Patent Grant
12054777
Applicants assert that the accompanying text copy of the Sequence Listing is identical to the Sequence Listing in computer readable form submitted in U.S. application Ser. No. 12/560,708. Applicants incorporate the contents of the sequence listing by reference in its entirety.
The present invention relates to the field of molecular diagnostics, and more particularly to the field of prenatal genetic diagnosis.
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. That is, certain components of the present invention may be described in greater detail in the materials discussed below. The discussion below should not be construed as an admission as to the relevance of the information to the claimed invention or the prior art effect of the material described.
Fetal aneuploidy and other chromosomal aberrations affect 9 out of 1000 live births (1). The gold standard for diagnosing chromosomal abnormalities is karyotyping of fetal cells obtained via invasive procedures such as chorionic villus sampling and amniocentesis. These procedures impose small but potentially significant risks to both the fetus and the mother (2). Non-invasive screening of fetal aneuploidy using maternal serum markers and ultrasound are available but have limited reliability (3-5). There is therefore a desire to develop non-invasive genetic tests for fetal chromosomal abnormalities.
Since the discovery of intact fetal cells in maternal blood, there has been intense interest in trying to use them as a diagnostic window into fetal genetics (6-9). While this has not yet moved into practical application (10), the later discovery that significant amounts of cell-free fetal nucleic acids also exist in maternal circulation has led to the development of new non-invasive prenatal genetic tests for a variety of traits (11, 12). However, measuring aneuploidy remains challenging due to the high background of maternal DNA; fetal DNA often constitutes <10% of total DNA in maternal cell-free plasma (13).
Recently developed methods for aneuploidy rely on detection focus on allelic variation between the mother and the fetus. Lo et al. demonstrated that allelic ratios of placental specific mRNA in maternal plasma could be used to detect trisomy 21 in certain populations (14).
Similarly, they also showed the use of allelic ratios of imprinted genes in maternal plasma DNA to diagnose trisomy 18 (15). Dhallan et al. used fetal specific alleles in maternal plasma DNA to detect trisomy 21 (16). However, these methods are limited to specific populations because they depend on the presence of genetic polymorphisms at specific loci. We and others argued that it should be possible in principle to use digital PCR to create a universal, polymorphism independent test for fetal aneuploidy using maternal plasma DNA (17-19).
An alternative method to achieve digital quantification of DNA is direct shotgun sequencing followed by mapping to the chromosome of origin and enumeration of fragments per chromosome. Recent advances in DNA sequencing technology allow massively parallel sequencing (20), producing tens of millions of short sequence tags in a single run and enabling a deeper sampling than can be achieved by digital PCR. As is known in the art, the term “sequence tag” refers to a relatively short (e.g., 15-100) nucleic acid sequence that can be used to identify a certain larger sequence, e.g., be mapped to a chromosome or genomic region or gene. These can be ESTs or expressed sequence tags obtained from mRNA.
Specific Patents and Publications
Science 309:1476 (2 Sep. 2005) News Focus “An Earlier Look at Baby's Genes” describes attempts to develop tests for Down Syndrome using maternal blood. Early attempts to detect Down Syndrome using fetal cells from maternal blood were called “just modestly encouraging.” The report also describes work by Dennis Lo to detect the Rh gene in a fetus where it is absent in the mother. Other mutations passed on from the father have reportedly been detected as well, such as cystic fibrosis, beta-thalassemia, a type of dwarfism and Huntington's disease. However, these results have not always been reproducible.
Venter et al., “The sequence of the human genome,” Science, 2001 Feb. 16; 291(5507):1304-51 discloses the sequence of the human genome, which information is publicly available from NCBI. Another reference genomic sequence is a current NCBI build as obtained from the UCSC genome gateway.
Wheeler et al., “The complete genome of an individual by massively parallel DNA sequencing,” Nature, 2008 Apr. 17; 452(7189):872-6 discloses the DNA sequence of a diploid genome of a single individual, James D. Watson, sequenced to 7.4-fold redundancy in two months using massively parallel sequencing in picolitre-size reaction vessels. Comparison of the sequence to the reference genome led to the identification of 3.3 million single nucleotide polymorphisms, of which 10,654 cause amino-acid substitution within the coding sequence.
Quake et al., US 2007/0202525 entitled “Non-invasive fetal genetic screening by digital analysis,” published Aug. 30, 2007, discloses a process in which maternal blood containing fetal DNA is diluted to a nominal value of approximately 0.5 genome equivalent of DNA per reaction sample.
Chiu et al., “Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic DNA sequencing of DNA in maternal plasma,” Proc. Natl. Acad. Sci. 105(51):20458-20463 (Dec. 23, 2008) discloses a method for determining fetal aneuploidy using massively parallel sequencing. Disease status determination (aneuploidy) was made by calculating a “z score.” Z scores were compared with reference values, from a population restricted to euploid male fetuses. The authors noted in passing that G/C content affected the coefficient of variation.
Lo et al., “Diagnosing Fetal Chromosomal Aneuploidy Using Massively Parallel Genomic Sequencing,” US 2009/0029377, published Jan. 29, 2009, discloses a method in which respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of massively parallel sequencing. It was found that the percentage representation of sequences mapped to chromosome 21 is higher in a pregnant woman carrying a trisomy 21 fetus when compared with a pregnant woman carrying a normal fetus. 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.
Lo et al., Determining a Nucleic Acid Sequence Imbalance,” US 2009/0087847 published Apr. 2, 2009, discloses a method for determining whether a nucleic acid sequence imbalance exists, such as an aneuploidy, the method comprising deriving a first cutoff value from an average concentration of a reference nucleic acid sequence in each of a plurality of reactions, wherein the reference nucleic acid sequence is either the clinically relevant nucleic acid sequence or the background nucleic acid sequence; comparing the parameter to the first cutoff value; and based on the comparison, determining a classification of whether a nucleic acid sequence imbalance exists.
The following brief summary is not intended to include all features and aspects of the present invention, nor does it imply that the invention must include all features and aspects discussed in this summary.
The present invention comprises a method for analyzing a maternal sample, e.g., from peripheral blood. It is not invasive into the fetal space, as is amniocentesis or chorionic villi sampling. In the preferred method, fetal DNA which is present in the maternal plasma is used. The fetal DNA is in one aspect of the invention enriched due to the bias in the method towards shorter DNA fragments, which tend to be fetal DNA. The method is independent of any sequence difference between the maternal and fetal genome. The DNA obtained, preferably from a peripheral blood draw, is a mixture of fetal and maternal DNA. The DNA obtained is at least partially sequenced, in a method which gives a large number of short reads. These short reads act as sequence tags, in that a significant fraction of the reads are sufficiently unique to be mapped to specific chromosomes or chromosomal locations known to exist in the human genome. They are mapped exactly, or may be mapped with one mismatch, as in the examples below. By counting the number of sequence tags mapped to each chromosome (1-22, X and Y), the over- or under-representation of any chromosome or chromosome portion in the mixed DNA contributed by an aneuploid fetus can be detected. This method does not require the sequence differentiation of fetal versus maternal DNA, because the summed contribution of both maternal and fetal sequences in a particular chromosome or chromosome portion will be different as between an intact, diploid chromosome and an aberrant chromosome, i.e., with an extra copy, missing portion or the like. In other words, the method does not rely on a priori sequence information that would distinguish fetal DNA from maternal DNA. The abnormal distribution of a fetal chromosome or portion of a chromosome (i.e., a gross deletion or insertion) may be determined in the present method by enumeration of sequence tags as mapped to different chromosomes. The median count of autosomal values (i.e., number of sequence tags per autosome) is used as a normalization constant to account for differences in total number of sequence tags is used for comparison between samples and between chromosomes The term “chromosome portion” is used herein to denote either an entire chromosome or a significant fragment of a chromosome. For example, moderate Down syndrome has been associated with partial trisomy 21q22.2→qter By analyzing sequence tag density in predefined subsections of chromosomes (subchromosomal sequences, e.g., 10 to 100 kb windows), a normalization constant can be calculated, and chromosomal subsections quantified (e.g., 21q22.2). With large enough sequence tag counts, the present method can be applied to arbitrarily small fractions of fetal DNA. It has been demonstrated to be accurate down to 6% fetal DNA concentration. Exemplified below is the successful use of shotgun sequencing and mapping of DNA to detect fetal trisomy 21 (Down syndrome), trisomy 18 (Edward syndrome), and trisomy 13 (Patau syndrome), carried out non-invasively using cell-free fetal DNA in maternal plasma. This forms the basis of a universal, polymorphism-independent non-invasive diagnostic test for fetal aneuploidy. The sequence data also allowed us to characterize plasma DNA in unprecedented detail, suggesting that it is enriched for nucleosome bound fragments. The method may also be employed so that the sequence data obtained may be further analyzed to obtain information regarding polymorphisms and mutations.
Thus, the present invention comprises, in certain aspects, a method of testing for an abnormal distribution of a specified chromosome portion in a mixed sample of normally and abnormally distributed chromosome portions obtained from a single subject, such as a mixture of fetal and maternal DNA in a maternal plasma sample. One carries out sequence determinations on the DNA fragments in the sample, obtaining sequences from multiple chromosome portions of the mixed sample to obtain a number of sequence tags of sufficient length of determined sequence to be assigned to a chromosome location within a genome and of sufficient number to reflect abnormal distribution. Using a reference sequence, one assigns the sequence tags to their corresponding chromosomes including at least the specified chromosome by comparing the sequence to reference genomic sequence. Often there will be on the order of millions of short sequence tags that are assigned to certain chromosomes, and, importantly, certain positions along the chromosomes. One then may determine a first number of sequence tags mapped to at least one normally distributed chromosome portion and a second number of sequence tags mapped to the specified chromosome portion, both chromosomes being in one mixed sample. The present method also involves correcting for nonuniform distribution sequence tags to different chromosomal portions. This is explained in detail below, where a number of windows of defined length are created along a chromosome, the windows being on the order of kilobases in length, whereby a number of sequence tags will fall into many of the windows and the windows covering each entire chromosome in question, with exceptions for non-informative regions, e.g., centromere regions and repetitive regions. Various average numbers, i.e., median values, are calculated for different windows and compared. By counting sequence tags within a series of predefined windows of equal lengths along different chromosomes, more robust and statistically significant results may be obtained. The present method also involves calculating a differential between the first number and the second number which is determinative of whether or not the abnormal distribution exists.
In certain aspects, the present invention may comprise a computer programmed to analyze sequence data obtained from a mixture of maternal and fetal chromosomal DNA. Each autosome (chr. 1-22) is computationally segmented into contiguous, non-overlapping windows. (A sliding window could also be used). Each window is of sufficient length to contain a significant number of reads (sequence tags, having about 20-100 bp of sequence) and not still have a number of windows per chromosome. Typically, a window will be between 10 kb and 100 kb, more typically between 40 and 60 kb. There would, then, for example, accordingly be approximately between 3,000 and 100,000 windows per chromosome. Windows may vary widely in the number of sequence tags that they contain, based on location (e.g., near a centromere or repeating region) or G/C content, as explained below. The median (i.e., middle value in the set) count per window for each chromosome is selected; then the median of the autosomal values is used to account for differences in total number of sequence tags obtained for different chromosomes and distinguish interchromosomal variation from sequencing bias from aneuploidy. This mapping method may also be applied to discern partial deletions or insertions in a chromosome. The present method also provides a method for correcting for bias resulting from G/C content. For example, some the Solexa sequencing method was found to produce more sequence tags from fragments with increased G/C content. By assigning a weight to each sequence tag based on the G/C content of a window in which the read falls. The window for GC calculation is preferably smaller than the window for sequence tag density calculation.
That is, in the left panels (A and C), one plots the distribution of reads per chromosome coordinate, i.e., chromosomal position in terms of number of reads within each 50 kb non-overlapping sliding window. Then, one determines the distribution of the number of sequence tags for each 50 kb window, and obtains a median number of sequence tags per chromosome for all autosomes and chromosome X (Examples of chr 1 [top] and chr 22 [bottom] are illustrated here). These results are referred to as M. The median of the 22 values of M (from all autosomes, chromosomes 1 through 22) is used as the normalization constant N. The normalized sequence tag density of each chromosome is M/N (e.g., chr 1: M1/N; chr 22: M22/N). Such normalization is necessary to compare different patient samples since the total number of sequence tags (thus, the sequence tag density) for each patient sample is different (the total number of sequence tags fluctuates between ˜8 to ˜12 million). The analysis thus flows from frequency of reads per coordinate (A and C) to # reads per window (B and D) to a combination of all chromosomes.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Generally, nomenclatures utilized in connection with, and techniques of, cell and molecular biology and chemistry are those well known and commonly used in the art. Certain experimental techniques, not specifically defined, are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. For purposes of the clarity, following terms are defined below.
“Sequence tag density” means the normalized value of sequence tags for a defined window of a sequence on a chromosome (in a preferred embodiment the window is about 50 kb), where the sequence tag density is used for comparing different samples and for subsequent analysis. A “sequence tag” is a DNA sequence of sufficient length that it may be assigned specifically to one of chromosomes 1-22, X or Y. It does not necessarily need to be, but may be non-repetitive within a single chromosome. A certain, small degree of mismatch (0-1) may be allowed to account for minor polymorphisms that may exist between the reference genome and the individual genomes (maternal and fetal) being mapped. The value of the sequence tag density is normalized within a sample. This can be done by counting the number of tags falling within each window on a chromosome; obtaining a median value of the total sequence tag count for each chromosome; obtaining a median value of all of the autosomal values; and using this value as a normalization constant to account for the differences in total number of sequence tags obtained for different samples. A sequence tag density as calculated in this way would ideally be about 1 for a disomic chromosome. As further described below, sequence tag densities can vary according to sequencing artifacts, most notably G/C bias; this is corrected as described. This method does not require the use of an external standard, but, rather, provides an internal reference, derived from al of the sequence tags (genomic sequences), which may be, for example, a single chromosome or a calculated value from all autosomes.
“T21” means trisomy 21.
“T18” means trisomy 18.
“T13” means trisomy 13.
“Aneuploidy” is used in a general sense to mean the presence or absence of an entire chromosome, as well as the presence of partial chromosomal duplications or deletions or kilobase or greater size, as opposed to genetic mutations or polymorphisms where sequence differences exist.
“Massively parallel sequencing” means techniques for sequencing millions of fragments of nucleic acids, e.g., using attachment of randomly fragmented genomic DNA to a planar, optically transparent surface and solid phase amplification to create a high density sequencing flow cell with millions of clusters, each containing ˜1,000 copies of template per sq. cm. These templates are sequenced using four-color DNA sequencing-by-synthesis technology. See, products offered by Illumina, Inc., San Diego, California. In the present work, sequences were obtained, as described below, with an Illumina/Solexa 1G Genome Analyzer. The Solexa/Illumina method referred to below relies on the attachment of randomly fragmented genomic DNA to a planar, optically transparent surface. In the present case, the plasma DNA does not need to be sheared. Attached DNA fragments are extended and bridge amplified to create an ultra-high density sequencing flow cell with 50 million clusters, each containing ˜1,000 copies of the same template. These templates are sequenced using a robust four-color DNA sequencing-by-synthesis technology that employs reversible terminators with removable fluorescent dyes. This novel approach ensures high accuracy and true base-by-base sequencing, eliminating sequence-context specific errors and enabling sequencing through homopolymers and repetitive sequences.
High-sensitivity fluorescence detection is achieved using laser excitation and total internal reflection optics. Short sequence reads are aligned against a reference genome and genetic differences are called using specially developed data analysis pipeline software.
Copies of the protocol for whole genome sequencing using Soelxa technology may be found at BioTechniques® Protocol Guide 2007 Published December 2006: p 29, www(dot)biotechniques.com/default.asp?page=protocol&subsection=article_display&id=112378. Solexa's oligonucleotide adapters are ligated onto the fragments, yielding a fully-representative genomic library of DNA templates without cloning. Single molecule clonal amplification involves six steps: Template hybridization, template amplification, linearization, blocking 3′ ends, denaturation and primer hybridization. Solexa's Sequencing-by-Synthesis utilizes four proprietary nucleotides possessing reversible fluorophore and termination properties. Each sequencing cycle occurs in the presence of all four nucleotides.
The presently used sequencing is preferably carried out without a preamplification or cloning step, but may be combined with amplification-based methods in a microfluidic chip having reaction chambers for both PCR and microscopic template-based sequencing. Only about 30 bp of random sequence information are needed to identify a sequence as belonging to a specific human chromosome. Longer sequences can uniquely identify more particular targets. In the present case, a large number of 25 bp reads were obtained, and due to the large number of reads obtained, the 50% specificity enabled sufficient sequence tag representation.
Further description of a massively parallel sequencing method, which employed the below referenced 454 method is found in Rogers and Ventner, “Genomics: Massively parallel sequencing,” Nature, 437, 326-327 (15 Sep. 2005). As described there, Rothberg and colleagues (Margulies, M. et al. Nature 437, 376-380 (2005)), have developed a highly parallel system capable of sequencing 25 million bases in a four-hour period—about 100 times faster than the current state-of-the-art Sanger sequencing and capillary-based electrophoresis platform. The method could potentially allow one individual to prepare and sequence an entire genome in a few days. The complexity of the system lies primarily in the sample preparation and in the microfabricated, massively parallel platform, which contains 1.6 million picoliter-sized reactors in a 6.4-cm2 slide. Sample preparation starts with fragmentation of the genomic DNA, followed by the attachment of adaptor sequences to the ends of the DNA pieces. The adaptors allow the DNA fragments to bind to tiny beads (around 28μ in diameter). This is done under conditions that allow only one piece of DNA to bind to each bead. The beads are encased in droplets of oil that contain all of the reactants needed to amplify the DNA using a standard tool called the polymerase chain reaction. The oil droplets form part of an emulsion so that each bead is kept apart from its neighbor, ensuring the amplification is uncontaminated. Each bead ends up with roughly 10 million copies of its initial DNA fragment. To perform the sequencing reaction, the DNA-template-carrying beads are loaded into the picoliter reactor wells—each well having space for just one bead. The technique uses a sequencing-by-synthesis method developed by Uhlen and colleagues, in which DNA complementary to each template strand is synthesized. The nucleotide bases used for sequencing release a chemical group as the base forms a bond with the growing DNA chain, and this group drives a light-emitting reaction in the presence of specific enzymes and luciferin. Sequential washes of each of the four possible nucleotides are run over the plate, and a detector senses which of the wells emit light with each wash to determine the sequence of the growing strand. This method has been adopted commercially by 454 Life Sciences.
Further examples of massively parallel sequencing are given in US 20070224613 by Strathmann, published Sept. 27, 2007, entitled “Massively Multiplexed Sequencing.” Also, for a further description of massively parallel sequencing, see US 2003/0022207 to Balasubramanian, et al., published Jan. 30, 2003, entitled “Arrayed polynucleotides and their use in genome analysis.”
Non-invasive prenatal diagnosis of aneuploidy has been a challenging problem because fetal DNA constitutes a small percentage of total DNA in maternal blood (13) and intact fetal cells are even rarer (6, 7, 9, 31, 32). We showed in this study the successful development of a truly universal, polymorphism-independent non-invasive test for fetal aneuploidy. By directly sequencing maternal plasma DNA, we could detect fetal trisomy 21 as early as 14th week of gestation. Using cell-free DNA instead of intact cells allows one to avoid complexities associated with microchimerism and foreign cells that might have colonized the mother; these cells occur at such low numbers that their contribution to the cell-free DNA is negligible (33, 34). Furthermore, there is evidence that cell-free fetal DNA clears from the blood to undetectable levels within a few hours of delivery and therefore is not carried forward from one pregnancy to the next (35-37).
Rare forms of aneuploidy caused by unbalanced translocations and partial duplication of a chromosome are in principle detectable by the approach of shotgun sequencing, since the density of sequence tags in the triplicated region of the chromosome would be higher than the rest of the chromosome. Detecting incomplete aneuploidy caused by mosaicism is also possible in principle but may be more challenging, since it depends not only on the concentration of fetal DNA in maternal plasma but also the degree of fetal mosaicism. Further studies are required to determine the effectiveness of shotgun sequencing in detecting these rare forms of aneuploidy.
The present method is applicable to large chromosomal deletions, such as 5p− Syndrome (five p minus), also known as Cat Cry Syndrome or Cri du Chat Syndrome. 5p− Syndrome is characterized at birth by a high-pitched cry, low birth weight, poor muscle tone, microcephaly, and potential medical complications. Similarly amenable disorders addressed by the present methods are p−, monosomy 9P, otherwise known as Alfi's Syndrome or 9P-, 22q11.2 deletion syndrome, Emanuel Syndrome, also known in the medical literature as the Supernumerary Der(22) Syndrome, trisomy 22, Unbalanced 11/22 Translocation or partial trisomy 11/22, Microdeletion and Microduplication at 16p11.2, which is associated with autism, and other deletions or imbalances, including those that are presently unknown.
An advantage of using direct sequencing to measure aneuploidy non-invasively is that it is able to make full use of the sample, while PCR based methods analyze only a few targeted sequences. In this study, we obtained on average 5 million reads per sample in a single run, of which ˜66,000 mapped to chromosome 21. Since those 5 million reads represent only a portion of one human genome, in principle less than one genomic equivalent of DNA is sufficient for the detection of aneuploidy using direct sequencing. In practice, a larger amount of DNA was used since there is sample loss during sequencing library preparation, but it may be possible to further reduce the amount of blood required for analysis.
Mapping shotgun sequence information (i.e., sequence information from a fragment whose physical genomic position is unknown) can be done in a number of ways, which involve alignment of the obtained sequence with a matching sequence in a reference genome. See, Li et al., “Mapping short DNA sequencing reads and calling variants using mapping quality score,” Genome Res., 2008 Aug. 19. [Epub ahead of print].
We observed that certain chromosomes have large variations in the counts of sequenced fragments (from sample to sample, and that this depends strongly on the G/C content (
Plasma DNA samples used in this study were obtained about 15 to 30 minutes after amniocentesis or chorionic villus sampling. Since these invasive procedures disrupt the interface between the placenta and maternal circulation, there have been discussions whether the amount of fetal DNA in maternal blood might increase following invasive procedures. Neither of the studies to date have observed a significant effect (41, 42).
Our results support this conclusion, since using the digital PCR assay we estimated that fetal DNA constituted less than or equal to 10% of total cell-free DNA in the majority of our maternal plasma samples. This is within the range of previously reported values in maternal plasma samples obtained prior to invasive procedures (13). It would be valuable to have a direct measurement addressing this point in a future study.
The average fetal DNA fraction estimated from sequencing data is higher than the values estimated from digital PCR data by an average factor of two (p<0.005, paired t-test on all male pregnancies that have complete set of data). One possible explanation for this is that the PCR step during Solexa library preparation preferentially amplifies shorter fragments, which others have found to be enriched for fetal DNA (22, 23). Our own measurements of length distribution on one sample do not support this explanation, but nor can we reject it at this point. It should also be pointed out that using the sequence tags we find some variation of fetal fraction even in the same sample depending on which chromosome we use to make the calculation (
Our sequencing data suggest that the majority of cell-free plasma DNA is of apoptotic origin and shares features of nucleosomal DNA. Since nucleosome occupancy throughout the eukaryotic genome is not necessarily uniform and depends on factors such as function, expression, or sequence of the region (30, 43), the representation of sequences from different loci in cell-free maternal plasma may not be equal, as one usually expects in genomic DNA extracted from intact cells. Thus, the quantity of a particular locus may not be representative of the quantity of the entire chromosome and care must be taken when one designs assays for measuring gene dosage in cell-free maternal plasma DNA that target only a few loci.
Historically, due to risks associated with chorionic villus sampling and amniocentesis, invasive diagnosis of fetal aneuploidy was primarily offered to women who were considered at risk of carrying an aneuploid fetus based on evaluation of risk factors such as maternal age, levels of serum markers, and ultrasonographic findings. Recently, an American College of Obstetricians and Gynecologists (ACOG) Practice Bulletin recommended that “invasive diagnostic testing for aneuploidy should be available to all women, regardless of maternal age” and that “pretest counseling should include a discussion of the risks and benefits of invasive testing compared with screening tests” (2).
A noninvasive genetic test based on the results described here and in future large-scale studies would presumably carry the best of both worlds: minimal risk to the fetus while providing true genetic information. The costs of the assay are already fairly low; the sequencing cost per sample as of this writing is about $700 and the cost of sequencing is expected to continue to drop dramatically in the near future.
Shotgun sequencing can potentially reveal many more previously unknown features of cell-free nucleic acids such as plasma mRNA distributions, as well as epigenetic features of plasma DNA such as DNA methylation and histone modification, in fields including perinatology, oncology and transplantation, thereby improving our understanding of the basic biology of pregnancy, early human development and disease.
Sequencing Methods
Commercially available sequencing equipment was used in the present illustrative examples, namely the Solexa/Illumina sequencing platform and the 454/Roche platform. It will be apparent to those skilled in the art that a number of different sequencing methods and variations can be used. One sequencing method that can be used to advantage in the present methods involves paired end sequencing. Fluorescently labeled sequencing primers could be used to simultaneously sequence both strands of a dsDNA template, as described e.g., in Wiemann et al. (Anal. Biochem. 224: 117 [1995]; Anal. Biochem. 234: 166 [1996]. Recent examples of this technique have demonstrated multiplex co-sequencing using the four-color dye terminator reaction chemistry pioneered by Prober et al. (Science 238: 336 [1987]). Solexa/Illumina offers a “Paired End Module” to its Genome Analyzer. Using this module, after the Genome Analyzer has completed the first sequencing read, the Paired-End Module directs the resynthesis of the original templates and the second round of cluster generation. The Paired-End Module is connected to the Genome Analyzer through a single fluidic connection. In addition, 454 has developed a protocol to generate a library of Paired End reads. These Paired End reads are approximately 84-nucleotide DNA fragments that have a 44-mer adaptor sequence in the middle flanked by a 20-mer sequence on each side. The two flanking 20-mers are segments of DNA that were originally located approximately 2.5 kb apart in the genome of interest.
By using paired end reads in the present method, one may obtain more sequence information from a given plasma DNA fragment, and, significantly, one may also obtain sequence information from both ends of the fragment. The fragment is mapped to the human genome as explained here elsewhere. After mapping both ends, one may deduce the length of the starting fragment. Since fetal DNA is known to be shorter than maternal DNA fragments circulating in plasma, one may use this information about the length of the DNA fragment to effectively increase the weight given to sequences obtained from shorter (e.g., about 300 bp or less) DNA fragments. Methods for weighting are given below.
Another method for increasing sensitivity to fetal DNA is to focus on certain regions within the human genome. One may use sequencing methods which select a priori sequences which map to the chromosomes of interest (as described here elsewhere, such as 18, 21, 13, X and Y). One may also choose to focus, using this method, on partial chromosomal deletions, such as 22q11 deletion syndrome. Other microdeletions and microduplications are set forth in Table 1 of US 2005/0181410, published Aug. 18, 2005 under the title “Methods and apparatuses for achieving precision genetic diagnosis.”
In sequencing selected subsequences, one may employ sequence-based methodologies such as sequencing by array, or capture beads with specific genomic sequences used as capture probes. The use of a sequencing array can be implemented as described in Chetverin et al., “Oligonucleotide arrays: new concepts and possibilities,” Biotechnology (N Y). 1994 November; 12(11):1093-9, as well as Rothberg, US 2002/0012930 A1 entitled “Method of Sequencing a Nucleic Acid,” and Reeve et al., “Sequencing by Hybridization,” U.S. Pat. No. 6,399,364. In these methods, the target nucleic acid to be sequenced may be genomic DNA, cDNA or RNA. The sample is rendered single stranded and captured under hybridizing conditions with a number of single stranded probes which are catalogued by bar coding or by physical separation in an array. Emulsion PCR, as used in the 454 system, the SOLiD system, and Polonator (Dover Systems) and others may also be used, where capture is directed to specific target sequences, e.g., genome sequences mapping uniquely to chromosome 21 or other chromosome of interest, or to a chromosome region such as 15q11 (Prader-Willi syndrome), or excessive CGG repeats in the FMR1 gene (fragile X syndrome).
The subsequencing method is in one aspect contrary to conventional massively parallel sequencing methodologies, which seek to obtain all of the sequence information in a sample. This alternative method selectively ignores certain sequence information by using a sequencing method which selectively captures sample molecules containing certain predefined sequences. One may also use the sequencing steps exactly as exemplified, but in mapping the sequence fragments obtained, give greater weight to sequences which map to areas known to be more reliable in their coverage, such as exons. Otherwise, the method proceeds as described below, where one obtains a large number of sequence reads from one or more reference chromosomes, which are compared to a large number of reads obtained from a chromosome of interest, after accounting for variations arising from chromosomal length, G/C content, repeat sequences and the like.
One may also focus on certain regions within the human genome according to the present methods in order to identify partial monosomies and partial trisomies. As described below, the present methods involve analyzing sequence data in a defined chromosomal sliding “window,” such as contiguous, nonoverlapping 50 Kb regions spread across a chromosome. Partial trisomies of 13q, 8p (8p23.1), 7q, distal 6p, 5p, 3q (3q25.1), 2q, 1q (1q42.1 and 1q21-qter), partial Xpand monosomy 4q35.1 have been reported, among others. For example, partial duplications of the long arm of chromosome 18 can result in Edwards syndrome in the case of a duplication of 18q21.1-qter (See, Mewar et al., “Clinical and molecular evaluation of four patients with partial duplications of the long arm of chromosome 18,” Am J Hum Genet. 1993 December; 53(6):1269-78).
Shotgun Sequencing of Cell-Free Plasma DNA
Cell-free plasma DNA from 18 pregnant women and a male donor, as well as whole blood genomic DNA from the same male donor, were sequenced on the Solexa/Illumina platform. We obtained on average ˜10 million 25 bp sequence tags per sample. About 50% (i.e., ˜5 million) of the reads mapped uniquely to the human genome with at most 1 mismatch against the human genome, covering ˜4% of the entire genome. An average of ˜154,000, ˜135,000, ˜66,000 sequence tags mapped to chromosomes 13, 18, and 21, respectively. The number of sequence tags for each sample is detailed in the following Table 1 and Table 2.
†For 454 sequencing, this number represents the number of reads with at least 90% accuracy and 90% coverage when mapped to hg18.
‡Insufficient materials were available for quantifying fetal DNA % with digital PCR for these samples (either no samples remained for analysis or there was insufficient sampling).
§Sequenced on Solexa/Illumina platform;
¶Sequenced on 454/Roche platform
∥Sample P13 was the first to be analyzed by shotgun sequencing. It was a normal fetus and the chromosome value was clearly disomic. However, there were some irregularities with this sample and it was not included in further analysis. This sample was sequenced on a different Solexa instrument than the rest of the samples of this study, and it was sequenced in the presence of a number of samples of unknown origin. The G/C content of this sample was lower than the G/C bias of the human genome, while the rest of the samples are above. It had the lowest number of reads, and also the smallest number of reads mapped successfully to the human genome. This sample appeared to be outlier in sequence tag density for most chromosomes and the fetal DNA fraction calculated from chromosomes X was not well defined. For these reasons we suspect that the irregularities are due to technical problems with the sequencing process.
In Table 1 and Table 2, each sample represents a different patient, e.g., P1 in the first row. The total number of sequence tags varied but was frequently was in the 10 million range, using the Solexa technology. The 454 technology used for P25 and P13 gave a lower number of reads.
We observed a non-uniform distribution of sequence tags across each chromosome. This pattern of intra-chromosomal variation was common among all samples, including randomly sheared genomic DNA, indicating the observed variation was most probably due to sequencing artifacts. We applied an arbitrary sliding window of 50 kb across each chromosome and counted the number of tags falling within each window. The window can be varied in size to account for larger numbers of reads (in which cases a smaller window, e.g., 10 kb, gives a more detailed picture of a chromosome) or a smaller number of reads, in which case a larger window (e.g., 100 kb) may still be used and will detect gross chromosome deletions, omissions or duplications. The median count per 50 kb window for each chromosome was selected. The median of the autosomal values (i.e., 22 chromosomes) was used as a normalization constant to account for the differences in total number of sequence tags obtained for different samples. The inter-chromosomal variation within each sample was also consistent among all samples (including genomic DNA control). The mean sequence tag density of each chromosome correlates with the G/C content of the chromosome (p<10−9) (
Detection of Fetal Aneuploidy
The distribution of chromosome 21 sequence tag density for all 9 T21 pregnancies is clearly separated from that of pregnancies bearing disomy 21 fetuses (p<10−5), Student's t-test) (
Plasma DNA of pregnant women carrying T18 fetuses (2 cases) and a T13 fetus (1 case) were also directly sequenced. Over-representation was observed for chromosome 18 and chromosome 13 in T18 and T13 cases respectively (
Fetal DNA Fraction in Maternal Plasma
Using digital Taqman PCR for a single locus on chromosome 1, we estimated the average cell-free DNA concentration in the sequenced maternal plasma samples to be ˜360 cell equivalent/ml of plasma (range: 57 to 761 cell equivalent/ml plasma) (Table 1), in rough accordance to previously reported values (13). The cohort included 12 male pregnancies (6 normal cases, 4 T21 cases, 1 T18 case and 1 T13 case) and 6 female pregnancies (5 T21 cases and 1 T18 case). DYS14, a multi-copy locus on chromosome Y, was detectable in maternal plasma by real-time PCR in all these pregnancies but not in any of the female pregnancies (data not shown). The fraction of fetal DNA in maternal cell-free plasma DNA is usually determined by comparing the amount of fetal specific locus (such as the SRY locus on chromosome Y in male pregnancies) to that of a locus on any autosome that is common to both the mother and the fetus using quantitative real-time PCR (13, 22, 23). We applied a similar duplex assay on a digital PCR platform (see Methods) to compare the counts of the SRY locus and a locus on chromosome 1 in male pregnancies. SRY locus was not detectable in any plasma DNA samples from female pregnancies. We found with digital PCR that for the majority samples, fetal DNA constituted § 10% of total DNA in maternal plasma (Table 2), agreeing with previously reported values (13).
The percentage of fetal DNA among total cell-free DNA in maternal plasma can also be calculated from the density of sequence tags of the sex chromosomes for male pregnancies. By comparing the sequence tag density of chromosome Y of plasma DNA from male pregnancies to that of adult male plasma DNA, we estimated fetal DNA percentage to be on average ˜19% (range: 4-44%) for all male pregnancies (Table 2, above,
We plotted in
Size Distribution of Cell-Free Plasma DNA
We analyzed the sequencing libraries with a commercial lab-on-a-chip capillary electrophoresis system. There is a striking consistency in the peak fragment size, as well as the distribution around the peak, for all plasma DNA samples, including those from pregnant women and male donor. The peak fragment size was on average 261 bp (range: 256-264 bp). Subtracting the total length of the Solexa adaptors (92 bp) from 260 bp gives 169 bp as the actual peak fragment size. This size corresponds to the length of DNA wrapped in a chromatosome, which is a nucleosome bound to a H1 histone (24). Because the library preparation includes an 18-cycle PCR, there are concerns that the distribution might be biased. To verify that the size distribution observed in the electropherograms is not an artifact of PCR, we also sequenced cell-free plasma DNA from a pregnant woman carrying a male fetus using the 454 platform. The sample preparation for this system uses emulsion PCR, which does not require competitive amplification of the sequencing libraries and creates product that is largely independent of the amplification efficiency. The size distribution of the reads mapped to unique locations of the human genome resembled those of the Solexa sequencing libraries, with a predominant peak at 176 bp, after subtracting the length of 454 universal adaptors (
Of particular interest is the size distribution of maternal and fetal DNA in maternal cell-free plasma. Two groups have previously shown that the majority of fetal DNA has size range of that of mono-nucleosome (<200-300 bp), while maternal DNA is longer. Because 454 sequencing has a targeted read-length of 250 bp, we interpreted the small peak at around 250 bp (
Cell-Free Plasma DNA Shares Features of Nucleosomal DNA
Since our observations of the size distribution of cell-free plasma DNA suggested that plasma DNA is mainly apoptotic of origin, we investigated whether features of nucleosomal DNA and positioning are found in plasma DNA. One such feature is nucleosome positioning around transcription start sites. Experimental data from yeast and human have suggested that nucleosomes are depleted in promoters upstream of transcription start sites and nucleosomes are well-positioned near transcription start sites (27-30). We applied a 5 bp window spanning +/−1000 bp of transcription start sites of all RefSeq genes and counted the number of tags mapping to the sense and antisense strands within each window. A peak in the sense strand represents the beginning of a nucleosome while a peak in the antisense strand represents the end. After smoothing, we saw that for most plasma DNA samples, at least 3 well-positioned nucleosomes downstream of transcription start sites could be detected, and in some cases, up to 5 well-positioned nucleosomes could be detected, in rough accordance to the results of Schones et al. (27) (
Correction for Sequencing Bias
Shown in
The examples below describe the direct sequencing of cell-free DNA from plasma of pregnant women with high throughput shotgun sequencing technology, obtaining on average 5 million sequence tags per patient sample. The sequences obtained were mapped to specific chromosomal locations. This enabled us to measure the over- and under-representation of chromosomes from an aneuploid fetus. The sequencing approach is polymorphism-independent and therefore universally applicable for the non-invasive detection of fetal aneuploidy. Using this method we successfully identified all 9 cases of trisomy 21 (Down syndrome), 2 cases of trisomy 18 and 1 case of trisomy 13 in a cohort of 18 normal and aneuploid pregnancies; trisomy was detected at gestational ages as early as the 14th week. Direct sequencing also allowed us to study the characteristics of cell-free plasma DNA, and we found evidence that this DNA is enriched for sequences from nucleosomes.
The study was approved by the Institutional Review Board of Stanford University. Pregnant women at risk for fetal aneuploidy were recruited at the Lucile Packard Children Hospital Perinatal Diagnostic Center of Stanford University during the period of April 2007 to May 2008. Informed consent was obtained from each participant prior to the blood draw. Blood was collected 15 to 30 minutes after amniocentesis or chorionic villus sampling except for 1 sample that was collected during the third trimester. Karyotype analysis was performed via amniocentesis or chorionic villus sampling to confirm fetal karyotype. 9 trisomy 21 (T21), 2 trisomy 18 (T18), 1 trisomy 13 (T13) and 6 normal singleton pregnancies were included in this study. The gestational age of the subjects at the time of blood draw ranged from 10 to 35 weeks (Table 1). Blood sample from a male donor was obtained from the Stanford Blood Center.
7 to 15 ml of peripheral blood drawn from each subject and donor was collected in EDTA tubes. Blood was centrifuged at 1600 g for 10 minutes. Plasma was transferred to microcentrifuge tubes and centrifuged at 16000 g for 10 minutes to remove residual cells. The two centrifugation steps were performed within 24 hours after blood collection. Cell-free plasma was stored at −80 C until further processing and was frozen and thawed only once before DNA extraction. DNA was extracted from cell-free plasma using QIAamp DNA Micro Kit (Qiagen) or NucleoSpin Plasma Kit (Macherey-Nagel) according to manufacturers' instructions. Genomic DNA was extracted from 200 μl whole blood of the donors using QIAamp DNA Blood Mini Kit (Qiagen). Microfluidic digital PCR (Fluidigm) was used to quantify the amount of total and fetal DNA using Taqman assays targeting at the EIF2C1 locus on chromosome 1 (Forward: 5′ GTTCGGCTTTCACCAGTCT 3′ (SEQ ID NO: 1); Reverse: 5′ CTCCATAGCTCTCCCCACTC 3′ (SEQ ID NO: 2); Probe: 5′ HEX-GCCCTGCCATGTGGAAGAT-BHQ1 3′ (SEQ ID NO: 3); amplicon size: 81 bp) and the SRY locus on chromosome Y (Forward: 5′ CGCTTAACATAGCAGAAGCA 3′(SEQ ID NO: 4); Reverse: 5′ AGTTTCGAACTCTGGCACCT 3′(SEQ ID NO: 5); Probe: 5′ FAM-TGTCGCACTCTCCTTGTTTTTGACA-BHQ1 3′(SEQ ID NO: 6); amplicon size: 84 bp) respectively. A Taqman assay targeting at DYS14 (Forward: 5′ ATCGTCCATTTCCAGAATCA 3′(SEQ ID NO: 7); Reverse: 5′ GTTGACAGCCGTGGAATC 3′ (SEQ ID NO: 8); Probe: 5′ FAM-TGCCACAGACTGAACTGAATGATTTTC-BHQ1 3′ (SEQ ID NO: 9); amplicon size: 84 bp), a multi-copy locus on chromosome Y, was used for the initial determination of fetal sex from cell-free plasma DNA with traditional real-time PCR. PCR reactions were performed with 1× iQ Supermix (Bio-Rad), 0.1% Tween-20 (microfluidic digital PCR only), 300 nM primers, and 150 nM probes. The PCR thermal cycling protocol was 95 C for 10 min, followed by 40 cycles of 95 C for 15 s and 60 C for 1 min. Primers and probes were purchased form IDT.
A total of 19 cell-free plasma DNA samples, including 18 from pregnant women and 1 from a male blood donor, and genomic DNA sample from whole blood of the same male donor, were sequenced on the Solexa/Illumina platform. ˜1 to 8 ng of DNA fragments extracted from 1.3 to 5.6 ml cell-free plasma was used for sequencing library preparation (Table 1). Library preparation was carried out according to manufacturer's protocol with slight modifications. Because cell-free plasma DNA was fragmented in nature, no further fragmentation by nebulization or sonication was done on plasma DNA samples.
Genomic DNA from male donor's whole blood was sonicated (Misonix XL-2020) (24 cycles of 30 s sonication and 90 s pause), yielding fragments with size between 50 and 400 bp, with a peak at 150 bp. ˜2 ng of the sonicated genomic DNA was used for library preparation. Briefly, DNA samples were blunt ended and ligated to universal adaptors. The amount of adaptors used for ligation was 500 times less than written on the manufacturer's protocol. 18 cycles of PCR were performed to enrich for fragments with adaptors using primers complementary to the adaptors. The size distributions of the sequencing libraries were analyzed with DNA 1000 Kit on the 2100 Bioanalyzer (Agilent) and quantified with microfluidic digital PCR (Fluidigm). The libraries were then sequenced using the Solexa 1G Genome Analyzer according to manufacturer's instructions.
Cell-free plasma DNA from a pregnant woman carrying a normal male fetus was also sequenced on the 454/Roche platform. Fragments of DNA extracted from 5.6 ml of cell-free plasma (equivalent to ˜4.9 ng of DNA) were used for sequencing library preparation. The sequencing library was prepared according to manufacturer's protocol, except that no nebulization was performed on the sample and quantification was done with microfluidic digital PCR instead of capillary electrophoresis. The library was then sequenced on the 454 Genome Sequencer FLX System according to manufacturer's instructions.
Electropherograms of Solexa sequencing libraries were prepared from cell-free plasma DNA obtained from 18 pregnant women and 1 male donor. Solexa library prepared from sonicated whole blood genomic DNA from the male donor was also examined. For libraries prepared from cell-free DNA, all had peaks at average 261 bp (range: 256-264 bp). The actual peak size of DNA fragments in plasma DNA is ˜168 bp (after removal of Solexa universal adaptor (92 bp)). This corresponds to the size of a chromatosome.
Shotgun Sequence Analysis
Solexa sequencing produced 36 to 50 bp reads. The first 25 bp of each read was mapped to the human genome build 36 (hg18) using ELAND from the Solexa data analysis pipeline. The reads that were uniquely mapped to the human genome having at most 1 mismatch were retained for analysis. To compare the coverage of the different chromosomes, a sliding window of 50 kb was applied across each chromosome, except in regions of assembly gaps and microsatellites, and the number of sequence tags falling within each window was counted and the median value was chosen to be the representative of the chromosome. Because the total number of sequence tags for each sample was different, for each sample, we normalized the sequence tag density of each chromosome (except chromosome Y) to the median sequence tag density among autosomes. The normalized values were used for comparison among samples in subsequent analysis. We estimated fetal DNA fraction from chromosome 21 for T21 cases, chromosome 18 from T18 cases, chromosome 13 from T13 case, and chromosomes X and Y for male pregnancies. For chromosome 21,18, and 13, fetal DNA fraction was estimated as 2*(x−1), where x was the ratio of the over-represented chromosome sequence tag density of each trisomy case to the median chromosome sequence tag density of the all disomy cases. For chromosome X, fetal DNA was estimated as 2*(1−x), where x was the ratio of chromosome X sequence tag density of each male pregnancy to the median chromosome X sequence tag density of all female pregnancies. For chromosome Y, fetal DNA fraction was estimated as the ratio of chromosome Y sequence tag density of each male pregnancy to that of male donor plasma DNA. Because a small number of chromosome Y sequences were detected in female pregnancies, we only considered sequence tags falling within transcribed regions on chromosome Y and subtracted the median number of tags in female pregnancies from all samples; this amounted to a correction of a few percent. The width of 99% confidence intervals was calculated for all disomy 21 pregnancies as t*s/vN, where N is the number of disomy 21 pregnancies, t is the t-statistic corresponding to a=0.005 with degree of freedom equals N−1, and s is the standard deviation. A confidence interval gives an estimated range of values, which is likely to include an unknown population parameter, the estimated range being calculated from a given set of sample data. (Definition taken from Valerie J. Easton and John H. McColl's Statistics Glossary v1.1)
To investigate the distribution of sequence tags around transcription start sites, a sliding window of 5 bp was applied from −1000 bp to +1000 bp of transcription start sites of all RefSeq genes on all chromosomes except chromosome Y. The number of sequence tags mapped to the sense and antisense strands within each window was counted. Moving average with a window of 10 data points was used to smooth the data. All analyses were done with Matlab.
We selected the sequence tags that mapped uniquely to the human genome with at most 1 mismatch (on average ˜5 million) for analysis. The distribution of reads across each chromosome was examined. Because the distribution of sequence tags across each chromosome was non-uniform (possibly technical artifacts), we divided the length of each chromosome into non-overlapping sliding window with a fixed width (in this particular analysis, a 50 kbp window was used), skipping regions of genome assembly gaps and regions with known microsatellite repeats. The width of the window is should be large enough such that there are a sufficient number of sequence tags in each window, and should be small enough such that there are sufficient number of windows to form a distribution. With increasing sequencing depth (i.e., increasing total number of sequence tags), the window width can be reduced. The number of sequence tags in each window was counted. The distribution of the number of sequence tags per 50 kb for each chromosome was examined. The median value of the number of sequence tags per 50 kb (or ‘sequence tag density’) for each chromosome was chosen in order to suppress the effects of any under- or over-represented regions within the chromosome. Because the total number of sequence tags obtained for each sample was different, in order to compare among samples, we normalized each chromosomal sequence tag density value (except chromosome Y) by the median sequence tag density among all autosomes (non-sex chromosomes).
For the 454/Roche data, reads were aligned to the human genome build 36 (hg18, see hyper text transfer protocol (http) genome.ucsc.edu/cgi-bin/hgGateway) using the 454 Reference Mapper. Reads having accuracy of greater than or equal to 90% and coverage (i.e., fraction of read mapped) greater than or equal to 90% were retained for analysis. To study the size distribution of total and fetal DNA, the number of retained reads falling within each 10 bp window between 50 bp to 330 bp was counted. The number of reads falling within different size ranges may be studied, i.e., reads of between 50-60 bp, 60-70 bp, 70-80 bp, etc., up to about 320-330 bp, which is around the maximum read length obtained.
Information regarding G/C content, location of transcription start sites of RefSeq genes, location of assembly gaps and microsatellites were obtained from the UCSC Genome Browser.
The distribution of sequence tags around transcription start sites (TSS) of RefSeq genes were analyzed (data not shown). The plots were similar to
i. With Digital PCR Taqman Assays
Digital PCR is the amplification of single DNA molecule. DNA sample is diluted and distributed across multiple compartments such that on average there is less than 1 copy of DNA per compartment. A compartment displaying fluorescence at the end of a PCR represents the presence of at least one DNA molecule.
Assay for Total DNA: EIF2C1 (Chromosome 1)
Assay for Fetal DNA: SRY (Chromosome Y)
The count of positive compartments from the microfluidic digital PCR chip of each assay is converted to the most probable count according to the method described in the supporting information of the following reference: Warren L, Bryder D, Weissman I L, Quake S R (2006) Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR. Proc Nat Acad Sci, 103: 17807-12.
Fetal DNA Fraction ε=(SRY count)/(EIF2C1 count/2)
ii. With Sequence Tags
From ChrX:
Let fetal DNA fraction be
Male pregnancies ChrX sequence tag density (fetal and maternal)=2(1−ε)+ε=2−ε
Female pregnancies ChrX sequence tag density (fetal and maternal)=2(1−ε)+2ε=2
Let x be the ratio of ChrX sequence tag density of male to female pregnancies. In this study, the denominator of this ratio is taken to be the median sequence tag density of all female pregnancies.
Thus, fetal DNA fraction ε=2(1−x)
From ChrY:
Fetal DNA fraction c=(sequence tag density of ChrY in maternal plasma/sequence tag density of ChrY in male plasma)
Note that in these derivations, we assume that the total number of sequence tags obtained is the same for all samples. In reality, the total number of sequence tags obtained for different sample is different, and we have taken into account such differences in our estimation of fetal DNA fraction by normalizing the sequence tag density of each chromosome to the median of the autosomal sequence tag densities for each sample.
Calculating Fetal DNA Fraction in Maternal Plasma of Aneuploid (Trisomy) Pregnancies:
Let fetal DNA fraction be
Trisomic pregnancies trisomic chromosome sequence counts (fetal and maternal)
=2(1−ε)+3ε=2+ε
Disomic pregnancies trisomic chromosome sequence counts (fetal and maternal)
=2(1−ε)+2ε=2
Let x be the ratio of trisomic chromosome sequence counts (or sequence tag density) of trisomic to disomic pregnancies. In this study, the denominator of this ratio is taken to be the median sequence tag density of all disomic pregnancies.
Thus, fetal DNA fraction ε=2(x−1).
This example shows a refinement of results indicating sequences mapping to different chromosomes and permitting the determination of the count of different chromosomes or regions thereof. That is, the results as shown in
Outlying chromosome sequence tag densities can be seen as significantly above a median sequence tag density; disomic chromosomes are clustered about a line running along a density value of about 1. As can be seen there, the results from chromosome 19 (far right, highest in G/C content), for example, show a similar value when disomic as other disomic chromosomes. The variations between chromosomes with low and high G/C content are eliminated from the data to be examined. Samples (such as P13 in the present study) which could not have been unambiguously interpreted now may be. Since G/C content is the opposite of A/T content, the present method will correct for both. Either G/C bias or A/T bias can result from different sequencing methods. For example, it has been reported by others that the Solexa method results in a higher number of reads from sequences where the G/C content is high. See, Dohm et al., “Substantial biases in ultra-short read data sets from high-throughput DNA sequencing,” Nuc. Acids Res. 36(16), e105; doi:10.1093/nar/gkn425. The procedure of the present example follows the following steps:
a. Calculate G/C content of the human genome. Calculate the G/C content of every 20 kb non-overlapping window of each chromosome of the human genome (HG18) using the hgG/CPercent script of the UCSC Genome Browser's “kent source tree,” which contains different utility programs, available to the public under license. The output file contains the coordinate of each 20 kb bin and the corresponding G/C content. It was found that a large number of reads were obtained higher G/C ranges (about 55-70%) and very few reads were obtained at lower G/C content percentages, with essentially none below about 30% G/C (data not shown). Because the actual length of a sequenced DNA fragment is not known (we only sequenced the first 25 bp of one end of a piece of DNA on the flow cell), and it's the G/C content of the entire piece of DNA that contributed to sequencing bias, an arbitrary window of known human genomic DNA sequence is chosen for determining G/C content of different reads. We chose a 20 kb window to look at the relationship between number of reads and GC content. The window can be much smaller e.g., 10 kb or 5 kb, but a size of 20 kb makes computation easier.
b. Calculate the relationship between sequence coverage and G/C content. Assign weight to each read according to G/C content. For each sample, the number of read per 20 kb bin is counted. The number of read is plotted against G/C content. The average number of read is calculated for every 0.1% G/C content, ignoring bins with no reads, bins with zero G/C percent, and bins with over-abundant reads. The reciprocal of the average number of reads for a particular G/C percent relative to the global median number of read is calculated as the weight. Each read is then assigned a weight depending on the G/C percent of the 20 kb window it falls into.
c. Investigate the distribution of reads across each autosome and chromosome X. In this step, the number of reads, both unweighted and weighted, in each non-overlapping 50 kb window is recorded. For counting, we chose a 50 kb window in order to obtain a reasonable number of reads per window and reasonable number of windows per chromosome to look at the distributions. Window size may be selected based on the number of reads obtained in a given experiment, and may vary over a wide range. For example, 30K-100K may be used. Known microsatellite regions are ignored. A graph showing the results of chr1 of P7 is shown in
d. Investigate the distribution of reads across chrY. Calculate the number of chrY reads in transcribed regions after applying weight to reads on chrY. Chromosome Y is treated individually because it is short and has many repeats. Even female genome sequence data will map in some part to chromosome Y, due to sequencing and alignment errors. The number of chrY reads in transcribed regions after applying weight to reads on chrY is used to calculate percentage of fetal DNA in the sample.
This example shows another refinement of results as obtained using the previous examples. In this case, multiple patient samples are analyzed in a single process.
Step 1: Calculate a t statistic for each chromosome relative to all other chromosome in a sample. Each t statistic tells the value of each chromosome median relative to other chromosomes, taking into account the number of reads mapped to each chromosome (since the variation of the median scales with the number of reads). As described above, the present analyses yielded about 5 million reads per sample. Although one may obtain 3-10 million reads per sample, these are short reads, typically only about 20-100 bp, so one has actually only sequenced, for example about 300 million of the 3 billion bp in the human genome. Thus, statistical methods are used where one has a small sample and the standard deviation of the population (3 billion, or 47 million for chromosome 21) is unknown and it is desired to estimate it from the sample number of reads in order to determine the significance of a numerical variation. One way to do this is by calculating Student's t-distribution, which may be used in place of a normal distribution expected from a larger sample. The t-statistic is the value obtained when the t-distribution is calculated. The formula used for this calculation is given below. Using the methods presented here, other t-tests can be used.
Step 2: Calculate the average t statistic matrix by averaging the values from all samples with disomic chromosomes. Each patient sample data is placed in a t matrix, where the row is chr1 to chr22, and the column is also chr1 to chr22. Each cell represents the t value when comparing the chromosomes in the corresponding row and column (i.e., position (2,1) in the matrix is the t-value of when testing chr2 and chr1) the diagonal of the matrix is 0 and the matrix is symmetric. The number of reads mapping to a chromosome is compared individually to each of chr1-22.
Step 3: Subtract the average t statistic matrix from the t statistic matrix of each patient sample. For each chromosome, the median of the difference in t statistic is selected as the representative value.
The t statistic for 99% confidence for large number of samples is 3.09. Any chromosome with a representative t statistic outside −3.09 to 3.09 is determined as non-disomic.
In this example, a method is presented that was used to calculate the minimum concentration of fetal DNA in a sample that would be needed to detect an aneuploidy, based on a certain number of reads obtained for that chromosome (except chromosome Y).
In
For large n (n>30), t statistic
where
half width of the confidence interval at confidence level governed by the value of t. Thus,
For every chromosome in every sample, we can calculate the value
which corresponds to the minimum over- or under-representation
that can be resolved with confidence level governed by the value of t. Note that
corresponds to the minimum fetal DNA % of which any over- or under-representation of chromosomes can be detected. We expect the number of reads mapped to each chromosome to play a role in determining standard deviation s1, since according to Poisson distribution, the standard deviation equals to the square root of the mean. By plotting
number of reads mapped to each chromosome in all the samples, we can evaluate the minimum fetal DNA % of which any over- or under-representation of chromosomes can be detected given the current sampling rate.
After correction of G/C bias, the number of reads per 50 kb window for all chromosomes (except chromosome Y) is normally distributed. However, we observed outliers in some chromosomes (e.g., a sub-region in chromosome 9 has near zero representation; a sub-region in chromosome 20 near the centromere has unusually high representation) that affect the calculation of standard deviation and the mean. We therefore chose to calculate confidence interval of the median instead of the mean to avoid the effect of outliers in the calculation of confidence interval. We do not expect the confidence interval of the median and the mean to be very different if the small number of outliers has been removed. The 99.9% confidence interval of the median for each chromosome is estimated from bootstrapping 5000 samples from the 50 kb read distribution data using the percentile method. The half width of the confidence interval is estimated as 0.5*confidence interval. We plot 2*(half width of confidence interval of median)/median*100% vs. number of reads mapped to each chromosome for all samples.
Bootstrap resampling and other computer-implemented calculations described here were carried out in MATLAB®, available from The Mathworks, Natick, MA
The above specific description is meant to exemplify and illustrate the invention and should not be seen as limiting the scope of the invention, which is defined by the literal and equivalent scope of the appended claims. Any patents or publications mentioned in this specification are intended to convey details of methods and materials useful in carrying out certain aspects of the invention which may not be explicitly set out but which would be understood by workers in the field. Such patents or publications are hereby incorporated by reference to the same extent as if each was specifically and individually incorporated by reference, as needed for the purpose of describing and enabling the method or material referred to.
This application is a continuation of and claims priority to pending U.S. patent application Ser. No. 15/195,135, filed on Jun. 28, 2016, which is a continuation of U.S. patent application Ser. No. 13/921,881, filed on Jun. 19, 2013, now U.S. Pat. No. 9,404,157, which is a continuation of U.S. patent application Ser. No. 13/218,317, filed on Aug. 25, 2011, abandoned, which is a continuation of U.S. patent application Ser. No. 12/560,708, filed on Sep. 16, 2009, abandoned, which claims priority from U.S. Provisional Patent Application No. 61/098,758, filed on Sep. 20, 2008, all of which are hereby incorporated by reference in their entireties.
This invention was made with U.S. Government support under NIH Director's Pioneer Award DP1 OD000251. The U.S. Government has certain rights in this invention.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4508625 | Graham | Sep 1985 | A |
| 4675286 | Calenoff | Jun 1987 | A |
| 4789628 | Nayak | Dec 1988 | A |
| 4800159 | Mullis et al. | Jan 1989 | A |
| 4971904 | Luddy | Nov 1990 | A |
| 4977078 | Niimura et al. | Dec 1990 | A |
| 5153117 | Simons | Oct 1992 | A |
| 5215926 | Etchells, III et al. | Jun 1993 | A |
| 5296375 | Kricka et al. | Mar 1994 | A |
| 5300779 | Hillman et al. | Apr 1994 | A |
| 5304487 | Wilding et al. | Apr 1994 | A |
| 5427663 | Austin et al. | Jun 1995 | A |
| 5427946 | Kricka et al. | Jun 1995 | A |
| 5432054 | Saunders et al. | Jul 1995 | A |
| 5447842 | Simons | Sep 1995 | A |
| 5486335 | Wilding et al. | Jan 1996 | A |
| 5498392 | Wilding et al. | Mar 1996 | A |
| 5529903 | Kübler et al. | Jun 1996 | A |
| 5556773 | Youmo | Sep 1996 | A |
| 5629147 | Asgari et al. | May 1997 | A |
| 5639669 | Ledley | Jun 1997 | A |
| 5641628 | Bianchi | Jun 1997 | A |
| 5646001 | Terstappen et al. | Jul 1997 | A |
| 5676849 | Sammons et al. | Oct 1997 | A |
| 5707799 | Hansmann et al. | Jan 1998 | A |
| 5709943 | Coleman et al. | Jan 1998 | A |
| 5715946 | Reichenbach | Feb 1998 | A |
| 5726026 | Wilding et al. | Mar 1998 | A |
| 5750339 | Smith | May 1998 | A |
| 5766843 | Asgari et al. | Jun 1998 | A |
| 5770029 | Nelson et al. | Jun 1998 | A |
| 5798042 | Chu et al. | Aug 1998 | A |
| 5837115 | Austin et al. | Nov 1998 | A |
| 5840502 | Van Vlasselaer | Nov 1998 | A |
| 5842787 | Koph-Sill et al. | Dec 1998 | A |
| 5858649 | Asgari et al. | Jan 1999 | A |
| 5866345 | Wilding et al. | Feb 1999 | A |
| 5879883 | Benson et al. | Mar 1999 | A |
| 5891651 | Roche et al. | Apr 1999 | A |
| 5928880 | Wilding et al. | Jul 1999 | A |
| 5952173 | Hansmann et al. | Sep 1999 | A |
| 5962234 | Golbus | Oct 1999 | A |
| 5962237 | Ts'o et al. | Oct 1999 | A |
| 5962332 | Singer et al. | Oct 1999 | A |
| 5972721 | Bruno et al. | Oct 1999 | A |
| 5994517 | Ts'o et al. | Oct 1999 | A |
| 5993665 | Terstappen et al. | Nov 1999 | A |
| 5994057 | Mansfield | Nov 1999 | A |
| 6007690 | Nelson et al. | Dec 1999 | A |
| 6008007 | Fruehauf et al. | Dec 1999 | A |
| 6013188 | Terstappen et al. | Jan 2000 | A |
| 6066449 | Ditkoff et al. | May 2000 | A |
| 6074827 | Nelson et al. | Jun 2000 | A |
| 6100029 | Lapidus et al. | Aug 2000 | A |
| 6159685 | Pinkel et al. | Oct 2000 | A |
| 6143496 | Brown et al. | Nov 2000 | A |
| 6143576 | Buechler | Nov 2000 | A |
| 6154707 | Livak et al. | Nov 2000 | A |
| 6156270 | Buechler | Dec 2000 | A |
| 6176962 | Soane et al. | Jan 2001 | B1 |
| 6184043 | Fodstad et al. | Feb 2001 | B1 |
| 6186660 | Koph-Sill et al. | Feb 2001 | B1 |
| 6197523 | Rimm et al. | Mar 2001 | B1 |
| 6200765 | Murphy et al. | Mar 2001 | B1 |
| 6210891 | Nyren et al. | Apr 2001 | B1 |
| 6214558 | Shuber et al. | Apr 2001 | B1 |
| 6235474 | Feinberg | May 2001 | B1 |
| 6258540 | Lo et al. | Jul 2001 | B1 |
| 6265229 | Fodstad et al. | Jul 2001 | B1 |
| 6300077 | Shuber et al. | Oct 2001 | B1 |
| 6344326 | Nelson et al. | Feb 2002 | B1 |
| 6361958 | Shieh et al. | Mar 2002 | B1 |
| 6365362 | Terstappen et al. | Apr 2002 | B1 |
| 6368871 | Christel et al. | Apr 2002 | B1 |
| 6376181 | Ramsey et al. | Apr 2002 | B2 |
| 6383759 | Murphy et al. | May 2002 | B1 |
| 6387707 | Seul et al. | May 2002 | B1 |
| 6391559 | Brown et al. | May 2002 | B1 |
| 6394942 | Moon et al. | May 2002 | B2 |
| 6399364 | Reeve et al. | Jun 2002 | B1 |
| 6432630 | Blankenstein | Aug 2002 | B1 |
| 6440706 | Vogelstein et al. | Aug 2002 | B1 |
| 6444461 | Knapp et al. | Sep 2002 | B1 |
| 6454938 | Moon et al. | Sep 2002 | B2 |
| 6479299 | Parce et al. | Nov 2002 | B1 |
| 6511967 | Weissleder et al. | Jan 2003 | B1 |
| 6517234 | Koph-Sill et al. | Feb 2003 | B1 |
| 6540895 | Spence et al. | Apr 2003 | B1 |
| 6566101 | Shuber et al. | May 2003 | B1 |
| 6576478 | Wagner et al. | Jun 2003 | B1 |
| 6582904 | Dahm | Jun 2003 | B2 |
| 6582969 | Wagner et al. | Jun 2003 | B1 |
| 6596144 | Regnier et al. | Jul 2003 | B1 |
| 6596545 | Wagner et al. | Jul 2003 | B1 |
| 6613525 | Nelson et al. | Sep 2003 | B2 |
| 6618679 | Loehriein et al. | Sep 2003 | B2 |
| 6632619 | Harrison et al. | Sep 2003 | B1 |
| 6632652 | Austin et al. | Oct 2003 | B1 |
| 6637463 | Lei et al. | Oct 2003 | B1 |
| 6645731 | Terstappen et al. | Nov 2003 | B2 |
| 6664056 | Lo et al. | Dec 2003 | B2 |
| 6664104 | Pourahmadi et al. | Dec 2003 | B2 |
| 6673541 | Klein et al. | Jan 2004 | B1 |
| 6674525 | Bardell et al. | Jan 2004 | B2 |
| 6685841 | Lopez et al. | Feb 2004 | B2 |
| 6689615 | Murto et al. | Feb 2004 | B1 |
| 6746503 | Benett et al. | Jun 2004 | B1 |
| 6753147 | Vogelstein et al. | Jun 2004 | B2 |
| 6783928 | Hvichia et al. | Aug 2004 | B2 |
| 6818184 | Fulwyler et al. | Nov 2004 | B2 |
| 6830936 | Anderson et al. | Dec 2004 | B2 |
| 6858439 | Xu et al. | Feb 2005 | B1 |
| 6875619 | Blackburn | Apr 2005 | B2 |
| 6893881 | Fodstad et al. | May 2005 | B1 |
| 6906182 | Ts'o et al. | Jun 2005 | B2 |
| 6911345 | Quake et al. | Jun 2005 | B2 |
| 6913697 | Lopez et al. | Jul 2005 | B2 |
| 6927028 | Dennis et al. | Aug 2005 | B2 |
| 6953668 | Israeli et al. | Oct 2005 | B1 |
| 6960449 | Wang et al. | Nov 2005 | B2 |
| 7115709 | Gray et al. | Oct 2006 | B1 |
| 7150812 | Huang et al. | Dec 2006 | B2 |
| 7171975 | Moon et al. | Feb 2007 | B2 |
| 7190818 | Ellis et al. | Mar 2007 | B2 |
| 7192698 | Kinch et al. | Mar 2007 | B1 |
| 7198787 | Fodstad et al. | Apr 2007 | B2 |
| 7208275 | Gocke et al. | Apr 2007 | B2 |
| 7212660 | Wetzel et al. | May 2007 | B2 |
| 7220594 | Foster et al. | May 2007 | B2 |
| 7227002 | Kufer et al. | Jun 2007 | B1 |
| 7229838 | Foster et al. | Jun 2007 | B2 |
| 7250256 | Reinhard et al. | Jul 2007 | B2 |
| 7252976 | Lin et al. | Aug 2007 | B2 |
| 7258987 | Lamorte et al. | Aug 2007 | B2 |
| 7262177 | Ts'o et al. | Aug 2007 | B2 |
| 7262269 | Lam et al. | Aug 2007 | B2 |
| 7264972 | Foster | Sep 2007 | B2 |
| 7272252 | De La Torre-Bueno et al. | Sep 2007 | B2 |
| 7276170 | Oakey et al. | Oct 2007 | B2 |
| 7332277 | Dhallan | Feb 2008 | B2 |
| 7442506 | Dhallan | Oct 2008 | B2 |
| 7476363 | Unger et al. | Jan 2009 | B2 |
| 7645576 | Lo et al. | Jan 2010 | B2 |
| 7655399 | Cantor et al. | Feb 2010 | B2 |
| 8195415 | Fan et al. | Jun 2012 | B2 |
| 8293470 | Quake et al. | Oct 2012 | B2 |
| 8296076 | Fan et al. | Oct 2012 | B2 |
| 8682594 | Fan et al. | Mar 2014 | B2 |
| 20010007749 | Feinberg | Jul 2001 | A1 |
| 20010051341 | Lo et al. | Dec 2001 | A1 |
| 20010053958 | Ried et al. | Dec 2001 | A1 |
| 20020006621 | Bianchi | Jan 2002 | A1 |
| 20020009738 | Houghton et al. | Jan 2002 | A1 |
| 20020012930 | Rothberg et al. | Jan 2002 | A1 |
| 20020012931 | Waldman et al. | Jan 2002 | A1 |
| 20020016450 | Laugharn et al. | Feb 2002 | A1 |
| 20020019001 | Light | Feb 2002 | A1 |
| 20020028431 | Julien | Mar 2002 | A1 |
| 20020058332 | Quake et al. | May 2002 | A1 |
| 20020076825 | Cheng et al. | Jun 2002 | A1 |
| 20020086329 | Shvets et al. | Jul 2002 | A1 |
| 20020110835 | Kumar | Aug 2002 | A1 |
| 20020123078 | Seul et al. | Sep 2002 | A1 |
| 20020137088 | Bianchi | Sep 2002 | A1 |
| 20020164816 | Quake | Nov 2002 | A1 |
| 20020166760 | Prentiss et al. | Nov 2002 | A1 |
| 20020172987 | Terstappen et al. | Nov 2002 | A1 |
| 20030004402 | Hitt et al. | Jan 2003 | A1 |
| 20030017514 | Pachmann et al. | Jan 2003 | A1 |
| 20030022207 | Balasubramanian | Jan 2003 | A1 |
| 20030033091 | Opalsky et al. | Feb 2003 | A1 |
| 20030044388 | Dennis et al. | Mar 2003 | A1 |
| 20030072682 | Kikinis | Apr 2003 | A1 |
| 20030077292 | Hanash et al. | Apr 2003 | A1 |
| 20030119077 | Ts'o et al. | Jun 2003 | A1 |
| 20030119724 | Ts'o et al. | Jun 2003 | A1 |
| 20030129676 | Terstappen et al. | Jul 2003 | A1 |
| 20030153085 | Leary et al. | Aug 2003 | A1 |
| 20030159999 | Oakey et al. | Aug 2003 | A1 |
| 20030165852 | Schueler et al. | Sep 2003 | A1 |
| 20030170631 | Houghton et al. | Sep 2003 | A1 |
| 20030170703 | Piper et al. | Sep 2003 | A1 |
| 20030175990 | Heyenga | Sep 2003 | A1 |
| 20030186255 | Williams et al. | Oct 2003 | A1 |
| 20030190602 | Pressman et al. | Oct 2003 | A1 |
| 20030199685 | Pressman et al. | Oct 2003 | A1 |
| 20030204331 | Whitney et al. | Oct 2003 | A1 |
| 20030206901 | Chen | Nov 2003 | A1 |
| 20030232350 | Afar et al. | Dec 2003 | A1 |
| 20040005582 | Shipwash | Jan 2004 | A1 |
| 20040009471 | Cao | Jan 2004 | A1 |
| 20040018116 | Desmond et al. | Jan 2004 | A1 |
| 20040018509 | Bianchi | Jan 2004 | A1 |
| 20040043506 | Haussecker et al. | Mar 2004 | A1 |
| 20040048360 | Wada et al. | Mar 2004 | A1 |
| 20040053352 | Ouyang et al. | Mar 2004 | A1 |
| 20040072278 | Chou et al. | Apr 2004 | A1 |
| 20040096892 | Wang et al. | May 2004 | A1 |
| 20040037470 | Dhallan | Jul 2004 | A1 |
| 20040137452 | Levett et al. | Jul 2004 | A1 |
| 20040137470 | Dhallan | Jul 2004 | A1 |
| 20040142463 | Walker et al. | Jul 2004 | A1 |
| 20040144651 | Huang et al. | Jul 2004 | A1 |
| 20040166555 | Braff et al. | Aug 2004 | A1 |
| 20040171091 | Lesko et al. | Sep 2004 | A1 |
| 20040185495 | Schueler et al. | Sep 2004 | A1 |
| 20040197797 | Inoko et al. | Oct 2004 | A1 |
| 20040203037 | Lo et al. | Oct 2004 | A1 |
| 20040209299 | Pinter et al. | Oct 2004 | A1 |
| 20040214240 | Cao | Oct 2004 | A1 |
| 20040232074 | Peters et al. | Nov 2004 | A1 |
| 20040241707 | Gao et al. | Dec 2004 | A1 |
| 20050014208 | Krehan et al. | Jan 2005 | A1 |
| 20050019792 | McBride et al. | Jan 2005 | A1 |
| 20050037388 | Antonarakis et al. | Feb 2005 | A1 |
| 20050042623 | Ault-Riche et al. | Feb 2005 | A1 |
| 20050042685 | Albert et al. | Feb 2005 | A1 |
| 20050049793 | Paterlini-Brechot | Mar 2005 | A1 |
| 20050061962 | Mueth et al. | Mar 2005 | A1 |
| 20050118591 | Tamak et al. | Jun 2005 | A1 |
| 20050129581 | McBride et al. | Jun 2005 | A1 |
| 20050145496 | Goodsaid et al. | Jul 2005 | A1 |
| 20050147977 | Koo et al. | Jul 2005 | A1 |
| 20050153342 | Chen | Jul 2005 | A1 |
| 20050158754 | Puffenberger et al. | Jul 2005 | A1 |
| 20050164241 | Hahn et al. | Jul 2005 | A1 |
| 20050175996 | Chen | Aug 2005 | A1 |
| 20050181353 | Rao et al. | Aug 2005 | A1 |
| 20050181410 | Shaffer et al. | Aug 2005 | A1 |
| 20050181463 | Rao et al. | Aug 2005 | A1 |
| 20050196785 | Quake | Sep 2005 | A1 |
| 20050207940 | Butler et al. | Sep 2005 | A1 |
| 20050211556 | Childers et al. | Sep 2005 | A1 |
| 20050214855 | Wagner et al. | Sep 2005 | A1 |
| 20050221341 | Shimkets et al. | Oct 2005 | A1 |
| 20050221373 | Enzelberger et al. | Oct 2005 | A1 |
| 20050239101 | Sukumar et al. | Oct 2005 | A1 |
| 20050244843 | Chen et al. | Nov 2005 | A1 |
| 20050250111 | Xie et al. | Nov 2005 | A1 |
| 20050250155 | Lesko et al. | Nov 2005 | A1 |
| 20050250199 | Anderson et al. | Nov 2005 | A1 |
| 20050252773 | McBride et al. | Nov 2005 | A1 |
| 20050255001 | Padmanabhan et al. | Nov 2005 | A1 |
| 20050262577 | Guelly et al. | Nov 2005 | A1 |
| 20050266433 | Kapur et al. | Dec 2005 | A1 |
| 20050272103 | Chen | Dec 2005 | A1 |
| 20050282196 | Costa | Dec 2005 | A1 |
| 20050282293 | Cosmen et al. | Dec 2005 | A1 |
| 20050287611 | Nugent et al. | Dec 2005 | A1 |
| 20060000772 | Sano et al. | Jan 2006 | A1 |
| 20060008807 | O'Hara et al. | Jan 2006 | A1 |
| 20060008824 | Ronaghi et al. | Jan 2006 | A1 |
| 20060024756 | Tibbe et al. | Feb 2006 | A1 |
| 20060046258 | Lapidus et al. | Mar 2006 | A1 |
| 20060051265 | Mohamed et al. | Mar 2006 | A1 |
| 20060051775 | Bianchi et al. | Mar 2006 | A1 |
| 20060060767 | Wang et al. | Mar 2006 | A1 |
| 20060072805 | Tsipouras et al. | Apr 2006 | A1 |
| 20060073125 | Clarke et al. | Apr 2006 | A1 |
| 20060094109 | Trainer | May 2006 | A1 |
| 20060121452 | Dhallan | Jun 2006 | A1 |
| 20060121624 | Huang et al. | Jun 2006 | A1 |
| 20060128006 | Gerhardt et al. | Jun 2006 | A1 |
| 20060134599 | Toner et al. | Jun 2006 | A1 |
| 20060160105 | Dhallan | Jul 2006 | A1 |
| 20060160150 | Seilhamer et al. | Jul 2006 | A1 |
| 20060160243 | Tang et al. | Jul 2006 | A1 |
| 20060183886 | Ts'o et al. | Aug 2006 | A1 |
| 20060205057 | Wayner et al. | Sep 2006 | A1 |
| 20060223178 | Barber et al. | Oct 2006 | A1 |
| 20060252054 | Lin et al. | Nov 2006 | A1 |
| 20060252061 | Zabeau et al. | Nov 2006 | A1 |
| 20060252068 | Lo et al. | Nov 2006 | A1 |
| 20060252071 | Lo et al. | Nov 2006 | A1 |
| 20060252087 | Tang et al. | Nov 2006 | A1 |
| 20070015171 | Bianchi et al. | Jan 2007 | A1 |
| 20070017633 | Tonkovich et al. | Jan 2007 | A1 |
| 20070026381 | Huang et al. | Feb 2007 | A1 |
| 20070026413 | Toner et al. | Feb 2007 | A1 |
| 20070026414 | Fuchs et al. | Feb 2007 | A1 |
| 20070026415 | Fuchs et al. | Feb 2007 | A1 |
| 20070026416 | Fuchs | Feb 2007 | A1 |
| 20070026417 | Fuchs et al. | Feb 2007 | A1 |
| 20070026418 | Fuchs et al. | Feb 2007 | A1 |
| 20070026419 | Fuchs et al. | Feb 2007 | A1 |
| 20070026469 | Fuchs et al. | Feb 2007 | A1 |
| 20070037172 | Chiu et al. | Feb 2007 | A1 |
| 20070037173 | Allard et al. | Feb 2007 | A1 |
| 20070037273 | Shuler et al. | Feb 2007 | A1 |
| 20070037275 | Shuler et al. | Feb 2007 | A1 |
| 20070042238 | Kim et al. | Feb 2007 | A1 |
| 20070042339 | Toner et al. | Feb 2007 | A1 |
| 20070042360 | Afar et al. | Feb 2007 | A1 |
| 20070042368 | Zehentner-Wilkinson et al. | Feb 2007 | A1 |
| 20070048750 | Peck et al. | Mar 2007 | A1 |
| 20070054268 | Sutherland et al. | Mar 2007 | A1 |
| 20070054287 | Bloch | Mar 2007 | A1 |
| 20070059680 | Kapur et al. | Mar 2007 | A1 |
| 20070059683 | Barber et al. | Mar 2007 | A1 |
| 20070059716 | Balis et al. | Mar 2007 | A1 |
| 20070059718 | Toner et al. | Mar 2007 | A1 |
| 20070059719 | Grisham et al. | Mar 2007 | A1 |
| 20070059737 | Baker et al. | Mar 2007 | A1 |
| 20070059774 | Grisham et al. | Mar 2007 | A1 |
| 20070059781 | Kapur et al. | Mar 2007 | A1 |
| 20070059785 | Bacus et al. | Mar 2007 | A1 |
| 20070065845 | Baker et al. | Mar 2007 | A1 |
| 20070065858 | Haley | Mar 2007 | A1 |
| 20070071762 | Ts'o et al. | Mar 2007 | A1 |
| 20070072228 | Brauch | Mar 2007 | A1 |
| 20070072290 | Hvichia | Mar 2007 | A1 |
| 20070077578 | Penning et al. | Apr 2007 | A1 |
| 20070092444 | Benos et al. | Apr 2007 | A1 |
| 20070092881 | Ohnishi et al. | Apr 2007 | A1 |
| 20070092917 | Guyon | Apr 2007 | A1 |
| 20070099207 | Fuchs et al. | May 2007 | A1 |
| 20070099219 | Teverovskiy et al. | May 2007 | A1 |
| 20070099289 | Irimia et al. | May 2007 | A1 |
| 20070105105 | Clelland et al. | May 2007 | A1 |
| 20070105133 | Clark et al. | May 2007 | A1 |
| 20070110773 | Asina et al. | May 2007 | A1 |
| 20070117158 | Coumans et al. | May 2007 | A1 |
| 20070122856 | Georges et al. | May 2007 | A1 |
| 20070122896 | Shuler et al. | May 2007 | A1 |
| 20070128655 | Obata | Jun 2007 | A1 |
| 20070131622 | Oakey et al. | Jun 2007 | A1 |
| 20070134658 | Bohmer et al. | Jun 2007 | A1 |
| 20070134713 | Cao | Jun 2007 | A1 |
| 20070135621 | Bourel et al. | Jun 2007 | A1 |
| 20070141587 | Baker et al. | Jun 2007 | A1 |
| 20070141588 | Baker et al. | Jun 2007 | A1 |
| 20070141717 | Carpenter et al. | Jun 2007 | A1 |
| 20070154928 | Mack et al. | Jul 2007 | A1 |
| 20070154960 | Connelly et al. | Jul 2007 | A1 |
| 20070160503 | Sethu et al. | Jul 2007 | A1 |
| 20070160974 | Sidhu et al. | Jul 2007 | A1 |
| 20070160984 | Huang et al. | Jul 2007 | A1 |
| 20070161064 | Kinch et al. | Jul 2007 | A1 |
| 20070166770 | Hsieh et al. | Jul 2007 | A1 |
| 20070170811 | Rubel | Jul 2007 | A1 |
| 20070172903 | Toner et al. | Jul 2007 | A1 |
| 20070178067 | Maier et al. | Aug 2007 | A1 |
| 20070178458 | O'Brien et al. | Aug 2007 | A1 |
| 20070187250 | Huang et al. | Aug 2007 | A1 |
| 20070196663 | Schwartz et al. | Aug 2007 | A1 |
| 20070196820 | Kapur et al. | Aug 2007 | A1 |
| 20070196840 | Roca et al. | Aug 2007 | A1 |
| 20070196869 | Perez et al. | Aug 2007 | A1 |
| 20070202106 | Palucka et al. | Aug 2007 | A1 |
| 20070202109 | Nakamura et al. | Aug 2007 | A1 |
| 20070202525 | Quake et al. | Aug 2007 | A1 |
| 20070202536 | Yamanishi et al. | Aug 2007 | A1 |
| 20070207351 | Christensen et al. | Sep 2007 | A1 |
| 20070207466 | Cantor et al. | Sep 2007 | A1 |
| 20070212689 | Bianchi et al. | Sep 2007 | A1 |
| 20070212698 | Bendele et al. | Sep 2007 | A1 |
| 20070212737 | Clarke et al. | Sep 2007 | A1 |
| 20070212738 | Haley et al. | Sep 2007 | A1 |
| 20070224613 | Strathmann | Sep 2007 | A1 |
| 20070231851 | Toner et al. | Oct 2007 | A1 |
| 20070238105 | Barrett et al. | Oct 2007 | A1 |
| 20070259424 | Toner et al. | Nov 2007 | A1 |
| 20070264675 | Toner et al. | Nov 2007 | A1 |
| 20070275402 | Lo et al. | Nov 2007 | A1 |
| 20080020390 | Mitchell et al. | Jan 2008 | A1 |
| 20080023399 | Inglis et al. | Jan 2008 | A1 |
| 20080026390 | Stoughton et al. | Jan 2008 | A1 |
| 20080038733 | Bischoff et al. | Feb 2008 | A1 |
| 20080050739 | Stoughton et al. | Feb 2008 | A1 |
| 20080070792 | Stoughton et al. | Mar 2008 | A1 |
| 20080071076 | Hahn et al. | Mar 2008 | A1 |
| 20080090239 | Shoemaker et al. | Apr 2008 | A1 |
| 20080096216 | Quake | Apr 2008 | A1 |
| 20080096766 | Lee | Apr 2008 | A1 |
| 20080124721 | Fuchs | May 2008 | A1 |
| 20080138809 | Kapur et al. | Jun 2008 | A1 |
| 20080153090 | Lo et al. | Jun 2008 | A1 |
| 20080182261 | Bianchi | Jul 2008 | A1 |
| 20080193927 | Mann et al. | Aug 2008 | A1 |
| 20080213775 | Brody et al. | Sep 2008 | A1 |
| 20080220422 | Shoemaker et al. | Sep 2008 | A1 |
| 20080299562 | Oeth et al. | Dec 2008 | A1 |
| 20090029377 | Lo et al. | Jan 2009 | A1 |
| 20090087847 | Lo et al. | Apr 2009 | A1 |
| 20090117538 | Hashimoto et al. | May 2009 | A1 |
| 20090170113 | Quake et al. | Jul 2009 | A1 |
| 20090170114 | Quake et al. | Jul 2009 | A1 |
| 20090215042 | Sella-Tavor et al. | Aug 2009 | A1 |
| 20090280492 | Stoughton et al. | Nov 2009 | A1 |
| 20090291443 | Stoughton et al. | Nov 2009 | A1 |
| 20090307181 | Colby et al. | Dec 2009 | A1 |
| 20100093835 | McSwiggen et al. | Apr 2010 | A1 |
| 20100112575 | Fan et al. | May 2010 | A1 |
| 20100112590 | Lo et al. | May 2010 | A1 |
| 20110319272 | Fan et al. | Dec 2011 | A1 |
| 20120003635 | Lo et al. | Jan 2012 | A1 |
| 20140051583 | Fan et al. | Feb 2014 | A1 |
| 20140329691 | Fan et al. | Nov 2014 | A1 |
| Number | Date | Country |
|---|---|---|
| 2009293354 | Mar 2010 | AU |
| 2737643 | Mar 2010 | CA |
| 2562268 | Feb 2013 | EA |
| 0637996 | Jul 1997 | EP |
| 0405972 | May 1999 | EP |
| 1262776 | Dec 2002 | EP |
| 0994963 | May 2003 | EP |
| 0970365 | Oct 2003 | EP |
| 783694 | Nov 2003 | EP |
| 1262776 | Jan 2004 | EP |
| 1388013 | Feb 2004 | EP |
| 0920627 | May 2004 | EP |
| 1418003 | May 2004 | EP |
| 0739240 | Jun 2004 | EP |
| 1462800 | Sep 2004 | EP |
| 0919812 | Oct 2004 | EP |
| 1524321 | Apr 2005 | EP |
| 1561507 | Aug 2005 | EP |
| 1409727 | Nov 2005 | EP |
| 1272668 | Feb 2007 | EP |
| 1754788 | Feb 2007 | EP |
| 1757694 | Feb 2007 | EP |
| 1409745 | Apr 2007 | EP |
| 1754788 | Apr 2007 | EP |
| 1770171 | Apr 2007 | EP |
| 1313882 | May 2007 | EP |
| 1803822 | Jul 2007 | EP |
| 951645 | Aug 2007 | EP |
| 1813681 | Aug 2007 | EP |
| 1832661 | Sep 2007 | EP |
| 1757694 | Feb 2008 | EP |
| 2161347 | Jun 2010 | EP |
| 2334812 | Jun 2011 | EP |
| WO 199006509 | Jun 1990 | WO |
| WO 199107660 | May 1991 | WO |
| WO 1991016452 | Oct 1991 | WO |
| WO 199322053 | Nov 1993 | WO |
| WO 199429707 | Dec 1994 | WO |
| WO 199509245 | Apr 1995 | WO |
| WO 199746882 | Dec 1997 | WO |
| WO 199802528 | Jan 1998 | WO |
| WO 199810267 | Mar 1998 | WO |
| WO 9922868 | May 1999 | WO |
| WO 199944064 | Sep 1999 | WO |
| WO 1999061888 | Dec 1999 | WO |
| WO 0040750 | Jul 2000 | WO |
| 0058507 | Oct 2000 | WO |
| WO 200062931 | Oct 2000 | WO |
| WO 200135071 | May 2001 | WO |
| WO 200151668 | Jul 2001 | WO |
| WO 1999061888 | Dec 2001 | WO |
| WO 200135071 | Feb 2002 | WO |
| WO 2002012896 | Feb 2002 | WO |
| WO 2002028523 | Apr 2002 | WO |
| WO 2002030562 | Apr 2002 | WO |
| WO 200231506 | Apr 2002 | WO |
| WO 200244318 | Jun 2002 | WO |
| WO 2002073204 | Sep 2002 | WO |
| WO 03020986 | Mar 2003 | WO |
| WO 2003018757 | Mar 2003 | WO |
| WO 2003019141 | Mar 2003 | WO |
| WO 2003020974 | Mar 2003 | WO |
| WO 2003023057 | Mar 2003 | WO |
| WO 2003031938 | Apr 2003 | WO |
| WO 03044217 | May 2003 | WO |
| WO 2003035894 | May 2003 | WO |
| WO 2003035895 | May 2003 | WO |
| WO 2003044224 | May 2003 | WO |
| 2003048295 | Jun 2003 | WO |
| WO 2003069421 | Aug 2003 | WO |
| WO 2003018757 | Sep 2003 | WO |
| WO 03044217 | Oct 2003 | WO |
| WO 2002073204 | Oct 2003 | WO |
| WO 2003031938 | Nov 2003 | WO |
| WO 2003093795 | Nov 2003 | WO |
| WO 2003023057 | Dec 2003 | WO |
| WO 2003069421 | Dec 2003 | WO |
| WO 2003035895 | Jan 2004 | WO |
| WO 2003035894 | Mar 2004 | WO |
| WO 2004025251 | Mar 2004 | WO |
| WO 2003019141 | Apr 2004 | WO |
| WO 2004029221 | Apr 2004 | WO |
| WO 2004029221 | May 2004 | WO |
| WO 2004037374 | May 2004 | WO |
| WO 2004044236 | May 2004 | WO |
| WO 2004056978 | Jul 2004 | WO |
| 2004065629 | Aug 2004 | WO |
| WO 2004076643 | Sep 2004 | WO |
| WO 2003093795 | Oct 2004 | WO |
| WO 2004037374 | Oct 2004 | WO |
| WO 2004088310 | Oct 2004 | WO |
| WO 2004025251 | Nov 2004 | WO |
| WO 2004101762 | Nov 2004 | WO |
| WO 2004113877 | Dec 2004 | WO |
| WO 2004101762 | Feb 2005 | WO |
| WO 2005028663 | Mar 2005 | WO |
| 2005039389 | May 2005 | WO |
| WO 2005042713 | May 2005 | WO |
| WO 2005043121 | May 2005 | WO |
| WO 2005047529 | May 2005 | WO |
| WO 2005047532 | May 2005 | WO |
| WO 2005023091 | Jun 2005 | WO |
| WO 2005049168 | Jun 2005 | WO |
| WO 2005058937 | Jun 2005 | WO |
| WO 2005061075 | Jul 2005 | WO |
| WO 2005049168 | Sep 2005 | WO |
| WO 2005084374 | Sep 2005 | WO |
| WO 2005084380 | Sep 2005 | WO |
| WO 2005085476 | Sep 2005 | WO |
| WO 2005085861 | Sep 2005 | WO |
| WO 2005098046 | Oct 2005 | WO |
| WO 2005108621 | Nov 2005 | WO |
| WO 2005109238 | Nov 2005 | WO |
| WO 2005028663 | Dec 2005 | WO |
| WO 2005098046 | Dec 2005 | WO |
| WO 2005116264 | Dec 2005 | WO |
| WO 2005118852 | Dec 2005 | WO |
| WO 2005121362 | Dec 2005 | WO |
| WO 2005085861 | Feb 2006 | WO |
| WO 2006010610 | Feb 2006 | WO |
| WO 2006023563 | Mar 2006 | WO |
| WO 2005121362 | Apr 2006 | WO |
| WO 2006041453 | Apr 2006 | WO |
| WO 2006043181 | Apr 2006 | WO |
| WO 2005109238 | Jun 2006 | WO |
| WO 2006010610 | Jun 2006 | WO |
| WO 2006043181 | Jun 2006 | WO |
| WO 2006076567 | Jul 2006 | WO |
| WO 2006078470 | Jul 2006 | WO |
| WO 2005043121 | Aug 2006 | WO |
| 2006097049 | Sep 2006 | WO |
| WO 2006076567 | Sep 2006 | WO |
| WO 2006078470 | Sep 2006 | WO |
| WO 2006100366 | Sep 2006 | WO |
| WO 2005042713 | Nov 2006 | WO |
| WO 2006023563 | Nov 2006 | WO |
| WO 2006120434 | Nov 2006 | WO |
| WO 2005084380 | Dec 2006 | WO |
| WO 2005116264 | Feb 2007 | WO |
| WO 2007020081 | Feb 2007 | WO |
| WO 2004076643 | Mar 2007 | WO |
| WO 2007024264 | Mar 2007 | WO |
| WO 2007028146 | Mar 2007 | WO |
| WO 2007030949 | Mar 2007 | WO |
| WO 2007033167 | Mar 2007 | WO |
| WO 2007034221 | Mar 2007 | WO |
| WO 2007035414 | Mar 2007 | WO |
| WO 2007024264 | Apr 2007 | WO |
| WO 2007036025 | Apr 2007 | WO |
| WO 2007038264 | Apr 2007 | WO |
| WO 2007041610 | Apr 2007 | WO |
| WO 2007044690 | Apr 2007 | WO |
| WO 2007048076 | Apr 2007 | WO |
| WO 2007030949 | May 2007 | WO |
| WO 2007034221 | May 2007 | WO |
| WO 2007050495 | May 2007 | WO |
| WO 2007053142 | May 2007 | WO |
| WO 2007053648 | May 2007 | WO |
| WO 2007053785 | May 2007 | WO |
| WO 2007059430 | May 2007 | WO |
| WO 2007062222 | May 2007 | WO |
| WO 2005058937 | Jun 2007 | WO |
| WO 2007067734 | Jun 2007 | WO |
| WO 2007048076 | Jul 2007 | WO |
| WO 2007053648 | Jul 2007 | WO |
| WO 2007075879 | Jul 2007 | WO |
| WO 2007076989 | Jul 2007 | WO |
| WO 2007079229 | Jul 2007 | WO |
| WO 2007079250 | Jul 2007 | WO |
| WO 2007080583 | Jul 2007 | WO |
| WO 2007082144 | Jul 2007 | WO |
| WO 2007082154 | Jul 2007 | WO |
| WO 2007082379 | Jul 2007 | WO |
| 2007092473 | Aug 2007 | WO |
| WO 2007050495 | Aug 2007 | WO |
| WO 2007075879 | Aug 2007 | WO |
| WO 2007087612 | Aug 2007 | WO |
| WO 2007089880 | Aug 2007 | WO |
| WO 2007089911 | Aug 2007 | WO |
| WO 2007090670 | Aug 2007 | WO |
| WO 2007092713 | Aug 2007 | WO |
| WO 2007098484 | Aug 2007 | WO |
| 2007100911 | Sep 2007 | WO |
| WO 2006100366 | Sep 2007 | WO |
| WO 2007100684 | Sep 2007 | WO |
| WO 2007101609 | Sep 2007 | WO |
| WO 2007033167 | Oct 2007 | WO |
| WO 2007038264 | Oct 2007 | WO |
| WO 2007044690 | Oct 2007 | WO |
| WO 2007053785 | Oct 2007 | WO |
| WO 2007059430 | Oct 2007 | WO |
| WO 2005084374 | Nov 2007 | WO |
| WO 2007035414 | Nov 2007 | WO |
| WO 2007044091 | Nov 2007 | WO |
| WO 2007089880 | Nov 2007 | WO |
| WO 2007126938 | Nov 2007 | WO |
| WO 2007132166 | Nov 2007 | WO |
| WO 2007132167 | Nov 2007 | WO |
| WO 2007082379 | Dec 2007 | WO |
| WO 2007098484 | Dec 2007 | WO |
| WO 2007062222 | Jan 2008 | WO |
| WO 2007100684 | Jan 2008 | WO |
| WO 2007075836 | Feb 2008 | WO |
| WO 2008017871 | Feb 2008 | WO |
| WO 2007089911 | May 2008 | WO |
| WO 2007028146 | Jun 2008 | WO |
| WO 2007067734 | Aug 2008 | WO |
| WO 2007126938 | Oct 2008 | WO |
| WO 2007082154 | Nov 2008 | WO |
| WO 2007087612 | Nov 2008 | WO |
| WO 2007082144 | Dec 2008 | WO |
| WO 2007092713 | Dec 2008 | WO |
| 2009013492 | Jan 2009 | WO |
| 2009013496 | Jan 2009 | WO |
| WO 2007079229 | Jan 2009 | WO |
| WO 2007080583 | Feb 2009 | WO |
| WO 2007079250 | Mar 2009 | WO |
| WO 2007041610 | Apr 2009 | WO |
| WO 2009019455 | Apr 2009 | WO |
| 2010033578 | Mar 2010 | WO |
| Entry |
|---|
| Shuji Ogino, et al. “Sensitive Sequencing Method for KRAS Mutation Detection by Pyrosequencing” Journal of Molecular Diagnostics, vol. 7, No. 3, Aug. 2005 (Year: 2005). |
| Ravinder Dhallan, et al. “A non-invasive test for prenatal diagnosis based on fetal DNA present in maternal blood: a preliminary study” The Lancet, vol. 369 Feb. 10, 2007. (Year: 2007). |
| Thomas J Albert, et al. “Direct selection of human genomic loci by microarray hybridization” Nature Methods, vol. 4, No. 11, Nov. 2007 p. 903-905 (Year: 2007). |
| Emily Hodges, et al., “Genome-wide in situ exon capture for selective resequencing” Nature Genetics, vol. 39, No. 12, Dec. 2007, p. 1522-1527. (Year: 2007). |
| Thomas Jarvie “Next generation sequencing technologies” Drug Discovery Today: Technologies vol. 2, Issue 3, Autumn 2005, pp. 255-260 (Year: 2005). |
| Ruiqiang Li, et al.“SOAP: short oligonucleotide alignment program” Bioinformatics, vol. 24 No. 5 2008, pp. 713-714 (Year: 2008). |
| David R. Bentley, et al “Accurate whole human genome sequencing using reversible terminator chemistry” Nature, vol. 456, No. Nov. 6, 2008. (Year: 2008). |
| Written Opinion, issued in SG 201102011-2 on Sep. 25, 2013, 14 pages. |
| Examination Report, issued in EP 09 815 105.3 on May 8, 2014, 4 pages. |
| Examination Report, issued in EP 12 183 946.8 on May 7, 2014, 5 pages. |
| Y. M. Dennis Lo et al., “Noninvasive Prenatal Diagnosis of Fetal Chromosomal Aneuploidies by Maternal Plasma Nucleic Acid Analysis,” Clinical Chemistry, 54:3, pp. 461-466 (2008). |
| Examination Report, issued in SG 2011020112 on Oct. 23, 2014, 9 pages. |
| Wiemann, S., et al., “Doublex' Fluorescent DNA Sequencing: Two Independent Sequences Obtained Simultaneously in One Reaction with Internal Labeling and Unlabeled Primers,” Analytical Blochimistry, vol. 234, 1996, pp. 166-174. |
| Solexa, “Protocol for Whole Genome Sequencing using Solexa Technology,” BioTechniques, Protocol Guide, 2007, p. 29. |
| ACOG Practice Bulletin No. 88, “Invasive prenatal testing for aneuploidy,” Obstet Gynecol vol. 110, Dec. 2007, pp. 1459-1467, abstract only. |
| Bianchi et al., “PCR Quantitation of Fetal Cells in Maternal Blood in Normal and Aneuploid Pregnancies,” Am. J. Hum. Genet., vol. 61, 1997, pp. 822-829. |
| Boyle, et al., “F-Seq: a feature density estimator for high-throughput sequence tags,” Bioinformatics Applications Note, vol. 24 No. 21; doi:10.1093/bioinformatics/btn480, Sep. 10, 2008, pp. 2537-2538. |
| Chetverin, et al., “Oligonucleotide Arrays: New Concepts and Possibilities,” Nature Biotechnology, vol. 12, Nov. 1994, pp. 1093-1099. |
| Cheung et al., “Prenatal diagnosis of sickle cell anaemia and thalassaemia by analysis of fetal cells in maternal blood,” Nat Genet, vol. 14, 1996, pp. 264-268, abstract only. |
| Cooper, G. M., et al., “Chapter 5,” The Cell: A Molecular Approach, Sinauer Associates, Inc., 2007, pp. 166-176, 22 pages. |
| Cunningham et al., “Williams Obstetrics 21st Ed,” McGraw-Hill Professional, Apr. 27, 2001, p. 942. |
| Dhallan et al., “A non-invasive test for prenatal diagnosis based on fetal DNA present in maternal blood: a preliminary study,” The Lancet, vol. 369, Feb. 10, 2007, pp. 474-481. |
| Giacona et al., “Cell-Free DNA in Human Blood Plasma: Length Measurements in Patients with Pancreatic Cancer and Healthy Controls,” Pancreas, vol. 17, No. 1, 1998, pp. 89-97. |
| Hamada et al., “Fetal nucleated cells in maternal peripheral blood: frequency and relationship to gestational age,” Hum Genet, vol. 91, 1993, pp. 427-432. |
| Harris, et al., “Single-Molecule DNA Sequencing of a Viral Genome,” Science, vol. 320, Apr. 4, 2008, 106-109 and Suppl. Materials 1-25. |
| Hillier et al., “Whole-genome sequencing and variant discovery in C. elegans,” Nat Methods, vol. 5, No. 2, Feb. 2008, pp. 183-188. |
| Jahr et al., “DNA Fragments in the Blood Plasma of Cancer Patients: Quantitations and Evidence for Their Origin from Apoptotic and Necrotic Cells,” Cancer Res, vol. 61, Feb. 15, 2001, pp. 1659-1665. |
| Kaiser, “An Earlier Look At Baby's Genes,” Science, vol. 309, Sep. 2, 2005, pp. 1476-1478. |
| Khosrotehrani, “Fetal cell microchimerism: helpful or harmful to the parous woman?,” Current Opinion in Obstetrics and Gynecology, vol. 15, 2003, pp. 195-199. |
| Lander, et al., “Initial sequencing and analysis of the human genome,” Nature, vol. 409, Feb. 15, 2001, pp. 860-921. |
| Lee et al., “A high-resolution atlas of nucleosome occupancy in yeast,” Nature Genetics, vol. 39, No. 10, Oct. 2007, pp. 1235-1244. |
| Lo, et al., “Increased Fetal DNA Concentrations in the Plasma of Pregnant Women Carrying Fetuses with Trisomy 21,” Clinical Chemistry 45:10, 1999, pp. 1747-1751. |
| Lo, et al., “Noninvasive Prenatal Diagnosis of Fetal Chromosomal Aneuploidies by Maternal Plasma Nucleic Acid Analysis,” Clin Chem. 54(3), 2008, pp. 461-466. |
| Lo, et al., “Rapid Clearance of Fetal DNA from Maternal Plasma,” Am. J. Hum. Genet. 64, 1999, pp. 218-224. |
| Malone et al., “First-Trimester or Second-Trimester Screening, or Both, for Down's Syndrome,” N Engl J Med vol. 353, No. 19, Nov. 10, 2005, pp. 2001-2011. |
| Mardis, “Next-Generation DNA Sequencing Methods,” Annual Review of Genomics and Human Genetics, vol. 9, 2008, pp. 387-402. |
| Mewar et al., “Clinical and Molecular Evaluation of Four Patients with Partial Duplications of the Long Arm of Chromosome 18,” Am. J. Hum. Genet., vol. 53, 1993, pp. 1269-1278. |
| Moore, et al., “Prenatal diagnosis of aneuploidy and deletion 22q11.2 in fetuses with ultrasound detection of cardiac defects,” American Journal of Obstetrics and Gynecology, vol. 191, 2004, pp. 2068-2073. |
| Nelson, “Your cells are my cells,” Scientific American, Feb. 2008, pp. 72-79. |
| Ozsolak et al., “High-throughput mapping of the chromatin structure of human promoters,” Nature Biotechnology, 33 vol. 25, No. 2, Feb. 2007, pp. 244-248. |
| Prober, et al., “A System for Rapid DNA Sequencing with Fluorescent Chain-Terminating Dideoxynucleotides,” Science, vol. 238, Oct. 16, 1987, pp. 336-341. |
| Rijnders et al., “Cell-Free Fetal DNA is Not Present in Plasma of Nonpregnant Mothers,” Clinical Chemistry, vol. 50, No. 3, 2004, pp. 679-681. |
| Rogers, “Massively parallel sequencing,” Nature, vol. 437, Sep. 15, 2005, pp. 326-327. |
| Samura et al., “Cell-free Fetal DNA in Maternal Circulation after Amniocentesis,” Clinical Chemistry, vol. 49, No. 7, 2003, pp. 1193-1195. |
| Schones et al., “Dynamic Regulation of Nucleosome Positioning in the Human Genome,” Cell, vol. 132, Mar. 7, 2008, pp. 887-898. |
| Segal et al., “A genomic code for nucleosome positioning,” Nature, vol. 442, Aug. 2006, pp. 772-778. |
| Smid et al., “No evidence of fetal DNA persistence in maternal plasma after pregnancy,” Hum Genet, vol. 112, 2003, pp. 617-618. |
| Tong, et al., “Noninvasive Prenatal Detection of Fetal Trisomy 18 by Epigenetic Allelic Ratio Analysis in Maternal Plasma: Theoretical and Empirical Considerations,” Clinical Chemistry 52:12, 2006, pp. 2194-2202. |
| Venter, et al., “The Sequence of the Human Genome,” Science, vol. 291, Feb. 16, 2001, pp. 1304-1351. |
| Wang, et al., “Digital karyotyping,” PNAS, vol. 99, No. 25, Dec. 10, 2002, pp. 16156-16161. |
| Wang, et al., “Digital karyotyping identifies thymidylate synthase amplification as a mechanism of resistance to 5-fluorouracil in metastatic colorectal cancer patients,” Proc Natl Acad Sci, 101(9); doi: 10.1073/pnas.0308716101, Mar. 2, 2004, pp. 3089-3094. |
| Wheeler, et al., “The complete genome of an individual by massively parallel DNA sequencing,” Nature. vol. 452, Apr. 17, 2008, pp. 872-876. |
| Wiemann et al., “Simultaneous On-line DNA Sequencing on Both Strands with Two Fluorescent Dyes,” Anal. Biochem., vol. 224, 1995, pp. 117-121. |
| Yuan et al., “Genome-Scale Identification of Nucleosome Positions in S. cerevisiae,” Science, vol. 309, Jul. 22, 2005, pp. 626-630. |
| Zhang et al., “A novel method to calculate the G+C content of genomic sequences,” J. Biomol Struct Dyn vol. 2, Abstract only, 2001. |
| Zheng, et al., “Fetal cell identifiers: Results of microscope slide—based immunocytochemical studies as a function of gestational age and abnormality,” Am J Obstet Gynecol, vol. 180, No. 5, May 1999, pp. 1234-1239. |
| Rebecca Sparkes, et al., “New Molecular Techniques for the Prenatal Detection of Chromosomal Aneuploidy,” JOGC, Jul. 2008, No. 210, 617-621. |
| Bernhard Zimmermann, “Molecular Diagnosis in Prenatal Medicine,” Ph.D. Thesis, 2004. |
| Jay Shendure, et al., “Next-generation DNA sequencing”, Nature, 2008, 26:1135-1145. |
| Juliane C. Dohm, et al., “Substantial biases in ultra-short read data sets from high-throughput DNA sequencing”, Nucleic Acids Research, 2008, 36: e105 doi: 10.1093\nar\gkn425. |
| Heng Li, et al., “Mapping short DNA sequencing reads and calling variants using mapping quality scores”, Genome Research, 2008, doi: 10.1101/gr.078212.108. |
| Rossa W. K. Chiu, et al., “Non-invasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in matemal plasma”, PNAS, 2008, 105: 20458-20463. |
| Melissa J. Fullwood, et al., “Next-generation DNA sequencing of paired-end tags (PET) for transcriptome and genome analyses”, Genomne Research, 2009, 19: 521-532. |
| Y.M. Dennis Lo, et al., “Digital PCR for the molecular detection of fetal chromosomal aneuploidy,” PNAS, Aug. 7, 2007, vol. 104, No. 32, 13116-13121. |
| Yuk-Ming Dennis Lo, “Noninvasive prenatal detection of fetal chromosomal aneuploidies by maternal plasma nucleic acid analysis: a review of the current state of the art,” BJOG, 2009, vol. 116, 152-157. |
| H. Christina Fan, et al., “Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood,” PNAS, Oct. 21, 2008, vol. 105, 16266-16271. |
| Fiona M. F. Lun, et al., “Microfluidics Digital PCR Reveals a Higher than Expected Fraction of Fetal DNA in Maternal Plasma,” Clinical Chemistry, 2008, vol. 54, No. 10, 1664-1672. |
| Richard A. White III, et al., “Digital PCR provides sensitive and absolute calibration for high throughput sequencing,” BMC Genomics, Mar. 19, 2009, 10:116. |
| Frank Diehl, et al., “Digital quantification of mutant DNA in cancer patients,” Curr Opin Oncol, 2007, 19:36-42. |
| Y-M. D. Lo, et al., “Prenatal Sex Determination by DNA Amplification from Maternal Peripheral Blood,” The Lancet, Dec. 9, 1989, 1363-1365. |
| Y-M. D. Lo, et al., “Detection of fetal RhD sequence from peripheral blood of sensitized RhD-negative pregnant women,” British Journal of Haematology, 1994, vol. 87, 658-660. |
| Y. M. Dennis Lo, et al., “Presence of fetal DNA in maternal plasma and serum,” The Lancet, Aug. 16, 1997, vol. 350, 485-487. |
| Jouni Uitto, et al., “Probing the fetal genome: progress in non-invasive prenatal diagnosis,” Trends in Molecular Medicine, Aug. 2003, vol. 9, No. 8, 339-343. |
| Sinuhe Hahn, et al., “Prenatal Diagnosis Using Fetal Cells and Cell-Free Fetal DNA in Maternal Blood: What is Currently Feasible?” Clinical Obstetrics and Gynecology, Sep. 2002, vol. 45, No. 3, 649-656. |
| Barbara Pertl, et al., “Fetal DNA in Maternal Plasma: Emerging Clinical Applications,” Obstetrics and Gynecology, Sep. 2001, vol. 98, No. 3, 483-490. |
| Leo L.M. Poon, et al., “Circulating fetal DNA in maternal plasma,” Clinical Chimica Acta, 2001, vol. 313, 151-155. |
| Y-M. D. Lo, et al., “Fetal DNA in Maternal Plasma,” Ann. N. Y. Acad. Sci, Apr. 2000, vol. 906, 141-147. |
| Y-M. D. Lo, et al., “Detection of single-copy fetal DNA sequence from maternal blood,” The Lancet, Jun. 16, 1990, vol. 335, 1463-1464. |
| Y.M. Dennis Lo, et al., “Quantitative Analysis of Fetal NA in Maternal Plasma and Serum: Implications for Noninvasive Prenatal Diagnosis,” Am J. Hum. Genet., 1998, vol. 62, 768-775. |
| Rossa W.K. Chiu, et al., “Effects of Blood-Processing Protocols on Fetal and Total DNA Quantification in Maternal Plasma,” Clinical Chemistry, 2001, vol. 47, No. 9, 1607-1613. |
| Enders K.O. Ng, et al., “The Concentration of Circulating Corticotropin-releasing Hormone mRNA in Maternal Plasma Is Increased in Preeclampsia,” Clinical Chemistry, 2003, vol. 49, No. 5, 727-731. |
| Ido Braslavsky, et al., “Sequence information can be obtained from single DNA molecules,” PNAS, Apr. 2003, vol. 100, No. 7, 3960-3964. |
| Jun Zhu, et al., “Single Molecule Profiling of Alternative Pre-mRNA Splicing,” Science, Aug. 2003, vol. 301, 836-838. |
| Devin Dressman, et al., “Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations,” PNAS, Jul. 2003, vol. 100, No. 15, 8817-8822. |
| Eugene Y. Chan, et al., “DNA Mapping Using Microfluidic Stretching and Single-Molecule Detection of Fluorescent Site-Specific Tags,” Genome Research, 2004, vol. 14, 1137-1146. |
| Jong Wook Hong, et al., “Molecular biology on a microfluidic chip,” Journal of Physics: Condensed Matter, 2006, vol. 18, S691-S701. |
| Elizabeth A. Ottesen, et al., “Microfluidic Digital PCR Enables Multigene Analysis of Individual Environmental Bacteria,” Science, Dec. 2006, vol. 314, 1464-1467. |
| Joshua S. Marcus, et al., “Parallel Picoliter RT-PCR Assays Using Microfluidics,” Analytical Chemistry, Feb. 1, 2006, vol. 78, No. 3, 956-958. |
| Y. M. Dennis Lo, et al., “Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection,” Nature Medicine, Jan. 2007, 1-6. |
| Joshua S. Marcus, et al., “Microfluidic Single-Cell mRNA Isolation and Analysis,” American Chemical Society, Mar. 2006, p. A-F. |
| Y. M. Dennis Lo, et al., “Prenatal diagnosis: progress through plasma nucleic acids,” Nature, Jan. 2007, vol. 8, 71-76. |
| Tetsuya S. Tanaka, et al., “Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray,” PNAS, Aug. 2000, vol. 97, No. 16, 9127-9132. |
| “Separation of RNA & DNA by Gel Filtration Chromatography,” Edvotek, 1987, 1-9. |
| Li, Y., et al., “Size Separation of Circulatory DNA in Maternal Plasma Permits Ready Detection of Fetal DNA Polymorphisms,” 2004, Clinical Chemistry, vol. 50, No. 6, pp. 1002-1011. |
| H. Christina Fan, et al., “Detection of Aneuploidy with Digital Polymerase Chain Reaction,” Analytical Chemistry, Oct. 1, 2007, vol. 79, No. 19, 7576-7579. |
| H. Christina Fan, et al., “Microfluidic digital POR enables rapid prenatal diagnosis of fetal aneuploidy,” American Journal of Obstetrics & Gynecology, May 2009, 543e1-543-e7. |
| Lo, et al., U.S. Appl. No. 60/951,438, “Determining a Nucleic Acid Sequence Imbalance” filed Jul. 23, 2007. |
| Giurato, et al., “An accurate pipeline for analysis of NGS data of small non-coding RNA,” EMBnetJournal vol. 18, pp. 100-101 (2012). |
| Green, et al., “Analysis of one million base pairs of Neanderthal DNA,” Nature, vol. 444, pp. 330-336 (2006). |
| Nannya, et al., “A robust algorithm for copy number detection using high-density oligonucleotide single nucleotide polymorphism genotyping arrays,” Cancer Res. vol. 65, pp. 6071-6079 (2005). |
| Noonan, et al., “Sequencing and Analysis of Neanderthal Genomic DNA,” Science vol. 314, pp. 1113-1118 (2006). |
| Seo, et al., “Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides,” Proc. Nat. Acad. Sci. vol. 102, No. 17, pp. 5926-5931 (2005). |
| Smith, et al., “Using quality scores and longer reads improves accuracy of Solexa read mapping,” BMC Bioinformatics, vol. 9, 128, pp. 1-8 (2008). |
| Thornley, “Analysis of Trace Data from Fluorescence Based Sanger Sequencing,” Thesis, University of London Imperial College of Science, Technology and Medicine Department of Computing, 1997. |
| Court Document dated Sep. 28, 2012, Case No. 3:12-cv-00865-SI, Invalidity Contentions. |
| Dohm et al., Nucleic Acids Research, 2008, vol. 36, pp. 1-10. |
| Braun et al., Statistical Science, 1998, vol. 13, pp. 142-162. |
| Extended European Search Report in EP 18156359.4 dated May 31, 2018, 10 pages. |
| Office Action issued in Canadian application No. 2737643 mailed Jul. 30, 2019, 4 pages. |
| Examination Report No. 1 issued for Australian patent application No. 2018201920 dated Oct. 18, 2019, 5 pages. |
| Australian Examination Report, issued in AU 2015202167 on Dec. 21, 2016, 4 pages. |
| Communication issued for European patent application No. 19172977.1 dated Dec. 9, 2019, 13 pages. |
| Zimmermann, B. G., et al., “Digital PCR: a powerful new tool for noninvasive prenatal diagnosis,” Prenatal Diagnosis 2008, vol. 28, pp. 1087-1093. |
| Evans M. I., et al., “Digital PCR for Noninvasive Detection of Aneuploidy: Power Analysis Equations for Feasibility,” Fetal Diagnosis and Therapy, 2012, vol. 31, pp. 244-247. |
| El Khattabi, L. A., et al. “Could Digital PCR Be an Alternative as a Non-Invasive Prenatal Test for Trisomy 21: A Proof of Concept Study,” PLOS ONE 2016, vol. 11(5), 13 pages. |
| Canadian Office Action issued in CA 2,737,643 on Nov. 21, 2016, 4 pages. |
| U.S. Appl. No. 11/825,298, filed Jul. 5, 2007, Lopez et al. |
| U.S. Appl. No. 11/825,677, filed Jul. 5, 2007, Lopez et al. |
| U.S. Appl. No. 11/909,959, filed Sep. 27, 2007, Duff. |
| U.S. Appl. No. 60/764,420, filed Feb. 2, 2005, Quake. |
| U.S. Appl. No. 60/949,227, filed Jul. 11, 2007, Kapur. |
| Adinolfi, et al. Gene Amplification to Detect Fetal Nucleated Cells in Pregnant Women. The Lancet. Aug. 5, 1989:328-329. |
| Adinolfi, et al. Rapid detection of aneuploidies by microsatellite and the quantitative fluorescent polymerase chain reaction. Prenat. Diagn. 1997; 17(13):1299-311. |
| Adinolfi, M. On a Non-Invasive Approach to Prenatel Diagnosis based on the detection of Fetal Nucleated Cells in Maternal Blood Samples. Prenatal Diagnosis. 1991;11:799-804. |
| Ahn, et al. A fully integrated micromachined magnetic particle separator. Journal of Microelectromechanical Systems. 1996; 5(3):151-158. |
| Andrews, et al. Enrichment of fetal nucleated cells from maternal blood: model test system using cord blood. Prenatal Diagnosis. 1995; 15:913-919. |
| Applicant's Amendment and Response dated Jun. 17, 2009 Non-Final Office Action of Jan. 28, 2009 re U.S. Appl. No. 11/701,686. |
| Ariga, et al. Kinetics of fetal cellular and cell-free DNA in the maternal circulation during and after pregnancy: implications for noninvasive prenatal diagnosis. Transfusion. 2001; 41:1524-1530. |
| Arnould, et al. Agreement between chromogenic in situ hybridisation (CISH) and FISH in the determination of HER2 status in breast cancer. Br J Cancer. 2003; 88(10):1587-91. (Abstract only). |
| Babochkina, et al. Direct detection of fetal cells in maternal blood: a reappraisal using a combination of two different Y chromosome-specific FISH probes and a single X chromosome-specific probe. Arch Gynecol Obstet. Dec. 2005;273(3):166-9. (Abstract only). |
| Babochkina, T. I. Ph. D. Dissertation—Fetal cells in maternal circulation: Fetal cell separation and FISH analysis. University of Basel, Switzerland. Dec. 8, 2005. (123 pages). |
| Balko, et al. Gene expression patterns that predict sensitivity to epidermal growth factor receptor tyrosine kinase inhibitors in lung cancer cell lines and human lung tumors. BMC Genomics. Nov. 1, 20060;7:289 (14 pages). |
| Barrett, et al. Comparative genomic hybridization using oligonucleotide microarrays and total genomic DNA. Proc Natl Acad Sci U S A. 2004; 101(51):17765-70. |
| Basch, et al. Cell separation using positive immunoselective techniques. Journal of Immunological Methods. 1983;56:269-280. |
| Bauer, J. Advances in cell separation: recent developments in counterflow centrifugal elutriation and continuous flow cell separation. Journal of Chromatography. 1999;722:55-69. |
| Becker, et al. Fabrication of Microstructures With High Aspect Ratios and Great Structural Heights by Synchrotron Radiation Lithography, Galvanoforming, and Plastic Moulding (LIGA Process). Microelectronic Engineering. 1986;4:35-56. |
| Becker, et al. Planar quartz chips with submicron channels for two-dimensional capillary electrophoresis applications. J. Micromech Microeng. 1998;9:24-28. |
| Beebe et al. Functional Hydrogel Structures for Autonomous Flow Control Inside Microfluidic Channels. Nature. 2000; 404:588-590. |
| Bennett, et al. Toward the 1,000 dollars human genome. Pharmacogenomics. 2005;6(4):373-82. |
| Berenson, et al. Cellular Immunoabsorption Using Monoclonal Antibodies. Transplantation.1984;38:136-143. |
| Berenson, et al. Positive selection of viable cell populations using avidin-biotin immunoadsorption. Journal of Immunological Methods. 1986;91:11-19. |
| Berg, H. C. Random Walks in Biology, Ch. 4. Princeton University Press. Princeton, NJ. 1993. pp. 48-64. |
| Berger, et al. Design of a microfabricated magnetic cell separator. Electrophoresis. Oct. 2001;22(18):3883-92. |
| Bianchi, et al. Isolation of fetal DNA from nucleated erythrocytes in maternal blood. Medical Sciences. 1990;87:3279-3283. |
| Bianchi, et al. Demonstration of fetal gene sequences in nucleated erythrocytes isolated from maternal blood. American Journal of Human Genetics. 1989;45:A252. |
| Bianchi, et al. Fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of Nifty I data. Prenatal Diagnosis. 2002; 22:609-615. |
| Bianchi, et al. Fetal nucleated erythrocytes (FNRBC) in maternal blood: erythroid-specific antibodies improve detection. The American Journal of Human Genetics. Oct. 1992. Supplemental to vol. 51, No. 4: 996. |
| Bianchi, et al. Isolation of Male Fetal DNA from Nucleated Erythrocytes (NRBC) in Maternal Blood. The American Pediatric Society and Society for Pediatric Research, (1989) Mar. 1989; 818:139A. |
| Bianchi, et al. Possible Effects of Gestational Age on the Detection of Fetal Nucleated Erythrocytes in Maternal Blood. Prenatal Diagnosis. 1991;11:523-528. |
| Bignell, et al. High-resolution analysis of DNA copy number using oligonucleotide microarrays. Genome Research. 2004; 14(2):287-295. |
| Blake, et al. Assessment of multiplex fluorescent PCR for screening single cells for trisomy 21 and single gene defects. Mol. Hum. Reprod. 1999; 5(12):1166-75. |
| Bode, et al. Mutations in the tyrosine kinase domain of the EGFR gene are rare in synovial sarcoma. Mod Pathol. Apr. 2006;19(4):541-7. |
| Boehm, et al. Analysis of Defective Dystrophin Genes with cDNA Probes: Rearrangement Polymorphism, Detection of Deletions in Carrier Females, and Lower Than Expected Frequency of Carrier Mothers in Isolated Cases of Delections. Pediatric Research. Apr. 1989: 139A-820. |
| Bohmer, et al. Differential Development of Fetal and Adult Haemoglobin Profiles in Colony Culture: Isolation of Fetal Nucleated Red Cells by Two-Colour Fluorescence Labelling. Br. J. Haematol. 1998; 103:351-60. |
| Brison, et al. General Method for Cloning Amplified DNA by Differential Screening with Genomic Probes. Molecular and Cellular Biology. 1982;2:578-587. |
| Brody, et al. Deformation and Flow of Red Blood Cells in a Synthetic Lattice: Evidence for an Active Cytoskeleton. Biophys. J. 68:2224-2232 (1995). |
| Caggana, M. Microfabricated devices for sparse cell isolation. CNF Project #905-00. Cornell NanoScale Facility. 2003; pp. 38-39. |
| Caggana, M. Microfabricated devices for sparse cell isolation. CNF Project #905-00. Cornell NanoScale Facility. 2004-2005; pp. 32-33. |
| Calin, et al. A microRNA signature Associated with prognosis and progression in chronic lymphocytic leukemia. New England Journal of Medicine. 2005; 353:1793-1801. |
| Cha, The utility of an erythroblast scoring system and gender-independent short tandem repeat (STR) analysis for the detection of aneuploid fetal cells in maternal blood. Prenat. Diagn. 2005; 25(7):586-91. |
| Chamberlain, et al. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Research. 1988;16:11141-11156. |
| Chan, et al. Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem. Jan. 2004;50(1):88-92. |
| Chang, et al. Biomimetic technique for adhesion-based collection and separation of cells in a microfluidic channel. Lab Chip. 2005; 5:64-73. |
| Cheung, et al. Development and validation of a CGH microarray for clinical cytogenetic diagnosis. Genet Med. 2005; 7(6):422-32. |
| Chiu, et al. Patterned Deposition of Cells and Proteins Onto Surfaces by Using Three-Dimensional Microfluidic Systems. Proceedings of the National Academy of Sciences of the United States of America. 2000; pp. 2408-2413. |
| Choesmel, et al. Enrichment methods to detect bone marrow micrometastases in breast carcinoma patients: clinical relevance. Breast Cancer Res. 2004;6(5):R556-569. |
| Choolani, et al. Characterization of First Trimester Fetal Erythroblasts for Non-Invasive Prenatal Diagnosis. Mol. Hum. Reprod. 2003; 9:227-35. |
| Chou, et al. A Microfabricated Device for Sizing and Sorting DNA Molecules. Proceedings of the National Academy of Sciences of the United States of America. 1999; pp. 11-13. |
| Chou, et al. Sorting by diffusion: An asymmetric obstacle course for continuous molecular separation. PNAS. 1999; 96(24):13762-13765. |
| Christel, et al. High aspect ratio silicon microstructures for nucleic acid extraction. Solid-state sensor and actuator workshop. Hilton Head, SC. Jun. 8-11, 1998; 363-366. |
| Christensen, et al. Fetal Cells in Maternal Blood: A Comparison of Methods for Cell Isolation and Identification. Fetal Diagn. Ther. 2005; 20:106-12. |
| Chueh, et al. Prenatal Diagnosis Using Fetal Cells from the Maternal Circulation. West J. Med. 159:308-311 (1993). |
| Chueh, et al. Prenatal Diagnosis Using Fetal Cells in the Maternal Circulation. Seminars in Perinatology. 1990;14:471-482. |
| Chueh, et al. The search for fetal cells in the maternal circulation. J Perinat Med. 1991;19:411-420. |
| Cirigliano, et al. “Clinical application of multiplex quantitative fluorescent polymerase chain reaction (QF-PCR) for the rapid prenatal detection of common chromosome aneuploidies,” Molecular Human Reproduction, 2001, vol. 7, No. 10, 1001-1006. |
| Clayton, et al. Fetal Erythrocytes in the Maternal Circulation of Pregnant Women. Obstetrics and Gynecology. 1964;23:915-919. |
| Collarini, et al. Comparison of methods for erythroblast selection: application to selecting fetal erythroblasts from maternal blood. Cytometry. 2001; 45:267-276. |
| Cremer, et al. Detection of chromosome aberrations in human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L1.84. Human Genetics. 1986;74:346-352. |
| Cremer, et al. Detection of chromosome aberaations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes. Human Genetics. 1988;80:235-246. |
| Das, et al. Dielectrophoretic segregation of different human cell types on microscope slides. Anal. Chem. 2005; 77:2708-2719. |
| De Alba, et al. Prenatal diagnosis on fetal cells obtained from maternal peripheral blood: report of 66 cases. Prenat Diagn. Oct. 1999; 19(10):934-40. |
| De Kretser, et al. The Separation of Cell Populations using Monoclonal Antibodies attached to Sepharose. Tissue Antigens. 1980;16:317-325. |
| Delamarche, et al. Microfluidic Networks for Chemical Patterning of Substrates: Design and Application to Bioassays. Journal of the American Chemical Society. 1998; 120:500-508. |
| Delamarche, et al. Patterned Delivery of Immunoglobulins to Surfaces Using Microfluidic Networks. Science. 1997; 276:779-781. |
| Deng, et al. Enumeration and microfluidic chip separation of circulating fetal cells early in pregnancy from maternal blood. American Journal of Obstetrics & Gynecology. Dec. 2008 (vol. 199, Issue 6, p. S134). |
| Deshmukh, et al. Continuous Micromixer With Pulsatile Micropumps. Solid-State Sensor and Actuator Workshop. Hilton Head Island, South Carolina; Jun. 4-8, 2000:73-76. |
| Di Naro, et al. Prenatal diagnosis of beta-thalassaemia using fetal erythroblasts enriched from maternal blood by a novel gradient. Mol Hum Reprod. 2000; 6(6):571-4. |
| Dilella, et al. Screening for Phenylketonuria Mutations by DNA Amplification with the Polymerase Chain Reaction. The Lancet. Mar. 5, 1988:497-499. |
| Doyle, et al. Self-Assembled Magnetic Matrices for DNA Separation Chips. Science 295:2237 (2002). |
| Eigen, et al. Sorting Single Molecules: Application to Diagnostics and Evolutionary Biotechnology. Proceedings of the National Academy of Sciences of the United States of America. 1994; 91:5740-5747. |
| Emanuel, et al. Amplification of Specific Gene Products from Human Serum. GATA, 1993, vol. 10, No. 6, 144-146. |
| European search report dated Nov. 9, 2009 for Application No. 7784442.1. |
| European search report dated Dec. 21, 2009 for Application No. 07798579.4. |
| European search report dated Dec. 22, 2009 for Application No. 07798580.2. |
| European search report dated Dec. 22, 2009 for Application No. 0778444.7. |
| European Search Report dated Jul. 31, 2009 for EP07763674.4. |
| Final Office Action dated Sep. 11, 2009 issued in U.S. Appl. No. 11/701,686. |
| Non-Final Office Action dated Jan. 28, 2009 issued in U.S. Appl. No. 11/701,686. |
| Falcidia, et al. Fetal Cells in maternal blood: a six-fold increase in women who have undergone mniocentesis and carry a fetus with Down syndrome: a multicenter study. Neuropediatrics. 2004; 35(6):321-324. (Abstract only). |
| Fan, et al. Highly parallel SNP genotyping. Cold Spring Harb. Symp. Quant. Biol. 2003; 68:69-78. |
| Fan, et al. Single cell degenerate oligonucleotide primer-PCR and comparative genomic hybridization with modified control reference. Journal of Ahejian University—Science A. 2001; 2(3):318-321. |
| Farber, et al. Demonstration of spontaneous XX/XY chimerism by DNA fingerprinting. Human Genetics. 1989;82:197-198. |
| Farooqui, et al. Microfabrication of Submicron Nozzles in Silicon Nitride. Journal of Microelectromechanical Systems. 1992; 1(2):86-88. |
| Fiedler, et al. Dielectrophoretic Sorting of Particles and Cells in a Microsystem. Analytical Chemistry. 1998; pp. 1909-1915. |
| Findlay, et al. Using MF-PCR to diagnose multiple defects from single cells: implications for PGD. Mol Cell Endocrinol. 2001; 183 Suppl 1:S5-12. |
| Freemantle, M. Downsizing Chemistry. Chemical analysis and synthesis on microchips promise a variety of potential benefits. Chemical & Engineering News. 1999; pp. 27-36. |
| Fu, et al. An integrated miscrofabricated cell sorter. Anal Chem. 2002;74:2451-2457. |
| Fu, et al. A Microfabricated Fluorescence-Activated Cell Sorter. Nature Biotechnology.1999; 17:1109-1111. |
| Fuhr, et al. Biological Application of Microstructures. Topics in Current Chemistry. 1997; 194:83-116. |
| Furdui, et al. Immunomagnetic T cell capture from blood for PCR analysis using microfluidic systems. Lab Chip. Dec. 2004;4(6):614-8. |
| Ganshirt-Ahlert, et al. Magnetic cell sorting and the transferrin receptor as potential means of prenatal diagnosis from maternal blood. Am J Obstet Gynecol. 1992;166:1350-1355. |
| Ganshirt-Ahlert, et al. Noninvasive prenatal diagnosis: Triple density gradient, magnetic activated cell sorting and FISH prove to be an efficient and reproducible method for detection of fetal aneuploidies from maternal blood. The American Journal of Human Genetics. Oct. 1992. Supplemental to vol. 51, No. 4: 182. |
| GenomeWeb. Immunicon inks biomarker assay, lab services deal with merck serona. Available at C:\Documents and Settings\fc3\Local Settings\Temporary Internet Files\OLK35E\141896-1.htm. Accessed on Sep. 11, 2007. |
| Ghia, et al. Ordering of human bone marrow B lymphocyte precursors by single-cell polymerase chain reaction analyses of the rearrangement status of the immunoglobulin H and L chain gene loci. J Exp Med. Dec. 1, 1996;184(6):2217-29. |
| Giddings, J. C. Chemistry 'Eddy' Diffusion in Chromatography. Nature. 1959;184:357-358. |
| Giddings, J. C. Field-Flow Fractionation: Analysis of Macromolecular, Colloidal, and Particulate Materials. Science. 1993;260:1456-1465. |
| Gonzalez, et al. Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environments. Environ Microbiol. 2005; 7(7):1024-8. |
| Graham. Efficiency comparison of two preparative mechanisms for magnetic separation of erthrocytes from whole blood. J. Appl. Phys. 1981; 52:2578-2580. |
| Greaves, et al. Expression of the OKT Monoclonal Antibody Defined Antigenic Determinants in Malignancy. Int. J. Immunopharmac. 1981;3:283-299. |
| Guetta, et al. Analysis of fetal blood cells in the maternal circulation: challenges, ongoing efforts, and potential solutions. Stem Cells Dev. 2004;13(1):93-9. |
| Gunderson, et al. A genome-wide scalable SNP genotyping assay using microarray technology. Nat Genet. 2005; 37(5):549-54. |
| Hahn, et al. Current applications of single-cell PCR. Cell. Mol. Life Sci. 2000; 57(1):96-105. Review. |
| Hamabe, et al. Molecular study of the Prader-Willi syndrome: deletion, RFLP, and phenotype analyses of 50 patients. Am J Med Genet. Oct. 1, 1991;41(1):54-63. |
| Han, et al. Separation of Long DNA Molecules in a Microfabricated Entropic Trap Array. Science. 2000;288:1026-1029. |
| Hardenbol, et al. Highly multiplexed molecular inversion probe genotyping: over 10,000 targeted SNPs genotyped in a single tube assay. Genome Res. 2005;15(2):269-75. |
| Hardenbol, et al. Multiplexed genotyping with sequence-tagged molecular inversion probes. Nat. Biotechnol. 2003; 21(6):673-8. |
| Hartmann, et al. Gene expression profiling of single cells on large-scale oligonucleotide arrays. Nucleic Acids Research. 2006; 34(21): e143. (11 pages). |
| Herzenberg, et al. Fetal cells in the blood of pregnant women: Detection and enrichment by flourescence-activated cell sorting. Proc. Natl. Acad. Sci. 1979;76:1453-1455. |
| Holzgreve, et al. Fetal Cells in the Maternal Circulation. Journal of Reproductive Medicine. 1992;37:410-418. |
| Hong, et al. A nanoliter-scale nucleic acid processor with parallel architecture. Nat. Biotechnol. 2004; 22(4):435-9. |
| Hosono, et al. Unbiased whole-genome amplification directly from clinical samples. Genome Res. May 2003;13(5):954-64. |
| Hromadnikova, et al. “Quantitative analysis of DNA levels in maternal plasma in normal and Down syndrome pregnancies.” Bio Med Central, May 2002, 1-5. |
| Huang, et al. A DNA prism for high-speed continuous fractionation of large DNA molecules. Nature Biotechnology. 2002;20:1048-1051. |
| Huang, et al. Continuous Particle Separation Through Deterministic Lateral Displacement. Science 304:987-90 (2004). |
| Huang, et al. Electric Manipulation of Bioparticles and Macromoledules on Microfabricated Electrodes. Analytical Chemistry. 2001; pp. 1549-1559. |
| Huang, et al. Role of Molecular Size in Ratchet Fractionation. 2002; 89(17):178301-1-178301-4. |
| Huh, et al. Gravity-driven microhydrodynamics-based cell sorter (microHYCS) for rapid, inexpensive, and efficient cell separation and size-profiling. 2nd Annual International IEEE-EMBS Special Topic Conference on Microtechnology in Medicine and Biology. Madison, Wisconsin USA; May 2-4, 2002:466-469. |
| Hviid T. In-Cell PCT method for specific genotyping of genomic DNA from one individual in a micture of cells from two individuals: a model study with specific relevance to prenatal diagnosis based on fetal cells in maternal blood. Molecular Diagnostics and Genetics. 2002; 48:2115-2123. |
| Hviid, T. In-cell polymerase chain reaction: strategy and diagnostic applications. Methods Mol Biol. 2006;336:45-58. |
| International search report and written opinion dated Mar. 16, 2010 for PCT Application No. US2009/57136. |
| International Search Report and Written Opinion dated Sep. 18, 2008 for PCT/US2007/003209, received Sep. 22, 2008. |
| International search report dated Jan. 16, 2008 for PCT Application No. US2007/71247. |
| International search report dated Jan. 25, 2008 for PCT Application No. US2007/71250. |
| International search report dated Nov. 15, 2007 for PCT Application No. US2007/71149. |
| International search report dated Nov. 26, 2007 for PCT Application No. US2007/71256. |
| International search report dated Feb. 25, 2008 for PCT Application No. US07/71148. |
| International search report dated Feb. 25, 2008 for PCT Application No. US2007/71248. |
| Iverson, et al. Detection and Isolation of Fetal Cells From Maternal Blood Using the Flourescence-Activated Cell Sorter (FACS). Prenatal Diagnosis 1981;1:61-73. |
| Jan, et al. Fetal Erythrocytes Detected and Separated from Maternal Blood by Electronic Fluorescent Cell Sorter. Texas Rep Biol Med. 1973;31:575. |
| Jeon, et al. Generation of Solution and surface Gradients Using Microfluidic Systems. Langmuir. 2000, pp. 8311-8316. |
| Jiang, et al. Genome amplification of single sperm using multiple displacement amplification. Nucleic Acids Res. 2005; 33(10):e91. (9 pages). |
| Kamholz, et al. Quantitative Analysis of Molecular Interaction in a Microfluidic Channel: the T-Sensor. Analytical Chemistry. 1999; pp. 5340-5347. |
| Kan, et al. Concentration of Fetal Red Blood Cells From a Mixture of Maternal and Fetal Blood by Anti-i Serum—An Aid to Prenatal Diagnosis of Hemoglobinopathies. Blood. 1974; 43:411-415. |
| Kasakov, et al. Extracellular DNA in the blood of pregnant women. Tsitologiia. 1995;37(3):232-6. (English translation only). |
| Kenis, et al. Microfabrication Inside Capillaries Using Multiphase Laminar Flow Patterning. Science. 1999; 285:83-85. |
| Kim, et al. Polymer microstructures formed by moulding in capillaries. Nature. 1995;376:581-584. |
| Kimura, et al. The DYRKIA gene, encoded in chromosome 21 Down syndrome critical region, bridges between (ß-amyloid production and tau phosphorylation in Alzheimer disease. Human Molecular Genetics, Nov. 29, 2008, vol. 16, No. 1, 15-23. |
| Klein, C. A. Single cell amplification methods for the study of cancer and cellular ageing. Mech. Ageing Dev. 2005; 126(1):147-51. |
| Klein, et al. Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells. Proc Natl Acad Sci U S A. 1999; 96(8):4494-9. |
| Kogan, et al. An Improved Method for Prenatal Diagnosis of Genetic Diseases by Analysis of Amplified DNA Sequences, Application to Hemophilia A. The New England Journal of Medicine. 1987;317:985-990. |
| Korenberg, et al. Down syndrome phenotypes: the consequences of chromosomal imbalance. PNAS 1994; 91:4997-5001. |
| Krabchi, et al. Quantification of all fetal nucleated cells in maternal blood between the 18th and 22nd weeks of pregnancy using molecular cytogenic techniques. Clin. Genet. 2001; 60:145-150. |
| Krivacic, et al. A rare-cell detector for cancer. PNAS. 2004; 101:10501-10504. |
| Kulozik, et al. Fetal Cell in the Maternal Circulation: Detection by Direct AFP-Immunoflourescence. Human Genetics. 1982;62:221-224. |
| Leutwyler, K. Mapping Chromosome 21. Available at http://www.scientificamerican.com/article.cfm?id=mapping-chromosome-21. Accessed Feb. 3, 2010. |
| Levett, et al. A large-scale evaluation of amnio-PCR for the rapid prenatal diagnosis of fetal trisomy. Ultrasound Obstet Gynecol. 2001; 17(2):115-8. |
| Li , et al. Transport, Manipulation, and Reaction of Biological Cells On-Chip Using Electrokinetic Effects. Analytical Chemistry., 1997; pp. 1564-1568. |
| Li, et al. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature. 1988;335:414-417. |
| Lichter , et al. Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hyridization using recombinant DNA libraries. Hum Genet. 1988;80:224-234. |
| Liu, et al. Development and validation of a T7 based linear amplification for genomic DNA. BMC Genomics. 2003; 4(1):19. (11 pages). |
| Loken , et al. Flow Cytometric Analysis of Human Bone Marrow: I. Normal Erythroid Development. Blood. 1987;69:255-263. |
| Mahr, et al. Fluorescence in situ hybridization of fetal nucleated red blood cells. The American Journal of Human Genetics. Oct. 1992. Supplemental to vol. 51, No. 4: 1621. |
| Maloney et al. “Microchimerism of maternal origin persists into adult life,” J. Clin. Invest. 104:41-47 (1999). |
| Margulies, et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature. 2005; 437:376-80. |
| Martin, et al. “A method for using serum or plasma as a source of NDA for HLA typing,” Human Immunology. 1992; 33:108-113. |
| Mavrou, et al. Identification of nucleated red blood cells in maternal circulation: A second step in screening for fetal aneuploidies and pregnancy complications. Prenat Diagn. 2007; 247:150-153. |
| McCabe, et al. DNA microextraction from dried blood spots on filter paper blotters: potential applications to newborn screening. Hum Genet.1987;75:213-216. |
| McCarley, et al. Patterning of surface-capture architectures in polymer-based microanalytical devices. In Kuther, et al. Eds. Royal Society of Chemistry Special Publication. 2005;130-132. (Abstract only). |
| Mehrishi , et al. Electrophoresis of cells and the biological relevance of surface charge. Electrophoresis.2002;23:1984-1994. |
| Melville, et al. Direct magnetic separation of red cells from whole blood. Nature. 1975; 255:706. |
| Mohamed, et al. A Micromachined Sparse Cell Isolation Device: Application in Prenatal Diagnostics. Nanotech 2006 vol. 2; 641-644. (Abstract only). |
| Mohamed, et al. Biochip for separating fetal cells from maternal circulation. J Chromatogr A. Aug. 31, 2007;1162(2):187-92. |
| Mohamed, et al. Development of a rare cell fractionation device: application for cancer detection. IEEE Trans Nanobioscience. 2004; 3(4): 251-6. |
| Moore, et al. Lymphocyte fractionation using immunomagnetic colloid and a dipole magnet flow cell sorter. J Biochem Biophys Methods. 1998;37:11-33. |
| Moorhead, et al. Optimal genotype determination in highly multiplexed SNP data. Eur. J. Hum. Genet. 2006;14(2):207-15. (published online Nov. 23, 2005). |
| Mueller , et al. Isolation of fetal trophoblast cells from peripheral blood of pregnant women. The Lancet. 1990;336:197-200. |
| Muller, et al. Moderately repeated DNA sequences specific for the short arm of the human Y chromosome are present in XX makes and reduced in copy number in an XY female. 1986;14:1325-1340. |
| Mullis, et al. Specific Enzymatic Amplification of DNA In Vitro: The Polymerase Chain Reaction. Cold Spring Harbor Symposia on Quantitative Biolgy 1986;51:263-273. |
| Murakami, et al. A novel single cell PCR assay: detection of human T lymphotropic virus type I DNA in lymphocytes of patients with adult T cell leukemia. Leukemia. Oct. 1998;12(10):1645-50. |
| Murthy, et al. Assessment of multiple displacement amplification for polymorphism discovery and haplotype determination at a highly polymorphic locus, MC1R. Hum. Mutat. 2005; 26(2):145-52. |
| Nagrath, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature. 2007; 450: 1235-1241 (with Supplemental pp. 1-10). |
| Nelson, et al. Genotyping Fetal DNA by Non-Invasive Means: Extraction From Maternal Plasma. Vox Sang. 2001;80:112-116. |
| Newcombe, R. G. Two-sided confidence intervals for the single proportion: comparison of seven methods. Statistics in Medicine. 1998; 17:857-872. |
| Oakey et al. Laminar Flow-Based Separations at the Microscale. Biotechnology Progress. 2002; pp. 1439-1442. |
| Office action dated Nov. 3, 2009 for U.S. Appl. No. 11/763,133. |
| Office action dated Dec. 1, 2009 for U.S. Appl. No. 11/763,426. |
| Office action dated Mar. 11, 10 for U.S. Appl. No. 11/763,245. |
| Office action dated Mar. 4, 2009 for U.S. Appl. No. 11/228,454. |
| Office action dated May 4, 2009 for U.S. Appl. No. 11/763,431. |
| Olson, et al. An In Situ Flow Cytometer for the Optical Analysis of Individual Particles in Seawater. Available at http://www.whoi.edu/science/B/Olsonlab/insitu2001.htm. Accessed Apr. 24, 2006. |
| Oosterwijk, et al. Prenatal diagnosis of trisomy 13 on fetal cells obtained from maternal blood after minor enrichment. Prenat Diagn. 1998;18(10):1082-5. |
| Owen, et al. High gradient magnetic separation of erythrocytes. Biophys. J. 1978; 22:171-178. |
| Pallavicini, et al. Analysis of fetal cells sorted from maternal blood using fluorescence in situ hybridization. The American Journal of Human Genetics. Oct. 1992. Supplemental to vol. 51, No. 4: 1031. |
| Parano, et al. Fetal Nucleated red blood cell counts in peripheral blood of mothers bearing Down Syndrome fetus. Neuropediatrics. 2001; 32(3):147-149. (Abstract only). |
| Parano, et al. Noninvasive Prenatal Diagnosis of Chromosomal Aneuploidies by Isolation and Analysis of Fetal Cells from Maternal Blood. Am. J. Med. Genet. 101:262-7 (2001). |
| Paterlini-Brechot, et al. Circulating tumor cells (CTC) detection: Clinical impact and future directions. Cancer Letter. 2007. (In press, 25 pages.) Available at www.sciencedirect.com. |
| Paul, et al. Single-molecule dilution and multiple displacement amplification for molecular haplotyping. Biotechniques. 2005; 38(4):553-4, 556, 558-9. |
| Pawlik, et al. Prodrug Bioactivation and Oncolysis of Diffuse Liver Metastases by a Herpes Simplex Virus 1 Mutant that Expresses the CYP2B1 Transgene. Cancer. 2002;95:1171-81. |
| Peixoto, et al. Quantification of multiple gene expression in individual cells. Genome Res. Oct. 2004;14(10A):1938-47. |
| Peng, et al. Real-time detection of gene expression in cancer cells using molecular beacon imaging: new strategies for cancer research. Cancer Res. 2005; 65(5):1909-17. |
| Petersen, et al. The Promise of Miniaturized Clinical Diagnostic Systems. IVD Technol. 4:43-49 (1998). |
| Pfaffl, et al. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. May 1, 2002;30(9):e36. |
| Pinkel, et al. Cytogenetic Analysis Using Quantitative, High-sensitivity, Fluorescence Hybridization. Genetics. 1986;83:2934-2938. |
| Pinkel, et al. Fluorescence in situ Hybridization with Human Chromosome-specific Libraries: Detection of Trisomy 21 and Translocations of Chromosome 4. Genetics. 1988;85:9138-9142. |
| Pinkel, et al. Detection of structural chromosome abberations in metaphase in metaphase spreads and interphase nuclei by in situ hybridization high complexity probes which stain entire human chromosomes. The American Journal of Human Genetics. Sep. 1988. Supplemental to vol. 43, No. 3: 0471. |
| Pinzani, et al. Isolation by size of epithelial tumor cells in peripheral blood of patients with breast cancer: correlation with real-time reverse transcriptase-polymerase chain reaction results and feasibility of molecular analysis by laser microdissection. Hum Pathol. 2006; 37(6):711-8. |
| Pohl et al. Principle and applications of digital PCR. Expert Rev Mol Diagn. Jan. 2004;4(1):41-7. |
| Potti, et al. Genomic signatures to guide the use of chemotherapeutics. Nat Med. 2006; 12(11):1294-1300. |
| Price, et al. Prenatal Diagnosis with Fetal Cells Isolated from Maternal Blood by Multiparameter Flow Cytometry. Am. J. Obstet. Gynecol. 1991; 165:1731-7. |
| Prieto, et al. Isolation of fetal nucleated red blood cells from maternal blood in normal and aneuploid pregnancies. Clin Chem Lab Med. Jul. 2002;40(7):667-72. |
| Product literature for GEM, a system for blood testing: GEM Premier 3000. Avaiable at http://www.ilus.com/premier_gem3000_iqm.asp. Accessed Apr. 24, 2006. |
| Purwosunu, et al. Clinical potential for noninvasive prenatal diagnosis through detection of fetal cells in maternal blood. Taiwan J Obstet Gynecol. Mar. 2006;45(1):10-20. |
| Raeburn, P. Fetal Cells Isolated In Women's Blood. Associated Press (Jul. 28, 1989) [electronic version]. |
| Rahil, et al. Rapid detection of common autosomal aneuploidies by quantitative fluorescent PCR on uncultured amniocytes, European Journal of Human Genetics, 2002, vol. 10, 462-466. |
| Rickman, et al. Prenatal diagnosis by array-CGH. European Journal of Medical Genetics. 2005; 48:232-240. |
| Rolle, et al. Increase in number of circulating disseminated epithelia cells after surgery for non-small cell lung cancer monitored by MAINTRAC is a predictor for relapse: a preliminary report. World Journal of Surgical Oncology. 2005; 9 pages. |
| Ruan, et al. Identification of clinically significant tumor antigens by selecting phage antibody library on tumor cells in situ using laser capture microdissection. Molecular & Cellular Proteomics. 2006; 5(12): 2364-73. |
| Sakhnini, et al. Magnetic behavior of human erythrocytes at different hemoglobin states. Eur Biophys J. Oct. 2001;30(6):467-70. |
| Samura, et al. Diagnosis of trisomy 21 in fetal nucleated erythrocytes from maternal blood by use of short tandem repeat sequences. Clin. Chem. 2001; 47(9):1622-6. |
| Samura, et al. Female fetal cells in maternal blood: use of DNA polymorphisms to prove origin. Hum. Genet. 2000;107(1):28-32. |
| Sato, et al. Individual and Mass Operation of Biological Cells Using Micromechanical Silicon Devices. Sensors and Actuators. 1990;A21-A23:948-953. |
| Schaefer, et al. The Clinical Relevance of Nucleated Red Blood Cells counts. Sysmex Journal International. 2000; 10(2):59-63. |
| Schröder, et al. Fetal Lymphocytes in the Maternal Blood. The Journal of Hematolog:Blood. 1972;39:153-162. |
| Sethu, et al. Continuous Flow Microfluidic Device for Rapid Erythrocyte Lysis. Anal. Chem. 76:6247-6253 (2004). |
| Shen, et al. High-throughput SNP genotyping on universal bead arrays. Mutat. Res. 2005; 573:70-82. |
| Shendure, et al. Accurate multiplex polony sequencing of an evolved bacterial genome. Science. 2005; 309:1728-32. |
| Sherlock, et al. Assessment of diagnostic quantitative fluorescent multiplex polymerase chain reaction assays performed on single cells. Ann. Hum. Genet. 1998; 62:9-23. |
| Sitar, et al. The Use of Non-Physiological Conditions to Isolate Fetal Cells from Maternal Blood. Exp. Cell. Res. 2005; 302:153-61. |
| Sohda, et al. The Proportion of Fetal Nucleated Red Blood Cells in Maternal Blood: Estimation by FACS Analysis. Prenat. Diagn. 1997; 17:743-52. |
| Stipp, D. IG Labs Licenses New Technology for Fetal Testing. The Wall Street Journal. Aug. 10, 1990:B5. |
| Stoecklein, et al. SCOMP is superior to degenerated oligonucleotide primed-polymerase chain reaction for global amplification of minute amounts of DNA from microdissected archival tissue samples. Am J Pathol. 2002; 161(1):43-51. |
| Stoughton, et al. Data-adaptive algorithms for calling alleles in repeat polymorphisms. Electrophoresis. 1997;18(1):1-5. |
| Sun, et al. Whole-genome amplification: relative efficiencies of the current methods. Leg Med. 2005; 7(5):279-86. |
| Sykes, et al. Quantitation of targets for PCR by use of limiting dilution. Biotechniques. Sep. 1992;13(3):444-9. |
| Tettelin, et al. The nucleotide sequence of Saccharomyces cerevisiae chromosome VII. Nature. May 29, 1997;387(6632 Suppl):81-4. |
| Thomas, et al. Specific Binding and Release of Cells from Beads Using Cleavable Tettrametric Antibody Complexes. Journal of Immunological Methods 1989; 120:221-231. |
| Tibbe, et al. Statistical considerations for enumeration of circulating tumor cells. Cytometry A. Mar. 2007;71(3):154-62. |
| Toner, et al. Blood-on-a-Chip. Annu. Rev. Biomed. Eng. 7:77-103, C1-C3 (2005). |
| Trask, et al. Detection of DNA Sequences and Nuclei in Suspension by In Situ Hybridization and Dual Beam Flow Cytometry. Science.1985;230:1401-1403. |
| Troeger, et al. Approximately half of the erythroblasts in maternal blood are of fetal origin. Mol Hum Reprod. 1999; 5(12):1162-5. |
| Van Raamsdonk, et al. Optimizing the detection of nascent transcripts by RNA fluorescence in situ hybridization. Nucl. Acids. Res. 2001; 29(8):e42. |
| Vogelstein, et al. “Digital PCR.” Proc Natl. Acad Sci. USA, Aug. 1999, vol. 96., 9236-9241. |
| Volkmuth, et al. DNA electrophoresis in microlithographic arrays. Nature. 1992; 358:600-602. |
| Volkmuth, et al. Observation of Electrophoresis of Single DNA Molecules in Nanofabricated Arrays. Presentation at joint annual meeting of Biophysical Society and the American Society for Biochemistry and Molecular Biology. Feb. 9-13, 1992. |
| Von Eggeling, et al. Determination of the origin of single nucleated cells in maternal circulation by means of random PCR and a set of length polymorphisms. Hum Genet. Feb. 1997;99(2):266-70. |
| Vona, et al. Enrichment, immunomorphological, and genetic characterization of fetal cells circulating in maternal blood. Am J Pathol. Jan. 2002;160(1):51-8. |
| Voullaire, et al. Detection of aneuploidy in single cells using comparative genomic hybridization. Prenat Diagn. 1999; 19(9):846-51. |
| Vrettou, et al. Real-time PCR for single-cell genotyping in sickle cell and thalassemia syndromes as a rapid, accurate, reliable, and widely applicable protocol for preimplantation genetic diagnosis. Human Mutation. 2004; 23(5):513-21. |
| Wachtel, et al. Fetal Cells in the Maternal Circulation: Isolation by Multiparameter Flow Cytometry and Confirmation by Polymerase Chain Reaction. Human Reproduction. 1991;6:1466-1469. |
| Wang, et al. Allele quantification using molecular inversion probes (MIP). Nucleic Acids Research. 2005; 33(21); e183 (14 pages). |
| Wapner, et al. First-trimester screening for trisomies 21 and 18. N. Engl. J. Med. 2003; 349:1405-1413. |
| Warren, et al. Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR. PNAS. Nov. 21, 2006; 103(47):17807-17812. |
| Washizu, et al. Handling Biological Cells Utilizing a Fluid Integrated Circuit. IEEE Industry Applications Society Annual Meeting Presentations. Oct. 2-7, 1988;: 1735-40. |
| Washizu, et al. Handling Biological Cells Utilizing a Fluid Integrated Circuit. IEEE Transactions of Industry Applications. 1990; 26: 352-8. |
| Weigl, et al. Microfluidic Diffusion-Based Separation and Detection. Science. 1999; pp. 346-347. |
| Williams, et al. Comparison of cell separation methods to entrich the proportion of fetal cells in material blood samples. The American Journal of Human Genetics. Oct. 1992. Supplemental to vol. 51, No. 4: 1049. |
| Xiong, et al. “A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences,” Nucleic Acids Research, Apr. 19, 2004, vol. 32, No. 12, e98. |
| Yang, et al. Prenatal diagnosis of trisomy 21 with fetal cells i maternal blood using comparative genomic hybridization. Fetal Diagn Ther. 2006; 21:125-133. |
| Yang, et al. Rapid Prenatal Diagnosis of Trisomy 21 by Real-time Quantitative Polymerase Chain Reaction with Amplification of Small Tandem Repeats and S100B in Chromosome 21. Yonsei Medical Journal, 2005, vol. 46, No. 2, 193-197. |
| Yu, et al. Objective Aneuploidy Detection for Fetal and Neonatal Screening Using Comparative Genomic Hybridization (CGH). Cytometry. 1997; 28(3): 191-197. (Absbract). |
| Zavala, et al. Genomic GC content prediction in prokaryotes from a sample of genes. Gene. Sep. 12, 2005;357(2):137-43. |
| Zhen, et al. Poly-FISH: a technique of repeated hybridizations that improves cytogenetic analysis of fetal cells in maternal blood. Prenat Diagn. 1998; 18(11):1181-5. |
| Zheng, et al. Fetal cell identifiers: results of microscope slide-based immunocytochemical studies as a function of gestational age and abnormality. Am J Obstet Gynecol. May 1999;180(5):1234-9. |
| Zimmerman, et al. Novel real-time quantitative PCR test for trisomy 21. Jan. 1, 2002. Clinical Chemistry, American Association for Clinical Chemistry. 48:(2) 362-363. |
| Zuska, P. Microtechnology Opens Doors to the Universe of Small Space, MD&DI Jan. 1997, p. 131. |
| Solexa, Biotechniques, 2007, Protocol Guide, 1 page. |
| Huse et al., Genome Biology, 2007, vol. 8, article 143, pp. 1-9. |
| Invitation to Respond to Second Written Opinion issued in SG 201102011-2 on Sep. 25, 2013, 14 pages. |
| Office Action issued in U.S. Appl. No. 12/560,708 on May 10, 2012, 72 pages. |
| Office Action issued in U.S. Appl. No. 12/560,708 on Jan. 10, 2013, 20 pages. |
| Calin, G.A et al., “MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias”, Proc. Natl. Acad. Sci. USA, vol. 101, No. 32, pp. 11755-11760 (Aug. 10, 2004). |
| Instrumentation Laboratory, “Critical Care Diagnostics: IL's GEM® PremierTM 3000 With iQM™,” Product Sheet, Apr. 24, 2006, 3 pages. |
| Mullis, K.B et al., “Specific Synthesis of DNA in Vitro via a Polymerase-Catalyzed Chain Reaction”, Methods in Enzymology, vol. 155, Recombinant DNA, part F, 1987, 19 page. |
| Sun, F. et al., “Whole genome amplification of single cells: mathematical analysis of PEP and tagged PCR”, Nucleic Acids Research, vol. 23, No. 15, pp. 3034-3040, (1995). |
| Number | Date | Country | |
|---|---|---|---|
| 20200270700 A1 | Aug 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61098758 | Sep 2008 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15195135 | Jun 2016 | US |
| Child | 16852663 | US | |
| Parent | 13921881 | Jun 2013 | US |
| Child | 15195135 | US | |
| Parent | 13218317 | Aug 2011 | US |
| Child | 13921881 | US | |
| Parent | 12560708 | Sep 2009 | US |
| Child | 13218317 | US |
