HK1219511B - Characterization of molecules in nanofluidics - Google Patents

Description

Characterization of molecules in nanofluids

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61767,219, filed February 20, 2013, which is incorporated herein by reference in its entirety.

SUMMARY OF THE INVENTION

According to some embodiments herein, a method for characterizing a sample is provided. The method may include labeling a plurality of sample molecules with at least a first marker, wherein the sample molecule comprises a polynucleotide sequence of a first genomic fragment of interest or a plurality of first genomic fragments of interest, and wherein a first genomic fragment of interest or a plurality of first genomic fragments of interest corresponds to a possible abnormal genomic region of the sample. The method may include providing a plurality of labeled reference molecules, wherein the reference molecule comprises a polynucleotide sequence of a reference genomic fragment or a plurality of reference genomic fragments, and wherein it is known that a reference genomic fragment or a plurality of reference genomic fragments do not correspond to a possible abnormal genomic region. As used herein, "corresponding to a possible abnormal genomic region" and variations of the root term include genomic fragments that overlap or are included in an abnormal chromosome region, including but not limited to duplication, deletion, inversion, translocation and/or aneuploid chromosomes or fragments thereof. In this way, a genomic fragment or fragment may correspond to an abnormal genomic region that exists (e.g., duplicated) or does not exist (e.g., deleted, for example, if the genomic fragment is included in a deleted region or overlaps with it). The method may include translocating a plurality of labeled sample molecules and a plurality of labeled reference molecules through a fluid channel. The method can include detecting a signal from a labeled sample molecule to determine at least one first pattern or patterns specific to a first genomic segment of interest or a plurality of first genomic segments of interest; and a second pattern or patterns specific to a reference genomic segment or a plurality of reference genomic segments. The method can include correlating the signal determining the first pattern or patterns with the signal determining the second pattern or patterns. In some embodiments, labeling comprises labeling the sample molecule with a first label, and wherein the reference molecule comprises a second label, wherein the first label is configured to produce a first pattern or patterns, and wherein the second label is configured to produce a second pattern or patterns, and wherein the first label and the second label are different from each other. In some embodiments, labeling comprises labeling with the first label, wherein the first pattern or patterns and the second pattern or patterns each comprise the first label, and wherein the first pattern or patterns and the second pattern or patterns are different from each other. In some embodiments, the method further comprises labeling the reference molecule to produce a labeled reference molecule, wherein the labeled reference molecule comprises the second pattern or patterns. In some embodiments, the labeled reference molecule and the sample molecule are from a sample. In some embodiments, the labeled reference molecule and the sample molecule are from different tissues of the same organism. In some embodiments, the labeled reference molecule and the sample molecule are from different organisms. In some embodiments, the signal from the labeled reference molecule comprises an electronically or optically stored value or set of values. In some embodiments, the method further comprises labeling a plurality of second sample molecules from a second sample with at least a first label, wherein the plurality of second sample molecules comprise a polynucleotide sequence of a first genomic fragment of interest or a plurality of first genomic fragments of interest, wherein the plurality of second sample molecules are known not to correspond to a chromosomal abnormality; translocating the plurality of second sample molecules through a fluid channel, and detecting the signal from the labeled sample molecule, thereby determining at least one first pattern or multiple first patterns unique to a first genomic fragment of interest or a plurality of first genomic fragments of interest, and a second pattern or multiple second patterns unique to a reference genomic fragment or a plurality of reference genomic fragments. In some embodiments, the method further comprises aligning the position of the pattern with the position of the pattern in the reference genome. In some embodiments, the sample molecule is from a sample containing a possible genomic abnormality, and a reference genomic fragment or a plurality of reference genomic fragments comprise a first chromosome or a fragment thereof, wherein the reference genomic fragment is from a second sample known not to contain a genomic abnormality. In some embodiments, a first genomic segment of interest or a plurality of first genomic segments of interest comprises a sex chromosome or at least one fragment thereof, and a reference genomic segment or a plurality of reference genomic segments comprises an autosome or at least one fragment thereof. In some embodiments, a first genomic segment of interest or a plurality of first genomic segments of interest comprises a first autosome or at least one fragment thereof selected from the group consisting of human chromosome 21, human chromosome 13, human chromosome 14, human chromosome 15, human chromosome 16, human chromosome 18, and human chromosome 22, and fragments thereof, and a reference genomic segment or a plurality of reference genomic segments comprises a second autosome or at least one fragment thereof, wherein the second autosome or fragment thereof is different from the first autosome or fragment thereof. In some embodiments, correlating the signals comprises using a ratio (K) between signals (S1, S2…Sn) generated by a plurality of labeled sample molecules or portions thereof and a signal (C) generated by a reference: K1=S1/C, K2=S2/C…Kn=Sn/C. In some embodiments, the first label comprises at least one of a fluorescent label, a radioactive label, a magnetic label, or a non-optical label. In some embodiments, the second label comprises at least one of a fluorescent label, a radioactive label, a magnetic label, or a non-optical label. In some embodiments, labeling comprises nicking one strand of the double-stranded DNA at a first sequence motif with a nicking endonuclease; and labeling the DNA. In some embodiments, the method further comprises repairing at least some of the nicks in the DNA. In some embodiments, the nicks are not repaired. In some embodiments, the label comprises a transcription terminator. In some embodiments, labeling with the first label comprises tagging at least one sequence motif of the sample molecule with a DNA binding entity and a methyltransferase, the DNA binding entity being selected from the group consisting of a non-cutting restriction enzyme, a zinc finger protein, an antibody, a transcription factor, a transcription activator-like domain, a DNA binding protein, a polyamide, a triple helix-forming oligonucleotide, and a peptide nucleic acid. In some embodiments, labeling with the first label comprises tagging at least one sequence motif of the sample molecule with a methyltransferase. In some embodiments, the method further comprises labeling the sample molecule with a non-sequence-specific label. The non-sequence-specific label may be different from the first label and the second label. In some embodiments, the potentially abnormal genomic region comprises at least one of a translocation, addition, amplification, transversion, inversion, aneuploidy, polyploidy, monosomy, trisomy, trisomy 21, trisomy 13, trisomy 14, trisomy 15, trisomy 16, trisomy 18, trisomy 22, triploidy, tetraploidy, or sex chromosome aneuploidy. In some embodiments, the genetic abnormality comprises at least one of trisomy or monosomy.

According to some embodiments herein, a method for characterizing a sample is provided. The method may include marking multiple sequence-specific positions on the polynucleotide sequence of the sample molecule. The method may include linearizing at least a portion of the sample molecule in a fluid channel. The method may include quantifying the signal from the mark on the sample molecule. The method may include comparing the number of signals from the sample molecule with the number of signals from a reference molecule. The method may include determining whether a genetic abnormality exists in the sample DNA when the number of signals from the sample molecule is different from the number of signals generated by the reference molecule. In some embodiments, the sample molecule and the reference molecule are from the same organism. In some embodiments, the sample molecule and the reference molecule are from different tissues of the same organism. In some embodiments, the sample molecule and the reference molecule are from different organisms. In some embodiments, the signal from the number of reference molecule signals includes an electronically or optically stored value or set of values. In some embodiments, the sample molecule comprises DNA. In some embodiments, the genetic abnormality comprises at least one of a translocation, an addition, an amplification, a transversion, an inversion, an aneuploidy, a polyploidy, a monosomy, a trisomy, trisomy 21, trisomy 13, trisomy 14, trisomy 15, trisomy 16, trisomy 18, trisomy 22, a triploidy, a tetraploidy, or a sex chromosome aneuploidy. In some embodiments, the genetic abnormality comprises at least one of a trisomy or a monosomy. In some embodiments, labeling comprises labeling the polynucleotide with at least one of a fluorescent label, a radioactive label, a magnetic label, or a non-optical label. In some embodiments, labeling comprises: nicking one strand of the double-stranded DNA at a first sequence motif with a nicking endonuclease; and labeling the DNA. In some embodiments, labeling further comprises repairing at least some of the nicks in the first DNA. In some embodiments, the nicks are not repaired. In some embodiments, the label comprises a transcription terminator. In some embodiments, labeling comprises tagging at least one sequence motif of the sample molecule with a DNA binding entity and a methyltransferase, the DNA binding entity being selected from the group consisting of a non-cutting restriction enzyme, a zinc finger protein, an antibody, a transcription factor, a transcription activator-like domain, a DNA binding protein, a polyamide, a triple helix-forming oligonucleotide, and a peptide nucleic acid. In some embodiments, labeling with a first label comprises tagging at least one sequence motif of the sample molecule with a methyltransferase.

According to some embodiments herein, a method for characterizing a sample is provided. The method may include labeling a sample nucleic acid molecule. The method may include translocating the labeled sample nucleic acid molecule through a fluid nanochannel, wherein the fluid nanochannel is configured to extend at least a portion of the sample nucleic acid molecule, and wherein the length of the fluid nanochannel is at least 10 nm and the cross-sectional diameter is less than 1000 nm. The method may include detecting a signal generated by the sample nucleic acid molecule in the fluid channel. The method may include determining the position of the label on the sample nucleic acid molecule. The method may include aligning the position of the label on the sample nucleic acid molecule with the position of the label in a reference genome, wherein the reference genome is obtained by a second sample from the same organism as the sample molecule.

In some embodiments, the fluid nanochannel of any of the methods herein includes a channel having a length of at least 10 nm and a cross-sectional diameter of less than 5000 nm. In some embodiments, the fluid channel includes a nanochannel. In some embodiments, the fluid channel is arranged parallel to the substrate surface. In some embodiments. In some embodiments, the translocation includes applying a driving force to the labeled sample, the driving force being selected from the group consisting of fluid flow, radiation field, electroosmotic force, electrophoretic force, electromotive force, temperature gradient, surface property gradient, capillary flow, pressure gradient, magnetic field, electric field, receding meniscus, surface tension, thermal gradient, pulling force, thrust, and combinations thereof.

In some embodiments, the sample of any method herein is selected from bacteria, virions, DNA molecules, RNA molecules, nucleic acid polymers, proteins, peptides and polysaccharides. In some embodiments, the sample of any method herein is derived from maternal blood, and wherein the reference molecule is derived from a maternal sample other than blood. In some embodiments, the sample of any method herein comprises nucleotides, and wherein at least two labels are located at either end of the region of interest in the nucleotides. In some embodiments, the sample of any method herein comprises circulating fetal cells, circulating tumor cells or body fluids or tissues.

In some embodiments, any of the methods herein include optical detection, which includes measuring physical count, intensity, wavelength, or the size of the label. In some embodiments, any of the methods herein include optical detection, which includes measuring the length of at least one labeled region in the sample. In some embodiments, any of the methods herein also includes measuring the signal generated by a pool comprising a sample or sample portion.

In some embodiments, any of the methods herein includes using a ratio (K) between a signal (S1, S2 ... Sn) generated by a plurality of samples or sample portions and a signal (C) generated by a reference: K1 = S1 / C, K2 = S2 / C ... Kn = Sn / C. In some embodiments, the difference between K1 and Kn is used to identify the presence of a fetal sample. In some embodiments, the difference between K1 and Kn is used to identify the presence of DNA from a tumor or other cancerous source. In some embodiments, The difference between K1 and Kn is used to determine the presence of a genetic abnormality in a sample. In some embodiments, the genetic abnormality is aneuploidy. In some embodiments, the genetic abnormality is a translocation, addition, amplification, inversion, or inversion.

In some embodiments, any of the methods herein include references derived from known diploid or haploid chromosomes. In some embodiments, any of the methods herein include associating the signal from the sample with a population distribution from a metagenomic or microbiome study. In some embodiments, any of the methods herein include generating a histogram distribution to reflect the depth of coverage of the sample.

In some embodiments, a system for characterizing a sample is provided. The system can include labeling one or more regions of sample molecules with at least two labels. The system can include a fluid channel for translocating the labeled sample molecules, wherein the fluid channel is configured to extend at least a portion of the sample molecules, and wherein the fluid channel has a length of at least 10 nm and a cross-sectional diameter of less than 5000 nm. The system can include a device for detecting a signal generated by the labeled sample in the fluid channel.

In some embodiments, a system for characterizing a sample is provided. The system may include one or more regions for labeling a sample nucleic acid molecule. The system may include a fluid nanochannel for translocating the labeled sample nucleic acid molecule, wherein the fluid nanochannel is configured to extend at least a portion of the sample nucleic acid molecule, and wherein the length of the fluid nanochannel is at least 10 nm and the cross-sectional diameter is less than 1000 nm. The system may include a device for detecting a signal generated by the sample nucleic acid molecule in the fluid channel.

In some embodiments, a system for characterizing a sample is provided. The system can include a region for labeling a plurality of sequence-specific positions on the sample DNA. The system can include a region for linearizing at least a portion of the sample DNA. The system can include a device for quantifying the signal generated by the labeling on the sample DNA.

In some embodiments, a system for characterizing a sample is provided. The system may include means for labeling sample molecules with at least two labels. The system may include means for linearizing the labeled sample molecules. The system may include means for detecting a signal generated by the labeled sample in a fluid channel.

In some embodiments, a system for characterizing a sample is provided. The system can include components for labeling sample nucleic acid molecules. The system can include components for linearizing the labeled sample nucleic acid molecules. The system can include components for detecting signals generated by the sample nucleic acid molecules in a fluid channel.

In some embodiments, a system for characterizing a sample is provided. The system can include components for labeling multiple sequence-specific positions on the sample DNA. The system can include components for linearizing at least a portion of the sample DNA. The system can include components for quantifying the signal generated by the labeling on the sample DNA.

In some embodiments, any system described herein can characterize a sample selected from bacteria, virions, DNA molecules, RNA molecules, nucleic acid polymers, proteins, peptides, and polysaccharides. In some embodiments, any system described herein can characterize a sample derived from maternal blood, and wherein the reference molecule is derived from a maternal sample other than blood. In some embodiments, any system described herein can characterize a sample comprising nucleotides, and wherein at least two labels are located at either end of the region of interest in the nucleotides. In some embodiments, any system described herein can characterize a sample comprising circulating fetal cells, circulating tumor cells, or body fluids or tissues.

In some embodiments, any system described herein may include a label selected from a fluorescent label, a radioactive label, a magnetic label, or a combination thereof. In some embodiments, any system described herein may be configured for optical detection, wherein optical detection includes measuring the size of the physical count, intensity, wavelength, or label. In some embodiments, optical detection includes measuring the length of the area of at least one label in the sample. In some embodiments, any system described herein may be configured to associate signals, wherein associating the signals includes measuring the signals generated by the collection of sample pools or sample portions. In some embodiments, any system described herein may be configured to associate signals, wherein associating the signals includes using the ratio (K) between the signals (S1, S2 ... Sn) generated by a variety of samples or sample portions and the signals (C) generated by the reference: K1 = S1 / C, K2 = S2 / C ... Kn = Sn / C. In some embodiments, the difference between K1 and Kn is used to identify the presence of fetal samples. In some embodiments, the difference between K1 and Kn is used to identify the presence of DNA from tumors or other cancerous sources. In some embodiments, the difference between K1 and Kn is used to determine the presence of a genetic abnormality in a sample. In some embodiments, the genetic abnormality is aneuploidy. In some embodiments, the genetic abnormality is a translocation, addition, amplification, transversion, or inversion.

In some embodiments, any of the systems described herein can include a reference derived from a known diploid or haploid chromosome.

In some embodiments, any of the systems described herein can correlate signals from a sample with population distributions from metagenomic or microbiome studies.

In some embodiments, the fluid channel of any of the systems described herein comprises a nanochannel. In some embodiments, the fluid channel of any of the systems described herein is arranged parallel to the substrate surface. In some embodiments, translocation comprises applying a driving force to the labeled sample, the driving force selected from the group consisting of fluid flow, radiation field, electroosmotic force, electrophoretic force, electromotive force, temperature gradient, surface property gradient, capillary flow, pressure gradient, magnetic field, electric field, receding meniscus, surface tension, thermal gradient, pulling force, pushing force, and combinations thereof.

In some embodiments, any of the systems described herein is configured to generate a histogram distribution to reflect the coverage depth of a sample.

In some embodiments, kits for performing any of the methods described herein are provided.

In some embodiments, kits for using any of the systems described herein are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

1 is a schematic diagram illustrating sample molecules or particles (ovoids) and reference or comparison molecules or particles (spheres) flowing through a nanofluidic channel, according to some embodiments herein.

2 is a schematic diagram illustrating an embodiment of an imaging setup for detecting signals emitted by labeled molecules or particles to chart the amount, intensity, and configuration of sample and reference molecules or particles.

Figure 3a illustrates a series of images of small double-stranded DNA fragments of known sizes (233 bp, 498 bp, and 834 bp) generated by PCR, fluorescently colored, flowed, and imaged in a single nanofluidic channel. Figure 3b shows the same double-stranded DNA fragments mixed together, flowed, and imaged in the same nanofluidic channel. The fluorescence signals are plotted in a histogram.

FIG4 is a series of diagrams illustrating Gaussian curves of photons emitted by individually labeled DNA molecules of known sizes (233 bp, 498 bp, and 834 bp). Total counts and intensities are linearly proportional to mass and/or molecular size. Within the linear dynamic range, unknown molecular sizes and quantities can be inferred by this method.

FIG5 is a series of scatter plots illustrating the estimation of the size and number of unknown molecules within the linear dynamic range using the information from FIG4.

Figure 6 is a histogram illustrating genomic DNA fragments plotted against a reference genome (Human Genome Version 19). The y-axis shows the depth of coverage of specific chromosomal regions. A uniform distribution across the genome was observed, except for regions lacking sequence signals (e.g., centromeres and telomeres).

FIG7 a is a diagram illustrating a diploid genomic fragment from a human male sample aligned with chromosome 1. The y-axis provides coverage number. The x-axis provides nucleotide position. The average depth of coverage is 5X. FIG7 b is a diagram showing a haploid sex chromosome X from the same male sample with an average depth of coverage of 2X-2.5X (roughly half the depth of a diploid autosome), showing quantitative measurements using the methods and platforms of some embodiments herein.

Detailed Description of the Invention

Small DNA fragments are shed from the fetus into the maternal bloodstream. Tumors have also been found to release DNA into the blood. The methods of some embodiments herein are for analyzing polynucleotide fragments such as DNA fragments in the blood to detect the presence of polynucleotides or cells from the fetus or tumor. The methods of some embodiments herein are also used to analyze fetal DNA in maternal blood to detect genetic abnormalities. In some preferred embodiments, the methods described herein require the use of a nanofluidic-based single molecule detection platform to identify genetic abnormalities. The methods and devices of some embodiments herein have the advantage of analyzing small or large molecules such as small or large DNA molecules. In some embodiments, a molecule or region of interest is marked with at least one pattern, and a reference molecule or region of interest is marked with at least one pattern. The molecule can be linearized in a microfluidic channel, and the depth of coverage of the molecule or region of interest can be compared with the depth of coverage of a reference molecule to determine the copy number of the molecule of interest.

It is estimated that about 3-15% of the short DNA in maternal blood is of fetal origin. This paper describes a method for easily detecting and quantifying small molecules including short DNA fragments using a method that incorporates fluidics. In some preferred embodiments, the method includes quantifying short DNA fragments without sequencing or assembly.

Existing prenatal testing involves needle puncture to draw amniotic fluid, which can lead to miscarriage and other complications. In addition, many current cancer detection methods also involve invasive procedures such as biopsies. According to some embodiments herein, non-invasive prenatal testing methods are provided. In some embodiments, the methods are used to test blood. In some embodiments, the methods only test blood samples and do not test samples from other tissues.

Also described herein are methods for detecting larger molecules comprising longer DNA fragments and tracing their origins using methods incorporating fluidics. For example, in some embodiments, DNA fragments can be traced back to other sources of tumors or cancers. In some preferred embodiments, the methods are used to trace the origins of DNA fragments to identify or characterize genetic abnormalities.

In a preferred embodiment, circulating DNA from a maternal blood sample is analyzed to identify or quantify fetal DNA relative to the maternal genome. In some embodiments, this information is used to determine fetal genomic health (such as trisomy 21) without invasive testing. Examples of suitable oligonucleotides for use in assays for detecting aneuploidy are provided in the HSA21 oligonucleotide assay described by Yahya-Graison et al. (Yahya-Graison et al., Classification of Human Chromosome 21 Gene-Expression Variations in Down Syndrome: Impact on Disease Phenotypes, Am J Hum Genet 2007, 81(3):475-491), which is incorporated herein by reference in its entirety.

In some embodiments, the sample of interest is compared with a reference sample. In some embodiments, the sample of interest is derived from a maternal blood sample. In some embodiments, the reference sample is a maternal sample from a source other than blood. In some embodiments, the maternal reference sample comprises polynucleotides such as DNA isolated from diploid tissues other than blood. In some embodiments, the maternal reference sample comprises an oral sample, a saliva sample, a urine sample, a sputum sample, or a tear sample. For example, in some embodiments, compared with a maternal oral sample, trisomy 21 in a maternal blood sample is detected.

In some embodiments, prior to performing the methods described herein, the sample of interest is enriched for fetal nucleic acid. For example, in some embodiments, fetal cells are enriched using a fetal cell-specific marker that can be pulled down by an antibody. In some embodiments, the sample of interest is size-fractionated. However, any enrichment method known to those skilled in the art can be used.

In some embodiments, the sample of interest is derived from a tumor cell or a suspected tumor cell or a tissue (e.g., blood) in fluid communication with a tumor cell. In some embodiments, the reference sample is a sample from a healthy cell. In some embodiments, the reference sample is from a healthy cell of the same organism as the tumor cell or suspected tumor cell. In some embodiments, the reference sample is selected from a tissue that hardly contains or is unlikely to contain a tumor cell or a nucleic acid from a tumor cell.

Those skilled in the art will recognize that the sample of interest may include nucleic acids from a variety of sources. In some embodiments, the sample of interest comprises bacteria or virions derived from environmental samples, animal or plant tissues, blood, or other body fluids. In some embodiments, DNA fragments are used to detect chromosomal abnormalities or cancer genomes.

As will be appreciated by those skilled in the art, the methods described herein can be used to prepare and analyze DNA from circulating fetal or tumor cells.For example, in some embodiments, cells are lysed to release the DNA of interest prior to analysis.

In some embodiments, the entire genome is tested or analyzed. In some embodiments, only a portion of the genome is tested or analyzed. In some embodiments, all chromosomes are tested or analyzed. In some embodiments, only a portion of chromosomes are tested or analyzed. In some embodiments, the entire gene is analyzed. In some embodiments, only a portion of the gene is tested or analyzed.

The signals described herein can include any suitable signal, including an optical signal, a fluorescent signal, a non-optical signal, a radiation signal, an electrical signal, a magnetic signal, a chemical signal, or any combination thereof. In some embodiments, the signal is generated by an electron spin resonance molecule, a fluorescent molecule, a chemiluminescent molecule, a radioisotope, an enzyme substrate, a biotin molecule, an avidin molecule, a charge transfer molecule, a semiconductor nanocrystal, a semiconductor nanoparticle, a colloidal gold nanocrystal, a ligand, a microbead, a magnetic bead, a paramagnetic particle, a quantum dot, a chromogenic substrate, an affinity molecule, a protein, a peptide, a nucleic acid, a carbohydrate, an antigen, a nanowire, a hapten, an antibody, an antibody fragment, a lipid, or a combination thereof.

In some embodiments, the signal is generated by inducing fluorescence, chemiluminescence, phosphorescence, bioluminescence, or any combination thereof using one or more excitation sources. Suitable excitation sources include lasers, visible light sources, infrared light sources, ultraviolet light sources, or any combination thereof.

In some embodiments, the detection of nucleotides or related signals (e.g., fluorophores) is quantitative. In some embodiments, the length of the nucleotides is quantified. In some embodiments, the molecular size is quantified. In some embodiments, the intensity of the signal is related to the length of the molecule. For example, as shown in Figure 3a, longer DNA molecules can produce stronger signals than short DNA molecules. In some embodiments, the intensity of the signal is related to the amount of DNA in the sample or fluid channel.

In some embodiments, samples are analyzed for copy number variation, e.g., as described in U.S. Patent Publication No. 20130034546, which is incorporated herein by reference in its entirety.

Specific molecules such as the quantity of DNA fragments derived from different chromosomes can be quantitatively measured in the methods provided herein. In some embodiments, it has been observed that the amount of genomic DNA derived from diploid autosomes is twice that derived from diploid sex chromosomes. In some embodiments, the quantity of this fragment reflects the copy number of the source chromosome. In some embodiments, two or three color markers are used.

In some embodiments, fragments of chromosome origin are detected and aneuploidy is identified using relative ratios. In some embodiments, the copy number of the nucleotide is calculated using the ratios K1=S1/C and K2=S2/C, where K1 is the signal ratio of the first sample to the control sample, and K2 is the signal ratio of the second sample to the control sample. It is expected that the copy number from the reference sample is an integer, and the difference between K1 and K2 can show an abnormality in a sample of interest. In some embodiments, the abnormality is detected by comparing the ratio of a specific sample with the average ratio from multiple samples. The method also anticipates that the reference genomic sequence includes different parts of each genome with a known total length, wherein the sequence of interest includes different parts of each normal gene with a known length, and wherein the significant difference between K1 and K2 indicates a genetic abnormality in the genome. In some embodiments, the nucleotide of interest may be associated with a trisomic linked chromosome, wherein the reference genomic sequence is from a chromosome other than the trisomic linked chromosome, and wherein a K1/K2 ratio of approximately 2:3 or 3:2 indicates a trisomic genotype. In some embodiments, the nucleotide sequence of interest comprises a deletion of a portion of the genome. In some embodiments, the nucleotide sequence of interest comprises a repetitive sequence. Thus, according to some embodiments herein, the copy number of a repetitive sequence can be determined. In some embodiments, the first sample comprises maternal blood (which, without being limited by any one theory, may include fetal nucleic acid), and the second sample comprises maternal tissue other than blood (preferably a tissue that contains little or no fetal nucleic acid).

In some embodiments, digital counting is carried out and is detected. In some embodiments, digital counting is carried out and is detected to particle (such as pearl), bacterium or virion particle. As those skilled in the art will appreciate, method described herein can be applied to various targets that can be uniquely labeled. In some embodiments, digital karyotyping is carried out. For example, in some embodiments, digital karyotyping is carried out to the chromosome with interested potential aneuploidy. Method described herein can be used for detecting any interested chromosomal variation, including translocation, addition, amplification, transversion, inversion, aneuploidy, polyploidy, monosomy, trisomy, 21 trisomy, 13 trisomy, 14 trisomy, 15 trisomy, 16 trisomy, 18 trisomy, 22 trisomy, triploid, tetraploid or sex chromosome abnormality, including but not limited to XO, XXY, XYY and XXX.

In some embodiments, methods are provided herein that are sensitive enough to detect "short" fragments that are about tens to hundreds of nucleotides in length. In some embodiments, the sample molecules described herein comprise "short" fragments of polynucleotides. For example, in some embodiments, the polynucleotide fragments are about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length. In some embodiments, the polynucleotide fragment is about 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 nucleotides in length. In some embodiments, the length of the molecule of interest is less than about 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 nucleotides. In some embodiments, the fragment is double-stranded. In some embodiments, the fragment comprises DNA. In some embodiments, the fragment comprises DNA hybridized to RNA. In some embodiments, the sensitivity is about as high as detecting a single fluorophore associated with the target fragment.

In some embodiments, the nucleotides of interest are fragments of at least about 500 nucleotides in length, e.g., about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 nucleotides, including ranges between any two of the recited values, such as about 500 to about 2000, about 500 to about 1500, about 500 to about 1000, about 500 to about 900, about 500 to about 700, about 700 to about 2000, about 700 to about 1500, about 700 to about 1000, about 700 to about 900, about 1000 to about 2000, about 1000 to about 1500, or about 1500 to about 2000 nucleotides in length.

Molecules suitable for use in the methods and systems described herein include polymers, double-stranded DNA, single-stranded DNA, RNA, DNA-RNA hybrids, polypeptides, biomolecules, proteins, etc. Suitable polymers include homopolymers, copolymers, block copolymers, random copolymers, branched copolymers, dendrimers, or any combination thereof.

In some embodiments, the methods described herein are sufficiently sensitive to detect fetal molecules that make up less than about 0.25%, 0.5%, 0.75%, 1%, 1.25%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, or 25% of the total number of molecules in a maternal blood sample.

In some embodiments, the label is directed to a sequence motif or a chemical moiety. Labeling can be performed using any technology known to those skilled in the art, including chemical or biochemical conjugation. In some embodiments, the label described herein is combined with a unique sequence motif. In some embodiments, the label described herein is combined with a chemical moiety. In some embodiments, the chemical moiety is related to a specific chromosome.

In some embodiments herein, each label is independently selected from a fluorophore, a quantum dot, a dendrimer, a nanowire, a bead, a hapten, a streptavidin, avidin, a neutravidin, biotin, and a reactive group. In some embodiments herein, the first and second labels are independently selected from a fluorophore or a quantum dot. In some embodiments herein, at least one of the labels comprises a non-optical label. In some embodiments herein, labeling is performed with a polymerase. In some embodiments herein, labeling is performed with a polymerase in the presence of dNTPs comprising the label. In some embodiments herein, the polymerase has a 5' to 3' exonuclease activity. In some embodiments herein, the polymerase leaves the flank region, and wherein the flank region is removed to restore the ligatable nick before repairing with a ligase. In some embodiments herein, the 5' to 3' exonuclease activity of the polymerase is used to remove the flank region under conditions where at least one nucleotide is present at a limiting concentration. In some embodiments herein, the 5' to 3' exonuclease activity of the polymerase is used to remove the flank region under conditions where at least one nucleotide is omitted from the reaction. In some embodiments herein, the flanking regions are removed with flank endonucleases. In some embodiments herein, labeling is performed with a polymerase under the conditions in which at least one dNTP exists. In some embodiments herein, at least one dNTP is a single type of dNTP. In some embodiments herein, the method described herein also includes regulating the activity of the polymerase during labeling by regulating temperature, dNTP concentration, cofactor concentration, buffer concentration, or any combination thereof. In some embodiments herein, making the first motif or the second motif produce a nick includes producing a nick with Nt.BspQI. In some embodiments herein, in addition to sequence-specific labels or multiple labels described herein, non-sequence-specific labels such as polynucleotide backbone labels have also been used.

In some embodiments, at least one label described herein includes a non-optical label. Various non-optical labels can be used in combination with the embodiments of this invention. In some embodiments, the non-optical label comprises an electronic label. Exemplary electronic labels include but are not limited to molecules with a strong charge, for example, ions such as metal ions, charged amino acid side chains or other cations or anions. When the label is placed in a detector, the electronic label can be detected, for example, by conductivity (or resistivity). In some embodiments, the nanochannel comprises an electrode, which is configured to determine whether the electronic label exists by measuring the conductivity or resistivity of the material provided in the channel. In some embodiments, the non-optical label comprises a metal, a metal oxide (such as a metal oxide) or a silicon oxide portion. In some embodiments, the non-optical label comprises a portion (such as a nanoparticle) containing a metal, a metal oxide or other oxide. The presence of a specific metal or oxide portion can be detected, for example, by nuclear magnetic resonance. In some embodiments, the label is configured to release a portion, such as a proton or anion, according to specific conditions (such as pH changes), and detect whether the released portion exists or does not exist.

In some embodiments, two or more marks are different from each other.For example, the first marker can be used to mark the first motif to produce the first unique pattern, and the second motif different from the first motif can be used to mark the second motif different from the first motif, to produce the second unique pattern. In some embodiments, two or more marks are identical. For example, the first marker can be used to mark the first motif, and the second motif different from the first motif can also be used to mark the same marker to produce a unique pattern. In some embodiments, a plurality of probes corresponding to the first chromosome interested or region are marked with the first marker, and a plurality of second probes corresponding to the chromosome interested or region (for example, with reference to chromosome or region) are marked with the second marker different from the first marker. Like this, the sample molecule comprising the mark from the first chromosome interested or the sequence in the region can be distinguished from the sample molecule comprising the sequence from the second chromosome interested or the region based on whether using the first marker or the second marker sample molecule.

The nucleotide with reversible terminator can form the first phosphodiester bond, but before the terminator is reversed, it is not possible to form (or have limited ability to form) the second phosphodiester bond. Therefore, the nucleotide with reversible terminator can be incorporated into polynucleotide (for example, at the nick site), but until the terminator is reversed, the nucleotide can form a downstream phosphodiester bond. Technology well known to those skilled in the art can be used to reverse. For example, the terminator can be connected to the nucleotide by a cleavable connexon that can be cut, for example, by electromagnetic radiation. If the labeled nucleotide comprising 3 ' reversible terminator is used to carry out nick repair, the nucleotide of a single label can be incorporated into nick, but the terminator can prevent the nucleotide of other labels from being incorporated into nick. Therefore, the nick mark can be limited to the nucleotide of a label of each nick. The nick mark is limited to a label portion of each nick, and the potential deviation of the multiple labels incorporated into the same nick can be minimized. For example, if a method is adopted to limit the mark to a label portion of each nick, two very close nicks (i.e., the possibility that two labels are simply incorporated into the same nick) can be resolved based on the relatively strong signal from the mark. For example, if a quantitative assessment of the number of cuts is desired, a one-label-per-cut approach can facilitate a direct correlation between the strength of the label signal and the number of cuts. Labels on nucleotides containing reversible terminators can be as described herein. In some embodiments, the nucleotides containing reversible terminators comprise quantum dots. In some embodiments, the nucleotides containing reversible terminators comprise fluorophores. In some embodiments, the nucleotides containing reversible terminators comprise non-optical labels.

In some embodiments, multiple markers mark a single sample molecule. In some embodiments, at least one marker comprises a sequence-specific marker. In some embodiments, at least one marker comprises a non-sequence-specific marker. In some embodiments, at least one marker comprises a sequence-specific marker, and at least one marker comprises a non-sequence-specific marker. In some embodiments, at least one marker does not cut a single or double strand of DNA. For example, in some embodiments, at least one marker is selected from a non-cutting restriction enzyme, a methyltransferase, a zinc finger protein, an antibody, a transcription factor, a DNA binding protein, a hairpin polyamide, an oligodeoxynucleotide forming a triple helix, a peptide nucleic acid, or a combination thereof. In some embodiments, neither the sequence-specific nor the non-sequence-specific marker cuts DNA.

In some embodiments, for example, if a fluorescent label is provided, the labeling is detected using a sensitive camera. In some embodiments, for example, if a non-optical label is provided, the labeling is detected electronically. However, any detection method suitable for the corresponding label can be used. The methods described herein may include binding to one or more regions of the molecules described herein with a fluorescent label, a radioactive label, a magnetic label, or any combination thereof. Binding can be accomplished where the label is specifically complementary to the molecule or at least a portion of the molecule or other region of interest.

In some embodiments, the nickase produces a sequence-specific nick subsequently labeled with, for example, nucleotides or nucleonucleotide analogs. In some embodiments, fluorescently labeled nucleotides or analogs. In some embodiments, DNA is linearized by restriction in nanochannels, thereby causing uniform linearization and allowing strict and accurate measurement of the distance between the nick marks comprising a characteristic pattern (signature pattern) on the DNA molecule. In some embodiments, a second nickase is used. In some embodiments, the second nickase is used together with a second marker color. The exemplary nickases that can be used according to the embodiments herein include but are not limited to Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BspQI, Nt.BstNBI, Nt.CviPII and combinations thereof. Examples of incision agents and experimental protocols are also provided in US Patent Application Publication No. 2011/0171634 and US Patent Application Publication No. 2012/0237936, the entire contents of which are incorporated herein by reference.

In some embodiments, by hybridizing probe with single-stranded polynucleotide, labeling polynucleotide such as RNA or DNA.This probe can be with the single strand of RNA or DNA or its partial complementarity.In some embodiments, probe and specific sequence motif complementarity.In some embodiments, provide multiple probe to be complementary with multiple specific sequence motif, for example at least 2,3,4,5,6,7, 8,9,10,20,30,40,50,60,70,80,90,100,200,300,400, 500,600,700,800,900,1000,5,000 or 10,000 probes, comprise the scope between any two kinds in the listed values.In some embodiments, probe has random sequence.In some embodiments, provide probe with multiple random sequence. In some embodiments, the probe comprises one or more of an organic fluorophore, a quantum dot, a dendrimer, a nanowire, a bead, an Au bead, a paramagnetic bead, a magnetic bead, a radiolabel, a polystyrene bead, a polyethylene bead, a peptide, a protein, a hapten, an antibody, an antigen, a streptavidin avidin, a neutravidin, a biotin, a nucleotide, an oligonucleotide, a sequence-specific binding factor such as an engineered restriction enzyme, a methyltransferase, a zinc finger binding protein, and the like. In some embodiments, the probe comprises a fluorophore-quencher pair. One configuration of the probe can include a fluorophore attached to a first end of the probe and a suitable quencher tied to a second end of the probe. Thus, when the probe is not hybridized, the quencher prevents the fluorophore from fluorescing, and when the probe is hybridized to the target sequence, the probe is linearized, thereby moving the quencher away from the fluorophore and allowing the fluorophore to fluoresce when excited by electromagnetic radiation of an appropriate wavelength. In some embodiments, the first probe comprises a first fluorophore of a FRET pair, and the second probe comprises a second fluorophore of the FRET pair. Thus, hybridization of the first probe and the second probe to a single flank or a pair of flanks within the FRET radius of each other can allow energy transfer via FRET. In some embodiments, the first probe comprises a first fluorophore of a FRET pair, and the label incorporated into the nucleotide to fill the corresponding gap can comprise a second fluorophore of the FRET pair. Thus, hybridization of the first probe to the flank and entry of the labeled nucleotide into the corresponding gap allow energy transfer via FRET.

In some embodiments, double-stranded DNA can be labeled by first melting the hydrogen bonds between the double strands of certain genomic regions by increasing the temperature or by manipulation with an organic solvent, so as to open the so-called D-loop, and then hybridizing with at least one specific probe with an affinity equal to or greater than that of the single-stranded region before annealing back to a relatively stable form. In this way, in some embodiments, double-stranded DNA can be labeled by the probes described herein without nicking or cleaving either strand. In some embodiments, multiple D-loops can be opened on single strands. In this way, multiple probes can anneal to specific double-stranded DNA.

In some embodiments, marking comprises transferring the mark to polynucleotide by methyltransferase. In some embodiments, methyltransferase specificity methylates sequence motifs. Like this, marking comprises transferring the mark to sequence motifs by methyltransferase. Exemplary applicable DNA methyltransferases (MTase) include but are not limited to M.BseCI (methylating adenine at N6 in 5'-ATCGAT-3' sequence), M.Taql (methylating adenine at N6 in 5'-TCGA-3' sequence) and M.Hhal (methylating the first cytosine at C5 in 5'-GCGC-3' sequence). In some embodiments, two or more methyltransferases provide two or more marks that can be identical or different.

In some embodiments, marking comprises transferring the mark to polynucleotide by methyltransferase.In some embodiments, methyltransferase specificity methylates sequence motif.Like this, marking comprises transferring the mark to sequence motif by methyltransferase.Exemplary applicable DNA methyltransferase (MTase) includes but is not limited to M.BseCI (methylates adenine at N6 in 5'-ATCGAT-3' sequence), M.Taql (methylates adenine at N6 in 5'-TCGA-3' sequence) and M.Hhal (methylates the first cytosine at C5 in 5'-GCGC-3' sequence).In some embodiments, two or more methyltransferases provide two or more marks that can be identical or different.

In some embodiments, the channel comprises a microchannel. In some embodiments, the channel comprises a nanochannel. The characteristic cross-sectional dimension of a suitable fluid nanochannel segment is less than about 1000 nm, less than about 500 nm, or less than about 200 nm, or less than about 100 nm, or even less than about 50 nm, about 10 nm, about 5 nm, about 2 nm, or even less than about 0.5 nm. Suitable are fluid nanochannel segments having a characteristic cross-sectional dimension less than about twice the radius of gyration of a molecule. In some embodiments, the characteristic cross-sectional dimension of a nanochannel is at least about the persistence length of a molecule. The length of a fluid nanochannel segment suitable for use in the present invention is at least about 100 nm, at least about 500 nm, at least about 1000 nm, at least about 2 microns, at least about 5 microns, at least about 10 microns, at least about 1 micron, or even at least about 10 mm. In some embodiments, the fluid nanochannel segment is present at a density of at least 1 fluid nanochannel segment per cubic decimeter.

Examples of fluidic channels can be found in U.S. Patent Publication No. 2008/0242556, the entire disclosure of which is incorporated herein by reference. In some embodiments, viral particles or bacterial particles are analyzed. For example, in some embodiments, microchannels are used to analyze bacterial cells. In some embodiments, the channels allow cells with diameters ranging from a few microns to tens of microns to flow through.

FIG1 is a schematic diagram illustrating a fluidic channel arrangement according to some embodiments herein. The arrangement may include a sample input chamber 10. The arrangement may include a fluidic channel array 12, such as a fluidic nanochannel array. The arrangement may include a sample output chamber 14. The output chamber may contain a buffer solution 16. The nanofluidic channel array 12 may be in fluid communication with the input chamber 10. The nanofluidic channel array 12 may be in fluid communication with the output chamber 14. Sample molecules or particles of interest 18 may be disposed in the nanofluidic channel array 10. Control or comparison molecules or particles of interest 18 may be disposed in the nanofluidic channel array 10. In some embodiments, the fluidic channel array 12 connects the input chamber 10 with the output chamber 14. In some embodiments, the sample molecules or particles of interest 18 and the control or comparison molecules or particles of interest 20 are loaded into the sample input chamber and travel through the nanofluidic channel array in the buffer solution 16. In some embodiments, the sample molecules or particles of interest 18 and the control or comparison molecules or particles of interest 20 are deposited from the fluidic channel array 12 into the sample output chamber 14.

FIG2 is a schematic diagram illustrating an arrangement for detecting sample molecules or particles of interest according to some embodiments herein. In some embodiments, the arrangement includes a first sample inlet or outlet 11, a second sample inlet or outlet 11, and at least one fluid channel 13 located therebetween and in fluid communication with each of the first and second inlets or outlets 11. As contemplated herein, if a sample is loaded into the first inlet or outlet 11, the first inlet or outlet 11 acts as an inlet, and the second inlet or outlet 11 can act as an outlet. As contemplated herein, if a sample is loaded into the second inlet or outlet 11, the second inlet or outlet 11 acts as an inlet, and the first inlet or outlet 11 can act as an outlet. In some embodiments, the sample comprises molecules or particles of interest 18, control or comparison particles 20 of interest, or a combination of the two. In some embodiments, molecules or particles of interest 18, control or comparison particles 20 of interest pass through fluid channel 13. In some embodiments, fluid channel 13 comprises nanochannels. In some embodiments, fluid channel 13 comprises microchannels. In some embodiments, fluid channel 13 comprises a detection zone 22. In some embodiments, the system includes a cover 24 disposed over the detection region 24. In some embodiments, the cover 24 comprises a transparent cover. In some embodiments, a detector 26 is located over the detection region 22 and the cover 24 (if present). In some embodiments, for example, if optical detection is used, the detector 26 comprises a photon detector/imager. In some embodiments, a lens 28 is positioned in optical communication with the detection region 22 and the detector 26. In some embodiments, the lens 28 is positioned between the detection region 22 and the detector 26. In some embodiments, a dichroic mirror 30 is positioned in optical communication with the detection region 22, the lens 28, the detector 26, and an excitation source 32 so that a fluorescent marker (if present) can be excited and fluorescence from the fluorescent marker (if present) can be detected.

In some embodiments, a comparison of a sample to a reference sample is provided in the form of a histogram. In some embodiments, the physical counts of molecules with a specific marker pattern that matches a reference or de novo electronic sequencing genome assembly are tabulated in the histogram distribution to reflect the depth of coverage. As in the case of aneuploidy in genetic diseases or structural variations in cancer, a higher or lower than average depth of coverage in a specific region or entire chromosome reflects a deviation from normal ploidy.

Other optional implementation options

Some embodiments described herein may include the following: a method of characterizing a sample, comprising: labeling a region of a sample molecule with at least two labels; translocating the labeled sample molecule through a fluid channel, wherein the fluid channel is configured to extend at least a portion of the sample molecule, and wherein the fluid channel has a length of at least 10 nm and a cross-sectional diameter of at least 5000 nm; detecting a signal in the fluid channel generated by the labeled sample; and correlating the signal generated by the labeled sample with a signal generated by a corresponding region of a reference molecule. The method may also include: labeling a region of a reference molecule corresponding to the region of the sample molecule; translocating the labeled reference sample molecule through a fluid channel, wherein the fluid channel is configured to extend at least a portion of the sample molecule, and wherein the fluid channel has a length of at least 10 nm and a cross-sectional diameter of less than 5000 nm; and detecting a signal in the fluid channel generated by the labeled reference sample, wherein the signal generated by the known corresponding region of the reference molecule is the signal generated by the labeled reference sample.

In some embodiments, a method for characterizing a sample is provided. The method may include: labeling a sample nucleic acid molecule; translocating the labeled sample nucleic acid molecule through a fluidic nanochannel, wherein the fluidic nanochannel is configured to extend at least a portion of the sample nucleic acid molecule, and wherein the fluidic nanochannel has a length of at least 10 nm and a cross-sectional diameter of less than 1000 nm; detecting a signal generated by the sample nucleic acid molecule in the fluidic channel; determining a position of the label on the sample nucleic acid molecule; and aligning the position of the label on the sample nucleic acid molecule with a position of the label in a reference genome.

In some embodiments, a method for characterizing a sample is provided. The method may include: treating a double-stranded DNA sample to generate a flank of a first strand of the double-stranded DNA sample that is displaced from the double-stranded DNA sample, wherein the flank has a length ranging from about 1 base to about 1000 bases, and wherein the flank causes a gap in the first strand of the double-stranded DNA sample corresponding to the flank; incorporating one or more bases into the double-stranded DNA to eliminate at least a portion of the gap; labeling at least a portion of the treated double-stranded DNA with one or more tags; and quantifying a signal generated by the tag on the double-stranded DNA; comparing the amount of the signal generated by the double-stranded DNA to the amount of the signal generated by a reference DNA; and determining the presence of a genetic abnormality in the double-stranded DNA when the amount of the signal generated by the double-stranded DNA is different from the amount of the signal generated by the reference DNA.

In some embodiments, a method of characterizing a sample is provided. The method may include labeling a plurality of sequence-specific positions on sample DNA; linearizing at least a portion of the sample DNA; quantifying a signal generated by the label on the sample DNA; comparing the amount of signal generated by the sample DNA to the amount of signal generated by a reference DNA; and determining the presence of a genetic abnormality in the sample DNA when the amount of signal generated by the sample DNA differs from the amount of signal generated by the reference DNA.

In some embodiments, a system for characterizing a sample is provided. The system may include: one or more regions for labeling sample molecules with at least two labels; a fluidic channel for translocating the labeled sample molecules, wherein the fluidic channel is configured to extend at least a portion of the sample molecules, and wherein the fluidic channel has a length of at least 10 nm and a cross-sectional diameter of less than 5000 nm; and means for detecting a signal generated by the labeled sample in the fluidic channel.

In some embodiments, a system for characterizing a sample is provided. The system may include: one or more regions for labeling sample nucleic acid molecules; a fluidic nanochannel for translocating the labeled sample nucleic acid molecules, wherein the fluidic nanochannel is configured to extend at least a portion of the sample nucleic acid molecules, and wherein the fluidic nanochannel has a length of at least 10 nm and a cross-sectional diameter of less than 1000 nm; and means for detecting a signal generated by the sample nucleic acid molecules in the fluidic channel.

In some embodiments, a system for characterizing a sample is provided. The system can include: one or more regions for treating a double-stranded DNA sample to generate flanks of a first strand of the double-stranded DNA sample that are displaced from the double-stranded DNA sample, wherein the flanks have a length ranging from about 1 base to about 1000 bases, and wherein the flanks induce gaps in the first strand of the double-stranded DNA sample corresponding to the flanks; one or more regions for incorporating one or more bases into the double-stranded DNA to eliminate at least a portion of the gap; one or more regions for labeling at least a portion of the treated double-stranded DNA with one or more tags; and a device for quantifying signals generated by the tags on the double-stranded DNA.

In some embodiments, a system for characterizing a sample is provided. The system may include: a region for labeling a plurality of sequence-specific positions on the sample DNA; a region for linearizing at least a portion of the sample DNA; and a device for quantifying a signal generated by the label on the sample DNA.

In some embodiments, a system for characterizing a sample is provided. The system may include: a component for labeling sample molecules with at least two labels; a component for linearizing the labeled sample molecules; and a component for detecting a signal generated by the labeled sample in a fluid channel.

In some embodiments, a system for characterizing a sample is provided. The system may include: a component for labeling sample nucleic acid molecules; a component for linearizing the labeled sample nucleic acid molecules; and a component for detecting a signal generated by the sample nucleic acid molecules in a fluid channel.

In some embodiments, a system for characterizing a sample is provided. The system may include: means for processing a double-stranded DNA sample to generate a flank of a first strand of the double-stranded DNA sample that is displaced from the double-stranded DNA sample, wherein the flank has a length ranging from about 1 to about 1000 bases, and wherein the flank causes a gap in the first strand of the double-stranded DNA sample corresponding to the flank; means for incorporating one or more bases into the double-stranded DNA to eliminate at least a portion of the gap; means for labeling at least a portion of the processed double-stranded DNA with one or more tags; and means for quantifying a signal generated by the tags on the double-stranded DNA.

In some embodiments, a system for characterizing a sample is provided. The system may include: a system for characterizing a sample, comprising a component for labeling a plurality of sequence-specific positions on the sample DNA; a component for linearizing at least a portion of the sample DNA; and a component for quantifying a signal generated by the label on the sample DNA.

According to some embodiments, there is provided a method or system as described herein, wherein the sample is selected from the group consisting of bacteria, virions, DNA molecules, RNA molecules, nucleic acid polymers, proteins, peptides, and polysaccharides.

According to some embodiments, there is provided a method or system as described herein, wherein the sample is derived from maternal blood, and wherein the reference molecule is derived from a maternal sample other than blood.

According to some embodiments, there is provided a method or system as described herein, wherein the sample comprises nucleotides, and wherein the at least two labels are located at either end of the region of interest in the nucleotides.

According to some embodiments, there is provided a method or system as described herein, wherein the label is selected from a fluorescent label, a radioactive label, a magnetic label, or a combination thereof.

According to some embodiments, there is provided a method or system as described herein, wherein optical detection comprises determining the species count, intensity, wavelength, or size of the marker.

According to some embodiments, there is provided a method or system as described herein, wherein the optical detection comprises determining the length of the at least one labeled region in the sample.

According to some embodiments, there is provided a method or system as described herein, wherein correlating the signal comprises measuring a signal produced by a collection of samples or a collection of portions of samples.

According to some embodiments, there is provided a method or system as described herein, wherein associating the signals comprises using a ratio (K) between signals (S1, S2 ... Sn) generated by a plurality of samples or sample portions and a signal (C) generated by a reference: K1 = S1 / C, K2 = S2 / C ... Kn = Sn / C. In some embodiments, the difference between K1 and Kn is used to identify the presence of a fetal sample. In some embodiments, the difference between K1 and Kn is used to identify the presence of DNA from a tumor or other cancerous source. In some embodiments, the difference between K1 and Kn is used to determine the presence of a genetic abnormality in a sample. In some embodiments, the genetic abnormality is aneuploidy. In some embodiments, the genetic abnormality is a translocation, addition, amplification, transversion, or inversion. In some embodiments, the reference is derived from a known diploid or haploid chromosome. In some embodiments, the signal from the sample is associated with a population distribution from a metagenomic or microbiome study.

According to some embodiments, the methods or systems described herein are provided, wherein the fluid channel is a nanochannel. In some embodiments, the fluid channel is arranged parallel to the substrate surface. In some embodiments,

According to some embodiments, a method or system as described herein is provided, further comprising generating a histogram distribution to reflect the coverage depth of the sample.

According to some embodiments, a method or system as described herein is provided, wherein the sample comprises circulating fetal cells, circulating tumor cells, or a bodily fluid or tissue.

According to some embodiments, a method or system as described herein is provided, wherein translocation comprises applying a driving force to the labeled sample, the driving force being selected from the group consisting of a fluid flow, a radiation field, an electroosmotic force, an electrophoretic force, an electrodynamic force, a temperature gradient, a surface property gradient, capillary flow, a pressure gradient, a magnetic field, an electric field, a receding meniscus, surface tension, a thermal gradient, a pulling force, a pushing force, and combinations thereof.

According to some embodiments, kits for performing the methods described herein are provided.

According to some embodiments, a kit for using the system of any one of the preceding claims is provided.

In the description provided herein, reference is made to the accompanying drawings which form a part of the specification. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter set forth herein. It should be readily understood that aspects of the present invention, as generally described herein and illustrated in the accompanying drawings, may be arranged, substituted, combined, or designed in a variety of different configurations, all of which are clearly contemplated and are a part of the present invention.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "channel" refers to a region defined by a boundary. Such boundaries can be physical, electrical, chemical, magnetic, etc. The term "nanochannel" is used to indicate that certain channels are considered to be nanometer-sized.

As used herein, the term "DNA" refers to DNA of any length (e.g., 0.1 Kb to 1 megabase). The DNA may be a highly purified preparation, raw material, or semi-raw material. The DNA may be from any biological source or may be synthesized.

As used herein, the term "nucleotide" refers to a molecule containing a deoxyribonucleic acid (e.g., DNA, mtDNA, gDNA, or cDNA), a ribonucleic acid (e.g., RNA, or mRNA), or any other variant of a nucleic acid known in the art. The term "labeled nucleotide" refers to a nucleotide comprising any detectable modification. This includes, but is not limited to, nucleotides having a reporter gene attached to a base. Reporter genes include, but are not limited to, fluorescent dyes, haptens, biotin molecules, or gold nanoparticles. The term "natural nucleotide" refers to an unmodified nucleotide, or has a slight modification that does not interfere with its incorporation into DNA. The terms "t," "c," "a," "g," and "u" refer to nucleotides in DNA.

The term "nick" refers to the break of a phosphodiester bond with a 3' hydroxyl terminus on one DNA strand or the other.

As used herein, the term "nicking endonuclease" refers to any naturally occurring or engineered enzyme that breaks the phosphodiester bond on a single strand of DNA, leaving a 3'-hydroxyl group at a defined sequence. Nicking endonucleases can be naturally occurring, engineered by modifying a restriction enzyme to eliminate the cleavage activity on one DNA strand, or generated by fusing a nicking subunit to a DNA binding domain, such as a zinc finger and a transcription activator-like effector DNA recognition domain.

The term "labeling site" as used herein refers to any DNA site with an exposed 3' hydroxyl group to which a polymerase can add nucleotides in a template-dependent manner. The labeling site can be generated by nicking endonucleases, hybridization probes, or any chemical or physical means of destroying the phosphodiester bond on any DNA strand. Means of destroying the phosphodiester bond can occur on DNA outside its biological source or before DNA extraction, for example, due to exposure of the biological sample to chemicals or external forces such as radiation. If the 3' end is not extendable, it can be repaired to restore the hydroxyl group, for example, by using the New England Biolabs PreCR kit.

As used herein, a "sample" may include, for example, blood, serum, plasma, saliva, lavage fluid, cerebrospinal fluid, urine, semen, sweat, tears, sputum, etc. As used herein, "blood," "plasma," and "serum" clearly include fractions or processed portions thereof, wherein the sample is obtained from a biopsy, swab, smear, etc., and "sample" clearly includes processed fractions or portions derived from a biopsy, swab, smear, etc.

As used herein, the term "chromosome" refers to the heredity-bearing gene carrier of a living cell that is derived from chromatin and comprises DNA and protein components (particularly histones).

Those skilled in the art will recognize that "translocation" can be used interchangeably with linearization in the context of passing a DNA molecule through a nanochannel.

The methods, devices, systems, and kits described herein may incorporate the methods, devices, systems, and kits described in any of the following references: U.S. Patent Application Publication No. 2009/0305273, PCT Publication No. WO/2008/079169, U.S. Patent Application Publication No. 2008/0242556, PCT Publication No. WO/2008/121828, U.S. Patent Application Publication No. 2011/0171634, PCT Publication No. WO/2010/002883, U.S. Patent Application Publication No. 2011/0296903, PCT Publication No. WO/2009/149362, U.S. Patent Application Publication No. 2011/0306504, PCT Publication No. WO/2010/059731, U.S. Patent Application Publication No. 2012/0097835, PCT Publication No. WO/2010/135323, PCT Application No. PCT/US11/57115, U.S. Patent Application Serial No. 13/606819, PCT Application No. PCT/US2012/054299, U.S. Patent Application Publication No. 2012/0244635, PCT Publication No. WO/2011/038327, U.S. Patent Application Publication No. 2012/0237936, U.S. Patent Application Serial No. 13/503307, PCT Publication No. WO/2011/050147, U.S. Patent Application Serial No. 61/734327, U.S. Patent Application Serial No. 61/761189, and U.S. Patent Application Serial No. 61/713862, the entire contents of which are incorporated herein by reference.

Example 1

Genomic fragments were generated from human male samples by PCR, labeled, and passed through a nanochannel. The detected fragments were then aligned to a single gene reference optical map for each chromosome. The molecules were classified based on the alignment starting point.

As shown in Figure 7a, the average depth of coverage observed for a diploid autosome (chromosome 1) is 5X and is evenly distributed across the chromosome. If the molecules were sampled evenly, the alignment start sites would be randomly distributed across the chromosome, resulting in a linear map.

As shown in Figure 7b, the average depth of coverage observed for haploid sex chromosomes (chromosome X) from the same male sample is 2X-2.5X (roughly half the depth of diploid autosomes), and is also evenly distributed across the chromosome. This example illustrates that quantitative measurements can be achieved using the methods and platforms described herein.

Claims (86)

1. A method for characterizing a sample for non-diagnostic purposes, the method comprising: labeling a plurality of sample molecules with at least a first label, wherein the sample molecules comprise a polynucleotide sequence of a genomic fragment of interest or a plurality of genomic fragments of interest; providing a plurality of labeled reference molecules, wherein the reference molecules comprise the polynucleotide sequence of the genomic fragment; linearizing a plurality of labeled sample molecules through a fluidic channel; linearizing a plurality of labeled reference molecules through the fluidic channel; detecting signals from the labeled sample molecules and the labeled reference molecules, the signals comprising a pattern characteristic of the genomic fragment of interest; comparing the pattern to a pattern of a reference genomic segment of interest; and determining the coverage depth of the sample molecule on the genomic fragment of interest; and the coverage depth of the reference molecule on the genomic fragment, To determine the copy number of the genomic fragment of interest in the sample. 2. The method of claim 1, wherein labeling comprises labeling the sample molecule with a first label, and wherein the reference molecule comprises a second label, wherein the first marker is configured to generate a first pattern or a plurality of first patterns, wherein the second marker is configured to produce a second pattern or a plurality of second patterns, and The first mark and the second mark are different from each other. 3. The method of claim 1 , wherein marking comprises marking with a first marker, wherein one first pattern or multiple first patterns and one second pattern or multiple second patterns each include the first mark, and The first mode or modes and the second mode or modes are different from each other. 4. The method of claim 1, further comprising labeling a reference molecule to produce the labeled reference molecule, wherein the labeled reference molecule comprises one or more second patterns. 5. The method of claim 1, wherein the labeled reference molecules and sample molecules are derived from the sample. The method of claim 1 , wherein the labeled reference molecule is derived from a different tissue from the same organism as the sample. 7. The method of claim 1, wherein the signal from the labeled reference molecule comprises an electronically or optically stored value or set of values. 8. The method of claim 1, further comprising: labeling a plurality of second sample molecules from a second sample with at least the first label, wherein the plurality of second sample molecules comprise the polynucleotide sequence of the first genomic fragment of interest or the plurality of first genomic fragments of interest, wherein the plurality of second sample molecules are known not to correspond to a chromosomal abnormality; The plurality of second sample molecules are translocated through the fluidic channel. 9. The method according to claim 1, wherein the first genomic segment of interest or the plurality of first genomic segments of interest comprise a sex chromosome or at least one segment thereof, and wherein the reference genome segment or segments comprise an autosome or at least one segment thereof. 10. The method according to claim 1, wherein the first genomic segment of interest or the plurality of first genomic segments of interest comprise a first autosome or at least one segment thereof selected from the group consisting of human chromosome 21, human chromosome 13, human chromosome 14, human chromosome 15, human chromosome 16, human chromosome 18 and human chromosome 22, and segments thereof, and wherein the reference genome segment or segments comprise a second autosome or at least one segment thereof, wherein the second autosome or segment thereof is different from the first autosome or segment thereof. 11. The method of claim 1 , wherein the sample molecules are from a sample containing a possible genomic abnormality, and wherein the reference genomic segment or segments comprise a first chromosome or segments thereof, and wherein the reference genomic segments are from a second sample known not to comprise the possible genomic abnormality. 12. The method of any one of claims 1-11, wherein the first label comprises at least one of a fluorescent label, a radioactive label, a magnetic label, or a non-optical label. 13. The method of claim 2, wherein the second label comprises at least one of a fluorescent label, a radioactive label, a magnetic label, or a non-optical label. 14. The method of any one of claims 1 to 11, wherein labeling comprises: nicking one strand of the double-stranded DNA at the first sequence motif using a nicking endonuclease; and The DNA is labeled. 15. The method of claim 14, further comprising repairing at least some of the nicks in the DNA. 16. The method of claim 14, wherein the incision is not repaired. 17. The method of claim 14, wherein the marker comprises a transcription terminator. 18. The method of any one of claims 1-11, wherein labeling with the first label comprises tagging at least one sequence motif of the sample molecule with a DNA binding entity and a methyltransferase, wherein the DNA binding entity is selected from the group consisting of a non-cutting restriction enzyme, a zinc finger protein, an antibody, a transcription factor, a transcription activator-like domain, a DNA binding protein, a polyamide, a triple helix-forming oligonucleotide, and a peptide nucleic acid. 19. The method of any one of claims 1-11, wherein labeling with the first label comprises tagging at least one sequence motif of the sample molecule with a methyltransferase. 20. The method of any one of claims 1-11, further comprising labeling the sample molecules with a non-sequence specific label. 21. The method of any one of claims 1-11, wherein the fluidic nanochannel comprises a channel having a length of at least 10 nm and a cross-sectional diameter of less than 5000 nm. 22. The method of any one of claims 1 to 11, wherein the sample is selected from bacteria, virions, DNA molecules, RNA molecules, nucleic acid polymers, proteins, peptides, and polysaccharides. 23. The method of any one of claims 1-11, wherein the sample is derived from maternal blood, and wherein the reference molecule is derived from a maternal sample other than blood. 24. The method of any one of claims 1-11, wherein the sample comprises nucleotides, and wherein at least two labels are located at either end of a region of interest in the nucleotides. 25. The method of any one of claims 1-11, wherein the detecting comprises determining the species count, intensity, wavelength, or size of the marker. 26. The method of any one of claims 1-11, wherein the detecting comprises determining the length of at least one labeled region in the sample. 27. The method of any one of claims 1-11, further comprising measuring a signal generated by a collection comprising the sample or the sample portion. 28. The method of any one of claims 1-11, wherein the comparison comprises using a ratio K between the signals S1, S2...Sn generated by a plurality of samples or sample portions and a signal C generated by a reference: K1=S1/C, K2=S2/C...Kn=Sn/C. 29. The method of claim 28, wherein the difference between K1 and Kn is used to identify the presence of a fetal sample. 30. The method of claim 28, wherein the difference between K1 and Kn is used to identify the presence of DNA from a tumor or other cancerous source. 31. The method of claim 28, wherein the difference between K1 and Kn is used to identify the presence of a genetic abnormality in the sample. 32. The method of any one of claims 1-11, wherein the reference genome segment of interest is derived from a known diploid or haploid chromosome. 33. The method of any one of claims 1-11, wherein the signal from the sample is correlated with population distribution from a metagenomic or microbiome study. 34. The method of any one of claims 1-11, wherein the fluidic channel is a nanochannel. 35. The method of any one of claims 1-11, wherein the fluid channel is positioned parallel to the substrate surface. 36. The method of any one of claims 1-11, further comprising generating a histogram distribution to reflect the coverage depth of the sample. 37. The method of any one of claims 1-11, wherein the sample comprises circulating fetal cells, circulating tumor cells, or a body fluid or tissue. 38. The method of any one of claims 1-11, wherein translocation comprises applying a driving force to the labeled sample, the driving force being selected from the group consisting of a fluid flow, a radiation field, an electroosmotic force, an electrophoretic force, an electrodynamic force, a temperature gradient, a surface property gradient, capillary flow, a pressure gradient, a magnetic field, an electric field, a receding meniscus, surface tension, a thermal gradient, a pulling force, a pushing force, and combinations thereof. 39. The method of any one of claims 1-11, wherein each of the sample molecules is less than 500 nucleotides in length. 40. A system for characterizing a sample, comprising: one or more regions for labeling sample molecules with at least two labels; a fluidic channel for translocating the labeled sample molecules, wherein the fluidic channel is configured to elongate at least a portion of the sample molecules, and wherein the fluidic channel has a length of at least 10 nm and a cross-sectional diameter of less than 5000 nm; and means for detecting a signal generated by said labeled sample in said fluid channel, The system is configured to generate a histogram to reflect the coverage depth of the sample to determine the copy number of the genomic fragment of interest in the sample. 41. A system for characterizing a sample, comprising: one or more regions for labeling sample nucleic acid molecules; a fluidic nanochannel for translocating the labeled sample nucleic acid molecules, wherein the fluidic nanochannel is configured to extend at least a portion of the sample nucleic acid molecules, and wherein the fluidic nanochannel has a length of at least 10 nm and a cross-sectional diameter of less than 1000 nm; and means for detecting a signal generated by said sample nucleic acid molecule in said fluid channel, The system is configured to generate a histogram to reflect the coverage depth of the sample to determine the copy number of the genomic fragment of interest in the sample. 42. A system for characterizing a sample, comprising: A region for marking multiple sequence-specific positions on sample DNA; a region for linearizing at least a portion of the sample DNA; and means for quantifying the signal generated by the label on the sample DNA, The system is configured to generate a histogram to reflect the coverage depth of the sample to determine the copy number of the genomic fragment of interest in the sample. 43. The system of any one of claims 40-42, wherein the sample is selected from the group consisting of bacteria, virions, DNA molecules, RNA molecules, nucleic acid polymers, proteins, peptides, and polysaccharides. 44. The system of any one of claims 40-42, wherein the sample is derived from maternal blood. 45. The system of any one of claims 40-42, wherein the sample comprises nucleotides and the at least two labels are located at either end of a region of interest in the nucleotides. 46. The system of any one of claims 40-42, wherein the label is selected from a fluorescent label, a radioactive label, a magnetic label, or a combination thereof. 47. The system of any one of claims 40-41, wherein detecting a signal comprises determining an instance count, intensity, wavelength, or size of the marker. 48. The system of any one of claims 40-41, wherein detecting the signal comprises determining the length of at least one labeled region in the sample. 49. The system of any one of claims 40-42, wherein correlating the signals comprises measuring signals generated by a collection of samples or a collection of sample portions. 50. A system as described in any of claims 40-42, wherein the system is configured to correlate signals, wherein the signals are correlated using the ratio K between the signals S1, S2...Sn generated by multiple samples or sample parts and the signal C generated by a reference: K1=S1/C, K2=S2/C...Kn=Sn/C. 51. The system of claim 50, wherein the difference between K1 and Kn is used to identify the presence of a fetal sample. 52. The system of claim 50, wherein the difference between K1 and Kn is used to identify the presence of DNA from a tumor or other cancerous source. 53. The system of claim 50, wherein the difference between K1 and Kn is used to determine the presence of a genetic abnormality in the sample. 54. The system of claim 53, wherein the genetic abnormality is aneuploidy. 55. The system of claim 53, wherein the genetic abnormality is a translocation, addition, amplification, transversion, or inversion. 56. The system of any one of claims 40-42, further comprising a reference derived from a known diploid or haploid chromosome. 57. The system of any one of claims 40-42, wherein the signal from the sample is correlated with population distribution from a metagenomic or microbiome study. 58. The system of any one of claims 40-42, wherein the fluidic channel is a nanochannel. 59. The system of any one of claims 40-42, wherein the fluid passage is positioned parallel to a substrate surface. 60. The system of any one of claims 40-42, wherein the sample comprises circulating fetal cells, circulating tumor cells, or a bodily fluid or tissue. 61. The system of any one of claims 40-42, wherein the translocation comprises applying a driving force to the labeled sample, the driving force being selected from the group consisting of a fluid flow, a radiation field, an electroosmotic force, an electrophoretic force, an electrodynamic force, a temperature gradient, a surface property gradient, capillary flow, a pressure gradient, a magnetic field, an electric field, a receding meniscus, surface tension, a thermal gradient, a pulling force, a pushing force, and combinations thereof. 62. The system of any one of claims 40-41, wherein the sample molecules are nucleic acids, each less than 500 nucleotides in length. 63. The system of claim 42, wherein the sample DNA is less than 500 nucleotides in length. 64. A method for characterizing a sample comprising a polynucleotide sequence for non-diagnostic purposes, the method comprising: labeling a plurality of sample molecules with at least a first label, wherein the sample molecules comprise polynucleotide sequences of a genomic segment of interest or a plurality of genomic segments of interest, and wherein the genomic segment of interest or the plurality of genomic segments of interest correspond to potentially abnormal genomic regions; Providing a plurality of labeled reference molecules, wherein the reference molecules comprise polynucleotide sequences of a reference genomic segment or segments, and wherein the reference genomic segment or segments are known not to correspond to the potentially abnormal genomic region; translocating the plurality of labeled sample molecules and the plurality of labeled reference molecules through one or more fluidic channels; Detecting signals from the labeled sample molecules and the labeled reference molecules to determine at least: a first pattern or patterns specific to the genomic segment or segments of interest; and a second pattern or patterns specific to the reference genome segment or segments; and The signal determining the one or more first patterns is associated with the signal determining the one or more second patterns, wherein the association includes comparing the coverage depth of the one or more genomic fragments of interest with the coverage depth of a reference genomic fragment or multiple reference genomic fragments to determine the copy number of the one or more genomic fragments of interest. 65. The method of claim 64, wherein the sample is derived from maternal blood, and wherein the reference molecule is derived from a maternal sample other than blood. 66. The method of claim 64, wherein the sample comprises circulating fetal cells. 67. The method of claim 64, further comprising generating a histogram distribution to reflect the depth of coverage of the sample. 68. The method of claim 64, further comprising labeling a reference molecule to produce the labeled reference molecule, wherein the labeled reference molecule comprises the second pattern or patterns. 69. The method of claim 64, wherein the signal from the labeled reference molecule comprises an electronically or optically stored value or set of values. 70. The method of claim 64, wherein the genomic segment of interest or the genomic segments of interest comprise a sex chromosome or at least one segment thereof, and wherein the reference genome segment or segments comprise an autosome or at least one segment thereof. 71. The method of claim 64, wherein the first label comprises at least one of a fluorescent label, a radioactive label, a magnetic label, or a non-optical label. 72. The method of claim 64, wherein marking comprises: nicking one strand of the double-stranded DNA at the first sequence motif using a nicking endonuclease, thereby creating a nick in the double-stranded DNA; and The DNA is labeled. 73. The method of claim 72, further comprising repairing at least some of the nicks in the double-stranded DNA. 74. The method of claim 64, wherein the plurality of labeled reference molecules comprises a label different from the first label. 75. The method of claim 64, wherein the plurality of labeled reference molecules comprises a label that is the same as the first label. 76. The method of claim 64, wherein labeling with the first label comprises tagging at least one sequence motif of the sample molecule with a methyltransferase. 77. The method of claim 64, further comprising labeling the sample molecules with a non-sequence specific label. 78. The method of any one of claims 64-77, wherein each of the sample molecules is less than 500 nucleotides in length. 79. A method for characterizing a sample comprising a polynucleotide sequence for non-diagnostic purposes, the method comprising: marking a plurality of sequence-specific positions on the polynucleotide sequence of the sample molecule; linearizing at least a portion of the sample molecules in a fluidic channel; quantifying a signal from the label on the sample molecule; Comparing the coverage depth of the polynucleotide sequence of the sample molecule with the coverage depth of the polynucleotide sequence of a reference molecule to determine the copy number of the sample molecule; and When the coverage depth of the sample molecule is different from the coverage depth of the reference molecule, it is determined whether a genetic abnormality is present in the sample molecule. 80. The method of claim 79, wherein the sample comprises circulating fetal cells. 81. The method of claim 79, further comprising generating a histogram distribution to reflect the depth of coverage of the sample. 82. The method of claim 79, wherein the sample molecule and the reference molecule are from different tissues of the same organism. 83. The method of claim 79, wherein the quantity of signal from the reference molecule comprises an electronically or optically stored value or set of values. 84. The method of claim 79, wherein the genetic abnormality comprises at least one of a translocation, an addition, an amplification, a transversion, an inversion, an aneuploidy, a polyploidy, a monosomy, a trisomy, a trisomy 21, a trisomy 13, a trisomy 14, a trisomy 15, a trisomy 16, a trisomy 18, a trisomy 22, a triploidy, a tetraploidy, and a sex chromosome aneuploidy. 85. The method of any one of claims 79-84, wherein the sample comprises plant tissue. 86. The method of any one of claims 79-84, wherein the sample comprises bacteria or virions derived from an environmental sample.