Download Focused Panel RNASeq Analysis of Urinary Exosomal RNA

Focused Panel RNASeq Analysis of Urinary Exosomal RNA Samples from
Prostate Cancer Patients
Kathryn Pellegrini1, Martin Sanda1, Maria Simbirsky2, Martin Pieprzyk2, Carlos S. Moreno1,
1Emory University School of Medicine, Atlanta, GA. 2Cellular Research Inc., Palo Alto, CA
PIPELINE & DATA PROCESSING
ABSTRACT
Cellular Research, Inc.
RESULTS
To begin development of non-invasive biomarkers that may predict aggressive prostate cancer, we
have performed a pilot study using PreciseTM Molecular IndexingTM technology, developed at Cellular
Research, Inc.
A set of eight pilot formalin-fixed paraffin-embedded (FFPE) prostatectomy RNA samples that have
already been sequenced by conventional methods, eight FFPE biopsy RNA samples, and nine urine
exosomal RNA specimens were sequenced using PreciseTM Molecular IndexingTM technology for
library preparation, with a panel of 109 genes. This custom gene panel included a recently published
(1) 24 gene RNASeq signature, predictive of biochemical recurrence.
Our preliminary results indicate that the PreciseTM Molecular IndexingTM technology robustly detects
65-73% of genes assayed in exosomal urinary RNA within a panel of 109 genes, and detected 22 of
the 24 signature genes in over 80% of the samples tested. Importantly, PreciseTM Molecular
IndexingTM technology also demonstrated segregation of benign samples from patients with
aggressive (Gleason Score 7 or higher) prostate cancer using 22 of the 24 genes in the RNASeq
signature.
INTRODUCTION & METHODS
It is well established that prostate cancers are heterogeneous in nature, and thus sampling of RNA
from urine specimens represents a non-invasive approach to assess the entire organ. Previous
sequence analysis of urinary exosomal RNA has shown that prostate-specific messages are five to
ten times more abundant than kidney or bladder mRNAs in urine samples (2).
We recently published a set of RNA biomarkers derived from RNASeq analysis of prostatectomy
specimens that is predictive of biochemical recurrence (1). To begin development of non-invasive
biomarkers that may predict aggressive prostate cancer, we have performed a pilot study using the
PreciseTM Molecular IndexingTM technology, developed at Cellular Research, Inc. This sequencing
assay, originally developed for single-cell sequencing analysis, is highly sensitive, requiring a
maximum RNA input of 1 ng. As such, it is well suited for the analysis of urine-derived RNA
specimens.
We have prepared exosomal-fraction RNA using ultrafiltration to recover the exosomes, and the
RNeasy Micro Kit from Qiagen to extract RNA. This method has yielded between 4.5 – 234.2 ng of
RNA from urine (mean = 76.0 ng) and 97% of RNA samples were of high enough concentration to
use the maximum amount of RNA input (1 ng) for the PreciseTM Assay. (Figure 4A) Moreover, this
RNA is of high quality, with a median RNA Integrity (RIN) > 6. (Figure 4B)
We have employed the methods of Fu et al (3) to generate molecularly indexed PCR-based
amplicon sequencing libraries that a) targets only the sequences of interest and b) controls for interassay variation in PCR amplification during library preparation by indexing each individual mRNA
molecule during reverse transcription. (Figure 1) In this pilot study, we used the PreciseTM Molecular
IndexingTM technology to generate indexed libraries for 150bp paired-end RNASeq analysis.
Sequencing was completed using an Illumina MiSeq instrument through the CLIA arm of the Emory
Integrated Genomics Core (EIGC). Automated data processing was performed on the Seven
BridgesTM Genomics Platform. (Figure 2,3)
Figure 2. Cellular Research Precise TM Assay Pipeline. This pipeline, available through
Seven Bridges TM Genomics, performs all of the required steps to de-multiplex and quantitate
expression levels using Cellular Research’s Precise TM Assay.
Inputs
• FastQ files (R01, R02)
• Illumina adaptor
FASTA
• Gene panel
sequences FASTA
Steps
Outputs
• FASTQC analysis
• Flexbar adapter
trimming
• Sample demultiplexing
• Gene mapping with
Bowtie2
• Read and Molecular
Indexing count
• Sample demultiplexing report
• Read mapping report
• Molecular Index
mapping report
Figure 5. Focused RNASeq analysis of 9 urinary exosomal RNA Specimens.
A) Cluster analysis of 109 genes tested on urinary exosome RNA with the Precise focused
RNASeq panel. B) Cluster analysis of the 24 signature genes tested in this assay
segregates benign from aggressive (Gleason 7+) prostate cancer.
Figure 3. Data processing steps. Sequencing data analysis is provided in a secure online
cloud environment through Seven BridgesTM Genomics. The report is output in a simple, easy-toread excel (or tabular) format consisting of read alignment summaries and molecular index
counts for each gene per each sample.
A set of eight pilot formalin-fixed paraffin-embedded (FFPE) prostatectomy RNA
samples that have already been sequenced by conventional methods, eight FFPE
biopsy RNA samples, and nine urine exosomal RNA specimens were sequenced using
the Cellular Research molecular indexing methodology with a panel of 109 genes
including the 24 gene RNASeq signature that we recently published. (Figure 5)
RESULTS
These preliminary results indicate that the exosomal urinary RNA robustly detects 65 73% of genes targeted in the overall assay of 109 genes, and detected 22 of the 24
signature genes in over 80% of the samples tested. Importantly, they also demonstrate
segregation of benign samples from patients with aggressive (Gleason Score 7 or
higher) prostate cancer using 22 of the 24 genes in the RNASeq signature. (Table 1)
LIBRARY PREPARATION
1. RT (96-well format)
mRNA
Figure 4: Urine exosomal RNA analysis. A) RNA yield from urinary exosomes. B) RNA
Integrity (RIN) of urinary exosomal RNA.
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TTTTTTTTTTTTTTTTTTTT
Molecular
Index
2. N1 PCR (pooled into 1 tube)
Sample
Barcode
Universal
Primer
Urinary Exosome
Biopsy
FFPE RP
% Genes Detected per sample
73.1%
29.5%
26.3%
% Genes Detected > 80% samples
64.7%
17.2%
19.0%
# Sig 24 genes detected > 80% samples
22
6
5
% Sig 24 genes detected > 80% samples
95.7%
26.1%
21.7%
cDNA
Library
Read 2
Read 1
PreciseTM
Figure 1. Precise assay steps. The
workflow can be completed with three simple
steps. After the reverse transcription step, the samples are combined into one tube for easy
downstream processing.
REFERENCES
1. Q. Long et al., Global transcriptome analysis of formalin-fixed prostate cancer specimens
identifies biomarkers of disease recurrence. Cancer Research 74, 3228-3237 (2014).
RNA Source
3. N2 PCR (second, nested PCR Step)
SUMMARY
Table 1. Focused RNASeq Analysis of urinary exosome, FFPE Biopsy and RP RNA
Specimens. Exosomal RNA has high % of genes detected, and detects 22 of 24 signature
genes in 80% or more patient samples (n=9). Genes with a molecular index count > 3
were classified as detected.
2. K. C. Miranda et al., Massively parallel sequencing of human urinary exosome/micro
vesicle RNA reveals a predominance of non-coding RNA. PloS One 9, e96094 (2014).
3. Fu, G. K., Wilhelmy, J., Stern, D., Fan, H. C. & Fodor, S. P. A. Digital encoding of cellular
mRNAs enabling precise and absolute gene expression measurement by single-molecule
counting. Anal. Chem. 86, 2867–2870 (2014).
FUNDING
This work was supported in part by NIH grants R44HG007130 to Glenn Fu, U01 CA113913-suppl
to Martin Sanda, and NCI Cancer Center Support Grant P30CA138292.