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`US 20130029852Al
`c19) United States
`
`
`c12) Patent Application Publication
`c10) Pub. No.: US 2013/0029852 Al
`(43) Pub. Date: Jan. 31, 2013
`Rava et al.
`
`(54)DETECTING AND CLASSIFYING COPY
`NUMBER VARIATION
`
`61/296,358, filed on Jan. 19, 2010, provisional appli­
`
`
`
`
`
`
`cation No. 61/360,837, filed on Jul. 1, 2010, provi­
`
`
`
`
`sional application No. 61/407,017, filed on Oct. 26,
`(75)Inventors: Richard P. Rava, Redwood City, CA
`
`
`
`
`
`
`
`2010, provisional application No. 61/455,849, filed on
`(US); B rian K. Rhees, Gilbert, AZ (US)
`Oct. 26, 2010.
`
`(73)Assignee: VERINATA HEALTH, INC., Redwood
`
`City, CA (US)
`
`
`
`Publication Classification
`
`
`
`(21) Appl. No.: 13/555,037
`
`(22)Filed:Jul. 20, 2012
`
`Related U.S. Application Data
`
`(51)Int. Cl.
`GOIN 33/50 (2006.01)
`C40B 60/10 (2006.01)
`G06F 19100 (2011.01)
`C40B 20100 (2006.01)
`
`
`
`(52) U.S. Cl. ................................. 506/2; 506/38; 702/20
`(63) Continuation-in-part of application No. 13/191,366,
`
`
`(57)
`ABST R ACT
`
`
`filed on Jul. 26, 2011, which is a continuation-in-part
`
`
`of application No. 12/958,352, filed on Dec. 1, 2010,
`The invention provides a method for determining copy num­
`
`
`
`
`
`Continuation-in-part of application No. 13/009,708,
`
`
`ber variations (CNV) ofa sequence ofinterest in a test sample
`
`filed on Jan. 19, 2011, Continuation-in-part of appli­
`
`
`
`that comprises a mixture of nucleic acids that are known or are
`
`cation No. 13/445,778, filed on Apr. 12, 2012, Con­
`
`
`
`suspected to differ in the amount of one or more sequence of
`
`
`tinuation-in-part of application No. 12/958,347, filed
`
`
`
`
`interest. The method comprises a statistical approach that
`
`
`on Dec. 1, 2010, Continuation-in-part of application
`
`
`
`
`accounts for accrued variability stemming from process-re­
`
`No. 12/958,356, filed on Dec. 1, 2010.
`
`
`
`lated, interchromosomal and inter-sequencing variability.
`(60) Provisional application No. 61/407,017, filed on Oct.
`
`
`
`
`
`The method is applicable to determining CNV of any fetal
`
`
`26, 2010, provisional application No. 61/296,464,
`
`
`
`aneuploidy, and CNVs known or suspected to be associated
`
`
`
`filed on Jan. 19, 2010, provisional application No.
`
`
`with a variety of medical conditions. CNV that can be deter­
`
`
`61/474,362, filed on Apr. 12, 2011, provisional appli­
`
`
`
`mined according to the method include trisomies and mono­
`
`
`
`cation No. 61/296,358, filed on Jan. 19, 2010, provi­
`
`somies of any one or more of chromosomes 1-22, X and Y,
`
`
`
`sional application No. 61/360,837, filed on Jul. 1,
`
`
`
`
`other chromosomal polysomies, and deletions and/or dupli­
`
`
`
`2010, provisional application No. 61/407,017, filed on
`
`
`
`cations of segments of any one or more of the chromosomes,
`
`
`
`Oct. 26, 2010, provisional application No. 61/455,849,
`
`
`which can be detected by sequencing only once the nucleic
`
`
`
`filed on Oct. 26, 2010, provisional application No.
`acids of a test sample.
`
`110 Obtain qualified samples
`
`115 Obtain test sample
`
`
`comprising nucleic acids
`
`corprising nucleic acids
`
`!
`
`125 Sequence at least a portion
`
`120 Sequence at least a portion
`
`of test nucleic acids
`
`
`of qualified nucleic acids
`!
`! 135 Determine
`test sequence
`
`
`130 Determine qualified sequence
`tag densities
`tag derities
`
`140 Determine qualified sequence
`!
`doses!
`
`
`
`145 Identify qualified normalizing sequence
`!
`
`
`150 Determine test sequence dose
`based on test normalizing
`sequence
`
`corresponding to identified
`qualified 1�
`
`normalizing sequence
`
`155 Determine thresholds
`�
`160 Compare test sequence dose
`
`
`to threshold value
`!
`
`
`165 Determine presence or absence
`of copy number variation
`
`�
`100
`
`

`

`
`Patent Application Publication
`
`
`Jan. 31, 2013 Sheet 1 of 75 US 2013/0029852 Al
`
`
`
`110 Obtain qualified samples
`
`115
`Obtain test sample
`
`
`comprising nucleic acids
`cor ,ising
`
`nucleic acids
`i
`125
`
`Sequence at least a portion
`
`120 Sequence at least a portion
`
`of test nucleic acids
`
`
`of qualified nucleic acids
`i
`i 135 Determine
`test sequence
`
`
`130 Determine qualified sequence
`tag densities
`1ag derties
`
`
`140 Determine qualified sequence
`l
`doses i
`
`
`
`145 Identify qualified normalizing sequence
`l 150 Determine test sequence dose
`
`
`based on test normalizing
`sequence
`qualified j
`
`corresponding to identified
`
`normalizing sequence
`
`155 Determine thresholds
`
`160
`+ Compare test sequence dose
`
`
`to threshold value
`
`
`i Determine presence or absence
`165
`of copy number variation
`
`� 100
`
`FIG.1
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 2 of 75
`
`US 2013/0029852 A1
`
`reNaTH [pceenomvexron|P [sATALWE] P [ting|P [Poaueuevne|P
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`amplify in
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`
`Amplify on solid
`surface and
`detached 1-step
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 3 of 75
`
`US 2013/0029852 Al
`
`x400
`
`Obtain a Biological Source Sample
`Comprising Genomic Nucleic Acids
`
`Combine Marker Nucleic Acids with
`Biological Source Sample
`
`
`
`Prepare Sequencing Libraries of
`Sample Genomic and Marker
`Nucleic Acids
`
`Perform Massively Parallel
`Singleplex Sequencing
`
`Analyze Sequencing Information
`
`Verify the Integrity of Samples
`
`410
`
`420
`
`430
`
`440
`
`450
`
`460
`
`FIG. 4
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 4 of 75
`
`US 2013/0029852 Al
`
`x 500
`
`510
`
`
`
`Obtain a Plurality of Biological
`Samples Comprising Genomic
`Nucleic Acids
`
`Combine Unique Marker Nucleic
`Acids with Biological Source
`
`Prepare Sequencing Libraries of
`Indexed Genomic Sample and
`
`Marker Nucleic Acids
`
`Sequencing
`
`520
`
`530
`
`540
`
`550
`
`560
`
`Verify the Integrity of Each of the
`Plurality of Sampies
`
`FIG. 5
`
`

`

`Patent Application Publication
`
`Jan. 31, 2013 Sheet 5 of 75
`
`US 2013/0029852 Al
`
`610
`
`620
`
`630
`
`640
`
`Obtain test sample comprising a mixture of fetal and
`maternal nucleic acids
`
`Enrich the mixture of nucleic acids for polymorphic target
`nucleic acids
`
`presence or absence of aneuploidy in the test sample
`
`Sequence the enriched mixture of nucleic acids
`
`Determine the fraction of fetal nucleic acids and the
`
`600
`
`FIG. 6
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 6 of 75
`
`US 2013/0029852 Al
`
`710
`
`Maternal Plasma Sample
`
`720
`
`730
`
`
`
`
`cfDNA purification
`
`Polymorphic Nucleic Acid
`Amplification
`
`
`
`
`
`
`
`
`
`740
`
`760
`
`Massively Parallel
`Sequencing
`
`Fetal Fraction
`Determination
`
`750
`
`Size Separation by
`Electrophoresis
`
`770
`
`Fetal Fraction
`Determination
`
`FIG. 7
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 7 of 75
`
`US 2013/0029852 Al
`
`Obtain test sample comprising a mixture of fetal and maternal
`nucleic acids
`
`
`
`810
`
`
`
`
`
`
`
`
`Amplify polymorphic target nucleic acids in a portion of the
`mixture of fetal and maternal nucleic acids in the test sample
`
`820
`
`Enrich the mixture of nucleic acids by combining the
`amplified polymorphic target nucleic acids
`
`830
`
`Purify the enriched mixture
`
`
`
`
`850
`
`860
`
`Sequence the enriched mixture of nucleic acids
`
`Determine the fraction of fetal nucleic acids and the presence
`or absence of aneuploidy in the test sample
`
`800
`
`FIG. 8
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 8 of 75
`
`US 2013/0029852 Al
`
`
`
`
`
`
`
`Obtain test sample comprising a mixture of fetal and maternal
`nucleic acids
`
`910
`
`
`
`Purify said mixture of fetal and maternal nucleic acids
`
`
`
`
`
`Amplify polymorphic target nucleic acids in a portion of the
`mixture of fetal and maternal nucleic acids in the purified
`sample
`
`940
`
`Enrich the mixture of nucleic acids by combining the amplified
`polymorphic target nucleic acids
`
`
`
`
`950
`
`960
`
`Sequencethe enriched mixture of nucleic acids
`
`Determine the fraction of fetal nucleic acids and the presence
`or absence of aneuploidy in the test sample
`
`900
`
`FIG. 9
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 9 of 75
`
`US 2013/0029852 Al
`
`Obtain test sample comprising a mixture of fetal and maternal
`nucleic acids
`
`4010
`
`Purify said mixture of fetal and maternal nucleic acids
`
`1020
`
`Amplify polymorphic target nucleic acids in a portion of the
`mixture of fetal and maternal nucleic acids in the purified sample
`
`1040
`
`Prepare sequencinglibrary of amplified polymorphic target
`nucleic acids
`
`
`
`
`Prepare sequencinglibrary of purified mixture of fetal and
`maternal nucleic acids
`
`41050
`
`
`
`Enrich the mixture of nucleic acids by combining the library of
`amplified palymorphic target nucleic acids
`
` Sequence the enriched mixture of nucleic acids
`
`Determinethe fraction of fetal nucleic acids and the presence
`or absence of aneuploidy in the test sample
`
`1070
`
`1080
`
`FIG. 10
`
`1030
`
`1000
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 10 of 75
`
`US 2013/0029852 Al
`
`
`
`Maternal Plasma Sample
`
`Polymorphic Nucleic Acid
`Amplification:
`
`Polymorphic Nucleic Acid
`Amplification
`
`
`
`
`
`
`
`
`
`
`
`
`| q
`
`
`ciDNA Purification,
`
`
`
`Polymorphic Nucleic Acid
`Amplification
`
`
`Sequencing Library
`Preparation
`
`Massively Parallel
`Sequencing
`
`y
`Sequencing Library
`
`Preparation
`
`FIG. 11
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 11 of 75
`
`US 2013/0029852 Al
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`Patent Application Publication
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`Jan. 31, 2013 Sheet 12 of 75
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`US 2013/0029852 A1
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`Patent Application Publication
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`Jan. 31, 2013 Sheet 13 of 75
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`US 2013/0029852 A1
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`Patent Application Publication
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`Jan. 31,2013 Sheet 14 of 75
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`US 2013/0029852 Al
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`FIG.15
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`Patent Application Publication
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`Jan. 31, 2013 Sheet 15 of 75
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`US 2013/0029852 A1
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`Patent Application Publication
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`Jan. 31, 2013 Sheet 16 of 75
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`Patent Application Publication
`
`Jan. 31,2013 Sheet 17 of 75
`
`US 2013/0029852 Al
`
`Calculate non-CNV
`fetal fraction (NCNFF)
`
`Calculate CNVfetal
`fraction (CNFF)
`(based on
`assumption about
`type of aneuploidy)
`
`Compare NCNFF and
`CNFF
`
`CNFF assumption
`regarding aneuploidy
`is correct
`
`1806
`
`Disagree
`
`
`
`
`No Conclusion
`
`Bin chromosome or
`
`segmentat issue and
`determine whether any
`bins have a DNA amount
`indicating aneuploidy
`
`
`
`1816
`
`Mosaic
`
`
`
`Partial Aneuploidy
`located in affected
`bin(s)
`
`Do any bins have a DNA amount
`indicating aneuploidy?
`
`FIG. 18
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 18 of 75
`
`US 2013/0029852 Al
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`FIG. 19
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`

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`Patent Application Publication
`
`Jan. 31, 2013 Sheet 19 of 75
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`US 2013/0029852 A1
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`Patent Application Publication
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`Jan. 31,2013 Sheet 20 of 75
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`US 2013/0029852 Al
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`FIG. 21A
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`FIG. 21B
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`Patent Application Publication
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`Jan. 31, 2013 Sheet 21 of 75
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`US 2013/0029852 A1
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`SSRISSSESSSIISSISSISSISSISSISSSSISSSSSSSASSSSSSASSO
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`FIG. 22A
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`Patent Application Publication
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`Jan. 31,2013 Sheet 22 of 75
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`US 2013/0029852 Al
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`13 5
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`FIG. 22C
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`Patent Application Publication
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`Jan. 31,2013 Sheet 23 of 75
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`US 2013/0029852 Al
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`%ChrK
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`FIG. 23B
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`Patent Application Publication
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`Jan. 31,2013 Sheet 24 of 75
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`US 2013/0029852 Al
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`Patent Application Publication
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`Jan. 31,2013 Sheet 25 of 75
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`US 2013/0029852 Al
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`green enna ene nen aang tenant n EEA EEE ERE DETTE DEE EEE EAE E DESERTED E ETERS TERETE ER EEE EEE DEER EET ET ERD E TERETE EERSTE TEETER ET OT ETE R SEER E OETA EE OE ERE T TERETE ERT SEER EERE
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`FIG. 25B
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`

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`Patent Application Publication
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`Jan. 31, 2013 Sheet 26 of 75
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`US 2013/0029852 Al
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`13 -
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`FIG. 25C
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`Patent Application Publication
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`Jan. 31, 2013 Sheet 27 of 75
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`US 2013/0029852 A1
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`FIG. 26B
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`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 28 of 75
`
`US 2013/0029852 Al
`
`(nM)}
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`Patent Application Publication
`
`Jan. 31,2013 Sheet 29 of 75
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`US 2013/0029852 Al
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`2000
`3000
`4000
`5000
`6000
`
`cfDNA(pg)
`
`

`

`Patent Application Publication
`
`Jan. 31, 2013 Sheet 30 of 75
`
`US 2013/0029852 Al
`
`
`
`indexedReads
`
`
`
`

`

`Patent Application Publication
`
`Jan. 31, 2013 Sheet 31 of 75
`
`US 2013/0029852 A1
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`mnOMOOMMFTMHNAOO
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`FIG. 30A
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`140
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`160
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`180
`
`200
`
`220
`
`240
`
`Chromosomesize (Mbp)
`
`FIG. 30B
`
`
`
`

`

`Patent Application Publication
`
`Jan. 31, 2013 Sheet 32 of 75
`
`US 2013/0029852 Al
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`0.025 ~
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`

`

`Patent Application Publication
`
`Jan. 31, 2013 Sheet 33 of 75
`
`US 2013/0029852 Al
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`Chramosame 24
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`~aeneo:nh23‘24$Fsa .a$hFsa:::2,sopehinncsebinitiieseis.*=:3-:uo‘3:4:he;35
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`FIG. 32A
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`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 34 of 75
`
`US 2013/0029852 Al
`
`Chromosame a
`
`FIG. 32B
`
`

`

`Patent Application Publication
`
`Jan. 31, 2013 Sheet 35 of 75
`
`US 2013/0029852 Al
`
`Chromosome 12
`2AECECACRRAACER
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`aaot
`
`ameBe
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`
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` SRTEf
`
`Bs 1
`
`FIG. 33A
`
`

`

`Patent Application Publication
`
`Jan. 31, 2013 Sheet 36 of 75
`
`US 2013/0029852 Al
`
`Chromosome18
`
`FIG. 33B
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 37 of 75
`
`US 2013/0029852 Al
`
`Chromista 25
`
`
`ee
`32eeaees
`
`
`
`
`
`
`
`
`
`
`
`FIG. 34A
`
`

`

`Patent Application Publication
`
`Jan. 31, 2013 Sheet 38 of 75
`
`US 2013/0029852 Al
`
`fhromaabrmne 45
`
`SORostioseres debeeoy
`
`FIG. 34B
`
`

`

`Patent Application Publication
`
`Jan. 31, 2013 Sheet 39 of 75
`
`US 2013/0029852 Al
`
`Chromosome =
`
`Deas
`
`FIG. 35A
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 40 of 75
`
`US 2013/0029852 Al
`
`Chromosome &.
`
`FIG. 35B
`
`

`

`Patent Application Publication
`
`Jan. 31, 2013 Sheet 41 of 75
`
`US 2013/0029852 A1
`
`acea
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`.ceceecceneiieeereattececetccigeneeeritcece
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`tts:
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`
`FIG. 36A
`
`
`
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 42 of 75
`
`US 2013/0029852 Al
`
`Chreemasnime ¥
`
`FIG. 36B
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 43 of 75
`
`US 2013/0029852 Al
`
`CVinnoneaflecteds
`
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` &
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`Denominahey chramiosons
`
`FIG. 37
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 44 of 75
`
`US 2013/0029852 Al
`
`fynoneaiectadis.
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`0.20
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`FIG. 38
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 45 of 75
`
`US 2013/0029852 Al
`
`
`
`Cournglatie:‘distributions
`
`
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 46 of 75
`
`US 2013/0029852 Al
`
`
`
`Chromesome11SegmentDasegp
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`ne
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`be]
`
`Qualified and Test Samples
`
`FIG. 40
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 47 of 75
`
`US 2013/0029852 Al
`
`Chromosome 21
`
`NormalizedDose
`
`A y
`
`e 8.
`
`a -
`
`Chromosome (denominator)
`N O
`T21 &
`
`FIG. 414A
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 48 of 75
`
`US 2013/0029852 Al
`
`Chromosome 18
`
`NormalizedDose
`
`Chromosome (denominator)
`N ©
`T18 &
`
`FIG. 41B
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 49 of 75
`
`US 2013/0029852 Al
`
`8 : A
`
`Chromosome 13
`
`verage(chrs 3-6)
`
`NormalizedDose
`
`Chromosome (denominator)
`NO
`T13 &
`
`FIG. 41C
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 50 of 75
`
`US 2013/0029852 Al
`
`Chromosome X
`
`NormalizedDose 4
`
`Chromosome (denominator)
`XX O
`XY &
`xO +
`
`FIG. 41D
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 51 of 75
`
`US 2013/0029852 Al
`
`Q©OQ 9 8 ° 0 A
`
`Chromosome Y
`
`verage(chrs 1-22,X)
`
`Chromosome (denominator)
`XY O
`XX 4s
`XO --
`
`FIG. 41E
`
`NormalizedDose
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 52 of 75
`
`US 2013/0029852 Al
`
`Values
`
`oO
`
`
`
`NormalizedChromosome
`
`
`
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 53 of 75
`
`US 2013/0029852 Al
`
`
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 54 of 75
`
`US 2013/0029852 Al
`
`
`
`Ww
`ay
`
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`
`2c
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`ow E
`E 3
`o 2
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`NormalizedChromosomeValues
`
`OChromasome 21. AChromosomei8 Chromosome 13
`
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` iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii
`
`iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii
`
`FIG. 45
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 55 of 75
`
`US 2013/0029852 Al
`
`
`sstnnnnnnnnnntnnnnnnnnnnnnnnnnnnnnninnnnnnnnnnnnnnnninERQepee
`
`FIG. 46B
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 56 of 75
`
`US 2013/0029852 Al
`
`OChromosome 21 AChromosomei8 Chromosome 13
`
`Values
`NormalizedChromosome
`
`OChromosome 9
`
`FIG. 47
`
`
`
`FIG. 48
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 57 of 75
`
`US 2013/0029852 Al
`
`Value
`
`NarmalixedChrotecsume
`
`FIG. 49
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 58 of 75
`
`US 2013/0029852 Al
`
`
`
`JY atigihle worrnan
`esth blaoed draw snd ciste
`
`
`n=Z,882
`
`ineligiole samples
`
`mehey
`
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`..
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`.
`
`No Ksryotypes
`n=-45
`
`
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`Mulisle Gastetiun
`eBS
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`ne3, 825
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`
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`Selected tar Testing
`ne5a4
`
`Samia Tracking issue
`
`
`“Analyzed Sarsples
`neG32
`
`
`
`FIG. 50A
`
`a
`Higitsle
`Samples
`4,825
`
`we
`
`eenSasTE
`ae
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`(Ansiyzed
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`
`
`Karyotyaes
`
`\
`
`Me,
`
`
`
`anes cen
`
`FIG. 50B
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 59 of 75
`
`US 2013/0029852 A1
`
`“
`“Chromosome 2
`t Analysis
`neS3e
`
`™
`a
`
`
`
`vr" Safe
`;
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`ie
`neg
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`J2E Adsent
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`
`
`FIG. 51A
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 60 of 75
`
`US 2013/0029852 Al
`
`Mos,.
`oe
`Let Chromasarie 18 Analysis
`
`Ne
`e832
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`a
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`
`
`FIG. 51B
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 61 of 75
`
`US 2013/0029852 Al
`
`ie
`my
`a
`ma
`ae Chromosome 13 Analysis ms
`*. a
`n=532
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`: Censored complex
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`
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`
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`
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`
`
`
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`
`
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` T13 Presentnelt 723 Absentn=O T13 Present
`
`
`
`ree3
`
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`neds
`
`
`
`FIG. 51C
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 62 of 75
`
`US 2013/0029852 Al
`
`a
`we
`
`Mey
`*
`
`on
`<
`a
`
`hyei
`Femate Analysis
`3532
`
`a,
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`wowe
`
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`
`s,
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`|
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`BEG
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`karyotypes*
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`
`
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`No ns20e
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`
`
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`
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`n=232
`
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`nat
`
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`ne}
`
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`ne4i99
`
`FIG. 51D
`
`Fernaie
`Karyotype
`n=233
`
`Femate
`Karyotype
`neG
`
`Not female
`Karyotype
`n-44
`
`
`
`

`

`Patent Application Publication
`
`Jan. 31, 2013 Sheet 63 of 75
`
`US 2013/0029852 A1
`
`Male Analysis
`ne$3Z
`
`Son,
`
`o
`
`o
`
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`detected
`SN ee
`
`
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`
`
`
`
`f
`(
`
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`
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`
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`a8
`~~
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`TOF
`Net Mave
`Karyotype
`meh
`
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`OU
`Not Male
`Karyotype
`n249
`
`
`
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`n=184
`
`Not Mate
`net
`
`Male
`neo
`
`Not Male
`neZ4a
`
`FIG. 51E
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 64 of 75
`
`US 2013/0029852 A1
`
`iaeoe
`a
`a Monosomy X Analysis
`oN,
`n=532
`.
`we
`.
`a
`
`peneey,
`YoNe fetal DNA™.,
`detected
`:
`.,
`tie A
`fon _
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`Censored complex
`karyotypes*
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`n=37
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`.
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`
`ae
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`ew
`
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`
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`
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`
`
`n=t MX Absent
` n=2 MX Presentnei5 MX Absent
`
`
`
`n=427
`
`Unaffected
`Karyotype
`
`
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`
`Unaffected
`Karyotype
`
`n=45
`
`MX Present
`
`ne4i6
`
`FIG. 51F
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 650f75
`
`US 2013/0029852 Al
`
`28
`
`ACY
`
`12
`
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`
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`
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`
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`
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`Euplosi th Treeay 18 Eupinkt 73 mes
`
`FIG. 52
`
`

`

`Patent Application Publication
`
`Jan. 31, 2013 Sheet 66 of 75
`
`US 2013/0029852 Al
`
` see:
`
`Male 4X Gin
`
`Female
`
`

`

`Patent Application Publication
`
`Jan. 31, 2013 Sheet 67 of 75
`
`US 2013/0029852 A1
`
`
`
`XAONOe
`
`°:
`
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`
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`
`FIG. 54
`
`
`
`
`

`

`Patent Application Publication
`
`Jan. 31, 2013 Sheet 68 of 75
`
`US 2013/0029852 A1
`
`
`
`
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`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 69 of 75
`
`US 2013/0029852 Al
`
`
`
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`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 70 of 75
`
`US 2013/0029852 Al
`
`4
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`Patent Application Publication
`
`Jan. 31,2013 Sheet 71 of 75
`
`US 2013/0029852 Al
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`MELISSA STUDY: Trisomy 21 samples
`Whole chromosome aneuploidy for Chr21 confirmed in affected “male-fetus” samples by
`
`the near-perfect correlation between estimated FF_X and FF_21
`
`

`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 72 of 75
`
`US 2013/0029852 Al
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`MELISSA STUDY: Trisomy 18 samples
`Whole chromosome aneuploidy for Chr18 confirmed in affected “male-fetus” samples by
`the near-perfect correlation between estimated FF_X and FF_18
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`

`Patent Application Publication
`
`Jan. 31,2013 Sheet 73 of 75
`
`US 2013/0029852 Al
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`MELISSA STUDY: Trisomy 13 samples
`Whole chromosome aneuploidy for Chri3 confirmed in affected “male-fetus” samples by
`the near-perfect correlation between estimated FF_X and FF_13
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`Patent Application Publication
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`Jan. 31,2013 Sheet 74 of 75
`
`US 2013/0029852 Al
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`Patent Application Publication
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`Jan. 31,2013 Sheet 75 of 75
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`US 2013/0029852 Al
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`US 2013/0029852 Al
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`Jan. 31, 2013
`
`DETECTING AND CLASSIFYING COPY
`NUMBERVARIATION
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`[0001] This application is a continuation-in-part of U.S.
`application Ser. No. 13/191,366, filed on Jul. 26, 2011, which
`is a continuation-in-part of U.S. application Ser. No. 12/958,
`352, filed on Dec. 1, 2010, which claims priority to U.S.
`Provisional Application No(s). 61/296,358 filed Jan. 19, 2010
`and 61/360,837 filed Jul. 1, 2010 and 61,407,017 and 61/455,
`849 both filed Oct. 26, 2010, all of which are incorporated by
`reference in their entireties. This application is also a continu-
`ation-in-part of U.S. application Ser. No. 13/009,708 filed
`Jan. 19, 2011, which claims benefit ofU.S. Provisional Patent
`Application No. 61/296,464filed Jan. 19, 2010, both ofwhich
`are incorporated herein by reference their entireties. This
`application is also a continuation-in-part of U.S. application
`Ser. No. 13/445,778 filed Apr. 12, 2012, which claims benefit
`of U.S. Provisional Patent Application No. 61/474,362 filed
`Apr. 12, 2011, both of which are incorporated herein by
`reference in their entireties. This application is also a continu-
`ation-in-part of U.S. application Ser. No. 12/958,347 filed
`Dec. 1, 2010, which claims benefit ofU.S. Provisional Patent
`Application No(s). 61/296,358 filed Jan. 19, 2010 and
`61/360,837 filed Jul. 1, 2010 and 61/407,017 and 61/455,849
`both filed Oct. 26, 2010, all of which are incorporated by
`reference in their entireties and for all purposes. This appli-
`cation is also a continuation-in-part of U.S. application Ser.
`No. 12/958,356 filed Dec. 1, 2010, which claims benefit of
`USS. Provisional Patent Application No(s). 61/296,358 filed
`Jan. 19, 2010 and 61/360,837 filed Jul. 1, 2010 and 61/407,
`017 and 61/455,849 both filed Oct. 26, 2010, all of which are
`incorporated by reference in their entireties and for all pur-
`poses.
`
`BACKGROUND
`
`[0002] One of the critical endeavors in human medical
`research is the discovery of genetic abnormalities that pro-
`duce adverse health consequences. In many cases, specific
`genes and/orcritical diagnostic markers have been identified
`in portions of the genomethat are present at abnormal copy
`numbers. For example, in prenatal diagnosis, extra or missing
`copies of whole chromosomes are frequently occurring
`geneticlesions. In cancer, deletion or multiplication of copies
`of whole chromosomes or chromosomal segments, and
`higher level amplifications of specific regions of the genome,
`are common occurrences.
`
`[0003] Most information about copy numbervariation has
`been provided by cytogenetic resolution that has permitted
`recognition of structural abnormalities. Conventional proce-
`dures for genetic screening and biological dosimetry have
`utilized invasive procedures e.g. amniocentesis, to obtain
`cells for the analysis ofkaryotypes. Recognizing the need for
`more rapid testing methods that do not require cell culture,
`fluorescence in situ hybridization (FISH), quantitative fluo-
`rescence PCR (QF-PCR) and array-Comparative Genomic
`Hybridization (array-CGH) have been developed as molecu-
`lar-cytogenetic methods for the analysis of copy number
`variations.
`
`[0004] The advent of technologies that allow for sequenc-
`ing entire genomesinrelatively short time, and the discovery
`of circulating cell-free DNA (cfDNA) have provided the
`
`opportunity to compare genetic material originating from one
`chromosome to be compared to that of another without the
`risks associated with invasive sampling methods. However,
`the limitations of the existing methods, which include insuf-
`ficient sensitivity stemming from the limited levels of
`cfDNA,and the sequencingbiasof the technology stemming
`from the inherent nature ofgenomic information, underlie the
`continuing need for noninvasive methods that would provide
`any or all of the specificity, sensitivity, and applicability, to
`reliably diagnose copy numberchangesina variety ofclinical
`settings.
`[0005] Embodiments disclosed herein fulfill some of the
`above needsandin particular offers an advantagein providing
`a reliable methodthat is applicable at least to the practice of
`noninvasive prenatal diagnostics, and to the diagnosis and
`monitoring of metastatic progression in cancerpatients.
`
`SUMMARY
`
`[0006] Methodsare provided for determining copy number
`variations (CNV)of a sequence ofinterest in a test sample
`that comprises a mixture ofnucleic acids that are knownor are
`suspected to differ in the amountof one or more sequence of
`interest. The method comprises a statistical approach that
`accounts for accrued variability stemming from process-re-
`lated,
`interchromosomal and inter-sequencing variability.
`The method is applicable to determining CNV of anyfetal
`aneuploidy, and CNVs knownor suspected to be associated
`with a variety of medical conditions. CNV that can be deter-
`mined according to the present method include trisomies and
`monosomiesof any one or more of chromosomes1-22, X and
`Y, other chromosomal polysomies, and deletions and/or
`duplications of segments of any one or more of the chromo-
`somes, which can be detected by sequencing only once the
`nucleic acids of a test sample. Any aneuploidy can be deter-
`mined from sequencing information that
`is obtained by
`sequencing only once the nucleic acids of a test sample.
`[0007]
`Inone embodiment, a methodis provided for deter-
`mining the presenceor absence of any four or more different
`complete fetal chromosomal aneuploidies in a maternaltest
`sample comprising fetal and maternal nucleic acid molecules.
`The steps of the method comprise (a) obtaining sequence
`information for the fetal and maternal nucleic acids in the
`
`maternal test sample; (b) using the sequence information to
`identify a number of sequence tags for each of any four or
`more chromosomesofinterest selected from chromosomes
`1-22, X and Y andto identify a numberof sequencetags for a
`normalizing chromosomesequencefor eachofthe any four or
`more chromosomes of interest; (c) using the number of
`sequence tags identified for each of the any four or more
`chromosomesof interest and the number of sequence tags
`identified for each normalizing chromosometo calculate a
`single chromosome dose for each of the any four or more
`chromosomes of interest; and (d) comparing each of the
`single chromosome doses for each of the any four or more
`chromosomesofinterest to a threshold value for each of the
`four or more chromosomesofinterest, and thereby determin-
`ing the presence or absence of any four or more complete
`different fetal chromosomalaneuploidies in the matern