`Leland Stanford Junior University
`
`Paper __
`
`By: R. Danny Huntington, Lead Counsel
`Sharon E. Crane, Ph.D., Backup Counsel
`Rothwell, Figg, Ernst & Manbeck, P .C.
`607 14th St., N.W., Suite 800
`Washington, DC 20005
`Telephone: 202-783-6040
`Facsimile: 202-783-6031
`E-mail: dhuntington@rfem.com
`scrane@rfem.com
`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`SEQUENOM, INC.
`Petitioner,
`
`v.
`
`THE BOARD OF TRUSTEES OF
`THE LELAND STANFORD JUNIOR UNIVERSITY
`Patent Owner.
`
`Case IPR2013-00390
`Patent 8,195,415
`
`DECLARATION OF HEI-MUN CHRISTINA FAN, PH.D.
`
`1
`
`STANFORD EXHIBIT 2132
`SEQUENOM v. STANFORD
`CASE IPR2013-00390
`
`
`
`
`
`The undersigned, Hei-Mun Christina Fan, Ph.D., does hereby declare
`
`and state that:
`
`1.
`
`I make the following declaration based upon my knowledge and
`
`belief.
`
`My educational and professional background
`
`2.
`
`I am one of the named inventors on the above-identified patent
`
`by Hei-Mun Christina Fan and Stephen Quake, namely U.S. Patent No.
`
`8,195,415, issued on June 5, 2012 (“the ‘415 patent”; Ex. 2011) from U.S.
`
`Application Serial No. 12/696,509, filed January 29, 2010 (“the ‘509
`
`application”; Ex. 2012), which is a divisional of U.S. Application Serial No.
`
`12/560,708, filed September 16, 2009 (“the ‘708 application”; Ex. 2014),
`
`which claims priority from U.S. Provisional Application No. 61/098,758,
`
`filed on September 20, 2008 (“the ‘758 provisional”; Ex. 2015).
`
`3.
`
`I am currently employed as a Staff Scientist at Cellular
`
`Research, Inc. in Palo Alto, California. Prior to my current position, in
`
`2011-2013, I was the Director of Technology Development at
`
`ImmunoMetrix LLC in Sunnyvale, California. In 2011, I was a postdoctoral
`
`scientist in the laboratory of Eddy Rubin at the Department of Energy Joint
`
`Genome Institute, Genomics Division, at the Lawrence Berkeley National
`
`Laboratory.
`
`2
`
`
`
`
`
`
`4.
`
`From 2006 to 2011, I was a graduate student in the laboratory
`
`of Stephen Quake in the Department of Bioengineering at Stanford
`
`University (Howard Hughes Medical Institute). During my time in Dr.
`
`Quake’s laboratory, he and I conceived and reduced to practice the invention
`
`claimed in the ‘415 patent.
`
`5.
`
`I am informed that a reduction to practice of an invention
`
`requires: (1) constructing an embodiment or performing a process meeting
`
`every limitation of the interference Count; and (2) demonstrating that
`
`embodiment or process operates for its intended purpose. I am also
`
`informed that reduction to practice may be an actual reduction or a
`
`constructive reduction to practice, which occurs when a patent application
`
`on the claimed invention is filed and that the filing of a patent application
`
`serves as conception and constructive reduction to practice of the subject
`
`matter described in the application.
`
`The present interference
`
`6.
`
`I understand that the ‘415 patent is involved in an interference
`
`with U.S. Application Serial No. 13/070,266, filed March 23, 2011 (“the
`
`‘266 application”; Ex. 2016) by Yuk-Ming Dennis Lo, Rossa Wai Kwun
`
`Chiu and Kwan Chee Chan (“Lo”).
`
`3
`
`
`
`
`
`
`7.
`
`I understand that the Count of an interference is the subject
`
`matter which is being contested in an interference.
`
`8.
`
`I understand that the Count in the ‘922 interference is Fan claim
`
`1, which recites:(cid:31)
`
`A method of testing for an abnormal distribution of a specified
`chromosome portion in a mixed sample of normally and
`abnormally distributed chromosome portions obtained from a
`subject, comprising:
`(a) sequencing DNA from the mixed sample to obtain
`sequences from multiple chromosome portions, wherein said
`sequences comprise a number of sequence tags of sufficient
`length of determined sequence to be assigned to a chromosome
`location within a genome;
`(b) assigning the sequence tags to corresponding
`chromosome portions including at least the specified
`chromosome by comparing the determined sequence of the
`sequence tags to a reference genomic sequence;
`(c) determining values for numbers of sequence tags
`mapping to chromosome portions by using a number of
`windows of defined length within normally and abnormally
`distributed chromosome portions to obtain a first value and a
`second value therefrom; and
`(d) using the values from step (c) to determine a
`differential, between the first value and the second value, which
`is determinative of whether or not the abnormal distribution
`exists.
`
`9.
`
`I also understand the ‘415 patent is involved in an Inter Partes
`
`Review designated IPR2013-00390, filed by Sequenom, Inc.
`
`10. Prior to filing the ‘758 provisional, Steve Quake and I filed an
`
`earlier provisional application, U.S. Provisional Application 60/764,420,
`
`filed on February 2, 2006 (“the ‘420 provisional”; Ex. 2005), and then U.S.
`
`4
`
`
`
`
`
`
`Application Serial No. 11/701,686, filed on February 2, 2007 (“the ‘686
`
`application”; Ex. 2004), which issued as U.S. Patent No. 7,888,017, issued
`
`February 15, 2011 (“the ‘017 patent”; Ex. 1046). These applications
`
`disclosed the non-invasive diagnosis of fetal aneuploidy using digital PCR
`
`and massively parallel sequencing.
`
`11. Toward the end of 2006 and through 2008, Steve Quake and I
`
`worked with Dr. Yair Blumenfeld in the Division of Maternal-Fetal
`
`Medicine in the Department of Obstetrics and Gynecology at Stanford
`
`University. Dr. Blumenfeld was interested in collaborating with us to
`
`provide us with maternal blood samples to use in optimizing our fetal
`
`aneuploidy diagnosis test. (Ex. 2103, 2104, 2106). Dr. Blumenfeld
`
`prepared the necessary protocol and consent forms for the Institutional
`
`Review Board (“IRB”) in order to collect the samples from patients. (Ex.
`
`2105, 2107, 2108). The IRB consent listed Dr. Usha Chitkara, Dr. Louanne
`
`Hudgins, Dr. Yair Blumenfeld and Dr. Quake.
`
`12. Throughout our studies and through the time we submitted our
`
`paper entitled “Noninvasive prenatal diagnosis of fetal chromosomal
`
`aneuploidy by massively parallel genomic sequencing of DNA in maternal
`
`plasma” (Proc. Natl. Acad. Sci. USA 105(51):20458-20643 [2008]; Ex.
`
`1036]), on which I was a coauthor with Drs. Quake, Blumenfeld, Chitkara
`
`5
`
`
`
`
`
`
`and Hudgins, we frequently corresponded by email with Dr. Blumenfeld to
`
`keep him updated on the progress of our research.
`
`13. As discussed in the Fan Priority Statement submitted as Paper
`
`No. 24 in the present interference, Stephen Quake and I conceived of the
`
`invention of the Count at least as early as December 18, 2007. On this date,
`
`I began preparing DNA from patient plasma samples (patients P27, P28,
`
`P30, P31, P32, P35, P36 and P37) containing a mixture of maternal and fetal
`
`DNA for the purpose of doing massively parallel sequencing to obtain
`
`sequence tags, assigning sequence tags to their corresponding chromosomes,
`
`determining how many sequence tags aligned to a chromosome of interest
`
`versus a reference chromosome, and comparing the number of sequence tags
`
`on the chromosome of interest and the reference chromosome to determine
`
`whether an abnormal distribution (indicative of a trisomy) existed. (Ex.
`
`2109, pp. 70-71).
`
`14. On January 2, 2008, Dr. Quake sent an email to Arend Sidow,
`
`Associate Professor of Pathology and of Genetics at Stanford University
`
`School of Medicine, asking Dr. Sidow if we could use his Solexa sequencer
`
`to sequence clinical samples for our “non-invasive test for Down syndrome
`
`based on the small amount of fetal dna that floats around the mother’s blood
`
`6
`
`
`
`
`
`
`stream.” Dr. Sidow responded to this email on January 3, 2008 to schedule a
`
`time to discuss. (Ex. 2118).
`
`15. On January 9, 2008, Dr. Quake contacted Joseph DeRisi, who
`
`was, and is, a Professor and Howard Hughes Investigator in the department
`
`of Biochemistry and Biophysics at the University of California at San
`
`Francisco (UCSF) School of Medicine, requesting to use Dr. DeRisi’s
`
`Solexa sequencer for use with our project on non-invasive diagnosis of fetal
`
`aneuploidy. Dr. DeRisi agreed and indicated that we should talk with
`
`Clement Chu to arrange the sequencing. Later in January of 2008, Drs.
`
`Quake and DeRisi further corresponded about a “reagent barter” where the
`
`Quake lab would provide reagents when Dr. DeRisi wanted to run
`
`sequencing reactions on the Quake 454 sequencer, and the DeRisi lab would
`
`provide reagents when we wanted to run sequencing reactions on their
`
`Solexa sequencer. (Ex. 2119).
`
`16. On January 14, 2008, I extracted and quantified DNA from
`
`additional patient plasma samples (patients P34, P38, P39, P40, P41, P42,
`
`P43, P44 and P45) containing a mixture of maternal and fetal DNA for the
`
`purpose of doing massively parallel sequencing. (Ex. 2109, pp. 72-73).
`
`17. On January 17, 2008, I prepared DNA from one additional
`
`patient plasma sample (patient P13) containing a mixture of maternal and
`
`7
`
`
`
`
`
`
`fetal DNA for the purpose of doing massively parallel sequencing. (Ex.
`
`2109, pp. 74-78).
`
`18. On January 29, 2008, Dr. Quake emailed Clement Chu at
`
`UCSF, referencing his discussions with Dr. DeRisi and inquiring as to how
`
`long it might take before we would be able to use the sequencer, and
`
`whether they had the sequencing reagents in stock. Dr. Chu responded that
`
`he was aware of our interest in using the sequencer, that they did not stock
`
`reagents in the facility, and that they could work us in within a week or so of
`
`when we had our libraries prepared. (Ex. 2121).
`
`19. Also on January 29, 2008, I emailed Dr. Chu separately
`
`indicating that I would be the person working on the sequencing project,
`
`noting that I had heard the reagents were back-ordered and wondering how
`
`we might try to get them faster. Dr. Chu responded that the lead time to get
`
`the reagents was about 3 weeks. (Ex. 2122).
`
`20. The P13 library was sequenced on a Solexa sequencer in the
`
`laboratory of Dr. Arend Sidow, with the assistance of Guang Shi, who was a
`
`post-doc in Dr. Sidow’s laboratory, and Norma Neff, who was a scientist
`
`responsible for running the instrument in Dr. Sidow’s laboratory, on March
`
`12, 2008. (Ex. 2109, pp. 147A).
`
`8
`
`
`
`
`
`
`21. On March 28, 2008, I entered into my laboratory notebook the
`
`results for the Solexa sequencing of the P13 sample. (Ex. 2109, pp. 147A-
`
`147U).
`
`22. The sequencing data from the P13 sequencing run was run
`
`through an alignment program using ELAND software on the Solexa
`
`sequencer, and sequence tags were aligned with each of the chromosomes.
`
`(Ex. 2109, pp. 147B).
`
`23.
`
`I then determined numbers of sequence tags mapping to
`
`chromosome portions using a number of windows of defined length across
`
`each chromosome. (Ex. 2109, pp. 147J-O).
`
`24.
`
`I used a number of windows of defined length within normally
`
`and abnormally distributed chromosome portions to determine the numbers
`
`of sequence tags mapping to chromosome portions and obtained a first value
`
`and a second value therefrom. (Ex. 2109, pp. 147J-O).
`
`25.
`
`I also assessed GC content of the sequence reads. (Ex. 2109,
`
`pp. 147R).
`
`26. On April 4, 2008, Dr. Quake emailed Dr. DeRisi indicating we
`
`were “gearing up for a full solexa run around may 1.” On April 5, 2008, Dr.
`
`DeRisi replied that they had reagents in stock for our Solexa run, and that we
`
`9
`
`
`
`
`
`
`should bring a 1 Tb portable hard drive when we came to do our sequencing.
`
`(Ex. 2120).
`
`27. Also on April 4, 2008, Dr. Quake emailed Dr. Chu indicating
`
`that we would be looking to make a full sequencing run on or about May 1.
`
`Dr. Chu responded to both of us indicating that he could get us in at that
`
`time, and that reagents were not in stock but could be obtained from
`
`Illumina in about two weeks. (Ex. 2123).
`
`28. On April 11, 2008, I began an experiment to extract plasma
`
`DNA from patient 25 (P25) to be used in a massively parallel sequencing
`
`experiment on a 454 sequencer. (Ex. 2110, p. 16). The sequencing run was
`
`performed on April 23, 2008 by Richard (“Rick”) White, III, who ran the
`
`454 sequencer in Dr. Quake’s laboratory. (Ex. 2110, p. 16A).
`
`29. Also on April 11, 2008, I emailed Clement Chu to confirm
`
`dates for Solexa sequencing and confirmed that Dr. DeRisi would provide us
`
`with the reagents. Dr. Chu responded that he was flexible as to dates. On
`
`April 17, 2008, I emailed Dr. Chu to say that I had obtained a library kit and
`
`would prepare libraries for seven total samples, and suggested having the
`
`sequencing run on April 30. Dr. Chu confirmed. (Ex. 2124).
`
`30. The sequencing data from the P25 sequencing run was run
`
`through an alignment program and sequence tags were aligned with each of
`
`10
`
`
`
`
`
`
`the chromosomes, the numbers of sequence tags mapping to chromosome
`
`portions was determined by using a number of windows of defined length
`
`across each chromosome, and the numbers of sequence tags mapping to
`
`chromosome portions were determined by using a number of windows (or
`
`“bins”) of defined length within normally and abnormally distributed
`
`chromosome portions to obtain a first value and a second value therefrom.
`
`On June 16, 2008, I entered into my laboratory notebook an analysis of the
`
`P25 sequence and alignment data. (Ex. 2110, pp. 16A-F, Q-X).
`
`31. GC content of the sequence reads was also assessed. (Ex.
`
`2110, p. 16G).
`
`32. We also assessed the size distribution of all sequences and
`
`evaluated the difference in size distribution between fetal DNA
`
`(chromosome Y fragments) vs. non-fetal DNA (non-chromosome Y
`
`fragments). (Ex. 2110, pp. 16H-P).
`
`33. The P13 and P25 sequencing and analysis confirmed that the
`
`method we had conceived for assessing the presence of fetal aneuploidy in a
`
`mixed maternal sample would work, and we began a larger scale experiment
`
`using DNA from multiple patients, some of whom carried trisomy 21.
`
`34. On April 21, I extracted DNA from the plasma of patients 26,
`
`40 and 42 (P 26, P40, P42) for Solexa sequencing and created sequencing
`
`11
`
`
`
`
`
`
`libraries from these plasma DNA samples using the library kits I had
`
`emailed Dr. Chu about on April 11, 2008 (¶ 29, above). (Ex. 2110, pp. 18-
`
`23).
`
`35. On April 24, I extracted DNA from the plasma of patients 1, 6,
`
`52 and 53 (P1, P6, P52 and P53) for Solexa sequencing. (Ex. 2110, pp. 24-
`
`25).
`
`36. On April 25, I created a library from the extracted DNA from
`
`the plasma of each of the patients 1, 6, 52 and 53 (P1, P6, P52 and P53) for
`
`Solexa sequencing, again using the library kits I had emailed Dr. Chu about
`
`on April 11, 2008 (¶ 29, above). (Ex. 2110, pp. 26-37).
`
`37. On April 29, 2008, I entered into my notebook quantitation of
`
`the sequencing libraries using digital PCR with primers designed to be
`
`complementary to the Solexa library adaptor, which adaptor was added to
`
`the ends of the DNA fragments extracted from the patient plasma. The
`
`quantitation was necessary for, and a standard part of the Illumina protocol
`
`when performing proper cluster generation on the Solexa flow cell to ensure
`
`that the density of clusters on the flow cell was not too low or too high. The
`
`primers had been designed by Rick White. (Ex. 2110, pp. 38-39).
`
`38. On April 30, 2008, I entered into my notebook that I was doing
`
`cluster generation (the first stage of the Solexa sequencing) for DNA
`
`12
`
`
`
`
`
`
`libraries generated from patients P1, P6, P52, P53, P26, P40 and P42 at
`
`University of California, San Francisco (“UCSF”). Dr. Joseph DeRisi had
`
`given us permission to perform our Solexa sequencing at his facility.
`
`Clement Chu assisted us with the actual sequencing run, and Dr. Kael
`
`Fischer, a post-doc in Dr. DeRisi’s laboratory, assisted us with the
`
`alignment. (Ex. 2110, p. 40).
`
`39. On May 20, 2008, Kael Fischer, a post-doc in Dr. DeRisi’s lab,
`
`sent me alignment data (GERALD run) from the sequencing run. He
`
`indicated that one gets fewer sequences mapped when “you use all the way
`
`out to cycle 42.” I responded that because my goal was to “count the
`
`number of reads mapped to each chromosome,” and because I had done an
`
`alignment at Stanford with 25 cycles, I suggested the same be done with the
`
`data from the run in Dr. DeRisi’s lab. (Ex. 2125).
`
`40. On May 24, 2008, Dr. Fischer forwarded additional links to
`
`completed alignments. On May 26, 2008, I asked if there was a way I could
`
`download the files over the internet. We corresponded on how best to do
`
`that. I also asked Dr. Fischer to confirm/explain various codes and
`
`numerical entries. (Ex. 2126).
`
`41. On May 28, 2008, I sent to Yair Blumenfeld and Stephen
`
`Quake a graph of the preliminary analysis of the full sequencing run, which
`
`13
`
`
`
`
`
`
`included four trisomy 21 samples and three non-aneuploid samples. I noted
`
`that they should “pay attention to the distribution of chr21,” and Dr.
`
`Blumenfeld responded that the results were “awesome.” (Ex. 2127). This
`
`analysis constituted an actual reduction to practice of all of the elements of
`
`the Count, and was corroborated by Dr. Blumenfeld.
`
`42. On June 3, 2008, I extracted plasma DNA from patients 2, 7,
`
`14, 19 and 31 for Solexa sequencing. Additionally, I extracted DNA from
`
`plasma and whole blood from a male blood donor and sonicated the DNA in
`
`those samples to obtain fragments. (Ex. 2110, pp. 40-52). These two
`
`samples from the male donors served as controls.
`
`43. On June 6, 2008, I prepared libraries from the plasma DNA
`
`from patients 2, 7, 14, 19 and 31, plasma DNA from a male donor, and
`
`genomic DNA from whole blood from a male donor, for Solexa sequencing.
`
`(Ex. 2110, pp. 53-61).
`
`44. On June 19, 2008, I sent to Steve Quake and Yair Blumenfeld a
`
`“rough draft of the non-invasive study,” (Ex. 2111, 2113) which was based
`
`on our first Solexa sequencing run and was to be submitted to the journal
`
`Proceedings of the National Academy of Sciences, USA, which ultimately
`
`published as the article “Noninvasive diagnosis of fetal aneuploidy by
`
`shotgun sequencing DNA from maternal blood,” Proc. Natl. Acad. Sci. USA
`
`14
`
`
`
`
`
`
`(2008) 105(42):16266-16271 (“first draft of the PNAS manuscript”; Ex.
`
`2113). I was listed as the first author of that paper. My co-authors were
`
`Yair Blumenfeld, Usha Chitkara, Louanne Hudgins and Stephen Quake.
`
`45. The June 19, 2008 draft (Ex. 2113) of the manuscript that
`
`ultimately became Ex. 1036 described the work that I had done, and
`
`demonstrated our reduction to practice, because it showed:
`
`
`
`The Count/Claim 1 of ‘415 Disclosure in the June 19, 2008 draft of the
`PNAS paper (Ex. 2113)
`This is also the first time that cell-free plasma
`DNA is sequenced in high-throughput, allowing
`us to study the physical properties of plasma
`DNA in unprecedented detail. (p. 2, ll. 22-24)
`
`A method of testing for an
`abnormal distribution of a
`specified chromosome
`portion in a mixed sample
`of normally and abnormally
`distributed chromosome
`portions obtained from a
`subject, comprising:
`
`
`“By counting the number of tags from each
`chromosome, the over- or under- representation
`of any chromosome in maternal plasma DNA
`contributed by an aneuploid fetus can be detected.
`We demonstrate here the successful
`use of massively parallel sequencing to detect
`fetal trisomy 21 (Down Syndrome) and trisomy
`18 (Edward Syndrome) non-invasively using cell-
`free fetal DNA in maternal plasma.” (p. 2, ll. 16-
`21)
`
`
`
`(a) sequencing DNA from
`
`“Figure 1. Density of sequence tags for each
`chromosome.” (p. 11; top)
`“This is also the first time that cell-free plasma
`
`15
`
`
`
`
`The Count/Claim 1 of ‘415 Disclosure in the June 19, 2008 draft of the
`PNAS paper (Ex. 2113)
`DNA is sequenced in high-throughput, allowing
`us to study the physical properties of plasma
`DNA in unprecedented detail.” (p. 2, ll. 22-24)
`
`
`
`“By counting the number of tags from each
`chromosome, the over- or under- representation
`of any chromosome in maternal plasma DNA
`contributed by an aneuploid fetus can be
`detected.” (p. 2, ll. 16-18)
`“A total of 20 cell-free plasma DNA samples,
`including 18 from pregnant women and 2 from
`healthy donors, and a genomic DNA sample from
`whole blood of the male donor, were sequenced
`on the Solexa/Illumina platform.” (p. 3, ll. 13-15)
`“Solexa sequencing produced 36 to 48bp reads.
`The first 25bp of each read was mapped to the
`human genome build 36 (hgl8) using ELAND
`built into the Solexa data analysis pipeline. The
`reads that were uniquely mapped to the human
`genome having at most 1 mismatch were retained
`for analysis.” (p. 3; first paragraph under “Data
`Analysis”)
`“Besides chromosome-wise variability of
`sequence tag coverage in plasma DNA samples,
`distribution of sequence tags within each
`chromosome is also non-uniform.” (p. 4, last two
`lines)
`“By counting the number of tags from each
`chromosome, the over- or under- representation
`of any chromosome in maternal plasma DNA
`contributed by an aneuploid fetus can be
`detected.” (p. 2, ll. 16-18)
`
`the mixed sample to obtain
`sequences from multiple
`chromosome portions,
`wherein said sequences
`comprise a number of
`sequence tags of sufficient
`length of determined
`sequence to be assigned to a
`chromosome location within
`a genome;
`
`
`
`
`
`
`
`
`(b) assigning the sequence
`tags to corresponding
`chromosome portions
`including at least the
`specified chromosome by
`
`16
`
`
`
`
`The Count/Claim 1 of ‘415 Disclosure in the June 19, 2008 draft of the
`PNAS paper (Ex. 2113)
`
`
`
`“Solexa sequencing produced 36 to 48bp reads.
`The first 25bp of each read was mapped to the
`human genome build 36 (hgl8) using ELAND
`built into the Solexa data analysis pipeline. The
`reads that were uniquely mapped to the human
`genome having at most 1 mismatch were retained
`for analysis.” (p. 3; first paragraph under “Data
`Analysis”)
`“Reads were aligned to the human genome build
`36 (hg18) using the 454 Mapper. The majority of
`reads retained for analysis had accuracy and
`coverage >90%.” (p. 4, ll. 9-10)
`“A sliding window of
`50kb was applied across each chromosome,
`ignoring regions of assembly gaps and micro
`satellites, and the number of tags falling within
`each window was counted. The median count per
`50kb window for each chromosome normalized
`to the genome-wide value is plotted (Figure 1).”
`(p. 4, ll. 24-28)
`
`“To compare the coverage of the different
`chromosomes, a sliding window of 50 kb was
`applied across each chromosome … and the
`number of sequence tags falling within each
`window was counted. To compare the
`chromosomal coverage among all samples, for
`each sample, the median number of sequence tags
`per 50 kb window of each chromosome was
`normalized to the genome-wide median number
`of sequence tags per 50 kb window.” (p. 3; first
`paragraph under “Data Analysis”)
`
`comparing the determined
`sequence of the sequence
`tags to a reference genomic
`sequence;
`
`
`
`
`(c) determining values for
`numbers of sequence tags
`mapping to chromosome
`portions by using a number
`of windows of defined
`length within normally and
`abnormally distributed
`chromosome portions to
`obtain a first value and a
`second value therefrom;
`and
`
`
`17
`
`
`
`
`
`
`The Count/Claim 1 of ‘415 Disclosure in the June 19, 2008 draft of the
`PNAS paper (Ex. 2113)
`“The distribution of chromosome 21 for T21
`pregnancies is clearly separated from that of
`normal pregnancies. The coverage
`of chromosome 21 for T21 cases is bout 12-16%
`higher than that of the normal cases.” (p. 4, ll.
`32-34)
`“Let the number of ChrX sequences from female
`plasma be N and fetal DNA fraction be ε. The
`number of ChrX sequences from maternal plasma
`
`
`
`
`
`is then N*(l- ε) + Nε/2, where the first and second
`
`terms are contributions from mother and fetus
`respectively. The ratio of ChrX coverage in
`maternal plasma: ChrX coverage in female
`plasma is equal to 2/(2-ε).” (p. 14,
`Supplementary Information)
`“The distribution of chromosome 21 for T21
`pregnancies is clearly separated from that of
`normal pregnancies. The coverage of
`chromosome 21 for T21 cases is about 12-16%
`higher than that of the normal cases.” (p. 4, ll.
`32-34)
`
`
`“Let the number of ChrX sequences from female
`plasma be N and fetal DNA fraction be ε. The
`number of ChrX sequences from maternal plasma
`
`is then N*(l- ε) + Nε/2, where the first and second
`
`terms are contributions from mother and fetus
`respectively. The ratio of ChrX coverage in
`maternal plasma: ChrX coverage in female
`plasma is equal to 2/(2-ε).” (p. 14,
`Supplementary Information)
`
`(d) using the values from
`step (c) to determine a
`differential, between the
`first value and the second
`value, which is
`determinative of whether or
`not the abnormal
`distribution exists.
`
`
`
`
`46. On June 20, 2008, we did a second Solexa sequencing run at
`
`UCSF on libraries prepared from patients P2, P7, P14, P19 and P31 and
`
`18
`
`
`
`
`
`
`male donor’s plasma DNA and whole blood genomic DNA. (Ex. 2110, p.
`
`62).
`
`47. On June 26, 2008, I assessed the Solexa libraries using digital
`
`PCR. (Ex. 2110, pp. 63-71).
`
`48. On June 28, 2008, I extracted plasma DNA from patient 23.
`
`(Ex. 2110, p. 62).
`
`49. On July 1, I sent a graph including new data points (3 Down
`
`syndrome [P2, P7, P14] and 2 normal males [P19, P31]) to Dr. Quake and
`
`Dr. Blumenfeld. (Ex. 2129).
`
`50. Dr. Quake and I also corresponded by email about going for
`
`one more full run that would start the week of July 21 due to skilled users of
`
`the Solexa sequencer being gone during the intervening days. (Ex. 2130).
`
`51. On July 1, 2008, Dr. Quake began corresponding with Leonard
`
`Herzenberg regarding communicating our PNAS manuscript (Ex. 1036) to
`
`the journal. Dr. Quake forwarded the draft manuscript to Dr. Herzenberg on
`
`July 9. (Ex. 2112).
`
`52. On July 7, 2008, Drs. Quake, Blumenfeld and I discussed
`
`revisions to the PNAS manuscript. (Ex. 2131).
`
`53. On July 14, 2008, I prepared libraries for a third sequencing run
`
`on the Solexa sequencer at UCSF. (Ex. 2110, pp. 96-108).
`
`19
`
`
`
`
`
`
`54. We performed the third Solexa sequencing run on July 23, 2008
`
`with patient samples P17, P20, P23, P57, P59 and P64. (Ex. 2110, pp. 86).
`
`55.
`
`I revised the first draft of the PNAS manuscript as a second
`
`draft of the PNAS manuscript. On July 9, 2008, that second draft of the
`
`PNAS manuscript was sent by Dr. Quake (copying me on that
`
`communication) to Len Herzenberg for publication in PNAS. (Ex. 2112)
`
`Len Herzenberg was a Professor Emeritus of Genetics at the Stanford
`
`University School of Medicine. Dr. Herzenberg passed away in October of
`
`2013. As indicated on the front page of the PNAS publication (Ex. 1036),
`
`the PNAS manuscript was received for review by PNAS reviewers on July
`
`13, 2008, communicated on August 22, 2008, and published on October 21,
`
`2008.
`
`56. The second draft of the PNAS paper (Ex. 2112) demonstrated
`
`our reduction to practice, because it showed:
`
`The Count/Claim 1 of ‘415
`
`A method of testing for an
`abnormal distribution of a
`specified chromosome portion in a
`mixed sample of normally and
`abnormally distributed
`chromosome portions obtained
`from a subject, comprising:
`
`
`Disclosure in the second draft of
`the PNAS paper (Ex. 2112)
`“We directly sequenced cell-free
`DNA from plasma of pregnant
`women…This enabled us to measure
`the over- and underrepresentation of
`chromosomes from an aneuploid
`fetus” (p. 2; Abstract)
`
`“By counting the number of
`sequence tags mapped to each
`chromosome, the over- or
`
`20
`
`
`
`
`
`
`Disclosure in the second draft of
`the PNAS paper (Ex. 2112)
`underrepresentation of any
`chromosome in maternal plasma
`DNA contributed by an aneuploid
`fetus can be detected.” (p. 4, ll. 9-
`11)
`We demonstrate here the successful
`use of massively parallel sequencing
`to detect fetal trisomy 21
`noninvasively using cell-free fetal
`DNA in maternal plasma.” (p. 4, ll.
`14-17)
`“An average of ~60,000 sequence
`tags mapped to chromosome 21.” (p.
`5; ll. 5-6)
`“We directly sequenced cell-free
`DNA with high-throughput shotgun
`sequencing technology… obtaining,
`on average, 5 million sequence tags
`per … sample” (p. 2; Abstract)
`
`
`“By counting the number of
`sequence tags mapped to each
`chromosome, the over- or
`underrepresentation of any
`chromosome in maternal plasma
`DNA contributed by an aneuploidy
`fetus can be detected.” (p. 4, ll. 9-
`11)
`“Cell-free plasma DNA from 13
`pregnant women…were sequenced
`on the Solexa/Illumina platform. We
`obtained on average ~8 million 25 bp
`sequence tags per sample.” (p. 5; ll.
`3-5)
`
`The Count/Claim 1 of ‘415
`
`
`
`
`
`(a) sequencing DNA from the
`mixed sample to obtain sequences
`from multiple chromosome
`portions, wherein said sequences
`comprise a number of sequence
`tags of sufficient length of
`determined sequence to be
`assigned to a chromosome location
`within a genome;
`
`
`
`
`21
`
`
`
`
`
`
`Disclosure in the second draft of
`the PNAS paper (Ex. 2112)
`“We observed a nonuniform
`distribution of sequence tags across
`each chromosome.” (p. 5, l. 10)
`“By counting the number of
`sequence tags mapped to each
`chromosome, the over- or
`underrepresentation of any
`chromosome in maternal plasma
`DNA contributed by an aneuploidy
`fetus can be detected.” (p. 4, ll. 9-
`11)
`“An average of ~60,000 sequence
`tags mapped to chromosome 21.” (p.
`5; ll. 5-6)
`“Solexa sequencing produced 36 to
`50bp reads. The first 25bp of each
`read was mapped to the human
`genome build 36 (hg18) by using
`ELAND from the Solexa data
`analysis pipeline. The reads that
`were uniquely mapped to the human
`genome having at most 1 mismatch
`were retained for analysis.” (p. 19;
`ll. 5-8)
`“For the 454/Roche data, reads were
`aligned to the human genome build
`36 (hg18) using the 454 Reference
`Mapper. Reads having accuracy of ≥
`90% and coverage (i.e. fraction of
`read mapped) ≥ 90% were retained
`for analysis.” (p. 20, ll. 11-13)
`“We applied a sliding window of 50
`kb across each chromosome and
`counted the number of tags falling
`within each window.” (p. 5; ll. 13-
`14)
`
`The Count/Claim 1 of ‘415
`
`
`
`(b) assigning the sequence tags to
`corresponding chromosome
`portions including at least the
`specified chromosome by
`comparing the determined
`sequence of the sequence tags to a
`reference genomic sequence;
`
`
`
`
`
`
`
`(c) determining values for
`numbers of sequence tags mapping
`to chromosome portions by using a
`number of windows of defined
`length within normally and
`abnormally distributed
`chromosome portions to obtain a
`
`22
`
`
`
`
`
`
`Disclosure in the second draft of
`the PNAS paper (Ex. 2112)
`
`“To compare the coverage of the
`different chromosomes, a sliding
`window of 50 kb was applied across
`each chromosome … and the number
`of sequence tags falling within each
`window were counted and the
`median value was chosen to be the
`representative of the chromosome.”
`(p. 19; ll. 8-12)
`“The distribution of chromosome 21
`sequence tag density for T21
`pregnancies is clearly separated from
`that of normal pregnancies …” (p. 6;
`ll. 9-10)
`“The coverage of chromosome 21 for
`T21 cases is ~4-8% higher (average
`~11%) than that of the disomy 21
`cases.” (p. 6; ll. 11-12)
`“Because the sequence tag density of
`chromosome 21 for T21 cases should
`be (1 + ε/2) of that of normal
`pregnancies, where ε is the fraction
`of total plasma DNA originating
`from the fetus (see SI for
`derivations), such increase in
`chromosome 21 coverage in T21
`cases corresponds to a fetal DNA
`fraction of ~16-35% (average
`~26%).” (p. 6; ll. 12-16).
`“The coverage of chromosome 21 for
`T21 cases is ~4-8% higher (average
`~11%) than that of the disomy 21
`cases.” (p. 6; ll. 11-12)
`
`The Count/Claim 1 of ‘415
`
`first value and a second value
`therefrom; and
`
`
`
`
`
`
`
`
`(d) using the values from step (c) to
`determine a differential, between
`the first value and the second
`value, which is determinative of
`whether or not the abnormal
`distribution exists.
`
`23
`
`
`
`
`The Count/Claim 1 of ‘415
`
`
`
`
`
`
`
`
`
`Disclosure in the second draft of
`the PNAS paper (Ex. 2112)
`“Because the sequence tag density of
`chromosome 21 for T21 cases should
`be (1 + ε/2) of that of normal
`pregnancies, where ε is the fraction
`of total plasma DNA originating
`from the fetus (see SI for
`derivations), such increase in
`chromosome 21 coverage in T21
`cases corresponds to a fetal DNA
`fraction of ~16-35% (average
`~26%).” (p. 6; ll. 12-16).
`“We constructed a 99% confidence
`interval of the distribution of
`chromosome 21 sequence tag density
`of normal pregancies. The