Patent Owner Illumina Cambridge Ltd. By

Filed on behalf of:
Patent Owner Illumina Cambridge Ltd.
By: Brenton R. Babcock
William R. Zimmerman (admitted pro hac vice)
Jonathan E. Bachand
KNOBBE, MARTENS, OLSON & BEAR, LLP
2040 Main Street, 14th Floor
Irvine, CA 92614
Tel.: (949) 760-0404
Fax: (949) 760-9502
Email:
[email protected]
UNITED STATES PATENT AND TRADEMARK OFFICE
__________________________________
BEFORE THE PATENT TRIAL AND APPEAL BOARD
__________________________________
INTELLIGENT BIO-SYSTEMS, INC.
Petitioner,
v.
ILLUMINA CAMBRIDGE LTD.
Patent Owner
Case IPR2013-00128 (LMG)
Patent 7,057,026
SUBSTITUTE DECLARATION OF ERIC VERMAAS ACCOMPANYING
PATENT OWNER’S MOTION TO AMEND
IPR2013-00128
IBS v. Illumina
1.
I, Eric Vermaas, am presently employed by Illumina, Inc., a Delaware
corporation with its principal place of business located at 5200 Illumina Way, San
Diego, CA 92122. Illumina, Inc. is the parent company of Illumina Cambridge
Limited, the Patent Owner in this case. I understand that the U.S. Patent and
Trademark Office Patent Trial and Appeal Board has instituted an inter partes
review of the patentability of the claims of U.S. Patent No. 7,056,026 (“the ’026
patent”) based upon a petition filed by Intelligent Bio-Systems, Inc.
2.
I received a B.A. in Genetics in 1989 from the University of
California at Berkeley.
3.
I am not being compensated for my time spent on this testimony
outside of my normal compensation as an employee of Illumina, Inc.
4.
My present position at Illumina, Inc. is Director of Consumables
Product Development. I have been employed by Illumina, Inc. since 2006. My
previous positions at Illumina, Inc. were Associate Director (2009-2013) and Staff
Scientist (2006-2009).
5.
The following is a description of a nucleotide sequencing experiment
that was performed under my supervision.
6.
The nucleotides utilized in this experiment were: FFT-N3-NR550SO,
FFA-N3-SS-NR550SO, FFC-[N3]2-PEG4-Biotin, and 3’-AZM-dGTP.
structures of these nucleotides are depicted below:
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The
IPR2013-00128
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FFT-N3-NR550SO
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IBS v. Illumina
FFA-N3-SS-NR550SO
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FFC-[N3]2-PEG4-Biotin
3’-AZM-dGTP
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7.
A streptavidin-NR550C4SO conjugate was also used in this
experiment. The streptavidin-NR550C4SO conjugate was prepared by conjugating
streptavidin to NR550C4SO dye as follows. Dye Activation: A reaction flask was
first washed with 100% acetonitrile (Sigma Aldrich), a stir bar was added to the
flask, and then the flask and the stir bar were dried in an oven overnight at 60˚ C.
The flask and stir bar were then allowed to cool to room temperature in a
desiccator under vacuum. NR550C4SO dye (Illumina, 25.8 mg) was also dried
overnight in a desiccator. The NR550C4SO dye and dimethylacetamide (Sigma
Aldrich, 1.2 mL) were combined in the flask. The flask was then closed and
allowed to stir until dissolution was completed.
N,N-Diisopropylethylamine
(Sigma Aldrich, 16.5 µL) was then added to the flask and the reaction mixture was
allowed
to
stir
for
5-10
seconds.
N,N,N′,N′-Tetramethyl-O-(N-
succinimidyl)uronium tetrafluoroborate (Sigma Aldrich, 14.01 mg) was then added
to the reaction mixture. The flask was closed and allowed to stir at 22˚ C for 90
minutes to afford an activated dye. Dye Conjugation: Streptavidin (Pierce, 56.1
mg) was then added to a 15 mL conical falcon tube and diluted with 7 mL of a 100
mM NaHCO3 buffer (pH 8.8). The activated dye (407 µL) was then added to the
tube containing the diluted streptavidin mixture. The tube was then rocked in the
dark for 120 minutes at 22˚ C. The tube was then stored at 4˚ C for > 12 hours.
5M NaCl was added to adjust the final NaCl concentration to 150 mM, followed
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IPR2013-00128
IBS v. Illumina
by storage at 4˚ C until purification. Conjugate purification: The dye conjugation
reaction was purified by gel filtration with a Thermo Scientific Fluorescent Dye
removal column (PN #22858). Two 5 ml resin columns were prepared by pouring
5 mL in each column and centrifuging at 2,000 rpm for 3 minutes. The columns
were then centrifuged again at 2,000 rpm for one minute. The conjugate reaction
(3.8 mL) was added to the top of each column and centrifuged for 5 min at 2,000
rpm. The columns were then centrifuged for an additional two minutes. The
obtained streptavidin-NR550C4SO conjugate was then quantitated using a
NanoDrop 2000 (Thermo Fisher) and diluted to a stock solution of 1 mg/mL
streptavidin-NR550C4SO conjugate in 5 mM Tris-HCl pH 7.5, 0.5 mM EDTA and
1 M NaCl.
8.
All nucleotide stock solutions contained 100 µM nucleotide in 10 mM
Tris Buffer, pH 8.0. Polymerase stock solution contained 600 µg/mL of High
Density polymerase Epicentre in buffer solution.
9.
Incorporation Mix (OIM) included the four nucleotide stock solutions,
polymerase stock solution and buffer to yield the final concentrations of 2 µM
FFT-N3-NR550SO (“dTTP analog”), 2 µM FFA-N3-SS-NR550SO (“dATP
analog”), 2 µM FFC-[N3]2-PEG4-Biotin (“dCTP analog”), 2 µM 3’-AZM-dGTP
(“dGTP analog”), and High Density polymerase in buffer solution.
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IPR2013-00128
IBS v. Illumina
10.
Cleavage
Mix
(OCM)
contained
50
mM
tris(hydroxymethyl)phosphine in pH 9.9 buffer solution.
11.
Scan Mix (SREOSM) contained buffer solution used for detection of
sequencing carried out on a MiSeqTM instrument.
12.
Streptavidin-Binding Mix (SBM) was prepared by diluting the
streptavidin-NR550C4SO stock solution to give a final concentration of 0.005
mg/mL streptavidin-NR550C4SO in 2 mM tris(hydroxymethyl)phosphine, 5 mM
Tris-HCl pH 7.4, 1 M NaCl, 0.5 mM EDTA and 0.005% Tween-20.
13.
Sequencing polymerase reactions were conducted as described below
using sequencing control PhiX v3 (Illumina, PN #FC-110-3001).
14.
PhiX Control DNA was clustered using the bridge amplification
technique described in Bentley et. al (Nature 2008 Nov 6;456(7218):53-9) on a
MiSeqTM flowcell (Illumina, PN #MS-102-2002) to yield approximately 307,302
clusters per mm2 containing approximately 1000 copies of template DNA per
cluster.
15.
The flowcell was primed and sequenced on a MiSeqTM sequencing
instrument (Illumina, Inc.) unit for 150 cycles. All sSteps were performed at 60˚
C, 65˚ C, or 22˚ C, as indicated below. All fluorescence measurements were
performed by irradiating with an LED source at about 52732 nm and measuring
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IBS v. Illumina
fluorescence in the range from about 583 nm to aboutat 63700 nm. The steps of
each sequencing cycle were as follows:
a. Adjust temperature to 60˚ C (15 second duration);
i. Wash (120 µL, 3.6 second duration);
ii. OCM (60 µL, 1.8 second duration);
iii. Wash (20 µL, 1 second duration);
iv. Dispense contents to waste (6 second duration);
v. Wash (250 µL, 7.5 second duration);
a.vi. OIM Delivery (60 µL, 1.860 seconds incubation);
vii. Wash (20 µL, 1 second duration);
b. Adjust temperature to 65˚ C (25 second duration);
i. Wash (5 µL, 1 second duration );
ii. Dispense contents to waste (30 second duration);
c. Adjust temperature to 22˚ C (15 second duration);
i. Wash (120 µL, 3.6 second duration);
b.ii. SREOSM, scan at 527 nm (60120 µL, 1.84 seconds incubation
+ image time of 2 minutes);
c.iii. Wash (120 µL, 0.6 seconds duration incubation);
iv. Dispense contents to waste (25 second duration);
v. Image (60 second duration);
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IBS v. Illumina
d. Adjust temperature to 60˚ C (15 second duration);
i. Wash (125 µL, 3.75 second duration);
d.ii. SBM (60 µL, 1.8 second duration and 520 seconds incubation);
e. Adjust temperature to 22˚ C (15 second duration);
i. Wash (60 µL, 1.8 second duration);
e.ii. SREOSM, scan at 527 nm (60 µL, 1.84 seconds incubation +
image time of 2 minutes);
f. OCM (27 seconds incubation);
g.iii. Wash (120 µL, 0.6 seconds incubation) duration;
iv. Dispense contents to waste (15 second duration); and
h.
Image (60 second duration). Repeat from step a.
v. The time for flowing solution through the flow cell between
each of steps (a) through (g) was about 5-10 seconds.
16.
Results from the sequencing experiment can be found in Figures 1-3.
17.
Figure 1 shows the Percent Error Rate for basecalls over the 150 cycle
sequencing run for tile #1101 (“tile 1”) of 12 tiles analyzed in the experiment. The
y-axis is the Percent Error Rate of a particular sequencing cycle, and the x-axis is
the Sequencing Cycle Number.
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Figure 1
Percent Error Rate for each cluster is the number of miscalls in the 150-cycle read
for each cluster divided by 150 (total number of cycles) and multiplied by 100 (to
obtain percentage).
Percent Error Rate was calculated by processing the run
through with RTA 1.18.11 (Illumina Analysis Software, proprietary) to generate
base calls and quality scores, then processing the reads through MiSeqTM Reporter
version 2.2.3 (Illumina Alignment Software). The reads were aligned using the
software tool bwa (Burrows-Wheeler Aligner) version 0.6.1-r104-tpx. The aligner
determines the location, within the sequence of the Phi X reference sequence, of
the experimental sequence determined by 150 basecalls measured for a particular
cluster.
Bwa was able to align 93.5% of reads.
A frequency of error of
approximately 0.43% over 150 cycles was measured for tile 1.
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IBS v. Illumina
18.
Figure 2 shows the frequency of errors for an expected base measured
as one of the other 3 bases in the detection for all tiles, where the frequency of
errors was calculated in accordance with paragraph 17. The twelve possible base
miscalls are listed. As an illustrative example, T->A is the percentage of A bases
miscalled as T bases. The red line represents the mean frequency of error, the blue
box represents the upper first quartile and lower first quartile of frequencies of
error, the black lines represent one standard deviation from the mean frequency of
error, and blue lines represent frequencies of error from each individual tile.
Figure 2
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Frequency of Errors
IPR2013-00128
IBS v. Illumina
19.
Figure 3 is a cloud plot of fluorescence at steps bc and e for all tiles.
The y-axis is the fluorescence intensity (arbitrary units) measured at step bc, and
the x-axis is the fluorescence intensity (arbitrary units) measured at step e.
Figure 3
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A signal along the x-axis greater than 700 and a signal along the y-axis greater than
600 was identified as the nucleotide residue T. A signal along the x-axis less than
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IBS v. Illumina
700 and a signal along the y-axis greater than 600 was identified as the nucleotide
residue A. A signal along the x-axis greater than 700 and a signal along the y-axis
less than 600 was identified as the nucleotide residue C. A signal along the x-axis
less than 700 and a signal along the y-axis less than 600 was identified as the
nucleotide residue G.
20.
I hereby declare that all statements made herein of my own
knowledge are true and that all statements made on information and belief are
believed to be true; and further that these statement were made with the knowledge
that willful false statements and the like so made are punishable by fine or
imprisonment, or both, under Section 1001 of Title 18 of the United States Code
and that such willful false statement may jeopardize the validity of the application
or any patent issued thereon.
Dated:___________________
By:_________________________________
Eric Vermaas
16911245
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