Application Note: Sequencing

MiSeq Focus: TruSeq® Targeted RNA Expression
Solution for the MiSeq® System
Flexible, simple, and fully optimized gene expression assay for the MiSeq System.

Highlights

Figure 1: TruSeq Targeted RNA Expression Workflow• Focused and Customizable Content for Maximum
Flexibility
Combine fixed panels focused on biological pathways or
disease-specific markers with custom content• Fully Integrated RNA-to-Data Solution
Streamlined, fully integrated workflow from library preparation
to final data analysis• Highly Multiplexed for Rapid, Efficient Results
Supports 384 samples per run and 12–1000 assays persample

Introduction
TruSeq Targeted RNA Expression on the MiSeq System delivers
a simple and accurate method for measuring gene expression
levels across thousands of targets in a single sequencing run.
Library preparation begins with standard RNA isolation and reverse
transcription reactions and is completed with one of several TruSeq
Targeted RNA Expression Panel Kits (Figure 1). Dual indexing duringlibrary preparation enables highly multiplexed gene expression profilingfor 12–1,000 assays* per sample and up to 384 samples per MiSeq
System run.

The TruSeq Targeted RNA Expression assay chemistry begins withreverse transcribing cDNA from purified total RNA. Two customdesigned oligonucleotide probes with adapter sequences hybridize up
and downstream of the region of interest. An extension-ligation reaction,
followed by amplification creates a new template strand. Templates are thenPCR amplified to add indexes, creating sequence-ready libraries.

With the highly multiplexed TruSeq Targeted RNA Expression Solution,
the amount of processing time and hands-on time is significantlyreduced. In a single run, the MiSeq System can generate 25 million
reads or 25,000 datapoints (for example, with 50 multiplexed samples
and 500 assays per sample) in less than two days (Figure 2).1 An
equivalent number of data points using traditional qPCR methods
would require 65 runs and ~16 days (using 384-well plates).
Additionally, all targets are amplified in a single reaction, minimizing
potential amplification bias compared to traditional methods.
TruSeq Targeted RNA Expression on the MiSeq System leverages
Illumina sequencing by synthesis (SBS) technology, the most highly
adopted and trusted next-generation sequencing technology in theindustry.2-4 With an intuitive touch-screen interface and step-by-stepguided workflows, Targeted RNA Expression panels offer a quickand easy solution for RNA expression profiling or gene expression
array validation.

Figure 2: MiSeq SystemThe MiSeq System leverages the latest advances in SBS chemistry andthe industry’s simplest workflow.

* Assays are defined as the probe set measuring the expression of a single region of interest.

Application Note: Sequencing

Flexible Options with Fixed Panels orCustom Content
TruSeq Targeted RNA Expression Library Preparation Kits are available
with fixed or custom panels. For disease-focused expression studies,
fixed panels offer pre-designed, ready-to-use assays for commonly
studied diseases, or molecular pathways (Table 1). Fixed content
panels are ideal for profiling many samples at once, screening cell
types quickly and economically, or validating microarray results.
Beyond the validated assays provided in fixed panels, content may
be expanded on any panel by adding 12–1000 custom assays.
Using DesignStudio™, a free, online tool accessible throughMyIllumina, simply upload a list of targets and choose from over
540,000 pre-designed assays based on RefSeq gene models inhuman, rat, or mouse. By quickly building a custom panel, adding
new targets to previously ordered custom panels, or choosing oneof many fixed panels, the TruSeq Targeted RNA Expression Solution
has the flexibility to meet specific research needs.
The flexible MiSeq System provides opportunities to switch easily
from one sequencing application to another in pursuit of evolving
research goals. Beyond RNA profiling, the MiSeq System is fullycompatible with a broad range of research applications, including denovo sequencing, 16S metagenomics, small genome sequencing,amplicon sequencing, HLA Typing, and more. While the TruSeq
Targeted RNA Expression application requires only 50 bp reads, theMiSeq System can produce 2 × 300 bp read lengths for additionalapplications.

Table 1: TruSeq RNA Expression Fixed Panels

Apoptosis

Neurodegeneration

Cardiotoxicity

NFκB Pathway

Cell Cycle

Stem Cell

Cytochrome P450

TP53 Pathway

Hedgehog Pathway

Wnt Pathway

Simple, Streamlined Data AnalysisData analysis, including alignment, quantitation, and data visualization,
takes place directly on the MiSeq instrument with MiSeq Reporter(Figure 3). Pairwise comparisons for relative expression between
samples or groups of samples is simple and intuitive. Customizablesignificance thresholds allow quick identification of differentiallyexpressed targets. From library preparation to final data analysis,
the MiSeq Targeted RNA Expression Solution offers a simple, fully
integrated workflow.

Figure 3: Visualization of TruSeq Targeted RNA Expression Data using MiSeq Reporter

Data visualization with MiSeq Reporter allows easy comparison of data sets.

Application Note: Sequencing

TruSeq Targeted RNA Expression and FFPESamples

Product Specifications

TruSeq Targeted RNA Expression panels have a robust design
compatible with high to low-quality or low-quantity RNA. The small
amplicon size and short read requirements allow successful target
detection with degraded or poor-quality samples. For additional
guidance using TruSeq Targeted RNA Expression Kits with formalinfixed, paraffin-embedded (FFPE) or highly degraded samples, see
the Expression Analysis of FFPE Samples Illumina Technical Note.5The low 50 ng RNA input requirement enables TruSeq Targeted RNA
Expression studies with precious samples or low-quantity samples,such as tumor RNA.

Specification

Summary
Designed for the MiSeq system, TruSeq Targeted RNA Expression
provides accurate and efficient RNA profiling and validation for gene
expression studies. Combine fixed panels focused on biologicalpathways or disease-specific markers with custom content for
maximum flexibility. In less than two days, go from RNA sample todata with a simple, integrated workflow.

Learn MoreFor more on TruSeq Targeted RNA Expression, visit:www.illumina.com/products/truseq-targeted-rna-expression-kits.ilmn
To learn more about read budget and normalization with TruSeq
Targeted RNA Expression Panels, visit:www.illumina.com/documents/products/technotes/technote_truseq_
targ_rna_design.pdf

References1.

Illumina (2014) MiSeq system specification sheet. (www.illumina.com/documents/products/datasheets/datasheet_miseq.pdf)

2.

Nakazato T, Ohta T, Bono H. (2013) Experimental design-based functional
mining and characterization of high-throughput sequencing data in thesequence read archive. PLoS One 22;8(10): e77910.

3.

Ross MG, Russ C, Costello M, Hollinger A, Lennon NJ, et al. (2013)
Characterizing and measuring bias in sequence data. Gen Biol 14: R51.

4.

Liu L, Li Y, Li S, Hu N, He Y, et al. (2012) Comparison of next-generation
sequencing systems. J Biomed Biotechnol 2012: 251364.

5.

Illumina (2014) Expression analysis of FFPE samples. (www.illumina.com/documents/products/technotes/technote-expression-analysis-ffpe-samples.
pdf)

Database content
Target types

Value> 540,000 designs(mouse, human, rat)Gene, transcript, exon,
splice junction, cSNP, fusion

Dynamic range

5 orders of magnitude

Time to answer

1.5 daysHands-on time

4 hours

RNA quality> 200 bp unfixed or FFPE

Join the Illumina Community
With a MiSeq System in their laboratory, researchers join aworldwide community of over 100,000 scientists using Illumina
technology for their research studies. Illumina schedules
community events throughout the year, bringing researchers
together to share ideas. User group meetings, scientificsymposiums, and blog forums provide venues to discuss new
research methods and breakthrough studies.
An integral part of the Illumina community is our dedicatedservice and support team, consisting of more than 300 people
worldwide, 75% of whom have advanced degrees. Illumina
technical support begins when the MiSeq System is delivered,with Illumina scientists and engineers assisting with systeminstallation and setup, and the training of laboratory personnel.
They are there 24/7 globally to answer questions every step of
the way, giving researchers the peace of mind to focus on theirnext research study.
As researchers’ needs change, new systems are brought into
the laboratory, or new methods are undertaken, the Illumina
support and training teams are there to provide assistance. In
addition to on-site support, training courses (via webinar or at anIllumina facility) are available to bring laboratory personnel quickly
up to speed.

Application Note: Sequencing
Ordering Information
Number ofSamples

Catalog No.

48

RT-101-1001

96

RT-102-1001

48

RT-801-1001

96

RT-802-1001

48

RT-201-1010

96

RT-202-1010

48

RT-201-1009

96

RT-202-1009

48

RT-201-1003

96

RT-202-1003

48

RT-201-1006

96

RT-202-1006

48

RT-201-1002

96

RT-202-1002

48

RT-201-1001

96

RT-202-1001

48

RT-201-1008

96

RT-202-1008

48

RT-201-1005

96

RT-202-1005

48

RT-201-1007

96

RT-202-1007

48

RT-201-1004

96

RT-202-1004

TruSeq Targeted RNA Index Kit

48

RT-401-1001

TruSeq Targeted RNA Index Kit A

96

RT-402-1001

TruSeq Targeted RNA Index Kit B

96

RT-402-1002

TruSeq Targeted RNA Index Kit C

96

RT-402-1003

TruSeq Targeted RNA Index Kit D

96

RT-402-1004

Product Name
TruSeq Targeted RNA Expression Custom Components
TruSeq Targeted RNA Custom Kit

TruSeq Targeted RNA Supplemental Content
TruSeq Targeted RNA Expression Fixed Panels
TruSeq Targeted RNA Apoptosis Panel Kit

TruSeq Targeted RNA Cardiotoxicity Panel Kit

TruSeq Targeted RNA Cell Cycle Panel Kit

TruSeq Targeted RNA Cytochrome p450 Panel Kit

TruSeq Targeted RNA Hedgehog Panel Kit

TruSeq Targeted RNA Neurodegeneration Panel Kit

TruSeq Targeted RNA NFκB Panel Kit

TruSeq Targeted RNA Stem Cell Panel Kit

TruSeq Targeted RNA TP53 Pathway Panel Kit

TruSeq Targeted RNA Wnt Pathway Panel Kit
TruSeq Targeted RNA Expression Index Kits

Illumina • 1.800.809.4566 toll-free (U.S.) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com
FOR RESEARCH USE ONLY
© 2014 Illumina, Inc. All rights reserved.
Illumina, DesignStudio, Epicentre, Genetic Energy, MiSeq, Powered by Illumina, TruSeq, 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 countries. All other names, logos, and other trademarks are
the property of their respective owners.
Pub. No. 770-2014-025 Current as of 25 June 2014