Illumina Experiment Manager User Guide 15031335 J

Illumina Experiment Manager User Guide 15031335 J
Illumina Experiment Manager
User Guide
FOR RESEARCH USE ONLY
Revision History
What is Illumina Experiment Manager?
Getting Started
Creating a Sample Plate
Creating a Sample Sheet
Technical Assistance
ILLUMINA PROPRIETARY
Part # 15031335 Rev. J
March 2015
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Part #
Revision
Date
15031335
J
March 2015
Updated software descriptions to IEM v1.9.
Added:
• NeoPrep Library Prep System sample sheet
• TruSight HLA application for MiSeq
• HiSeq 3000 and 4000 sequencing system
Removed unnecessary checkboxes for NextSeq custom
primers
Nextera XT v2 sample sheets can be created for 384 samples
in a single sheet.
15031335
H
June 2014
Updated software descriptions to IEM v1.8.
Added TruSeq Synthetic Long-Read DNA as a sample prep
kit option to HiSeq FASTQ Only application run settings
Illumina Experiment Manager User Guide
Description of Change
3
Revision History
Revision History
Part #
Revision
Date
15031335
G
April 2014
4
Description of Change
Updated software descriptions to IEM v1.7.
The following changes were made to the software:
• Added NextSeq instrument option for use with the
NextSeq FASTQ application
• Added Nextera XT v2 Set A, B, C, and D as sample prep kit
options
• Removed the wording, "Recommended for cancer
samples" that appears next to the Use Somatic Variant
Caller checkbox
• Added the option to save and edit CASAVA sample sheets
for the HiSeq to use with CASAVA or bcl2fastq v1.8.4
software. This functionality was removed in v1.5 and v1.6
• Underscores can now be used in the text you enter in
sample sheets
• Added an export to gVCF option in the workflow-specific
settings for PCR Amplicon, TruSeq Amplicon, and
Enrichment
• Added a genus-level classification option to Metagenomics
workflow-specific settings
• Added the TruSight Enrichment sample prep kit to HiSeq
Enrichment application run settings
• Cycle counts are no longer reset to the default of 2 x 150
when you edit an existing sample sheet.
• IEM now supports samples with different reference
genomes within the same sample sheet
• IEM no longer checks the base pair diversity in the indexes
specified for MiSeq Targeted Resequencing in the TruSeq
Amplicon workflow. MiSeq RTA v1.17.28 and higher
processes low-plexity index reads for the TruSeq Amplicon
workflow, which makes base pair diversity checks in IEM
unnecessary.
• Index 517 is now an option for the Nextera, Nextera XT,
and Nextera Rapid Capture Enrichment sample prep kit.
The following changes were made to the user guide:
• Added sample sheet run settings parameters for use with
CASAVA.
• Added supported Sample Prep Kit information for
CASAVA.
• Added a description of the Export to gVCF option in
workflow-specific settings for the MiSeq
• Added a description of the Genus-level Classification
option to workflow-specific settings for the MiSeq
• Added TruSight Enrichment to the list of sample prep kits
supported for the HiSeq Enrichment application
• Added information on downloading target and probe
manifest files for Nextera Rapid Capture Custom
Enrichment
Part # 15031335 Rev. J
Revision
Date
15031335
F
May 2013
Illumina Experiment Manager User Guide
Description of Change
Updated software descriptions to IEM v1.6.
The following changes were made to the software:
• Added the Amplicon - DS workflow option to the MiSeq
Targeted Resequencing category
• When creating a sample sheet, IEM automatically checks
that all samples with the same manifest also have the same
reference genome. Otherwise, the software displays an
error message if this is not the case
• Improved the error messages provided when attempting
to edit an invalid sample sheet
• The Genome Folder and Manifest columns for Applications
that require the fields to be populated were moved to the
left of the Sample Project and Description columns for
increased usability
• IEM now generates HiSeq sample sheets that are in the
correct format for use with the HiSeq Analysis Software
analysis package. These sample sheets are fully compatible
with HCS 2.0
• Corrected the indexes available for use with the Nextera
Mate Pair kit
• For some analysis options under the MiSeq and HiSeq
Enrichment categories, additional kits were incorrectly
listed. These kits could not actually be analyzed with the
currently available downstream MiSeq and HiSeq analysis
software and these kit names were removed
• For the MiSeq TruSeq Custom Amplicon analysis, the
workflow value listed in the resulting sample sheet is now
set to Custom Amplicon. Previously it was Amplicon
The following changes were made to the user guide:
• Added the Amplicon - DS workflow description to the
MiSeq Applications and Sample Prep Kits section
• Added the Amplicon - DS workflow as an option in the
Targeted Resequencing section
5
Revision History
Part #
Part #
Revision
Date
15031335
E
March 2013
6
Description of Change
Updated software descriptions to IEM v1.5.
The following changes were made to the software:
• Creating sample sheets for use with CASAVA or AVC is
no longer supported
• Manifests are no longer created as part of Create Sample
Plate
• Manifests must be Illumina provided or created using the
Create Sample Sheet function, depending on the workflow
• Removed Project Name from the Sample Sheet Workflow
Parameters
• Added Nextera Mate Pair, Nextera Rapid Capture,
Targeted RNA Expression, and TruSight Enrichment to
sample prep kit types
• Removed TruSeq Amplicon - Cancer Panel from the
TruSeq Amplicon application for the MiSeq sample sheet
• Added Nextera Rapid Capture and TruSight Enrichment to
the Enrichment and FASTQ Only applications for the MiSeq
sample sheet
• Added Nextera Mate Pair to the Resequencing, Assembly,
Library QC, and FASTQ Only applications for the MiSeq
sample sheet
• Added TruSeq Amplicon to the FASTQ Only application for
the MiSeq sample sheet
• Added a Targeted RNA application to the RNA Sequencing
Category for the MiSeq sample sheet and added Targeted
RNA Expression to the application
• Added Indel Realignment GATK, Run Picard Hs Metrics,
Reverse Compliment, and K-mer size workflow-specific
settings
• FASTA reference files are no longer required for the PCR
Amplicon and Enrichment applications for MiSeq sample
sheets
• Added the HiSeq 2500 label to the Create Sample Sheet
function
• Renamed the Workflow Selection screen to Application
Selection
• Added applications to the Create Sample Sheet function for
HiSeq, HiScanSQ, and Genome Analyzer™
• Removed Sample Sheet Name, Flowcell ID, Operator, and
Recipe fields from the HiSeq, HiScanSQ, and Genome
Analyzer sample sheet run settings parameters.
• Added Reagent Cartridge Barcode, Experiment Name,
Investigator Name, Description, Date, Read Type, and
Cycles Read as well as workflow-specific settings to the
HiSeq, HiScanSQ, and Genome Analyzer sample sheet run
settings parameters.
• TruSeq Small RNA Sample Preparation, TruSeq Custom
Amplicon, and TruSeq Amplicon - Cancer Panel Library
Preparation sample sheets can now be created for HiSeq,
HiScanSQ, and Genome Analyzer.
Part # 15031335 Rev. J
Revision
Date
15031335
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(continued)
March 2013
15031335
D
January 2013
Illumina Experiment Manager User Guide
Description of Change
The following changes were made to the user guide:
• Added instructions for downloading Illumina provided
manifests in Getting Started
• Moved the MiSeq Applications and Sample Prep Kits
section into the Creating a Sample Sheet section
• Added a HiSeq, HiScanSQ, and Genome Analyzer
Applications and Sample Prep Kits section into the
Creating a Sample Sheet section
Updated software descriptions to IEM v1.4.
The following changes were made to the software:
• Added Variant Filter Quality cutoff parameter to
Resequencing, PCR Amplicon, Custom Amplicon, and
Enrichment applications
• Added Flag PCR Duplicates option to Resequencing,
Library QC, PCR Amplicon, and Enrichment workflows
• Added Use Somatic Variant Caller option to Resequencing,
PCR Amplicon, and Enrichment workflows
The following changes were made to the user guide:
• Added Resequencing, PCR Amplicon, and Enrichment
workflows to the Use Somatic Variant Caller description as
options.
• Added Flag PCR Duplicates description in the Creating a
MiSeq-Compatible Sample Sheet section.
Added the following new sections:
• Creating a Custom Sample Prep Kit Type
• Creating a Custom Sample Prep Kit
7
Revision History
Part #
Part #
Revision
Date
15031335
C
September
2012
8
Description of Change
Updated software descriptions to IEM v1.3.
The following changes were made to the software:
• Added the Nextera Enrichment to kit options
• Modified TruSeq DNA v2 and TruSeq RNA v2 sample prep
kit assay and workflow options to be called TruSeq LT
• Enrichment application now uses GenerateFASTQ
workflow
• Displays the index sequence in the sample sheet sample
table (read only) and outputs it to the sample sheet
• Changed order of sample sheet columns
• When creating more than one manifest per sample sheet,
all samples no longer need to be aligned to the same
reference genome.
The following user interface changes were made:
• Reset default installer option to be Just Me rather than All
Users
• Changed Application to Category and Workflow to
Application in the MiSeq Workflow Selection screen
• Changed name of TruSeq DNA v2 and TruSeq RNA v2 kit
options to TruSeq LT
• Prompt to select Assay changed to select Sample Prep Kit
Type
• Changed Custom Amplicon to TruSeq Amplicon in the
workflow select panel
• Changed Custom Enrichment to Enrichment in the
workflow select panel
• Enrichment now maps to the PCR Amplicon workflow
• Renamed Amplicon Manifest column to Nextera Manifest
in the plate editor
• Print function added to Plate Samples screen
• Select All, Add Blank Row and Remove Selected Rows
functions added to Sample Selection screen
• Genome Folder now selected from a drop-down menu
when creating a sample sheet
The following changes were made to the user guide:
• Removed kit-specific sections and created a quick reference
card for each user interface workflow. The cards provide
specific options and settings.
• The guide now provides detailed general information on
how to use the IEM application and definitions of the
sample sheet applications.
Part # 15031335 Rev. J
Revision
Date
15031335
B
June 2012
Updated software descriptions to IEM v1.2
Added the following sample and library preparation kits to
assay and workflow options:
• Nextera XT Library Preparation Kits
• TruSeq Amplicon - Cancer Panel Preparation Kits
Added the following new procedures and sections:
• Genome Repository Preferences
• Workflows and Sample Prep Kits table listing all the
workflows, the associated sample prep kits, and the
analysis workflow written to the sample sheet.
Updated the following information:
• Support for the new MiSeq Reporter FASTQ Only
workflow.
• Option to specify the use of custom primers and associated
ports.
15031335
A
December
2011
Initial release
Illumina Experiment Manager User Guide
Description of Change
9
Revision History
Part #
The Illumina® Experiment Manager (IEM) software enables you to create and edit wellformed sample sheets for Illumina instruments and analysis software.
Create your sample sheet before starting sample or library preparation. IEM can detect
and warn you if an index combination specified is suboptimal. By creating the sample
sheet before sample or library preparation, you can try a different index combination
without risking your samples.
There are two steps to creating a sample sheet with the IEM for Illumina sequencing
systems:
} First, you create a sample plate. The sample plate stores information about the
samples in each well of a 96-well (or fewer) plate, including: the type of sample prep
that is performed, the plate name, and sample indexes.
} Second, you create a sample sheet based on the information previously defined on
the sample plate. The sample sheet passes run and sample information in the correct
file format (*.csv) to the instrument control and analysis software.
Creating a sample sheet for the NeoPrep™ Library Prep System makes sure that the
sample sheet passes run and sample information in the correct file format (*.csv) to the
NeoPrep Control Software.
This guide describes detailed general information on how to use the IEM application. For
instructions specific to the sample or library preparation kit you are using, see the IEM
quick reference cards.
NOTE
You can download the IEM quick reference cards from the Illumina website at
www.illumina.com. Go to the Illumina Experiment Manager support page or the support
page for your kit and select the Documentation & Literature tab.
Illumina Experiment Manager User Guide
10
What is Illumina Experiment Manager?
What is Illumina Experiment Manager?
Getting Started
Downloading the Software
You can download the IEM software from the Illumina website at
support.illumina.com/sequencing/sequencing_software/experiment_manager.ilmn. From
the Experiment Manager Support page, select Downloads. A MyIllumina account is
required.
Installing the Software
The IEM software can be run on any Windows platform. To start the installation, doubleselect the Setup.exe file. The installation wizard opens. Click Next to accept all default
settings.
Starting the Software
To start the software, go to Start | All Programs | Illumina | Illumina Experiment
Manager, or select the IEM icon on your desktop.
Setting your Preferences
You can specify where your sample plates and sample sheets are saved by selecting
Folder Settings on the main screen of the IEM software. Click Browse to navigate to the
desired folders for each type of file, and then select OK.
TIP
To make sure that the files are accessible from the sequencing instrument in your lab, save
the files in a shared network folder. Alternatively sample sheets and manifests can be
copied to a USB drive on an individual basis.
Downloading Manifests
Illumina provides manifests for sample prep kits that use the TruSeq® Amplicon,
Enrichment, or Targeted RNA applications. Download the manifests from the Illumina
website at www.illumina.com.
} For sample prep kits that use the TruSeq Amplicon or Enrichment applications, go to
the support page for the sample prep kit that you are using, and then select
Downloads. Save manifests in the IEM Manifest Repository specified in the Folder
Settings.
} For sample prep kits that use the Targeted RNA application, log in to your
MyIllumina account. Follow the links in the Custom Products tab to access the
Product Files for TruSeq Targeted RNA Expression. To download a manifest, select
Download next to the file name on the Product Files page. If you ordered an add-on
project, the two manifest files created for the project must be merged using the Merge
Files function on the Product Files page. Save manifests in the IEM Manifest
Repository specified in the Folder Settings.
} For Nextera® Rapid Capture Custom Enrichment, download target and probe
manifest files from the Export Manifest function in the Project Dashboard of your
DesignStudio project and the Product Files section of Custom Products in your
MyIllumina account.
11
Part # 15031335 Rev. J
The IEM requires a reference genome for the creation of a sample sheet, particularly for
runs that are analyzed using the PCR Amplicon workflow. When the IEM is installed, a
series of folders mimicking the genomes folder structure on the MiSeq® are created on
your computer. The folder structure that is created is a copy of the latest genome build.
Download the latest genomes from MyIllumina. To save space on the computer that the
IEM is installed on, these folders are blank except for a genomesize.xml file. The
genomesize.xml file provides the IEM with the genome information necessary for the
creation of the sample sheet.
If you are using a custom reference genome not included in the default IEM installed
folder structure, follow these steps:
1
Copy your custom genome to the local directory that the IEM is using.
IEM recognizes the directory structure of the custom genome folder when it is the
following: The path{repository}/genome_name exists.
At least one FASTA file is required in the folder.
a If your custom genome has a genomesize.xml file, you can delete the *.fa files in
the folder.
b If your custom genome does NOT have a genomesize.xml file, the IEM creates
one the first time a PCR Amplicon manifest is created. After the genomesize.xml
file is created, you can delete the *.fa files in the folder.
NOTE
If the genome is ever edited, repeat step b.
Loading a Custom Genome
To load a custom genome into the Genome Repository folder, follow these steps:
1
Make that the genome is in a FASTA format.
2
Place the FASTA file in a folder labeled with the 'genome' name inside the genome
folder in the genome repository.
3
Close and reopen the IEM.
4
Create a manifest file and select the new genome.
5
When asked if you want to convert the file to an xml file, select Yes.
Creating a Custom Sample Prep Kit Type
WARNING
Any non-Illumina sample prep kits are required to meet the specified parameters (e.g.
Index Read number and Index Read length) for the chosen application. If these parameters
do not match, there is a risk of an issue with the recipe when setting up the run. For
example, when using the Default recipe file, there is only a single Index Read supported.
Illumina is unable to provide guidance or support for sample prep kits that Illumina does
not make, or for custom recipes on Illumina sequencing systems.
1
Go to your Program Files folder and find the Illumina\Illumina Experiment
Manager\SamplePrepKits folder.
2
Copy an existing sample prep kit *.txt file and rename the file a unique name for the
custom sample prep kit.
3
Open the new *.txt file in a text editor.
Illumina Experiment Manager User Guide
12
Getting Started
Genome Repository Preferences
4
Under the [Name] section, rename the entry the same unique name you named the
file.
5
Update the sequences accordingly.
6
Save and close *.txt file. If you have the IEM open, close it and reopen the IEM.
7
After you reopen the IEM and select Create Sample Plate your new custom Sample
Prep Kit is a new option.
Creating a Custom Sample Prep Kit
NOTE
Create a sample prep kit type before you create a custom Sample Prep Kit. Only new
sample prep kit types added to the Illumina\Illumina Experiment
Manager\SamplePrepKits folder can be added to existing Applications.
13
1
Go to your Program Files folder and find the Illumina\Illumina Experiment
Manager\Applications folder.
2
Copy an existing Sample Prep Kit .txt file and rename the file a unique name for the
custom sample prep kit.
3
Open the existing Application .txt file of where you want your custom sample prep
kit to display.
4
Under the [Compatible Sample Prep Kits] section, add the file name of a new
custom Sample Prep Kit Type you have already created.
5
Save and close .txt file. If you have the IEM open close and relaunch the IEM.
6
After you reopen the IEM, your new custom Sample Prep Kit is an option in the
Sample Prep Kit drop-down list of the selected Application.
Part # 15031335 Rev. J
NOTE
These instructions describe the creation of a new sample plate. You can also adapt them to
edit an existing sample plate by selecting Edit Sample Plate and navigating to the sample
plate you want to edit.
1
On the main screen, select Create Sample Plate.
2
On the Sample Prep Kit Selection screen, select the appropriate kit, and then select
Next.
3
On the Assay Parameters screen, do the following:
a In the Unique Plate Name field, type a name for the sample plate.
b In the Index Reads field, select the number of indexes you run for the samples
on this plate: 0, 1, or 2 (if applicable).
c Click Next.
4
On the Plate Samples screen, select the Table or the Plate tab, depending on your
preference. You can enter information for the wells in your plate using either view.
• Table tab displays the wells on a 96-well plate in a list, identifiable by the
column and row (A01, A02... B01, B02... etc.) All information for a well is visible
at a time, horizontally across the row: Sample Name, Sample ID, Index 1 (if
applicable), Index 2 (if applicable), Sample Project, and Description.
• Plate tab mimics the layout of a 96-well plate, with wells laid out in columns AH and rows 1–12. Only 1 type of information for a well is visible at a time, as
determined by your selection in the Currently Displaying drop-down menu.
5
For each well containing sample, do the following:
a Enter a unique sample ID.
This sample ID is used to track the sample from preparation through sequencing
and analysis. The ID is often a barcode, but any value is acceptable.
Illumina Experiment Manager User Guide
14
Creating a Sample Plate
Creating a Sample Plate
TIP
Use the IEM Fill feature to populate any type of data for the wells in your sample sheet
quickly. The Fill feature can copy data as-is from cell to cell or can automatically increase
numerical data by 1 to create sequential data. The IEM determines which method is
appropriate based on what type of data are in the cell or cells being copied.
• To generate sequential data, enter sequential data in two adjacent wells, then highlight
the other cells you want to populate. Right-select the highlighted area and select Fill
Down or Fill Right. The highlighted cells are numbered in sequential order.
• To copy data, populate 1 cell, then highlight, right-select, and fill the other cells you
want to populate with the same information.
• Content can be pasted into the Sample ID column directly from an excel table.
b
c
6
15
If you selected 1 or 2 index reads on the Assay Parameters screen, specify what
index adapter you use for each index read.
Use combinations that result in at least 1 A or C base (red) and at least 1 G or T
base (green) at every cycle.
After entering a sample ID and index adapter, the gray shading is removed from
the well, indicating that there is enough information for the well to populate a
sample sheet.
— Illumina provides a recommended default index layout. Click Apply Default
Index Layout to auto-populate the indexes for all index reads for 96 wells.
After the index adapters are populated, you can edit them if you want to try
a different combination of adapters.
— If you want to use your modified index layout in the future, select Save As
Default Index Layout. The next time you create a sample plate, select Apply
Default Index Layout to use your version.
— You can always restore the Illumina layout as the default later by selecting
Restore Illumina Default Index Layout.
If you want to capture more detailed information about the plate, enter a sample
name, project, description.
[Optional] Click the Plate Graphic tab to view the sample ID and index adapter in
each well.
Part # 15031335 Rev. J
b
7
If you want to copy an image of your sample plate for use in a presentation or
paper, go the Plate Graphics tab and select Copy to Clipboard.
You can paste the image into any graphics-enabled program, such as Paint,
Microsoft PowerPoint, Microsoft Word, and Adobe Photoshop.
[Optional] If you want to print an image of your sample plate, go the Plate
Graphics tab, and select Print....
Click Finish and save the sample plate file in the desired folder.
WARNING
You can use any file naming convention that makes sense for your organization, but
the file must contain the default *.plt file extension. If you use a different file
extension, the IEM does not recognize the sample plate when it comes time to
generate a sample sheet.
For the default file extension specific to the sample or library preparation kit you are
using, see the IEM appropriate quick reference card for your kit.
Illumina Experiment Manager User Guide
16
Creating a Sample Plate
a
Creating a Sample Sheet
A sample sheet can be generated for the following types of instruments:
}
}
}
}
}
}
MiSeq
HiSeq®
HiScanSQ™
Genome Analyzer
NextSeq in standalone mode with the bcl2fastq2 software package
NeoPrep
The sample sheet can be used to set up secondary analysis. For a HiSeq run, the loaded
samples are specified for each lane. The analysis workflows available in IEM vary by
sequencing system.
} For analysis of MiSeq sequencing data, see MiSeq-Compatible Sample Sheets on page
17.
} For analysis of HiSeq, HiScanSQ, or Genome Analyzer sequencing data, see HiSeq-,
HiScanSQ-, or Genome Analyzer-Compatible Sample Sheets on page 28.
} For analysis of NextSeq sequencing data, see NextSeq-Compatible Sample Sheets on
page 33.
The sample sheet can be used to support a NeoPrep library prep run. See NeoPrepCompatible Sample Sheets on page 36.
MiSeq-Compatible Sample Sheets
This section provides instructions for creating a sample sheet for the analysis of MiSeq
sequencing data.
MiSeq Applications and Sample Prep Kits
To process a sequencing run fully on the MiSeq, MiSeq Reporter requires that an analysis
application is specified in the sample sheet.
17
Part # 15031335 Rev. J
Creating a Sample Sheet
The following categories and their applications are supported:
Category
Application
Description
Targeted
Resequencing
TruSeq
Amplicon
RNA expression analysis by sequencing and
quantification of cDNA amplicons prepared using a
TruSeq Targeted RNA Expression kit. A manifest file
from DesignStudio is required for alignment. Reads
are aligned against one or more manifest files
specified in the sample sheet.
PCR
Amplicon
Sequencing of PCR amplicon samples prepared
using the Nextera XT Sample Prep kit. The PCR
amplicons are generated from primers targeting
particular genome positions (up to approximately
384 loci from up to 96 samples). Reads are aligned
against the specified reference genome. A usergenerated manifest file (specified in the sample
sheet) gives the targeted regions; variant calling is
performed only within these regions, and coverage
statistics are reported for the target regions.
Metagenomics
16s rRNA
Sequencing of genetic material from uncultured
samples. No genomic reference is required for a
Metagenomics workflow. Reads are classified using
a database of 16S rRNA data included with the
MiSeq Reporter software.
Enrichment
Sequencing of DNA obtained from Nextera (hybrid
capture-based) enrichment. Reads are aligned
against the specified reference genome. An Illuminaprovided manifest file (specified in the sample sheet)
gives the targeted regions; variant calling is
performed only within these regions, and coverage
statistics are reported for the target regions.
Clone
Checking
Verification of clone production by sequencing
typically done with custom primers. A pseudogene
(in FASTA format) representing the clone of interest
is required for mapping and alignment.
Amplicon DS
Small
Genome
Sequencing
Designed explicitly for the TruSight® Tumor assay, the
Amplicon - DS workflow is uniquely suited for
detection of somatic mutations in formalin-fixed
paraffin-embedded (FFPE) samples. This workflow
independently processes variants from the forward
and reverse strands of the sample material, and then
algorithmically reconciles the calls.
Resequencing
Sequencing of a small genome, such as E. coli. Reads
are aligned against the reference and variants are
reported.
Plasmids
Sequencing of entire plasmid DNA. A reference
plasmid genome is required for mapping and
alignment. Output files are *.bam and *.vcf.
Assembly
Assembly of small (< 20 Mb) genomes from reads
without the use of a genomic reference. If a genomic
reference is included, a dot-plot is generated with
regarding the reference.
Illumina Experiment Manager User Guide
18
Category
Application
Description
RNA
Sequencing
Targeted
RNA
Sequencing of TruSeq Targeted RNA Expression
libraries. A target reference is needed as well as a
manifest file from DesignStudio. Reads are aligned
against one or more manifest files specified in the
sample sheet.
Small RNA
Sequencing of cDNA following reverse transcription
of small RNA. Annotation backed by mirBase and
Rfam. FASTQ files and stats files are available for
subsequent downstream analysis.
RNA-Seq
Sequencing of cDNA following reverse transcription
and library preparation. FASTQ files are generated
and can be used with third-party software for
subsequent analysis.
TruSight HLA
Sequencing of Long Range PCR HLA gene
amplicons prepared using Nextera XT sample prep
technology.
Library QC
Fast resequencing of a reference genome to QC the
DNA library and generate per-sample statistics.
FASTQ Only
Generation of demultiplexed FASTQ files from any
type of library.
ChIP-Seq
Sequencing of TruSeq ChIP libraries. FASTQ files are
generated and can be used with third-party
software for subsequent analysis.
Other
The following tables list the applications of each category and the sample prep kits
supported for each. It also indicates the associated analysis workflow written to the
sample sheet.
Targeted Resequencing
Application in IEM
19
Sample Prep Kit
Supported
Analysis Workflow
TruSeq Amplicon
TruSeq Custom
Amplicon
Amplicon
PCR Amplicon
Nextera
Nextera XT
Nextera XT v2
Nextera XT v2 Set A
Nextera XT v2 Set B
Nextera XT v2 Set C
Nextera XT v2 Set D
PCR Amplicon
in Sample Sheet
Part # 15031335 Rev. J
Sample Prep Kit
Supported
Analysis Workflow
Metagenomics 16S rRNA
Nextera
Nextera XT
Nextera XT v2
Nextera XT v2 Set A
Nextera XT v2 Set B
Nextera XT v2 Set C
Nextera XT v2 Set D
TruSeq HT
TruSeq LT
Metagenomics
Enrichment
Nextera Rapid Capture
Enrichment
TruSight Enrichment
Enrichment
Clone Checking
Nextera
Nextera XT
Nextera XT v2
Nextera XT v2 Set A
Nextera XT v2 Set B
Nextera XT v2 Set C
Nextera XT v2 Set D
TruSeq HT
TruSeq LT
GenerateFASTQ
Amplicon - DS
TruSight Tumor
Amplicon - DS
Creating a Sample Sheet
Application in IEM
in Sample Sheet
Small Genome Sequencing
Application in IEM
Resequencing
Illumina Experiment Manager User Guide
Sample Prep Kit
Supported
Analysis Workflow
Nextera
Nextera Mate Pair
Nextera XT
Nextera XT v2
Nextera XT v2 Set A
Nextera XT v2 Set B
Nextera XT v2 Set C
Nextera XT v2 Set D
TruSeq HT
TruSeq LT
Resequencing
in Sample Sheet
20
Application in IEM
Sample Prep Kit
Supported
Analysis Workflow
Plasmids
Nextera
Nextera XT
Nextera XT v2
Nextera XT v2 Set A
Nextera XT v2 Set B
Nextera XT v2 Set C
Nextera XT v2 Set D
TruSeq HT
TruSeq LT
GenerateFASTQ
Assembly
Nextera
Nextera Mate Pair
Nextera XT
Nextera XT v2
Nextera XT v2 Set A
Nextera XT v2 Set B
Nextera XT v2 Set C
Nextera XT v2 Set D
TruSeq HT
TruSeq LT
Assembly
Sample Prep Kit
Supported
Analysis Workflow
Targeted RNA
Targeted RNA Expression
Targeted RNA
Small RNA
TruSeq Small RNA
SmallRNA
RNA-Seq
TruSeq HT
TruSeq LT
GenerateFASTQ
in Sample Sheet
RNA Sequencing
Application in IEM
in Sample Sheet
Other Workflows
Application in IEM
Sample Prep Kit Supported
Analysis Workflow
in Sample Sheet
TruSight HLA
21
Nextera XT v2
Nextera XT v2 Set A
Nextera XT v2 Set B
Nextera XT v2 Set C
Nextera XT v2 Set D
GenerateFASTQ
Part # 15031335 Rev. J
Sample Prep Kit Supported
Analysis Workflow
in Sample Sheet
LibraryQC
Nextera
Nextera Mate Pair
Nextera XT
Nextera XT v2
Nextera XT v2 Set A
Nextera XT v2 Set B
Nextera XT v2 Set C
Nextera XT v2 Set D
TruSeq HT
TruSeq LT
LibraryQC
FASTQ Only
Nextera
Nextera Mate Pair
Nextera Rapid Capture
Enrichment
Nextera XT
Nextera XT v2
Nextera XT v2 Set A
Nextera XT v2 Set B
Nextera XT v2 Set C
Nextera XT v2 Set D
TruSeq Amplicon
TruSeq Small RNA
TruSeq HT
TruSeq LT
TruSight Enrichment
GenerateFASTQ
ChIP-Seq
TruSeq LT
GenerateFASTQ
Creating a MiSeq Sample Sheet
NOTE
These instructions describe the creation of a new sample sheet. You can also adapt them to
edit an existing sample sheet by selecting Edit Sample Sheet and navigating to the sample
sheet you want to edit.
1
On the main screen, select Create Sample Sheet.
2
On the Instrument Selection screen, select MiSeq, and then select Next.
3
On the MiSeq Application Selection screen, select the desired category and
application and for your kit, and then select Next. Reference MiSeq Applications and
Sample Prep Kits on page 17.
4
On the Workflow Parameters screen, do the following:
a In the Reagent Cartridge Barcode field, enter the barcode number of the MiSeq
reagent cartridge. The barcode number is on the cartridge label.
Illumina Experiment Manager User Guide
22
Creating a Sample Sheet
Application in IEM
b
From the Sample Prep Kit drop-down menu, select the appropriate option for the
kit you are using.
NOTE
For the appropriate option for your kit, reference the appropriate IEM quick
reference card for your kit.
c
d
e
f
g
h
23
In the Index Reads field, select the number of indexes you run for the samples
on this plate: 0, 1, or 2 (if applicable). The same number of indexes you selected
when you created the sample plate must be selected here. The number of Index
Cycles is selected automatically based on the selection you make in the Index
Reads field.
Type an experiment name, investigator name, and description.
Select the date from the calendar.
Select Paired End or Single Read sequencing run, depending on the options
available.
Select the number of cycles for each read in your sequencing run, plus 1.
If you are performing a paired-end sequencing run, Illumina recommends
performing the same number of cycles in both reads.
Depending on which application you selected in step 3, check or uncheck the
appropriate checkboxes or specify the following settings for your sequencing run:
WorkflowSpecific Setting
Description
Custom Primer
for Read 1
Available for all applications except TruSeq Amplicon.
Select this checkbox if you used a custom Read 1 primer and not an
Illumina-provided Read 1 primer.
Custom Primer
for Index
Available for all applications except TruSeq Amplicon.
Select this checkbox if you used a custom Index primer and not an
Illumina-provided Index primer.
Custom Primer
for Read 2
Available for all applications except TruSeq Amplicon.
Select this checkbox if you used a custom Read 2 primer and not an
Illumina-provided Read 2 primer.
Use Somatic
Variant Caller
Available in the TruSeq Amplicon, PCR Amplicon, Enrichment,
and Resequencing applications.
Select this checkbox if you are using the somatic variant caller.
Somatic variant caller is an Illumina-developed variant calling
algorithm specifically for TruSeq Amplicon - Cancer Panel to detect
low frequency mutations (even below 5%) in a mixed cell
population.
For more information, see the Somatic Variant Caller Tech Note,
which you can download from the Illumina website at
www.illumina.com.
Indel
Realignment
GATK
Available in the Resequencing and Enrichment applications.
Select this checkbox to perform a local realignment of reads
around indels such that number of mismatches is minimized.
Part # 15031335 Rev. J
Description
Flag PCR
Duplicates
Available in the Resequencing, PCR Amplicon, Enrichment, and
Library QC applications.
Select this checkbox to flag apparent PCR duplicates in the BAM
files and not used for variant calling. PCR duplicates are defined as
two clusters from a paired-end run where both clusters have the
exact same alignment positions for each read.
Variant Quality
Filter
Available in the TruSeq Amplicon, PCR Amplicon,
Resequencing, and Enrichment applications.
Cutoff parameter. The default setting is 30.
For more information, see the MiSeq Sample Sheet Quick Reference
Guide, which you can download from the Illumina website at
www.illumina.com.
Use Adapter
Trimming
Available for all applications except TruSeq Amplicon. This setting
is recommended when sequencing libraries prepared with
Nextera, Nextera XT, Nextera Rapid Capture Enrichment, or
TruSight Enrichment kits.
It is possible that some clusters can sequence beyond the sample
DNA and read bases from the adapter.
Select this checkbox to cause MiSeq Reporter to mask the adapter
sequence, improving accuracy and speed during secondary
analysis.
The default adapter sequence is the adapter present in all Nextera
libraries. If a different adapter sequence is used, edit the sequence
that is displayed in the final sample sheet.
For TruSight HLA, do not change the default.
Use Adapter
Trimming Read 2
Available in the Plasmids, Assembly, RNA-Seq, Library QC,
FASTQ Only, and ChIP-Seq applications.
Select this checkbox to set the AdapterRead2 setting in the sample
sheet to trim a different adapter sequence in read2.
Run Picard
HsMetrics
Available in the Enrichment application.
Select this checkbox to perform Picard hybrid selection (HS)
analysis of the *.bam file.
Reverse
Complement
Available in the Resequencing, Library QC, FASTQ Only, and
Assembly applications.
Select this checkbox to convert Nextera Mate Pair libraries from a
mate pair to a paired-end orientation as required by BWA and
Velvet.
K-mer size
Available in the Assembly application.
Set the K-mer sized used for assembly. The range is 2–255. Larger
k-mer sizes require more memory and can dramatically affect the
stability and performance of the analysis. K-mer optimization is
suggested for optimal assemblies.
Illumina Experiment Manager User Guide
24
Creating a Sample Sheet
WorkflowSpecific Setting
WorkflowSpecific Setting
i
5
Description
Genome
Available in the Small RNA application.
1. Select from the Genome drop-down menu
2. Provide the relative or absolute path to the following reference
sequence folders. The typical settings for human runs are in
parentheses:
• Contaminants (HumanRNAContaminants)
• RNA (HumanRNA)
• miRNA (HumanRNAMature)
Export to gVCF
Available in the PCR Amplicon, TruSeq Amplicon, and Enrichment
applications. Select the checkbox to enable gVCF files to output to
the run folder.
Genus-Level
Classification
Available in the Metagenomics application. Select the checkbox to
enable genus-level classification, which overrides the species-level
taxonomic classification default.
Click Next.
On the Sample Selection screen, select Select Plate and navigate to a sample plate
you created previously.
TIP
Uncheck Maximize to view the sample plate and sample sheet portion of the screen.
NOTE
If you have not yet created a sample plate, you can do so now by selecting New
Plate. For more information, see Creating a Sample Plate on page 14.
6
Click Select All to include all wells in this sequencing run or highlight the wells you
want to include in this sequencing run.
7
Click Add Selected Samples.
8
[Optional] Click Add Blank Row to add rows and manually enter the sample
information.
TIP
Use the IEM Fill feature to populate any type of data for the wells in your sample sheet
quickly. The Fill feature can copy data as-is from cell to cell or can automatically increase
numerical data by one to create sequential data. The IEM determines which method is
appropriate based on what type of data are in the cell or cells being copied.
• To generate sequential data, enter sequential data in two adjacent wells, then highlight
the other cells you want to populate. Right-select the highlighted area and select Fill
Down or Fill Right. The highlighted cells are numbered in sequential order.
• To copy data, populate one cell, then highlight, right-select, and fill the other cells you
want to populate with the same information.
• Content can be pasted into the Sample ID column directly from an excel table.
9
25
[Optional]Highlight one or more fields, and then select Remove Selected Rows to
remove the entire rows that contain those fields.
Part # 15031335 Rev. J
TIP
Check Maximize to view only the sample sheet portion of the screen.
11 For the following applications that use a genomic reference, select where the FASTA
reference files are saved from the Genome Folder drop-down menu.
Category
Application
Targeted Resequencing
TruSeq Amplicon
PCR Amplicon
Enrichment
Amplicon - DS
Small Genome Sequencing
Resequencing
Assembly
Other
LibraryQC
12 Do one of the following:
• If you are using the PCR Amplicon application, proceed to Creating a Manifest
File on page 26.
• For all other applications, proceed to Finish Creating a MiSeq Sample Sheet on page
28.
Creating a Manifest File
The manifest file is required for analysis using the PCR Amplicon workflow. The
manifest contains information about each sample that focuses analysis results only to
user-defined regions of interest. There can be multiple manifests per sample sheet.
Manifest creation requires input of:
• Reference genome used for alignment.
• Chromosome coordinates (start, stop) of each amplicon/ROI.
• Length of each PCR primer used for generation of the PCR amplicon.
1
Select one of the following from the Nextera Manifest drop-down menu:
• Select New to create a manifest.
• Select Edit to choose an existing manifest.
Illumina Experiment Manager User Guide
26
Creating a Sample Sheet
10 Type a sample name, sample project, and description for each sample.
a
If you are creating a new manifest file, the Create New Amplicon Manifest screen
opens.
b
Select a single reference genome for each new manifest from the Genomes dropdown menu. The genome must be located in your IEM genome repository
directory. Each sample sheet you are creating must have the same reference
genome for all manifests associated with it.
TIP
Make sure that the reference genome matches the reference genome used to
design the PCR amplicon primers.
NOTE
The IEM genome repository directory should also be your MiSeq Reporter
Genomes location, where the actual reference genome is located.
27
c
d
Add a blank row for each ROI and name the Amplicon.
Choose the appropriate chromosome and input the amplicon coordinates (start
and end-including primers). The amplicon start and end should include the
primer lengths.
e
Specify the length of each primer (upstream and downstream). This allows
variants called in these regions to be flagged during analysis.
Part # 15031335 Rev. J
f
Name your manifest file to save it into the Manifest Repository.
NOTE
Copy the new manifest file into the MCS Manifest Repository location on the
MiSeq instrument before starting your run.
Finish Creating a MiSeq Sample Sheet
1
If you are using the Enrichment application, enter the name of the Manifest file
Illumina provided for your assay or control. Leave off the .txt file extension part of
the file name.
TIP
If you do not see a manifest file for your sample prep kit in the drop-down menu, go
to the Manifest Repository folder for IEM. Copy an Illumina-provided manifest file
there. See Downloading Manifests on page 11.
2
Click Finish and save the sample sheet file in the desired folder.
3
When prompted, select Yes if you want to review the sample sheet in Microsoft
Excel or No to exit the sample sheet wizard without reviewing the sample sheet.
HiSeq-, HiScanSQ-, or Genome Analyzer-Compatible Sample Sheets
This section provides instructions for creating a sample sheet for the analysis of HiSeq,
HiScanSQ, or Genome Analyzer sequencing data.
HiSeq, HiScanSQ, and Genome Analyzer Applications and Sample
Prep Kits
To process a sequencing run fully on HiSeq, HiScanSQ, or Genome Analyzer, an analysis
application is specified in the sample sheet.
The following categories and their applications are supported:
Application
Human
Genome
Resequencing
Description
Sequencing of a human genome. Reads are aligned against the hg19 reference
and variants are reported.
HiSeq
FASTQ Only
Generation of demultiplexed FASTQ files from any type of library.
HiSeq
Enrichment
Sequencing of DNA obtained from Nextera (hybrid capture-based)
enrichment. Reads are aligned against the specified reference genome. An
Illumina provided manifest file (specified in the sample sheet) gives the
targeted regions; variant calling is performed only within these regions, and
coverage statistics are reported for the target regions.
HiSeq
CASAVA
Sequencing of human and other genomes available from Illumina iGenomes.
Outputs include aligned reads and variant calls. For more information on
iGenomes, see support.illumina.com/sequencing/sequencing_
software/igenome.ilmn.
Illumina Experiment Manager User Guide
28
Creating a Sample Sheet
TIP
All content needed for a new manifest file can be copied from an Excel table into
IEM. Make sure that the columns contain the appropriate contents after pasting
in from the Excel table. The upstream and downstream probe length will be the
PCR primers used to generate the amplicon.
The following tables list the applications and the sample prep kits supported for each. It
also indicates the associated analysis workflow written to the sample sheet.
Application in IEM
Sample Prep Kit Supported
Analysis
Workflow
in Sample Sheet
29
Human Genome
Resequencing
Nextera
Nextera Mate Pair
Nextera XT
Nextera XT v2
Nextera XT v2 Set A
Nextera XT v2 Set B
Nextera XT v2 Set C
Nextera XT v2 Set D
TruSeq HT
TruSeq LT
Resequencing
HiSeq FASTQ Only
Nextera
Nextera Mate Pair
Nextera Rapid Capture
Enrichment
Nextera XT
Nextera XT v2
Nextera XT v2 Set A
Nextera XT v2 Set B
Nextera XT v2 Set C
Nextera XT v2 Set D
TruSeq Amplicon
TruSeq Small RNA
TruSeq HT
TruSeq LT
TruSight Enrichment
TruSeq Synthetic Long-Read DNA
GenerateFASTQ
HiSeq Enrichment
Nextera Rapid Capture
Enrichment
TruSight Enrichment
Enrichment
HiSeq CASAVA
Nextera
Nextera Mate Pair
Nextera XT
Nextera XT v2
Nextera XT v2 Set A
Nextera XT v2 Set B
Nextera XT v2 Set C
Nextera XT v2 Set D
TruSeq Amplicon
TruSeq Small RNA
TruSeq HT
TruSeq LT
TruSight Enrichment
Not applicable
Part # 15031335 Rev. J
NOTE
• These instructions describe the creation of a new sample sheet. You can also adapt
them to edit an existing sample sheet by selecting Edit Sample Sheet and navigating
to the sample sheet you want to edit.
• This function does not support TruSeq Small RNA Sample Preparation and TruSeq
Custom Amplicon and TruSeq Amplicon - Cancer Panel Library Preparation Kits.
1
On the main screen, select Create Sample Sheet.
2
On the Instrument Selection screen, select HiSeq, HiScanSQ, GA, and then select
Next.
3
On second the Instrument Selection screen, select HiSeq 3000, 4000, HiSeq 1500,
2500, HiSeq 1000, 2000, HiScanSQ, or Genome Analyzer, and then select Next.
4
On the HiSeq Application Selection screen, select the desired application and for
your kit, and then select Next. Reference HiSeq, HiScanSQ, and Genome Analyzer
Applications and Sample Prep Kits on page 28.
5
On the Workflow Parameters screen, do the following:
a In the Reagent Kit Barcode field, enter the reagent kit ID from the label of either
box 1 or box 2 of the SBS kit.
b From the Sample Prep Kit drop-down menu, select the appropriate option for the
kit you are using.
NOTE
For the appropriate option for your kit, reference the appropriate IEM quick
reference card for your kit.
c
In the Index Reads field, select the number of indexes you run for the samples
on this plate: 0, 1, or 2 (if applicable). The same number of indexes you selected
when you created the sample plate must be selected here. The number of Index
Cycles is selected automatically based on the selection you make in the Index
Reads field.
6
If creating a sample sheet for Human Genome Resequencing, HiSeq FASTQ Only, or
HiSeq Enrichment, do the following:
a Type an experiment name, investigator name, and description.
b Select the date from the calendar.
c Select a Paired End or Single Read sequencing run, depending on the options
available.
d Select the number of cycles for each read in your sequencing run, plus 1.
If you are performing a paired-end sequencing run, perform the same number of
cycles in both reads.
7
If creating a sample sheet for HiSeq CASAVA, do the following:
a In the Flow Cell ID field, enter the barcode number of the flow cell.
b In the Operator field, enter the name of the person running the sequencing
instrument.
c In the Recipe field, enter the name of the recipe to be used for the run of the
sequencing instrument.
8
Depending on which application you selected in step 4, check or uncheck the
appropriate checkboxes or specify the following settings for your sequencing run:
Illumina Experiment Manager User Guide
30
Creating a Sample Sheet
Creating a HiSeq-, HiScanSQ-, or Genome Analyzer Sample Sheet
31
WorkflowSpecific Setting
Description
Custom Primer
for Read 1
Available for the HiSeq FASTQ Only application.
Select this checkbox if you used a custom Read 1 primer and not an
Illumina-provided Read 1 primer.
Custom Primer
for Index
Available for the HiSeq FASTQ Only application.
Select this checkbox if you used a custom Index primer and not an
Illumina-provided Index primer.
Custom Primer
for Read 2
Available for the HiSeq FASTQ Only application.
Select this checkbox if you used a custom Read 2 primer and not an
Illumina-provided Read 2 primer.
Indel
Realignment
GATK
Available for the HiSeq Enrichment application.
Select this checkbox to perform a local realignment of reads
around indels such that number of mismatches is minimized.
Flag PCR
Duplicates
Available for the Human Genome Resequencing and HiSeq
Enrichment applications.
Select this checkbox to flag apparent PCR duplicates in the BAM
files and not used for variant calling. PCR duplicates are defined as
two clusters from a paired-end run where both clusters have the
exact same alignment positions for each read.
Variant Quality
Filter
Available for the HiSeq Enrichment application.
Cutoff parameter. The default setting is 30.
Use Adapter
Trimming
Available for the Human Genome Resequencing, HiSeq FASTQ
Only, and HiSeq Enrichment applications.
This setting is recommended when sequencing libraries prepared
with Nextera, or Nextera XT kits.
It is possible that some clusters can sequence beyond the sample
DNA and read bases from the adapter.
Selecting this checkbox causes the analysis software to mask the
adapter sequence, improving accuracy and speed during
secondary analysis.
The default adapter sequence is the adapter present in all Nextera
libraries. If a different adapter sequence is used, edit the sequence
that is displayed in the final sample sheet.
Use Adapter
Trimming Read 2
Available for the Human Genome Resequencing and HiSeq
FASTQ Only applications.
Select this checkbox to set the AdapterRead2 setting in the sample
sheet to trim a different adapter sequence in read2.
Run Picard
HsMetrics
Available in the HiSeq Enrichment application.
Select this checkbox to perform Picard hybrid selection (Hs)
analysis of the *.bam file.
Reverse
Complement
Available for the HiSeq FASTQ Only application.
Select this checkbox to convert Nextera Mate Pair libraries from a
mate pair to a paired-end orientation as required by BWA and
Velvet.
Flow Cell ID
Available for the HiSeq CASAVA Only application.
Enter the barcode number of the flow cell.
Part # 15031335 Rev. J
Description
Operator
Available for the HiSeq CASAVA Only application.
Enter the name of the person running the sequencing instrument.
Recipe
Available for the HiSeq CASAVA Only application.
Enter the name of the recipe to be used for the run on the
sequencing instrument.
Click Next.
10 On the Sample Selection screen, select Select Plate and navigate to the sample plate
you created previously.
TIP
Uncheck Maximize to view the sample plate and sample sheet portion of the screen.
NOTE
If you have not yet created a sample plate, you can do so now by selecting New
Plate. For more information, see Creating a Sample Plate on page 14.
11 For each lane you are using in the flow cell, do the following:
a Click the lane number: 1 through 8 on the sample sheet area of the screen.
b Click Select All to include all wells in this sequencing run or highlight the wells
you want to include in this sequencing run.
c Click Add Selected Samples.
d [Optional]Click Add Blank Row to add rows and manually enter the sample
information.
TIP
Use the IEM Fill feature to populate any type of data for the wells in your sample
sheet quickly. The Fill feature can copy data as-is from cell to cell or can automatically
increase numerical data by one to create sequential data. The IEM determines which
method is appropriate based on what type of data are in the cell or cells being copied.
• To generate sequential data, enter sequential data in two adjacent wells, then
highlight the other cells you want to populate. Right-select the highlighted area
and select Fill Down or Fill Right. The highlighted cells are numbered in
sequential order.
• To copy data, populate one cell, then highlight, right-select, and fill the other cells
you want to populate with the same information.
• Content can be pasted into the Sample ID column directly from an excel table.
e
[Optional]Highlight one or more fields, and then select Remove Selected Rows
to remove the entire rows that contain those fields.
12 Type a sample name, sample reference, sample project, and description for each
sample in each lane.
TIP
Check Maximize to view only the sample sheet portion of the screen.
13 When the wells and samples for every lane in the flow cell have been defined, select
Finish and save the sample sheet file in the desired folder.
Illumina Experiment Manager User Guide
32
Creating a Sample Sheet
9
WorkflowSpecific Setting
14 When prompted, select Yes if you want to review the sample sheet in Microsoft
Excel or No to exit the sample sheet wizard without reviewing the sample sheet.
NextSeq-Compatible Sample Sheets
This section provides instructions for creating a sample sheet for the analysis of NextSeq
sequencing data.
NOTE
The NextSeq sample sheets generated by IEM are for use when operating the NextSeq in
standalone mode, with the bcl2fastq2 software package.
NextSeq Applications and Sample Prep Kits
To process a sequencing run on NextSeq, an analysis application is specified in the
sample sheet.
The following categories and their applications are supported:
Application
Description
NextSeq FASTQ Only
Generation of demultiplexed FASTQ files from any type of
library.
The following tables list the applications and the sample prep kits supported for each. It
also indicates the associated analysis workflow written to the sample sheet.
Application in IEM
Sample Prep Kit Supported
Analysis Workflow
in Sample Sheet
NextSeq FASTQ Only
Nextera
Nextera Mate Pair
Nextera Rapid Capture
Enrichment
Nextera XT
Nextera XT v2
Nextera XT v2 Set A
Nextera XT v2 Set B
Nextera XT v2 Set C
Nextera XT v2 Set D
TruSeq Amplicon
TruSeq Small RNA
TruSeq HT
TruSeq LT
TruSight Enrichment
GenerateFASTQ
Creating a NextSeq Sample Sheet
NOTE
These instructions describe the creation of a new sample sheet. You can also adapt
them to edit an existing sample sheet by selecting Edit Sample Sheet and navigating
to the sample sheet you want to edit.
33
1
On the main screen, select Create Sample Sheet.
2
On the Instrument Selection screen, select NextSeq, and then select Next.
Part # 15031335 Rev. J
On the NextSeq Application Selection screen, select the desired application and for
your kit, and then select Next. Reference NextSeq Applications and Sample Prep Kits on
page 33.
4
On the Workflow Parameters screen, do the following:
a In the Reagent Kit Barcode field, enter the reagent kit ID from the label of either
box 1 or box 2 of the SBS kit.
b From the Sample Prep Kit drop-down menu, select the appropriate option for the
kit you are using.
NOTE
For the appropriate option for your kit, reference the appropriate IEM quick
reference card for your kit.
c
d
e
f
g
5
In the Index Reads field, select the number of indexes you run for the samples
on this plate: 0, 1, or 2 (if applicable). The same number of indexes you selected
when you created the sample plate must be selected here. The number of Index
Cycles is selected automatically based on the selection you make in the Index
Reads field.
Type an experiment name, investigator name, and description.
Select the date from the calendar.
Select a Paired End or Single Read sequencing run, depending on the options
available.
Select the number of cycles for each read in your sequencing run, plus 1.
If you are performing a paired-end sequencing run, perform the same number of
cycles in both reads.
Check or uncheck the appropriate checkboxes or specify the following settings for
your sequencing run:
WorkflowSpecific Setting
Description
Custom Primer
for Read 1
Available for the NextSeq FASTQ Only application.
Select this checkbox if you used a custom Read 1 primer and not an
Illumina-provided Read 1 primer.
Custom Primer
for Index
Available for the NextSeq FASTQ Only application.
Select this checkbox if you used a custom Index primer and not an
Illumina-provided Index primer.
Custom Primer
for Read 2
Available for the NextSeq FASTQ Only application.
Select this checkbox if you used a custom Read 2 primer and not an
Illumina-provided Read 2 primer.
Illumina Experiment Manager User Guide
34
Creating a Sample Sheet
3
WorkflowSpecific Setting
Description
Use Adapter
Trimming
Available for the NextSeq FASTQ Only application.
This setting is recommended when sequencing libraries prepared
with Nextera, or Nextera XT kits.
It is possible that some clusters can sequence beyond the sample
DNA and read bases from the adapter.
Selecting this checkbox causes the analysis software to mask the
adapter sequence, improving accuracy and speed during
secondary analysis.
The default adapter sequence is the adapter present in all Nextera
libraries. If a different adapter sequence is used, edit the sequence
that is displayed in the final sample sheet.
Use Adapter
Trimming Read 2
Available for the NextSeq FASTQ Only applications.
Select this checkbox to set the AdapterRead2 setting in the sample
sheet to trim a different adapter sequence in read2.
6
Click Next.
7
On the Sample Selection screen, select Select Plate and navigate to the sample plate
you created previously.
TIP
Uncheck Maximize to view the sample plate and sample sheet portion of the screen.
NOTE
If you have not yet created a sample plate, you can do so now by selecting New
Plate. For more information, see Creating a Sample Plate on page 14.
8
Click Select All to include all wells in this sequencing run or highlight the wells you
want to include in this sequencing run.
9
Click Add Selected Samples.
10 [Optional]Click Add Blank Row to add rows and manually enter the sample
information.
TIP
Use the IEM Fill feature to populate any type of data for the wells in your sample
sheet quickly. The Fill feature can copy data as-is from cell to cell or can automatically
increase numerical data by one to create sequential data. The IEM determines which
method is appropriate based on what type of data are in the cell or cells being copied.
• To generate sequential data, enter sequential data in two adjacent wells, then
highlight the other cells you want to populate. Right-select the highlighted area
and select Fill Down or Fill Right. The highlighted cells are numbered in
sequential order.
• To copy data, populate one cell, then highlight, right-select, and fill the other cells
you want to populate with the same information.
• Content can be pasted into the Sample ID column directly from an excel table.
11 [Optional] Highlight one or more fields, and then select Remove Selected Rows to
remove the entire rows that contain those fields.
35
Part # 15031335 Rev. J
TIP
Check Maximize to view only the sample sheet portion of the screen.
13 When the wells and samples have been defined, select Finish and save the sample
sheet file in the desired folder.
14 When prompted, select Yes if you want to review the sample sheet in Microsoft
Excel or No to exit the sample sheet wizard without reviewing the sample sheet.
NeoPrep-Compatible Sample Sheets
This section provides instructions for creating a sample sheet for a NeoPrep Library Prep
System run.
Creating a NeoPrep Sample Sheet
NOTE
These instructions describe the creation of a new sample sheet. You can also adapt them to
edit an existing sample sheet by selecting Edit Sample Sheet and navigating to the sample
sheet you want to edit. On the IEM main screen, select Create Sample Sheet.
1
On the Instrument Selection screen, select NeoPrep and then select Next.
2
On the Workflow Parameters screen, do the following:
a From the Sample Prep Kit drop-down menu, select the appropriate option for the
kit that you are using.
NOTE
For the appropriate option for your kit, reference the IEM NeoPrep quick
reference card.
b
c
d
e
Type a run name, operator name, and notes.
Select the date.
Select the number of samples. The default is 16. This number is used to define
the default wells and indexes in the Sample Selection screen.
For TruSeq DNA, select a 350, 550, or Mixed insert size. The default is 350.
3
Select Next.
4
On the Sample Selection screen:
a Type a sample name for each sample.
b The default well, index adapter, and index sequence are displayed for each
sample. The default index adapters correspond to the default adapter layout in
the kit reagent plate. For information on the default and optional index adapters,
see the library prep guide for the kit that you are using.
c To edit the defaults, do the following:
— Select the Well cell and select a well number from the drop-down menu.
— Select the Index1 (i7) cell and select an index from the drop-down menu. The
index sequence is automatically displayed in the i7 Sequence column.
d For TruSeq DNA, the selected insert size is displayed.
— To change the insert size, select the Insert Size cell and select a size from the
drop-down menu.
— If Mixed Insert Size was selected in step 2, select the Insert Size cell and
select a size from the drop-down menu for each sample.
Illumina Experiment Manager User Guide
36
Creating a Sample Sheet
12 Type a sample name, sample reference, sample project, and description for each
sample.
37
5
[Optional] Select Add Blank Row to add rows and manually enter the sample
information for each sample.
a Type a sample name.
b Select the Well cell and select a well number from the drop-down menu.
c Select the Index1 (i7) cell and select an index from the drop-down menu.
The index sequence is automatically displayed in the i7 Sequence column.
6
Select Finish and save the sample sheet file.
7
When prompted, select Yes if you want to review the sample sheet in Microsoft
Excel or No to exit the sample sheet wizard without reviewing the sample sheet.
Part # 15031335 Rev. J
For technical assistance, contact Illumina Technical Support.
Table 1 Illumina General Contact Information
Website
Email
www.illumina.com
[email protected]
Table 2 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Italy
Australia
1.800.775.688
Netherlands
Austria
0800.296575
New Zealand
Belgium
0800.81102
Norway
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
United Kingdom
Ireland
1.800.812949
Other countries
Contact Number
800.874909
0800.0223859
0800.451.650
800.16836
900.812168
020790181
0800.563118
0800.917.0041
+44.1799.534000
Safety Data Sheets
Safety data sheets (SDSs) are available on the Illumina website at
support.illumina.com/sds.html.
Product Documentation
Product documentation in PDF is available for download from the Illumina website. Go
to support.illumina.com, select a product, then select Documentation & Literature.
Illumina Experiment Manager User Guide
Technical Assistance
Technical Assistance
*15031335*
Part # 15031335 Rev. J
Illumina
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com
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