JP2013541347A - Analysis of fragmented genomic DNA in droplets - Google Patents

Abstract

  A method for analyzing genomic DNA. Genomic DNA containing the target may be obtained. Genomic DNA may be intentionally fragmented to produce fragmented DNA. The fragmented DNA may be passed through the droplet forming unit to form an aqueous droplet containing the fragmented DNA. An assay may be performed on the droplet to measure the level of the target. In some embodiments, the droplets may comprise genomic DNA at a concentration of at least about 5 nanograms / microliter and the droplets may be formed with a droplet formation frequency of at least about 50 droplets / second. The droplets may have an average volume of each droplet of less than about 10 nanoliters, the droplets may be formed at a flow rate greater than about 50 nanoliters / second, or any combination thereof There may be.

Description

This application claims priority to the following earlier applications: U.S. Provisional Patent Application No. 61 / 409,106 filed on November 1, 2010 and December 22, 2010. No. 12 / 976,827 published on Sep. 8, 2011 as U.S. Patent Application Publication No. 2011/0217712 (A1). The entire contents of both patent applications are incorporated herein by reference for all purposes.

This application incorporates the following documents by reference for all purposes: US Pat. No. 7,041,481, issued May 9, 2006, July 2010 U.S. Patent Application Publication No. 2010/0173394 (A1) published on the 8th, PCT International Publication No.WO2011 / 120024 published on September 29, 2011, and Joseph R. Lakowicz, PRINCIPLES OF FLUORESCENCE SPECTROSCOPY (No. 2) Edition, 1999).

Introduction Many biomedical applications rely on high-throughput assays of samples for target nucleic acids. For example, in research and clinical applications, high-throughput genetic testing using target-specific reagents enables accurate and accurate target nucleic acid quantification, especially for drug discovery, biomarker discovery and clinical diagnostics Is possible.

  Emulsions have great expectations that will revolutionize target high-throughput assays. Emulsification techniques can create many aqueous droplets that function as independent reaction vessels for biochemical reactions. For example, an aqueous sample (e.g., 20 microliters) can be divided into multiple droplets (e.g., 20,000 droplets of 1 nanoliter each), so that each droplet can be used to target Individual tests can be performed.

  Aqueous droplets can be suspended in oil to create a water-in-oil emulsion (W / O). Stabilizing the emulsion with a surfactant can reduce the aggregation of the droplets during heating, cooling and movement, thereby enabling thermal cycling. Thus, emulsions have been used to perform single copy amplification of target nucleic acid molecules in droplets using the polymerase chain reaction (PCR). The ability to detect the presence of individual target molecules in a droplet enables a digital assay.

  In an exemplary droplet-based digital assay, the sample is divided into a set of droplets at the target limiting dilution (ie, some droplets do not contain the target molecule). If the target molecules are randomly distributed between the droplets, there will be exactly 0, 1, 2, 3 or more target molecules in the droplets based on a predetermined average concentration of the target in the droplets The probability of being found is indicated by the Poisson distribution. Conversely, the concentration of the target molecule in the droplet (and thus in the sample) may be calculated from the probability that a predetermined number of molecules are found in the droplet.

  The probability that a target molecule is not found and the probability that one or more target molecules are found may be measured in a digital assay. In a binary approach, each droplet may be tested to determine whether the droplet is positive and therefore contains at least one target molecule, or whether the droplet is negative and therefore does not contain a target molecule. . The probability that a target molecule is not found in a droplet is the fraction that is negative of the tested droplet (`` negative fraction ''), and the probability that at least one target molecule is found is the fraction that is positive for the tested droplet (`` It can be approximated by "positive fraction"). The positive or negative fraction value can then be used in a Poisson algorithm to calculate the target concentration in the droplet. In other cases, the digital assay may produce more than two values of data. For example, the assay may assess how many target molecules are present in each droplet with a resolution greater than negative (0) or positive (> 0) (e.g., 0, 1 or> 1 molecule; 0, 1, 2 or> 2 molecules).

  In order for various sample droplet-based DNA assays to combine high throughput and accuracy, the droplets must be rapidly formed in uniform sizes (ie, monodisperse droplets). However, the sample component may interfere with the ability of the droplets to separate from the bulk sample phase, particularly when the droplet formation frequency is high. As a result, the size of the droplets formed or even the ability to form droplets can vary from sample to sample, reducing the reliability of the assay.

US Provisional Patent Application No. 61 / 409,106 U.S. Patent Application No. 12 / 976,827 U.S. Patent No. 7,041,481 US Patent Application Publication No. 2010/0173394 (A1) PCT International Publication No.WO2011 / 120024

Joseph R. Lakowicz, PRINCIPLES OF FLUORESCENCE SPECTROSCOPY (2nd edition, 1999)

  There is a need for new approaches that provide reliable and consistent droplet formation with high formation frequency.

  The present disclosure provides a method for analyzing genomic DNA. Genomic DNA containing the target may be obtained. Genomic DNA may be intentionally fragmented to produce fragmented DNA. The fragmented DNA may be passed through a droplet generator to form an aqueous droplet containing the fragmented DNA. A digital assay may be performed on the droplet to measure the level of the target. In some embodiments, the droplets may comprise genomic DNA at a concentration of at least about 5 nanograms / microliter, and the droplets have a droplet generation frequency of at least about 50 droplets / second. The droplets may be formed, the average volume of each droplet may be less than about 10 nanoliters, the droplets may be formed at a sample flow rate greater than about 50 nanoliters / second, or Any combination thereof may be used.

2 is a flowchart illustrating an exemplary method of genomic DNA analysis according to aspects of the present disclosure. FIG. 5 is a matrix diagram made from photographs of droplet formations processing three different samples at each of four different driving pressures. FIG. 6 is a graph of droplet volume graphed as a function of droplet formation frequency for samples containing no genomic DNA or digested (EcoRI) or undigested genomic DNA (Raji or Coriell). FIG. 4 is a drop volume graph graphed as a function of sample flow rate for each sample of FIG. FIG. 4 is a graph of maximum elongation plotted as a function of droplet formation frequency for each sample of FIG. FIG. 4 is a graph of maximum elongation graphed as a function of sample flow rate for each sample of FIG.

  The present disclosure provides a method for analyzing genomic DNA. Genomic DNA containing the target may be obtained. Genomic DNA may be intentionally fragmented to produce fragmented DNA. The fragmented DNA may be passed through the droplet forming unit to form an aqueous droplet containing the fragmented DNA. An assay may be performed on the droplet to measure the level of the target. In some embodiments, the droplets may comprise genomic DNA at a concentration of at least about 5 nanograms / microliter and the droplets may be formed with a droplet formation frequency of at least about 50 droplets / second. The droplets may have an average volume of each droplet of less than about 10 nanoliters, the droplets may be formed at a flow rate greater than about 50 nanoliters / second, or any combination thereof There may be.

  As disclosed herein, methods for analyzing genomic DNA in droplets have significant advantages over other droplet-based approaches. Advantages include forming droplets with high frequency, high monodispersity, high DNA content, and / or substantially less interference from genomic DNA.

  For these and other aspects of the present disclosure, the following sections: (I) an overview of exemplary methods of genomic DNA analysis, (II) exemplary data for drop formation tests and (III) selected implementations Described in the form.

I. Overview of an Exemplary Method of Genomic DNA Analysis FIG. 1 shows a flow chart illustrating an exemplary method 20 of genomic DNA analysis. The presented steps may be performed in any suitable combination in any suitable order.

Genomic DNA may be obtained, and this is 22. DNA can also be obtained from any suitable organism, including mammals (e.g., humans, mice, rats, monkeys, etc.), non-mammalian vertebrates, invertebrates, yeasts or fungi, plants, protozoa, bacteria, etc. Good. DNA may be obtained by any suitable method, such as commercially purchased, received as a gift, obtained by extraction from cells or body fluids, or received as a clinical sample. DNA may be obtained in a relatively high molecular weight state, for example, in particular, having a molecular weight of at least about 10 ⁴ , 10 ⁵ or 10 ⁶ kilodaltons (e.g., an average length of at least about 25, 50, 100, 200 , 500 or 1,000 km base).

  Genomic DNA may be fragmented prior to the formation of the droplets, which is 24. Fragmentation may be performed intentionally, i.e. deliberately. Fragmentation generally includes any procedure that substantially reduces the molecular weight of genomic DNA, such as by cutting or breaking the DNA strand. By fragmentation, the average molecular weight and / or length may be reduced, in particular, to any suitable amount, such as at least about 5, 10, 20, 50 or 1/100. An exemplary approach to fragmenting genomic DNA includes digestion with restriction enzymes (eg, enzymes with, among other things, 4, 5, 6 or 8 nucleotide recognition sites). The target may not include a restriction enzyme recognition site to avoid any cleavage of the target molecule. Restriction enzyme digestion may be complete or partial digestion. Alternatively or in addition, an aqueous sample of genomic DNA may be heated to fragment the DNA. Typical heating to fragment the DNA may be in particular at a temperature of at least 95 ° C. for at least about 10, 15, 20 or 30 minutes. In other cases, the DNA may be fragmented by shearing, sonication, spraying, irradiation, and the like.

  Genomic DNA may include a target, generally the sequence under test. Fragmentation of genomic DNA may be performed without substantially destroying the target, which is less than half the target sequence of genomic DNA destroyed (e.g. break or cleaved) by the fragmentation process Means that.

  A droplet containing fragmented DNA may be formed, which is designated 26. You may form a droplet continuously by each of one or several droplet formation part. The fragmented DNA may be passed through at least one droplet forming part to form droplets. Generally, the fragmented DNA is placed in an aqueous sample, and the aqueous sample and the immiscible continuous phase pass through the droplet former to form aqueous droplets containing the fragmented DNA, which are in the continuous phase enter. Further embodiments of droplet formation and emulsion phases that may be appropriate are described in the literature cited above in cross-reference, which are incorporated herein by reference, in particular in 2010. US Patent Application Publication No. 2010/0173394 (A1) published on July 8, and PCT International Publication No. WO2011 / 120024 published on September 29, 2011.

  The droplets can be of any suitable size. For example, the average volume of the droplets may be less than about 1 μL, 100 nL, 10 nL, 1 nL, 100 pL, 10 pL, or 1 pL, among others. Alternatively or additionally, the average volume of the droplets may be in particular greater than about 10 fL, 100 fL, 1 pL, 10 pL or more than 100 pL. In some cases, the average volume of the droplets may be in particular about 1 pL to 100 nL, 1 pL to 10 nL, or 0.1 to 10 nL. The droplets may be monodispersed.

  The droplet may contain any suitable concentration of fragmented DNA. For example, fragmented DNA may be placed in droplets, particularly at a concentration of at least about 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50 ng / L. In some cases, the concentration may be about 0.1-50 or 0.2-20 ng / L. By fragmenting DNA, the content of DNA incorporated in the droplet can be further increased. Fragmented DNA may be present in an average of less than about 2 genome equivalents per droplet. The target may be present at an average of less than about 2 molecules per droplet.

  The droplets may be formed with any suitable droplet formation frequency, particularly at least about 10, 20, 50, 100, 200, 500 or 1,000 Hz (droplets / second). In general, the droplet formation frequency is inversely proportional to the size of the formed droplet, and the smaller the droplet, the higher the droplet formation frequency.

  The aqueous sample (containing fragmented DNA) used to form the droplets may be passed through and / or converted to droplets at any suitable flow rate. Exemplary flow rates that may be suitable include, among others, at least about 1, 5, 10, 20, 50, 100, 200, 500, 1,000, 5,000, or 10,000 nL / sec. In general, the sample flow rate is directly related to the size of the droplet being formed, with larger droplets allowing faster flow rates.

  The values (or ranges) for exemplary droplet volume, DNA concentration, droplet formation frequency and flow rate may be combined in any suitable combination (s) listed above.

  An assay may be performed on the droplet, which is 28. The assay may be a digital assay that detects individual target molecules in each droplet. Digital assays may include, in particular, amplification of target molecules such as by PCR or ligase chain reaction. Digital assays may also include detection of fluorescence from the droplets. The assay may further include measuring the level (eg, concentration) of the target in each droplet using the Poisson algorithm.

II. Exemplary Data for Drop Formation Tests This section presents representative data for drop formation tests involving fragmented or unfragmented genomic DNA. See Figures 2-6.

  The formation of droplets in a microfluidic device may depend on the flow rate at which the sample moves to the droplet formation section and the frequency with which the droplets are formed. At high flow rates, the sample stream may jet into the immiscible continuous phase and be unable to form droplets. At a formation rate close to the jetting limit, the sample begins to extend down the discharge channel before the droplet is formed. The stretch length can be used to determine how close the sequence of formation conditions is to the ejection limit.

  FIG. 2 shows a matrix diagram made from a photograph of a droplet formation unit 30 processing three different aqueous samples 32-36 at each of four different driving pressures, and hence flow rates. Three samples were (a) control sample 32 without DNA (PCR buffer without template), (b) undigested, high molecular weight (MW) human genomic DNA (Raji, 18.75 ng / L). Aqueous sample 34 and (c) Aqueous sample 36 of human genomic DNA (Raji, 18.75 ng / L) digested with restriction enzymes and reduced in molecular weight. The four drive pressures (1, 2, 3 and 4) are negative pressures (depressurization) applied downstream of the drop formation, expressed in pounds per square inch (psi), and 1 psi is approximately 6.9 kilopascals. It corresponds to. Depending on the level of vacuum, the sample flow rate, overall flow rate, and droplet formation frequency may be controlled.

  The droplet forming unit 30 may be formed by a channel network including a sample injection channel 38, at least one or a set of oil injection channels 40, 42, and a discharge channel 44. The injection channel 38 sends the bulk aqueous phase 46 of the aqueous sample 32, 34 or 36 to the droplet formation section. The injection channels 40 and 42 send the continuous phase 48 (for example, oil and surfactant) to the droplet forming unit. The discharge channel 44 sends the droplet 50 from the channel intersection 52 to the continuous phase 48.

  The top row shows the droplet formation of control sample 32 without genomic DNA. The droplet 50 is approximately 1 nL and does not change much in size at different decompression levels.

  The middle row shows droplet formation with sample 34 containing undigested genomic DNA. Genomic DNA significantly reduces droplet formation, and droplet formation occurs only at the lowest vacuum level tested (1 psi). Even at the lowest level, the bulk aqueous phase 46 that has passed through the flow path intersection 52 is considerably elongated as indicated by the arrow 54, and the droplet 50 is also large. At higher decompression levels (e.g., comparing 1 psi to 2-4 psi) and flow rates, the sample flow rapidly flows into the discharge channel 44 without splitting into droplets as indicated by the arrows at 56. Is not formed. Thus, the presence of human genomic DNA can greatly impede droplet formation and may require the use of low DNA concentrations, slow flow rates and low droplet formation frequencies. As a result, sample processing may be significantly slower. In addition, the appearance rate of droplets containing the target in the emulsion may be substantially reduced (due to the low DNA concentration), and more droplets are needed to achieve the same confidence level for the measured target level. Will need to be analyzed.

  The bottom row shows droplet formation with sample 36 containing the same genomic DNA concentration (mass per unit volume) as sample 34, but after the DNA has been digested with restriction enzymes into short fragments. . At the pressures (and flow rates) shown here, the fragmented genomic DNA does not reduce the formation of droplets as appreciable. This sample produces a droplet 50 similar to the control sample 32 that does not contain DNA.

  Further studies were conducted to quantitatively evaluate the relationship between formation pressure reduction, sample flow rate, drop formation frequency, velocity in the discharge channel, drop size and maximum sample extension during drop formation. . The aqueous samples used were Spectral Dye Buffer (same control sample as in Figure 2, but without DNA polymerase), Raji human genomic DNA (Loftstrand Laboratories) (`` Raji '') and 19205 human DNA (Coriell Institute) ( "Coriell"). DNA samples were either undigested or digested with the restriction enzyme EcoRI. DNA was digested with genomic DNA at a final concentration of 200 ng / μL using NEB # 4 buffer solution of EcoRI (New England Biolabs) at a concentration of 20 U / μL. The mixture was incubated at 37 ° C. for 1 hour and then diluted to various final concentrations.

  The graphs in FIGS. 3-6 show Master Mix (without DNA), 18.75 ng / μL Raji DNA digested with EcoRI, 18.75 ng / μL Coriell 19205 DNA digested with EcoRI, 18.75 ng / μL undigested Shows the results of a drop formation experiment performed with samples of Raji DNA and undigested 18.75 ng / μL Coriell 19205 DNA. FIG. 3 shows a graph of droplet volume graphed as a function of droplet formation frequency for each sample. FIG. 4 shows a graph of drop volume graphed as a function of sample flow rate for each sample. FIG. 5 shows a graph of maximum elongation graphed as a function of droplet formation frequency for each sample. FIG. 6 shows a graph of maximum elongation graphed as a function of sample flow rate for each sample of FIG.

  For undigested human genomic DNA, only very low droplet formation frequencies or very slow sample flow rates were possible before jetting occurred. For example, for Coriell 19205 DNA, the maximum was 60 Hz and 83 nL / sec. For Raji DNA, the maximum was 120 Hz and 162 nL / sec. However, even below these limits, the volume of the formed droplet was larger than that without DNA, and the droplet was formed with the sample extending much longer into the discharge channel.

  These graphs also show that no effect of DNA on droplet formation was observed with the same concentration of digested DNA. No squirt or long sample elongation is observed at any flow rate or frequency of formation tested, and the volume of the droplet is the same as the sample without DNA.

  These results indicate that droplet formation is significantly inhibited in the presence of undigested human DNA, but not after digestion with restriction enzymes.

III. Selected Embodiments This section describes selected embodiments of the present disclosure as a series of headings. These embodiments do not limit the full scope of the disclosure.

  A. (i) obtaining genomic DNA containing a target; (ii) intentionally fragmenting the genomic DNA to produce fragmented DNA; (iii) at least one droplet of the fragmented DNA Passing through a formation to form an aqueous droplet containing the fragmented DNA; and (iv) performing a digital assay on the droplet to measure the level of the target. A method of analyzing DNA.

  B. The method of paragraph A, wherein the droplets have an average volume of less than about 10 nanoliters.

  C. The method of paragraph A, wherein the droplet comprises the genomic DNA at a concentration of at least about 5 nanograms / microliter.

  D. The genomic DNA is placed in an aqueous sample and the droplet is formed at a flow rate of the aqueous sample through the droplet formation of greater than about 50 nanoliters / second. the method of.

  E. The method of any of paragraphs A through D, wherein the droplets are formed with a droplet formation frequency of at least about 50 droplets / second.

  F. The method of paragraph A, wherein the droplets have an average volume of less than about 10 nanoliters and contain the genomic DNA at a concentration of at least about 5 nanograms / microliter.

  G. The droplets have an average volume of less than about 10 nanoliters, the genomic DNA is placed in an aqueous sample, and the droplets have a flow rate of the aqueous sample through the droplet formation of about 50 The method of paragraph A, formed at a flow rate in excess of nanoliters / second.

  H. The droplet comprises the genomic DNA at a concentration of at least about 5 nanograms / microliter, the genomic DNA is placed in an aqueous sample, and the droplet is passed through the droplet formation portion The method of paragraph A, wherein the flow rate of is formed at a flow rate greater than about 50 nanoliters / second.

  I. The genomic DNA is placed in an aqueous sample and the droplet is formed at a flow rate of the aqueous sample through the droplet formation of greater than about 50 nanoliters / second. the method of.

  J. The method of paragraph F, wherein the droplets are formed with a droplet formation frequency of at least about 50 droplets / second.

  K. The method of paragraph G, wherein the droplets are formed with a droplet formation frequency of at least about 50 droplets / second.

  L. The method of paragraph H, wherein the droplets are formed with a droplet formation frequency of at least about 50 droplets / second.

  M. The method of paragraph L, wherein the droplets have an average volume of less than about 10 nanoliters.

  N. The method of any of paragraphs A to M, wherein the fragmentation step comprises digesting the genomic DNA with a restriction enzyme.

  O. The method of paragraph N, wherein the restriction enzyme cleaves the genomic DNA on average less than about once per kilobase.

  P. The method of any of paragraphs A through M, wherein the fragmenting step comprises shearing the genomic DNA.

  Q. The method of any of paragraphs A to M, wherein the fragmenting step comprises sonicating the genomic DNA.

  R. The method of any of paragraphs A through Q, wherein the droplets contain an average of less than about 2 copies of the target per droplet.

  S. The method of any of paragraphs A through R, wherein the droplets contain an average of less than about 2 genomic equivalents of the genomic DNA per droplet.

  T. The method of any of paragraphs A through S, wherein the fragmentation step does not substantially destroy the target.

  U. The method of any of paragraphs A through T, wherein performing the digital assay comprises amplifying the target in the droplet.

  V. The method of paragraph U, wherein the target is amplified by PCR.

  W. The method of any of paragraphs A through V, wherein performing the digital assay comprises detecting fluorescence of the droplet.

  X. The method of any of paragraphs A through W, wherein performing the digital assay comprises measuring the level of the target using a Poisson algorithm.

  Y. The method of any of paragraphs A through X, wherein the droplets have an average volume of about 0.1 to 10 nanoliters.

  Z. (i) obtaining a sample comprising DNA at a concentration of at least about 5 ng / microliter; (ii) intentionally fragmenting the DNA to produce fragmented DNA; and (iii) the sample To form an aqueous droplet containing the fragmented DNA, wherein the droplet is formed with a droplet formation frequency of at least about 50 droplets / second and has an average volume A method of dividing an aqueous sample containing DNA into droplets, comprising a step of less than about 10 nanoliters.

  A1. (I) obtaining a sample containing genomic DNA; (ii) intentionally fragmenting the DNA to produce fragmented DNA; and (iii) passing the sample through a droplet formation section. Forming aqueous droplets comprising the fragmented DNA, wherein the droplets are formed with a droplet formation frequency of at least about 50 droplets / second and an average volume of less than about 10 nanoliters A method of dividing an aqueous sample containing DNA into droplets, wherein the passing of the genomic DNA is performed under the same conditions without fragmenting the DNA with the genomic DNA A method that is at a concentration that prevents droplet formation in some cases.

  The above disclosure may include a number of different inventions that have independent utility. While each of these inventions has been disclosed in a preferred form (s), the specific embodiments disclosed and illustrated herein are not limited to the numerous variations that are possible. Should not be considered in meaning. The subject matter of the present invention includes all novel and non-obvious combinations and subcombinations of the various elements, features, functions and / or properties disclosed herein. The appended claims particularly point to specific combinations and subcombinations that are novel and not obvious. Inventions embodied in other combinations and subcombinations of features, functions, elements and / or properties may be claimed in an application claiming priority from this or a related application. Such claims may be directed to different inventions or the same invention, but may be broad, narrow, equivalent or different to the original claims, which are also within the scope of the inventive subject matter of this disclosure. Is considered to be contained within. In addition, indications such as first, second, or third order for a particular element are used to distinguish each element, except where specifically stated otherwise. It does not indicate a particular position or order of the elements.

30 Droplet formation part
32 samples
34 samples
36 samples
38 Sample injection flow path
40 Oil injection flow path
42 Oil injection flow path
44 Discharge flow path
46 Bulk aqueous phase
48 Continuous phase
50 droplets
52 Channel crossing

Claims (27)

Obtaining genomic DNA containing the target;
Voluntarily fragmenting the genomic DNA to generate fragmented DNA;
Passing the fragmented DNA through at least one droplet forming unit to form an aqueous droplet containing the fragmented DNA; and performing a digital assay on the droplet to determine the level of the target A method for analyzing genomic DNA comprising the step of measuring.   The method of claim 1, wherein the droplets have an average volume of less than about 10 nanoliters.   2. The method of claim 1, wherein the droplet comprises the genomic DNA at a concentration of at least about 5 nanograms / microliter.   2. The genomic DNA is placed in an aqueous sample, and the droplets are formed at a flow rate that exceeds about 50 nanoliters / second through the droplet formation portion. Method.   5. The method of any one of claims 1-4, wherein the droplets are formed with a droplet formation frequency of at least about 50 droplets / second.   2. The method of claim 1, wherein the droplets have an average volume of less than about 10 nanoliters and contain the genomic DNA at a concentration of at least about 5 nanograms / microliter.   The droplets have an average volume of less than about 10 nanoliters, the genomic DNA is placed in an aqueous sample, and the droplets have a flow rate of the aqueous sample through the droplet formation of about 50 nanoliters. The method of claim 1, wherein the method is formed at a flow rate in excess of / second.   The droplets contain the genomic DNA at a concentration of at least about 5 nanograms / microliter, the genomic DNA is placed in an aqueous sample, and the droplets flow through the droplet forming portion. 2. The method of claim 1, wherein is formed at a flow rate greater than about 50 nanoliters / second.   7. The genomic DNA is placed in an aqueous sample, and the droplets are formed at a flow rate that the aqueous sample flow rate through the droplet formation exceeds about 50 nanoliters / second. Method.   7. The method of claim 6, wherein the droplets are formed with a droplet formation frequency of at least about 50 droplets / second.   8. The method of claim 7, wherein the droplets are formed with a droplet formation frequency of at least about 50 droplets / second.   9. The method of claim 8, wherein the droplets are formed with a droplet formation frequency of at least about 50 droplets / second.   The method of claim 12, wherein the droplets have an average volume of less than about 10 nanoliters.   2. The method according to claim 1, wherein the fragmentation step comprises digesting the genomic DNA with a restriction enzyme.   15. The method of claim 14, wherein the restriction enzyme cleaves the genomic DNA on average less than about once per kilobase.   The method of claim 1, wherein the fragmenting step comprises shearing the genomic DNA.   2. The method of claim 1, wherein the fragmenting step comprises sonicating the genomic DNA.   The method of claim 1, wherein the droplet comprises an average of less than about 2 copies of the target per droplet.   The method of claim 1, wherein the droplet comprises an average of less than about 2 genome equivalents of the genomic DNA per droplet.   The method of claim 1, wherein the fragmentation step does not substantially destroy the target.   The method of claim 1, wherein performing the digital assay comprises amplifying the target in the droplet.   24. The method of claim 21, wherein the target is amplified by PCR.   24. The method of claim 21, wherein performing the digital assay comprises detecting fluorescence from the droplet.   24. The method of any one of claims 21 to 23, wherein performing the digital assay comprises measuring the level of the target using a Poisson algorithm.   The method of claim 1, wherein the droplets have an average volume of about 0.1 to 10 nanoliters. Obtaining a sample containing DNA at a concentration of at least about 5 ng / microliter;
A step of intentionally fragmenting the DNA to generate fragmented DNA; and a step of passing the sample through a droplet formation unit to form an aqueous droplet containing the fragmented DNA. A method of splitting an aqueous sample containing DNA into droplets, wherein the droplets are formed with a droplet formation frequency of at least about 50 droplets / second and the average volume is less than about 10 nanoliters. Obtaining a sample containing genomic DNA;
A step of intentionally fragmenting the DNA to generate fragmented DNA; and a step of passing the sample through a droplet formation unit to form an aqueous droplet containing the fragmented DNA. A method of dividing an aqueous sample comprising DNA into droplets, comprising droplets formed at a droplet formation frequency of at least about 50 droplets / second and having an average volume of less than about 10 nanoliters, comprising:
The method wherein the genomic DNA is at a concentration that prevents droplet formation when the passing step is performed under the same conditions without fragmenting the DNA.

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