U.S. patent application number 14/087525 was filed with the patent office on 2014-09-11 for diagnosing fetal chromosomal aneuploidy using massively parallel genomic sequencing.
This patent application is currently assigned to THE CHINESE UNIVERSITY OF HONG KONG. The applicant listed for this patent is Kwan Chee Chan, Rossa Wai Kwun Chiu, Yuk-Ming Dennis Lo. Invention is credited to Kwan Chee Chan, Rossa Wai Kwun Chiu, Yuk-Ming Dennis Lo.
Application Number | 20140256560 14/087525 |
Document ID | / |
Family ID | 39798126 |
Filed Date | 2014-09-11 |
United States Patent
Application |
20140256560 |
Kind Code |
A1 |
Lo; Yuk-Ming Dennis ; et
al. |
September 11, 2014 |
DIAGNOSING FETAL CHROMOSOMAL ANEUPLOIDY USING MASSIVELY PARALLEL
GENOMIC SEQUENCING
Abstract
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 chromosome and of background chromosomes are
determined from results of the sequencing. A parameter derived from
these amounts (e.g. a ratio) is compared to one or more cutoff
values, thereby determining a classification of whether a fetal
chromosomal aneuploidy exists.
Inventors: |
Lo; Yuk-Ming Dennis;
(Kowloon, HK) ; Chiu; Rossa Wai Kwun; (New
Territories, HK) ; Chan; Kwan Chee; (New Territories,
HK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Lo; Yuk-Ming Dennis
Chiu; Rossa Wai Kwun
Chan; Kwan Chee |
Kowloon
New Territories
New Territories |
|
HK
HK
HK |
|
|
Assignee: |
THE CHINESE UNIVERSITY OF HONG
KONG
New Territories
HK
|
Family ID: |
39798126 |
Appl. No.: |
14/087525 |
Filed: |
November 22, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12178181 |
Jul 23, 2008 |
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14087525 |
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60951438 |
Jul 23, 2007 |
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Current U.S.
Class: |
506/2 |
Current CPC
Class: |
G16B 30/00 20190201;
C12Q 2600/154 20130101; C12Q 1/6827 20130101; Y02A 90/10 20180101;
G16B 20/10 20190201; C12Q 2600/156 20130101; G01N 2800/387
20130101; C12Q 2600/112 20130101; G16B 20/00 20190201; C12Q 1/6883
20130101; C12Q 1/6888 20130101; C12Q 1/6827 20130101; C12Q 2521/331
20130101; C12Q 2537/16 20130101 |
Class at
Publication: |
506/2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1-23. (canceled)
24. A method for determining a presence or absence of a fetal
aneuploidy in a fetus for each of a plurality of maternal blood
samples obtained from a plurality of different pregnant women, said
maternal blood samples comprising fetal and maternal cell-free
genomic DNA, said method comprising: (a) obtaining a fetal and
maternal cell-free genomic DNA sample from each of the plurality of
maternal blood samples; (b) selectively enriching a plurality of
non-random polynucleotide sequences of each fetal and maternal
cell-free genomic DNA sample of (a) to generate a library derived
from each fetal and maternal cell-free genomic DNA sample of
enriched and indexed fetal and maternal non-random polynucleotide
sequences, wherein each library of enriched and indexed fetal and
maternal non-random polynucleotide sequences includes an indexing
nucleotide sequence which identifies a maternal blood sample of the
plurality of maternal blood samples, wherein said plurality of
non-random polynucleotide sequences comprises at least 100
different non-random polynucleotide sequences selected from a first
chromosome tested for being aneuploid and at least 100 different
non-random polynucleotide sequences selected from a reference
chromosome, wherein the first chromosome tested for being aneuploid
and the reference chromosome are different, and wherein each of
said plurality of non-random polynucleotide sequences is from 10 to
1000 nucleotide bases in length, (c) pooling the libraries
generated in (b) to produce a pool of enriched and indexed fetal
and maternal non-random polynucleotide sequences; (d) performing
massively parallel sequencing of the pool of enriched and indexed
fetal and maternal non-random polynucleotide sequences of (c) to
produce sequence reads corresponding to enriched and indexed fetal
and maternal non-random polynucleotide sequences of each of the at
least 100 different non-random polynucleotide sequences selected
from the first chromosome tested for being aneuploid and sequence
reads corresponding to enriched and indexed fetal and maternal
non-random polynucleotide sequences of each of the at least 100
different non-random polynucleotide sequences selected from the
reference chromosome; (e) based on the indexing nucleotide
sequence, for each of the plurality of maternal blood samples,
enumerating sequence reads corresponding to enriched and indexed
fetal and maternal non-random polynucleotide sequences selected
from the first chromosome tested for being aneuploid and sequence
reads corresponding to enriched and indexed fetal and maternal
non-random polynucleotide sequences selected from the reference
chromosome; and (f) for each of the plurality of maternal blood
samples, determining the presence or absence of a fetal aneuploidy
comprising using a number of enumerated sequence reads
corresponding to the first chromosome and a number of enumerated
sequence reads corresponding to the reference chromosome of
(e).
25. The method of claim 24, wherein for each of the plurality of
maternal blood samples determining the presence or absence of a
fetal aneuploidy comprises comparing the number of enumerated
sequence reads corresponding to the first chromosome tested for
being aneuploid with the number of enumerated sequence reads
corresponding to the reference chromosome.
26. The method of claim 24, wherein said plurality of non-random
polynucleotide sequences comprises at least 300 different
non-random polynucleotide sequences selected from the first
chromosome tested for being aneuploid and at least 300 different
non-random polynucleotide sequences selected from the reference
chromosome.
27. The method of claim 26, wherein said plurality of non-random
polynucleotide sequences comprises at least 500 different
non-random polynucleotide sequences selected from the first
chromosome tested for being aneuploid and at least 500 different
non-random polynucleotide sequences selected from the reference
chromosome.
28. The method of claim 24, wherein each of said plurality of
non-random polynucleotide sequences is from 10 to 500 nucleotide
bases in length.
29. The method of claim 24, wherein each of said plurality of
non-random polynucleotide sequences is from 50 to 150 nucleotide
bases in length.
30. The method of claim 24, wherein said first chromosome tested
for being aneuploid is selected from the group consisting of
chromosome 13, chromosome 18, chromosome 21, chromosome X, and
chromosome Y.
31. The method of claim 30, wherein said fetal aneuploidy comprises
fetal aneuploidy of a chromosome selected from the group consisting
of chromosome 13, chromosome 18, chromosome 21, chromosome X, and
chromosome Y.
32. The method of claim 31, wherein said fetal aneuploidy is
selected from the group consisting of trisomy 21, trisomy 18,
trisomy 13, and monosomy X.
33. The method of claim 24, wherein said reference chromosome is
selected from the group consisting of chromosome 1, chromosome 2,
chromosome 3, chromosome 13, chromosome 18, and chromosome 21.
34. The method of claim 24, wherein said fetal aneuploidy comprises
monosomy, trisomy, tetrasomy, or pentasomy of the first
chromosome.
35. The method of claim 24, wherein said selectively enriching of
(b) comprises performing polymerase chain reaction (PCR)
amplification.
36. The method of claim 35, wherein for each fetal and maternal
cell-free genomic DNA sample PCR amplification comprises
hybridizing at least two oligonucleotides to each of the at least
100 different non-random polynucleotide sequences selected from the
first chromosome tested for being aneuploid and each of the at
least 100 different non-random polynucleotide sequences selected
from the reference chromosome.
37. The method of claim 36, wherein said oligonucleotides do not
hybridize to non-random polynucleotide sequences comprising one or
more polymorphisms.
38. The method of claim 36, wherein each of said oligonucleotides
has a substantially similar melting temperature.
39. The method of claim 24, wherein said massively parallel
sequencing generates at least 30 nucleotide bases per sequence
read.
40. The method of claim 24, wherein said fetal aneuploidy comprises
partial monosomy or partial trisomy.
41. The method of claim 24, wherein said plurality of non-random
polynucleotide sequences comprises no more than 1000 different
non-random polynucleotide sequences selected from the first
chromosome tested for being aneuploid and no more than 1000
different non-random polynucleotide sequences selected from the
reference chromosome.
42. A method for determining a presence or absence of a fetal
aneuploidy in a fetus for each of a plurality of maternal blood
samples obtained from a plurality of different pregnant women, said
maternal blood samples comprising fetal and maternal cell-free
genomic DNA, said method comprising: (a) obtaining a fetal and
maternal cell-free genomic DNA sample from each of the plurality of
maternal blood samples; (b) selectively enriching a plurality of
non-random polynucleotide sequences of each fetal and maternal
cell-free genomic DNA sample of (a) to generate a library derived
from each fetal and maternal cell-free genomic DNA sample of
enriched and indexed fetal and maternal non-random polynucleotide
sequences, wherein each library of enriched and indexed fetal and
maternal non-random polynucleotide sequences includes an indexing
nucleotide sequence which identifies a maternal blood sample of the
plurality of maternal blood samples, wherein said plurality of
non-random polynucleotide sequences comprises at least 100
different non-random polynucleotide sequences selected from at
least one chromosome region tested for being aneuploid and at least
100 different non-random polynucleotide sequences selected from at
least one chromosome control region, wherein the at least one
chromosome region tested for being aneuploid and the at least one
chromosome control region are different, and wherein each of said
plurality of non-random polynucleotide sequences is from 10 to 1000
nucleotide bases in length; (c) pooling the libraries generated in
(b) to produce a pool of enriched and indexed fetal and maternal
non-random polynucleotide sequences; (d) performing massively
parallel sequencing of the pool of enriched and indexed fetal and
maternal non-random polynucleotide sequences of (c) to produce
sequence reads corresponding to enriched and indexed fetal and
maternal non-random polynucleotide sequences of each of the at
least 100 different non-random polynucleotide sequences selected
from the at least one chromosome region tested for being aneuploid
and sequence reads corresponding to enriched and indexed fetal and
maternal non-random polynucleotide sequences of each of the at
least 100 different non-random polynucleotide sequences selected
from the at least one chromosome control region; (e) based on the
indexing nucleotide sequence, for each of the plurality of maternal
blood samples, enumerating sequence reads corresponding to enriched
and indexed fetal and maternal non-random polynucleotide sequences
selected from the at least one chromosome region tested for being
aneuploid and sequence reads corresponding to enriched and indexed
fetal and maternal non-random polynucleotide sequences selected
from the at least one chromosome control region; and (f) for each
of the plurality of maternal blood samples, determining the
presence or absence of a fetal aneuploidy comprising using a number
of enumerated sequence reads corresponding to the at least one
chromosome region tested for being aneuploid and a number of
enumerated sequence reads corresponding to the at least one
chromosome control region of (e).
43. The method of claim 42, wherein for each of the plurality of
maternal blood samples determining the presence or absence of a
fetal aneuploidy comprises comparing the number of enumerated
sequence reads corresponding to the at least one chromosome region
tested for being aneuploid with the number of enumerated sequence
reads corresponding to the at least one chromosome control
region.
44. The method of claim 42, wherein said plurality of non-random
polynucleotide sequences comprises at least 300 different
non-random polynucleotide sequences selected from the at least one
chromosome region tested for being aneuploid and at least 300
different non-random polynucleotide sequences selected from the at
least one chromosome control region.
45. The method of claim 42, wherein each of said plurality of
non-random polynucleotide sequences is from 10 to 500 nucleotide
bases in length.
46. The method of claim 42, wherein the at least one chromosome
region tested for being aneuploid is selected from at least one
chromosome selected from the group consisting of chromosome 13,
chromosome 18, chromosome 21, chromosome X, and chromosome Y.
47. The method of claim 46, wherein said fetal aneuploidy comprises
fetal aneuploidy of a chromosome selected from the group consisting
of chromosome 13, chromosome 18, chromosome 21, chromosome X, and
chromosome Y.
48. The method of claim 47, wherein said fetal aneuploidy is
selected from the group consisting of trisomy 21, trisomy 18,
trisomy 13, and monosomy X.
49. The method of claim 42, wherein the at least one chromosome
control region is selected from at least one chromosome selected
from the group consisting of chromosome 1, chromosome 2, chromosome
3, chromosome 13, chromosome 18, and chromosome 21.
50. The method of claim 42, wherein said fetal aneuploidy comprises
monosomy, trisomy, tetrasomy, or pentasomy of at least one
chromosome.
51. The method of claim 42, wherein said selectively enriching of
(b) comprises performing polymerase chain reaction (PCR)
amplification.
52. The method of claim 51, wherein for each fetal and maternal
cell-free genomic DNA sample PCR amplification comprises
hybridizing at least two oligonucleotides to each of the at least
100 different non-random polynucleotide sequences selected from the
at least one chromosome region tested for being aneuploid and each
of the 100 different non-random polynucleotide sequences selected
from the at least one chromosome control region.
53. The method of claim 42, wherein said plurality of non-random
polynucleotide sequences comprises no more than 1000 different
non-random polynucleotide sequences selected from the at least one
chromosome region tested for being aneuploid and no more than 1000
different non-random polynucleotide sequences selected from the at
least one chromosome control region.
Description
CLAIM OF PRIORITY
[0001] The present application claims priority from and is a
non-provisional application of U.S. Provisional Application No.
60/951,438, entitled "DETERMINING A NUCLEIC ACID SEQUENCE
IMBALANCE" filed Jul. 23, 2007 (Attorney 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 concurrently
filed non-provisional application entitled "DETERMINING A NUCLEIC
ACID SEQUENCE IMBALANCE," (Attorney Docket No. 016285-005210US) the
entire contents of which are herein incorporated by reference for
all purposes.
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
identification 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 chromosomal region. The
abnormal dose(s) can be abnormally high, e.g. the presence of an
extra chromosome 21 or chromosomal region in trisomy 21; or
abnormally low, e.g. 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 sampling of
fetal materials by invasive procedures such as amniocentesis or
chorionic villus sampling, which pose a finite 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 definitive invasive diagnostic procedures. However, these
screening methods typically measure epiphenomena that are
associated with the chromosomal aneuploidy, e.g., trisomy 21,
instead of the core chromosomal abnormality, and thus have
suboptimal diagnostic accuracy and other disadvantages, such as
being highly influenced by gestational age.
[0006] The discovery of circulating cell-free fetal DNA in maternal
plasma in 1997 offered new possibilities for noninvasive 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 N Engl J Med 346,
1502) and certain single gene disorders (Lo, Y M D et al. 1998 N
Engl J Med 339, 1734-1738), its application to the prenatal
detection of fetal chromosomal aneuploidies has represented a
considerable challenge (Lo, Y M D and Chiu, R W K 2007, supra).
First, fetal nucleic acids co-exist in maternal plasma with a high
background 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 J Hum Genet 62, 768-775). Second, fetal nucleic acids
circulate in maternal plasma predominantly in a cell-free form,
making it difficult to derive dosage information of genes or
chromosomes within the fetal genome.
[0007] Significant developments overcoming these challenges have
recently been made (Benachi, A & Costa, J M 2007 Lancet 369,
440-442). One approach detects fetal-specific nucleic acids in the
maternal plasma, thus overcoming the problem of maternal background
interference (Lo, Y M D and Chin, 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-specific alleles to non-fetal-specific alleles for single
nucleotide polymorphisms (SNPs) on chromosome 21. SNP ratios were
similarly computed for a reference chromosome. An imbalance of
fetal chromosome 21 was then inferred by detecting a statistically
significant difference between the SNP ratios for chromosome 21 and
those of the reference chromosome, where significant is defined
using a fixed p-value of .ltoreq.0.05. To ensure high population
coverage, more than 500 SNPs were targeted per chromosome. 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 chromosome, the power of the statistical test for SNP ratio
comparison would be variable from case to case (Lo, Y M D &
Chiu, R W K. 2007 Lancer 369, 1997). Furthermore, since these
approaches depend on the detection of genetic polymorphisms, they
are limited to fetuses heterozygous for these polymorphisms.
[0009] Using polymerase chain reaction (PCR) and DNA quantification
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 difference
has been the limit of conventional real-time PCR. To achieve finer
degrees of quantitative discrimination, alternative 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 amplification
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.
Specific nucleic acid targets are amplified with sequence-specific
primers to generate specific amplicons by digital PCR. The nucleic
acid loci to be targeted and the species of or panel of
sequence-specific 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 classify the experimental results as
being suggestive of the presence 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 fluctuations.
[0013] It is therefore desirable that noninvasive tests have high
sensitivity and specificity 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 biological sample
received from a pregnant female is analyzed to perform a prenatal
diagnosis of a fetal chromosomal aneuploidy. 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 sufficient
for accurate diagnosis yet not overly excessive so as to contain
costs and the amount of input biological sample required.
[0016] Based on the sequencing, a first amount of a first
chromosome is determined from sequences identified as originating
from the first chromosome. A second amount of one or more second
chromosomes is determined from sequences identified as originating
from one of the second chromosomes. A parameter from the first
amount and the second amount is then compared to one or more cutoff
values. Based on the comparison, a classification of whether a
fetal chromosomal aneuploidy exists for the first 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 background nucleic acids.
[0017] According to one exemplary embodiment, a biological sample
received from a pregnant female is analyzed to perform a prenatal
diagnosis of a fetal chromosomal aneuploidy. The biological sample
includes nucleic acid molecules. A percentage of fetal DNA in the
biological sample is identified. 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 biological sample are randomly sequenced.
[0018] Based on the random sequencing, a first amount of a first
chromosome is determined from sequences identified as originating
from the first chromosome. A second amount of one or more second
chromosomes is determined from sequences identified as originating
from one of the second chromosomes. A parameter from the first
amount and the second amount is then compared to one or more cutoff
values. Based on the comparison, a classification of whether a
fetal chromosomal aneuploidy exists for the first chromosome is
determined. The random 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 background
nucleic acids.
[0019] Other embodiments of the invention are directed to systems
and computer readable media associated with methods 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 drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a flowchart 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 flowchart 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 cuploid 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 microfluidics 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 (%) in percentage
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 fractional
fetal DNA concentrations in maternal plasma involving trisomy 21
fetuses according to an embodiment of the present invention.
[0028] FIG. 6 shows a table of a portion of human genomic 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 according 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
specifically limited, the term encompasses nucleic acids containing
known analogs of natural nucleotides that have similar binding
properties as the reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides. Unless otherwise
indicated, a particular nucleic acid sequence also implicitly
encompasses conservatively modified variants thereof (e.g.,
degenerate codon substitutions), alleles, orthologs, SNPs, and
complementary sequences as well as the sequence explicitly
indicated. Specifically, 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., J. Biol. Chem. 260:2605-2608
(1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
The term nucleic acid is used interchangeably 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 "reaction" is
an amplification 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 predetermined location within a confined
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 corresponding 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 and Y. 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 amplified
in a malignant tumor, e.g. sequences in which loss of
heterozygosity or gene duplication occur. In some embodiments,
multiple clinically relevant nucleic acid sequences, or
equivalently multiple makers of the clinically relevant nucleic
acid sequence, can be used to provide data for detecting the
imbalance. For instance, data from five non-consecutive sequences
on chromosome 21 can be used in an additive fashion for the
determination of possible chromosomal 21 imbalance, effectively
reducing the need of sample volume to 1/5.
[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 heterozygosity.
In another example, the background nucleic acid sequence is one
allele that is heterozygous 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 concentration 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 number of wells that show a
fluorescent 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 first amount of a first 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 classification for a biological
sample. For example, if a parameter is greater than the cutoff
value, a first classification of the quantitative data is made
(e.g. diseased state); or if the parameter is less than the cutoff
value, a different classification of the quantitative data is made
(e.g. non-diseased state).
[0045] The term "imbalance" as used herein means any significant
deviation as defined 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
3/5, 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 specifically identified or targeted before the sequencing
procedure. Sequence-specific primers to target specific gene loci
are not required. The pools of nucleic acids sequenced vary from
sample to sample and even from analysis to analysis for the same
sample. The identities of the sequenced nucleic acids are only
revealed from the sequencing output generated. In some embodiments
of the present invention, the random sequencing may be preceded by
procedures to enrich a biological sample with particular
populations of nucleic acid molecules sharing certain common
features. 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 "portion 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 number 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 minimal 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 classification.
[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 (e.g. a gene) may exist
separately (i.e. not connected) 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 chromosomal 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 relation 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 imbalance), 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 correspondence),
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 first type of cells and from
one or more second types of cells. For example, fetal nucleic acid
sequences originating from fetal/placental cells are present in a
biological sample, such as maternal plasma, which contains a
background of maternal nucleic acid sequences originating from
maternal cells. In one embodiment, the cutoff value is determined
based at least in part on a percentage of the first type of cells
in a biological sample. Note the percentage of fetal sequences in a
sample may be determined by any fetal-derived loci and not limited
to measuring the clinically-relevant nucleic acid sequences. In
another embodiment, the cutoff value is determined at least in part
on the percentage of tumor sequences in a biological sample, such
as plasma, serum, saliva or urine, which contains a background of
nucleic acid sequences derived from the non-malignant cells within
the body.
I. General Method
[0053] FIG. 1 is a flowchart 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.
[0054] In step 110, a biological sample from the pregnant female is
received. The biological sample may be plasma, urine, serum, or any
other suitable sample. The sample contains nucleic acid molecules
from the fetus and the pregnant female. For example, the nucleic
acid molecules may be fragments from chromosomes.
[0055] In step 120, at least a portion of a plurality of the
nucleic acid molecules contained in the biological sample are
sequenced. The portion sequenced represents a fraction of the human
genome. In one embodiment, the nucleic acid molecules are fragments
of respective chromosomes. One end (e.g. 35 basepairs (bp)), both
ends, or the entire fragment may be sequenced. All of the nucleic
acid molecules in the sample may be sequenced, or just a subset may
be sequenced. This subset may be randomly chosen, as will be
described in more detail later.
[0056] In one embodiment, the sequencing is done using massively
parallel sequencing. Massively parallel sequencing, such as that
achievable on the 454 platform (Roche) (Margulies, M. et al. 2005
Nature 437, 376-380), Illumina Genome Analyzer (or Solexa platform)
or SOLiD System (Applied Biosystems) or the Helicos True Single
Molecule DNA sequencing technology (Harris T D et al. 2008 Science,
320, 106-109), the single molecule, real-time (SMRT.TM.) technology
of Pacific Biosciences, and nanopore sequencing (Soni G V and
Meller A. 2007 Clin Chem 53: 1996-2001), allow the sequencing of
many nucleic acid molecules isolated from a specimen at high orders
of multiplexing in a parallel fashion (Dear Brief Funct Genomic
Proteomic 2003; 1: 397-416). Each of these platforms sequences
clonally expanded or even non-amplified single molecules of nucleic
acid fragments.
[0057] As a high number of sequencing reads, in the order of
hundred thousands to millions or even possibly hundreds of millions
or billions, are generated from each sample in each run, the
resultant sequenced reads form a representative profile of the mix
of nucleic acid species in the original specimen. For example, the
haplotype, trascriptome and methylation profiles of the sequenced
reads resemble those of the original specimen (Brenner et al Nat
Biotech 2000; 18: 630-634; Taylor et al Cancer Res 2007; 67:
8511-8518). Due to the large sampling of sequences from each
specimen, the number of identical sequences, such as that generated
from the sequencing of a nucleic acid pool at several folds of
coverage or high redundancy, is also a good quantitative
representation of the count of a particular nucleic acid species or
locus in the original sample.
[0058] In step 130, based on the sequencing (e.g. data from the
sequencing), a first amount of a first chromosome (e.g. the
clinically relevant chromosome) is determined. The first amount is
determined from sequences identified as originating from the first
chromosome. For example, a bioinformatics procedure may then be
used to locate each of these DNA sequences to the human genome. It
is possible that a proportion of such sequences will be discarded
from subsequent analysis because they are present in the repeat
regions of the human genome, or in regions subjected to
inter-individual variations, e.g. copy number variations. An amount
of the chromosome of interest and of one or more other chromosomes
may thus be determined.
[0059] In step 140, based on the sequencing, a second amount of one
or more second chromosomes is determined from sequences identified
as originating from one of the second chromosomes. In one
embodiment, the second chromosomes are all of the other chromosomes
besides the first one (i.e. the one being tested). In another
embodiment, the second chromosome is just a single other
chromosome.
[0060] There are a number of ways of determining the amounts of the
chromosomes, including but not limited to counting the number of
sequenced tags, the number of sequenced nucleotides (basepairs) or
the accumulated lengths of sequenced nucleotides (basepairs)
originating from particular chromosome(s) or chromosomal
regions.
[0061] In another embodiment, rules may be imposed on the results
of the sequencing to determine what gets counted. In one aspect, an
amount may be obtained based on a proportion of the sequenced
output. For example, sequencing output corresponding to nucleic
acid fragments of a specified size range could be selected after
the bioinformatics analysis. Examples of the size ranges are about
<300 bp, <200 bp or <100 bp.
[0062] In step 150, a parameter is determined from the first amount
and the second amount. The parameter may be, for example, a simple
ratio of the first amount to the second amount, or the first amount
to the second amount plus the first amount. In one aspect, each
amount could be an argument to a function or separate functions,
where a ratio may be then taken of these separate functions. One
skilled in the art will appreciate the number of different suitable
parameters.
[0063] In one embodiment, a parameter (e.g. a fractional
representation) of a chromosome potentially involved in a
chromosomal aneuploidy, e.g. chromosome 21 or chromosome 18 or
chromosome 13, may then be calculated from the results of the
bioinformatics procedure. The fractional representation may be
obtained based on an amount of all of the sequences (e.g. some
measure of all of the chromosomes including the clinically-relevant
chromosome) or a particular subset of chromosomes (e.g. just one
other chromosome than the one being tested.)
[0064] In step 150, the parameter is compared to one or more cutoff
values. The cutoff values may be determined from any number of
suitable ways. Such ways include Bayesian-type likelihood method,
sequential probability ratio testing (SPRT), false discovery,
confidence interval, receiver operating characteristic (ROC).
Examples of applications of these methods and sample-specific
methods are described in concurrently filed application
"DETERMINING A NUCLEIC ACID SEQUENCE IMBALANCE," (Attorney Docket
No. 016285-005210US), which is incorporated by reference.
[0065] In one embodiment, the parameter (e.g. the fractional
representation of the clinically relevant chromosome) is then
compared to a reference range established in pregnancies involving
normal (i.e. cuploid) fetuses. It is possible that in some variants
of the procedure, the reference range (i.e. the cutoff values)
would be adjusted in accordance with the fractional concentration
of fetal DNA (f) in a particular maternal plasma sample. The value
of f can be determined from the sequencing dataset, e.g. using
sequences mappable to the Y chromosome if the fetus is male. The
value of f may also be determined in a separate analysis, e.g.
using fetal epigenetic markers (Chan K C A et al 2006 Clin Chem 52,
2211-8) or from the analysis of single nucleotide
polymorphisms.
[0066] In step 160, based on the comparison, a classification of
whether a fetal chromosomal aneuploidy exists for the first
chromosome is determined. In one embodiment, the classification is
a definitive yes or no. In another embodiment, a classification may
be unclassifiable or uncertain. In yet another embodiment, the
classification may be a score that is to be interpreted at a later
date, for example, by a doctor.
II. Sequencing Aligning, and Determining Amounts
[0067] As mentioned above, only a fraction of the genome is
sequenced. In one aspect, even when a pool of nucleic acids in a
specimen is sequenced at <100% genomic coverage instead of at
several folds of coverage, and among the proportion of captured
nucleic acid molecules, most of each nucleic acid species is only
sequenced once. Also, dosage imbalance of a particular chromosome
or chromosomal regions can be quantitatively determined. In other
words, the dosage imbalance of the chromosome or chromosomal
regions is inferred from the percentage representation of the said
locus among other mappable sequenced tags of the specimen.
[0068] This is contrasted from situations where the same pool of
nucleic acids is sequenced multiple times to achieve high
redundancy or several folds of coverage whereby each nucleic acid
species is sequenced multiple times. In such situations, the number
of times a particular nucleic acid species have been sequenced
relative to that of another nucleic acid species correlate with
their relative concentrations in the original sample. The
sequencing cost increases with the number of fold coverage required
to achieve accurate representation of the nucleic acid species.
[0069] In one example, a proportion of such sequences would be from
the chromosome involved in an aneuploidy such as chromosome 21 in
this illustrative example. Yet other sequences from such a
sequencing exercise would be derived from the other chromosomes. By
taking into account of the relative size of chromosome 21 compared
with the other chromosomes, one could obtain a normalized
frequency, within a reference range, of chromosome 21-specific
sequences from such a sequencing exercise. If the fetus has trisomy
21, then the normalized frequency of chromosome 21-derived
sequences from such a sequencing exercise will increase, thus
allowing the detection of trisomy 21. The degree of change in the
normalized frequency will be dependent on the fractional
concentration of fetal nucleic acids in the analyzed sample.
[0070] In one embodiment, we used the Illumina Genome Analyzer for
single-end sequencing of human genomic DNA and human plasma DNA
samples. The Illumina Genome Analyzer sequences clonally-expanded
single DNA molecules captured on a solid surface termed a flow
cell. Each flow cell has 8 lanes for the sequencing of 8 individual
specimens or pools of specimens. Each lane is capable of generating
.about.200 Mb of sequence which is only a fraction of the 3 billion
basepairs of sequences in the human genome. Each genomic DNA or
plasma DNA sample was sequenced using one lane of a flow cell. The
short sequence tags generated were aligned to the human reference
genome sequence and the chromosomal origin was noted. The total
number of individual sequenced tags aligned to each chromosome were
tabulated and compared with the relative size of each chromosome as
expected from the reference human genome or non-disease
representative specimens. Chromosome gains or losses were then
identified.
[0071] The described approach is only one exemplification of the
presently described gene/chromosome dosage strategy. Alternatively,
paired end sequencing could be performed. Instead of comparing the
length of the sequenced fragments from that expected in the
reference genome as described by Campbell et al (Nat Genet 2008;
40: 722-729), the number of aligned sequenced tags were counted and
sorted according to chromosomal location. Gains or losses of
chromosomal regions or whole chromosomes were determined by
comparing the tag counts with the expected chromosome size in the
reference genome or that of a non-disease representative specimen.
As paired end sequencing allows one to deduce the size of the
original nucleic acid fragment, one example is to focus on the
counting of the number of paired sequenced tags corresponding to
nucleic acid fragments of a specified size, such as <300 bp,
<200 bp or <100 bp.
[0072] In another embodiment, the fraction of the nucleic acid pool
that is sequenced in a run is further sub-selected prior to
sequencing. For example, hybridization based techniques such as
oligonucleotide array could be used to first sub-select for nucleic
acid sequences from certain chromosomes, e.g. a potentially
aneuploid chromosome and other chromosome(s) not involved in the
aneuploidy tested. Another example is that a certain sub-population
of nucleic acid sequences from the sample pool is sub-selected or
enriched prior to sequencing. For example, as discussed above, it
has been reported that fetal DNA molecules in maternal plasma are
comprised of shorter fragments than the maternal background DNA
molecules (Chan et al Clin Chem 2004; 50: 88-92). Thus, one may use
one or more methods known to those of skill in the art to
fractionate the nucleic acid sequences in the sample according to
molecule size, e.g. by gel electrophoresis or size exclusion
columns or by microfluidics-based approach. Yet, alternatively, in
the example of analyzing cell-free fetal DNA in maternal plasma,
the fetal nucleic acid portion could be enriched by a method that
suppresses the maternal background, such as by the addition of
formaldehyde (Dhallan et al JAMA 2004; 291: 1114-9). In one
embodiment, a portion or subset of the pre-selected pool of nucleic
acids is sequenced randomly.
[0073] Other single molecule sequencing strategies such as that by
the Roche 454 platform, the Applied Biosystems SOLiD platform, the
Helicos True Single Molecule DNA sequencing technology, the single
molecule, real-time (SMRT.TM.) technology of Pacific Biosciences,
and nanopore sequencing could similarly be used in this
application.
III. Determining Amounts of Chromosomes from Sequencing Output
[0074] After the massively parallel sequencing, bioinformatics
analysis was performed to locate the chromosomal origin of the
sequenced tags. After this procedure, tags identified as
originating from the potentially aneuploid chromosome, i.e.
chromosome 21 in this study, are compared quantitatively to all of
the sequenced tags or tags originating from one of more chromosomes
not involved in the aneuploidy. The relationship between the
sequencing output from chromosome 21 and other non-21 chromosomes
for a test specimen is compared with cut-off values derived with
methods described in the above section to determine if the specimen
was obtained from a pregnancy involving a euploid or trisomy 21
fetus.
[0075] A number of different amounts include but not limited to the
following could be derived from the sequenced tags. For example,
the number of sequenced tags, i.e. absolute count, aligned to a
particular chromosome could be compared to the absolute count of
sequenced tags aligned to other chromosomes. Alternatively, the
fractional count of the amount of sequenced tags from chromosome 21
with reference to all or some other sequenced tags could be
compared to that of other non-aneuploid chromosomes. In the present
experiment, because 36 bp were sequenced from each DNA fragment,
the number of nucleotides sequenced from a particular chromosome
could easily be derived from 36 bp multiplied by the sequenced tag
count.
[0076] Furthermore, as each maternal plasma specimen was only
sequenced using one flow cell which could only sequence a fraction
of the human genome, by statistics, most of the maternal plasma DNA
fragment species would only each have been sequenced to generate
one sequenced tag count. In other words, the nucleic acid fragments
present in the maternal plasma specimen were sequenced at less than
1-fold coverage. Thus, the total number of sequenced nucleotides
for any particular chromosome would mostly correspond to the
amount, proportion or length of the part of the said chromosome
that has been sequenced. Hence, the quantitative determination of
the representation of the potentially aneuploid chromosome could be
derived from a fraction of the number or equivalent length of
nucleotides sequenced from that chromosome with reference to a
similarly derived quantity for other chromosomes.
IV. Enrichment for Pools of Nucleic Acids for Sequencing
[0077] As mentioned above and established in the example section
below, only a portion of the human genome needs to be sequenced to
differentiate trisomy 21 from cuploid cases. Thus, it would be
possible and cost-effective to enrich the pool of nucleic acids to
be sequenced prior to random sequencing of a fraction of the
enriched pool. For example, fetal DNA molecules in maternal plasma
are comprised of shorter fragments than the maternal background DNA
molecules (Chan et al Clin Chem 2004; 50: 88-92). Thus, one may use
one or more methods known to those of skill in the art to
fractionate the nucleic acid sequences in the sample according to
molecule size, e.g. by gel electrophoresis or size exclusion
columns or by microfluidics-based approach.
[0078] Yet, alternatively, in the example of analyzing cell-free
fetal DNA in maternal plasma, the fetal nucleic acid portion could
be enriched by a method that suppresses the maternal background,
such as by the addition of formaldehyde (Dhallan et al JAMA 2004;
291: 1114-9). The proportion of fetal derived sequences would be
enriched in the nucleic acid pool comprised of shorter fragments.
According to FIG. 7, the number of sequenced tags required for
differentiating euploid from trisomy 21 cases would reduce as the
fractional fetal DNA concentration increases.
[0079] Alternatively, sequences originating from a potentially
aneuploid chromosome and one or more chromosomes not involved in
the aneuploidy could be enriched by hybridization techniques for
example onto oligonucleotide microarrays. The enriched pools of
nucleic acids would then be subjected to random sequencing. This
would allow the reduction in sequencing costs.
V. Random Sequencing
[0080] FIG. 2 is a flowchart of a method 200 for performing
prenatal diagnosis of a fetal chromosomal aneuploidy using random
sequencing according to an embodiment of the present invention. In
one aspect for the massively parallel sequencing approach,
representative data from all of the chromosomes may be generated at
the same time. The origin of a particular fragment is not selected
ahead of time. The sequencing is done at random and then a database
search may be performed to see where a particular fragment is
coming from. This is contrasted from situations when a specific
fragment from chromosome 21 and another one from chromosome 1 are
amplified.
[0081] In step 210, a biological sample from the pregnant female is
received. In step 220, the number N of sequences to be analyzed is
calculated for a desired accuracy. In one embodiment, a percentage
of fetal DNA in the biological sample is first identified. This may
be done by any suitable means as will be known to one skilled in
the art. The identification may simply be reading a value that was
measured by another entity. In this embodiment, the calculation of
the number N of sequences to be analyzed is based on the
percentage. For example, the number of sequences needed to be
analyzed would be increased when the fetal DNA percentage drops,
and could be decreased when the fetal DNA rises. The number N may
be a fixed number or a relative number, such as a percentage. In
another embodiment, one could sequence a number N that is known to
be adequate for accurate disease diagnosis. The number N could be
made sufficient even in pregnancies with fetal DNA concentrations
that are at the lower end of the normal range.
[0082] In step 230, at least N of a plurality of the nucleic acid
molecules contained in the biological sample are randomly
sequenced. A feature of this described approach is that the nucleic
acids to be sequenced are not specifically identified or targeted
before sample analysis, i.e. sequencing. Sequence-specific primers
to target specific gene loci are not needed for sequencing. The
pools of nucleic acids sequenced vary from sample to sample and
even from analysis to analysis for the same sample. Furthermore,
from the below descriptions (FIG. 6), the amount of sequencing
output required for case diagnosis could vary between the tested
specimens and the reference population. These aspects are in marked
contrast to most molecular diagnostic approaches, such as those
based on fluorescence in situ hybridization, quantitative
florescence PCR, quantitative real-time PCR, digital PCR,
comparative genomic hybridization, microarray comparative genomic
hybridization and so on, where gene loci to be targeted require
prior pre-determination, thus requiring the use of locus-specific
primers or probe sets or panels of such.
[0083] In one embodiment, random sequencing is performed on DNA
fragments that are present in the plasma of a pregnant woman, and
one obtains genomic sequences which would originally have come from
either the fetus or the mother. Random sequencing involves sampling
(sequencing) a random portion of the nucleic acid molecules present
in the biological sample. As the sequencing is random, a different
subset (fraction) of the nucleic acid molecules (and thus the
genome) may be sequenced in each analysis. Embodiments will work
even when this subset varies from sample to sample and from
analysis to analysis, which may occur even using the same sample.
Examples of the fraction are about 0.1%, 0.5%, 1%, 5%, 10%, 20%, or
30% of the genome. In other embodiments, the fraction is at least
any one of these values.
[0084] The rest of the steps 240-270 may proceed in a similar
manner as method 100.
VI. Post-Sequencing Selection of Pools of Sequenced Tags
[0085] As described in examples II and III below, a subset of the
sequenced data is sufficient to distinguish trisomy 21 from euploid
cases. The subset of sequenced data could be the proportion of
sequenced tags that passed certain quality parameters. For example,
in example II, sequenced tags that were uniquely aligned to the
repeat-masked reference human genome were used. Alternatively, one
may sequence a representative pool of nucleic acid fragments from
all of the chromosomes but focus on the comparison between data
relevant to the potentially aneuploid chromosome and data relevant
to a number of non-aneuploid chromosomes.
[0086] Yet alternatively, a subset of the sequencing output
encompassing sequenced tags generated from nucleic acid fragments
corresponding to a specified size window in the original specimen
could be sub-selected during the post-sequencing analysis. For
example, using the Illumina Genome analyzer, one could use
paired-end sequencing which refers to sequencing the two ends of
nucleic acid fragments. The sequenced data from each paired-end are
then aligned to the reference human genome sequence. The distance
or number of nucleotides spanning between the two ends could then
be deduced. The whole length of the original nucleic acid fragment
could also be deduced. Alternatively, sequencing platforms such as
the 454 platform and possibly some single molecule sequencing
techniques are able to sequence the full length of short nucleic
acid fragments, for example 200 bp. In this manner, the actual
length of the nucleic acid fragment would be immediately known from
the sequenced data.
[0087] Such paired-end analysis is also possible using other
sequencing platforms, e.g. the Applied Biosystems SOLiD system. For
the Roche 454 platform, because of its increased read length
compared with other massively parallel sequencing systems, it is
also possible to determine the length of a fragment from its
complete sequence.
[0088] The advantage of focusing the data analysis on the subset of
sequenced tags corresponding to short nucleic acid fragments in the
original maternal plasma specimen because the dataset would
effectively be enriched with DNA sequences derived from the fetus.
This is because the fetal DNA molecules in maternal plasma are
comprised of shorter fragments than the maternal background DNA
molecules (Chan et al Clin Chem 2004; 50: 88-92). According to FIG.
7, the number of sequenced tags required for differentiating
euploid from trisomy 21 cases would reduce as the fractional fetal
DNA concentration increases.
[0089] The post-sequencing selection of subsets of nucleic acid
pools is different from other nucleic acid enrichment strategies
which are performed prior to specimen analysis, such as the use gel
electrophoresis or size exclusion columns for the selection of
nucleic acids of particular sizes, which require the physical
separation of the enriched pool from the background pool of nucleic
acids. The physical procedures would introduce more experimental
steps and may be prone to problems such as contamination. The
post-sequencing in silico selection of subsets of sequencing output
would also allow one to vary the selection depending on the
sensitivity and specificity required for disease determination.
[0090] The bioinformatics, computational and statistical approaches
used to determine if a maternal plasma specimen is obtained from a
pregnant woman conceived with a trisomy 21 or cuploid fetus could
be compiled into a computer program product used to determine
parameters from the sequencing output. The operation of the
computer program would involve the determining of a quantitative
amount from the potentially aneuploid chromosome as well as
amount(s) from one or more of the other chromosomes. A parameter
would be determined and compared with appropriate cut-off values to
determine if a fetal chromosomal aneuploidy exists for the
potentially aneuploid chromosome.
Examples
[0091] The following examples are offered to illustrate, but not to
limit the claimed invention,
I. Prenatal Diagnosis of Fetal Trisomy 21
[0092] Eight pregnant women were recruited for the study. All of
the pregnant women were in the 1.sup.st or 2.sup.nd trimester of
gestation and had a singleton pregnancy. Four of them were each
carrying a fetus with trisomy 21 and the other four were each
carrying a euploid fetus. Twenty milliliters of peripheral venous
blood was collected from each subject. Maternal plasma was
harvested after centrifugation at 1600.times.g for 0 minutes and
further centrifuged at 16000.times.g for 10 minutes. DNA was then
extracted from 5-10 mL of each plasma sample. The maternal plasma
DNA was then used for massively parallel sequencing by the Illumina
Genome Analyzer according to manufacturer's instructions. The
technicians performing the sequencing were blinded from the fetal
diagnoses during the sequencing and sequence data analysis.
[0093] Briefly, approximately 50 ng of maternal plasma DNA was used
for DNA library preparation. It is possible to start with lesser
amounts such as 15 ng or 10 ng of maternal plasma DNA. Maternal
plasma DNA fragments were blunt-ended, ligated to Solexa adaptors
and fragments of 150-300 bp were selected by gel purification.
Alternatively, blunt-ended and adaptor-ligated maternal plasma DNA
fragments could be passed through columns (e.g. AMPure, Agencourt)
to remove unligated adaptors without size-selection before cluster
generation. The adaptor-ligated DNA was hybridized to the surface
of flow cells, and DNA clusters were generated using the Illumina
cluster station, followed by 36 cycles of sequencing on the
Illumina Genome Analyzer. DNA from each maternal plasma specimen
was sequenced by one flow cell. Sequenced reads were compiled using
Solexa Analysis Pipeline. All reads were then aligned to the
repeat-masked reference human genomic sequence, NCBI 36 assembly
(GenBank accession numbers: NC.sub.--000001 to NC.sub.--000024),
using the Eland application.
[0094] In this study, to reduce the complexity of the data
analysis, only sequences that have been mapped to a unique location
in the repeat-masked human genome reference are further considered.
Other subsets of or the entire set of the sequenced data could
alternatively be used. The total number of uniquely mappable
sequences for each specimen was counted. The number of sequences
uniquely aligned to chromosome 21 was expressed as a proportion to
the total count of aligned sequences for each specimen. As maternal
plasma contains fetal DNA among a background of DNA of maternal
origin, the trisomy 21 fetus would contribute extra sequenced tags
originating from chromosome 21 due to the presence of an extra copy
of chromosome 21 in the fetal genome. Hence, the percentage of
chromosome 21 sequences in maternal plasma from a pregnancy
carrying a trisomy 21 fetus would be higher than that from a
pregnancy with a euploid fetus. The analysis does not require the
targeting of fetal-specific sequences. It also does not require the
prior physical separation of fetal from maternal nucleic acids. It
also does not require the need to distinguish or identify fetal
from maternal sequences after sequencing.
[0095] FIG. 3A shows the percentage of sequences mapped to
chromosome 21 (percentage representation of chromosome 21) for each
of the 8 maternal plasma DNA samples. The percentage representation
of chromosome 21 was significantly higher in maternal plasma of
trisomy 21 pregnancies than in that of euploid pregnancies. These
data suggest that noninvasive prenatal diagnosis of fetal
aneuploidy could be achieved by determining the percentage
representation of the aneuploid chromosome compared to that of a
reference population. Alternatively, the chromosome 21
over-representation could be detected by comparing the percentage
representation of chromosome 21 obtained experimentally with the
percentage representation of chromosome 21 sequences expected for a
euploid human genome. This could be done by masking or not masking
the repeat regions in the human genome.
[0096] Five of the eight pregnant women were each carrying a male
fetus. The sequences mapped to the Y chromosome would be
fetal-specific. The percentage of sequences mapped to the
Y-chromosome was used to calculate the fractional fetal DNA
concentration in the original maternal plasma specimen. Moreover,
the fractional fetal DNA concentration was also determined by using
microfluidics digital PCR involving the zinc finger protein,
X-linked (ZFX) and zinc finger protein, Y-linked (ZFY) paralogous
genes.
[0097] FIG. 3B shows the correlation of the fractional fetal DNA
concentrations as inferred by the percentage representation of Y
chromosome by sequencing and that determined by ZFY/ZFX
microfluidics digital PCR. There was a positive correlation between
the fractional fetal DNA concentrations in maternal plasma
determined by these two methods. The coefficient of correlation (r)
was 0.917 in the Pearson correlation analysis.
[0098] The percentages of maternal plasma DNA sequences aligned to
each of the 24 chromosomes (22 autosomes and X and Y chromosomes)
for two representative cases are shown in FIG. 4A. One pregnant
woman was carrying a trisomy 21 fetus and the other was carrying a
euploid fetus. The percentage representation of sequences mapped to
chromosome 21 is higher in the pregnant woman carrying a trisomy 21
fetus when compared with the pregnant woman carrying a normal
fetus.
[0099] The differences (%) of the percentage representation per
chromosome between the maternal plasma DNA specimens of the above
two cases is shown in FIG. 4B. The percentage difference for a
particular chromosome is calculated using the formula below:
Percentage difference(%)=(P.sub.21-P.sub.E)/P.sub.E.times.100%,
where
P.sub.21=percentage of plasma DNA sequences aligned to the
particular chromosome in the pregnant woman carrying a trisomy 21
fetus and; P.sub.E=percentage of plasma DNA sequences aligned to
the particular chromosome in the pregnant woman carrying a euploid
fetus.
[0100] As shown in FIG. 4B, there is an over-representation of
chromosome 21 sequences by 11% in the plasma of the pregnant woman
carrying a trisomy 21 fetus when compared with the pregnant woman
carrying a euploid fetus. For the sequences aligned to other
chromosomes, the differences between the two cases were within 5%.
As the percentage representation for chromosome 21 is increased in
the trisomy 21 compared with the euploid maternal plasma samples,
the difference (%) could be alternatively referred as the degree of
over-representation in chromosome 21 sequences. In addition to
differences (%) and absolute differences between the chromosome 21
percentage representation, ratios of the counts from test and
reference samples could also be calculated and would be indicative
of the degree of chromosome 21 over-representation in trisomy 21
compared with cuploid samples.
[0101] For the four pregnant women each carrying a euploid fetus, a
mean of 1.345% of their plasma DNA sequences were aligned to
chromosome 21. In the four pregnant women carrying a trisomy 21
fetus, three of their fetuses were males. The percentage
representation of chromosome 21 was calculated for each of these
three cases. The difference (%) in chromosome 21 percentage
representation for each of these three trisomy 21 cases from the
mean chromosome 21 percentage representation derived from values of
the four cuploid cases were determined as described above. In other
words, the mean of the four cases carrying a euploid fetus was used
as the reference in this calculation. The fractional fetal DNA
concentrations for these three male trisomy 21 cases were inferred
from their respective percentage representation of Y chromosome
sequences.
[0102] The correlation between the degree of over-representation
for chromosome 21 sequences and the fractional fetal DNA
concentrations is shown in FIG. 5. There was a significant positive
correlation between the two parameters. The coefficient of
correlation (r) was 0.898 in the Pearson correlation analysis.
These results indicate that the degree of over-representation of
chromosome 21 sequences in maternal plasma is related to the
fractional concentration of fetal DNA in the maternal plasma
sample. Thus, cut-off values in the degree of chromosome 21
sequence over-representation relevant to the fractional fetal DNA
concentrations could be determined to identify pregnancies
involving trisomy 21 fetuses.
[0103] The determination of the fractional concentration of fetal
DNA in maternal plasma can also be done separate to the sequencing
run. For example, the Y chromosome DNA concentration could be
pre-determined using real-time PCR, microfluidics PCR or mass
spectrometry. For example, we have demonstrated in FIG. 3B that
there is good correlation between the fetal DNA concentrations
estimated based on the Y-chromosome count generated during the
sequencing run and the ZFY/ZFX ratio generated external to the
sequencing run. In fact, fetal DNA concentration could be
determined using loci other than the Y chromosome and applicable to
female fetuses. For example, Chan et al showed that fetal-derived
methylated RASSF1A sequences would be detected in the plasma of
pregnant women in the background of maternally derived unmethylated
RASSF1A sequences (Chan et al, Clin Chem 2006; 52:2211-8). The
fractional fetal DNA concentration can thus be determined by
dividing the amount of methylated RASSF1A sequences by the amount
of total RASSF1A (methylated and unmethylated) sequences.
[0104] It is expected that maternal plasma would be preferred over
maternal serum for practicing our invention because DNA is released
from the maternal blood cells during blood clotting. Thus, if serum
is used, it is expected that the fractional concentration of fetal
DNA will be lower in maternal plasma than maternal serum. In other
words, if maternal serum is used, it is expected that more
sequences would need to be generated for fetal chromosomal
aneuploidy to be diagnosed, when compared with a plasma sample
obtained from the same pregnant woman at the same time.
[0105] Yet another alternative way of determining the fractional
concentration of fetal DNA would be through the quantification of
polymorphic differences between the pregnant women and the fetus
(Dhallan R, et al. 2007 Lancet, 369, 474-481). An example of this
method would be to target polymorphic sites at which the pregnant
woman is homozygous and the fetus is heterozygous. The amount of
fetal-specific allele can be compared with the amount of the common
allele to determine the fractional concentration of fetal DNA.
[0106] In contrast to the existing techniques for detecting
chromosomal aberrations, including comparative genomic
hybridization, microarray comparative genomic hybridization,
quantitative real-time polymerase chain reaction, which detect and
quantify one or more specific sequence(s), massively parallel
sequencing is not dependent on the detection or analysis of
predetermined or a predefined set of DNA sequences. A random
representative fraction of DNA molecules from the specimen pool is
sequenced. The number of different sequenced tags aligned to
various chromosomal regions is compared between specimens
containing or not containing the DNA species of interest.
Chromosomal aberrations would be revealed by differences in the
number (or percentage) of sequences aligned to any given
chromosomal region in the specimens.
[0107] In another example the sequencing technique on plasma
cell-free DNA may be used to detect the chromosomal aberrations in
the plasma DNA for the detection of a specific cancer. Different
cancers have a set of typical chromosomal aberrations. Changes
(amplifications and deletions) in multiple chromosomal regions may
be used. Thus, there would be an increased proportion of sequences
aligned to the amplified regions and a decreased proportion of
sequences aligned to decreased regions. The percentage
representation per chromosome could be compared with the size for
each corresponding chromosome in a reference genome expressed as
percentage of genomic representation of any given chromosome in
relation to the whole genome. Direct comparisons or comparisons to
a reference chromosome may also be used.
II. Sequencing Just a Fraction of the Human Genome
[0108] In the experiment described in example I above, maternal
plasma DNA from each individual specimen was sequenced using one
flow cell only. The number of sequenced tags generated from each of
the tested specimens by the sequencing run is shown in FIG. 6. T21
denote a sample obtained from a pregnancy involving a trisomy 21
fetus.
[0109] As 36 bp were sequenced from each of the sequenced maternal
plasma DNA fragments, the number of nucleotides/basepairs sequenced
from each specimen could be determined by 36 bp multiplied by the
sequenced tag count and are also shown in FIG. 6. As there are
approximately 3 billion basepairs in the human genome, the amount
of sequencing data generated from each maternal plasma specimen
represented only a fraction, ranging from some 10% to 13%.
[0110] Furthermore, in this study, only the uniquely mappable
sequenced tags, termed U0 in nomenclature from the Eland software,
were used to demonstrate the presence of over-representation in the
amount of chromosome 21 sequences in the maternal plasma specimens
from pregnancies each carrying a fetus with trisomy 21, as
described in example I above. As shown in FIG. 6, U0 sequences only
represent a subset of all the sequenced tags generated from each
specimen and further represent an even smaller proportion, some 2%,
of the human genome. These data indicate that the sequencing of
only a portion of the human genomic sequences present in the tested
specimen is sufficient to achieve the diagnosis of fetal
aneuploidy.
III. Determination of Number of Sequences Required
[0111] The sequencing result of the plasma DNA from a pregnant
woman carrying a cuploid male fetus is used for this analysis. The
number of sequenced tags that can be mapped without mismatches to
the reference human genome sequence was 1,990,000. Subsets of
sequences were randomly chosen from these 1,990,000 tags and the
percentage of sequences aligned to chromosome 21 was calculated
within each subset. The number of sequences in the subsets was
varied from 60,000 to 540,000 sequences. For each subset size,
multiple subsets of the same number of sequenced tags were compiled
by random selection of the sequenced tags from the total pool until
no other combination was possible. The mean percentage of sequences
aligned to chromosome 21 and its standard deviation (SD) were then
calculated from the multiple subsets within each subset size. These
data were compared across different subset sizes to determine the
effect of subset size on the distribution of the percentage of
sequences aligned to the chromosome 21. The 5.sup.th and 95.sup.th
percentiles of the percentages were then calculated according to
the mean and SD.
[0112] When a pregnant woman is carrying a trisomy 21 fetus, the
sequenced tags aligned to chromosome 21 should be over-represented
in the maternal plasma due to an extra dose of chromosome 21 from
the fetus. The degree of over-representation is dependent on the
fetal DNA percentage in the maternal plasma DNA sample following
the equation below:
Per.sub.T21=Per.sub.Eu.times.(1+f/2)
where Per.sub.T21 represents the percentage of sequences aligned to
chromosome 21 in a woman with a trisomy 21 fetus; and Per.sub.Eu
represents the percentage of sequences aligned to chromosome 21 in
a woman with a euploid fetus; and f represents the fetal DNA
percentage in maternal plasma DNA
[0113] As shown in FIG. 7, the SD for the percentages of sequences
aligned to chromosome 21 decreases with increasing number of
sequences in each subset. Therefore, when the number of sequences
in each subset increases, the interval between the 5.sup.th and
95.sup.th percentiles decreases. When the 5%-95% interval for the
euploid and trisomy 21 cases do not overlap, then the
differentiation between the two groups of cases would be possible
with an accuracy of >95%.
[0114] As shown in FIG. 7, the minimal subset size for the
differentiation of trisomy 21 cases from euploid cases is dependent
on the fetal DNA percentage. The minimal subset sizes for
differentiating trisomy 21 from euploid cases were 120,000, 180,000
and 540,000 sequences for fetal DNA percentages of 20%, 10% and 5%,
respectively. In other words, the number of sequences needed to be
analyzed would be 120,000 for determining whether a fetus has
trisomy 21 when a maternal plasma DNA sample contains 20% fetal
DNA. The number of sequences needed to be analyzed would be
increased to 540,000 when the fetal DNA percentage drops to 5%.
[0115] As the data were generated using 36 basepair sequencing,
120,000, 180,000 and 540,000 sequences correspond to 0.14%, 0.22%
and 0.65% of the human genome, respectively. As the lower range of
fetal DNA concentrations in maternal plasma obtained from early
pregnancies were reported to be some 5% (Lo, Y M D et al. 1998 Am J
Hum Genet 62, 768-775), the sequencing of about 0.6% of the human
genome may represent the minimal amount of sequencing required for
diagnosis with at least 95% accuracy in detecting fetal chromosomal
aneuploidy for any pregnancy.
IV. Random Sequencing
[0116] To illustrate that the sequenced DNA fragments were randomly
selected during the sequencing run, we obtained the sequenced tags
generated from the eight maternal plasma samples analyzed in
example 1. For each maternal plasma specimen, we determined the
starting positions in relation to the reference human genome
sequence, NCBI assembly 36, of each of the 36 bp sequenced tags
that were aligned uniquely to chromosome 21 without mismatches. We
then ordered the starting position number for the pools of aligned
sequenced tags from each specimen in ascending order. We performed
a similar analysis for chromosome 22. For illustrative purpose, the
top ten starting positions for chromosome 21 and chromosome 22 for
each of the maternal plasma specimens are shown in FIGS. 8A and 8B,
respectively. As can be appreciated from these Tables, the
sequenced pools of DNA fragments were non-identical between
samples.
[0117] Any of the software components or functions described in
this application, may be implemented as software code to be
executed by a processor using any suitable computer language such
as, for example, Java, C++ or Perl using, for example, conventional
or object-oriented techniques. The software code may be stored as a
series of instructions, or commands on a computer readable medium
for storage and/or transmission, suitable media include random
access memory (RAM), a read only memory (ROM), a magnetic medium
such as a hard-drive or a floppy disk, or an optical medium such as
a compact disk (CD) or DVD (digital versatile disk), flash memory,
and the like. The computer readable medium may be any combination
of such storage or transmission devices.
[0118] Such programs may also be encoded and transmitted using
carrier signals adapted for transmission via wired, optical, and/or
wireless networks conforming to a variety of protocols, including
the Internet. As such, a computer readable medium according to an
embodiment of the present invention may be created using a data
signal encoded with such programs. Computer readable media encoded
with the program code may be packaged with a compatible device or
provided separately from other devices (e.g., via Internet
download). Any such computer readable medium may reside on or
within a single computer program product (e.g. a hard drive or an
entire computer system), and may be present on or within different
computer program products within a system or network. A computer
system may include a monitor, printer, or other suitable display
for providing any of the results mentioned herein to a user.
[0119] An example of a computer system is shown in FIG. 9. The
subsystems shown in FIG. 9 are interconnected via a system bus 975.
Additional subsystems such as a printer 974, keyboard 978, fixed
disk 979, monitor 976, which is coupled to display adapter 982, and
others are shown. Peripherals and input/output (I/O) devices, which
couple to I/O controller 971, can be connected to the computer
system by any number of means known in the art, such as serial port
977. For example, serial port 977 or external interface 981 can be
used to connect the computer apparatus to a wide area network such
as the Internet, a mouse input device, or a scanner. The
interconnection via system bus allows the central processor 973 to
communicate with each subsystem and to control the execution of
instructions from system memory 972 or the fixed disk 979, as well
as the exchange of information between subsystems. The system
memory 972 and/or the fixed disk 979 may embody a computer readable
medium.
[0120] The above description of exemplary embodiments of the
invention has been presented for the purposes of illustration and
description. It is not intended to be exhaustive or to limit the
invention to the precise form described, and many modifications and
variations are possible in light of the teaching above. The
embodiments were chosen and described in order to best explain the
principles of the invention and its practical applications to
thereby enable others skilled in the art to best utilize the
invention in various embodiments and with various modifications as
are suited to the particular use contemplated.
[0121] All publications, patents, and patent applications cited
herein are hereby incorporated by reference in their entirety for
all purposes.
* * * * *