Paper No.
`
`Filed on behalf of: Party QUAKE
`
`By:
`
`R. Danny Huntington, Esq.
`Sharon E. Crane, Ph.D., Esq.
`ROTHWELL, F IGG, ERNST & MANBECK, RC.
`607 1411‘ St., NW, Suite 800
`Washington, DC 20005
`dhuntington@rfem.com
`scrane@rfem.com
`Main Telephone: (202) 783-6040
`Main Facsimile: (202) 783-6031
`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`STEPHEN QUAKE and HEI—MUN CHRISTINA FAN
`Senior Party
`(Patent 8,008,018).
`v
`
`YUK-MING DENNIS LO, ROSSA WAI KWUN CHIU, and KWAN CHEE CHAN
`
`Junior Party
`(Application 131070275),
`
`YUK—MING DENNIS LO, ROSSA WA] KWUN CHIU, and KWAN CHEE CHAN
`
`Junior Party
`(Applications 12f178,181, 1311070240, ӣ614,350, 13f070,251) (Application 1311411119),
`v
`
`STEPHEN QUAKE and HEI-MUN CHRISTINA FAN
`
`Senior Party
`(Application [2893,833) (Application 12893333).
`
`Patent Interference Nos. 105,920, 105,923, 105,924 (DK)
`(Technology Center 1600)
`
`DECLARATION OF JOHN CHRISTOPHER BETTER, PHD.
`
`SEQUENOM EXHIBIT 1086
`SEQUENOM EXHIBIT 1086
`Sequenom v. Stanford
`Sequenom V. Stanford
`IPR2013-00390
`
`IPR2013-00390
`
`

`

`Paper No.
`
`Filed on behalf of: Party QUAKE
`
`By:
`
`R. Danny Huntington, Esq.
`Sharon E. Crane, Ph.D., Esq.
`ROTHWELL, FIGG, ERNST & MANBECK, RC.
`607 14th St., N.W., Suite 800
`Washington, DC 20005
`dhuntington@rfem.com
`scrane@rfem.com
`Main Telephone: (202) 783—6040
`Main Facsimile: (202) 783-6031
`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`STEPHEN QUAKE and HEI-MUN CHRISTINA FAN
`Senior Party
`(Patent 8,008,018).
`v
`
`YUK-MING DENNIS LO, ROSSA WAI KWUN CHIU, and KWAN CHEE CHAN
`
`Junior Party
`(Application 13/070,275),
`
`YUK-MING DENNIS LO, ROSSA WAI KWUN CHIU, and KWAN CHEE CHAN
`
`Junior Party
`(Applications 12/178,181, 13/070,240, 12/614,350, 13/070,251) (Application 13/417,119),
`v
`
`STEPHEN QUAKE and HEI-MUN CHRISTINA FAN
`Senior Party
`(Application 12/393,833) (Application 12/393,833).
`
`Patent Interference Nos. 105 ,920, 105,923, 105,924 (DK)
`(Technology Center 1600)
`
`DECLARATION OF JOHN CHRISTOPHER DETTER, PH.D.
`
`EXHIBIT 2049
`
`QUAKE v. LO, Interference No. 105,920
`FAN v. LO, Interference No. 105,922
`
`LO v. QUAKE, Interference No. 105,923
`LO v. QUAKE, Interference No. 105,924
`
`

`

`Interference Nos. 105,920, 105,923, 105,924
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`MAIL STOP INTERFERENCE
`United States Patent and Trademark Office
`Patent Trial and Appeal Board
`Madison Building East
`600 Dulany Street
`Alexandria, Virginia 22313
`
`
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`The undersigned, John Christopher (“Chris”) Detter, Ph.D., does hereby declare and state
`
`that:
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`1.
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`I make the following declaration based upon my knowledge and belief.
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`My educational and professional background
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`2.
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`I earned my B.S. in Biology at Baylor University in 1995 and my Ph.D. in
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`medical sciences (molecular genetics and microbiology) at University of Florida in Gainesville
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`in 1999.
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`3.
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`After receiving my Ph.D., I worked as the Team and Technical Leader for the
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`Research and Development Genomic Technology Group at the Lawrence Livermore National
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`Laboratory (“LLNL”) as part of the Joint Genome Institute from September, 1999 until May,
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`2001, where I developed DNA library creation protocols and methods for high-throughput
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`sequencing for the human genome sequencing project.
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`4.
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`From May, 2001 until September, 2005, I was the Group Leader in the
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`Genomics/Cloning Technology Group at LLNL, during which time I managed the high-
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`throughput library creation process for the Joint Genome Institute.
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`5.
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`From September, 2005 until February, 2009, I was the Team Leader for the
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`Sequencing Technology Team at the Joint Genome Technology Group (B-6) at the Los Alamos
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`National Laboratory (“LANL”), during which time I managed a highly automated sequencing
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`team focused on the genome finishing process. Also, from October, 2006 until April, 2007, I
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`

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`Interference Nos. 105,920, 105,923, 105,924
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`was acting Group Leader for the Joint Genome Institute-Genome Technology Group (B-5) in the
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`Bioscience Division of LANL, where I managed a diverse genomic sequencing and
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`computational biology group.
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`6.
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`Since February, 2008, I have been the Genome Sciences Center Director and
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`Group Leader (B-6) at LANL, where I manage a diverse genomic sequencing and computational
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`biology group of about 45 individuals. In addition, since September, 2011, I have been the
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`Acting Bioscience Deputy Divisional Leader at LANL, in which position I assist the Division
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`Leader in programmatic, strategic and tactical mission areas for Bioscience. As of October
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`2012, I am now the BioThreat, BioDefense Program Director for LANL.
`
`7.
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`I received additional technical training in automated DNA sequencing at Perkin-
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`Elmer Corporation in 1997, at Amersham in 1999, at Applied Biosystems in 2000, at Affymetrix
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`in 2000, at 454 Life Sciences in 2006, and I consider myself to be an expert on high-throughput
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`sequencing on the Roche 454, Illumina and PacBio platforms.
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`8.
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`I have published numerous scientific research papers in the areas of chromosomal
`
`mapping and genome sequencing, as listed on my curriculum vitae, which is being submitted
`
`with this declaration as Exhibit 2051.
`
`9.
`
`I am informed that three patent interferences, identified above, have been declared
`
`as follows:
`
`(cid:120)
`
`Interference 105,920 (“the ‘920 interference”) was declared between: (1) U.S. Patent
`
`No. 8,008,018, which issued on August 30, 2011 (“the Quake ’018 patent”; Exhibit
`
`2001) to Stephen Quake and Hei-Mun Christina Fan (“Quake”); and (2) U.S.
`
`Application Serial No. 13/070,275, filed on March 23, 2011 (“the Lo ’275
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`Interference Nos. 105,920, 105,923, 105,924
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`application”; Exhibit 2006), of Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, and
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`Kwan Chee Chan (“Lo”; Paper Nos. 1, 43);
`
`(cid:120)
`
`Interference No 105,923 (“the ‘923 interference”) was declared between: (1) U.S.
`
`Application Serial No. 12/393,833, which was filed on February 26, 2009 (“the
`
`Quake ’833 application”; Exhibit 2018) of Stephen Quake and Hei-Mun Christina
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`Fan (“Quake”); and (2) U.S. Application Serial Nos. 12/178,181, filed on July 23,
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`2008 (“the Lo ’181 application”; Exhibit 2009), 13/070,240, filed March 23, 2011
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`(“the Lo ‘240 application”; Exhibit 2069), 12/614,350, filed November 6, 2009 (“the
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`Lo ‘350 application”; Exhibit 2070) and 13/070,251, filed March 23, 2011 (“the Lo
`
`‘251 application”; Exhibit 2071), all to Yuk-Ming Dennis Lo, Rossa Wai Kwun
`
`Chiu, and Kwan Chee Chan (“Lo”; Paper Nos. 1, 20, 23); and
`
`(cid:120)
`
`Interference No 105,924 (“the ‘924 interference”) was declared between: (1) the
`
`Quake ’833 application (Exhibit 2018); and (2) U.S. Application Serial No.
`
`13/417,119, filed on March 9, 2012 (“the Lo ‘119 application”; Exhibit 2022), Yuk-
`
`Ming Dennis Lo, Rossa Wai Kwun Chiu, and Kwan Chee Chan (“Lo”; Paper No. 1).
`
`10.
`
`I am informed that in the ‘920 interference, Quake seeks benefit of the ‘686
`
`application (Exhibit 2004) and the ‘420 provisional (Exhibit 2005); and that in the ‘923 and ‘924
`
`interferences, Quake seeks benefit of the ‘420 provisional (Exhibit 2005).
`
`Independent Witness
`
`11.
`
`I have been retained in this matter by Rothwell, Figg, Ernst & Manbeck, P.C. of
`
`Washington, D.C. (“Rothwell”).
`
`12. My main contacts at Rothwell are Sharon Crane, Ph.D., Esq., Seth Cockrum,
`
`Ph.D., Esq. and Danny Huntington, Esq.
`
`
`
`

`

`Interference Nos. 105,920, 105,923, 105,924
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`13.
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`14.
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`I have had no previous contact with Rothwell or these main contacts.
`
`I understand that the real parties in interest for party Quake and party Fan is The
`
`Board of Trustees of the Leland Stanford University (“Stanford”), Fluidigm Corporation
`
`(“Fluidigm”), Verinata Health, Inc. (“Verinata”) and Illumina, Inc. (“Illumina”) and that the real
`
`parties in interest for party Lo are the Chinese University of Hong Kong (“the Chinese
`
`University”) and Sequenom, Inc. (“Sequenom”).
`
`15.
`
`I do not have any financial interest in any of the real parties in interest or in the
`
`outcome of this proceeding.
`
`16.
`
`I have had no previous personal contact with the Quake inventors or the Lo
`
`inventors.
`
`17.
`
`I believe that I can provide an independent witness opinion in this matter as
`
`independent expert witness.
`
`My opinion and its bases
`
`18.
`
`I have been asked to give my opinion on (1) whether the Quake ‘686 application
`
`(Exhibit 2004) provides a written description of at least one embodiment within the scope of the
`
`‘920 Count; (2) whether the Quake ‘686 application provides sufficient information that one of
`
`ordinary skill in the art would be able to practice at least one embodiment of the ‘920 Count
`
`without undue experimentation; (3) whether the Quake ‘420 provisional (Exhibit 2005) provides
`
`a written description of at least one embodiment within the scope of the ‘920, 923 and ‘924
`
`Counts; and (4) whether the Quake ‘420 provisional (Exhibit 2005) provides sufficient
`
`information that one of ordinary skill in the art would be able to practice at least one embodiment
`
`of the ‘920, ‘923 and ‘924 Counts without undue experimentation.
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`Interference Nos. 105,920, 105,923, 105,924
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`19.
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`I have been informed that an assessment of whether an application contains a
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`written description to support a claim is based on whether a person of ordinary skill in the art
`
`would appreciate from the description of the claimed invention that the inventors were in
`
`possession of the claimed invention at the time the application was filed. I also understand that
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`“possession” may be shown in various ways, including by describing distinguishing identifying
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`characteristics of the claimed invention, and that determining possession depends on the
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`particular facts of the case and with the nature of the invention.
`
`20.
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`I also understand that information which is well known in the art does not need
`
`not to be described in detail in the specification, and that one can rely on patents and printed
`
`publications in the art to determine what the level of knowledge and skill is in the art.
`
`21.
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`I have been informed that several factors (the “Wands factors”) may be
`
`considered when determining whether a disclosure calls for undue experimentation on the part of
`
`one reasonably skilled in the art: (1) the quantity of experimentation necessary, (2) the amount of
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`direction or guidance presented, (3) the presence or absence of working examples, (4) the nature
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`of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the
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`predictability or unpredictability of the art, and (8) the breadth of the claims.
`
`22.
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`I also understand that a patent need not teach, and preferably omits, what is well
`
`known in the art, and that any part of the specification can be used in the determination of
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`whether the specification supports the claims such that undue experimentation would not be
`
`necessary.
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`23.
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`I am informed that when a patent or applications teaches at least one species
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`within the scope of the Count, that it is considered a constructive reduction to practice of the
`
`Count.
`
`
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`

`

`Interference Nos. 105,920, 105,923, 105,924
`
`
`24.
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`As a part of these opinions, I considered the level of ordinary skill in the art in
`
`February 2006, the date the provisional application to which the ’017 and ’018 patents claim
`
`priority was filed. One of ordinary skill in the art relevant to the ’017 and ’018 patents would
`
`have a multi-disciplinary background. That person would have at least a bachelor’s degree in a
`
`life sciences area (e.g., biology, cell biology, genetics, molecular biology) and understand both
`
`the operation and application of massively parallel DNA sequencing platforms. Further, one of
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`ordinary skill in the art would understand the operation and application of basic bioinformatics
`
`techniques, including at least techniques for aligning sequence reads to a reference genome. One
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`of skill in the art could acquire an understanding of such techniques by, for example, earning a
`
`degree in computational biology or a related discipline or through carrying out relevant research
`
`activities that involve the use of such bioinformatics techniques.
`
`25.
`
`I have reviewed the Declaration of Stacey Polk Gabriel submitted in Lo U.S.
`
`Application Serial No. 13/070,275 (Exhibit 2048). I note that in paragraph 48 of that
`
`Declaration, Dr. Gabriel defines a person of ordinary skill in the art as someone with:
`
` “at least a bachelor’s degree in a life sciences area (e.g., biology, cell biology,
`genetics, molecular biology) and at least a master’s degree or Ph.D. in
`computational biology, mathematics or statistics, or equivalent training. One of
`ordinary skill in the art should understand both the operation and application of
`massively parallel DNA sequencing platforms, and have significant direct
`experience at performing and applying these techniques. Further, one of ordinary
`skill in the art should understand and have experience with techniques for aligning
`sequence reads generated by massively parallel sequencing to a reference
`genome.”
`
`26.
`
` I have also reviewed Dr. Gabriel’s education, experience and list of publications.
`
`Given my education, experience and additional training discussed above, I consider myself at
`
`least as qualified to speak to what a person of ordinary skill in the art associated with the Counts
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`in the interferences as Dr. Gabriel.
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`

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`27.
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` I have been informed that the relevant time frame for the matters I have been
`
`Interference Nos. 105,920, 105,923, 105,924
`
`
`asked to address is just prior to February, 2006 and/or February, 2007.
`
`The subject matter of the interference
`
`28.
`
`I understand that the Count of an interference is the subject matter which is being
`
`contested in an interference.
`
`29.
`
`I understand that the Count in the ‘920 interference is as follows:
`
`Lo claim 24
`
`A method for determining presence or absence of fetal aneuploidy in a
`maternal biological sample comprising fetal and maternal genomic DNA, wherein
`the method comprises:
`
`a.
`obtaining a mixture of fetal and maternal genomic DNA from said
`maternal biological sample;
`
`b.
`conducting massively parallel DNA sequencing of DNA fragments
`randomly selected from the mixture of fetal and maternal genomic DNA of step a)
`to determine the sequence of said DNA fragments;
`
`c.
`identifying chromosomes to which the sequences obtained in step
`b) belong;
`using data of step c) to compare an amount of at least one first
`d.
`chromosome in said mixture of maternal and fetal genomic DNA to an amount of
`at least one second chromosome in said mixture of maternal and fetal genomic
`DNA, wherein said at least one first chromosome is presumed to be euploid in the
`fetus, wherein said at least one second chromosome is suspected to be aneuploid
`in the fetus,
`thereby determining the presence or absence of said fetal
`aneuploidy.
`
`30.
`
`I understand that the Count in the ‘923 interference is as follows:
`
`Quake ‘833 application claim 25
`
`A method for performing prenatal diagnosis of a fetal chromosomal
`aneuploidy from a plasma or serum sample of a female subject pregnant with at
`least one fetus, wherein the plasma or serum sample includes cell-free genomic
`DNA molecules from the female subject and from the at least one fetus, the
`method comprising:
`massively parallel sequencing cell-free genomic DNA molecules
`contained in the plasma or serum sample to obtain random nucleic acid sequences
`from the genomic DNA molecules of the female subject(cid:31)and of the at least one
`fetus;
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`36
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`
`
`

`

`Interference Nos. 105,920, 105,923, 105,924
`
`
`identifying at least a portion of the nucleic acid sequences as belonging to
`a first specific human chromosome and at least one second specific human
`chromosome;
`determining a first amount of the nucleic acid sequences identified as
`being uniquely present on the first specific human chromosome and
`determining a second amount of the nucleic acid sequences identified as
`being uniquely present on the at least one second specific human chromosome;
`determining a ratio based on the first amount and the second amount,
`thereby determining a ratio of the amount of the nucleic acid sequences identified
`as being uniquely present on the first specific human chromosome to the amount
`of the nucleic acids being uniquely present on the at least one second specific
`chromosome;
`determining whether the ratio is statistically significant; and
`correlating a statistically significant result with the presence of a fetal
`chromosomal aneuploidy on the first chromosome.
`
`Or(cid:31)Lo ‘181 application claim 64
`
`
`A method for performing prenatal diagnosis of a fetal chromosomal
`aneuploidy from a plasma or serum sample of a female subject pregnant with at
`least one fetus, wherein the plasma or serum sample includes cell-free genomic
`DNA molecules from the female subject and from the at least one fetus, the
`method comprising:
`random sequencing of cell-free genomic DNA molecules contained in the
`plasma or serum sample to obtain sequenced tags from the genomic DNA
`molecules of the female subject and of the at least one fetus;
`aligning at least a portion of the sequenced tags to a first human
`chromosome and at least one second human chromosome;
`determining a first amount of the sequenced tags identified as being uniquely
`aligned to the first human chromosome; and
`determining a second amount of the sequenced tags identified as being
`uniquely aligned to the at least one second human chromosome;
`determining a ratio based on the first amount and the second amount,
`thereby determining a ratio of the amount of the sequenced tags identified as
`being uniquely aligned to the first human chromosome to the amount of the
`sequenced tags being uniquely aligned to the at least one second human
`chromosome;
`determining whether the ratio is statistically significant; and
`correlating a statistically significant result with the presence of a fetal
`chromosomal aneuploidy on the first human chromosome.
`
`I understand that the Count in the ‘924 interference is as follows:
`
`31.
`
`Quake ‘833 application claim 25
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`43
`44
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`
`
`

`

`Interference Nos. 105,920, 105,923, 105,924
`
`
`A method for performing prenatal diagnosis of a fetal chromosomal
`aneuploidy from a plasma or serum sample of a female subject pregnant with at
`least one fetus, wherein the plasma or serum sample includes cell-free genomic
`DNA molecules from the female subject and from the at least one fetus, the
`method comprising:
`massively parallel sequencing cell-free genomic DNA molecules
`contained in the plasma or serum sample to obtain random nucleic acid sequences
`from the genomic DNA molecules of the female subject(cid:31)and of the at least one
`fetus;
`
`identifying at least a portion of the nucleic acid sequences as belonging to
`a first specific human chromosome and at least one second specific human
`chromosome;
`determining a first amount of the nucleic acid sequences identified as
`being uniquely present on the first specific human chromosome and
`determining a second amount of the nucleic acid sequences identified as
`being uniquely present on the at least one second specific human chromosome;
`determining a ratio based on the first amount and the second amount,
`thereby determining a ratio of the amount of the nucleic acid sequences identified
`as being uniquely present on the first specific human chromosome to the amount
`of the nucleic acids being uniquely present on the at least one second specific
`chromosome;
`determining whether the ratio is statistically significant; and
`correlating a statistically significant result with the presence of a fetal
`chromosomal aneuploidy on the first chromosome.
`
`Or Lo ‘119 application claim 13
`
`A method for identifying a fetal aneuploidy in a maternal biological
`sample that includes cell-free fetal DNA from the genome of a fetus
`and(cid:31)maternal DNA from the genome of the mother of the fetus, the method
`comprising:
`a. obtaining the maternal biological sample;
`b. performing random sequencing of DNA fragments from the genome of
`the mother and from the genome of the fetus contained in the maternal biological
`sample to obtain a plurality of sequenced tags, wherein the obtained sequenced
`tags include sequenced tags corresponding to cell-free maternal DNA from the
`genome of the mother and sequenced tags corresponding to cell-free fetal DNA
`from the genome of the fetus;
`c. identifying the chromosomes from which the sequenced tags obtained in
`step b) originate by aligning, with a computer system, the sequenced tags to a
`human genome;
`d. using data of step c) to determine:
`a first amount of sequenced tags identified as originating from at
`least one first chromosome in the maternal biological sample and not
`originating from a second chromosome of the human genome, and
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`40
`41
`42
`43
`44
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`
`
`

`

`Interference Nos. 105,920, 105,923, 105,924
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`a second amount of sequences sequenced tags identified as
`originating from a second chromosome in the maternal biological sample
`and not originating from the least one first chromosome, wherein the at
`least one first chromosome is presumed to be euploid in the fetus, wherein
`the second chromosome is potentially aneuploid in the fetus;
`e. measuring a proportion of cell-free nucleic acid molecules in the
`biological sample that are from the second chromosome, the measuring including
`calculating a ratio of the first amount relative to the second amount; and
`f. comparing the proportion to one or more cutoff values, thereby
`determining whether a fetal aneuploidy exists for the second chromosome,
`wherein the one or more cutoff values take into account a size of the second
`chromosome relative to a size of the at least one first chromosome.
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`Background of DNA Sequencing
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`32.
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`The Counts in the ‘920, ‘923 and ‘924 interferences refer to the use of massively
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`parallel sequencing. A brief background on massively parallel sequencing may be helpful to
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`understand my opinions in this case.
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`33. Massively parallel sequencing with commercially available DNA sequencing
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`systems typically involves four steps:
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`a. Library preparation (optionally including amplification);
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`b. Strand separation (optionally including amplification);
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`c. Sequencing; and
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`d. Post-sequencing Analysis
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`34.
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`To illustrate these four steps, I will use the Illumina (Solexa) sequencing system
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`mentioned in the ‘018 patent (at 19:67) as an example. But many of these steps are common
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`amongst massively parallel sequencing systems that are commercially available today.
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`Library Preparation
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`35.
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`Library preparation is the most variable portion of the massively parallel
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`sequencing process within a given sequencing platform (e.g. within an Illumina sequencing
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`platform). Library preparation is often a key factor associated with the type of test being run on
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`Interference Nos. 105,920, 105,923, 105,924
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`a sequencing instrument.
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`36.
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`Lo and Quake appear to dispute whether the Quake patent discloses “targeted
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`sequencing” or “random sequencing.” (Ex. 2057, 2058). That distinction – random or targeted –
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`is typically associated with this library preparation phase. In targeted sequencing, specific
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`sequences are pre-selected in the library preparation phase, whereas in random sequencing there
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`is no such pre-selection in the library preparation phase.
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`37.
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`Care should be taken not to confuse “targeted sequencing,” a term of art in the
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`field, from the concept of “sequencing targets.” The specific phrase “targeted sequencing” often
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`implies the pre-selection of specific sequences. However, the term “target” or “sequencing
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`targets” on its own does not. In the field of sequencing, a target is “typically an unknown portion
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`[of a template] to be sequenced” (Ex. 2060, p. 32 – “Template” definition).. I understand that
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`Sequenom’s expert in the concurrent litigation, Dr. Michael Metzker, agreed with this
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`understanding of the term “target.” See, e.g., Metzker Deposition Transcript at p. 236, line 13 to
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`p. 239, line 18 (stating, e.g., “A target sequence which I'm defining as just the thing to be
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`sequenced … Q. Okay. And that's -- and in terms of the term "target sequence," the way that's
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`used by persons of skill in the art in the context of massively parallel sequencing is that's the
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`unknown portion of the nucleic acid that you -- that's to be sequenced, that you want to discover,
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`that's the standard usage? … [A.] That's as it exists in a template. … Q. But that's a yes, right? A.
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`Well -- well, with the caveat as it exists as a template, the answer is yes.”)
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`38. With these distinctions in mind, the following describe a number of ways that are
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`commercially available today to perform library preparation for the Illumina sequencing
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`platform. I note that many of the targeted approaches were not commercially available from
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`Illumina in 2007.
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`39.
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`Illumina’s standard library preparation protocol (which was generally available as
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`of 2006) involves adding forked adapters to a piece of DNA. The adapters carry priming
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`sequences need for later amplification in its strand separation step (step b) and also include the
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`sequencing primer sequence. Illumina’s forked adapter library preparation process is generally
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`described in U.S. Patent No. 7,741,463.
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`40.
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`Specifically, the process involves taking DNA from a sample. If the sample is not
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`already fragmented or contains long pieces of DNA, the DNA may first need to be
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`sheared/nebulized into shorter pieces. In the case of DNA from maternal serum, the DNA is
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`already fragmented. (See, e.g., Ex. , Col. 2, ll. 30-38). The fragments of DNA have their ends
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`“repaired” such that the strands are blunt ended; that is, so one strand does not hang over the
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`other. The third step then involves adding an “A” base to the 3’ ends of each strand of the DNA.
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`These first steps are illustrated in the following diagram:
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`41.
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`This process of preparing the DNA works on all of the DNA in the sample
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`simultaneously. In the next step, a forked adapter is added to each end of the prepared
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`fragments. The forked adapter generally has the structure:
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`As seen in the picture, there is a hybridized gray section where the two strands of the
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`forked adapter are connected. There are also unhybridized orange (5’) and green (3’) sections.
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`The green section includes the P7rev sequence needed for strand separation (step b), while the
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`orange section includes the P5 sequence needed for strand separation (step b) as well as the
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`sequencing primer sequence (SBS3) needed for sequencing (step c).
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`42.
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`The forked adapter is added by ligation, resulting in prepared DNA connected to
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`an adapter on both sides. Notably, each strand of the double-stranded DNA from the prepared
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`mixture is connected to both the green and orange sequences.1
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`1 I note that in 2006 and 2007, the commercially available Solexa/Illumina forked adapter process was
`slightly more involved. The process had a first step where shorter forked adapters were added by ligation to both
`ends of the fragments as described here. These shorter forked adapters had the sequencing primer sequence in them,
`and had universal priming sequences, but did not have the P5 and P7 sequences as part f those universal priming
`sequences. Intead, the P5 and P7 sequences were added to the adapter-containing fragments in a PCR amplification
`step involving the universal primers that were included in the adapter, which primers had overhangs that added the
`P5 and P7 sequences.
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`43.
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`These adapter-containing strands are optionally amplified by PCR using the P5
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`(orange) and P7 (green) sequences that are universal to all of the prepared fragments in the
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`mixture (i.e. such that all prepared fragments get amplified).
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`44.
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`The prepared, and optionally amplified, fragments are then typically subjected to
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`a size selection process (size being the number of individual bases making up the strand). This
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`generally involves putting the mixture on an electrophoresis gel where the fragments in the
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`mixture are separated by size. One size band on the gel was chosen for use in the strand
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`separation step (step b) and sequencing (step c).
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`45.
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`In random sequencing, the library preparation process would end here – with a
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`size-selected set of strands. Fragments from this prepared, size selected library would then be
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`randomly selected subjected to strand separation and sequencing.
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`46.
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`A first, common, method of targeted sequencing makes use of this same library
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`preparation process used for random sequencing, but adds an additional step – pulling out
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`specific sequences of interest before moving on to the strand separation and sequencing steps.
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`47.
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`One example of a kit that could be used for a type of targeted sequencing (“exon
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`sequencing”) is the Illumina TruSeq Exome Enrichment Kit. (Ex. 2052). Although I note that
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`this kit was only first sold by Illumina in November of 2010. (Ex. 2053).
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`Interference Nos. 105,920, 105,923, 105,924
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`48.
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`The TruSeq Exome Enrichment Kit operates by first preparing a library as
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`discussed above for random sequencing. The set of prepared fragments includes specific
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`sequences of interest (shown in purple) and others that are not of interest (shown in black). The
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`first step in this enrichment process is to turn the double-stranded molecules into single-stranded
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`molecules.
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`49.
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`Next, probes (shown in blue) that hybridize to specific sequences are added. The
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`probes are connected to a non-fluorescent biotin molecule (shown in red). The blue probes
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`Beads having streptavidin are added. The biotin on the probes bind to the
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`streptavidin on the beads, thus binding the probes to the beads. The specific sequences of
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`interest (pink) are hybridized to the probe, so are also retained on the beads.
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`51.
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`The beads and their associated DNA are separated from the other DNA using a
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`magnet that attracts the beads. This leaves just the magnetic beads with the probes (blue)
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`attached to the specific sequences of interest. The DNA having the specific sequences of interest
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`are separated from (denatured from) the blue probes, leaving just the molecules containing the
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`sequences of interest (in pink).
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`52.
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`In this targeted sequencing approach, now the library at the end of library creation
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`only includes the DNA molecules having the specific sequences of interest (the pink strands).
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