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TruSight RNA Pan-Cancer Panel
Comprehensive assessment of cancer-related RNA transcripts and fusion detection in FFPEtissues and other oncology samples.
HighlightslComprehensive View of Cancer Pathways
Illumina RNA enrichment chemistry enables interrogation ofgene expression levels, variants, and gene fusions

lLow-Input Protocol for Most Sample Types
Optimized for sequencing RNA from many sample types,
including formalin-fixed, paraffin-embedded (FFPE) tissues

lFocus on Relevant Transcripts and Fusion GenesIndustry validated content for a comprehensive view of 1,385
cancer-related RNA transcripts

lEconomical, Targeted RNA-Seq on a Desktop
Sequencer
Enables RNA sequencing on the MiniSeq™ or MiSeq®
Systems for affordable, sensitive analysis

Introduction
To date, at least 138 known driver genes for cancer have been
discovered.1 However, large numbers of variants are often detectedin DNA of unexpressed genes, highlighting that RNA expression canprovide key insights into the functionally relevant genes in cancer.
Cancer can arise from epigenetic changes, expression levelchanges, and gene fusions that are undetectable by standard
sequencing.2,3 RNA sequencing (RNA-Seq) using next-generation
sequencing (NGS) offers the ability to capture all relevant transcripts
in a single assay. Compared to traditional array-based approaches,targeted RNA-Seq is a highly sensitive approach, with a broader
dynamic range, that robustly detects RNAs of low abundance. In
addition, RNA-Seq can identify gene fusions from both known andnovel fusion gene partners.
To help clinical researchers gain a deeper understanding of the gene
expression patterns in cancer classification and progression, theTruSight RNA Pan-Cancer Panel offers in-depth assessment ofcancer-related RNA transcripts, including measurement ofexpression levels and detection of fusion genes. The panel includes
1,385 genes that have been cited in public databases and implicatedin cancer, including solid tumors, soft tissue cancers, andhematological malignancies. TruSight RNA Pan-Cancer Panel
enables cost-effective analysis of limited and degraded samples,such as FFPE tumor tissue, on a desktop sequencer.

For Research Use Only. Not for use in diagnostic procedures.

Comprehensive Coverage of Relevant Genes
The content of the TruSight RNA Pan-Cancer Panel represents
1385 genes implicated in cancer pathways (Table 1). In a single
assay, researchers can assess all relevant RNA transcripts for
multiple cancer types, regardless of origin. The focus on a subset ofrelevant genes enables RNA-Seq with high sensitivity on a desktop
sequencer, allowing cost effective access to NGS for any lab.
TruSight RNA Pan-Cancer Panel can be used for gene expression
profiling and variant calling, highlighting functionally relevant
mutations. The panel also detects fusion genes that most DNApanels would not detect. The TruSight RNA Pan-Cancer Panelprovides a comprehensive assessment of cancer-related RNAtranscripts for a more detailed view of cancer biology.
Table 1: Coverage Details
Parameter

Value

Number of target genes

1,385

Targeted exonic regions

21,043

Number of probese

57,010

Simple, Scalable Workflow
The TruSight RNA Pan-Cancer workflow first creates RNA-Seq
libraries. This method adds unique oligonucleotide indexes to eachlibrary, tagging them for downstream multiplexed sequencing(Figure 1A). Libraries are hybridized to biotin-labeled probes specificfor targeted RNA regions (Figure 1B). These targets are then captured
by adding streptavidin beads that bind to the biotinylated probes(Figure 1C). Magnets pull the bound fragments from solution(Figure 1D). Captured fragments are eluted from the beads and asecond hybridization and elution are performed. After amplification, atargeted library is ready for cluster generation and sequencing.
The TruSight RNA Pan-Cancer Panel is optimized to provide a simple
workflow that can be scaled according to the number of samples.
RNA samples can be multiplexed and sequenced on a desktop
system to maximize lab budgets. The integrated workflow includes
library preparation, sequencing, and easy data analysis using
BaseSpace ® Apps for RNA (Figure 2).

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Figure 2: Simple TruSight RNA Pan-Cancer Workflow—The TruSight RNA PanCancer Panel follows a simple workflow that is fully integrated and scalable tolarge sample numbers.

Efficient Analysis of Difficult Samples

Figure 1: TruSight Pan-Cancer Capture Chemistry—The TruSight RNA PanCancer Panel provides a simple, streamlined method for isolating targeted regionsof interest from total RNA.

Archival FFPE tissues provide a valuable repository of information forcancer research, but the RNA preserved within these samples isoften highly degraded. This degradation poses a challenge whencreating libraries for NGS. To overcome these challenges, theTruSight RNA Pan-Cancer Panel is optimized for high performancefrom both high and low quality RNA sample types, such as bonemarrow or FFPE tumor tissue. Libraries can be prepared from as little
as 10 ng total RNA, or 20 ng FFPE RNA. This low input requirement
makes the TruSight RNA Pan-Cancer Panel ideal for reliable targeted
analysis of limited samples (Figure 3).

Figure 3: Highly reproducible expression data obtained from poor quality FFPE samples—Degraded FFPE RNA samples of poor, medium, and high quality (as determinedby DV 200 values) were sequenced and data reproducibility measured by R-squared of technical replicate FPKM. DV 200 = the percentage of RNA fragments > 200 nucleotides.
FPKM = fragments per kilobase of transcript per million mapped reads. Each error bar is constructed using the minimum and maximum data values.

For Research Use Only. Not for use in diagnostic procedures.

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Highly Sensitive and EconomicalTargeted Sequencing
By focusing on key genes of interest, targeted sequencing enables
researchers to study high-value content enriched with cancerassociated transcripts, while retaining the ability to discover novel
somatic variants and gene fusions. This approach yields moresequencing reads for the regions of interest, delivering expression
level quantification with the sensitivity needed to detect raretranscripts and fusions. Gene fusion detection is highly quantitative,
as shown with spike-in experiments using synthetic fusion constructs(Figure 4). Novel fusions can be detected even when one of thefusion partners is not targeted in the panel (Figure 5). With thefocused content of the TruSight RNA Pan-Cancer Panel, RNA-Seq is
now possible on the Illumina family of desktop sequencers.
Sequencing focused content generates a smaller data set with easierdata analysis and data management. TruSight RNA Pan-Cancer
data sets can be analyzed using the new RNA-Seq Alignment app in
the BaseSpace environment (Table 2). Providing expert-preferreddata analysis tools packaged in an intuitive interface designed forbiologists, the RNA-Seq Alignment app provides gene expression
profiles, identifies gene fusions, and reports single nucleotide variants
and small indels. Simple-to-follow prompts guide users through theentire process, starting from selecting files generated by thesequencer, to filtering analyzed data, and visualizing results(Figure 6).

Figure 4: Quantitative detection of synthetic fusions—In-vitro transcribed fusions
were spiked into 10 ng MCF7 control RNA, and the number of fusion-supporting
reads is shown. Fusion supporting reads decrease linearly with decreasing spike-in
amounts and can be detected as low as concentrations equivalent to 1x10-7
pmoles, or ~13 fusion copies per cell (based on 500 cells/10 ng input).

Table 2: Features of RNA-Seq Data Analysis App
BaseSpace App

Description• RNA-Seq alignment using STAR-Manta,
and cSNP calling using Isaac Variant Caller• Works with many samples, including fresh,frozen, and FFPE• Results seamlessly integrated into theCufflinks Assembly and DifferentialExpression AppRNA-Seq Alignment• Read mapping, variant calling (SNVs andsmall indels), and gene fusion calling withSTAR-MantaFigure 5: Identification of novel gene fusion partners—(A) The size distribution of total RNA from 3 different FFPE samples derived from melanoma are shown on aBioanalyzer. (B) Table shows the detection of the gene fusion partner in FFPE samples when only one gene (highlighted) of the fusion pair is targeted.

For Research Use Only. Not for use in diagnostic procedures.

P rep are Li b rary | Seq uence | Analyze DataFigure 6: Screenshot of BaseSpace software—The RNA-Seq Alignment App provides easy access to sample results including a report of detected fusion genes.
Highlighted genes represent those that the panel targets.

SummaryThe TruSight RNA Pan-Cancer Panel enables comprehensive
detection of gene fusions and gene expression changes, providing
researchers with a focused view of the functionally relevant changes
occurring in cancer. The panel is compatible with FFPE tissue andaccommodates as little as 10 ng fresh-frozen total RNA, or 20 ng
FFPE RNA input. The TruSight RNA Pan-Cancer Panel provides asensitive, reproducible, and economical solution for studies ofexpression dynamics and functional mechanisms in cancer.

Learn More
For more information about the TruSight RNA Pan-Cancer Panel, visit
www.illumina.com/RNAPanCancer.

References
1.

Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr, Kinzler
KW. Cancer genome landscapes. Science. 2013;339:1546-1558.

2.

Green MR, Vicente-Duenas C, Romero-Camarero I, et al. Transient
expression of BCL6 is sufficient for oncogenic function and induction ofmature B-cell lymphoma. Nat Commun. 2014;5:3904
doi:10.1038/ncomms4904.

3.

Piskol R, Ramaswami G, Li JB. Reliable identification of genomic variantsfrom RNA-seq data. Am J Hum Genet. 2013;93(4):641-651

Ordering Information
Product
TruSight RNA Pan-Cancer Panel Set A
Includes library preparation consumables and oligos for 48 samples with 12 indexes
TruSight RNA Pan-Cancer Panel Set B
Includes library preparation consumables and oligos for 48 samples with 12 indexes
TruSight RNA Pan-Cancer Set A MiniSeq Kit
Includes library preparation consumables and oligos for 48 samples with 12 indexes, plus 6 MiniSeq High Output Kits (150 Cycles)

Illumina, Inc. • 1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com
For Research Use Only. Not for use in diagnostic procedures.
© 2015 Illumina, Inc. All rights reserved. Illumina, BaseSpace, MiSeq, TruSight, and the pumpkin orange color 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. 1170-2015-004-B

Catalog No.
RS-303-1002
RS-303-1003
20005611