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: -2- The IPR2013-00128 IBS v. Illumina FFT-N3-NR550SO -3- IPR2013-00128 IBS v. Illumina FFA-N3-SS-NR550SO -4- IPR2013-00128 IBS v. Illumina FFC-[N3]2-PEG4-Biotin 3’-AZM-dGTP -5- IPR2013-00128 IBS v. Illumina 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 -6- 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. -7- 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 -8- IPR2013-00128 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); -9- IPR2013-00128 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. -10- IPR2013-00128 IBS v. Illumina 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. -11- IPR2013-00128 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 -12- 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 -13- IPR2013-00128 IBS v. Illumina 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 -14- IPR2013-00128 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 -15-
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