Illumina Genome Analyzer Maintenance Guide

Sequencing User Guide iii

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.

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© 2007–2008 Illumina, Inc. All rights reserved.

Sequencing User Guide v

Revision History

Revision history for part # 1006747:

Revision Date Description of Change

A November 2008 Initial release.

Sequencing User Guide vii

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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Audience and Purpose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Technical Assistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Genome Analyzer System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Process and Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Related Documentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Paired-End Sequencing Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Key Differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Protocol Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Sequencing Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Site Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Instrument Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Reagent Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Sequencing Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Lab Tracking Forms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Software and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Quick Reference and Experienced User Cards . . . . . . . . . . . . . . . . . . . 10

User-Supplied Consumables and Equipment . . . . . . . . . . . . . . . . . . . . . . . 11Cluster Generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Chapter 2 Cluster Generation on the Cluster Station. . . . . . . . 13

Cluster Generation Workflow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Cluster Generation Protocols and Recipes . . . . . . . . . . . . . . . . . . . . . . . . . 15

Standard Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15One-Step Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Recipe Names . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Protocol Times. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

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Safe Stopping Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Reagent Volumes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Preparing DNA Template for Cluster Generation . . . . . . . . . . . . . . . . . . . . 18Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Template Mix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Performing Cluster Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Wash the Cluster Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Load Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Perform Cluster Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Wash the Cluster Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Performing Linearization, Blocking, and Primer Hybridization. . . . . . . . . . . 24Wash the Cluster Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Load Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Perform Linearization, Blocking, and Primer Hybridization . . . . . . . . . . 26Wash the Cluster Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Chapter 3 Sequencing on the Genome Analyzer . . . . . . . . . . . 29

Sequencing Workflows. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Sequencing Protocols and Recipes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

36-Cycle Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3151-Cycle Runs and 76-Cycle Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Paired-End Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Recipe Names . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Performing a Pre-Run Wash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Loading Reagents on the Genome Analyzer . . . . . . . . . . . . . . . . . . . . . . . . 35Load Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35Track Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Priming Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38Continuing the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Replace Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40Reset Reagent Volumes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41Weigh Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41Transfer Data for Paired-End Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Preparing for Read 2 Using the Paired-End Module . . . . . . . . . . . . . . . . . . 42Load Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42Prime the Paired-End Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Prepare for Read 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44Measure Reagent Volumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Sequencing Read 2 on the Genome Analyzer . . . . . . . . . . . . . . . . . . . . . . . 46Single-Folder Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46Two-Folder Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Replace Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Performing a Post-Run Wash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

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List of Figures

Figure 1 Paired-End Protocol Workflow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Figure 2 Cluster Generation Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Figure 3 Reagent Positions on the Cluster Station. . . . . . . . . . . . . . . . . . . . . . . . . . . 21Figure 4 Reagent Positions on the Cluster Station. . . . . . . . . . . . . . . . . . . . . . . . . . . 25Figure 5 Workflow Diagram for Single-Read and Paired-End Sequencing . . . . . . . . 30Figure 6 Reagent Positions on the Genome Analyzer . . . . . . . . . . . . . . . . . . . . . . . . 36Figure 7 Current Reagent Volumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Figure 8 Reset Reagent Volumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Figure 9 Updated Current Reagent Volumes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Figure 10 Reagent Barcodes Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Figure 11 Reagent Positions on the Paired-End Module. . . . . . . . . . . . . . . . . . . . . . . 42

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Sequencing User Guide xi

List of Tables

Table 1 Illumina Customer Support Contacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Table 2 Cluster Station Recipes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Table 3 Protocol Times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Table 4 Safe Stopping Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Table 5 Reagent Volumes Per Recipe. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Table 6 Reagent Positions for Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Table 7 Reagent Volumes for Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Table 8 Reagent Positions for Linearization, Blocking, and Primer Hybridization . . 25Table 9 Reagent Volumes for Linearization, Blocking, and Primer Hybridization . . . 27

Table 10 Sequencing Recipes Used on the Genome Analyzer. . . . . . . . . . . . . . . . . . 32Table 11 Reagent Positions on the Genome Analyzer . . . . . . . . . . . . . . . . . . . . . . . . 35Table 12 Reagent Positions on the Paired-End Module . . . . . . . . . . . . . . . . . . . . . . . 43Table 13 Reagent Volumes after Priming and Read 2 Preparation. . . . . . . . . . . . . . . 45

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Sequencing User Guide 1

Chapter 1

Overview

Topics2 Introduction

2 Audience and Purpose

3 Technical Assistance

4 Genome Analyzer System

6 Paired-End Sequencing Overview

9 Sequencing Documentation

11 User-Supplied Consumables and Equipment

2 CHAPTER 1Overview

Part # 1006747 Rev. A

Introduction

This guide describes the workflows for performing single-read and paired-end 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 Preparation—See 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 AnalyzerCluster Generation—See 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 deliverySequencing—See 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.

Technical Assistance 3

Sequencing User Guide

Technical Assistance

For technical assistance, contact Illumina Customer Support.

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.

Table 1 Illumina Customer Support Contacts

Contact Number

Toll-free Customer Hotline 1-800-809-ILMN (1-800-809-4566)

International Customer Hotline 1-858-202-ILMN (1-858-202-4566)

Illumina Website http://www.illumina.com

Email [email protected]

4 CHAPTER 1Overview

Part # 1006747 Rev. A

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 whole-genome 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.

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.

NOTEFor more information about the Illumina Genome Analyzer System, visit http://www.illumina.com/sequencing.

Genome Analyzer System 5


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.

6 CHAPTER 1Overview

Part # 1006747 Rev. A

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 walk-away 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 KitPaired-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 preparation—Sample 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 cell—Paired-end sequencing uses a modified, paired-end-enabled flow cell. A standard flow cell cannot perform both reads of the paired-end experiment.Two new linearization methods—Clusters 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 primers—Two hybridization events use a different sequencing primer for each read.Combined blocking steps—Clusters prepared using the Illumina paired-end 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 Kit—This 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 Module—This 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 recipes—Paired-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.

Paired-End Sequencing Overview 7


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.

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 1—Selectively linearizes one of the two strands.Blocking—Prevents non-specific sites from being sequenced.Denaturation and hybridization—Performs standard denaturation and hybridization of the first sequencing primer.

Paired-End Cluster AmplificationPerformed on the Cluster Station

Linearization, Blocking, andPrimer Hybridization

Performed on the Cluster Station

Read 1Performed on the Genome Analyzer

Read 2 PreparationPerformed on the Genome Analyzer

with the Paired-End Module attached

Read 2Performed on the Genome Analyzer

8 CHAPTER 1Overview

Part # 1006747 Rev. A

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 Dehybridization—Removes the extended sequencing primer used in Read 1.Deprotection—Prepares the flow cell for the next step.Resynthesis—Regenerates the previously linearized strand.Linearization 2—Linearizes the strand that was sequenced in Read 1 to allow hybridization of the second sequencing primer to the newly synthesized DNA strand.Blocking—Prevents non-specific sites from being sequenced.Denaturation and hybridization—Denatures 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.

Sequencing Documentation 9


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

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 DNAPreparing Samples for ChIP Sequencing of DNAPreparing Samples for Paired-End SequencingPreparing Samples for Gene-Expression-Tag Profiling with NlaIIIPreparing Samples for Gene-Expression-Tag Profiling with DpnIIPreparing Samples for Analysis of Small RNAPreparing Samples for Sequencing mRNAPreparing 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 GuideGenome Analyzer II Operations Guide—This 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 StationUsing the Paired-End Cluster Generation Kit v2 on the Cluster Station and Paired-End Module.Using the SBS Sequencing Kit v2 on the Genome Analyzer—This booklet includes instruction for the 18-Cycle SBS Kit and the 36-Cycle SBS Kit.Using the SBS Sequencing Kit v3 on the Genome Analyzer—This 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 1Overview

Part # 1006747 Rev. A

Sequencing Workflow

Sequencing workflow guides contain task-oriented instruction for cluster generation on the Cluster Station and sequencing on the Genome Analyzer.

Sequencing User Guide—This comprehensive guide includes information about single-read sequencing, paired-end sequencing, and sequencing multiplexed samples.Multiplexed Sequencing on the Genome Analyzer—This 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 FormPaired-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 GuideGenome Analyzer Pipeline Software User GuidePipeline to Maq to GBrowse User GuidePipeline to ChIP-Seq User GuideFocalAssist User GuideSCS 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.

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.

Consumables3-amino-1-propanol (> 99%)Tris-Cl 10 mM, pH 8.5

Sequencing ConsumablesImmersion oil, refractive index 1.473 (Cargille, catalog # 19570)Ethanol absoluteMilliQ 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)

12 CHAPTER 1Overview

Part # 1006747 Rev. A


Chapter 2

Cluster Generation on the Cluster Station

Topics14 Cluster Generation Workflow

15 Cluster Generation Protocols and Recipes

18 Preparing DNA Template for Cluster Generation

20 Performing Cluster Amplification

24 Performing Linearization, Blocking, and Primer Hybridization

14 CHAPTER 2Cluster Generation on the Cluster Station

Part # 1006747 Rev. A

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 single-read sequencing and paired-end sequencing.

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.

Prepare Template DNA

Perform Linearization, Blocking, andPrimer Hybridization

on the Cluster Station

Perform Amplificationon the Cluster Station

Sequence the Flow Cellon the Genome Analyzer

within 4 hours

Prepare and Load Reagentson the Cluster Station

Cluster Generation Protocols and Recipes 15


Cluster Generation Protocols and Recipes

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.

One-Step Protocol

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.

Recipe Names 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.

Table 2 Cluster Station Recipes

Protocol Application Recipe Names

Single-Read Standard Genomic DNA SR_Amplification_onlySR_Linearization_Blocking_PrimerHyb

Single-Read One-Step Genomic DNA SR_Amplification_Linearization_Blocking_PrimerHyb

Single-Read Multi-Primer Gene expression and small RNA

SR_Amplification_onlySR_Linearization_Blocking_MultiHyb

Single-Read Multi-Primer One-Step

Gene expression and small RNA

SR_Amplification_Linearization_Blocking_MultiHyb

Paired-End Standard Genomic DNA PE_Amplification_only_v<#>PE_2P_R1prep_Linearization_CombinedBlocking_PrimerHyb

Paired-End One-Step Genomic DNA PE_Amplification_Linearization_CombinedBlocking_PrimerHyb


Part # 1006747 Rev. A

Protocol Times The following table lists the approximate duration of each step in a protocol.

Safe Stopping Points

After the amplification step, a flow cell may be left on the Cluster Station overnight or stored indefinitely at 4°C.

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 4°C for up to one month. Be sure to use a new manifold from a sealed bag to prevent cross-contamination 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 3 Protocol Times

Step in ProtocolStep Duration (Single-Read Recipes)

Step Duration (Paired-End Recipes)

Instrument Wash 15 minutes 15 minutes

Template Hybridization 40 minutes 40 minutes

Amplification 2 hours 25 minutes 2 hours 25 minutes

Linearization 15 minutes 35 minutes

Blocking 55 minutes 55 minutes

Sequencing Primer Hybridization 20 minutes 20 minutes

Instrument Wash 15 minutes 15 minutes

Table 4 Safe Stopping Points

Step in Protocol Single-Read Flow Cell Paired-End Flow Cell

After Amplification Leave overnight or store at 4°C for 3 months

Leave overnight or store at 4°C for 3 months

After Linearization and Blocking Store at 4°C for up to one month Do not store flow cell

After Primer Hybridization Sequence within 4 hours Sequence within 4 hours

Cluster Generation Protocols and Recipes 17


Reagent Volumes

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.

Table 5 Reagent Volumes Per Recipe

Recipe Descriptions

Position/Label

Reagent Amp onlyLin-Block-Primer Hyb

Lin-Block-Multi Hyb

One Step (Standard)

One Step (Multi-Primer)

1 AMX (Amplification Mix) 15 ml 15 ml 15 ml

3 HT2 (Wash Buffer) 10 ml 7 ml (approx.) 7 ml (approx.) 10 ml 10 ml

4 BMX (Blocking Mix) 2 ml 2 ml 2 ml 2 ml

7 Sequencing Primer 1.3 ml 1.3 ml

9 AT1 (Formamide) 8 ml 8 ml 8 ml

11 APM1 (AMX1 Premix) 10 ml 10 ml 10 ml

12 HT1 (Hybridization Buffer) 15 ml 13 ml (approx.)

13 ml (approx.)

15 ml 15 ml

14 LMX1 (Linearization Mix 1)(for paired-end recipes)

1.3 ml 1.3 ml

15 LS1 (Linearization Solution 1)(for single-read recipes)

1.8 ml 1.8 ml 1.8 ml 1.8 ml

17 Diluted HP3 (0.1 N NaOH) 1.5 ml 1.5 ml

A HT1 (Hybridization Buffer) 140 μl/tube 140 μl/tube 140 μl/tube

B Denatured Template DNA 120 μl/tube 120 μl/tube 120 μl/tube

C HT2 (Wash Buffer) 100 μl/tube 100 μl/tube 100 μl/tube

D APM1 (AMX1 Premix) 100 μl/tube 100 μl/tube 100 μl/tube

E EMX (Extension Mix) 120 μl/tube 120 μl/tube 120 μl/tube

F Diluted HP3 (0.1 N NaOH) 100 μl/tube 100 μl/tube

G HT2 (Wash Buffer) 100 μl/tube 100 μl/tube

H Sequencing Primer (specific to your application)

100 μl/tube 100 μl/tube

I HT2 (Wash Buffer) 100 μl/tube 100 μl/tube

J HT1 (Hybridization Buffer) 100 μl/tube 100 μl/tube


Part # 1006747 Rev. A

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-SuppliedTris-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 1–4 pM, leading to a cluster density of approximately 40–180K/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 μl)• Tris-Cl 10 mM, pH 8.5 (17 μl)• HP3 (2 N NaOH) (1 μl)The total volume should be 20 μl (template final concentration 1 nM).

2. Vortex briefly to mix the template solution.

3. Pulse centrifuge the solution.

Preparing DNA Template for Cluster Generation 19


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 μl 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:

2. Invert several times to mix the template solution.

3. Pulse centrifuge the solution.

4. Dispense 120 μl 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 μl 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.

Required Final Concentration

1 pM 2 pM 4 pM 8 pM

1.0 nM Denatured DNA

1 μl 2 μl 4 μl 8 μl

Pre-chilled HT1

999 μl 998 μl 996 μl 992 μl


Part # 1006747 Rev. A

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

1. From the Illumina Cluster Station Software, select File | Open Recipe.

2. Open one of the following recipes and click Run.

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.

Workflow Recipe Name

Single-Read Standard Protocol SR_Amplification_only_v5.0.xml

Single-Read One-Step Protocol SR_Amplification_Linearization_Blocking_PrimerHyb_v5.0.xml

Paired-End Standard Protocol PE_Amplification_only_v5.0.xml

Paired-End One-Step Protocol PE_Amplification_Linearization_CombinedBlocking_PrimerHyb_v5.0.xml

Performing Cluster Amplification 21


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.

Figure 3 Reagent Positions on the Cluster Station

9 10 1811 12 13 14 15 16 17

1 8765432

Eight-Tube Strip Holder

Table 6 Reagent Positions for Amplification

Position Reagent Tube Size

1 AMX1 (Amplification Mix) 50 ml

3 HT2 (Wash Buffer) 15 ml

9 AT1 (Formamide) 50 ml

11 APM1 (AMX1 Premix) 50 ml

12 HT1 (Hybridization Buffer) 50 ml

A HT1 (Hybridization Buffer) 0.2 ml eight-tube strip

B Denatured DNA Template Mix 0.2 ml eight-tube strip

C HT2 (Wash Buffer) 0.2 ml eight-tube strip


Part # 1006747 Rev. A

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 20°C, 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.


10. When prompted, remove tube strip “C” and load the tube strip labeled “D” (AMX1 Premix) into the tube strip holder.


12. When prompted, remove tube strip “D” and load the tube strip labeled “E” (Extension Mix) into the tube strip holder.


14. When prompted, remove the manifold inlets from the tube strip to drain inlets.


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.

D APM1 (AMX1 Premix) 0.2 ml eight-tube strip

E EMX (Extension Mix) 0.2 ml eight-tube strip

Table 6 Reagent Positions for Amplification (Continued)

Position Reagent Tube Size

Performing Cluster Amplification 23



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 4°C until you are ready to proceed.

Table 7 Reagent Volumes for Amplification

Position Reagent Initial Volume Expected Volume After Amplification

1 AMX1 (Amplification Mix) 15 ml 1.8 ml

3 HT2 (Wash Buffer) 10 ml 7 ml

9 AT1 (Formamide) 8 ml 2.6 ml

11 APM1 (AMX1 Premix) 10 ml 1.3 ml

12 HT1 (Hybridization Buffer) 15 ml 12 ml

NOTEAt this point in the protocol, you can store the flow cell at 2° to 8°C or continue to linearization, blocking, and primer hybridization.


Part # 1006747 Rev. A

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.


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.

3. Install the washing bridge and load containers filled with water in the following positions:• Single-read recipe—Load positions 3, 4, 7, 12, 15, and 17.• Paired-end recipe—Load 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 recipe—Load positions 3, 4, 7, 12, 15, and 17.• Paired-end recipe—Load 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 tubes into place. This prevents crimping and twisting of the lines.

CAUTIONDo not linearize, block, and primer hybridize a paired-end flow cell until the day of use.

Workflow Recipe Name

Single-Read Standard Protocol SR_Linearization_Blocking_PrimerHyb_v5.0.xml

Paired-End Standard Protocol PE_2P_R1Prep_Linearization_CombinedBlocking_PrimerHyb_v5.0.xml

Performing Linearization, Blocking, and Primer Hybridization 25


Reagent Positions

The following figure illustrates reagent tube and bottle placement along with the number associated with each position.

Figure 4 Reagent Positions on the Cluster Station

Table 8 Reagent Positions for Linearization, Blocking, and Primer Hybridization

Position Reagent (Standard Protocol) Reagent (Multi-Primer Protocol) Tube Size

3 HT2 (Wash Buffer) HT2 (Wash Buffer) 15 ml

4 BMX (Blocking Mix) BMX (Blocking Mix) 15 ml

7 Sequencing Primer 2 ml

12 HT1 (Hybridization Buffer) HT1 (Hybridization Buffer) 50 ml

14 LMX (Linearization Mix 1)(for paired-end recipe)

2 ml

15 LS1 (Linearization Solution 1)(for single-read recipe)

LS1 (Linearization Solution 1) 2 ml

9

1 8765432

Eight-Tube Strip Holder

10 1811 12 13 14 15 16 17


Part # 1006747 Rev. A

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 recipe—Load positions 3, 4, 7, 12, 15, and 17.• Paired-end recipe—Load 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.

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.

17 Diluted HP3 (0.1 N NaOH) 2 ml

F Diluted HP3 (0.1 N NaOH) 0.2 ml eight-tube strip

G HT2 (Wash Buffer) 0.2 ml eight-tube strip

H Sequencing Primers 0.2 ml eight-tube strip

I HT2 (Wash Buffer) 0.2 ml eight-tube strip

J HT1 (Hybridization Buffer) 0.2 ml eight-tube strip

Table 8 Reagent Positions for Linearization, Blocking, and Primer Hybridization (Continued)

Position Reagent (Standard Protocol) Reagent (Multi-Primer Protocol) Tube Size

CAUTIONDo not store the flow cell at this point for long periods of time. Sequencing must be performed on the flow cell within four hours.

Performing Linearization, Blocking, and Primer Hybridization 27



1. Click OK to resume the recipe.

2. Attach the washing bridge and load containers filled with water in the following positions:• Single-read recipe—Load positions 3, 4, 7, 12, 15, and 17.• Paired-end recipe—Load 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.

Table 9 Reagent Volumes for Linearization, Blocking, and Primer Hybridization

Position Reagent Initial Volume Expected Volume After Lin/Block/PrimerHyb

3 HT2 (Wash Buffer) 7 ml (approx.) 3 ml (approx.)

4 BMX (Blocking Mix) 2 ml 0.53 ml

7 HP1 (Sequencing Primer Mix 1) 1.3 ml 0.35 ml

12 HT1 (Hybridization Buffer) 13 ml (approx.) 10 ml (approx.)

14 LMX1 (Linearization Mix 1)(for paired-end recipe)

1.3 ml 0.3 ml

15 LS1 (Linearization Solution 1)(for single-read recipe)

1.8 ml 0.3 ml

17 Diluted HP3 (0.1 N NaOH) 1.5 ml 0.46 ml

NOTE

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.


Part # 1006747 Rev. A


Chapter 3

Sequencing on the Genome Analyzer

Topics30 Sequencing Workflows

31 Sequencing Protocols and Recipes

33 Performing a Pre-Run Wash

35 Loading Reagents on the Genome Analyzer

38 Priming Reagents

39 Continuing the Run

42 Preparing for Read 2 Using the Paired-End Module

46 Sequencing Read 2 on the Genome Analyzer

49 Performing a Post-Run Wash

30 CHAPTER 3Sequencing on the Genome Analyzer

Part # 1006747 Rev. A

Sequencing Workflows

This chapter describes the workflow for single-read sequencing and paired-end 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.

Figure 5 Workflow Diagram for Single-Read and Paired-End Sequencing

Perform Pre-Run Instrument Wash

Weigh Reagents and PerformPost-Run Instrument Wash

Clean and Install PrismClean and Install Flow Cell

Apply OilFirst-Base Incorporation

Load Flow Cell with Scan MixAdjust Focus

Check Quality Metrics

See Genome Analyzer Operations Guide:

Continue the Sequencing Run

Read 2 PreparationUsing the Paired-End Module

Sequence Read 2

For Two-Folder Paired-End Method,Transfer Data Before Read 2

For Long Runs, Prepare toReplace Reagents andReset Reagent Tracking

Prepare and Load Reagentson the Genome Analyzer

See Using the SBS Sequencing Kiton the Genome Analyzer

See Using the Paired-End Cluster Generation Kiton the Cluster Station and Paired-End Module:

Prepare and Load Reagentson the Paired-End Module

Prepare and Load Reagentson the Genome Analyzer

See Using the SBS Sequencing Kiton the Genome Analyzer

Single-Read and Paired-End Sequencingon the Genome Analyzer

Paired-End Sequencingon the Genome Analyzerand Paired-End Module

For Long Runs, Prepare toReplace Reagents andReset Reagent Tracking

For single reads For paired ends

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 48–72 hours.

Single-read sequencing requires one 36-Cycle SBS Sequencing Kit. Paired-end sequencing requires two 36-Cycle SBS Sequencing Kits and the Paired-End 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.

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.

Number of Cycles and Reads36-Cycle SBS Sequencing Kit v3

18-Cycle SBS Sequencing Kit v3

51-Cycle single-read runs 1 1

51-Cycle paired-end runs 3

76-Cycle single-read runs 2

76-Cycle paired-end runs 4


Part # 1006747 Rev. A

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 Single-Read SequencingPaired-End SequencingTwo-Folder Method

Paired-End SequencingSingle-Folder Method

Pre-Run Wash GA2_PreWash_<v#> GA2-PEM_PreWash_<v#> GA2-PEM_PreWash_<v#>

Prime Reagents on the Genome Analyzer

GA2_Prime_<v#> GA2-PEM_Prime_<v#> GA2-PEM_Prime_<v#>

First-Base Incorporation GA1_FirstBase_<v#> GA2_FirstBase_<v#>

36-Cycle Sequencing GA2_36Cycle_SR_<v#> GA2_36Cycle_PE_<v#> GA2-PEM_2x36Cycle_<v#>



Prime Reagents on the Paired-End Module

PEM_R2Prime_<v#>

Prepare for Read 2 on the Paired-End Module

PEM_R2Prep_<v#>

First-Base Incorporation GA2_FirstBase_<v#>

36-Cycle Read 2 GA2_36Cycle_PE_v<#>



Post-Run Wash GA2_PostWash_<v#> GA2-PEM_PostWash_<v#>

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 paired-end 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-SuppliedPW1

User-SuppliedLens cleaning tissue(4) 125 ml Nalgene bottles (ThermoFisher Scientific, catalog # 2019-0125)(3) 50 ml conical tubesMilliQ water or laboratory grade water (for washing the Paired-End Module)

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: PumpTo: Flow cellSolution: 8 (single-read sequencing); 28 (paired-end sequencing)Volume: 0 Aspiration Rate: 250Dispense Rate: 2500With 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

CAUTIONRotate the tubes while holding the caps stationary, to prevent crimps and twisting in the liquid delivery lines.


Part # 1006747 Rev. A

5. [Paired-End Module] Place at least 5 ml of laboratory grade water in each Falcon tube in positions 9–21 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.

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.

Recipe Name Total Run TimeTotal Volume Expected

GA2_PreWash_v<#>.xml 15 minutes 7 ml

GA2-PEM_PreWash_v<#>.xml 40 minutes 21 ml

NOTE

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.

Loading Reagents on the Genome Analyzer 35


Loading Reagents on the Genome Analyzer

Always perform a pre-run wash before loading reagents onto the Genome Analyzer.

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 4°C 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.

CAUTION

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).

Table 11 Reagent Positions on the Genome Analyzer

Solution # Size Contents

1 50 ml Amber Tube IMX

2 125 ml Bottle PW1

3 50 ml Amber Tube SMX

4 125 ml Bottle PR1

5 125 ml Bottle PR2

6 50 ml Amber Tube CMX or CLM

7 125 ml Bottle PR3


Part # 1006747 Rev. A

Figure 6 Reagent Positions on the Genome Analyzer

Track Reagents 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.

Figure 7 Current Reagent Volumes

2. Click Reset Volumes. The Reset Reagent Volumes dialog box appears.

Figure 8 Reset Reagent Volumes

4 5 2 7 1 6 3

Loading Reagents on the Genome Analyzer 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 num-ber 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.


Part # 1006747 Rev. A

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.

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.


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: PumpTo: Flow cellSolution: 8 (single-read sequencing); 28 (paired-end sequencing)Volume: 0Aspiration Rate: 250Dispense 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.

NOTEPriming volumes are a key indicator of a stable fluid delivery system. The measured volumes must be within 10% of normal for optimal sequencing performance.

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.

1. Depending on the sequencing method you are using, open one of the following recipes or resume one of the single-folder recipes:

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.

NOTE

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 μl of Scan Mix must be pumped prior to resuming the run.

Single-Read SequencingPaired-End SequencingTwo-Folder Method

Paired-End SequencingSingle-Folder Method

GA2_36Cycle_SR_<v#> GA2_36Cycle_PE_<v#> GA2-PEM_2x36Cycle_<v#>



NOTE

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.


Part # 1006747 Rev. A

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.

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. IMXb. PR1c. PR2d. PR3e. 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.

Sequencing Method 51-Cycle Run 76-Cycle Run

Single-Read Run Cycle 34–37 Cycle 34–37

Paired-End Run, Read 1 (single-folder method or two-folder method)

Cycle 34–37 Cycle 34–37

Paired-End Run, Read 2 (single-folder method)

Cycle 70–74 Cycle 77 and Cycle 112–116

Paired-End Run, Read 2 (two-folder method)

Cycle 20–24 Cycle 1 and Cycle 34–37

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.

Continuing the Run 41


Reset Reagent Volumes

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.

Weigh Reagents 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.

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.

4. Proceed to Preparing for Read 2 Using the Paired-End Module on page 42.

NOTEIllumina recommends that you use the RoboCopy script rather than manually copying files.

CAUTIONDo 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.


Part # 1006747 Rev. A

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 tubes into place. This prevents crimping and twisting of the lines.

Load Reagents Reagent Positions

The following figure illustrates the reagent positions on the Paired-End Module and the number associated with each position.

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.

21

16

15

10

13

19

12

11

14

Preparing for Read 2 Using the Paired-End Module 43


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.

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.

Table 12 Reagent Positions on the Paired-End Module

Position Reagent

10 RMX (Resynthesis Mix)

11 LMX2 (Linearization Mix 2)

12 BMX (Blocking Mix)

13 AMX2 (Amplification Mix 2)

14 APM2 (AMX2 Premix)

15 AT2 (Formamide)

16 HP2 (Sequencing Primer Mix 2)

19 Diluted HP3 (0.1 N NaOH)

21 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.

CAUTIONWhen you prepare and load these reagents onto the Paired-End Module, you must use them the same day.


Part # 1006747 Rev. A

Two-Folder Method

1. Open the PEM_R2Prime_v5.0.xml recipe.

2. Click OK.The following prompt appears when priming is complete: “Priming com-plete. 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.

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.

CAUTION

Waste produced during Read 2 preparation on the Paired-End 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.

Preparing for Read 2 Using the Paired-End Module 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 Reagent Initial Volume Expected Volume After Priming

Expected Volume After Read 2 Preparation

10 RMX (Resynthesis Mix) 2 ml 1.4 ml 0.74 ml

11 LMX2 (Linearization Mix 2) 2 ml 1.4 ml 0.96 ml

12 BMX (Blocking Mix) 2 ml 1.4 ml 0.2 ml

13 AMX2 (Amplification Mix 2) 9 ml 8.4 ml 0.66 ml

14 APM2 (AMX2 Premix) 5 ml 4.25 ml 1.2 ml

15 AT2 (Formamide) 8 ml 7.25 ml 4.2 ml

16 HP2 (Sequencing Primer Mix 2) 1.5 ml 0.75 ml 0.19 ml

19 Diluted HP3 (0.1 N NaOH) 3 ml 2.25 ml 1.1 ml

21 HT2 (Wash Buffer) 10 ml 9.25 ml 2.7 ml


Part # 1006747 Rev. A

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.


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.

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: PumpTo: Flow cellSolution: 3Volume: 100Aspiration Rate: 250Dispense 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.

CAUTION

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

Waste produced during Read 2 preparation on the Paired-End 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.

CAUTIONDo not reprime reagents through the flow cell.

Sequencing Read 2 on the Genome Analyzer 47



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.

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.

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.

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: PumpTo: Flow cellSolution: 3Volume: 100Aspiration Rate: 250Dispense 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.

CAUTIONDo not reprime reagents through the flow cell.

NOTE

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.

CAUTIONIt is critical to introduce Scan Mix to the flow cell before adjusting the focal plane.


Part # 1006747 Rev. A

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


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.

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 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.

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 paired-end recipe is approximately 60 minutes.

Consumables Illumina-SuppliedPW1

User-SuppliedLens cleaning tissue(4) 125 ml Nalgene bottles (ThermoFisher Scientific, catalog # 2019-0125)(3) 50 ml conical tubes MilliQ water or laboratory grade water (for washing the Paired-End Module)

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 9–21 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.


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.

CAUTIONUsing 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.


Part # 1006747 Rev. A


Index

Ccluster densities 18cluster generation 5

linearization, blocking, primer hy-bridization 24

preparing template 18protocol times 16recipes 15workflow 14

Cluster Stationoverview 5stopping safely 16

consumables, user-supplied 11cross-contamination 20, 24, 35customer support 3

Ddata transfer, paired-end runs 41documentation 3, 9

Fflow cells

overview 4paired-end 6storage 16

GGenome Analyzer

overview (system) 4, 5Pipeline software 5

Hhelp

documentation 9technical 3

Llab tracking forms 10

loading reagentsCluster Station 20, 24Genome Analyzer 35Paired-End Module 42

Mmaterial safety data sheet (MSDS) 3

Oone-step protocols 15

Ppaired-end

data transfer 41flow cell 6overview 6protocols 31Read 2 preparation 44recipes 32single-folder method 46two-folder method 47workflow 7

Paired-End Module 5Pipeline 5, 10post-run wash 49pre-run wash 33priming Genome Analyzer reagents 38protocols, cluster generation

multi-primer 15one-step 15protocol times 16standard 15

protocols, sequencing 31

Rreagents, Cluster Station

loading 20, 24positions for amplification 21positions for linearization, blocking,

and primer hybridization 25

52 Index

Part # 1006747 Rev. A

volumes for amplification 22volumes for linearization, blocking,

and primer hybridization 26volumes per recipe 17

reagents, Genome Analyzerbarcodes 36loading 35positions 35priming 38replacing during long runs 40reset volumes 36, 41tracking 36weighing 41

reagents, Paired-End Moduleloading 42positions 42priming 43volumes for Read 2 preparation 45

recipescluster generation 15sequencing 32

RoboCopy 32, 41

Ssample preparation 4scheduling, protocol times 16sequencing

protocols 31recipes 32workflow 30

sequencing by synthesis (SBS) 4single-folder method 31, 46software

Pipeline 5stopping points, Cluster Station 16

Ttechnical assistance 3tips

avoiding cross-contamination on Genome Analyzer 35

loading reagents safely 20, 24, 35, 42

two-folder method 31, 47

Wwashes

post-run wash 49pre-run wash 33

workflowscluster generation 14sequencing 30

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]

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Illumina Genome Analyzer Maintenance Guide

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