Design Content | Prepare Library | Sequence | Analyze Data

Optimizing RNA input to detect gene fusions
with TruSight™ Oncology 500 High-Throughput
TruSight Oncology 500 High-Throughput and the NovaSeq™ 6000 System provide a sensitive
solution for detecting gene fusions.
Introduction
Gene fusions, known to drive tumorigenesis across many tumor types,
are important cancer-related biomarkers included in key guidelines andclinical studies. Currently, more than 20,000 gene fusions have been
discovered across 33 cancer types.1 Many of these oncogenic fusion
genes, such as NTRK, ALK, and RET, have multiple fusion partners,making it imperative that tumor profiling assays detect these genes
agnostic of the fusion partner. To meet this challenge, Illumina offers
TruSight Oncology 500 and TruSight Oncology 500 High-Throughput.
These assays provide a proven next-generation sequencing (NGS)
solution, using both RNA and DNA, for accurate analysis of multiple
tumor types and biomarkers, including gene fusions, in a single assay.

Fusion-agnostic strategy
TruSight Oncology 500 and TruSight Oncology 500 High-Throughputtake advantage of a hybrid-capture strategy to capture target
fragments. This strategy uses probes that align to the gene of interest
with sequencing proceeding in the 3' and 5' directions, capturing any
adjacent sequence. Using this method, it’s possible to identify andcharacterize novel and known fusions agnostic of the fusion partner
without additional confirmation steps (Figure 1). In addition, hybridcapture offers the sensitivity needed when working with degradedsamples, such as FFPE tissue.
Other assays employ an amplicon-based approach that uses primer
pairs to target a specific gene, or sequence, of interest. However,
amplicon assays can be prone to dropouts when mutations occur
at primer binding sites, yielding unreliable results and often requiringconfirmatory testing.
In addition, while effective for identifying known fusions, the primer-pairdesign of traditional amplicon methods prevents them from amplifying
novel events (Figure 1). Some amplicon assay providers havedeveloped an indirect approach to uncovering novel fusions that relies
on detecting a 5'/3' sequencing coverage imbalance. This method
increases the likelihood of identifying a novel fusion, but requires aconfirmatory test, such as fluorescent in situ hybridization (FISH),adding time and expense to a study. Furthermore, the fusion partner
will remain uncharacterized.

Assay differences
TruSight Oncology 500 and TruSight Oncology 500 High-Throughput
provide the same content and use the same hybrid-capture strategy
for tumor profiling. The differences lie in the intended sequencing
platforms, sample throughput, some reagents, minor workflow
changes, and secondary analysis (Table 1).

Figure 1: The hybrid-capture strategy detects novel fusions missed by anamplicon-based approach—An amplicon-based can use an indirect method
based on 5'/3' imbalance to detect a novel fusion. This approach typicallyrequires a confirmatory test and does not characterize the novel fusion partner.

Table 1: Differences between TruSight Oncology 500 and
TruSight Oncology 500 High-Throughput
TruSight Oncology 500

TruSight Oncology 500
High-Throughput

SystemNextSeq 550 and
NextSeq 550DxaSystemsNovaSeq 6000 System;compatible with NextSeq
550 and 550Dxa Systems

Run cycle

2 × 101 bp, 2 × 8 bp
(indexing)2 × 101 bp, 2 × 10 bp(indexing)

Throughput

up to 8 samples per run

16-192 samples per run

Library prep indexes

SUA1 (Short Universal
Adapters 1), UMI1
(Unique MolecularIdentifier 1), UPxx
(Unique Index Primer)

IDT for Illumina UMI DNA
Index Anchors–Set A andSet B

Enrichment buffer

TCA1 (Target Capture
Additives 1) and TCB1
(Target Capture Buffer 1)

EHB (Enrichment
Hybridization Buffer)

Recommended RNA
input amount40 ng

40-80 ng

End-repair/A-tailingtemperature

30°C/72°C

30°C/65°C

Library prep SPRI
elution volume

27.5 µl22.5 µl

Hybridization reaction

20 µl library + 15 µl
TCB1 + 10 µl TCA1+ 5 µl probes

20 µl library + 25 µl EHB+ 5 µl probes

Variant calling

Local Docker or oninstrument with Local
Run Manager (LRM)

Local Docker

Parameter
Sequencing system

Reagents

Workflowa. NextSeq 550Dx System in Research Mode

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

Pub. No. 1170-2020-003-A | 1Design Content | Prepare Library | Sequence | Analyze DataRNA input amounts
Both assays achieve highly sensitive results; however, a lower countof unique reads covering a gene fusion has been observed whenusing TruSight Oncology 500 High-Throughput. While 40 ng RNA canbe sufficient for both assays in specific situations (Table 2), Illumina
recommends using 40-80 ng RNA with TruSight Oncology 500
High-Throughput. It is highly recommended to use 80 ng RNA whenworking with low-quality RNA.
Although the update from the NextSeq™ System to the NovaSeq™
System is significant, this change is not causing the difference inRNA input performance. Both assays were run on both sequencing
systems, producing concordant results between platforms fordetecting RNA fusions. These studies rule out the sequencing platform
as the source of the performance difference.
To ensure higher levels of multiplexing, TruSight Oncology 500 HighThroughput has adopted a more diverse set of indexes, allowing forsequencing of up to 192 samples (DNA + RNA) in a single run. To
achieve this, the length of the index sequences was increased andcorresponding buffers and UMI index anchors were revised. During
development of TruSight Oncology 500 High Throughput, the input
requirement was increased to ensure that all relevant fusions weredetected using the modified library preparation chemistry.

Sequencing
Prepared libraries were sequenced on a NovaSeq 6000 S2 flow cell ata read length of 2 × 101 bp at full multiplexing capacity.
Analysis
Data was processed locally using the TruSight Oncology 500 v2.1
Local App. For RNA fusions, the count of unique supporting reads
for the fusion event was considered, with a minimum of 5 uniquesupporting reads required to make a positive fusion call.

Results
Comparing TruSight Oncology 500 toTruSight Oncology 500 High-Throughput
Both TruSight Oncology 500 and TruSight Oncology 500 HighThroughput detected RNA fusions and splice variants in a control
sample known to contain multiple RNA variants (Table 2). In mostcases, the fusion or splice variant was detected above the threshold
cutoff (5 unique supporting reads for fusions and 10 unique supporting
reads for splice variants) for calling the variant.
Table 2: A comparison of the number of unique supporting
sequencing reads generated across assays
TruSight
Oncology 500

Optimizing results
Even with the decrease in read coverage, TruSight Oncology 500
High-Throughput detects gene fusions with high analytical sensitivity.
To achieve the best results, it is important to optimize RNA input
amounts, using a range from 40-80 ng, depending on RNA sample
quality. This technical note demonstrates the impact of RNA inputs
ranging from 20-80 ng on the number of sequencing reads produced
when using TruSight Oncology 500 High-Throughput on the NovaSeq
6000 System.

Methods
TruSight Oncology 500 vs TruSight Oncology 500
High-Throughput
Analytical performance between TruSight Oncology 500 and TruSight
Oncology 500 High-Throughput was compared using an in-house
control sample containing a mix of pre-fragmented cell lines withmultiple RNA variants to mimic clinical FFPE RNA samples. TruSight
Oncology 500 was sequenced on the NextSeq 550 System. TruSight
Oncology 500 High-Throughput was sequenced on the NovaSeq
6000 System.

TruSight Oncology 500
High-Throughput

Fusion

40 ng

20 ng

40 ng

60 ng

80 ng

ABL1-BCR

10618424374

ALK-EML4

160778ALK-TRMT61B

8053

7

COL14A1FGFR2607

91

224449511

ERG-TMPRSS2662

108

228

338

416

PTPN3-ALK

23381511

RET-CCDC698

16529078

ROS1;GOPCSLC34A2

215

3684

99

117

TACC3-FGFR3162

61

135

192

111

RPS6KB1VMP125775

183

200

234

207

3275

92138

Splice variant
MET exon 14
skipping

Comparison performed using an in-house control sample. TruSight Oncology 500 wassequenced on the NextSeq 550 System. TruSight Oncology 500 High-Throughput wassequenced on the NovaSeq 6000 System.

RNA titration for TruSight Oncology 500 High-Throughput
Sample preparation
RNA was isolated from formalin-fixed paraffin-embedded (FFPE) tissue
samples using the QIAGEN AllPrep DNA/RNA Mini Kit (QIAGEN,Catalog no. 80204). RNA amounts of 20 ng, 40 ng, 60 ng, or 80 ng
from the same parent same were added to 8.5 µl buffer and usedin library preparation with TruSight Oncology 500 High-Throughput,
following the provided assay protocol.

2 | Pub. No. 1170-2020-003-A

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

Design Content | Prepare Library | Sequence | Analyze Data

Figure 2: Total On-Target Reads increase with input RNA amounts—For most samples, an increase was in Total On-Target Reads, which measures the number ofreads specific to a region, as the amount of input RNA increased. The solid line represents 9 million reads, which is the recommended QC performance metric.

RNA titration for TruSight Oncology 500 High-Throughput

Table 3: Fusion detection at varying RNA input levels

Quality control
Post sequencing, all samples used in this study passed the following
quality control (QC) standards: Median CV Gene 500× ≥ 0.93, MedianInsert Size ≥ 80 bp, and Total On-Target Reads ≥ 9 million. Thesemetrics were not heavily impacted by the decrease in the input
requirement; however, in most cases, the number of Total On-Target
Reads, which measures the number of reads specific to the region ofinterest, increased as the amount of input RNA increased (Figure 2).

FusionRNA input amounts
This study focused on a subset of RNA fusions with clinical relevance.
It is important to note that the protocol recommends using 40-80 ng
input RNA with TruSight Oncology 500 High-Throughput to obtain
optimal results. Many fusions were detected at a level passing thethreshold cutoff (minimum of 5 reads) with as little as 20 ng input RNA,
while all passed with the recommended 80 ng input (Table 3). Fusions
were detected agnostic of the fusion partner; targeted RNA sequenceis bolded. This allows researchers using the TruSight Oncology 500
assays to generate data on all fusion partners in a given sample. Twotargeted splice variants were also successfully detected as part of thestudy (Table 4).

No. unique supporting reads

TissueDetected

20 ng

40 ng

60 ng

80 ng

BRCA2-NRXN3√6336084

Bone

FLT3-GTF2F2√091014

Bone

FLT3-LCP1√0123247

Bone

FLT3-SMOX√

21507254

Bone

FLT3-VWA8√12295169

Bone

EGFR-METTL1√

29258471

Brain

EGFR-RAB3IP

√

75919

Brain

MYC-MRPL13√

7273552

Breast

MYC-STK3√

7113928

Breast

CCDC170-ESR1

√

27122

59168

KidneyALK-EML4√11152140Lung

ALK-MRPL33;
BRE√

02166Lung

ROS1;GOPCCD74√0057Lung

ROS1;GOPCCD74√4510492141Lung

ROS1;GOPCENC1√23325393Lung

ROS1;GOPCHEXB√0007Lung

ROS1;GOPCARSI√2574Lung

ALK-BRE√

1975112128

Sarcoma

EWSR1-ATF1√6193032Sarcoma

EWSR1-CBY1√

17443097

Sarcoma

NTRK3-SEMA6A√

1071625Skin

RET-NCOA4√

537478154

Thyroid

ANKUB1;RNF13ETV5;DGKG√11294572Uterus

BRCA1-MPP2√5252829Unknown

Data generated using RNA isolated from FFPE samples of the indicated tissue type andsequenced using TruSight Oncology 500 High-Throughput on the NovaSeq 6000 System.
Target RNA sequence is bolded. Duplicate fusions are from unique samples.

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

Pub. No. 1170-2020-003-A | 3

Design Content | Prepare Library | Sequence | Analyze Data
Table 4: Splice variant detection at varying input levels
Splice variantNo. unique supporting reads
40 ng

60 ng

80 ng

TissueDetected

20 ng

EGFR v3

√343

567

884

937

EGFR v3

√802

1249

1614

2049

Brain

ARv7

√

11263846

BreastBrain

Data generated using RNA isolated from FFPE samples of the indicated tissue type andsequenced using TruSight Oncology 500 High-Throughput on the NovaSeq 6000 System.
Duplicate fusions are from unique samples.

SummaryTruSight Oncology 500 High-Throughput and the NovaSeq 6000
System offer a sensitive solution for gene fusion detection. While it is in
the best interest of each lab to test varying levels of RNA to optimize
results, Illumina recommends using 40-80 ng RNA to detect variants
expressed at mid to high levels.* When sufficient RNA is available, 80ng input helps maximize analytical sensitivity for fusions present at very
low concentrations.

References
1.

*Hu X, Wang Q, Tang M, et al. TumorFusions: an integrative resource
for cancer-associated transcript fusions. Nucleic Acids Res. 2018;
46:D1144-D1149.

In-house assay validation was performed using 80 ng RNA input.

Illumina • 1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com© 2020 Illumina, Inc. All rights reserved. All trademarks are the property of Illumina, Inc. or their respective owners. For specific trademark
information, see www.illumina.com/company/legal.html. Pub. No. 1170-2020-003-A QB9628

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

Pub. No. 1170-2020-003-A | 4