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MiSeq® Sample Sheet
Quick Reference Guide
FOR RESEARCH USE ONLY
Revision History
3
Introduction
4
Sample Sheet Parameters
5
Sample Sheet Settings
10
Manifests
14
Analysis Workflows
16
Editing the Sample Sheet for Custom Primers
18
Naming the Sample Sheet
19
Technical Assistance
ILLUMINA PROPRIETARY
Part # 15028392 Rev. E
November 2012
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All other brands and names contained herein are the property of their respective owners.
Part #
Revision
Date
15028392
E
November
2012
15028392
D
July 2012
Added the following information:
• Added the PCR Amplicon analysis workflow for Nextera
XT libraries and information about the manifest file
• Noted that adapter trimming is recommended for longer
read lengths up to 250 cycles
• Added descriptions of sample sheet settings for
PercentTilesToScan and StrandBiasFilter
• Changed Setup Options screen to Run Options screen per
MCS v1.2
15028392
C
April 2012
Updated the following information:
• Updated name of Amplicon workflow to Custom
Amplicon
• Updated name of DenovoAssembly workflow to
Assembly
• Added GenerateFASTQ workflow
• Listed genome folder as required for amplicon sequencing
in Sample Sheet Parameters
15028392
B
December
2011
Updated the steps in Setting Up the Sample Sheet.
Listed manifest files as required for TruSeq Custom Amplicon
libraries.
15028392
A
September
2011
Initial release
MiSeq Sample Sheet Quick Reference Guide
Description of Change
Added the following information:
• Description of Enrichment workflow, manifest, and data
section requirements
• Data section requirements for PCR Amplicon workflow
• Descriptions of sample sheet settings AdapterRead2 and
QualityScoreTrim
• Note about supported option for listing genome references
for multiple species in the same sample sheet when using
MiSeq Reporter v2.1
Updated the following information:
• Organized sample sheet settings into settings for
sequencing, analysis, and variant calling
• Updated Small RNA workflow to list the genome folder as
required in the sample sheet
• Updated sample sheet settings for variant calling to add
VariantMinimumGQCutoff, and to update StandBiasFilter
and MinumumCoverageDepth for the Enrichment
workflow
3
Revision History
Revision History
Introduction
The sample sheet is a comma separated values (*.csv) file that stores information
needed to set up, perform, and analyze a sequencing run.
Illumina recommends that you create your sample sheet prior to preparing your
sample libraries. You can create your sample sheet using the Illumina Experiment
Manager or create it manually using a text editor, such as Excel or Notepad.
Before starting the run, make sure that the sample sheet is accessible to the
instrument by copying the sample sheet to a network location accessible from the
instrument computer, or copy the sample sheet from a USB flash drive to the
instrument computer using the Manage Files feature in MiSeq Control Software.
When the run begins, the software copies the sample sheet from the designated
sample sheet folder to the root of the output folder, and then copies it to the root of
the analysis folder. At the end of the run, the sample sheet is used for secondary
analysis by the MiSeq Reporter software.
For more information, see the MiSeq System User Guide, Part # 15027617.
Illumina Experiment Manager
The Illumina Experiment Manager is a wizard-based application that guides you
through the steps to create your sample sheet.
Using the Illumina Experiment Manager not only reduces syntax errors, but also
prompts you to provide information for sections that apply to your sample type and
analysis workflow. It provides a feature for recording parameters for your sample
plate, such as sample ID, project name, dual indices, and barcode information. Using
the Illumina Experiment Manager, you can import sample plate parameters to your
sample sheet.
The Illumina Experiment Manager can be run on any Windows platform. You can
download a copy from the Illumina website at www.illumina.com. Go to the MiSeq
support page and click Downloads. A MyIllumina login is required.
For more information, see the Illumina Experiment Manager User Guide, Part #
15031335.
Sample Sheet Workflow
4
1
Create your sample sheet using one of the following methods:
• Illumina Experiment Manager—See the Illumina Experiment Manager User
Guide, Part # 15031335.
• Excel or Notepad—See Sample Sheet Parameters on page 5.
2
Name your sample sheet with the reagent cartridge barcode number that you will
use for your run followed by *.csv. This enables the software to automatically
associate your sample sheet with the run. For more information, see Naming the
Sample Sheet on page 19.
3
Copy the sample sheet to the network location specified in Run Options in MCS,
or copy it to the instrument computer using a USB flash drive and the Manage
Files feature. For more information, see the MiSeq System User Guide.
Part # 15028392 Rev. E
The following sections describe the anatomy of a sample sheet and list requirements
for different analysis workflows. Many of these descriptions and requirements are
automated in Illumina Experiment Manager. If you are using Illumina Experiment
Manager to create your sample sheet, see the Illumina Experiment Manager User Guide,
Part # 15031335.
The sample sheet is organized by sections titled Header, Reads, Manifests, Data, and
Settings. Section headings, which are case-sensitive, are shown in brackets [ ] in
following example:
Figure 1 Sample Sheet Example in Excel
Not all sections of the sample sheet apply to every analysis workflow. Some sections
are required, while others are optional or do not apply at all.
} The Manifests section is required for the Custom Amplicon and PCR Amplicon
workflows only. For more information, see Manifests on page 14.
} The Data section is arranged in columns, and each analysis workflow requires
specific columns. For more information, see Data Section on page 6.
} The Settings section is optional for all workflows. For more information, see
Sample Sheet Settings on page 10.
Header Section
Parameter
Description
Investigator Name
Your name.
Project Name
Project name of your preference.
Experiment Name
Experiment name of your preference.
Date
Date of your experiment.
Workflow
Required
The workflow field must contain one of the following options:
Resequencing, Custom Amplicon, PCR Amplicon, Assembly,
SmallRNA, GenerateFASTQ, Metagenomics, or LibraryQC. For
more information, see Analysis Workflows on page 16.
MiSeq Sample Sheet Quick Reference Guide
5
Sample Sheet Parameters
Sample Sheet Parameters
Parameter
Description
Assay
The name of the assay used to prepare your samples.
Chemistry
This field identifies the recipe fragments used to build the runspecific recipe. If this field is blank, the system uses the default
recipe fragments.
Optional—For TruSeq RNA or TruSeq DNA libraries, this field
can be blank or use the default setting.
Required—For any workflows that use dual indexing,
specifically Nextera and TruSeq Custom Amplicon, the
chemistry field is required. Enter amplicon in this field.
Reads Section
Parameter
Description
Number of cycles
in Read 1
Required
Number of cycles
in Read 2
Required for paired-end runs.
NOTE
The number of cycles for the index read is indicated by the index sequence
defined in the Data section.
Data Section
Column Heading
Description
SampleID
Required
Every sample must have a unique sample ID.
This is usually a barcode but can have any value.
At least one sample must be listed. List one sample per line.
Sample_Name
Optional
The sample name is used in reporting and file naming.
Index
Required for multi-sample assays with single or dual indexing.
Nucleotide sequence—Valid characters are A, C, G, T, and N,
where N matches any base. Enter the index sequence of i7.
Index2
Required for multi-sample assays with dual indexing.
Nucleotide sequence—Valid characters are A, C, G, T, and N,
where N matches any base. Enter the index sequence of i5.
NOTE
For the appropriate index sequences, see the user guide for your sample
preparation kit.
The following tables summarizes the required Data section columns for each analysis
workflow. Column order is not important.
6
Part # 15028392 Rev. E
Data Columns
Assembly
Required: SampleID
Optional: Index, Index 2, GenomeFolder
Custom Amplicon
Required: SampleID, Manifest, GenomeFolder
Optional: Index, Index 2
Enrichment
Required: SampleID, Manifest, GenomeFolder
Optional: Index, Index 2
GenerateFASTQ
Required: SampleID
Optional: Index, Index 2
LibraryQC
Required: SampleID, GenomeFolder
Optional: Index, Index 2
Metagenomics
Required: SampleID
Optional: Index, Index 2
PCR Amplicon
Required: SampleID, Manifest, GenomeFolder
Optional: Index, Index 2
Resequencing
Required: SampleID, GenomeFolder
Optional: Index, Index 2
SmallRNA
Required: SampleID, GenomeFolder, Contaminants, miRNA, RNA
Optional: Index, Index 2
Sample Sheet Parameters
Analysis
Workflow
NOTE
You must enter the full path (UNC path) to the GenomeFolder in the sample
sheet. Do not enter the path using a mapped drive.
NOTE
Introduced in MiSeq Reporter v2.1, you can specify genome references for
multiple species in the same sample sheet for all workflows except the Small
RNA workflow.
Data Columns for Assembly Workflow
Column Heading
Description
GenomeFolder
Optional
If provided, MiSeq Reporter compares the de novo assembly
against the reference genome, and generates a dot-plot that
graphically summarizing the results.
• If the folder listed does not exist, Illumina Experiment
Manager combines the GenomePath configuration setting
with the genome string.
• If the path does not exist, Illumina Experiment Manager
stops processing.
MiSeq Sample Sheet Quick Reference Guide
7
Data Columns for Custom Amplicon Workflow
Column Heading
Description
GenomeFolder
Required
The reference genome folder containing the FASTA files to be
used in the alignment step. Must be the same genome used to
generate the manifest file. This is used to provide variant
annotations and set the chromosome sizes in the *.bam file
output.
Manifest
Required
Specify the manifest key for this sample, which is located in the
first column of the Manifests section of the sample sheet.
Data Columns for Enrichment Workflow
Column Heading
Description
GenomeFolder
Required
The reference genome folder containing the FASTA files to be
used in the alignment step.
Manifest
Required
Specify the manifest key for this sample, which is located in the
first column of the Manifests section of the sample sheet.
Data Columns for Library QC Workflow
Column Heading
Description
GenomeFolder
Required
The reference genome folder containing the FASTA files to be
used in the alignment step.
Data Columns for PCR Amplicon Workflow
8
Column Heading
Description
GenomeFolder
Required
The reference genome folder containing the FASTA files to be
used in the alignment step.
Manifest
Required
Specify the manifest key for this sample, which is located in the
first column of the Manifests section of the sample sheet.
Part # 15028392 Rev. E
Sample Sheet Parameters
Data Columns for Resequencing Workflow
Column Heading
Description
GenomeFolder
Required
The reference genome folder containing the FASTA files to be
used in the alignment step.
The GenomeFolder can be a network location or the MiSeq local
drive. For optimal results store Genomes on the local drive or
use BaseSpace.
The GenomeFolder location should be the folder containing the
FASTA files. An example file path: D:\Illumina\MiSeq
Reporter\Genomes\PhiX\Illumina\RTA\Sequence\Chromosomes
Data Columns for Small RNA Workflow
Column Heading
Description
GenomeFolder
Required
If provided, reads are aligned against the full reference genome.
Contaminants
Required
The path to the folder containing the FASTA files of
contaminants.
miRNA
Required
The path to the folder containing FASTA files of mature
miRNAs.
RNA
Required
The path to the folder containing FASTA files of small RNAs.
MiSeq Sample Sheet Quick Reference Guide
9
Sample Sheet Settings
You can specify settings in the Settings section of the sample sheet to control various
sequencing and analysis parameters. Each line in the Settings section contains a
setting name in the first column and a value in the second column.
Sample Sheet Settings for Sequencing
You can specify settings in the Settings section of the sample sheet to control various
analysis parameters. Each line in the Settings section contains a setting name in the
first column and a value in the second column.
Parameter
Description
CustomRead1PrimerMix
CustomIndexPrimerMix
CustomRead2PrimerMix
Create one line for each custom primer used and indicate C1
for the Read 1 primer, C2 for the Index primer, or C3 for the
Read 2 primer. Custom primers are supported for Read 1,
Index 1 Read, and Read 2 only.
For more information, see Editing the Sample Sheet for Custom
Primers on page 18.
PercentTilesToScan
If set to the default value of 1, 100% of the tiles are scanned.
Valid values are 0 through 1.
• If set to 0, the software will round up to one tile.
• For all other settings, the software will round down. For
example, a value of 0.99 results in 27 tiles (dual-surface) or 13
tiles (single-surface).
For more information about dual-surface scanning, see the
MiSeq System User Guide, Part # 15027617.
Sample Sheet Settings for Analysis
10
Parameter
Description
Adapter
Specify the 5' portion of the adapter sequence to
prevent reporting sequence beyond the sample
DNA.
For more information about adapter settings, see
the MiSeq Reporter User Guide, Part # 15028784.
• Nextera libraries—Illumina recommends
adapter trimming using BWA for Nextera
libraries (adapter sequence
CTGTCTCTTATACACATCT).
• Small RNA libraries—Adapter masking is
performed by default using Eland (adapter
sequence
TGGAATTCTCGGGTGCCAAGGC). This
adapter is masked if nothing is specified in the
sample sheet.
For other libraries, see the associated sample
prep documentation.
Part # 15028392 Rev. E
Description
AdapterRead2
Specify the 5' portion of the Read 2 adapter
sequence to prevent reporting sequence beyond
the sample DNA. Use this setting to specify a
different adapter other than the one specified in
the Adapter setting.
Aligner
For Resequencing and Library QC
workflows—Specify the method for aligning
reads against the reference genome. Options are
BWA (default) or Eland. The BWA aligner
provides improved speed, accuracy, and
throughput relative to Eland. When BWA is
specified, GATK is used for variant calling, by
default.
CustomAmpliconAlignerMaxIndelSize
For Custom Amplicon workflow—The
maximum detectable indel size using the Custom
Amplicon workflow. The default is 10.
Setting this to a larger value increases the
sensitivity to larger indels, but requires more
time to complete alignment.
FilterPCRDuplicates
Settings are 0 or 1. Default is 1, filtering.
If set to 1, PCR duplicates are flagged 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.
Kmer
For the Assembly workflow—Use this setting
to override the k-mer size used by Velvet.
Default is 31; odd-numbered values up to 63 are
supported.
QualityScoreTrim
If set to a value > 0, then the 3' ends of nonindexed reads with low quality scores are
trimmed. Trimming is automatically applied by
default for a cutoff of 15 when using BWA for
alignment.
TaxonomyFile
For the Metagenomics workflow—Use this
setting to override the taxonomy database
(default is taxonomy.dat).
VariantCaller
For the Custom Amplicon workflow—Specify
one of the following variant caller settings:
• GATK (default)
• Somatic (for tumor samples)
• Starling (legacy variant caller)
• None (no variant calling)
Sample Sheet Settings for Variant Calling
Some sample sheet settings specify parameters for variant calling. Most settings and
default values are specific to a variant caller.
MiSeq Sample Sheet Quick Reference Guide
11
Sample Sheet Settings
Parameter
12
Setting Name
Description
FilterOutSingleStrandVariants
This setting filters variants if they are only found
in one read-direction.
This setting applies to the Resequencing and PCR
Amplicon workflows only; it does not apply to the
Custom Amplicon workflow.
Default value and variant caller:
• On—Somatic Variant Caller
IndelRepeatFilterCutoff
This setting filters indels if the reference has a 1base or 2-base motif over eight times (by default)
next to the variant.
Default value and variant caller:
• 8—Somatic Variant Caller
• 8—GATK
• 8—Starling
MinimumCoverageDepth
This setting does not report variants if the
coverage depth at that location is less than the
specified threshold.
Default value and variant caller:
• 10—Somatic Variant Caller
• 20—GATK (Enrichment workflow only; 0
otherwise)
MinQScore
This setting specifies the minimum base Q-score to
use as input to variant calling.
Default value and variant caller:
• 20—Somatic Variant Caller
• 0—Starling
StrandBiasFilter
This setting filters variants with a significant bias in
read-direction. Variants filtered out in this way
will have sb in the filter column of the VCF file,
instead of PASS.
Default value and variant caller:
• 0.5—Somatic Variant Caller
• -10—GATK (Enrichment workflow only; no
filter otherwise)
VariantFilterQualityCutoff
This setting filters variants if the variant quality
score is less than the specified threshold.
Default value and variant caller:
• 30—Somatic Variant Caller
• 30—GATK
• 20—Starling
VariantFrequencyEmitCutoff
This setting does not report variants with a
frequency less than the specified threshold.
Default value and variant caller:
• 0.01—Somatic Variant Caller
Part # 15028392 Rev. E
Description
VariantFrequencyFilterCutoff
This setting filters variants with a frequency less
than the specified threshold.
Default value and variant caller:
• 0.01—Somatic Variant Caller
• 0.20—GATK
• 0.20—Starling
VariantMinimumGQCutoff
This setting filters sites if the genotype quality
(GQ) is less than the threshold.
Default value and variant caller:
• 30—Somatic Variant Caller
• 30—GATK
• 20—Starling
MiSeq Sample Sheet Quick Reference Guide
13
Sample Sheet Settings
Setting Name
Manifests
A manifest is a file that specifies the alignments to a reference and the targeted
reference regions used in the workflow.
A manifest file is required for the following analysis workflows:
} Custom Amplicon—The manifest file for the Custom Amplicon workflow is
provided by Illumina with your custom assay (CAT) and uses a *.txt file format.
You can obtain a manifest file for your TruSeq Custom Amplicon experiment
from the Illumina website. Log in to MyIllumina, and click Custom Products.
From the options for TruSeq Custom Amplicon, click Product Files.
} PCR Amplicon—The manifest file for the PCR Amplicon workflow is generated
using the Illumina Experiment Manager and uses a *.AmpliconManifest file
format. The genome reference must be specified in the manifest file. For more
information see the Illumina Experiment Manager User Guide, Part # 15031335.
} Enrichment—Like the PCR Amplicon workflow, the manifest file for the
Enrichment workflow is generated using the Illumina Experiment Manager and
uses a *.AmpliconManifest file format. For more information see the Illumina
Experiment Manager User Guide, Part # 15031335.
14
Parameter
Description
Name of manifest file
Name of your manifest file.
More than one manifest can be specified. Multiple manifests
are defined as keys in the first column in the Manifest
section of the sample sheet, such as A and B.
Two types of manifest formats are used for sequencing and
analysis depending on the analysis workflow specified in
the sample sheet:
• Custom Amplicon workflow—Requires the format *.txt.
Including the file extension (*.txt) in the sample sheet is
optional.
• PCR Amplicon or Enrichment workflow—Requires the
format *.AmpliconManifest. Including the file extension
(*.AmpliconManifest) in the sample sheet is
recommended.
Part # 15028392 Rev. E
Manifests
Figure 2 Sample Sheet Example with Two Manifests Specified
Load your manifest file onto the instrument using the Manage Files feature to the
specified location. For more information see the MiSeq System User Guide, Part #
15027617.
MiSeq Sample Sheet Quick Reference Guide
15
Analysis Workflows
The analysis workflow is a procedure performed by MiSeq Reporter. One analysis
workflow must be specified in the sample sheet for each sequencing run. When the
run is complete, MiSeq Reporter performs secondary analysis according to that
workflow.
16
Analysis Workflow
Applications
Output
Assembly
Assembles small genomes from reads
without the use of a genomic
reference.
If a genomic reference is specified, a
dot-plot is generated with respect to
the reference of genome position vs
assembled contigs.
Contigs in FASTA
format.
Custom Amplicon
Sequences TruSeq Custom amplicon
from probes targeting particular
genome positions (up to approx. 384
loci from up to approx. 96 samples).
Aligns reads against a manifest file
specified in the sample sheet. Two
manifests can be specified: one for the
control and one for the sample.
Aligned reads in
BAM format.
Variant calls in VCF
format.
Enrichment
Analyzes DNA that has been enriched
for particular target sequences using a
pulldown assay.
Aligns reads against the whole
genome reference, and performs
variant analysis for the regions of
interest specified in the manifest file.
Reporting accumulates coverage and
other statistics for each amplicon.
Generate FASTQ
Generates intermediate analysis files in
FASTQ format and then exits the
workflow. Enables the use of thirdparty tools to analyze sequencing data.
Sequence files in
FASTQ format.
Library QC
Aligns reads against reference
genomes specified in the sample sheet,
and then generates a sample report in
LibraryQC.html.
Per-sample
summary statistics.
Metagenomics
Classifies bacteria from a metagenomic
sample by amplifying specific regions
in 16S ribosomal RNA. No genomic
reference is required for the
metagenomics workflow. Reads are
classified using a database of 16S rRNA
data. For paired-end runs, each cluster
is classified using base calls from both
reads.
Read classifications
by taxonomic group
from kingdom
through genus.
Aligned reads in
BAM format.
Variant calls in
VCF format.
Part # 15028392 Rev. E
Applications
Output
PCR Amplicon
Sequences any number of PCR
amplicons that have been fragmented
using Nextera tagmentation.
Aligns reads against the reference
genomes specified in the sample sheet.
Performs variant analysis for the
regions of interest specified in the
manifest file.
Aligned reads in
BAM format.
Variant calls in
VCF format.
Resequencing
Sequences a small genome, such as E.
coli.
Aligns reads against the reference
genomes specified in the sample sheet
and performs variant analysis.
Aligned reads in
BAM format.
Variant calls in VCF
format.
Small RNA
Sequences miRNA.
Aligns reads against databases for
contaminants, mature miRNA, small
RNA, and a genomic reference, in that
order.
Reports on the
relative abundance
of each record.
MiSeq Sample Sheet Quick Reference Guide
17
Analysis Workflows
Analysis Workflow
Editing the Sample Sheet for Custom Primers
You can set up your sample sheet for custom primers using the Illumina Experiment
Manager v1.2, or later. For more information, see the Illumina Experiment Manager User
Guide, Part # 15031335.
If you edit your sample sheet manually, list the custom primers in the Settings section
of the sample sheet, as follows:
1
Open your sample sheet for editing in Excel or Notepad.
2
Locate the Settings section. If your sample sheet does not have a Settings section,
create one after the Reads section and before the Data section.
Figure 3 Example of Custom Primers in Settings Section
3
In the Settings section, add a line for each of your custom primers using the
following example:
Settings
CustomRead1PrimerMix
CustomIndexPrimerMix
CustomRead2PrimerMix
C1
C2
C3
Make sure that you enter C1 for the Read 1 primer, C2 for the Index Read primer,
and C3 for the Read 2 primer.
You only need to add an entry for the custom primers you are using. For
example, if you are using Illumina primers for Read 1 and Read 2, and a custom
primer for the Index Read, edit the Settings section to list only one custom primer,
as follows:
Settings
CustomIndexPrimerMix
C2
For information about preparing and loading custom primers, see the MiSeq
System User Guide, Part # 15027617.
18
Part # 15028392 Rev. E
During the run setup steps using MCS, the software automatically looks for a sample
sheet with a name that matches the barcode number of the reagent cartridge loaded
on the instrument. Therefore, Illumina recommends that you name your sample sheet
with the barcode number of the reagent cartridge that you will use for your run,
followed by *.csv extension. The barcode number is located on the reagent cartridge
label directly below the barcode.
In the following example, the sample sheet name is MS2000006-500.csv. You do not
need to include the kit version in the sample sheet name.
Figure 4 Reagent Cartridge Label
If you do not know which reagent cartridge you will use for your run, name your
sample sheet according to your preference followed by *.csv. When the software
cannot locate your sample sheet during the run setup steps, you can browse to the
appropriate sample sheet.
MiSeq Sample Sheet Quick Reference Guide
19
Naming the Sample Sheet
Naming the Sample Sheet
Notes
For technical assistance, contact Illumina Technical Support.
Table 1 Illumina General Contact Information
www.illumina.com
Illumina Website
[email protected]
Email
Table 2 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
Contact Number
North America
1.800.809.4566
Italy
800.874909
Austria
0800.296575
Netherlands
0800.0223859
Belgium
0800.81102
Norway
800.16836
Denmark
80882346
Spain
900.812168
Finland
0800.918363
Sweden
020790181
France
0800.911850
Switzerland
0800.563118
Germany
0800.180.8994
United Kingdom
0800.917.0041
Ireland
1.800.812949
Other countries
+44.1799.534000
MSDSs
Material safety data sheets (MSDSs) are available on the Illumina website at
www.illumina.com/msds.
Product Documentation
Additional product documentation in PDF is available for download from the
Illumina website. Go to www.illumina.com/support and select a product. A
MyIllumina login is required. To register for a MyIllumina account, please visit
my.illumina.com/Account/Register.
MiSeq Sample Sheet Quick Reference Guide
Technical Assistance
Technical Assistance
Illumina
Headquartered in San Diego, California, U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com