(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2009/0029377 A1
`(43) Pub. Date:
`Jan. 29, 2009
`Lo et al.
`
`US 20090029377A1
`
`(54) DIAGNOSING FETAL CHROMOSOMAL
`ANEUPLOIDY USING MASSIVELY
`PARALLEL GENOMIC SEQUENCING
`
`(75) Inventors:
`
`Yuk-Ming Dennis Lo, HoWloon
`(HK); Rossa Wai KWun Chiu, New
`Territories (HK); KWan Chee
`Chan, KOWlOOIl
`
`Correspondence Address:
`TOWNSEND AND TOWNSEND AND CREW,
`LLP
`TWO EMBARCADERO CENTER, EIGHTH
`FLOOR
`SAN FRANCISCO’ CA 94111-3834 (Us)
`
`(73) Assignee:
`
`The Chinese University of Hong
`Kong, NeW Territories (HK)
`
`(21) Appl. No.:
`
`12/178,181
`
`(22) Filed:
`
`Jul. 23, 2008
`
`Related US. Application Data
`
`(60) Provisional application No. 60/ 951,438, ?led on Jul.
`23, 2007.
`Publication Classi?cation
`
`(51) Int_ CL
`C1 2Q 1/68
`G06F 19/00
`
`(2006.01)
`(2006.01)
`
`435/6; 702/20
`
`ABSTRACT
`
`(57)
`Embodiments of this invention provide methods, systems,
`and apparatus for determining Whether a fetal chromosomal
`aneuploidy exists from a biological sample obtained from a
`pregnant female. Nucleic acid molecules of the biological
`sample are sequenced, such that a fraction of the genome is
`sequenced. Respective amounts of a clinically-relevant chro
`mosome and of background chromosomes are determined
`from results of the sequencing. A parameter derived from
`these amounts (eg a ratio) is compared to one or more cutoff
`values, thereby determining a classi?cation of Whether a fetal
`chromosomal aneuploidy exists.
`
`El Trisomy 21
`
`12 3 4 5 6 7 8 910111213141516171819202122X Y
`
`Ariosa Exhibit 1045
`pg. 1
`
`

`
`Patent Application Publication
`
`Jan. 29, 2009 Sheet 1 0f 9
`
`US 2009/0029377 A1
`
`110 A Receive sample
`
`120
`
`\ Sequence fraction of genome in
`
`7
`
`'
`
`'
`
`sample
`
`'
`
`'
`
`130
`
`V
`"
`\ Based on sequencing, determine ?rst
`amount of a ?rst chromosome
`
`140
`
`V
`\ Determine second amount of one
`or more second chromosomes
`
`V
`150 \ Determine parameter from ?rst
`amount and second amount
`
`160
`
`\ Based on the comparison, a classi?cation of
`whether a fetal chromosomal aneuploidy exists
`for the ?rst chromosome is determined
`
`i
`
`'
`
`.
`
`100 j
`
`FIG. 1
`
`Ariosa Exhibit 1045
`pg. 2
`
`

`
`Patent Application Publication
`
`Jan. 29, 2009 Sheet 2 0f 9
`
`US 2009/0029377 A1
`
`210
`
`\ Receive sample
`
`220
`
`V
`\ Calculate number of sequences
`needed
`
`V
`230 \ Randomly sequence fraction of
`
`genome
`
`‘
`
`V
`Based on sequencing, determine ?rst
`240 S
`amount of a ?rst chromosome _
`
`V
`250 \ Determine second amount of one
`or more second chromosomes
`
`7
`260 —\ Determine parameter from ?rst
`amount and second amount
`
`l
`270 \ Based on the comparison, a classi?cation of
`whether a fetal chromosomal aneuploidy exists
`for the ?rst chromosome is determined
`
`200 / FIG. 2
`
`Ariosa Exhibit 1045
`pg. 3
`
`

`
`Patent Application Publication
`
`Jan. 29, 2009 Sheet 3 0f 9
`
`US 2009/0029377 A1
`
`0
`
`-'
`V
`
`I
`1
`Trisomy 21
`Normal
`Status of the fetus
`
`FIG. 3A
`
`1.52
`
`1.50 -
`
`1.48 -
`
`1.46 -
`
`1.44 -
`
`1.42 -
`
`1.40 -
`
`1.38 —
`
`1.36 —
`
`1.34 -
`
`1.32
`
`13
`
`Percentage of sequences mapped to chromosome 21 (%)
`
`percentage of Y chromosome using massively parallel sequencing (%) sequences Fetal DNA percentage determined by the
`
`
`
`
`
`
`
`
`
`
`
`
`8
`
`8
`
`I
`I
`I
`I
`10.
`12
`14
`16
`Fetal DNA percentage determined by micro?uidics digital PCR
`ZFY/ZFX assays (%)
`
`18
`
`FIG. 3B
`
`Ariosa Exhibit 1045
`pg. 4
`
`

`
`Patent Application Publication
`
`Jan. 29, 2009 Sheet 4 0f 9
`
`US 2009/0029377 A1
`
`El Trisomy 21
`
`12 3 4 5 6 7 8 910111213141516171819202122X Y
`
`FIG. 4A
`
`Percentage difference in chromosomal representation (%)
`
`1 1
`
`
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`12 3 4 5 6 7 8 910111213141516171819202122
`
`Chromosome
`
`FIG. 4B
`
`
`
`
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`oEowoEoEo Q5 5223 356:6 wmmEwewm
`
`Ariosa Exhibit 1045
`pg. 5
`
`

`
`Patent Application Publication
`
`Jan. 29, 2009 Sheet 5 0f 9
`
`US 2009/0029377 A1
`
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`
`FIG. 5
`
`Ariosa Exhibit 1045
`pg. 6
`
`

`
`Patent Application Publication
`
`Jan. 29, 2009 Sheet 6 of 9
`
`US 2009/0029377 A1
`
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`Ariosa Exhibit 1045
`
`pg. 7
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`Ariosa Exhibit 1045
`pg. 7
`
`

`
`Patent Application Publication
`
`Jan. 29, 2009 Sheet 7 0f 9
`
`US 2009/0029377 A1
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`Ariosa Exhibit 1045
`pg. 8
`
`

`
`Patent Application Publication
`
`Jan. 29, 2009 Sheet 8 0f 9
`
`US 2009/0029377 A1
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`Ariosa Exhibit 1045
`pg. 9
`
`

`
`Patent Application Publication
`
`Jan. 29, 2009 Sheet 9 0f 9
`
`US 2009/0029377 A1
`
`Ariosa Exhibit 1045
`pg. 10
`
`

`
`US 2009/0029377 A1
`
`Jan. 29, 2009
`
`DIAGNOSING FETAL CHROMOSOMAL
`ANEUPLOIDY USING MASSIVELY
`PARALLEL GENOMIC SEQUENCING
`
`CLAIM OF PRIORITY
`
`[0001] The present application claims priority from and is a
`non-provisional application of US. Provisional Application
`No. 60/951,438, entitled “DETERMINING A NUCLEIC
`ACID SEQUENCE IMBALANCE” ?led Jul. 23, 2007 (At
`torney Docket No. 016285-005200US), the entire contents of
`Which are herein incorporated by reference for all purposes.
`
`CROSS-REFERENCES TO RELATED
`APPLICATIONS
`
`[0002] The present application is also related to concur
`rently ?led non-provisional application entitled “DETER
`MINING A NUCLEIC ACID SEQUENCE IMBALANCE,”
`(Attorney Docket No. 016285-005210US) the entire contents
`of Which are herein incorporated by reference for all pur
`poses.
`
`FIELD OF THE INVENTION
`
`[0003] This invention generally relates to the diagnostic
`testing of fetal chromosomal aneuploidy by determining
`imbalances betWeen different nucleic acid sequences, and
`more particularly to the identi?cation of trisomy 21 (DoWn
`syndrome) and other chromosomal aneuploidies via testing a
`maternal sample (e.g. blood).
`
`BACKGROUND
`
`[0004] Fetal chromosomal aneuploidy results from the
`presence of abnormal dose(s) of a chromosome or chromo
`somal region. The abnormal dose(s) can be abnormally high,
`eg the presence of an extra chromosome 21 or chromosomal
`region in trisomy 21; or abnormally loW, eg the absence of a
`copy of chromosome X in Turner syndrome.
`[0005] Conventional prenatal diagnostic methods of a fetal
`chromosomal aneuploidy, e.g., trisomy 21, involve the sam
`pling of fetal materials by invasive procedures such as amnio
`centesis or chorionic villus sampling, Which pose a ?nite risk
`of fetal loss. Non-invasive procedures, such as screening by
`ultrasonography and biochemical markers, have been used to
`risk- stratify pregnant Women prior to de?nitive invasive diag
`nostic procedures. HoWever, these screening methods typi
`cally measure epiphenomena that are associated With the
`chromosomal aneuploidy, e.g., trisomy 21, instead of the core
`chromosomal abnormality, and thus have suboptimal diag
`no stic accuracy and other disadvantages, such as being highly
`in?uenced by gestational age.
`[0006] The discovery of circulating cell-free fetal DNA in
`maternal plasma in 1997 offered neW possibilities for nonin
`vasive prenatal diagnosis (Lo, Y M D and Chiu, R W K 2007
`Nat Rev Genet 8, 71-77). While this method has been readily
`applied to the prenatal diagnosis of sex-linked (Costa, J M et
`al. 2002 NEngl JMed 346, 1502) and certain single gene
`disorders (Lo, Y M D et al. 1998 NEngl JMed 339, 1734
`1738), its application to the prenatal detection of fetal chro
`mosomal aneuploidies has represented a considerable chal
`lenge (Lo, Y M D and Chiu, R W K 2007, supra). First, fetal
`nucleic acids co-exist in maternal plasma With a high back
`ground of nucleic acids of maternal origin that can often
`interfere With the analysis of fetal nucleic acids (Lo, Y M D et
`al. 1998 Am JHum Genet 62, 768-775). Second, fetal nucleic
`
`acids circulate in maternal plasma predominantly in a cell
`free form, making it di?icult to derive dosage information of
`genes or chromosomes Within the fetal genome.
`[0007] Signi?cant developments overcoming these chal
`lenges have recently been made (Benachi, A & Costa, J M
`2007 Lancet 369, 440-442). One approach detects fetal-spe
`ci?c nucleic acids in the maternal plasma, thus overcoming
`the problem of maternal background interference (Lo, Y M D
`and Chiu, R W K 2007, supra). Dosage of chromosome 21
`Was inferred from the ratios of polymorphic alleles in the
`placenta-derived DNA/RNA molecules. HoWever, this
`method is less accurate When samples contain loWer amount
`of the targeted nucleic acid and can only be applied to fetuses
`Who are heterozygous for the targeted polymorphisms, Which
`is only a subset of the population if one polymorphism is
`used.
`[0008] Dhallan et al (Dhallan, R, et al. 2007, supra Dhallan,
`R, et al. 2007 Lancet 369, 474-481) described an alternative
`strategy of enriching the proportion of circulating fetal DNA
`by adding formaldehyde to maternal plasma. The proportion
`of chromosome 21 sequences contributed by the fetus in
`maternal plasma Was determined by assessing the ratio of
`paternally-inherited fetal-speci?c alleles to non-fetal-speci?c
`alleles for single nucleotide polymorphisms (SNPs) on chro
`mosome 21. SNP ratios Were similarly computed for a refer
`ence chromosome. An imbalance of fetal chromosome 21
`Was then inferred by detecting a statistically signi?cant dif
`ference betWeen the SNP ratios for chromosome 21 and those
`of the reference chromosome, Where signi?cant is de?ned
`using a ?xed p-value of 20.05. To ensure high population
`coverage, more than 500 SNPs Were targeted per chromo
`some. HoWever, there have been controversies regarding the
`effectiveness of formaldehyde to enrich fetal DNA to a high
`proportion (Chung, G T Y, et al. 2005 Clin Chem 51, 655
`658), and thus the reproducibility of the method needs to be
`further evaluated. Also, as each fetus and mother Would be
`informative for a different number of SNPs for each chromo
`some, the poWer of the statistical test for SNP ratio compari
`son Would be variable from case to case (Lo, Y M D & Chiu,
`R W K. 2007 Lancet 369, 1997). Furthermore, since these
`approaches depend on the detection of genetic polymor
`phisms, they are limited to fetuses heterozygous for these
`polymorphisms.
`[0009] Using polymerase chain reaction (PCR) and DNA
`quanti?cation of a chromosome 21 locus and a reference
`locus in amniocyte cultures obtained from trisomy 21 and
`euploid fetuses, Zimmermann et al (2002 Clin Chem 48,
`362-363) Were able to distinguish the tWo groups of fetuses
`based on the 1.5-fold increase in chromosome 21 DNA
`sequences in the former. Since a 2-fold difference in DNA
`template concentration constitutes a difference of only one
`threshold cycle (Ct), the discrimination of a 1.5-fold differ
`ence has been the limit of conventional real-time PCR. To
`achieve ?ner degrees of quantitative discrimination, altema
`tive strategies are needed.
`[0010] Digital PCR has been developed for the detection of
`allelic ratio skeWing in nucleic acid samples (Chang, H W et
`al. 2002 J Natl Cancer Inst 94, 1697-1703). Digital PCR is an
`ampli?cation based nucleic acid analysis technique Which
`requires the distribution of a specimen containing nucleic
`acids into a multitude of discrete samples Where each sample
`containing on average not more than about one target
`sequence per sample. Speci?c nucleic acid targets are ampli
`?ed With sequence-speci?c primers to generate speci?c
`
`Ariosa Exhibit 1045
`pg. 11
`
`

`
`US 2009/0029377 A1
`
`Jan. 29, 2009
`
`amplicons by digital PCR. The nucleic acid loci to be targeted
`and the species of or panel of sequence-speci?c primers to be
`included in the reactions are determined or selected prior to
`nucleic acid analysis.
`[0011] Clinically, it has been shoWn to be useful for the
`detection of loss of heteroZygosity (LOH) in tumor DNA
`samples (Zhou, W. et al. 2002 Lancet 359, 219-225). For the
`analysis of digital PCR results, sequential probability ratio
`testing (SPRT) has been adopted by previous studies to clas
`sify the experimental results as being suggestive of the pres
`ence of LOH in a sample or not (El Karoui at al. 2006 Stat
`Med 25, 3124-3133).
`[0012] In methods used in the previous studies, the amount
`of data collected from the digital PCR is quite loW. Thus, the
`accuracy can be compromised due to the small number of data
`points and typical statistical ?uctuations.
`[0013] It is therefore desirable that noninvasive tests have
`high sensitivity and speci?city to minimiZe false negatives
`and false positives, respectively. HoWever, fetal DNA is
`present in loW absolute concentration and represent a minor
`portion of all DNA sequences in maternal plasma and serum.
`It is therefore also desirable to have methods that alloW the
`noninvasive detection of fetal chromosomal aneuploidy by
`maximizing the amount of genetic information that could be
`inferred from the limited amount of fetal nucleic acids Which
`exist as a minor population in a biological sample containing
`maternal background nucleic acids.
`
`BRIEF SUMMARY
`
`[0014] Embodiments of this invention provide methods,
`systems, and apparatus for determining Whether a nucleic
`acid sequence imbalance (e.g., chromosome imbalance)
`exists Within a biological sample obtained from a pregnant
`female. This determination may be done by using a parameter
`of an amount of a clinically-relevant chromosomal region in
`relation to other non-clinically-relevant chromosomal
`regions (background regions) Within a biological sample. In
`one aspect, an amount of chromosomes is determined from a
`sequencing of nucleic acid molecules in a maternal sample,
`such as urine, plasma, serum, and other suitable biological
`samples. Nucleic acid molecules of the biological sample are
`sequenced, such that a fraction of the genome is sequenced.
`One or more cutoff values are chosen for determining
`Whether a change compared to a reference quantity exists (i.e.
`an imbalance), for example, With regards to the ratio of
`amounts of tWo chromosomal regions (or sets of regions).
`[0015] According to one exemplary embodiment, a bio
`logical sample received from a pregnant female is analyZed to
`perform a prenatal diagnosis of a fetal chromosomal aneup
`loidy. The biological sample includes nucleic acid molecules.
`A portion of the nucleic acid molecules contained in the
`biological sample are sequenced. In one aspect, the amount of
`genetic information obtained is su?icient for accurate diag
`nosis yet not overly excessive so as to contain costs and the
`amount of input biological sample required.
`[0016] Based on the sequencing, a ?rst amount of a ?rst
`chromosome is determined from sequences identi?ed as
`originating from the ?rst chromosome. A second amount of
`one or more second chromosomes is determined from
`sequences identi?ed as originating from one of the second
`chromosomes. A parameter from the ?rst amount and the
`second amount is then compared to one or more cutoff values.
`Based on the comparison, a classi?cation of Whether a fetal
`chromosomal aneuploidy exists for the ?rst chromosome is
`
`determined. The sequencing advantageously maximiZes the
`amount of genetic information that could be inferred from the
`limited amount of fetal nucleic acids Which exist as a minor
`population in a biological sample containing maternal back
`ground nucleic acids.
`[0017] According to one exemplary embodiment, a bio
`logical sample received from a pregnant female is analyZed to
`perform a prenatal diagnosis of a fetal chromosomal aneup
`loidy. The biological sample includes nucleic acid molecules.
`A percentage of fetal DNA in the biological sample is iden
`ti?ed. A number N of sequences to be analyZed based on a
`desired accuracy is calculated based on the percentage. At
`least N of the nucleic acid molecules contained in the biologi
`cal sample are randomly sequenced.
`[0018] Based on the random sequencing, a ?rst amount of a
`?rst chromosome is determined from sequences identi?ed as
`originating from the ?rst chromosome. A second amount of
`one or more second chromosomes is determined from
`sequences identi?ed as originating from one of the second
`chromosomes. A parameter from the ?rst amount and the
`second amount is then compared to one or more cutoff values.
`Based on the comparison, a classi?cation of Whether a fetal
`chromosomal aneuploidy exists for the ?rst chromosome is
`determined. The random sequencing advantageously maxi
`miZes the amount of genetic information that could be
`inferred from the limited amount of fetal nucleic acids Which
`exist as a minor population in a biological sample containing
`maternal background nucleic acids.
`[0019] Other embodiments of the invention are directed to
`systems and computer readable media associated With meth
`ods described herein.
`[0020] A better understanding of the nature and advantages
`of the present invention may be gained With reference to the
`folloWing detailed description and the accompanying draW
`mgs.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0021] FIG. 1 is a ?owchart of a method 100 for performing
`prenatal diagnosis of a fetal chromosomal aneuploidy in a
`biological sample obtained from a pregnant female subject
`according to an embodiment of the present invention.
`[0022] FIG. 2 is a ?owchart of a method 200 for performing
`prenatal diagnosis of a fetal chromosomal aneuploidy using
`random sequencing according to an embodiment of the
`present invention.
`[0023] FIG. 3A shoWs a plot of percentage representation
`of chromosome 21 sequences in maternal plasma samples
`involving trisomy 21 or euploid fetuses according to an
`embodiment of the present invention.
`[0024] FIG. 3B shoWs a correlation betWeen maternal
`plasma fractional fetal DNA concentrations determined by
`massively parallel sequencing and micro?uidics digital PCR
`according to an embodiment of the present invention.
`[0025] FIG. 4A shoWs a plot of percentage representation
`of aligned sequences per chromosome according to an
`embodiment of the present invention.
`[0026] FIG. 4B shoWs a plot of difference (%) inpercentage
`representation per chromosome betWeen the trisomy 21 case
`and euploid case shoWn in FIG. 4A.
`[0027] FIG. 5 shoWs a correlation betWeen degree of over
`representation in chromosome 21 sequences and the frac
`tional fetal DNA concentrations in maternal plasma involving
`trisomy 21 fetuses according to an embodiment of the present
`invention.
`
`Ariosa Exhibit 1045
`pg. 12
`
`

`
`US 2009/0029377 A1
`
`Jan. 29, 2009
`
`[0028] FIG. 6 shows a table of a portion of human genome
`that Was analyzed according to an embodiment of the present
`invention. T21 denote a sample obtained from a pregnancy
`involving a trisomy 21 fetus.
`[0029] FIG. 7 shoWs a table of a number of sequences
`required to differentiate euploid from trisomy 21 fetuses
`according to an embodiment of the present invention.
`[0030] FIG. 8A shoWs a table of top ten starting positions of
`sequenced tags aligned to chromosome 21 according to an
`embodiment of the present invention.
`[0031] FIG. 8B shoWs a table of top ten starting positions of
`sequenced tags aligned to chromosome 22 according to an
`embodiment of the present invention.
`[0032] FIG. 9 shoWs a block diagram of an exemplary
`computer apparatus usable With system and methods accord
`ing to embodiments of the present invention.
`
`DEFINITIONS
`
`[0033] The term “biological sample” as used herein refers
`to any sample that is taken from a subject (e.g., a human, such
`as a pregnant Woman) and contains one or more nucleic acid
`molecule(s) of interest.
`[0034] The term “nucleic acid” or “polynucleotide” refers
`to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)
`and a polymer thereof in either single- or double-stranded
`form. Unless speci?cally limited, the term encompasses
`nucleic acids containing knoWn analogs of natural nucle
`otides that have similar binding properties as the reference
`nucleic acid and are metabolized in a manner similar to natu
`rally occurring nucleotides. Unless otherWise indicated, a
`particular nucleic acid sequence also implicitly encompasses
`conservatively modi?ed variants thereof (e. g., degenerate
`codon substitutions), alleles, orthologs, SNPs, and comple
`mentary sequences as Well as the sequence explicitly indi
`cated. Speci?cally, degenerate codon substitutions may be
`achieved by generating sequences in Which the third position
`of one or more selected (or all) codons is substituted With
`mixed-base and/or deoxyinosine residues (Batzer et al.,
`Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., .1. Biol.
`Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell.
`Probes 8:91-98 (1994)). The term nucleic acid is used inter
`changeably With gene, cDNA, mRNA, small noncoding
`RNA, micro RNA (miRNA), PiWi-interacting RNA, and
`short hairpin RNA (shRNA) encoded by a gene or locus.
`[0035] The term “gene” means the segment of DNA
`involved in producing a polypeptide chain. It may include
`regions preceding and folloWing the coding region (leader
`and trailer) as Well as intervening sequences (introns)
`betWeen individual coding segments (exons).
`[0036] The term “reaction” as used herein refers to any
`process involving a chemical, enzymatic, or physical action
`that is indicative of the presence or absence of a particular
`polynucleotide sequence of interest. An example of a “reac
`tion” is an ampli?cation reaction such as a polymerase chain
`reaction (PCR). Another example of a “reaction” is a
`sequencing reaction, either by synthesis or by ligation. An
`“informative reaction” is one that indicates the presence of
`one or more particular polynucleotide sequence of interest,
`and in one case Where only one sequence of interest is present.
`The term “Well” as used herein refers to a reaction at a pre
`determined location Within a con?ned structure, e. g., a Well
`shaped vial, cell, or chamber in a PCR array.
`[0037] The term “clinically relevant nucleic acid sequence”
`as used herein can refer to a polynucleotide sequence corre
`
`sponding to a segment of a larger genomic sequence Whose
`potential imbalance is being tested or to the larger genomic
`sequence itself. One example is the sequence of chromosome
`21. Other examples include chromosome 18, 13, X andY. Yet
`other examples include mutated genetic sequences or genetic
`polymorphisms or copy number variations that a fetus may
`inherit from one or both of its parents. Yet other examples
`include sequences Which are mutated, deleted, or ampli?ed in
`a malignant tumor, e.g. sequences in Which loss of heterozy
`gosity or gene duplication occur. In some embodiments, mul
`tiple clinically relevant nucleic acid sequences, or equiva
`lently multiple makers of the clinically relevant nucleic acid
`sequence, can be used to provide data for detecting the imbal
`ance. For instance, data from ?ve non-consecutive sequences
`on chromosome 21 can be used in an additive fashion for the
`determination of possible chromosomal 21 imbalance, effec
`tively reducing the need of sample volume to 1/s.
`[0038] The term “background nucleic acid sequence” as
`used herein refers to a nucleic acid sequence Whose normal
`ratio to the clinically relevant nucleic acid sequence is knoWn,
`for instance a 1-to-1 ratio. As one example, the background
`nucleic acid sequence and the clinically relevant nucleic acid
`sequence are tWo alleles from the same chromosome that are
`distinct due to heterozygo sity. In another example, the back
`ground nucleic acid sequence is one allele that is heterozy
`gous to another allele that is the clinically relevant nucleic
`acid sequence. Moreover, some of each of the background
`nucleic acid sequence and the clinically relevant nucleic acid
`sequence may come from different individuals.
`[0039] The term “reference nucleic acid sequence” as used
`herein refers to a nucleic acid sequence Whose average con
`centration per reaction is knoWn or equivalently has been
`measured.
`[0040] The term “overrepresented nucleic acid sequence”
`as used herein refers to the nucleic acid sequence among tWo
`sequences of interest (e.g., a clinically relevant sequence and
`a background sequence) that is in more abundance than the
`other sequence in a biological sample.
`[0041] The term “based on” as used herein means “based at
`least in part on” and refers to one value (or result) being used
`in the determination of another value, such as occurs in the
`relationship of an input of a method and the output of that
`method. The term “derive” as used herein also refers to the
`relationship of an input of a method and the output of that
`method, such as occurs When the derivation is the calculation
`of a formula.
`[0042] The term “quantitative data” as used herein means
`data that are obtained from one or more reactions and that
`provide one or more numerical values. For example, the num
`ber of Wells that shoW a ?uorescent marker for a particular
`sequence Would be quantitative data.
`[0043] The term “parameter” as used herein means a
`numerical value that characterizes a quantitative data set and/
`or a numerical relationship betWeen quantitative data sets. For
`example, a ratio (or function of a ratio) betWeen a ?rst amount
`of a ?rst nucleic acid sequence and a second amount of a
`second nucleic acid sequence is a parameter.
`[0044] The term “cutoff value” as used herein means a
`numerical value Whose value is used to arbitrate betWeen tWo
`or more states (e.g. diseased and non-diseased) of classi?ca
`tion for a biological sample. For example, if a parameter is
`greater than the cutoff value, a ?rst classi?cation of the quan
`titative data is made (e. g. diseased state); or if the parameter
`
`Ariosa Exhibit 1045
`pg. 13
`
`

`
`US 2009/0029377 A1
`
`Jan. 29, 2009
`
`is less than the cutoff value, a different classi?cation of the
`quantitative data is made (e.g. non-diseased state).
`[0045] The term “imbalance” as used herein means any
`signi?cant deviation as de?ned by at least one cutoff value in
`a quantity of the clinically relevant nucleic acid sequence
`from a reference quantity. For example, the reference quantity
`could be a ratio of 3/s, and thus an imbalance Would occur if
`the measured ratio is 1:1.
`[0046] The term “chromosomal aneuploidy” as used herein
`means a variation in the quantitative amount of a chromosome
`from that of a diploid genome. The variation may be a gain or
`a loss. It may involve the Whole of one chromosome or a
`region of a chromosome.
`[0047] The term “random sequencing” as used herein refers
`to sequencing Whereby the nucleic acid fragments sequenced
`have not been speci?cally identi?ed or targeted before the
`sequencing procedure. Sequence-speci?c primers to target
`speci?c gene loci are not required. The pools of nucleic acids
`sequenced vary from sample to sample and even from analy
`sis to analysis for the same sample. The identities of the
`sequenced nucleic acids are only revealed from the sequenc
`ing output generated. In some embodiments of the present
`invention, the random sequencing may be preceded by pro
`cedures to enrich a biological sample With particular popula
`tions of nucleic acid molecules sharing certain common fea
`tures. In one embodiment, each of the fragments in the
`biological sample have an equal probability of being
`sequenced.
`[0048] The term “fraction of the human genome” or “por
`tion of the human genome” as used herein refers to less than
`100% of the nucleotide sequences in the human genome
`Which comprises of some 3 billion basepairs of nucleotides.
`In the context of sequencing, it refers to less than 1-fold
`coverage of the nucleotide sequences in the human genome.
`The term may be expressed as a percentage or absolute num
`ber of nucleotides/basepairs. As an example of use, the term
`may be used to refer to the actual amount of sequencing
`performed. Embodiments may determine the required mini
`mal value for the sequenced fraction of the human genome to
`obtain an accurate diagnosis. As another example of use, the
`term may refer to the amount of sequenced data used for
`deriving a parameter or amount for disease classi?cation.
`[0049] The term “sequenced tag” as used herein refers to
`string of nucleotides sequenced from any part or all of a
`nucleic acid molecule. For example, a sequenced tag may be
`a short string of nucleotides sequenced from a nucleic acid
`fragment, a short string of nucleotides at both ends of a
`nucleic acid fragment, or the sequencing of the entire nucleic
`acid fragment that exists in the biological sample. A nucleic
`acid fragment is any part of a larger nucleic acid molecule. A
`fragment (eg a gene) may exist separately (i.e. not con
`nected) to the other parts of the larger nucleic acid molecule.
`
`DETAILED DESCRIPTION
`
`[0050] Embodiments of this invention provide methods,
`systems, and apparatus for determining Whether an increase
`or decrease (diseased state) of a clinically-relevant chromo
`somal region exists compared to a non-diseased state. This
`determination may be done by using a parameter of an
`amount of a clinically-relevant chromosomal region in rela
`tion to other non-clinically-relevant chromosomal regions
`(background regions) Within a biological sample. Nucleic
`acid molecules of the biological sample are sequenced, such
`that a fraction of the genome is sequenced, and the amount
`
`may be determined from results of the sequencing. One or
`more cutoff values are chosen for determining Whether a
`change compared to a reference quantity exists (i.e. an imbal
`ance), for example, With regards to the ratio of amounts of tWo
`chromosomal regions (or sets of regions).
`[0051] The change detected in the reference quantity may
`be any deviation (upWards or doWnWards) in the relation of
`the clinically-relevant nucleic acid sequence to the other non
`clinically-relevant sequences. Thus, the reference state may
`be any ratio or other quantity (e. g. other than a 1-1 correspon
`dence), and a measured state signifying a change may be any
`ratio or other quantity that differs from the reference quantity
`as determined by the one or more cutoff values.
`[0052] The clinically relevant chromosomal region (also
`called a clinically relevant nucleic acid sequence) and the
`background nucleic acid sequence may come from a ?rst type
`of cells and