Case 1:20-cv-01580-LPS Document 40-1 Filed 03/05/21 Page 1 of 268 PageID #: 8144
`Case 1:20-cv-01580-LPS Document 40-1 Filed 03/05/21 Page 1 of 268 PageID #: 8144
`
`EXHIBIT 51
`
`EXHIBIT 51
`
`

`

`Case 1:20-cv-01580-LPS Document 40-1 Filed 03/05/21 Page 2 of 268 PageID #: 8145
`
`(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`28 July 2011 (28.07.2011)
`
`(10) International Publication Number
`_
`.
`.
`. ..
`.
`.
`.
`WO 2011/091046 Al
`
`(51) International Patent Classification:
`CI2Q 1/68 (2006.01)
`
`(21) International Application Number:
`PCT/US201 1/021729
`
`(22) International Filing Date:
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`19 January 201 1 (19.01 .201 1)
`
`English
`
`English
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
`NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD,
`SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR,
`TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`US (84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`US
`us
`GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG,
`ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`(71) Applicant (for all designated States except US): VERI-
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`NATA HEALTH, INC. [US/US]; 153 1 Industrial Road,
`EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU,
`San Carlos, CA 94070 (US).
`LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK,
`SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, ML, MR, NE, SN, TD, TG).
`
`(30) Priority Data:
`61/296,358
`61/360,837
`12/958,347
`
`19 January 2010 (19.01 .2010)
`1 July 2010 (01 .07.2010)
`1 December 2010 (01 .12.2010)
`
`(72) Inventor; and
`(for US only): RAVA, Richard, P.
`(75) Inventor/ Applicant
`[US/US]; 771 Edgewood Road, Redwood City, CA Published:
`94062 (US).
`— with international search report (Art. 21(3))
`
`Jeffrey
`(74) Agents: SED3EL,
`Sonsini
`al; Wilson
`et
`Goodrich & Rosati, 650 Page Mill Road, Palo Alto, CA
`94304-1050 (US).
`
`o ©
`
`(54) Title:
`
`o GENOME SEQUENCING
`
`IDENTIFICATION OF POLYMORPHIC SEQUENCES IN MIXTURES OF GENOMIC DNA BY WHOLE
`
`(57) Abstract: The present invention relates to methods comprising whole genome sequencing for identifying polymorphisms in
`samples comprising mixtures of genomes, and for determining and/or monitoring the presence or absence of disorders associated
`with the identified polymorphisms.
`
`A0623
`
`

`

`Case 1:20-cv-01580-LPS Document 40-1 Filed 03/05/21 Page 3 of 268 PageID #: 8146
`
`IDENTIFICATION OF POLYMORPHIC SEQUENCES IN MIXTURES OF GENOMIC DNA BY
`
`WHOLE GENOME SEQUENCING
`
`CROSS-REFERENCE
`
`This Application claims priority to U.S. Provisional Application Serial No. 61/296,358, filed on
`
`January 19, 2010; and U.S. Provisional Application Serial No. 61/360,837, filed on July 1, 2010 and U.S.
`
`Patent Application 12/958,347 filed on December 1, 2010, which applications are incorporated herein by
`
`reference in their entirety.
`
`FIELD OF THE INVENTION
`
`[0001] The invention is applicable to the field of medical diagnostics and particularly relates to whole
`
`genome sequencing methods for identifying polymorphisms in samples comprising mixtures of genomes.
`
`BACKGROUND OF THE INVENTION
`
`[0002] Prenatal screening and diagnosis are a routine part of antenatal care. Currently, prenatal diagnosis of
`
`genetic and chromosomal conditions involves invasive testing, such as amniocentesis or chorionic villus
`
`sampling (CVS), performed from 11weeks gestation and carrying a ~1% risk of miscarriage. The existence
`
`of circulating cell-free DNA in maternal blood (Lo et al., Lancet 350:485-487 [1997]) is being exploited for
`
`developing noninvasive processes that use fetal nucleic acids from a maternal peripheral blood sample to
`
`determine fetal chromosomal aneuploidies e.g. trisomy 2 1 (Fan C and Quake S Ana! Chem 79:7576-7579
`
`[2007]; Fan et al, Proc Natl Acad Sci 105:16266-16271 [2008]). These methods offer an alternative and
`
`safer source of fetal genetic material for prenatal diagnosis, and could effectively pronounce the end of
`
`invasive procedures.
`
`[0003] Next Generation Sequencing (NGS) technologies have been used to determine entire human genome
`
`sequences (Levy et al. PLoS Biol 55, e254 [2007]; Wheeler et al. Nature 452:872-876 [2008]; Bentley et al,
`
`Nature 456:53-59 [2008]), and a broad interest exists in using NGS technologies for whole genome
`
`sequencing (WGS) to better understand human genetic variation and genome-related diseases, and ultimately
`
`to guide discoveries and decisions about the health of individuals. The extensive public genome-wide
`
`database of patterns of common human sequence variation provided by the International HapMap Project, and
`
`the increasing accessibility to whole genome sequencing technologies, will lead to the identification of new
`
`therapeutic targets and the development of targeted interventions for an individuals' medical care.
`
`[0004] An additional need that remains is for identifying the disorder-associated genetic variations when two
`
`or more individual genomes are intermixed in a clinical sample e.g. mixtures of fetal and maternal genomes in
`
`biological fluid samples obtained from the mother, and mixtures of euploid and aneuploid genomes derived
`
`from cells of cancer patients.
`
`A0624
`
`

`

`Case 1:20-cv-01580-LPS Document 40-1 Filed 03/05/21 Page 4 of 268 PageID #: 8147
`
`[0005] The present invention addresses the need by providing a method for identifying polymorphisms in
`
`mixtures of genomes present in samples that can be obtained by noninvasive means. The method can be used
`
`for the non-invasive identification of multiple disease-associated polymorphisms in a variety of fields
`
`including but not limited to prenatal diagnostics, and oncology.
`
`SUMMARY OF THE INVENTION
`
`[0006] The present invention relates to methods comprising whole genome sequencing for identifying
`
`polymorphisms in samples comprising mixtures of genomes, and for determining and/or monitoring the
`
`presence or absence of disorders associated with the identified polymorphisms.
`
`[0007]
`
`In one embodiment, the invention provides a method for identifying multiple polymorphisms in a first
`
`genome of a blood sample comprising a mixture of cfDNA of a first and a second genome, comprising: (a)
`
`whole genome sequencing at least a portion of the mixture of cfDNA, thereby obtaining a plurality of
`
`sequence tags, wherein the mixture is unenriched for the multiple polymorphisms; (b) comparing the
`
`sequence of the plurality of tags to the sequence of multiple reference polymorphisms; (c) identifying the
`
`multiple polymorphisms in the first and second genome of the mixture; and (d) associating the multiple
`
`polymorphisms identified in step (c) with the first and second genome, thereby identifying the multiple
`
`polymorphisms in the first genome of the mixture. The blood sample can be unenriched for polymorphic
`
`target sequences in the mixture of first and second genomes. Sequencing is massively parallel sequencing of
`
`clonally amplified cfDNA molecules or of single cfDNA molecules. In some embodiments, sequencing is
`
`performed using massively parallel sequencing-by-synthesis with reversible dye terminators. In other
`
`embodiments, sequencing is performed using massively parallel sequencing-by-ligation.
`
`In some
`
`embodiments, the sample is a plasma sample.
`
`[0008]
`
`In another embodiment, the method for identifying multiple polymorphisms in a first genome of a
`
`blood sample comprising a mixture of cfDNA of a first and a second genome, comprises: (a) whole genome
`
`sequencing at least a portion of the mixture of cfDNA, thereby obtaining a plurality of sequence tags, wherein
`
`the mixture is unenriched for the multiple polymorphisms; (b) comparing the sequence of the plurality of tags
`
`to the sequence of multiple reference polymorphisms; (c) identifying the multiple polymorphisms in the first
`
`and second genome of the mixture; and (d) associating the multiple polymorphisms identified in step (c) with
`
`the first and second genome, thereby identifying the multiple polymorphisms in the first genome of the
`
`mixture. Step (c) comprises genotyping the second genome in a sample that is substantially free of the first
`
`genome. The blood sample can be unenriched for polymorphic target sequences in the mixture of first and
`
`second genomes. Sequencing is massively parallel sequencing of clonally amplified cfDNA molecules or of
`
`single cfDNA molecules. In some embodiments, sequencing is performed using massively parallel
`
`sequencing-by-synthesis with reversible dye terminators.
`
`In other embodiments, sequencing is performed
`
`using massively parallel sequencing-by-ligation.
`
`In some embodiments, the sample is a plasma sample.
`
`A0625
`
`

`

`Case 1:20-cv-01580-LPS Document 40-1 Filed 03/05/21 Page 5 of 268 PageID #: 8148
`
`[0009]
`
`In another embodiment, the method for identifying multiple polymorphisms in a first genome of a
`
`blood sample comprising a mixture of cfDNA of a first and a second genome, comprises: (a) whole genome
`
`sequencing at least a portion of the mixture of cfDNA, thereby obtaining a plurality of sequence tags, wherein
`
`the mixture is unenriched for the multiple polymorphisms; (b) comparing the sequence of the plurality of tags
`
`to the sequence of multiple reference polymorphisms; (c) identifying the multiple polymorphisms in the first
`
`and second genome of the mixture; and (d) associating the multiple polymorphisms identified in step (c) with
`
`the first and second genome, thereby identifying the multiple polymorphisms in the first genome of the
`
`mixture, wherein step (c) comprises counting sequence tags mapped to the multiple reference polymorphisms.
`
`The blood sample can be unenriched for polymorphic target sequences in the mixture of first and second
`
`genomes. Sequencing is massively parallel sequencing of clonally amplified cfDNA molecules or of single
`
`cfDNA molecules. In some embodiments, sequencing is performed using massively parallel sequencing-by-
`
`synthesis with reversible dye terminators. In other embodiments, sequencing is performed using massively
`
`parallel sequencing-by-ligation.
`
`In some embodiments, the sample is a plasma sample.
`
`[0010]
`
`In another embodiment, the method for identifying multiple polymorphisms in a first genome of a
`
`blood sample comprising a mixture of cfDNA of a first and a second genome, comprises: (a) whole genome
`
`sequencing at least a portion of the mixture of cfDNA, thereby obtaining a plurality of sequence tags, wherein
`
`the mixture is unenriched for the multiple polymorphisms; (b) comparing the sequence of the plurality of tags
`
`to the sequence of multiple reference polymorphisms; (c) identifying the multiple polymorphisms in the first
`
`and second genome of the mixture; and (d) associating the multiple polymorphisms identified in step (c) with
`
`the first and second genome, thereby identifying the multiple polymorphisms in the first genome of the
`
`mixture, wherein the multiple polymorphisms in the first genome are associated with at least one disorder.
`
`The blood sample can be unenriched for polymorphic target sequences in the mixture of first and second
`
`genomes. Sequencing is massively parallel sequencing of clonally amplified cfDNA molecules or of single
`
`cfDNA molecules. In some embodiments, sequencing is performed using massively parallel sequencing-by-
`
`synthesis with reversible dye terminators. In other embodiments, sequencing is performed using massively
`
`parallel sequencing-by-ligation.
`
`In some embodiments, the sample is a plasma sample.
`
`[0011]
`
`In another embodiment, the method for identifying multiple polymorphisms in a first genome of a
`
`blood sample comprising a mixture of cfDNA of a first and a second genome, comprises: (a) whole genome
`
`sequencing at least a portion of the mixture of cfDNA, thereby obtaining a plurality of sequence tags, wherein
`
`the mixture is unenriched for the multiple polymorphisms; (b) comparing the sequence of the plurality of tags
`
`to the sequence of multiple reference polymorphisms; (c) identifying the multiple polymorphisms in the first
`
`and second genome of the mixture; and (d) associating the multiple polymorphisms identified in step (c) with
`
`the first and second genome, wherein step (c) comprises genotyping said second genome in a sample that is
`
`substantially free of said first genome, and wherein the multiple polymorphisms in the first genome are
`
`associated with at least one disorder. The blood sample can be unenriched for polymorphic target sequences
`
`A0626
`
`

`

`Case 1:20-cv-01580-LPS Document 40-1 Filed 03/05/21 Page 6 of 268 PageID #: 8149
`
`in the mixture of first and second genomes. Sequencing is massively parallel sequencing of clonally
`
`amplified cfDNA molecules or of single cfDNA molecules. In some embodiments, sequencing is performed
`
`using massively parallel sequencing-by-synthesis with reversible dye terminators. In other embodiments,
`
`sequencing is performed using massively parallel sequencing-by-ligation.
`
`In some embodiments, the sample
`
`is a plasma sample.
`
`[0012]
`
`In another embodiment, the method for identifying multiple polymorphisms in a first genome of a
`
`blood sample comprising a mixture of cfDNA of a first and a second genome, comprises: (a) whole genome
`
`sequencing at least a portion of the mixture of cfDNA, thereby obtaining a plurality of sequence tags, wherein
`
`the mixture is unenriched for the multiple polymorphisms; (b) comparing the sequence of the plurality of tags
`
`to the sequence of multiple reference polymorphisms; (c) identifying the multiple polymorphisms in the first
`
`and second genome of the mixture; and (d) associating the multiple polymorphisms identified in step (c) with
`
`the first and second genome, thereby identifying the multiple polymorphisms in the first genome of the
`
`mixture, wherein step (c) comprises counting sequence tags mapped to the multiple reference polymorphisms,
`
`and wherein the multiple polymorphisms in the first genome are associated with at least one disorder. The
`
`blood sample can be unenriched for polymorphic target sequences in the mixture of first and second genomes.
`
`Sequencing is massively parallel sequencing of clonally amplified cfDNA molecules or of single cfDNA
`
`molecules. In some embodiments, sequencing is performed using massively parallel sequencing-by-synthesis
`
`with reversible dye terminators.
`
`In other embodiments, sequencing is performed using massively parallel
`
`sequencing-by-ligation.
`
`In some embodiments, the sample is a plasma sample.
`
`[0013]
`
`In another embodiment, the method for identifying multiple polymorphisms in a first genome of a
`
`blood sample comprising a mixture of cfDNA of a first and a second genome, comprises: (a) whole genome
`
`sequencing at least a portion of the mixture of cfDNA, thereby obtaining a plurality of sequence tags, wherein
`
`the mixture is unenriched for the multiple polymorphisms; (b) comparing the sequence of the plurality of tags
`
`to the sequence of multiple reference polymorphisms; (c) identifying the multiple polymorphisms in the first
`
`and second genome of the mixture; and (d) associating the multiple polymorphisms identified in step (c) with
`
`the first and second genome, thereby identifying the multiple polymorphisms in the first genome of the
`
`mixture. The blood sample is obtained from a pregnant woman, and it can be unenriched for cfDNA of the
`
`first or second genome. The first genome is a fetal genome and the second genome is a maternal genome.
`
`Optionally, the method further comprises identifying the multiple polymorphisms in a paternal genome.
`
`Sequencing is massively parallel sequencing of clonally amplified cfDNA molecules or of single cfDNA
`
`molecules. In some embodiments, sequencing is performed using massively parallel sequencing-by-synthesis
`
`with reversible dye terminators.
`
`In other embodiments, sequencing is performed using massively parallel
`
`sequencing-by-ligation.
`
`In some embodiments, the sample is a plasma sample.
`
`[0014]
`
`In another embodiment, the method for identifying multiple polymorphisms in a first genome of a
`
`blood sample comprising a mixture of cfDNA of a first and a second genome, comprises: (a) whole genome
`
`A0627
`
`

`

`Case 1:20-cv-01580-LPS Document 40-1 Filed 03/05/21 Page 7 of 268 PageID #: 8150
`
`sequencing at least a portion of the mixture of cfDNA, thereby obtaining a plurality of sequence tags, wherein
`
`the mixture is unenriched for the multiple polymorphisms; (b) comparing the sequence of the plurality of tags
`
`to the sequence of multiple reference polymorphisms; (c) identifying the multiple polymorphisms in the first
`
`and second genome of the mixture; and (d) associating the multiple polymorphisms identified in step (c) with
`
`the first and second genome, thereby identifying the multiple polymorphisms in the first genome of the
`
`mixture., wherein step (c) comprises genotyping the second genome in a sample that is substantially free of
`
`the first genome. The blood sample is obtained from a pregnant woman, and it can be unenriched for cfDNA
`
`of the first or second genome. The first genome is a fetal genome and the second genome is a maternal
`
`genome. Optionally, the method further comprises identifying the multiple polymorphisms in a paternal
`
`genome. Sequencing is massively parallel sequencing of clonally amplified cfDNA molecules or of single
`
`cfDNA molecules. In some embodiments, sequencing is performed using massively parallel sequencing-by-
`
`synthesis with reversible dye terminators. In other embodiments, sequencing is performed using massively
`
`parallel sequencing-by-ligation.
`
`In some embodiments, the sample is a plasma sample.
`
`[0015]
`
`In another embodiment, the method for identifying multiple polymorphisms in a first genome of a
`
`blood sample comprising a mixture of cfDNA of a first and a second genome, comprises: (a) whole genome
`
`sequencing at least a portion of the mixture of cfDNA, thereby obtaining a plurality of sequence tags, wherein
`
`the mixture is unenriched for the multiple polymorphisms; (b) comparing the sequence of the plurality of tags
`
`to the sequence of multiple reference polymorphisms; (c) identifying the multiple polymorphisms in the first
`
`and second genome of the mixture; and (d) associating the multiple polymorphisms identified in step (c) with
`
`the first and second genome, thereby identifying the multiple polymorphisms in the first genome of the
`
`mixture, wherein step (c) comprises counting sequence tags mapped to the multiple reference polymorphisms.
`
`The blood sample is obtained from a pregnant woman, and it can be unenriched for cfDNA of the first or
`
`second genome. The first genome is a fetal genome and the second genome is a maternal genome.
`
`Optionally, the method further comprises identifying the multiple polymorphisms in a paternal genome.
`
`Sequencing is massively parallel sequencing of clonally amplified cfDNA molecules or of single cfDNA
`
`molecules. In some embodiments, sequencing is performed using massively parallel sequencing-by-synthesis
`
`with reversible dye terminators.
`
`In other embodiments, sequencing is performed using massively parallel
`
`sequencing-by-ligation.
`
`In some embodiments, the sample is a plasma sample.
`
`[0016]
`
`In another embodiment, the method for identifying multiple polymorphisms in a first genome of a
`
`blood sample comprising a mixture of cfDNA of a first and a second genome, comprises: (a) whole genome
`
`sequencing at least a portion of the mixture of cfDNA, thereby obtaining a plurality of sequence tags, wherein
`
`the mixture is unenriched for the multiple polymorphisms; (b) comparing the sequence of the plurality of tags
`
`to the sequence of multiple reference polymorphisms; (c) identifying the multiple polymorphisms in the first
`
`and second genome of the mixture; and (d) associating the multiple polymorphisms identified in step (c) with
`
`the first and second genome, thereby identifying the multiple polymorphisms in the first genome of the
`
`A0628
`
`

`

`Case 1:20-cv-01580-LPS Document 40-1 Filed 03/05/21 Page 8 of 268 PageID #: 8151
`
`mixture. The sample is a blood sample obtained from a subject that is known or suspected of having cancer.
`
`The blood sample can be unenriched for polymorphic target sequences in the mixture of first and second
`
`genomes. Sequencing is massively parallel sequencing of clonally amplified cfDNA molecules or of single
`
`cfDNA molecules. In some embodiments, sequencing is performed using massively parallel sequencing-by-
`
`synthesis with reversible dye terminators. In other embodiments, sequencing is performed using massively
`
`parallel sequencing-by-ligation.
`
`In some embodiments, the sample is a plasma sample.
`
`[0017]
`
`In another embodiment, the method for identifying multiple polymorphisms in a first genome of a
`
`blood sample comprising a mixture of cfDNA of a first and a second genome, comprises: (a) whole genome
`
`sequencing at least a portion of the mixture of cfDNA, thereby obtaining a plurality of sequence tags, wherein
`
`the mixture is unenriched for the multiple polymorphisms; (b) comparing the sequence of the plurality of tags
`
`to the sequence of multiple reference polymorphisms; (c) identifying the multiple polymorphisms in the first
`
`and second genome of the mixture; and (d) associating the multiple polymorphisms identified in step (c) with
`
`the first and second genome, thereby identifying the multiple polymorphisms in the first genome of the
`
`mixture., wherein step (c) comprises genotyping the second genome in a sample that is substantially free of
`
`the first genome. The sample is a blood sample obtained from a subject that is known or suspected of having
`
`cancer. The blood sample can be unenriched for polymorphic target sequences in the mixture of first and
`
`second genomes. Sequencing is massively parallel sequencing of clonally amplified cfDNA molecules or of
`
`single cfDNA molecules. In some embodiments, sequencing is performed using massively parallel
`
`sequencing-by-synthesis with reversible dye terminators.
`
`In other embodiments, sequencing is performed
`
`using massively parallel sequencing-by-ligation.
`
`In some embodiments, the sample is a plasma sample.
`
`[0018]
`
`In another embodiment, the method for identifying multiple polymorphisms in a first genome of a
`
`blood sample comprising a mixture of cfDNA of a first and a second genome, comprises: (a) whole genome
`
`sequencing at least a portion of the mixture of cfDNA, thereby obtaining a plurality of sequence tags, wherein
`
`the mixture is unenriched for the multiple polymorphisms; (b) comparing the sequence of the plurality of tags
`
`to the sequence of multiple reference polymorphisms; (c) identifying the multiple polymorphisms in the first
`
`and second genome of the mixture; and (d) associating the multiple polymorphisms identified in step (c) with
`
`the first and second genome, thereby identifying the multiple polymorphisms in the first genome of the
`
`mixture, wherein step (c) comprises counting sequence tags mapped to the multiple reference polymorphisms.
`
`The sample is a blood sample obtained from a subject that is known or suspected of having cancer. The blood
`
`sample can be unenriched for polymorphic target sequences in the mixture of first and second genomes.
`
`Sequencing is massively parallel sequencing of clonally amplified cfDNA molecules or of single cfDNA
`
`molecules. In some embodiments, sequencing is performed using massively parallel sequencing-by-synthesis
`
`with reversible dye terminators. In other embodiments, sequencing is performed using massively parallel
`
`sequencing-by-ligation.
`
`In some embodiments, the sample is a plasma sample.
`
`A0629
`
`

`

`Case 1:20-cv-01580-LPS Document 40-1 Filed 03/05/21 Page 9 of 268 PageID #: 8152
`
`[0019]
`
`In another embodiment, the invention provides a method for determining the presence or absence of
`
`multiple disorders in a blood sample comprising a mixture of cfDNA of a first and a second genome,
`
`comprising: (a) whole genome sequencing at least a portion of the mixture of cfDNA, thereby providing a
`
`plurality of sequence tags; (b) identifying multiple polymorphisms in the plurality of sequence tags, wherein
`
`the multiple polymorphisms are associated with the number of disorders; and (c) associating the multiple
`
`polymorphisms with the first and/or second genome in the mixture, wherein the mixture is unenriched for the
`
`multiple polymorphisms. Sequencing is massively parallel sequencing of clonally amplified cfDNA
`
`molecules or of single cfDNA molecules. In some embodiments, sequencing is performed using massively
`
`parallel sequencing-by-synthesis with reversible dye terminators.
`
`In other embodiments, sequencing is
`
`performed using massively parallel sequencing-by-ligation.
`
`In some embodiments, the sample is a plasma
`
`sample.
`
`[0020]
`
`In another embodiment, the method for determining the presence or absence of multiple disorders in a
`
`blood sample comprising a mixture of cfDNA of a first and a second genome, comprises: (a) whole genome
`
`sequencing at least a portion of the mixture of cfDNA, thereby providing a plurality of sequence tags; (b)
`
`identifying multiple polymorphisms in the plurality of sequence tags, wherein the multiple polymorphisms are
`
`associated with the number of disorders; and (c) associating the multiple polymorphisms with the first and/or
`
`second genome in the mixture, wherein the mixture is unenriched for the multiple polymorphisms. The
`
`method further comprises comparing the sequence of the plurality of tags to the sequence of multiple
`
`reference polymorphisms, thereby identifying the multiple polymorphisms in the mixture of cfDNA.
`
`Sequencing is massively parallel sequencing of clonally amplified cfDNA molecules or of single cfDNA
`
`molecules. In some embodiments, sequencing is performed using massively parallel sequencing-by-synthesis
`
`with reversible dye terminators.
`
`In other embodiments, sequencing is performed using massively parallel
`
`sequencing-by-ligation.
`
`In some embodiments, the sample is a plasma sample.
`
`[0021]
`
`In another embodiment, the method for determining the presence or absence of multiple disorders in a
`
`blood sample comprising a mixture of cfDNA of a first and a second genome, comprises: (a) whole genome
`
`sequencing at least a portion of the mixture of cfDNA, thereby providing a plurality of sequence tags; (b)
`
`identifying multiple polymorphisms in the plurality of sequence tags, wherein the multiple polymorphisms are
`
`associated with the number of disorders; and (c) associating the multiple polymorphisms with the first and/or
`
`second genome in the mixture, wherein the mixture is unenriched for the multiple polymorphisms. In some
`
`embodiments, step (b) comprises counting sequence tags mapped to the multiple polymorphisms.
`
`Alternatively, step (b) comprises genotyping the second genome in a sample that is substantially free of the
`
`first genome. Sequencing is massively parallel sequencing of clonally amplified cfDNA molecules or of
`
`single cfDNA molecules. In some embodiments, sequencing is performed using massively parallel
`
`sequencing-by-synthesis with reversible dye terminators.
`
`In other embodiments, sequencing is performed
`
`using massively parallel sequencing-by-ligation.
`
`In some embodiments, the sample is a plasma sample.
`
`A0630
`
`

`

`Case 1:20-cv-01580-LPS Document 40-1 Filed 03/05/21 Page 10 of 268 PageID #: 8153
`
`[0022]
`
`In another embodiment, the method for determining the presence or absence of multiple disorders in a
`
`blood sample comprising a mixture of cfDNA of a first and a second genome, comprises: (a) whole genome
`
`sequencing at least a portion of the mixture of cfDNA, thereby providing a plurality of sequence tags; (b)
`
`identifying multiple polymorphisms in the plurality of sequence tags, wherein the multiple polymorphisms are
`
`associated with the number of disorders; and (c) associating the multiple polymorphisms with the first and/or
`
`second genome in the mixture, wherein the mixture is unenriched for the multiple polymorphisms, wherein
`
`the first genome is a genome of an unaffected cell and the second genome is a genome from an affected cell.
`
`Sequencing is massively parallel sequencing of clonally amplified cfDNA molecules or of single cfDNA
`
`molecules. In some embodiments, sequencing is performed using massively parallel sequencing-by-synthesis
`
`with reversible dye terminators.
`
`In other embodiments, sequencing is performed using massively parallel
`
`sequencing-by-ligation.
`
`In some embodiments, the sample is a plasma sample.
`
`[0023]
`
`In another embodiment, the method for determining the presence or absence of multiple disorders in a
`
`blood sample comprising a mixture of cfDNA of a first and a second genome, comprises: (a) whole genome
`
`sequencing at least a portion of the mixture of cfDNA, thereby providing a plurality of sequence tags; (b)
`
`identifying multiple polymorphisms in the plurality of sequence tags, wherein the multiple polymorphisms are
`
`associated with the number of disorders; and (c) associating the multiple polymorphisms with the first and/or
`
`second genome in the mixture, wherein the mixture is unenriched for the multiple polymorphisms, and
`
`wherein the first genome is a genome of an unaffected cell and the second genome is a genome from an
`
`affected cell. The method further comprises comparing the sequence of the plurality of tags to the sequence
`
`of multiple reference polymorphisms, thereby identifying the multiple polymorphisms in the mixture of
`
`cfDNA. Sequencing is massively parallel sequencing of clonally amplified cfDNA molecules or of single
`
`cfDNA molecules. In some embodiments, sequencing is performed using massively parallel sequencing-by-
`
`synthesis with reversible dye terminators. In other embodiments, sequencing is performed using massively
`
`parallel sequencing-by-ligation.
`
`In some embodiments, the sample is a plasma sample. Sequencing is
`
`massively parallel sequencing of clonally amplified cfDNA molecules or of single cfDNA molecules.
`
`In
`
`some embodiments, sequencing is performed using massively parallel sequencing-by-synthesis with
`
`reversible dye terminators. In other embodiments, sequencing is performed using massively parallel
`
`sequencing-by-ligation.
`
`In some embodiments, the sample is a plasma sample.
`
`[0024]
`
`In another embodiment, the method for determining the presence or absence of multiple disorders in a
`
`blood sample comprising a mixture of cfDNA of a first and a second genome, comprises: (a) whole genome
`
`sequencing at least a portion of the mixture of cfDNA, thereby providing a plurality of sequence tags; (b)
`
`identifying multiple polymorphisms in the plurality of sequence tags, wherein the multiple polymorphisms are
`
`associated with the number of disorders; and (c) associating the multiple polymorphisms with the first and/or
`
`second genome in the mixture, wherein the mixture is unenriched for the multiple polymorphisms, and
`
`wherein the first genome is a genome of an unaffected cell and the second genome is a genome from an
`
`A0631
`
`

`

`Case 1:20-cv-01580-LPS Document 40-1 Filed 03/05/21 Page 11 of 268 PageID #: 8154
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`affected cell. In some embodiments, step (b) comprises counting sequence tags mapped to the multiple
`
`polymorphisms. Alternatively, step (b) comprises genotyping the second genome in a sample that is
`
`substantially free of the first genome. Sequencing is massively parallel sequencing of clonally amplified
`
`cfDNA molecules or of single cfDNA molecules. In some embodiments, sequencing is performed using
`
`massively parallel sequencing-by-synthesis with reversible dye terminators. In other embodiments,
`
`sequencing is performed using massively parallel sequencing-by-ligation.
`
`In some embodiments, the sample
`
`is a plasma sample.
`
`[0025]
`
`In another embodiment, the method for determining the presence or absence of multiple disorders in a
`
`blood sample comprising a mixture of cfDNA of a first and a second genome, comprises: (a) whole genome
`
`sequencing at least a portion of the mixture of cfDNA, thereby providing a plurality of sequence tags; (b)
`
`identifying multiple polymorphisms in the plurality of sequence tags, wherein the multiple polymorphisms are
`
`associated with the number of disorders; and (c) associating the multiple polymorphisms with the first and/or
`