Create TruSeq Amplicon Sample Plates and Sample Sheets Quick Reference Card (1000000006182 v01)

Create TruSeq Amplicon Sample Plates and Sample Sheets
with IEM
For Research Use Only. Not for use in diagnostic procedures.
(A or B). Each unique DNA sample must have a unique
sample ID for pool A and pool B.
} Enter pool A sample IDs in cells A01 to A06, B01 to
B06, C01 to C06, and so on to populate the left side
of the plate.
} Enter pool B sample IDs in cells A07 to A12, B07 to
B12, C07 to C12, and so on to create a mirror image
on the right side of the plate.
} Enter the same Sample Name for matched pool A and
pool B samples.
This quick reference card describes how to use the Illumina®
Experiment Manager (IEM) to create a sample plate for
TruSeq® Custom Amplicon, TruSeq Custom Amplicon Low
Input, and TruSeq Amplicon - Cancer Panel. It also describes
how to create and edit sample sheets compatible with your
Illumina sequencing system and analysis software.
For more information about how to use the IEM application
and definitions of the sample sheet applications, see the
Illumina Experiment Manager User Guide (part # 15031335). New
or less experienced users are advised to read the guide before
using this quick reference card.
For information about other workflow options, see the MiSeq
Reporter TruSeq Amplicon Workflow Guide (document #
15042314).
Create a Sample Plate
Open the IEM software.
2
On the IEM main screen, select Create Sample Plate.
3
On the Library Prep Kit Selection screen, select TruSeq
Amplicon, and then select Next.
4
On the Assay Parameters screen, do the following:
a
b
c
On the Plate Samples screen, select the Table tab.
6
[Standard protocol] Do the following:
a
b
c
d
7
Enter a unique sample ID.
Specify the index adapter you intend to use for each
Index Read.
Enter the name of the Illumina Manifest file for your
assay or control. Do not include the .txt file extension
in the name.
To capture more information about the plate, enter a
sample name, project, and description.
[Dual strand protocol] Do the following:
} For each well that contains a sample, enter a unique
sample ID to identify the DNA sample and the pool Document # 1000000006182 v01
Sample 1—Pool A
Sample001A
DNASample001
Sample 1—Pool B
Sample001B
DNASample001
8
[Optional] Select the Plate Graphic tab to view the sample
ID and index adapter in each well.
} [Dual strand protocol] Make sure that pool A samples
appear in columns 1 to 6 and pool B samples appear in
columns 7 to 12.
} To copy an image of the sample plate, select Copy to
Clipboard, and then paste the image into any graphicsenabled program (Microsoft® Word, PowerPoint, Paint,
and so on).
} To print an image of the sample plate, select Print.
9
Select Finish, and save the sample plate file with a
*.amp28.plt file extension.
In the Unique Plate Name field, type a name for the
sample plate.
In the Index Reads field, 2 is automatically selected.
Select Next.
5
Sample Name
} Select the Index1 (I7) and Index2 (I5) values. The index
pair must be unique for each well.
} Enter the name of the Illumina Manifest file for pool A
samples and for pool B samples. Do not include the .txt
file extension in the name.
} To capture more information about the plate, enter
Sample Project and Description.
A sample plate is not required to generate a sample sheet, but
it can be useful for organizing samples.
1
Sample ID
Create a Sample Sheet
This section provides instructions for creating a sample sheet
for the analysis of MiSeq® or NextSeq® sequencing data.
Create a MiSeq-Compatible Sample Sheet
1
On the IEM main screen, select Create Sample Sheet.
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Create TruSeq Amplicon Sample Plates and Sample Sheets with IEM
2
On the Instrument Selection screen, select MiSeq, and
then select Next.
3
On the MiSeq Application Selection screen, select the
category and application for your kit and protocol design:
} [Standard protocol] The following combinations are
supported:
Category
Targeted Resequencing
Other
Application
TruSeq Amplicon
FASTQ Only
} [Dual strand protocol] Select Other and FASTQ Only.
4
Select Next.
5
On the Workflow Parameters screen, select your run
settings as follows:
a
b
c
d
e
f
g
6
In the Reagent Cartridge Barcode field, enter the
barcode number of the MiSeq reagent cartridge.
From the Library Prep Kit drop-down menu, select
TruSeq Amplicon.
In the Index Reads field, 2 is automatically selected.
To capture more information about the run, type an
experiment name, investigator name, and description.
Select the date.
The Paired End read type is automatically selected.
Select the number of cycles for each read in your
sequencing run, plus 1. Make sure that you select the
same number of cycles for both reads.
Select or clear the Workflow-Specific Settings checkboxes,
as desired.
} If you are creating a sample sheet for the TruSeq
Amplicon application for TruSeq Amplicon - Cancer
Panel, make sure that the Use Somatic Variant Caller
checkbox is selected.
7
Select Next.
8
Add sample information to the sample sheet. Choose from
the following:
} To import an existing sample plate, go to step 9.
} [Standard protocol] To enter sample information
manually, go to step 10.
} [Dual strand protocol] To enter sample information
manually, go to step 11.
9
Import an existing sample plate.
a
b
c
Clear the Maximize checkbox.
In the Sample Plate area, select Select Plate, browse to
and select the sample plate, and then select Open.
Select the wells you want to include in this run, or
select Select All to include all wells.
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d
Select Add Selected Samples.
10 [Standard protocol] Enter sample information manually.
} Select Add Blank Row.
} Enter the Sample ID.
} Select the Index1 (I7) and Index2 (I5) values.
} Enter the sample name and plate and well information,
as desired.
} If you are creating a sample sheet for the TruSeq
Amplicon application:
} Enter the name of the Illumina Manifest file for your
assay or control. Do not include the .txt file extension
in the name.
} From the Genome Folder drop-down menu, select
the location where the reference genome files (*.fasta)
are saved.
11 [Dual strand protocol] Enter sample information
manually.
} Select Add Blank Row. Add 2 rows per DNA sample,
1 row for each pool, A and B.
} Enter a unique sample ID to identify the DNA sample
and the pool (A or B). Each unique DNA sample must
have a unique sample ID for pool A and pool B.
} Enter the same Sample Name for matched pool A and
pool B samples.
Sample ID
Sample Name
Sample 1—Pool A
Sample001A
DNASample001
Sample 1—Pool B
Sample001B
DNASample001
} Select the Index1 (I7) and Index2 (I5) adapters from the
drop-down lists. The index pair must be unique for
each well.
} Select the Illumina Manifest file for pool A samples and
for pool B samples. Do not include the .txt file extension
in the name.
} From the Genome Folder drop-down menu, select the
location where the reference genome files (*.fasta) are
saved.
} To capture more information about the run, enter Plate,
Well, Sample Project, and Description.
12 Select Finish, and save the sample sheet file to the default
location.
13 When prompted to view the sample sheet in Microsoft
Excel, select Yes to review it or No to bypass the review
and exit the sample sheet wizard.
Document # 1000000006182 v01
Create TruSeq Amplicon Sample Plates and Sample Sheets with IEM
Create a NextSeq-Compatible Sample Sheet
1
On the IEM main screen, select Create Sample Sheet.
2
On the Instrument Selection screen, select NextSeq, and
then select Next.
3
On the NextSeq Application Selection screen, select the
NextSeq FASTQ Only application, and then select Next.
4
On the Workflow Parameters screen, select your run
settings as follows:
a
b
c
d
e
f
g
In the Reagent Kit Barcode field, enter the reagent kit
ID from the label on box 1 or box 2 of the SBS kit.
From the Library Prep Kit drop-down menu, select
TruSeq Amplicon.
In the Index Reads field, 2 is automatically selected.
To capture more information about the run, type an
experiment name, investigator name, and description.
Select the date.
In the Read Type field, select Paired End.
Select the number of cycles for your sequencing runs,
plus 1. Select the same number of cycles for both
reads.
5
Select Next.
6
Add sample information to the sample sheet. Choose from
the following:
} To import an existing sample plate, go to step 7.
} [Standard protocol] To enter sample information
manually, go to step 8.
} [Dual strand protocol] To enter sample information
manually, go to step 9.
7
Import an existing sample plate.
a
b
c
d
Clear the Maximize checkbox.
In the Sample Plate area, select Select Plate, browse to
and select the sample plate, and then select Open.
Select the wells you want to include in this run, or
select Select All to include all wells.
Click Add Selected Samples.
8
[Standard protocol] Enter sample information manually.
} Select Add Blank Row.
} Enter the Sample ID.
} Select the Index1 (I7) and Index2 (I5) values.
} To capture more information about the run, enter Plate,
Well, and Sample Project, and Description.
9
[Dual strand protocol] Enter sample information
manually.
} Select Add Blank Row. Add 2 rows per DNA sample,
1 row for each pool, A and B.
} Enter a unique sample ID to identify the DNA sample
and the pool (A or B). Each unique DNA sample must
have a unique sample ID for pool A and pool B.
} Enter the same Sample Name for matched pool A and
pool B samples.
Sample ID
Sample Name
Sample 1—Pool A
Sample001A
DNASample001
Sample 1—Pool B
Sample001B
DNASample001
} Select the Index1 (I7) and Index2 (I5) adapters from the
drop-down lists. The index pair must be unique for
each well.
} Select the Illumina Manifest file for pool A samples and
for pool B samples. Do not include the .txt file extension
in the name.
} To capture more information about the run, enter Plate,
Well, Sample Project, and Description.
10 Select Finish, and save the sample sheet file.
11 When prompted to view the sample sheet in Microsoft
Excel, select Yes to review it or No to bypass the review
and exit the sample sheet wizard.
Technical Assistance
For questions, see Illumina Experiment Manager on
www.illumina.com. If you do not find the information you
need there, contact Illumina Technical Support by email or
phone.
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© 2016 Illumina, Inc. All rights reserved.
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iScan, iSelect, MiniSeq, MiSeq, MiSeqDx, MiSeq FGx, NeoPrep,
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TruSeq, TruSight, Understand Your Genome, UYG, VeraCode, verifi,
VeriSeq, the pumpkin orange color, and the streaming bases design are
trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other
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their respective owners.
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