The presence of circulating (cell-free) nucleic acids in the bloodstream offers a potential non-invasive approach to monitor disease status and guide treatment options. In past years, increasing interest has been shown for circulating RNA; especially circulating small RNAs for their potential application as biomarkers that may lead toward more effective diagnosis and prognosis in the future. However, widespread inconsistencies have been observed among the studies due to biases generated during sample collection, handling, RNA extraction and analysis. We have developed a complete workflow that includes blood collection, plasma preparation, circulating RNA extraction, followed by expression analysis and gene fusion detection on Ion TorrentTM Next-Generation Sequencing platforms. Blood plasma research samples from normal research samples were utilized for circulating RNA isolation following a TRIzolTM LS Reagent and mirVanaTM miRNA Isolation Kit-based method to maximize circulating RNA recovery. Ion AmpliSeqTM library preparation was performed on purified circulating RNA using either Ion AmpliSeq Transcriptome panel for expression profiling of 21K coding and non-coding genes, or an Ion AmpliSeq panel targeting fusion transcript detection from RNA. Ion AmpliSeq Transcriptome data was analyzed using ampliSeqRNA plugin in Torrent Suite™ Software. ~3000 genes were detected in cfDNA from plasma research samples with high correlation (r>0.8) observed between normal research samples. Ion Reporter™ Software was used to analyze fusion transcript panel data. Detection of fusion gene transcripts was demonstrated by spiking trace amounts of RNA from a fusion positive cell line into circulating RNA from normal research samples, indicating high sensitivity of the detection system. In summary, this study demonstrated the feasibility of gene expression profiling and gene fusion detection from circulating RNA in plasma research samples on Ion Torrent NGS platforms.

1. Jian Gu, Kristi Lea, Xingwang Fang, Hannah Saunders, Kelli Bramlett
Thermo Fisher Scientific, 2130 Woodward St., Austin, TX 78744
I. ABSTRACT
The presence of circulating (cell-free) nucleic acids in the
bloodstream offers a potential non-invasive approach to
monitor disease status and guide treatment options. In past
years, increasing interest has been shown for circulating RNA;
especially circulating small RNAs for their potential application
as biomarkers that may lead toward more effective diagnosis
and prognosis in the future. However, widespread
inconsistencies have been observed among the studies due to
biases generated during sample collection, handling, RNA
extraction and analysis.
We have developed a complete workflow that includes blood
collection, plasma preparation, circulating RNA extraction,
followed by expression analysis and gene fusion detection on
Ion TorrentTM Next-Generation Sequencing platforms. Blood
plasma research samples from normal research samples were
utilized for circulating RNA isolation following a TRIzolTM LS
Reagent and mirVanaTM miRNA Isolation Kit-based method to
maximize circulating RNA recovery. Ion AmpliSeqTM library
preparation was performed on purified circulating RNA using
either Ion AmpliSeq Transcriptome panel for expression
profiling of 21K coding and non-coding genes, or an Ion
AmpliSeq panel targeting fusion transcript detection from RNA.
Ion AmpliSeq Transcriptome data was analyzed using
ampliSeqRNA plugin in Torrent Suite™ Software. ~3000 genes
were detected in cfDNA from plasma research samples with
high correlation (r>0.8) observed between normal research
samples. Ion Reporter™ Software was used to analyze fusion
transcript panel data. Detection of fusion gene transcripts was
demonstrated by spiking trace amounts of RNA from a fusion
positive cell line into circulating RNA from normal research
samples, indicating high sensitivity of the detection system. In
summary, this study demonstrated the feasibility of gene
expression profiling and gene fusion detection from circulating
RNA in plasma research samples on Ion Torrent NGS
platforms.
II. MATERIALS AND METHODS
Plasma preparation: Blood samples were collected into EDTA
or Streck RNA tubes following manufacturer’s instructions.
Plasma from blood samples was obtained by centrifugation at
1600 x g for 10 min at 4C, followed by another spin at 6,000 x
g for 30 min at 4 C to remove any residual blood cells.
cfRNA extraction: An optional Proteinase K treatment was
performed according to experiment design. In brief, 4-mL
plasma was mixed with 60 µL 20 mg/µL Proteinase K and 200
mL 20% SDS and incubated at 60 C for 20 minutes.
Proteinase-treated samples were then transferred to 50-mL
conical tube for TRIzolTM LS Reagent extraction. After phase
separation, aqueous phase was transferred to a clean tube for
RNA precipitation. RNA pellets were resuspended in lysis buffer
from mirVanaTM miRNA Isolation Kit, followed by RNA binding
and washing on RNAqueous Micro Filter Cartridge. RNA
samples were eluted in 16 µL elution solution.1 µL cfRNA
sample was loaded on Bioanalyzer Pico chip for quantitation.
Alternatively, QiaAmpTM Circulating Nucleic Acid Kit was used
for cfRNA isolation following manufacturer's instruction.
Library prep: Ion AmpliSeq™ Transcriptome Libraries were
generated using Ion AmpliSeq™ Transcriptome kit targeting
21,000 genes following standard user guide. Ion AmpliSeq™
RNA fusion libraries were generated using Ion AmpliSeq™
library kit 2.0 and OCP143 RNA fusion panel. Libraries were
quantified by qPCR and pooled for sequencing.
Sequencing: Sequencing template preparation and Ion 318™
Chip loading were performed on the Ion Chef™ Instrument
using the Ion PGM™ Hi-Q™ Chef Kit or Ion Proton ™ Hi-Q ™
Chef Kit. Sequencing was performed on the Ion PGM™ System
with the Ion PGM™ Hi-Q™ Sequencing Kit, or on the Ion Proton
™ System with the Ion Proton™ Hi-Q ™ Sequencing Kit.
Data analysis:For Ion AmpliSeq transcriptome data , analysis
was performed on Torrent Server using ampliSeqRNA plugin in
Torrent Suite™ Software. For Ion AmpliSeq RNA data, data
were uploaded to Ion Reporter ™ Server and analyzed using
Ion Reporter ™ Software v4.4.
Fusion Gene Detection and Gene Expression Analysis
of Circulating RNA in Plasma Research Samples
IV. SUMMARY
•We have established a workflow for cfRNA RNA fusion
detection and gene expression profiling using TRIzolTM LS
Reagent and mirVanaTM miRNA Isolation Kit followed by Ion
AmpliSeqTM library preparation.
•It is strongly recommended to use Streck RNA tube as
blood collection method for gene expression analysis and
RNA fusion detection. EDTA tube is only recommended if
plasma can be prepared immediately after blood collection
to minimize the chance of blood cell lysis.
•RNA fusion can be detected when positive control RNA
was spiked into cfRNA background at 2.5% or higher. LOD
varies depending on expression level of the fusion transcript
in the fusion positive control cell line.
For Research Use Only. Not for use in diagnostic procedures.
© 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are
the property of Thermo Fisher Scientific and its subsidiaries unless
otherwise specified. TRIzol is a trademark of Molecular Research Center,
Inc. QIAamp is a trademark of Qiagen GmbH.
III. RESULTS
III.A Recommended workflow for cfRNA analysis on
Ion Platforms
cfRNA
isolation
Library
Prep
Templating
Sequencing
Data
Analysis
TRIzol LS and mirVana kit/
QiaAmp Circulating
Nucleic Acid Kit
Ion Chef™ Instrument
Ion PGM™ Hi-Q™ Chef Kit
Ion AmpliSeq™ RNA
fusion panels and Ion
AmpliSeq Library Kit 2.0
Ion PGM™ 318™ Chip
Ion PGM™ Hi-Q™
Sequencing Kit
Figure 2: (A) Bioanalyzer trace of fusion positive control RNA. (B-D)
Bioanalyzer traces of cfRNA isolated from different normal research
samples. Blood research samples were collected in Streck RNA
tubes before plasma preparation. cfRNA yield varies from ~250 pg- 5
ng per mL plasma.
Figure 3: Ion Reporter output for fusion detection. EZR-ROS1 fusion
gene was detected when fusion positive control was spiked into cfRNA
at 2.5% or higher.
Plasma
Collection
Streck RNA tubes Streck RNA tubes
TRIzol LS and mirVana kit/
QiaAmp Circulating
Nucleic Acid Kit
Ion AmpliSeq™
Transcriptome Kit and
Panel
Ion Chef™ Instrument
Ion Proton™ Hi-Q™ Chef
Kit
Ion Proton™ P1™ Chip
Ion Proton™ Hi-Q™
Sequencing Kit
Torrent Suite™
ampliSeqRNA plugin
Ion Reporter™
RNA Fusion Gene Expression
III.B Gene expression analysis on cfRNA samples
using different blood collection methods
Input RNA
Blood
Sample
Tube PK
Library Yield
(nM)
Targets
detected
Plasma 1E
Blood
Sample
1
EDTA No 49 62.81%
Plasma 1S Streck No 0.044 13.92%
Plasma 1SP Streck Yes 0.209 15.43%
Plasma 2E
Blood
Sample
2
EDTA No 48 58.92%
Plasma 2S Streck No 0.109 15.79%
Plasma 2SP Streck Yes 0.063 11.66%
Table 1: Experiment design and library information on 6 gene expression
samples. Normal blood samples were collected using both EDTA and
Streck RNA tubes. cfRNA was extracted from plasma researchsamples
following TRIzol LS and mirVana protocol with or without optional
Proteinase K (PK) treatment. Ion AmpliSeq Transcriptome libraries were
constructed following standard User Guide and library yield was
quantitated using qPCR kit. Percentage targets detected indicates
numbers of expressed genes (equal to or more than 10 counts) in the
samples(out of ~21,000 total target genes).
Figure 1: Pairwise correlation between 6 cfRNA samples on. ~3,000 genes
were expressed in all the samples. Poor correlations were observed
between samples collected using different tubes (r<0.25). This, along with
significant library yield differences, suggests high background noise (RNA
released from blood cells) introduced when EDTA tubes were used for
collection. No significant difference was observed between samples with
or without Proteinase K treatment.
III.C Fusion gene detection on cfRNA samples using
Ion AmpliSeqTM OCP143TM RNA Fusion panel
Table 2: Experiment design for fusion positive control titration. 2.5 ng
total RNA input was used with positive control RNA spiked at 1-10% into
cfRNA background. Ion AmpliSeq libraries were prepared using OCP143
RNA fusion panel and Ion AmpliSeq Library Kit 2.0.
Input cfRNA
Control
RNA
Mix
Total
Input
Spike-
in%
Collection
Tube
OCP143
Yield (pM)
2.25 ng 250 pg
2.5 ng
10%
Streck
RNA
229
2.38 ng 125 pg 5%
Streck
RNA
256
2.44 ng 62.5 pg 2.5%
Streck
RNA
213
2.48 ng 25 pg 1%
Streck
RNA
182
A B
C D
10%spike-in
5%spike-in
2.5%spike-in
1%spike-in