U.S. patent application number 15/697277 was filed with the patent office on 2017-12-21 for means and methods for non-invasive diagnosis of chromosomal aneuploidy.
This patent application is currently assigned to LifeCodexx. The applicant listed for this patent is LifeCodexx. Invention is credited to Marcus BENZ, Wera HOFMANN, Christoph Von KALLE, Thomas POHL.
Application Number | 20170363628 15/697277 |
Document ID | / |
Family ID | 43216609 |
Filed Date | 2017-12-21 |
United States Patent
Application |
20170363628 |
Kind Code |
A1 |
BENZ; Marcus ; et
al. |
December 21, 2017 |
MEANS AND METHODS FOR NON-INVASIVE DIAGNOSIS OF CHROMOSOMAL
ANEUPLOIDY
Abstract
The invention relates to a prenatal diagnostic method for the
determination of a fetal chromosomal aneuploidy in a biological
sample obtained from a pregnant woman, which method comprises
enrichment and quantification of selected cell-free
deoxyribonucleic acid sequences showing consensus nucleosome
binding regions.
Inventors: |
BENZ; Marcus; (Konstanz,
DE) ; HOFMANN; Wera; (Konstanz, DE) ; POHL;
Thomas; (Konstanz, DE) ; KALLE; Christoph Von;
(Schallstadt, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LifeCodexx |
Konstanz |
|
DE |
|
|
Assignee: |
LifeCodexx
Konstanz
DE
|
Family ID: |
43216609 |
Appl. No.: |
15/697277 |
Filed: |
September 6, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13502498 |
Sep 7, 2012 |
9784742 |
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PCT/EP2010/066161 |
Oct 26, 2010 |
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15697277 |
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61272722 |
Oct 26, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6851 20130101;
G01N 33/574 20130101; G01N 2800/385 20130101; C12Q 1/6851 20130101;
C12Q 1/6806 20130101; G16B 30/00 20190201; C12Q 1/6806 20130101;
C12Q 2537/157 20130101; G01N 2800/368 20130101; C12Q 2525/107
20130101; C12Q 2545/101 20130101; C12Q 2525/121 20130101; C12Q
2525/107 20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574; C12Q 1/68 20060101 C12Q001/68 |
Claims
1.-14. (canceled)
15. A method for determining a chromosomal aberration in a
biological sample obtained from an individual, wherein the
biological sample includes nucleic acid molecules, the method
comprising: (a) selecting for and isolating from the biological
sample of an individual one or more target sequences of DNA
molecules present in the biological sample, wherein the target
sequences comprise DNA sequences leaving consensus nucleosome
binding regions; (b) amplifying the selected target sequences; (c)
sequencing the amplified selected target sequences and allotting
them to the chromosomes of the genome and identifying the unique
allotted target sequences; (d) determining a first amount for each
of one or more first chromosomes identified on the basis of the
unique allotted target sequences originating from the one or more
first chromosomes; (e) determining a second amount for each of one
or more second chromosomes identified on the basis of the unique
allotted target sequences originating from the one or more second
chromosomes; and (f) determining based on the first and second
amounts a chromosomal abberation.
16. The method of claim 15, wherein step (f) further comprises: (i)
determining a parameter from the first amount relative to the
second amount; (ii) comparing the parameter to a corresponding cut
off control value; and (iii) based upon the comparison in (ii),
determining whether or not there is a difference allowing for the
prediction of a chromosomal aberration in one or more of the first
chromosomes.
17. The method of claim 15, wherein the chromosomal aberration is
indicative of a predisposition for cancer.
18. The method of claim 15, wherein the individual is a human.
19. The method of claim 15. wherein the biological sample comprises
a cell suspected to be a cancer cell.
20. A computer programme product which when executed on a computer
or data processing device is capable of carrying out the following
steps: (a) allotting amplified selected and isolated target
sequences having consensus nucleosome binding regions from a
biological sample of an individual to chromosomes of the genome and
identifying unique allotted target sequences; (b) determining a
first amount for each of one or more first chromosomes identified
on the basis of the unique allotted target sequences originating
from the one or more first chromosomes; and (c) determining a
second amount for each of one or more second chromosomes identified
on the basis of the unique allotted target sequences originating
from the one or more second chromosomes; and d) determining based
on the first and second amounts a fetal chromosomal aneupoidy of
one or more of the first chromosomes or a chromosomal
aberration.
21. The computer programme product of claim 20, wherein the
determining in step (d) further comprises: (i) determining a
parameter from the first amount relative to the second amount; (ii)
comparing the parameter to a corresponding cut off control value;
and (iii) based upon the comparison in (ii), determining whether or
not there is a difference allowing for the prediction of a
chromosomal aberration in one or more of the first chromosomes or
determining whether or not there is a difference allowing for the
prediction of a fetal chromosomal aneuploidy of one or more of the
first chromosomes.
22. A device for carrying out the method of claim 15, comprising:
(a) an analyzing unit comprising a subunit capable of selecting for
and isolating from the biological sample of an individual one or
more target sequences of DNA molecules present in the biological
sample, wherein the target sequences comprise DNA sequences having
consensus nucleosome binding regions, a subunit for amplification
of the target DNA molecules, and a subunit for sequencing of
amplified target sequences; and (b) an evaluation unit comprising a
receiving subunit for the sequence data and a computer or data
processing subunit executing a computer programme product, wherein
the computer programme product which when executed on a computer or
data processing device is capable of carrying out the following
steps: (i) allotting amplified selected and isolated target
sequences having consensus nucleosome binding regions from a
biological sample of an individual to chromosomes of the genome and
identifying unique allotted target sequences; (ii) determining a
first amount for each of one or more first chromosomes identified
on the basis of the unique allotted target sequences originating
from the one or more first chromosomes; and (iii) determining a
second amount for each of one or more second chromosomes identified
on the basis of the unique allotted target sequences originating
from the one or more second chromosomes; and (iv) determining based
on the first and second amount a fetal chromosomal aneupoidy of one
or more of the first chromosomes or a chromosomal aberration.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/502,498, filed Sep. 7, 2012, which is the National
Stage of International Patent Application No. PCT/EP2010/066161,
filed Oct. 26, 2010, which claims priority from U.S. Provisional
Patent Application No. 61/272,722, filed Oct. 26, 2009. The
contents of these applications are incorporated herein by reference
in their entirety.
[0002] The present invention relates to a non-invasive prenatal
detection method in a biological sample obtained from a pregnant
woman. In particular, the invention relates to a method for
non-invasive prenatal diagnosis (NIPD) by enrichment and
quantification of selected cell-free deoxyribonucleic acid
sequences in a maternal blood sample, to detect fetal chromosomal
abnormalities, e.g. fetal chromosomal aneuploidies.
[0003] In many countries prenatal diagnosis of fetal aneuploidies
as well as single gene Mendelian disorders, such as cystic
fibrosis, are typically part of public health programs. The
obstetrics care includes particularly non-invasive prenatal testing
such as first trimester screening or second trimester screening to
risk-stratify pregnant women prior to invasive diagnostic
procedures. These screening tests usually include a blood test
estimating biochemical markers and an ultrasound examination to
measure fetal nuchal translucency with an accuracy of up to 90%
(Malone et al. 2005 N Eng J Med 353, 2001-2011). Invasive
procedures, including amniocentesis, chorionic villus sampling or
cordocentesis, are used to obtain direct fetal genetic material
(and perform karyotype analysis) and achieve over 99% accuracy.
However, these invasive procedures pose a potential risk of fetal
loss. There is currently great interest in the development of
methods for direct but non-invasive diagnosis of fetal genetic
disease with the absence of risk of miscarriage.
[0004] The discovery of cell-free fetal nucleic acids in maternal
plasma has opened up new possibilities for non-invasive prenatal
diagnosis. (Lo, Y M D et al. 1997 Lancet 350, 485-487 Lo, Y M D and
Chiu, R W K 2007 Nat Rev Genet 8, 71-77). Over the last few years,
this approach has been 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). Other applications involve the enrichment of fetal
DNA by a method that suppresses the maternal background, such as by
the addition of formaldehyde (Dhallan, R et al. 2004 JAMA291,
1114-1119) or the targeting of fetal-specific nucleic acid
molecules, including fetal-specific epigenetic markers and
placenta-specific mRNA markers (Chan, K C et al. Clin Chem 2006;
52:2211-8).
[0005] Recent reports have indicated that the development of highly
discriminatory quantitative methods for chromosome dosage analysis
using digital polymerase chain reaction technology may be useful
for the non-invasive detection of fetal aneuploidies by the
analysis of cell-free DNA and RNA in maternal plasma or serum (Fan,
H C and Quake, SR 2007 Anal Chem 79, 7576-7579; Lo, Y M D et al.
2007 PNAS 104, 13116-13121). But the low fraction of fetal nucleic
acids co-existing in maternal plasma with a high background of
nucleic acids of maternal origin can often interfere with these
analyses (Lo, Y M D et al. 1998 Am J Hum Genet 62, 768-775).
[0006] With the rapid development of high-throughput sequencing
technology allowing massively parallel sequencing of tens of
millions of short sequencing tags, the possibility of detecting the
presence of trisomy fetal genomes in the maternal plasma DNA sample
obtained in the first trimester of pregnancy has recently been
explored. (Fan, H C et al. 2008 PNAS 105, 16266-16271; Chiu, R W et
al. 2008 PNAS 105, 20458-20463). This shotgun sequencing technology
enables a deeper sampling than can be achieved by digital PCR.
However, the costs of DNA sequencing using shotgun sequencing are
too high to bring this approach in a routine medical test as part
of prenatal care.
[0007] In addition, a technical drawback of random shotgun
sequencing is the potential risk to introduce sequence bias. The
random shotgun sequencing cannot output a perfectly even read
distribution across a genome of interest so that statistically
there will always be some stretches of DNA that are read out more
than others.
[0008] WO 2009/013496 refers to a non-invasive method for prenatal
diagnostic testing of a fetal chromosomal aneuploidy by determining
imbalances between different nucleic acid sequences in a maternal
sample by massively parallel genomic sequencing (MPGS).
[0009] Accordingly, there is a strong need for a less costly and
less time intensive non-invasive method for prenatal diagnosis of a
fetal chromosomal abnormality e.g. a fetal chromosomal aneuploidy,
in a biological sample obtained from a pregnant woman, which method
has a high sensitivity and specificity and does not introduce any
further sequence bias.
[0010] It is thus an objective of the present invention to provide
an alternative, improved method for performing prenatal
diagnosis.
[0011] More specifically, the invention relates to a method for the
determination of a fetal chromosomal aneuploidy in a biological
sample obtained from a pregnant female individual, wherein the
biological sample includes nucleic acid molecules, the method
comprising: [0012] a) selecting for and isolating from a biological
sample of a pregnant female individual one or more target sequences
of DNA molecules contained in the biological sample, wherein said
target sequences comprise DNA sequences having consensus nucleosome
binding regions; [0013] b) amplifying said selected target
sequences; [0014] c) sequencing said amplified selected target
sequences and allotting them to chromosomes of the genome and
identifying the unique allotted target sequences; [0015] d)
determining a first amount each of one or more first chromosomes
identified on the basis of said amplified unique target sequences
originating from said one or more first chromosomes; [0016] e)
determining a second amount each of one or more second chromosomes
identified on the basis of said amplified unique target sequences
originating from said one or more second chromosomes; and [0017] f)
determining based on the said first and second amount a fetal
chromosomal aneupoidy of one or more of said first chromosomes.
[0018] The aforementioned method may or may not comprise additional
steps prior, after or in between the explicitly mentioned essential
steps of the method. Preferably, the method encompasses steps of
obtaining the biological sample from a pregnant female individual.
Moreover, the method may preferably encompass sample pre-treatment
steps which, e.g., aim to preserve the nucleic acids present in the
sample or which aim to release or isolate the nucleic acids from
cellular material present in the sample The method may be,
preferably, assisted by automation. The steps a) to c) may be
assisted by a robotic device, the remaining steps evaluating the
sequence data may be assisted by a computer or data processing
device having tangibly embedded an algorithm for determining the
amounts of the sequences and for carrying, out the comparison.
[0019] The term "fetal chromosomal aneuploidy" as used herein
refers to chromosomal aberration which is characterized by a number
of chromosomes which differs from the physiological number of
chromosomes which are expected to be present in a fetus, i.e. an
abnormal number of chromosomes. Such a difference in the number is
also referred to as numerical aberration. The term encompasses an
increased number of a chromosome as well as a decreased number of a
chromosome present in the fetal genome. A chromosomal aneuploidy as
referred to in the context of the present invention may be (i) a
monosomy, i.e. the lack of chromosome of a chromosome pair, (ii) a
disomy in cases where the individual to be examined is normally
triploid, tetraploid or even more, or an uniparenteral disomy, (ii)
a trisomy, i.e. the presence of three instead of two chromosomes
which are physiologically present for each chromosome in a diploid
organism, (iv) a tetrasomy, i.e. the presence of four chromosomes
instead of two chromosomes which are physiologically present for
each chromosome in a diploid organism or even (v) a pentasomy, i.e.
the presence of five chromosomes instead of two chromosomes which
are physiologically present for each chromosome in a diploid
organism. The aneuploidy can affect either the autosomes or the
gonosomes. In some cases a chromosome pair may be even entirely
missing in the genome. Moreover, an aneuploidy as used herein also
encompasses a numerical aberration with respect to a part of a
chromosome, i.e. a case where a part of one of the chromosomes of a
chromosome pair is deleted or present in one or more extra copies.
A part of a chromosome may be present in an inappropriate number of
copies as a result of of a structural aberration, e.g., unbalanced
translocations, contiguous gene syndromes or chromosomal deletion
syndromes. Fetal chromosomal aneupoidy usually results in severe
disorders or diseases affecting the fetus and its development.
Preferred fetal chromosomal aneuploidies and accompanying diseases
or disorders which can be determined by the method of the present
invention include Turner syndrome (gonosomal monosomy), Klinefelter
syndrome (XXY gonosomes), triple-X syndrome (XXX gonosomes), Down
syndrome (Trisomy 21), Edwards syndrome (Trisomy 18) or Patau
syndrome (Trisomy 13). Uniparenteral disomy is known for chromosome
15 as Prader-Willi-Syndrome. If such a uniparenteral disomy is to
be detected, the analysed DNA must also be distinguished in to
paternally inherited or maternally inherited DNA. Unbalanced
translocations as used herein encompass, preferably, unbalanced
Robertson trisomy, rob(13q:14q). Other structural aberrations which
can be preferably determined by the method of the invention include
4q-deletion (Wolf-Hirschhorn syndrome), 5q-deletion (cri du chat
syndrome) or microdeletion syndromes, in particular, 17q11.2
deletion (Smith-Magenis syndrome) or 22q11.2 deletion (DiGeorge
syndrome).
[0020] The phrase "determination of a fetal chromosomal aneuploidy"
as used herein refers to assessing the probability according to
which a subject is suffering or will suffer from a disease or
condition referred to in this specification. As will be understood
by those skilled in the art, such an assessment is usually not
intended to be correct for 100% of the subjects to be diagnosed.
The term, however, requires that a statistically significant
portion of subjects can be correctly diagnosed to suffer from the
disease or condition. Whether a portion is statistically
significant can be determined without further ado by the person
skilled in the art using various well known statistic evaluation
tools, e.g., determination of confidence intervals, p-value
determination, Student's t-test, Mann-Whitney test etc.. Details
are found in Dowdy and Wearden, Statistics for Research, John Wiley
& Sons, New York 1983. Preferred confidence intervals are at
least 90%, at least 95%, at least 97%, at least 98% or at least
99%. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or
0.0001. Preferably, the probability envisaged by the present
invention allows that the diagnosis will be correct for at least
60%, at least 70%, at least 80%, or at least 90% of the subjects of
a given cohort or population.
[0021] The term "individual" as used herein refers to a
multicellular organism which is capable of carrying its offspring
inside an uterus. More preferably, said organism refers to eutheric
organisms and, more preferably, to mammals. Most preferably, the
individual is a human. It will be understood that the individual a
sample of which is applied in the method of the present invention
shall be a pregnant female carrying an embryo or fetus inside its
uterus. During pregnancy and particular the fetal stages of the
embryonic development, fetal genomic DNA is released into the body
of the female individual. In particular, such fetal gemoic DNA
occurs in the blood of the female individual.
[0022] Thus, the term "biological sample" as used herein refers to
a sample from a individual, preferably, to a sample of a body
liquid obtained from a pregnant woman. More preferably, said body
liquid is selected from the group consisting of a blood sample, a
urine sample and a saliva sample. The blood sample referred to
herein encompasses a whole blood sample, a plasma sample or a serum
sample. It will be understood that the sample to be used in the
method of the present invention shall comprise nucleic acids and,
in particular, DNA molecules being representative for the genome of
the developing fetus. Preferably, the said fetal genome derived DNA
molecules equally represent each chromosome present in the said
fetal genome in a statistical manner. Thus, the more copies of a
given chromosome are present in the fetal genome, the more DNA
molecules will reflect these chromosomes in the sample and vice
versa.
[0023] The DNA molecules present in the sample including the fetal
genome derived DNA molecules are specifically selected for DNA
molecules comprising target sequences allowing for a meaningful an
efficient sequence analysis in the context of the present
invention. Suitable target sequences comprise consensus sequences
for nucleosome binding regions as specified elsewhere herein or
matrix attachment regions of the chromosomal DNA. The term
"consensus nucleosome binding region" as used herein refers to DNA
sequence patterns at or around transcription start sites (TSS) of
transcribed genes that are associated with well-positioned
nucleosomes located at or near such transcription start sites.
Suitable consensus sequences are known in the art and are described
in Schones et al. or Fan et al. (Schones 2008, Cell 132(5):
887-898, Fan 2008, Proc. Natl. Acad. Sci. USA 105:
16266-16271).
[0024] Selecting and isolating such DNA molecules comprising target
sequences for the sequence analysis as used herein includes the
provision of suitable sequences for primers used for DNA
amplification. Preferably, suitable sequences are provided by
ligating adapter oligonucleotides comprising suitable sequences for
DNA amplification to the DNA molecules. Preferably, the adapter
oligonucleotides can also comprise additional sequences,
preferably, barcode or index tag sequences which allow for
identification of a pool of identical DNA molecule species. (see
Parameswaran 2007, Nucl. Acids Res. 35(19): e130). By using such
adaptor oligonucleotides comprising barcode or index tag sequences,
it is possible to process a pool of DNA molecules of different
samples in the method of the present invention in parallel.
Moreover, the term also encompasses isolation and enrichment
procedures for DNA molecules comprising such target sequences
using, e.g., affinity chromatograph or batch-wise enrichment using
labled nucleic acid baits which allow for enrichment and isolation
of hybridized DNA molecules by affinity purification, e.g., via
magnetic beads. In a preferred embodiment of the method of the
present invention, biotinylated RNA bait molecules are brought into
contact with the DNA molecules comprised by the sample. The RNA
bait molecules are specifically designed to have a sequence which
is capable of hybridizing to the target sequence of the desired DNA
molecules, in particular, they have sequences capable of
hybridizing to the consensus sequences for nucleosome binding
regions as specified elsewhere herein or matrix attachment regions
of the chromosomal DNA. After hybridization, the chimeric DNA/RNA
hybrid molecules are isolated from the remaining sample by capture
of the biotin lable via streptavidin. Preferably, magnetic beads
with immobilized streptaviding can be applied for the isolation.
The isolated DNA molecules can be released from the magnetic beads.
Depending on how the release is carried out, enrichment of the DNA
molecules may also occur. A particular suitable techniques for
isolating and enriching the desired DNA molecules is the
commercially available SureSelect Target Enrichment System from
Agilent Technologies, Inc., US. Details are also found in Gnirke
2009, Nat Biotechnology 27(2): 182-189 hereby also incorporated by
reference. Subsequently, the said isolated DNA molecules comprising
the target sequence can be further processed according to the
method of the invention. Another particular technique is the
four-color DNA sequencing-by-synthesis technology from Illumina,
Inc., US (see Johnson 2007, Science 316: 1497-1502; Robertson 2007,
Nature Methods 4: 651-657; Barski 2007, Cell 129: 823-837;
Mikkelsen 2007, Nature 448: 553-560; Fields 2007, Science 316:
1441-1442).
[0025] Amplification of the target sequence comprising DNA
molecules can be achieved by the well known methods for nucleic
acid amplification. Preferably, amplification is carried out by
PCR. Sequencing of the amplification products can be done by the
well known techniques for nucleic acid sequencing. Quality of the
analyzed samples may be assured after amplification. Moreover, DNA
molecules in the sample may be allocated into DNA molecules with
sequences of paternal or maternal origin.
[0026] Preferably, paternal sequences are analyzed when fetal
genomes shall be analyzed by the method of the present invention.
By analyzing such paternally inherited DNA molecules, the
reliability of the method may be further improved since such
sequence unambiguously originate from the fetal genomic DNA.
[0027] The sequences identified by the sequencing of the amplified
DNA molecules comprising the desired target sequences can be
allocated to chromosomes or parts thereof by the presence of
characteristic sequences of said chromosomes. Suitable sequences
which allow for allocation are genetic marker sequences, for
example, by single sequence polymorphism markers, repetitive DNA
marker sequences such as single sequence repeats, short tandem
repeats, variable number tandem repeats or microsatellite sequences
in general. Alternatively, the allocation can be made by sequence
comparision with consensus sequences for chromosomes such as the
hg18 or hg19 consensus sequences for the human genome. After the
target sequences have been allocated to a chromosome, i.e. after
mapping of the target sequences to the chromosomes, the unique
alloted target sequences, i.e. the target sequences which have only
once been allocated to a chromosome, among the mapped target
sequences are identified. Suitable algorithms for sequence
comparison and allocations to chromosomes include Efficient Local
Alignment of Nucleotide Data (ELAND; an alignment tool integrated
in Illumina-Solexa data processing package, enable ungapped
alignment for reads with size up to 32 bp; Cox, unpublished) or
Burrows-Wheeler Aligner (BWA; Li and Durbin 2009, Bioinformatics
25:1754-1760).
[0028] In a subsequent step, a first amount for each of one or more
first chromosomes identified on the basis of said unique allotted
target sequences originating from said one or more first
chromosomes is determined. Said amount can be any number or value
which reflects or correlates to the number of the unique target
sequences being allocated to a chromosome which have been obtained
from the DNA molecules. The amount may be an absolute or relative
amount, i.e. a normalized amount, e.g., with respect to the total
number of amplified target sequences and/or the total amount of
nucleic acids found in the sample. Simultaneously or separately, a
second amount for each of one or more second chromosomes identified
on the basis of said unique alloted target sequences originating
from said one or more second chromosomes is determined.
[0029] Based on the said first and second amount a fetal
chromosomal aneuploidy of one or more of said first chromosomes can
be determined. This can be done for, e.g., a diploid individual by
comparing a relative first amount of a first chromosome to a
relative second amount of a second chromosome. In case of a diploid
individual the relative first and second amounts shall be
essentially identical. A increased relative first amount with
respect to the second amount, shall be indicative for an increased
number of the first chromosome and, thus, an aneuploidy of the
first chromosome in the fetal genome. The same applies mutatis
mutandis for a reduced first amount. Preferably, in humans,
chromosomes 1, 2, 3, 5 to 15, 18 and 21 can be used for deriving a
second amount as referred to above.
[0030] Also preferably, a parameter can be determined based on the
first and the second amounts. Such a parameter shall be
statistically more robust and allows for comparison between
different individuals or collectives of individuals.
[0031] Thus, in a preferred embodiments of the method of the
present invention, said method further comprises: [0032] i)
determining a parameter from said first amount relative to said
second amount; [0033] ii) comparing the parameter to a
corresponding cut off control value; and based on the comparison,
determining whether or not there is a difference allowing for the
prediction of a fetal chromosomal aneuploidy of one or more of said
first chromosomes.
[0034] The term "parameter" as used herein refers to a numerical
value that characterizes a quantitative data set and/or a numerical
relationship between quantitative data sets. More specifically,
said parameter may be calculated as a ratio of the first amount and
the second amount or may be a value derived from said ration such
as a percentage value. Preferably, the parameter reflects the
amount of a first chromosome to be investigated with respect to the
amount(s) of at least one other second chromosome and, preferably,
with respect to the remaining chromosomes as determined by the
target sequences. Moreover, the parameter may be even made more
robust with respect to individual variations and, thus, suitable
for comparison with parameters from reference individuals or groups
thereof. To this end, a parameter which has been calculated as set
forth above (reference parameter) may be, preferably, subtracted
from the determined parameter and divided by the standard deviation
for the reference parameter. The resulting transformed parameter,
also termed "z-score" (see Chiu 2008, Proc Natl Acad Sci USA
105(51):20458-63), can then be compared to a transformed parameter
from a reference individual or group of individuals known to
exhibit the chromosomal aneuploidy or known not exhibit the said
aneuploidy. The reference parameter thus constitutes a cut off
value and based on the comparison, it is possible to determine
whether or not there is a difference in the number of chromosomes
of the fetal genome allowing for the prediction of a fetal
chromosomal aneuploidy of one or more of said first chromosomes.
The term "cut off value" or "cut off control value" as used herein,
preferably, refers to a numerical value calculated as the ratio of
two quantities of chromosomes relative to each other, i.e. the
ratio of the amount of one or more first chromosomes relative to
the amount of one or more second chromosomes of an average or
pooled blood, urine or saliva sample of one or more healthy female
donors, and wherein the number of said second chromosomes is
identical with the number of said first chromosomes to be compared
with. The cut off value allows to distinguish between a diseased
and non-diseased state of an experimental biological sample.
Further techniques for determining parameters and comparing them
between different individuals or groups thereof which can be
preferably applied in the method of the present invention are those
that have been described in EP 2 183 692 A1, EP 2 183 693 A1 or
WO2010/033578.
[0035] It was recently found by Fan et al. (Fan 2008, loc.cit) that
cell-free plasma DNA is mainly apoptotic in origin and shares
features of nucleosomal DNA. Thus, according to the present
invention the non-invasive method for prenatal diagnosis disclosed
in WO 2009/013496 was further developed and improved by suggesting
carrying out an additional enrichment step before the step of
sequencing to capture and specifically enrich selected DNA regions
showing consensus nucleosome positions on the chromosomes of
interest. By this modification of the state-of-the-art method, the
cost- and process efficiency can be substantially improved and
particularly as compared to massively parallel genomic sequencing
using random shotgun sequencing. The present target enrichment
process offers significant increase in speed and additional
advantages in scalability over current polymerase chain reaction
(PCR) techniques in most next-generation sequencing workflows. It
is based on an efficient hybrid selection technique which improves
cost and process efficiency of the so-called "massively parallel
genomic sequencing" workflow (Gnirke, A et al. 2009 Nat Biotechnol
27, 182-189). The analysis of multiple sets of specific target loci
is used to increase the amount of data obtainable from a sample
without increasing the number of (digital) PCR sampling performed
for subsequent massively parallel genomic sequencing, such as that
achievable by 454 technology (Roche).
The method according to the present invention is suitable for
determining in a blood sample from a pregnant individual the
chromosomal aneuploidy of the developing offspring. In particular,
a chromosomal aneuploidy of a developing fetus can be determined,
particularly, as from the 6th week of gestation onwards.
[0036] The present invention also relates to a method for the
determination of a chromosomal aberration in a biological sample
obtained from an individual, wherein the biological sample includes
nucleic acid molecules, the method comprising: [0037] a) selecting
for and isolating from said biological sample of an individual one
or more target sequences of DNA molecules contained in the
biological sample, wherein said target sequences comprise DNA
sequences having consensus nucleosome binding regions; [0038] b)
amplifying said selected target sequences; [0039] c) sequencing
said amplified selected target sequences and allotting them to
chromosomes of the genome and identifying the unique allotted
target sequences; [0040] d) determining a first amount each of one
or more first chromosomes identified on the basis of said unique
allotted target sequences originating from said one or more first
chromosomes; [0041] e) determining a second amount each of one or
more second chromosomes identified on the basis of said unique
allotted target sequences originating from said one or more second
chromosomes; and [0042] f) determining based on the said first and
second amount a chromosomal abberation.
[0043] The term "chromosomal aberration" as used herein,
preferably, refers to numerical aberrations as defined elsewhere
herein.
[0044] Preferably, step f) of said method further comprises: [0045]
i) determining a parameter from said first amount relative to said
second amount; [0046] ii) comparing the parameter to a
corresponding cut off control value; and based on the comparison,
determining whether or not there is a difference wherein the
presence of a difference is indicative for a chromosomal aberration
in one or more of said first chromosomes.
[0047] More preferably, the chromosomal aberration shall be
indicative for a predisposition for cancer.
[0048] The term "predisposition" as used herein refers to an
increased risk that an individual will develop cancer within a
certain predictive window in the future. Preferably, said window is
up to 1 year, up to 5 years, up to 10 years or the entire life span
of the individual.
[0049] The term "cancer" as used herein encompasses any malignant
neoplasm. Different cancer types are well known in the art.
Preferably, the cancer referred to in accordance with the present
invention is associated with chromosomal aberration. In particular,
the following cancers have been reported to be frequently
associated with chromosomal aberrations are selected from the group
consisting of: human leukemias such as acute myeloid leukaemia or
chronic myeloid leukemia (see, e.g. Mitelman Database of Chromosome
Aberrations and Gene Fusions in Cancer (2010). Mitelman F,
Johansson B and Mertens F (Eds.),
cgap.nci.nih.gov/Chromosomes/Mitelman).
[0050] Preferably, said individual in context of the aforementioned
method is an individual as defined elsewhere herein and, most
preferably, it is a human.
[0051] Preferably, said biological sample comprises a cell
suspected to be a cancer cell. Suitable samples can be obtained
from body fluids in the case of non-solid tumors, e.g., blood in
the case of hematopoietic cancers, or from tissue biopsy samples.
Depending on the type of cancer, the person skilled in the art
knows how to obtain a sample comprising cells suspected to be
cancer cells.
[0052] The present invention contemplates a computer programme
product which when executed on a computer or data processing device
is capable of carrying out the following steps: [0053] a) allotting
amplified selected and isolated target sequences having consensus
nucleosome bindining regions from a biological sample of an
individual to chromosomes of the genome and identifying unique
allotted target sequences; [0054] b) determining a first amount for
each of one or more first chromosomes identified on the basis of
said unique allotted target sequences originating from said one or
more first chromosomes; and [0055] c) determining a second amount
for each of one or more second chromosomes identified on the basis
of said unique allotted target sequences originating from said one
or more second chromosomes; and [0056] d) determining based on the
said first and second amount a fetal chromosomal aneupoidy of one
or more of said first chromosomes or a chromosomal aberration.
[0057] In a preferred embodiment of said computer programme product
of said determining in step d) further comprises: [0058] i)
determining a parameter from said first amount relative to said
second amount; [0059] ii) comparing the parameter to a
corresponding cut off control value; and based on the comparison,
determining whether or not there is a difference allowing for the
prediction of a chromosomal aberration in one or more of said first
chromosomes or determining whether or not there is a difference
allowing for the prediction of a fetal chromosomal aneuploidy of
one or more of said first chromosomes.
[0060] Finally, the present invention contemplates a device for
carrying out a method of the present invention comprising: [0061]
a) an analyzing unit comprising a subunit capable of selecting for
and isolating from said biological sample of an individual one or
more target sequences of DNA molecules contained in the biological
sample, wherein said target sequences comprise DNA sequences having
consensus nucleosome binding regions, a subunit for amplification
of said target DNA molecules and a subunit for sequencing of
amplified target sequences; and [0062] b) an evaluation unit
comprising a receiving subunit for the sequence data and a computer
or data processing subunit executing the computer programme product
of the present invention.
[0063] The term "device" as used herein relates to a system
comprising at least the aforementioned analyzing unit and the
evaluation unit operatively linked to each other. How to link the
units of the device in an operating manner will depend on the type
of units included into the device. For example, where units for
automatic analysis of a sample are applied, the data obtained by
said automatically operating analyzing unit can be processed by the
computer program product of the evaluation unit in order to obtain
the desired diagnostic results. Preferably, the units are comprised
by a single device in such a case. However, the device may also
comprise physically separate units which are connected by wire,
wireless or via the internet.
[0064] All references cited in this specification are herewith
incorporated by reference with respect to their entire disclosure
content and the disclosure content specifically mentioned in this
specification.
FIGURES
[0065] FIG. 1 shows a flowchart delineating the principle workflow
of a method for performing prenatal diagnosis for the determination
of a fetal chromosomal aneuploidy in a biological sample obtained
from a pregnant woman according to the present invention.
[0066] FIG. 2 (A) shows a plot of percentage representation of
reads uniquely mapped without any mismatch to chromosome 13 in
selected cell-free DNA samples according to an embodiment of the
present invention; (B) shows a plot of percentage representation of
reads uniquely mapped without any mismatch to chromosome 21 in
selected cell-free DNA samples according to an embodiment of the
present invention.
[0067] FIG. 3 (A) shows a plot of percentage representation of
sequence reads uniquely mapped to bait regions on chromosome 13
(150 bp upstream-150 bp downstream) without any mismatch in
selected cell-free DNA samples according to an embodiment of the
present invention; (B) shows a plot of percentage representation of
sequence reads uniquely mapped to bait regions on chromosome 21
(150 bp upstream-150 bp downstream) without any mismatch in
selected cell-free DNA samples according to an embodiment of the
present invention.
[0068] FIG. 4 (A) shows a plot of percentage representation of
sequence reads uniquely mapped to TSS (500 bp upstream-1500 bp
downstream) on chromosome 13 without any mismatch in selected
cell-free DNA samples according to an embodiment of the present
invention; (B) shows a plot of percentage representation of
sequence reads uniquely mapped to TSS (500 bp upstream-1500 bp
downstream) on chromosome 21 without any mismatch in selected
cell-free DNA samples according to an embodiment of the present
invention.
[0069] FIG. 5 shows a plot of percentage representation of
chromosome 13 sequence reads in selected cell-free DNA samples
according to earlier results using shotgun sequencing strategy
without enrichment process.
[0070] In order that the invention described herein may be more
fully understood, the following example is set forth. It is for
illustrative purposes only and shall not be construed as limiting
this invention in any respect.
[0071] It is further understood that the present invention shall
also comprise variations of the expressly disclosed embodiments to
an extent as would be contemplated by a person of ordinary skill in
the art.
EXAMPLES
Example 1
Performance of Prenatal Diagnosis for Detection of Fetal
Chromosomal Disorders by Target Enrichment Technology
[0072] Firstly, up to 15 ml of peripheral blood are taken from a
pregnant woman and collected in tubes containing EDTA. Cell-free
plasma is obtained by centrifugation of the blood sample. The
cell-free plasma DNA is extracted from the plasma by using the
QIAamp DSP DNA Blood Mini Kit (Qiagen) or QIAamp DNA Micro Kit
(Qiagen).
[0073] After DNA extraction a step of target enrichment is carried
out. More specifically, one or more specific DNA sequences
comprising consensus nucleosome binding regions around
transcriptional start sites of protein-coding genes of the
chromosome(s) of interest are selectively enriched by
solution-based hybrid selection technique. For this specific
enrichment the SureSelect Target Enrichment System (Agilent
Technologies) may be used according to the user's manual. For
initiating sample preparation up to 10 ng of cell-free plasma DNA
are used for prepped library production specific to the sequencing
platform utilized downstream such as the Illumina sequencing
instrument. The subsequent library preparation is carried out
according to the corresponding manufacturer's protocol with the
modification that no fragmentation by nebulization or sonication is
done on the cell-free plasma DNA sample.
[0074] In parallel with library production a specific SureSelect
kit containing a mixture of designed SureSelect RNA
oligonucleotides is created on Agilent's web-based design tool.
Table 1 shows an example of a customized kit design suitable for
the detection of chromosomal aneuploidies and sex-linked genetic
diseases caused by a mutation on the X chromosome. In detail, for
each chromosome of interest including reference chromosome(s)
hybridization probes for the regions around the transcriptional
start sites (approximately 1.5 kb) of all known protein-coding
genes on the respective chromosome are generated resulting in an
amount of enriched DNA of about 4 Mb.
TABLE-US-00001 TABLE 1 Example of a customized kit design for
detection of chromosomal aneuploidies and X- linked genetic
diseases Protein-coding Genes approximately enriched (corresponding
to number of kb-length per 1.5 kb hybridisation probes) (Ensembl
around transcription Syndrome or release 55-July 2009; start site
of protein- chromosomal Chromosome www.ensembl.org/index.html)
coding genes disorder 13 359 520 Patau syndrome (trisomy 13) 16
1038 1250 trisomy 16 18 315 470 Edwards syndrome (trisomy 18) 21
265 390 Down syndrome (trisomy 21) X 883 1250 Turner syndrome,
Triple X syndrome, X-1inked genetic diseases Y 86 120 XYY syndrome,
sex- linked genetic diseases 4 Mb
[0075] To perform the DNA-capture the size-selected library is
incubated with the designed SureSelect RNA oligonucleotides, and
the thereby generated RNA-DNA-hybrids are incubated with
streptavidin-labeled magnetic beads in order to allow for capturing
the RNA-DNA hybrids by linking them to the beads. After collecting
the loaded beads by attracting them onto a magnet the beads are
washed and the RNA oligonucleotides are digested whereupon only the
remaining enriched DNA of interest is harvested. After final DNA
amplification and estimation of the quality of the PCR products
using, for example, Agilent 2100 Bioanalyzer (Agilent Technologies)
the enriched pool of target DNA sequences is then subjected to
sequencing by massive parallel sequencing using, for example, the
454 platform (Roche) (Margulies, M et al. 2005 Nature 437,
376-380), Illumina Genome Analyzer or SOLiD System (Applied
Biosystems), which allows for sequencing of many nucleic acid
molecules isolated from one human plasma DNA sample in a parallel
fashion.
[0076] The subsequent bioinformatics procedure is then used to
locate each of these DNA sequences on the human genome. More
specifically, the short reads are collected from the sequencing
instrument and aligned to the human reference genome (hg18, NCBI
build 36 (GenBank accession numbers: NC_000001 to NC_000024) using
several bioinformatic tools such as ELAND (Efficient Large-Scale
Alignment of Nucleotide Databases). To ensure a high quality of the
results, it is preferred that only those reads are considered for
further analysis which are located in pre-selected genomic regions
comprising consensus nucleosome binding regions of the
chromosome(s) of interest and which are uniquely mapped to the
human genome with only one or two mismatches against the human
genome.
[0077] The resulting digital readout of nucleic acid molecules is
then used for the detection of fetal chromosomal aneuploidies, e.g.
trisomy 13, 18 or 21, and can likewise be used for the
determination of the gender of the fetus.
[0078] An imbalance such as a chromosomal aneuploidy in a given
experimental sample is revealed by differences in the number or
percentage of sequences aligned to a given chromosomal region of
interest as compared to the corresponding number or percentage of
such sequences expected or pre-determined for a euploid human
genome sample.
[0079] The present method is suitable for the detection of one or
more chromosomal aneuploidies in one run, wherein the affected
chromosome is typically selected from the group consisting of
chromosome 21 (trisomy 21), chromosome 18 (trisomy 18), chromosome
13 (trisomy 13), and chromosome X (Turner syndrome).
[0080] The selective target enrichment sequencing method according
to the present invention may also be applicable to other diagnostic
applications involving qualitative and/or quantitative evaluation
of serum or plasma nucleic acid contents, e.g., in oncology and
transplantation medicine. For example, the afore described
selective target enrichment sequencing technique on cell-free DNA
may also be used to detect tumor-specific chromosomal alterations
associated with specific cancer.
[0081] The principle of the invention is further described in the
independent claim hereinafter, the various embodiments of the
invention being the subject matter of the dependent claims.
Example 2
Performance of Prenatal Diagnosis for Detection of Trisomy 21 and
Trisomy 13 by Target Enrichment Technology and Multiplexed Barcode
Sequencing
[0082] For the study maternal blood samples were selected from 4
singleton pregnancies. One of the pregnant women was carrying an
euploid male fetus and the other three were carrying a fetus with
trisomy 21, trisomy 13 and trisomy 18, respectively (Table 2,
below). Up to 15 ml of peripheral venous blood were taken from
these pregnant women and collected in EDTA tubes. The plasma was
obtained from the blood samples by centrifugation at 1600 g at
4.degree. C. for 10 min. To remove residual cells the plasma was
additionally centrifuged at 16000 g at 4.degree. C. for 10 min.
From plasma samples the cell-free DNA was extracted from 0.8-1 ml
of plasma by using the QIAamp Circulating Nucleic Acid Kit (Qiagen)
according to manufacture's protocol.
[0083] Up to 10 ng of cell-free plasma DNA was then used to
construct Illumina sequencing libraries. The DNA library
preparation followed the Illumina standard sample preparation
protocol for paired-end sequencing with a few modifications.
Briefly, no fragmentation by nebulization or sonication was done on
the cell-free plasma DNA samples. The library preparation was
carried out according to the beta Chromatin Immunoprecipitation
Sequencing (ChIP-Seg) sample preparation protocol (Illumina; Part
#11257047 Rev. A) using enzymes from Fermentas (T4 DNA Polymerase,
Klenow DNA Polymerase, T4 polynucleotide kinase, DNA Ligase) as
well as from Finnzymes Oy (Phusion* Polymerase). The products were
end-repaired and 3' non-template A's were added. To make
multiplexed barcode sequencing available, DNA libraries were
"tagged" with different identifiers (barcodes) during paired-end
adaptor ligation. The first adaptor contained the sequencing primer
sites for application read 1 and a 4 bp-identifier as well as a `T`
which is necessary for adaptor ligation.
[0084] The second adaptor contained the sequencing primer sites for
application read 2 and a `T` too. The DNA libraries with barcoded
samples were then additionally amplified using a 12-cycle PCR and
primers containing the attachment sites for the flow cells. The
adapter-ligated DNA fragments were size selected in the range of
150-300 bp using 2% agarose gel electorphoresis. A quality control
and quantification of libraries were done using a High Sensitivity
DNA kit on the Agilent 2100 Bioanalyzer according to the
manufacturer's instructions.
[0085] To target all human exons and their associated human genomic
regions corresponding to the transcription start sites (TSSs), 500
ng of libraries were incubated with the SureSelect Human All Exon
Kit (Agilent Technologies) and enriched according to the
manufacturer's protocol. After elution of the captured DNA
fragments, the libraries were reamplified for 12-14 cycles of PCR
with SureSelect Illumina-specific primers. Amplification enables
accurate quantification using the Bioanalyzer High Sensitivity chip
before sequencing.
[0086] The four different barcoded samples are then pooled into a
single tube and clonal clusters were generated using cBOT clonal
amplification system with the cBOT Paired-End Cluster Generation
Kit. Following Illuminas sequencing workflow the amplified
single-molecule DNA templates were sequenced using massive parallel
synthesis on Illumina Genome Analyzer IIx.
[0087] The subsequent bioinformatics procedure included image
analysis, base calling and alignment by using Illumina's pipeline
software. For individual downstream analysis a semi-automated tag
sorting strategy identified each uniquely barcoded sample. The
first 32-bp of each read of each sample were aligned to the
repeat-masked human genomic reference sequence NCBI build 36 (also
known as hg18; GenBank accession numbers: NC_000001 to NC_000024)
downloaded from UCSC Genome Browser using ELAND alignment software
(GAPipeline-1.4.0 software) provided by Illumina.
[0088] The resulting digital readouts of nucleic acid molecules
were then used for the detection of fetal chromosomal aneuploidies,
e.g. trisomy 13 or 21, and can likewise be used for the
determination of the gender of the fetus.
[0089] Initially, the total number of sequenced reads for each
sample were counted. Subsequently, only sorted reads that had
uniquely mapped to one location in the repeat-masked human genomic
reference sequence and without any nucleotide mismatch were used
for further analysis (see Table 2, below).
[0090] In the first place, an imbalance such as trisomy 21 and
trisomy 13 in the given experimental samples was revealed by
differences in the number or percentage of repeat-masked uniquely
mapped reads without any mismatch of interest (originating from
chromosomes 13 and 21, respectively) as compared to the
corresponding number or percentage of such sequences determined for
the euploid human genome sample. The expected percentage of
representation of each chromosome was obtained by dividing the
number of repeat-masked uniquely mapped reads without any mismatch
per chromosome by the number of total repeat-masked uniquely mapped
reads without any mismatch of all chromosomes. As shown in FIG. 2A,
the percentage of reads uniquely mapped to chromosome 13 from
sample S_T13 was higher than that from sample S_euploid with an
euploid fetus as well as from sample S_T21 carrying a fetus with
Trisomie 21. The percentage of reads uniquely mapped to chromosome
21 from sample S_T21 was also higher than that from sample
S_euploid with an euploid fetus as well as from sample S_T13
carrying a fetus with Trisomie 13 (FIG. 2B).
[0091] In the second place, an imbalance such as trisomy 21 and
trisomy 13 in the given experimental samples was revealed by
differences in the number or percentage of repeat-masked uniquely
mapped reads without any mismatch aligned to a given chromosomal
region of interest compared to the corresponding number or
percentage of such sequences determined for the euploid human
genome sample. The chromosomal region of interest was characterized
by the predetermined 120 bp-bait regions (available at eArray
platform by Agilent Technologies) of the SureSelect Human All Exon
Kit (Agilent Technologies) plus flanking 150 bp-regions upstream
and downstream located of bait regions. The expected percentage of
representation of each chromosome was then obtained as described
before. An overrepresentation of uniquely mapped reads was observed
for chromosomes 13 and chromosomes 21 in T13 and T21 cases,
respectively (FIGS. 3A and 3B).
[0092] In the third place, an imbalance such as trisomy 21 and
trisomy 13 in the given experimental samples was revealed by
differences in the number or percentage of repeat-masked uniquely
mapped reads without any mismatch aligned to a given consensus
nucleosome binding region as compared to the corresponding number
or percentage of such sequences expected or pre-determined for the
euploid human genome sample. The consensus nucleosome binding
region as used herein included sequence regions from 500 bp
upstream the TSS to 1500 bp downstream the TSS. The percentage of
reads uniquely mapped to chromosome 13 from sample S_T13 was higher
than that from sample S_euploid with an euploid fetus as well as
that from sample S_T13 (FIG. 4A). The percentage of reads uniquely
mapped to chromosome 21 from sample S_T21 was only higher than that
from sample S_T13 and not higher than that from sample S_euploid
with an euploid fetus (FIG. 4B).
[0093] The present method was suitable for the detection of one or
more chromosomal aneuploidies in one run, wherein the affected
chromosome is typically selected from the group consisting of
chromosome 21 (trisomy 21), chromosome 18 (trisomy 18), chromosome
13 (trisomy 13), and chromosome X (Turner syndrome).
[0094] In comparison to previous experiments (FIG. 5 and Table 3,
below) using solely shotgun sequencing this method was appropriate
to even detect a trisomy 13.
[0095] Furthermore, the described method tends to result in
reduction of storage capacities for raw data as well as for mapping
and alignment of generated sequence reads.
TABLE-US-00002 TABLE 2 (i) Summary of clinical data and number of
sequence reads of Example 2 " Performance of prenatal diagnosis for
detection of Trisomy 21 and Trisomy 13 by target enrichment
technology and multiplexed barcode sequencing" Gestational Total
no. of Total no. of Age sequence sequence Sample Karyotype (weeks +
days) reads_read1 reads_read2 S_T13 47XY + 13 13 + 5 5005094
5005094 S_T21 47XY + 21 13 + 0 6739231 6739231 S_euploid 46XY 16 +
0 3415786 3415786 No. of uniquely No. of uniquely No. of uniquely
No. of uniquely Total no. Total no. mapped reads mapped reads
mapped reads mapped reads of uniquely of uniquely without any
without any without any without any mapped reads mapped reads
mismatch of mismatch of mismatch of mismatch of without any without
any chromosome chromosome chromosome chromosome Sample
mismatch_read1 mismatch_read2 13_read1 13_read1 21_read1 21_read2
(ii) Summary of number of sequence reads of Example 2 S_T13 2856635
2745340 8643 83057 32290 31010 S_T21 3913173 3761195 109842 105110
45647 44115 S_euploid 1959738 1895396 56616 54658 2191 21186 (iii)
Summary of number of sequence reads of a chromosomal region of
interest (characterized by the predetermined 120 bp-bait regions of
the SureSelect Human All Exon Kit plus flanking 150 bp-regions
upstream and downstream located of bait regions) of Example 2 S_T13
1599317 1530800 42125 40162 17060 16256 S_T21 2215860 2120634 54244
51598 24433 23345 S_euploid 1091265 1051800 27601 26577 11679 11303
(iv) Summary of number of sequence reads aligned to a given
consensus nucleosome binding region including sequence regions from
500 bp upstream the TSS to 1500 bp downstream the TSS (Example 2)
S_T13 202723 196213 4254 4087 2755 2652 S_T21 282640 273834 5520
5408 3951 3814 S_euploid 135892 131693 2665 2567 1880 1857
TABLE-US-00003 TABLE 3 Summary of number of sequence reads of
previous experiments using shotgun sequencing method Total no. of
No. of uniquely No. of uniquely Total uniquely mapped reads mapped
reads no. of mapped reads without any without any sequence without
any mismatch of mismatch of Sample reads mismatch chromosome 13
chromosome 21 S_T13 16611762 5992776 215666 73885 S_T21 26137898
10752119 402319 141130 S_euploid 20289419 7928946 285433 99306
* * * * *
References