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Fully Automated Sample Preparation Methods for High Throughput Transcriptome Analysis by Arrays and NGS
Joe Don Heath and Wieland Keilholz, NuGEN Technologies, Inc., San Carlos, CA 94070
A B S T RACT
ME TH O D S and MATE RIA L S
NuGEN® Technologies is a provider of innovative sample preparation solutions for genomic assays. NuGEN offers a wide
palette of systems optimized for a variety of nucleic acid sample types such as whole blood RNA, small quantity RNA/DNA
and highly degraded RNA and DNA, i.e. from FFPE specimens. Powered by SPIA® (Single Primer Isothermal Amplification)
technology, these systems allow rapid linear amplification of input RNA from samples as small as a single cell and generate
microgram amounts of cDNA sufficient for analysis by qPCR, microarray, or sequencing. Reagent modules to further
prepare the SPIA product for subsequent analyses on microarrays or massive parallel sequencing complement NuGEN’s
amplification systems. NuGEN cDNA targets are compatible with all leading microarray formats and NGS platforms.
RNA samples were obtained from different sources:
Hyb-ready cDNA targets for Affymetrix HG-U133 Plus 2.0 Expression Arrays were generated using the Encore Biotin
Module.
• UHR (Stratagene) is a mixture of total RNA extracted from 10 different human cell lines.
Automation of sample preparation workflow not only increases sample throughput, it also minimizes variability introduced
by manual handling during the assay. NuGEN’s Ovation® and Encore™ lines of sample preparation assays were developed
and designed intentionally to be amenable to automated workflow strategies. A number of NuGEN’s genomic assays have
been successfully automated on a variety of liquid handling robotics platforms (Caliper, Hamilton and Beckman) and are in
daily use in many laboratories around the world. The assays are scripted in a modular form allowing highest flexibility with
regard to both the number of samples processed at a time as well as integration into the laboratory workflow. Starting from
total RNA, the entire process through target preparation for downstream analysis on microarrays or sequencing platforms
can be completed within two convenient workdays.
• Human Brain Reference RNA (Ambion) is pooled from multiple donors and several brain regions.
1) The Hamilton MicroLab STAR liquid handling system. This system is equipped with a 96 probe head, an eight channel
device for independent pipetting of up to 1 mL per channel, the iSWAP robotic hand and a Multiflex cooling module.
• FFPE sample type 1 is a mixture of RNA extracted from lung tumor tissue embedded in FFPE blocks.
The automated Ovation RNA-Seq assay and Encore NGS Multiplex Library System for generating sequence-ready libraries
are available for this robot from NuGEN in a modular form that allows both assays to run independently.
Access to both FFPE sample types was arranged through a collaborator.
Figure 1 shows a schematic overview of the whole transcript amplification chemistry applied in both the Ovation RNA-Seq
and the WT-Ovation FFPE RNA Amplification System. While there are substantial differences between the assay types, the
underlying basic concept remains the same. Briefly, total RNA is converted into ds-cDNA which has a unique RNA/DNA
heteroduplex tag segment incorporated at one end. The SPIA amplification step uses an additional chimeric primer, DNA
polymerase and RNAse H in an isothermal reaction. This leads to the rapid accumulation of amplified cDNA. In case the
analytical platform requires double-stranded cDNA, the single-stranded SPIA products can be easily converted by the postSPIA modification step.
2) The GCAS is a special configuration of a Caliper Sciclone robot, integrated with a Twister II. It is equipped with an ondeck thermal cycler, a 96 High Volume Head, an eight channel device for individual pipetting (Z8), a Gripper unit and Peltier
cooling element.
The automated WT-Ovation FFPE RNA Amplification System and the Encore Biotin Module for generating targets from
FFPE total RNA for analysis on Affymetrix arrays are available on this robot. Again, the modular form allows both assays to
run independently.
BG
3/5 Ratio
GAPDH
% Present
CV%
Number
Replicates
UHR
4.76
29.70
1.28
68.09
3.5
11*
Brain
7.51
28.41
1.57
65.73
2.3
3
FFPE Sample Type 1
55.17
26.00
2.70
32.06
9.6
11
FFPE Sample Type 2
16.7
27.67
3.82
44.39
1.6
12
FFPE Sample Type 2
14.86
28.01
3.28
46.25
1.3
12
FFPE Sample Type 2
25.67
27.11
3.55
38.47
2.6
12
Sample Type
Table 1: Average performance metrics of different sample types on Affymetrix HG-U133 Plus 2.0 arrays. FFPE type 2 samples were processed on two
robotic runs with 6 replicates each. A strong correlation can be observed between scaling factor, average %P and the associated CV% across the replicates. *
11 replicates of UHR through 3 different robotic runs
This study describes the results of automating two different sample preparation strategies:
1) The Ovation® RNA-Seq System and Encore™ NGS Multiplex System I on the Hamilton STAR liquid handling robotic
platform. The Ovation RNA-Seq System is designed for preparing amplified cDNA from as little as 500 picograms total RNA
for transcriptome sequencing applications. The method does not require an up-front ribosomal RNA reduction step, thereby
reducing the number and time of hands-on manipulation steps and therefore the potential for introducing additional bias or
degradation of the transcriptome.
SF
(TGT = 500)
The described assays were automated on two different robotics platforms:
• FFPE samples type 2 are sarcoma samples from 9 individual FFPE blocks.
I N T RODUCTION
Table 1
Illumina sequencing libraries are generated using NuGEN’s Encore NGS Library system.
Ovation RNA-Seq System
WT-Ovation FFPE RNA Amplification System
Encore NGS Multiplex Library
Prep Module
Encore Biotin Module
Fig. 8
150
80
2) The WT-Ovation FFPE RNA Amplification System and Encore Biotin Module is specifically targeted for preparing and
labeling amplified cDNA from total RNA derived from archival formalin-fixed paraffin-embedded (FFPE) tissues. As little as
50 ng of highly degraded RNA are processed to yield a hyb cocktail ready for analysis on microarrays.
TTTT
First Strand cDNA
RT
Determine
number
of samples
Set up deck
(and prepare
master mixes)
NNNNNN
NNNNNN
AAAA
TTTT
RT
cDNA Primer
(DNA/RNA,
random, poly-T)
SPIA Primer (DNA/RNA)
STEP 2:
Second Strand cDNA Synthesis
RT
Pol DNA Polymerase
Reverse Transcriptase
H RNAseH
STEP 3:
SPIA Amplification
Primer annealing
65° - 2’
4° - hold
2nd strand incubation
4° - 1’
25° - 10’
50° - 30’
80° - 20’
4° - hold
3 µL
Add first strand
master mix, mix
(TV = 10 µL)
1st strand incubation
4° - 1’
25° - 10’
42° - 10’
70° - 15’
4° - hold
32 µL
Add RNAClean XP
beads, mix
(TV = 52 µL)
Transfer to
magnetic plate
Determine
number
of samples and
adaptors for
multiplexing
2 µL
Transfer A1 primer to
PCR plate, mix
(TV = 7 µL)
10 µL
Add second strand
master mix, mix
(TV = 20 µL)
Set up deck
(and prepare
master mixes)
Start
14 µL
400 ng fragmented
DNA in PCR plate
(~29 ng/µL)
Wash beads 3X with
200 µL 70% EtOH
Remove sup to waste
(30 µL)
Wash beads 2X with
200 µL 70% EtOH
Air dry 15–20’
Air dry 5’
SPIA incubation
4° - 1’
47° - 60’
95° - 5’
4° - hold
Transfer to magnetic
plate, incubate
5 minutes to
clear all beads
Transfer 35 µL
cleared supernatant
to new PCR plate
5 µL
Add E1 Primer mix,
mix
(TV = 40 µL)
Mod I incubation
98° - 3’
4° - hold
10 µL
Add 10 µL
Post-SPI Mod II
master mix, mix
Mod II incubation
4° - 1’
30° - 10’
42° - 15’
75° - 10’
4° - hold
Add 10 µL
Ligation Master Mix,
mix gently and slowly
(TV=25 µL)
Ligation
25° - 20’
4° - hold
Library amplification
72°- 2’
5x (94° - 30’’, 55° - 30’’, 72° - 1’)
10x (94° - 30’’, 63° - 30’’, 72° - 1’)
72° - 5’
10° - hold
30 µL
Add 1X TE, mix
(TV = 30 µL)
Wash beads 2X with
200 µL 70% EtOH
RT - 5’
Transfer to
magnetic plate
Add 33 µL
1X TE to beads,
mix to resuspend
Clear - 2’
Air dry 2’
Seal and store or
quantitate cDNA
and normalize for
library prep
SPIA Primer
Add 24 µL
nuclease-free water
to beads,
mix to resuspend
Remove sup to waste
Transfer to
magnetic plate
Pol DNA Polymerase
Add 24 µL
RNAClean XP beads,
mix
(TV = 44 µL)
End repair
RT - 30’
4° - hold
Remove sup to waste
30 + 10 µL
Add SPIA master mix
(30 µL) and SPIA
enzyme (10 µL), mix
(TV = 40 µL)
RT - 10’
Pol Pol Pol
RT - 5’
RT - 10’
90 µL
Add RNAClean XP
beads, mix
(TV = 140 µL)
H RNAseH
Linear Amplification
5 µL
RNA
(500 pg–10 ng)
in PCR plate
Start
Pol DNA Polymerase
Double-stranded cDNA
Add 6 µL End Repair
master mix to
PCR plate, mix
(TV=20 µL)
Remove cleared
supernatant
containing cDNA
to a new plate
Incubate
Fig. 5
RT - 10’
Transfer to
magnetic plate
Clear - 5’
Add 72 µL
Amplification
master mix to
fresh PCR plate
(TV=72 µL)
Add 80 µL
RNAClean XP beads,
mix
(TV = 160 µL)
Carefully remove
30 µL to fresh
PCR plate
Transfer to
magnetic plate
-117
Transfer 10 µL
to fresh PCR plate
Add 5 µL Adaptor Mix,
mix
(TV = 15 µL)
-140
-190
-151
-113
-75
-37
PC #1 21.8%
0
38
Add 8 µL
ligation product to
Amplification master
mix in PCR plate, mix
(TV=80 µL)
Remove sup to waste
(140 µL)
Wash beads 2X with
200 µL 70% EtOH
Air dry 5’
Figure 5: User Interface of the automated WT-Ovation FFPE RNA Amplification Protocol on GCAS. Clear instructions supported by a
graphical deck layout help to minimize user-introduced errors. Setting up a run for the Encore Biotin Module follows the same concept.
Continue with
qualitative and
quantitative
assessment
of library
Transfer to
magnetic plate
Fig. 3
Fig. 6
500
15
10
300
Figure 3: cDNA yields generated from different input amounts of model RNAs UHR and Brain. Each bar represents the average of 4 technical replicates. These data demonstrate that even 50 pg of total RNA generate sufficient SPIA Product for library preparation as the Encore
NGS Multiplex Library Prep Module requires only 200 ng of double-stranded cDNA as input.
5 ng UHR
30
20
20
10
0
Figure 1: Schematic overview of the WT-Ovation RNA Amplification Process. After generation of the amplified single stranded cDNA (SPIA Product), additional reagent modules ensure compatibility with the analytical method of choice. For details, see the Materials and Methods section.
200
1000
4000
UHR
RIN = 8.0
25
[FU]
10
0
200
1000
4000
200
1000
4000
[nt]
FFPE
RIN = 1.0
500 pg Brain
20
2
200
1000
2
Ty
p
e
2
FF
PE
Ty
p
e
2
PE
FF
FF
PE
Ty
p
e
1
Ty
p
5
4
4000
Increased throughput:
A single robot can easily process three plates (288 samples) per week through the entire workflow.
2
20 25 30 35 40 45 50 55 60 65
sec
20 25 30 35 40 45 50 55 60 65
sec
6
5
4
2
[nt]
Figure 4: Bioanalyzer traces of the SPIA product after amplification by the automated RNA-Seq assay (Agilent RNA Nano Chip).
0
20 25 30 35 40 45 50 55 60 65
sec
2
0
20 25 30 35 40 45 50 55 60 65
sec
CONCLUSIONS
NuGEN has automated strategies for sample preparation on different robotics platforms.
0
0
0
10
25
e
1
FF
PE
Ty
p
PE
FF
0
ng
Br
e
ai
n
ai
Br
10
ng
ng
n
Hybridization Target
[nt]
0
25
SPIA Product
20 25 30 35 40 45 50 55 60 65
sec
30
20
0
4
0
10
[nt]
5 ng Brain
30
[FU]
Massive
Parallel Sequencing
Total RNA
0
25
Expression Arrays
Fig. 7
[FU]
Encore NGS Library
Prep Module
30
Array QC metrices demonstrate the correlation between sample type and array performance as can be shown e.g. by the
%P value. A very low coefficient of variation is observed over technical replicates, even between different runs. This again
demonstrates the robustness and reliability of both, the liquid handling system and the assay.
The individuality of different samples from the same biological type but different individuals is clearly shown by a PCA plot.
6
500 pg UHR
[FU]
Post-SPIA Modification
[FU]
Encore Biotin Module
Fig. 4
10
Figure 6: cDNA yields generated from different sample types and input amounts. Depending on the sample type, each bar represents the
average of 7 (model RNA) to 13 (FFPE type 1) replicates. In all cases, the amount of SPIA product generated was sufficient to allow for hybridization on Affymetrix expression arrays.
50 pg
UHR
[FU]
50 pg
Brain
[FU]
0.5 ng
UHR
[FU]
0.5 ng
Brain
[FU]
5 ng
UHR
[FU]
5 ng
Brain
50
R
UH
ng
50
Pol
0
This study demonstrates that sufficient SPIA product is generated from as little as 50 ng of highly degraded RNA extracted
from FFPE specimens. The amplified SPIA product reflects the grade of RNA degradation, however, the fragmented and
labeled hybridization target shows a size distribution independent of the input material and hence underlines the robustness
of the fragmentation reaction as such.
0
100
SPIA Product
2) In addition to other protocols, the automated WT-Ovation FFPE RNA Amplification System and the Encore Biotin Module
is currently available on the GCAS, a liquid handling system based on Caliper‘s Sciclone. The user interface guides the user
through the initial deck layout hence minimizing the risk of user-introduced errors.
5
200
Primer Extension by
DNA Polymerase
Figure 8: PCA plot of 9 different FFPE blocks in replicates. Samples are color coded and numbered according to their origin and replicates represent different slices and hence different RNA extractions from the same block. Sample 1 (red), 7 (brown) and 9 (grey) were processed in triplicates per slice and applied in two different robotic runs yielding a total of 12 replicates for each of these blocks.
The size distribution of the double-stranded cDNA looks as expected and even 50 pg of good quality total RNA give
sufficient yield to allow library production.
R
µg cDNA
400
Primer Extension by Strand
Displacement DNA Synthesis
76
104
After preparation of master mixes and an initial deck layout, the robot performs all reagent addition and mixing steps as well
as the necessary cleanups. Little user intervention is required for transportation of the plate to an off-deck thermal cycler.
UH
H RNAseH
1
1) The automated Ovation RNA-Seq and Encore NGS Multiplex Library Prep Module are currently in development and will
soon become available on two different robotics platforms, namely the Hamilton Microlab STAR and the Caliper Sciclone
NGS. This study shows preliminary data from a robotics run on the Hamilton STAR.
25
600
Pol
PC #3 7.77%
R ESULTS
RT - 5’
µg cDNA
RNAseH Cleavage to Free
Primer Hybridization Site
8
-95
Clear - 5’
Figure 2: Schematic overview of the RNA-Seq FFPE System workflow (left) and the Encore NGS Multiplex Library Prep Module (right)
automated on the Hamilton STAR.
Pol
Pol
2
-51
20
Pol
9
-73
SPIA Product
Reverse Transcriptase
4
6
DNA
RT
3
-30
RNA
STEP 1:
First Strand cDNA Synthesis
-83 -110
-7
Fig. 2
NNNNNN
-60
-37
5
14
CALIPER GCAS
NNNNNN
72
-5
36
Fig. 1
AAAA
98
58
HAMILTON MICROLAB STAR
Total RNA
124
20
46
20 25 30 35 40 45 50 55 60 65
sec
Figure 7: Typical Bioanalyzer traces. The average fragment size of SPIA product is 500 for UHR and below 200 for FFPE reflecting the degraded input RNA. cDNA target is fragmented to 50–70 bp for both sample types regardless of the SPIA product length.
Increased flexibility:
Any number from eight to 96 RNA samples can be processed from total RNA through to either sequence- or hybridizationready targets in one and a half working days. The modular design allows for the convenient separation of the amplification
and the labeling or library preparation part of the workflow. For increased throughput and flexibility in sequencing, libraries
may be barcoded with sixteen non-ambiguous multiplex adaptors.
Increased sensitivity:
Challenging RNA samples, such as clinical samples derived by LCM, cell-sorting, FNA or archived FFPE material can be
processed for RNA-Seq transcriptome, Microarray and qPCR analysis.
Increased reliability:
The high quality liquid handling systems as well as the robust assay performance introduce a reduced level of noise. This
is reflected by a high concordance of technical replicates as well as a clear separation of biologically related but distinct
sample types.
© 2011, NuGEN Technologies Inc. The Ovation® System family of products and methods is covered by U.S. Patent Nos. 6,692,918,
6,251,639, 6,946,251 and 7,354,717, and other issued and pending patents in the US and other countries. NuGEN, Ovation, SPIA, RiboSPIA, WT-Ovation, FL-Ovation, Encore, Applause and Imagine More From Less are trademarks or registered trademarks of NuGEN
Technologies, Inc. Other marks appearing in these materials are marks of their respective owners.
www.nugeninc.com