tab. c. In the Pump area, set the values as follows to pump Scan Mix: Command: Pump To: Flow cell Solution: 3 Volume: 100 Aspiration Rate: 250 Dispense Rate: 2500 d. With the cursor in the Dispense Rate box, press Enter. e. Perform manual focus and recalibrate the autofocus laser. See the Genome Analyzer II Operations Guide for instructions. f. Click OK to resume sequencing Read 2. 5. When imaging is complete, evaluate the first base report data for Read 2.
Part # 1006747 Rev. A
Sequencing Read 2 on the Genome Analyzer
47
6. If you are performing a 51-cycle or 76-cycle run, prepare to replace reagents at the halfway point. See Replace Reagents on page 48 for details. 7. Click OK to complete the sequencing of Read 2.
Two-Folder Method
1. Exchange the reagents used for Read 1 with fresh reservoirs from the reagents supplied in the SBS Sequencing Kit.
Do not reprime reagents through the flow cell. CAUTION
2. Open the GA2_FirstBase_v<#>.xml recipe. 3. Click OK to run the recipe. The software automatically makes a copy of the recipe file and stores it in the current Run folder. If you need to stop work at any point, you can reopen the recipe from that location and continue from where you left off.
In the two-folder workflow, the focus calibration from the first read can be used for the second read. You must introduce Scan Mix, but not recalibrate focus. This is possible as long as the Genome Analyzer or the software has not been restarted in between the two reads.
NOTE
4. Click Cancel to dismiss the Autofocus Calibration dialog box. The next step is to apply Scan Mix, and then determine the focal plane of the flow cell. This enables the software to automatically adjust the focus during the run.
It is critical to introduce Scan Mix to the flow cell before adjusting the focal plane.
CAUTION
5. Load the flow cell with Scan Mix. a. Click the Manual Control/Setup tab. b. In the Pump area, set the values as follows to pump Scan Mix: Command: Pump To: Flow cell Solution: 3 Volume: 100 Aspiration Rate: 250 Dispense Rate: 2500 c. With the cursor in the Dispense Rate box, press Enter. 6. Perform manual focus and recalibrate the autofocus laser. See the Genome Analyzer II Operations Guide for instructions. Reset only the Z axis as needed. Do not adjust the X axis or XY tilt. 7. Click OK to resume Read 2 sequencing.
48
8. Depending on the number of cycles you are running, open one of the following recipes: GA2_36Cycle_PE_v<#>.xml GA2_51Cycle_PE_v<#>.xml GA2_76Cycle_PE_v<#>.xml
9. If you are performing a 51-cycle or 76-cycle run, prepare to replace reagents at the halfway point. See Replace Reagents on page 48 for details. 10. Click OK to complete Read 2.
Replace Reagents
In order to successfully complete long sequencing runs, you must replace the first set of sequencing reagents with the second set at some point in the run. For recommended cycle times and instructions, see Replace Reagents on page 40.
Replace the reagents at a time when they are not being pumped through the flow cell. For example, replace IMX, PR1, PR2, PR3, and CLM during the flow cell imaging step (e.g., Cycle 38 imaging). These reagents are not used by the instrument during imaging. Replace SMX early in the chemistry step since it is used again at the end of chemistry, immediately before imaging restarts.
CAUTION
Part # 1006747 Rev. A
Performing a Post-Run Wash
49
Performing a Post-Run Wash

After every single-read run or after the second read of a paired-end run, you must perform a thorough instrument wash. The wash flushes 4 ml of wash solution through each reagent port on the Genome Analyzer and 1 ml through each reagent port on the Paired-End Module. Run time varies depending on the recipe you are using. The run time for the single-read recipe is approximately 45 minutes. The run time for the pairedend recipe is approximately 60 minutes.
Consumables
Illumina-Supplied ` PW1 User-Supplied ` Lens cleaning tissue ` (4) 125 ml Nalgene bottles
` (3) 50 ml conical tubes ` MilliQ water or laboratory grade water

Procedure
1. Load the Genome Analyzer with PW1 solution as follows: 10 ml PW1 into port positions 1, 6, and 3 40 ml PW1 into port positions 4, 5, 2, and 7
2. [Paired-End Module] Place at least 5 ml of laboratory grade water in each Falcon tube in positions 921 on the Paired-End Module. 3. Loosen and remove the Genome Analyzer waste tubing from the waste container. 4. Bundle the Genome Analyzer waste tubes with parafilm, making sure to keep the ends even. 5. Place the bundled tube ends into a pre-weighed 50 ml conical tube. 6. Click the Run tab. 7. Select File | Open Recipe. 8. Open one of the following recipes: For single-read sequencing, open GA2_PostWash_v<#>.xml. For paired-end sequencing, open GA2-PEM_PostWash_v<#>.xml.
9. Click Start and enter a file name.

Using wash reagents other than the PW1 solution in the Sequencing Kit, or failing to perform the wash cycle at the recommended intervals, may void the warranty.
CAUTION
50
Part # 1006747 Rev. A
Index
C
cluster densities 18 cluster generation 5 linearization, blocking, primer hybridization 24 preparing template 18 protocol times 16 recipes 15 workflow 14 Cluster Station overview 5 stopping safely 16 consumables, user-supplied 11 cross-contamination 20, 24, 35 customer support 3
loading reagents Cluster Station 20, 24 Genome Analyzer 35 Paired-End Module 42
M
material safety data sheet (MSDS) 3
O
one-step protocols 15
P
paired-end data transfer 41 flow cell 6 overview 6 protocols 31 Read 2 preparation 44 recipes 32 single-folder method 46 two-folder method 47 workflow 7 Paired-End Module 5 Pipeline 5, 10 post-run wash 49 pre-run wash 33 priming Genome Analyzer reagents 38 protocols, cluster generation multi-primer 15 one-step 15 protocol times 16 standard 15 protocols, sequencing 31
D
data transfer, paired-end runs 41 documentation 3, 9
F
flow cells overview 4 paired-end 6 storage 16
G
Genome Analyzer overview (system) 4, 5 Pipeline software 5
H
help documentation 9 technical 3
R
reagents, Cluster Station loading 20, 24 positions for amplification 21 positions for linearization, blocking, and primer hybridization 25
L
lab tracking forms 10
51
52
Index
volumes for amplification 22 volumes for linearization, blocking, and primer hybridization 26 volumes per recipe 17 reagents, Genome Analyzer barcodes 36 loading 35 positions 35 priming 38 replacing during long runs 40 reset volumes 36, 41 tracking 36 weighing 41 reagents, Paired-End Module loading 42 positions 42 priming 43 volumes for Read 2 preparation 45 recipes cluster generation 15 sequencing 32 RoboCopy 32, 41
protocols 31 recipes 32 workflow 30 sequencing by synthesis (SBS) 4 single-folder method 31, 46 software Pipeline 5 stopping points, Cluster Station 16
T
technical assistance 3 tips avoiding cross-contamination on Genome Analyzer 35 loading reagents safely 20, 24, 35, 42 two-folder method 31, 47
W
washes post-run wash 49 pre-run wash 33 workflows cluster generation 14 sequencing 30
S
sample preparation 4 scheduling, protocol times 16 sequencing
Part # 1006747 Rev. A
Illumina, Inc. 9885 Towne Centre Drive San Diego, CA 92121-1975 +1.800.809.ILMN (4566) +1.858.202.4566 (outside North America) [email protected] www.illumina.com

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Sequencing User Guide

Revision history for part # 1006747:

Sequencing User Guide

Cluster Generation on the Cluster Station. . . . . . . . 13

Sequencing User Guide

Sequencing on the Genome Analyzer . . . . . . . . . . . 29

Part # 1006747 Rev. A

Sequencing User Guide

Part # 1006747 Rev. A

Sequencing User Guide

Part # 1006747 Rev. A

Sequencing User Guide

` Cluster GenerationSee the Cluster Station Operations Guide for

` SequencingSee the Genome Analyzer II Operations Guide for

See Sequencing Documentation on page 9 for descriptions of other related documentation.

Audience and Purpose

Illumina Customer Support Contacts

Sequencing User Guide

Genome Analyzer System

Process and Components

Part # 1006747 Rev. A

Genome Analyzer System

Cluster Generation on the Cluster Station

Sequencing on the Genome Analyzer

Paired-End Sequencing Using the Paired-End Module

Data Analysis Using the Genome Analyzer Pipeline Software

Sequencing User Guide

Paired-End Sequencing Overview

` Two new linearization methodsClusters are prepared for sequencing

` Two sequencing primersTwo hybridization events use a different

` Paired-End Cluster Generation KitThis kit contains the reagents

` Paired-End ModuleThis module is an external valve attachment to the

` Paired-end recipesPaired-end sequencing recipes are essentially

Part # 1006747 Rev. A

Paired-End Sequencing Overview

Paired-End Cluster Amplification Performed on the Cluster Station

Linearization, Blocking, and Primer Hybridization Performed on the Cluster Station

Read 1 Performed on the Genome Analyzer

Read 2 Performed on the Genome Analyzer

Linearization, Blocking, and Primer Hybridization

Sequencing User Guide

Preparation for Read 2

Part # 1006747 Rev. A

` Genome Analyzer II Operations GuideThis guide also contains an

` Using the Paired-End Cluster Generation Kit v2 on the Cluster Station

` Using the SBS Sequencing Kit v2 on the Genome AnalyzerThis booklet

` Using the SBS Sequencing Kit v3 on the Genome AnalyzerThis booklet

Sequencing User Guide

` Multiplexed Sequencing on the Genome AnalyzerThis booklet

Lab Tracking Forms

` Paired-End Sequencing Lab Tracking Form

Software and Analysis

Quick Reference and Experienced User Cards

Part # 1006747 Rev. A

User-Supplied Consumables and Equipment

User-Supplied Consumables and Equipment

Consumables ` 3-amino-1-propanol (> 99%) ` Tris-Cl 10 mM, pH 8.5

` (4) 125 ml Nalgene bottles

Sequencing User Guide