Download Use of BAC aCGH for molecular karyotyping of whole

Application Note
Use of BAC aCGH for molecular karyotyping of
whole genome amplified FFPE DNA samples
Christopher Williams, Product Specialist, PerkinElmer Life and Analytical Sciences
Steve Michalik, Product Manager, Sigma-Aldrich Biotechnology
Introduction
Archival, formalin-fixed, paraffin-embedded
(FFPE) tissues represent an invaluable source
of material for molecular genetic studies.
There is a growing desire to profile FFPE
samples via array comparative genomic
hybridization (aCGH), however many of these
samples are unsuitable for microarray analysis
due to nucleic acid damage introduced
during the fixation process. Several methods
have been shown to be effective in the
assessment of DNA quality from sources
of damaged DNA, with PCR-based assays
being the most widely accepted (1, 2, 3). We
apply a simple, gel-based, multiplex PCR
assay to FFPE tissues prior to whole genome
amplification (WGA) and aCGH analysis
to assess DNA quality and predict aCGH
feasibility. In addition, there are currently
two commercially available technologies
for the application of WGA. One is Sigma’s
GenomePlex WGA product line that is
based on a modified DOP-PCR technology
(4), and the other is multiple displacement
amplification (MDA) WGA technology (5).
Several groups have applied MDA-based
WGA to FFPE samples, but the requirement
of full-length template DNA appears to
preclude this method from use with archived
samples (6, 7, 8). Here we show that Sigma’s
GenomePlex WGA platform and PerkinElmer
Spectral Chip™ 2600 BAC array platform
work effectively for generating high quality
aCGH data from archival samples when
starting with 10ng FFPE genomic DNA or
~1mg FFPE tissue. FFPE tissue can be used
directly with the GenomePlex Tissue Whole
Genome Amplification Kit and analyzed for
chromosomal gains or losses via PerkinElmer
Spectral Chip™ 2600 BAC arrays to generate
high quality aCGH data.
The Spectral Chip™ 2600 1 Mb
BAC Array Platform
Array-based comparative genomic
hybridization (aCGH) yields data that is
analogous to traditional karyotyping but with
significantly greater throughput, resolution
and ease of use. Traditional karyotyping
methods for detecting genomic variation, such
as FISH (Fluorescence in situ Hybridization)
or G-banding are well established for
detecting chromosomal aberrations, but they
do not provide the speed, throughput, or
resolution required for today’s cytogenetic
and research laboratories. The SpectralChip™
2600 array allows the researcher to perform
the equivalent, of thousands of FISH
experiments simultaneously in a 24-hour
protocol. Additionally, the SpectralChip™
array provides greater resolution and
improved data analysis tools.
www.perkinelmer.com
The 2605 non-overlapping bacterial
artificial chromosome (BAC) clones on the
SpectralChip™ array span the entire genome.
The BAC clones are spaced at approximately 1
megabase intervals, as compared to traditional
karyotyping, that has approximately
10 megabase resolution. The clones are
covalently coupled to glass microscope slides,
spotted in duplicate. Sample and reference
DNA are labeled in a two color, ratiometric
experiment. A fluorescent scanner captures
data from the array and SpectralWare®
software converts the scanner output data
into an intensity ratio profile. The software
analyzes copy number changes and displays
the location of the changes within the
genome. The aCGH process combines the
whole genome perspective of chromosome
karyotypmg with the increased resolution
of FISH and the high throughput of DNA
arrays. The SpectralChip™ array can generate
molecular profiles in a single experiment.
Hybridization can be completed in 16 hours,
and data are available in </=24 hours.
Methods
FFPE tissues were WGA amplified directly
using Sigma’s GenomePlex Tissue WGA kit
(Cat # WGA5), whereas purified genomic
DNA was WGA amplified using Sigma’s
GenomePlex WGA kit (Cat # WGA2) per
manufacturer’s recommendations. BAC aCGH
profiles were generated using 1µg WGA
DNA on PerkinElmer Spectral Chip™ 2600
BAC array (Cat # 4027-0020) platform. Array
CGH data was analyzed using PerkinElmer
SpectralWare® aCGH Analysis Software (Cat #
5007-1010) using default settings. Oligo aCGH
was performed by a certified service provider
(UHN Microarray center, Toronto, Canada).
Gel-based multiplex PCR assay for
DNA quality assessment
A single reaction, multiplex PCR assay
containing a mixture of five primer sets was
used to determine FFPE DNA quality and
WGA/aCGH outcome. High quality FFPE
DNA produces all five amplicons, whereas
lower quality DNA produces no amplicons, or
a subset of the amplicons (figures 1 to 3).
Figure 1. Primer information for multiplex PCR assay. Primer sequences were derived from the
UniSTS database, and the UniSTS accession numbers, primer sequences, amplicon sizes, and
chromosomal locations of the amplicons are provided.
2
Figure 2. Gel of multiplex amplicons generated
from FFPE lysates. Approximately 1mg of tissue
was collected from five FFPE tissue samples
followed by processing with the GenomePlex
Tissue Whole Genome Amplification Kit
(Sigma Cat. WGA5) as outlined in the technical
bulletin. 5 µl of undiluted WGA5 tissue lysate
was subjected to multiplex PCR amplification
as described at http://www.sigmaaldrich.com/
img/assets/3090/Advisor_FFPE_DNA_
diagnostic.pdf, and 5 µl of each reaction was
resolved on a 4% agarose gel. All five bands
were amplified in lanes 1 and 2 indicating that
these FFPE tissue lysates contain high quality
genomic DNA, whereas lanes 3, 4 and 5 contain
low quality DNA since all, or most, of the
multiplex PCR fragments were not amplifiable.
Similar results were observed when purified
DNA or amplified WGA product derived from
these FFPE tissues were used directly in the
multiplex qPCR assay (data not shown).
Figure 3. aCGH of results derived from Samples 1 and 5. Amplified DNA (1ug) from samples 1
and 5 were analyzed using the PerkinElmer BAC aCGH platform. Sample 1 shows a normal X
chromosome profile as predicted by the multiplex PCR assay (Figure 2), whereas Sample 5 shows
a very poor X chromosome profile due to low quality DNA predicted from the multiplex PCR assay.
3
Validation of GenomePlex Tissue
WGA Kit and Spectral Chip 2600
BAC aCGH platform using a model
system
We generated a series of GenomePlex
amplified aCGH profiles from male and
female FFPE tissues and female DNA known
to contain gains on the X-chromosome,
and hybridized these against GenomePlex
amplified normal female control DNA from
Promega. Briefly, FFPE tissue or genomic
DNA was whole genome amplified per the
Sigma product bulletins (Sigma Cat# WGA5
and WGA2, respectively). 1µg of resulting
products was used for BAC aCGH analysis
using PerkinElmer Labeling Reagents
(Cat # 4040-0020), Hybridization Reagents
(Cat # 4050-0010), Spectral Chip™ 2600 BAC
array and Wash Buffer Pack (Cat # 40380020) per manufacturer’s recommendations.
(Figures 4 to 7) The ideograms generated
using PerkinElmer SpectralWare® aCGH
analysis software below show varying X and
Y chromosome dosage effects in amplified
aCGH. The genotypes for each sample and
reference are given in the figure legends,
whereas array metrics are provided in the
tables below each plot. Mixed male and
female DNA samples (1:1) are indicated as
XY+XX, whereas chromosome trisomy X DNA
is indicated as XXX.
Figure 4. GenomePlex Amplified XY vs XX (1:2 loss of X; 1:0 gain of Y).
4
Figure 5. GenomePlex Amplified XY vs XY (1:1 ratio of X and Y).
Figure 6. GenomePlex Amplified XXX vs XX (3:2 gain of X).
5
Figure 7. GenomePlex Amplified XY +XX vs XX (1.5:2 dilution of X).
FFPE DNA analysis via BAC aCGH
and oligo aCGH
aCGh profiles were generated using the
PerkinElmer Spectral Chip 266 BAC array
platform and using an established oligo
based array product. The BAC arrays were
performed using the supplied manufacturers
protocol and the oligo arrays were performed
by a certified service provider. Figures
8-11 illustrate the design and results of
experiments performed to compare results
using fresh frozen and FFPE multiple
myeloma samples.
Figure 8. Diagram of the experimental
workflow comparing PerkinElmer BAC aCGH
and oligo aCGH analysis of GenomePlex
amplified paired fresh frozen and FFPE
myloma samples and GenomePlex amplified
control DNA (Promega Corp.). Hybridizations
were performed against control DNA of
the opposite sex. All hybridizations were
performed in quadruplicate.
6
Figure 9. SpectralChip 2600 ideograms of chromosome 7 comparing paired frozen and FFPE
myeloma samples. The whole genome data shown here was generated using PerkinElmer
SpectralWare® aCGH Analysis Software. These whole genome results of chromosome 7 are from
1 representative sample, and they show excellent concordance between fresh frozen and FFPE
samples.
Figure 10. Oligo aCGH of frozen and FFPE multiple myeloma samples. Representative
ideograms of comparison data from four sample hybridizations on chromosomes 12, 15, 18, and
22 are shown. When comparing the ”Fresh” and ”FFPE” samples there is little or no correlation
between the samples.
7
Figure 11. Oligo array and BAC array
ideograms of chromosome 7 comparing
results from fresh frozen and FFPE multiple
myeloma samples. Note the high correlation
of data in the BAC array results and the low
level of correlation in the oligo array data.
Conclusion
•
•
•
•
FFPE samples can now be a valuable source of archival material for genomic data analysis
DNA is extracted and WGA amplified directly from tissue with Sigma’s optimized WGA5 kit
DNA quality is predicted with a simple, gel-based, multiplex PCR test and linked to aCGH success
SpectralChip™ 2600 BAC array data correlates well when comparing fresh frozen samples and
FFPE samples
• Oligo-probe aCGH data did not correlate well when comparing fresh frozen samples and FFPE
samples prepared in the same manner.
References (general)
1) E.H. Van Beers et al. A multiplex PCR predictor for aCGH success of FFPE samples.
Br. J. Cancer, 2006, 94:333-337.
2) H.M. Hansen et al. DNA quantification of whole genome amplified samples for genotyping on a
multiplexed bead array platform. Cancer Epidemiol. Biomarkers Prev. 2007, 16:1686–1690.
3) F. Wang et al. DNA degradation test predicts success in whole genome amplification from diverse
clinical samples. J. Mol. Diag. 2007, 9:441-451.
4) E. Mueller. Genomic analysis of formalin-fixed paraffin embedded (FFPE) tissues through the use of
whole genome amplification (WGA).
http://www.sigmaaldrich.com/sigma/general%20information /ffpewhitepaper. October, 2007.
5) F.B. Dean et al. Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and
Multiply-Primed Rolling Circle Amplification. Genome Res, 2001, 11:1095-1099.
6) S.E. Little et al. Array CGH using whole genome amplification of fresh-frozen and formalin-fixed
paraffin-embedded tumor DNA. Genomics, 2006, 87:298-306.
7) K. Iwamoto et al. Evaluation of whole genome amplification methods using postmortem brain
samples. J. Neuroscience Methods, 2007, 165:104-110.
8) M.I.L. Sjoholm et al. Comparison of archival plasma and formalin-fixed paraffin-embedded tissue
for genotyping in hepatocellular carcinoma. Cancer Epidemiol. Biomarkers Prev, 2005, 14:251-255.
PerkinElmer, Inc.
940 Winter Street
Waltham, MA 02451 USA
Phone: (800) 762-4000 or
(+1) 203-925-4602
www.perkinelmer.com
PerkinElmer Genetic Screening
Center of Excellence
Wallac Oy, PO Box 10
20101 Turku, Finland
Phone: + 358 2 2678 111
Fax: + 358 2 2678 357
For a complete listing of our global offices, visit www.perkinelmer.com/lasoffices
©2008 PerkinElmer, Inc. All rights reserved. Spectral Chip is a trademark and Scan Array, SpectralWare and PerkinElmer are registered trademarks of PerkinElmer, Inc.
All trademarks depicted are the property of their respective holders or owners. PerkinElmer reserves the right to change this document at any time and disclaims liability for editorial,
pictorial or typographical errors. All PerkinElmer diagnostic products may not be available in all countries. For information on availability please contact your local representative.
1244-9857, June 2008
Printed in Finland by Offset House Oy Naantali 2008
www.perkinelmer.com