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Automated TruSeq RNA Sample Preparation
from FFPE tissue specimens utilizing the Biomek
FXP Liquid Handler
Michaela Bowden PhD
Dana Farber Cancer Institute
21st March 2013
Prepared by Michaela Bowden Ph.D
Dana Farber Cancer Institute
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Founded in 1947 for Sidney Farber MD
Mission: to offer cancer patients the best treatment available today,
while developing tomorrow’s cures through research
DFCI holds > 400 NIH and NCI grants and is one of the most successful
institutions in the country for securing cancer-related funding due to the
quality of research being conducted there
DFCI, as a non-profit organization, also benefits from the generosity of
individuals, corporations, foundations, and organizations
– ~350k clinical visits / annum
– 4000 DFCI employees between clinical, research and administrative staff
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Centre for Molecular Oncologic Pathology is a DFCI/BWH institute within
the division of Medical Oncology.
– CMOP supports collaborative pathology-based research projects that have
the potential to result in applications for targeted cancer therapy with a
particular focus on prostate cancer
Prepared by Michaela Bowden Ph.D
Prostate Cancer
• 238,590 new cases of prostate cancer will be diagnosed in
2013^
• 29,720 deaths are projected for 2013^
– Second-leading cause of cancer death in men
• Higher rate of incidence and mortality in African Americans
• Only established risk factor is AGE (~60% diagnosis in men
older than 65 years)
• Standard practice is routine PSA testing for early detection
The major challenge in clinical research of prostate cancer is
distinguishing between indolent and aggressive types with a
tendency to recur. Most research activities in the field focus on
better understanding this distinction.
^ American Cancer Society. Cancer Facts & Figures 2013. Atlanta: American Cancer Society; 2013
Prepared by Michaela Bowden Ph.D
Formalin-Fixed Paraffin Embedded Tissue
“20 million archival tissue samples are
stored annually in US as FFPE blocks but
RNA degradation during fixation and
storage has prevented their use for
transcriptional profiling”
Clin Cancer Res; 18(22) November 15, 2012
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Routine preservation method for archiving tumors
Archived collections are stored for decades and are an invaluable tool for
biomedical research
Large cohorts of samples are required for meaningful clinical discovery
Extensive RNA fragmentation and cross-linking have been limiting factors
for clinical translation applications leading to biomarker discovery
Prepared by Michaela Bowden Ph.D
Formalin-Fixed Paraffin Embedded Tissue
Diagnosis
Cohort Studies
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Globally large scale cohorts
for PrCa have been archived
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In-house (Collaboration with
HSPH)
2 specimen collections types
Fine need aspirate
– Less invasive
– Small # cells collected
– H&E and cell block only
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Core biopsy
– Large tissue core punch
• Frozen: Difficult to assess
morphologically
• Fixed: IHC, stored at RT, no extra cost
– Obligated to archive for 25 year, large
accumulation of preserved blocks
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PrCa cannot be diagnosed on frozen
sections due to the difficulty in
assessing the morphology
– > 3000 PrCa tissue specimens in
biorepository
• Physician Health Study
(PHS)
• Physicians Health Follow-up
Study (PHFS)
• Swedish Watchful Waiting
Cohort (SWWC)
Prepared by Michaela Bowden Ph.D
Formalin-Fixed Paraffin Embedded Tissue
• Factors influencing RNA degradation in FFPE tissues
– Fixation*
• Thickness of tissue specimen
• Duration of fixation process
• Volume of the formalin solution
– Age
– Water
• Retention of endogenous H2O during tissue processing
• Humid environment resulting in ambient H2O effect
– Temperature
• Effect of humidity is temperature Dependent
– Air
• Oxygenation
* “Unlocking your FFPE archive: Critical factors for molecular analysis of FFPE samples” – Qiagen FFPE Brochure 09/2010
Prepared by Michaela Bowden Ph.D
Project Goals
• To implement an automated method for
preparation of RNA-seq libraries from archival FFPE
tissues utilizing the TrueSeq kit
– To optimize optimal RNA input requirements
– To establish library size from degraded FFPE
– To establish purification methods for FFPE library
clean-up
– To compare manual library preparation and
automated method
Prepared by Michaela Bowden Ph.D
Collaboration Workflow
1.
2.
3.
4.
5.
6.
7.
8.
9.
Sample Selection
RNA Extraction
RNA QC and Quantitation
Biomek TruSeq Library Preparation
Library Purification
Manual TruSeq Library Preparation
Library Purification
RNA Sequencing at Centre for Computational Cancer Biology [CCCB]
Sequencing Performance Summary
Prepared by Michaela Bowden Ph.D
Sample Selection
• 3 paired Tumor / Normal FFPE tissue specimens were
selected after review by DFCI pathologist
– Gleason Score 3+4
• Sample ID’s have been masked to protect patient
confidentiality
– Prostate Tumor A / Benign A
– Prostate Tumor B / Benign B
– Prostate Tumor C / Benign C
• Tumor or Benign Regions of Interest [ROI] were identified
from H&E stains and cores were collected for RNA
extraction
Prepared by Michaela Bowden Ph.D
RNA Input
Extraction
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Assay: Agencourt®FormaPure® Kit,
Beckman Coulter® [Catalog #
A33343]
B
Tumor A
T
Quantitation
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Assay: Quant-iT™ RiboGreen® RNA
Assay Kit, Invitrogen™ [Catalog #
R11490]
Instrumentation: Perkin-Elmer
Victor™ X3
Benign A
Qualitative Analysis
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Assay: Agilent RNA 6000 Nano Kit,
Agilent Technologies [Catalog #
5067‐ 1511]
Instrumentation: Agilent
Bioanalyzer 2100
RNA concentrations ranged from 24.4
– 61.1 ng/l, of which 500 ng of total
RNA was utilized for each library as
the input amount.
Prepared by Michaela Bowden Ph.D
TruSeq Method
• The TruSeq RNA method comprises
multiple steps
• For adaptation to an automated
format, these steps can be broken into
three parts that correlate to stop points:
– mRNA purification, fragmentation and
cDNA synthesis
– cDNA library construction (end repair,
A-tailing and adaptor ligation)
– PCR amplification
purification
and
product
Prepared by Michaela Bowden Ph.D
TruSeq cDNA Library
• cDNA library yields are higher from automated preparations
Prepared by Michaela Bowden Ph.D
SPRIselect Fragment Size Selection
Benign A -Biomek
240
Prostate Benign A - Pre
Prostate Benign A - Post
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140
126bp
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90
High mol
wt marker:
10380bp
40
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-10
32
52
72
92
112
132
Time [s]
Tumor A - Biomek
340
Prostate Tumor A - Pre
Prostate Tumor A - Post
126bp
290
> 400bp
240
Fluorescence Units
Low
mol wt
marker:
35bp
Fluorescence Units
190
190
140
90
40
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Initial library had peak max for the
amplified
cDNA
library
at
approximately 260bp
Non-specific adaptor amplified peak
at 120bp present in all 12 samples.
An additional fragment >400bp also
present in some samples
SPRIselect – Size selection with
magnetic beads
– STEP 1: 0.7X ratio of SPRIselect bead
solution to cDNA library volume.
Fragments >500bp DNA binds to
beads and is discarded.
– STEP 2: The remaining supernatant
treated with a 1.1X volume of
SPRIselect beads to rebind the DNA
fragments between 150bp-400bp.
-10
32
52
72
92
112
132
Time [s]
Prepared by Michaela Bowden Ph.D
Sequencing Performance
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Sequencing was performed on the Illumina HiSeq platform.
Multiplexed sequencing was performed in such that each lane contained 3
samples.
50bp single end reads were mapped using Tophat
Transcript abundance was calculated using Cufflinks in FPKM units (Fragments per
Kilobase of mRNA per 106 reads)
Prepared by Michaela Bowden Ph.D
Biomek v Manual
Prepared by Michaela Bowden Ph.D
Endogenous Controls
12
GAPDH
y = 1.0951x - 0.8565
R² = 0.9765
10.0
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Log2[FKPM]
8.0
10
6.0
4.0
2.0
0.0
Robot
8
Manual
OAZ1
Log2[FKPM]
6.0
6
5.0
4.0
3.0
2.0
1.0
0.0
Robot
4
•
Manual
G6PD
3.0
2.5
Log2[FKPM]
Biomek [Log2]
7.0
2
2.0
1.5
1.0
0.5
0.0
Robot
0
0
2
4
6
“Normalizers”
are
commonly employed for
quantitative
gene
expression analysis in
clinical tumor samples to
correct
for
cellular
differences and RNA
quality variance from
sample to sample
Utilizing a panel of 27
housekeeping genes the
Biomek and Manual
Library
preparative
methods were compared
Manual
8
10
12
Manual [Log2]
Prepared by Michaela Bowden Ph.D
Conclusions
• Generated RNASeq libraries
utilizing adapted TruSeq
method from 500ng RNA of
FFPE origin
• Automated cDNA libraries
had a better yield than
those prepared manually
• At the transcript level
matched Biomek and
manual prepped samples
showed good concordance
• TruSeq Method is designed for
high quality RNA and works
extremely robustly for RNA of
cell or fresh frozen origin
(Routinely used in-house)
• No optimized Illumina protocol
is available for FFPE specimens
• Ribosomal RNA removal not an
option with TruSeq for FFPE
unless large RNA input is utilized
(not possible)
– rRNA removal highly desirable for
FFPE
Prepared by Michaela Bowden Ph.D
Acknowledgements
• Dana Farber Cancer Institute
– Ashley Rossi [Molecular Technician]
– Svitlana Tyekucheva [Bioinformatics]
– Massimo Loda [CMOP Director]
• Beckman Coulter
– Beena Lee [Senior Scientist]
– Sarah Simons [Field Application Specialist]
Prepared by Michaela Bowden Ph.D