Download High-Throughput Strand-Specific mRNA Library

High-Throughput Strand-Specific mRNA Library
Preparation for Illumina Sequencing from Total RNA
Isolated from Normal and Cancerous Ovary Tissue
Shanavaz Nasarabadi*, Jacob Berger, Paul Butler, Charles Troup, Tom Olenic, Mary Trounstine, Sayali Salodkar, Stephanie H.I. Yeung
and Monika Tomczyk
Reagents R&D Group, IntegenX Inc., *Corresponding author: [email protected]
Abstract
Materials and Methods
The cost benefits of whole genome and transcriptome next
generation sequencing (NGS) have revolutionized genetic
analysis. Determination of regulatory changes in cancer cells
by whole transcriptome analysis has proven useful for tailoring
treatment options for patients. An efficient, consistent and
reliable whole transcriptome library preparation method is
necessary for successful transition of the NGS technologies from
research to clinical applications. Due to the labile nature of mRNA
and the tedium of processing large numbers of patient samples,
automation would greatly improve the reliability and throughput
of whole transcriptome library preparation.
Schematic Overview of Directional Library Preparation
We have explored the automation of whole transcriptome library
preparation from total RNA of normal and cancerous ovary
tissue. The Apollo 324™ System (IntegenX Inc.) was used to
isolate polyA mRNA from six to 48 samples of total RNA and
prepare strand-specific mRNA libraries for sequencing on the
Genome Analyzer IIx (Illumina). The cDNA output was amplified
on a bench thermocycler to yield whole transcriptome libraries
in a single eight-hour work day. While most conventional
RNA-Seq library preparation methods convert mRNA to
cDNA, in our strand specific library preparation, we ligated
the adapters directly to fragmented mRNA to preserve strand
polarity. Preserving strand polarity of the transcript reduces the
bioinformatics bottleneck. A commercially available manual
strand-specific library was used as the bench control for
comparison and validation of our library preparations.
Materials for library prep
Total RNA from Normal and Cancer Ovary tissue (Clontech);
mRNA isolation kits: PrepX™ PolyA Isolation Kit (IntegenX) and
Ribo-Zero Magnetic Kit (Epicentre); Library preparation kits:
PrepX RNA-Seq Library Kit for Illumina (IntegenX) and TruSeq
Stranded mRNA Sample Preparation Kit (Illumina).
The gene expression profile of up-regulated and down-regulated
genes was equivalent between the high-throughput libraries and
the published data. The isolated polyA mRNA had an average of
0.3% rRNA contamination from 500 ng of total RNA. There was
a 75% time reduction for automated library preparation from total
RNA compared with conventional library preparation methods.
There was 90% correlation of gene expression between the
automated and bench library preparation methods for up to 48
samples.
1
Workflow for automated directional library preparation from normal and cancer ovary total RNA
Automated mRNA isolation (oligo dT25 or
ribosome depleted method) on Apollo 324
Hybridization
Wash
Elution of mRNA
Hands-on time: ~15 minutes
Script time: ~2 hours
Automated library preparation on Apollo 324
mRNA Fragmentation
Adapter ligation
cDNA synthesis
Cleanup
Hands-on time: ~30 minutes
Script time: ~6 hours
PCR amplification and quantification
Cleanup
Illumina GA IIx sequencing
36 bp single read
Figure 1: Process time for automated sample library preparation from total RNA to
mRNA for 48 samples vs. manual preparation for eight samples
2
Results
Messenger RNA isolation on the Apollo 324 System
Figure 2: Automated mRNA isolation on the Apollo 324 system with Ribo-Zero method has ~3% rRNA. Automated mRNA isolation
on the Apollo 324 system with PolyA methods has 0% rRNA.
Manual and automated library preparation
Figure 3: Bioanalyzer plots of manual library preparation using TruSeq Stranded mRNA Sample Preparation Kit with 15 cycles of
PCR and automated mRNA library preparation with PrepX RNA-Seq Library Kit with 12 cycles of PCR.
3
Gene expression analysis of manual and automated library preparation
Figure 4: Comparison of gene expression analysis for normal and cancer ovary RNA library preparations with PrepX RNA-Seq Library
Kit for Illumina on the Apollo 324 and manual TruSeq Stranded mRNA Sample Preparation Kit methods showed >90% correlation.
4
Gene Regulation of Normal and Cancer Ovary
Manual TruSeq library preparation and automated PrepX library preparation methods showed the same up-regulation and downregulation patterns for the genes in cancer cells.
Figure 5a: Up-regulated genes, RPL39, RPS3 and MALAt1, in cancer cells are comparable for the manual TruSeq and the
automated PrepX library preparation methods.
Figure 5b: Down-regulated genes, DCN and RPL5, in cancer cells are comparable for the manual TruSeq and the automated PrepX
library preparation methods.
5
Comparison of sample preparation and sequencing metrics
Table 1: Sequencing metrics.
Sequencing Metrics
Library prep with normal and
cancer mRNA
Linear
regression
of gene
expression
Total reads
(millions)
%
Confident
reads
% Repeats
% rRNA
Normal
0.999
9
78
13.4
0.51
Cancer
0.998
18
74
12.5
2.4
PrepX RNA-Seq
Normal
0.952
10
83
8.5
0.51
library prep
Cancer
0.987
9
77
10.1
3.4
Tissue type
TruSeq library prep
Conclusion
• An automated strand-specific RNA-Seq method was developed.
• Sequence analysis of the automated mRNA library prep from total RNA on the Apollo 324 system compared with manual library
preparation methods show a >90% correlation of gene expression.
• The automated and manual methods showed agreement of up-regulated and down-regulated genes for the normal and cancer
cells.
• PCR-ready whole transcriptome libraries from 48 total RNA samples were prepared in one 8-hour work day using the automated
high-throughput sample preparation method.
© 2013 IntegenX Inc. IntegenX, IntegenX design, Apollo 324, BeadX and PrepX are trademarks of IntegenX Inc.
All other trademarks are the sole property of their respective owners.
5720 Stoneridge Drive, Suite 300
Pleasanton, CA 94588
www.integenX.com
6