Patent Description:
Without random access at a molecular level, accessing any data from a DNA data store could require sequencing the entire DNA pool and then performing random access using conventional digital computer techniques. For small DNA data stores this may be possible. However, as the scale of these systems increases, sequencing the entire DNA pool for every data request quickly becomes unworkable.

One technique for performing random access at the molecular level makes use of polymerase chain reaction (PCR) and specific primer pairs to selectively amplify portions of a DNA pool. With this technique, the DNA strands in the DNA pool have payload regions that encode digital data and the payload regions are flanked by primer binding sites. The DNA pool as a whole may be designed with a correspondence between the primer binding sites and encoded data. For example, all nucleotide sequences encoding data from the same computer file may be flanked by the same primer binding sites. Thus, the DNA encoding a specific computer file may be selectively amplified using a specific primer pair. The amplification product is sequenced and decoded thereby achieving random access.

However, random-access using PCR and specific primer pairs has inefficiencies and shortcomings that become more significant as the scale of DNA data storage systems increase. PCR and the subsequent sequencing of the amplification products are fundamentally biological processes that include variations and inconsistent behavior. Thus, random access based on selective PCR amplification, particularly when many random-access requests are combined, may waste reagents and time as well as generate unreliable sequence data leading to potential data loss. This disclosure is made with respect to these and other considerations. <NPL>- No Abstract Available
<CIT> describes that the present disclosure relates to normalization of biological samples, particularly samples comprising nucleic acids to be sequenced. The normalization protocols described herein may be utilized across multiple samples to cap total stoichiometric input and minimize variations in transcript abundance on a per-sample basis in a multiplexed fashion to dramatically increase the accuracy, capacity and efficiency of nucleic acid sequencing. <CIT> describes that this disclosure describes an efficient method to copy all polynucleotides encoding digital data of digital files in a polynucleotide storage container while maintaining random access capabilities over a collection of files or data items in the container. The disclosure further describes a process whereby random-access and sequencing of the polynucleotides are combined in a single step. <NPL> - No Abstract Available
<CIT> describes that a bottleneck in the Next Generation Sequencing (NGS) workflow is the quantification of libraries for accurate pooling and loading of the sequencing instrument flow cell or chip. Disclosed herein are methods that improve performance and reduce time compared to existing methods. <CIT> describes that a microfluidic device includes a plurality of reaction wells; and a plurality of solid supports, and each of the solid supports has a reagent attached thereto. The reagent is attached to the solid support via a labile reagent/support bond such that the reagent is configured to be cleaved from the support via a cleaving operation. <CIT> describes that compositions and methods are described for standardizing the DNA amplification efficiencies of a highly heterogeneous set of oligonucleotide primers as may typically be used to amplify a heterogeneous set of DNA templates that contains rearranged lymphoid cell DNA encoding T cell receptors (TCR) or immunoglobulins (IG). The presently disclosed embodiments are useful to overcome undesirable bias in the utilization of a subset of amplification primers, which leads to imprecision in multiplexed high throughput sequencing of amplification products to quantify unique TCR or Ig encoding genomes in a sample. Provided is a template composition comprising a diverse plurality of template oligonucleotides in substantially equimolar amounts, for use as a calibration standard for amplification primer sets. Also provided are methods for identifying and correcting biased primer efficiency during amplification.

This disclosure provides methods and apparatus for efficient random-access to DNA-encoded data. The apparatus is not part of the claimed invention. The efficiencies and improvements provided by this disclosure relate to the steps of extracting DNA from storage and sequencing the DNA strands. A DNA data store may receive multiple random-access requests for specific data from one or more DNA pools. A random-access request such as for a specific computer file, for example, may be translated into a request to query a specific DNA pool using a specific primer pair. The translation may be performed by a digital computer that maintains a record of correspondence between digital data and molecular storage locations.

Processing these random-access requests in parallel (e.g., together in batch processes) is more efficient than processing each request separately. Selective PCR amplification of DNA sequences using specific primer pairs is performed by grouping multiple singleplex PCR reactions together. Multiple isolated reaction volumes each containing DNA strands from a DNA pool and multiple single-stranded oligonucleotides with the sequences of each member of a primer pair are sent through the same rounds of thermocycling. As used herein, "primer pair" refers to multiple molecules (e.g., many millions) of each of the two primers in a primer pair. Each of the isolated reaction volumes may contain DNA strands from a different DNA pool and/or a different primer pair. Thus, the amplification product of each may be different. This allows for the contents of each reaction volume to be independent of the other reaction volumes as in singleplex PCR, yet all of the reaction volumes are thermocycled together as in multiplex PCR.

The isolated reaction volumes may be microdroplets formed as water-in-oil emulsions. Microdroplets may also be formed by other techniques such as by encasing aqueous solutions in calcium alginate shells. Each microdroplet contains DNA from a DNA pool, a single primer pair, and PCR master mix. Multiple microdroplets may be placed in a thermocycler under conditions that allow PCR amplification to occur in the aqueous core of each microdroplet.

Wells on a plate may also be used to create isolated reaction volumes. All of the wells on a plate may be filled with the same solution of DNA from a DNA pool and PCR master mix. The variation across the individual reaction volumes is achieved by supplying different primer pairs to the wells. The primers can be supplied to each well on beads coated with single-stranded DNA that include the sequences of the primers. Each bead may be coated with the DNA sequences of both primers of a given primer pair. To prevent multiple different PCR reactions from occurring in the same well, the beads and wells may be sized so that only a single bead fits into a well. Thus, each well will include only a single primer pair. The surface of the plate may be coated with a thin layer of oil to prevent transfer between the wells. The entire plate may be sequentially heated and cooled so that the thermocycling necessary for PCR occurs in every well. In some implementations, the thermocycling may be spatially addressable so that each well can be subjected to a separate series of temperature changes.

Performing multiple PCR reactions in parallel makes the step of extracting DNA from a DNA pool more efficient but does not necessarily improve the efficiency of the subsequent sequencing. DNA sequencing can, like PCR, be performed on batches of molecules together. This is called multiplex sequencing. Multiplex sequencing is more efficient than sequencing each sample separately. However, the efficiency of multiplex sequencing can be increased further by controlling the quantity of DNA in the samples provided to a multiplex DNA sequencer.

Variations in the quantity of DNA in the samples analyzed in a single multiplex sequencing run may cause a multiplex DNA sequencer to perform unnecessary work when sequencing samples with higher quantities of DNA and fail to accurately sequence samples with lower quantities of DNA. This can be addressed through copy normalization-maintaining an approximately equal quantity of DNA across all of the samples sequenced together in the same sequencing run. Copy normalization may be necessary because of unequal PCR amplification. Due to differences in nucleotide sequences, different primer pairs can produce different amounts of amplification product even under identical PCR conditions. The quantity of DNA in each isolated reaction volume such as a microdroplet or well may vary due to differences in primer efficiency.

The quantity of DNA in the isolated reaction volumes can be measured to determine if, and to what extent, copy normalization is necessary. The quantity of DNA in a sample may be measured, for example, by adding a dye that fluoresces in proportion to the amount of DNA present. The amount of DNA in isolated reaction volumes with low levels of DNA (e.g., below a threshold level) may be increased by performing additional cycles of PCR. Microdroplets with low quantities of DNA may be routed back to a thermocycler for additional cycles of heating and cooling. Individual wells with low quantities of DNA may be subject to additional rounds of heating and cooling while other wells in the same plate are not.

Copy normalization of DNA quantity may also be performed by selectively grouping samples with approximately the same quantities of DNA into the same multiplex sequencing run. Microdroplets may be sorted into batches based on DNA quantity and all of the samples used in a single multiplex sequencing run may then be drawn from a batch of microdroplets with similar DNA quantities.

This Summary is not intended to identify key features or essential features of the claimed subject matter nor is it intended to be used to limit the scope of the claimed subject matter. The term "techniques," for instance, may refer to system(s) and/or method(s) as permitted by the context described above and throughout the document.

The Detailed Description is set forth with reference to the accompanying figures. Structures shown in the figures are representative and not necessarily to scale.

This disclosure provides techniques and systems for efficiently fulfilling random access requests sent to DNA data stores by generating samples of DNA with normalized DNA quantities for multiplex sequencing. Synthetic polynucleotides such as DNA may be used to store digital information by designing a sequence of nucleotide bases-adenine (A), cytosine (C), guanine (G), and thymine (T)-that encodes the zeros and ones of digital information. Advantages of using DNA rather than another storage media for storing binary data include information density and longevity. The sequence of nucleotide bases is designed on a computer and then DNA molecules with that sequence are generated by an oligonucleotide synthesizer. The DNA may be stored, selectively retrieved from storage, read by a DNA sequencer, and then decoded to retrieve the binary data.

Proof of concept systems and techniques for storing data in DNA have been previously demonstrated. See <NPL>) and <NPL>). As DNA data storage systems increase in size and complexity the ability to efficiently respond to random-access requests will become increasingly important. Techniques for performing random-access using selective PCR amplification are described in Organick, supra and <CIT>.

Random access of digital data stored in DNA strands can be achieved using PCR to selectively amplify DNA that encodes the requested digital data. PCR amplification of DNA increases by several orders of magnitude the number of copies of the target DNA sequences. Selective amplification increases the number of copies of the DNA strands encoding the desired digital data much more than other DNA strands in the same pool. For example, DNA strands encoding digital data for two or more different data files can be stored together in the same container: a DNA pool. Request for the digital data corresponding to just one of those files, a random-access request, begins with obtaining the sequence of DNA strands encoding the selected digital data without sequencing all the DNA strands in the DNA pool.

Selective amplification through PCR increases the number of DNA strands encoding the desired digital data by many orders of magnitude relative to other DNA strands in the same DNA pool. The amplification product can be sequenced by a DNA sequencer and the reads produced from sequencing are then decoded to reproduce the original bits of the requested digital data. Although the other DNA strands from the DNA pool are still present in the amplification products, the probability of sequencing these DNA strands is low because there are so many fewer copies. Thus, selective amplification provides specification through dilution.

The correlation between primer pairs and digital data may be implemented by assigning a unique group identifier to each DNA strand that contains data for a particular data file. The individual group identifier may be encoded as a specific sequence of nucleotides in the DNA strands. In some implementations, this group identifier may be a primer binding site. With this design, DNA that amplifies using a primer that hybridizes to the primer binding site will be DNA that encodes digital data from that particular data file. In this way, the DNA strands that encode the digital data being requested can be selectively amplified and subsequently sequenced and decoded to provide the requested digital data.

PCR amplification can be used to selectively "pullout" specific sequences of DNA from a DNA pool. Different primer pairs are used to respond to requests for different sets of data. As the scale of a DNA storage system grows, there will likely be a very large number of different primer pairs used. Different primer pairs inherently have different sequences which can result in uneven amplification. However, PCR performed with different primer pairs may generate different quantities of DNA even if all other variables are constant. This is likely due to variation in primer binding strength, non-specific binding, primer-dimer formation, and other factors. Thus, without copy-normalization, otherwise similar random-access requests can generate different amounts of amplified DNA.

One subcategory of microfluidics is droplet-based microfluidics which creates discrete volumes with the use of immiscible phases. The ultrahigh-throughput generation of uniform droplets with nL to pL volume greatly enhances the capability of microfluidics to perform a large number of reactions without increasing device size or complexity. Microfluidic droplet technology has the advantages of compartmentalizing reactions into discrete volumes, performing highly parallel reactions in monodisperse droplets, reducing cross-contamination between microdroplets, eliminating PCR bias and nonspecific amplification, as well as enabling fast amplification with rapid thermocycling.

Copy-normalization sequencing is the process of equalizing the quantity of DNA in samples for a multiplex sequencing run in which multiple DNA samples that contain or should contain the same sequence of nucleotides are sequenced. In next-generation sequencing (NGS) multiplexing is performed by loading multiple-often thousands-of separate samples on a single flow cell. This increases the efficiency of NGS and reduces costs. But uneven quantities of DNA from different random-access requests when combined in the same flow cell can lead to inconsistencies in quality of the sequence data output by the DNA sequencer. Variations in DNA quantity for samples placed in different flow cells do not cause these deficiencies.

Samples with high quantities of DNA are likely to be overrepresented on a flow cell while those with low quantities are likely underrepresented. Overrepresentation may not affect accuracy because it increases read depth. However, this wastes capacity and leads to inefficient use of multiplex DNA sequencing machines and consumes additional reagents which increases costs. Underrepresentation might result in poor read depth and unreliable sequence data, wasting capacity and potentially making it impossible to accurately decode the sequence into the original binary data. Therefore, normalizing DNA quantities or DNA copy number prior to sequencing improves the accuracy and efficiency of multiplex sequencing which improves the accuracy and efficiency of random-access requests for DNA-encoded data. Identical quantities of DNA across samples not required, but the extent of variation in DNA quantities between samples should be minimized.

In this disclosure, oligonucleotides, which are also referred to as polynucleotides, include both DNA, RNA, and hybrids containing mixtures of DNA and RNA. DNA includes nucleotides with one of the four natural bases cytosine (C), guanine (G), adenine (A), or thymine (T) as well as unnatural bases, noncanonical bases, and/or modified bases. RNA includes nucleotides with one of the four natural bases cytosine, guanine, adenine, or uracil (U) as well as unnatural bases, noncanonical bases, and/or modified bases. Nucleotides include both deoxyribonucleotides and ribonucleotides covalently linked to one or more phosphate groups. Although DNA may be referred to specifically as an illustrative oligonucleotide this is not limiting and it is to be understood that other oligonucleotides may be used instead of DNA.

Detail of procedures and techniques not explicitly described in this or other processes disclosed of this application are understood to be performed using conventional molecular biology techniques and knowledge readily available to one of ordinary skill in the art. Specific procedures and techniques may be found in reference manuals such as, for example, <NPL>).

<FIG> shows a first illustrative random-access system <NUM> for generating microdroplets <NUM> with samples of DNA <NUM> and primer pairs <NUM> that have normalized DNA quantities. The random-access system <NUM> creates a large number of microdroplets <NUM> that can each contain a unique combination of DNA <NUM> and a primer pair <NUM> together with the other reagents necessary for PCR in a PCR master mix <NUM>. Each of the microdroplets <NUM> provides an isolated reaction volume that prevents interaction between the components in separate microdroplets <NUM>. This dramatically reduces hardware complexity of the system <NUM> and allows for a much higher density of separate reactions as well as for easier automated manipulation than other techniques for singleplex PCR such as multiple flip-top tubes in a conventional thermocycler.

The microdroplets <NUM> may be created by forming an emulsion of oil and water. The water-in-oil microdroplets <NUM> may be created with a T-junction <NUM>. T-junction <NUM> geometries contain a continuous phase main channel <NUM> and a disperse phase inlet channel <NUM>, perpendicular to each other, which looks like the two branches of the "T. " A droplet formation cycle starts with the stream of the disperse phase (aqueous DNA-containing PCR solution) penetrating into the main channel (an immiscible oil such as mineral oil), and a microdroplet <NUM> begins to grow. The pressure gradient, the shear force, and the interfacial tension at the fluid-fluid interface distort and elongate the microdroplet <NUM> in the downstream direction, until the neck of the disperse phase becomes thin and eventually breaks. This releases the microdroplet <NUM> downstream into the main channel <NUM>. Then the tip of the disperse phase retracts to the end of inlet and the process repeats. Empirically, the size of microdroplet <NUM> and its generation process are highly dependent on the capillary number, the flow rates, the viscosity ratio, and the channel geometry. See <NPL>).

The microdroplets <NUM> may alternatively be created by encapsulation of an aqueous solution in a membrane. The membrane may be formed from a material such as calcium alginate. Calcium alginate (calcium β-D-mannopyranuronosyl-(<NUM>→<NUM>)- α-L-gulopyranuronosyl -(<NUM>-<NUM>)- α-L-gulopyranuronate) is a water-insoluble, gelatinous, cream-colored substance that can be created through the addition of aqueous calcium chloride to aqueous sodium alginate. An aqueous solution containing the DNA <NUM>, primer pair <NUM>, and PCR master mix <NUM> may be provided through the inlet channel <NUM> while the calcium alginate is provided through the main channel of the T-junction <NUM>. Calcium alginate forms shells around water or aqueous solutions. The calcium alginate shells themselves may be suspended in an alcohol solution which prevents evaporation of water and reduces the adhesion of the shells to each other. Techniques for encapsulating DNA in calcium alginate shells are described in <NPL>).

In an implementation, the microdroplets <NUM> may also be nested microdroplets that include two or more layers of encapsulation surrounding an aqueous core that holds the DNA <NUM>. For example, calcium alginate spheres may be placed into a water-in-oil emulsion creating two layers of isolation between reaction volumes. Each reaction volume containing DNA <NUM>, a primer pair <NUM>, and PCR master mix <NUM> is encapsulated within a calcium alginate shell which itself is within a water droplet surrounded by oil.

Varying the inputs into the inlet channel <NUM> controls the contents of the microdroplets <NUM>. Each microdroplet <NUM> may represent a response to a different random-access request. The DNA <NUM> is obtained from one of one or more DNA pools. Each DNA pool is a separate container holding many thousands, millions, or more individual DNA strands that encode digital data. DNA from one of the DNA pools is converted to an aqueous solution, if not in that form already, and a small portion is removed and used as the DNA <NUM> introduced into the inlet channel <NUM>.

The primer pair <NUM> may be obtained from a collection of pre-synthesized primer pairs. The sequences of the primer pairs that could potentially be used to amplify DNA fragments from one of the DNA pools may be known based on the design of the DNA strands use for encoding digital data. For example, for a first DNA Pool may contain DNA strands that have primer binding sites corresponding to one of <NUM> different primer pairs. The primer pair <NUM> may also be synthesized on-demand using an oligonucleotide synthesizer or other techniques for synthesis of short single-stranded polynucleotides. The same primer pair <NUM> may be used with different DNA pools to fulfill different random-access requests. For example, primer pair (<NUM>) may be used to amplify DNA corresponding to a first file from DNA pool (<NUM>) while the same primer pair (<NUM>) would be used to amplify DNA corresponding to a second file if combined with the DNA of DNA pool (<NUM>).

The PCR master mix <NUM> includes a DNA polymerase, deoxyribonucleotide triphosphates (dNTPs), in a reaction buffer. Techniques for selection and creation of suitable PCR master mixes are known to those of ordinary skill in the art. Many suitable master mixes are also commercially available such as, for example, the Gibson Assembly® master mix available from New England BioLabs, Inc.

The DNA <NUM> from a DNA pool, the primer pair <NUM>, and the PCR master mix <NUM> may be mixed by any automated or manual technique such as pipetting, microfluidics, laboratory robotics, etc. One automated system that may be used for mixing these, or other reagent discussed elsewhere in this disclosure, is a digital microfluidics device such as the "PurpleDrop" device described in <NPL>) and <NPL>).

The T-junction <NUM> is connected to a reaction chamber <NUM>. The reaction chamber <NUM> the temperature-controlled chamber such as a chamber of a thermocycler. The temperature in the reaction chamber <NUM> can be precisely controlled to thermocycle the microdroplet <NUM> under conditions that will result in PCR amplification. Specific temperatures and timings for PCR reactions are known to those of ordinary skill in the art and may be performed using any conventional protocol. For example, one protocol is (<NUM>) <NUM> for <NUM>, (<NUM>) <NUM> for <NUM>, (<NUM>) <NUM> for <NUM>, (<NUM>) <NUM> for <NUM>, (<NUM>) go to step <NUM> a varying number of times, and (<NUM>) <NUM> for <NUM>.

Thus, during thermocycling a different PCR reaction may occur in each microdroplet <NUM>. That is, each microdroplet <NUM> may contain a unique combination of DNA <NUM> and primer pair <NUM>. However, it is also possible that multiple microdroplets <NUM> may contain the same combination of DNA <NUM> and primer pair <NUM>. Because the DNA <NUM> may be taken from any one of a number of different DNA pools, the use of microdroplets <NUM> to create isolated reaction volumes allows for amplification of DNA from a mix of different DNA pools in the same reaction chamber <NUM>. Mixing multiple DNA pools while maintaining specificity of amplification products may not be possible in conventional multiplex PCR because the primer pairs <NUM> would have access to DNA strands from all of the multiple DNA pools. The reaction chamber <NUM> may hold many thousands or tens of thousands of microdroplets <NUM>.

The system <NUM> may also include a sensor <NUM> that detects DNA concentrations in individual microdroplets <NUM>. The sensor <NUM> may be positioned on a portion of the system <NUM> such as narrow tube in which single microdroplets <NUM> pass before the sensor <NUM>. The sensor <NUM> may be implemented as an ultraviolet (UV) light and corresponding UV photosensor to measure DNA quantity by the amount of UV absorption.

The sensor <NUM> may be implemented as a laser and fluorescence detector that excite and detect fluorescence emitted from a DNA-binding dye such as an intercalating dye. Examples of fluorescent dyes that may be used for detecting DNA include EvaGreen® available from Biotium, PicoGreen, and SYBR Green. The DNA binding dye may be included in the PCR master mix <NUM> or separately added to the inlet channel <NUM>. The sensor <NUM> may be adapted from devices described in <NPL>).

In an implementation, sensing could be performed in the reaction chamber <NUM> by configuring the reaction chamber <NUM> to include a plate with multiple wells each sized to hold a single microdroplet <NUM>. For example, the plate may be created using techniques for fabricating semiconductors in order to create wells with the dimensions that hold only a single microdroplet <NUM>. The plate may contain individually addressable heating elements under each well. The heating elements (coupled with a system for cooling all or part of the plate) may provide the temperature cycling used for PCR. Thus, PCR amplification is performed for the DNA in each microdroplet in its respective well. The microdroplets <NUM> may contain a fluorescent dye that is used as described above to monitor the quantity of DNA in each well. Normalization of DNA quantities are achieved by selectively providing additional cycles of PCR to those wells with DNA quantities that are below a threshold level. This can normalize the quantity of DNA in each well <NUM>. Thus, one or more sensors <NUM> may be configured to detect fluorescence levels in the wells as PCR is being performed. Examples of suitable plates and aspects of this technique for DNA quantity normalization are discussed below in the section describing <FIG>.

Measurement of DNA quantities in individual microdroplet <NUM> makes it possible to process the microdroplet <NUM> differentially based on DNA quantity. Individual microdroplets <NUM> may be routed through different pathways within the system <NUM> based on DNA quantity using microfluidics and/or cell-sorting techniques such as electrostatic sorting.

A return pathway <NUM> may return microdroplet <NUM> with low levels of DNA (e.g., levels of DNA below a specified threshold) to the reaction chamber <NUM> or to a separate reaction chamber (not shown) for additional cycles of PCR amplification. Providing the microdroplet <NUM> with additional heating and cooling cycles will further increase the quantity of DNA produced by PCR amplification.

A branch point <NUM> may be used to sort the microdroplets <NUM> into two or more different batches based on DNA quantity. For example, the microdroplets <NUM> may be sorted into batches of high, medium, and low DNA quantities and routed to different pathways <NUM>, <NUM>, and <NUM> respectively. However, the microdroplets <NUM> may be divided into more than three different batches. The cutoff thresholds of DNA quantities for placing a given microdroplet <NUM> in a DNA quantity-sorted batch may be derived from real-time DNA quantities measured by the sensor <NUM> or from previously collected data. The remainder of pathways <NUM> and <NUM> (not shown) may be the same as pathway <NUM>.

In some implementations, both the return pathway <NUM> and the branch point <NUM> may be used together. For example, the return pathway <NUM> may be an additional pathway off of the branch point <NUM> and microdroplets <NUM> with DNA quantities that are below a threshold level may be routed to the return pathway <NUM> for further PCR amplification. For example, the branch point <NUM> may separate the microdroplets <NUM> into groups of high, medium, low, and very low DNA quantities with the microdroplets <NUM> having very low DNA quantities being routed to the return pathway <NUM>.

After additional rounds of PCR or batching based on DNA quantity, the microdroplets <NUM> moving beyond the branch point <NUM> (e.g., through pathway <NUM>) will have normalized quantities of DNA. The DNA quantities will not necessarily be identical in every microdroplet <NUM> at this point but the variation in DNA quantities will be much less than in the microdroplets <NUM> inside the reaction chamber <NUM>.

Prior to DNA sequencing the oil is separated from the amplified DNA products. The emulsion may be broken by adding an alcohol such as <NUM>-butanol through inlet <NUM> prior to mixing with a mixer <NUM>. The mixer <NUM> may be any type of mixer suitable for mixing liquids without shearing DNA strands such as a magnetic stirrer or a vortex mixer. Mixing the emulsion and alcohol causes an organic phase <NUM> that contains the oil. The organic phase <NUM> may be discarded or processed and reused.

For microdroplets <NUM> formed with calcium alginate, the amplified DNA is released from the microdroplets <NUM> by mechanically disrupting the calcium alginate shells. Disruption may be performed by using microneedles, magnetic beads, or sonification. Alternatively, heating the microdroplets <NUM> to about <NUM> may also disrupt the calcium alginate shells. After the microdroplets <NUM> are broken, the remains of the calcium alginate shells are still present in an aqueous solution that contains the amplified DNA products.

The resulting aqueous phase contains the amplified DNA from the microdroplets <NUM>. The DNA corresponding to different random-access requests is no longer physically separated by the microdroplets <NUM>. Prior to sequencing the DNA is cleaned. The DNA may be adsorbed on a membrane <NUM> such as a silica or controlled pore glass (CPG) membrane. One or more DNA wash solutions can be added through inlet <NUM> to wash out contaminants and impurities that may negatively affect sequencing. Wash solutions for DNA purification are well known to those of ordinary skill in the art and may include solutions of chaotropic salts and/or ethanol. The wash solution(s) may remove remnants of calcium alginate shells. The wash solution(s) may flow through outlet <NUM> to a waste collection system. After washing an elution reagent is flowed from inlet <NUM> through the membrane <NUM> to release the DNA. The elution reagent may be an elution buffer or unbuffered water such as molecular water or distilled water. Elution buffers for DNA purification are well known to those of ordinary skill in the art and may include, for example, <NUM> Tris at pH <NUM>-<NUM>, TE buffer containing <NUM> Tris and <NUM> EDTA.

After the DNA is cleaned, the system <NUM> may include a component for selecting DNA by size (not shown). PCR may create side products which will typically have different lengths than the desired amplification products. A size-selection step may be used to separate the desired amplification products from the side products. Size selection may be performed by gel or capillary electrophoresis. Techniques for performing gel or capillary electrophoresis of DNA are well known to those of ordinary skill in the art.

The DNA released from the membrane <NUM> flows out of the system <NUM> through outlet <NUM> where the DNA may be stored or sent to a DNA sequencer such as a multiplex DNA sequencer. The DNA may be stored for a relatively short time in an aqueous solution such as the elution buffer. The DNA may be stored for a relatively longer period of time as a lyophilized pellet, encased in a protective coating, dried onto filter paper, or by another technique that preserves the structure of the DNA.

The outflow from outlet <NUM> contains amplification products in response to multiple different random-access requests but the amplification products are copy-normalized DNA in which every DNA strand amplified by PCR is present in about the same number of copies. Thus, approximately equal quantities of DNA are provided to the DNA sequencer in response to each random-access request. This improves efficiency and accuracy of multiplex sequencing.

<FIG> shows a second illustrative random-access system <NUM> for that uses a plate <NUM> with a plurality of wells <NUM> to generate PCR amplification products with normalized quantities of DNA. In this illustration, the plate <NUM> contains <NUM> wells; however, in practice the plate <NUM> may include any number of wells and will typically include many more such as, for example, <NUM> wells. The plate <NUM> is formed out of insulating material that inhibits heat transfer between the wells <NUM>. In one implementation, plate <NUM> may be formed from silicon dioxide. Creation of the plate <NUM> and the wells <NUM> may be performed using techniques adapted from semiconductor fabrication. Creating the plate <NUM> semiconductor chip allows for creation of nanometer-scale structures such as wells with diameters in the range of single micrometers.

Immobilized primer pairs are provided on beads <NUM>. The beads <NUM> may be, for example, amino-silanized CPG beads. The primer pairs are anchored to the beads either directly or via linkers. With both primers immobilized, PCR proceeds with bridge amplification similar to the technique used in sequencing-by-synthesis. Example techniques for performing PCR with primers immobilized on beads are provided in <NPL>).

In an implementation, the beads <NUM> and the wells <NUM> may be sized such that one and only one bead <NUM> can fit into each well. Thus, by flowing the beads <NUM> over the surface of the plate <NUM> each well <NUM> will be filled with a single primer pair <NUM>. Some wells <NUM> may remain empty depending on the quantity of beads <NUM> and the technique used to provide the beads to the well <NUM>. It may also be unknown which bead <NUM> occupies which well <NUM>.

The DNA <NUM> from a single DNA pool and the PCR master mix <NUM> may be added to all the wells <NUM> in the plate <NUM>. Because in some implementations a single aqueous solution is flowed into all of the wells <NUM>, the DNA <NUM> may be limited to DNA from only a single DNA pool. At this point, many or all of the wells <NUM> contains a single primer pair <NUM>, DNA <NUM> and the PCR master mix <NUM>.

The surface of the plate <NUM> may be coated with an oil <NUM> such as mineral oil. The oil <NUM> may contain surfactants (e.g., Tween <NUM> or Abil® Em <NUM>). The oil <NUM> forms a coating over the openings of the wells <NUM> creating isolated reaction volumes in each well <NUM>.

The thermocycling necessary for PCR may be performed by heating the entire plate <NUM> in a thermocycler. In some implementations, the heating of each individual well <NUM> may be controlled separately. For example, the plate <NUM> may be fabricated such that there is a separately-addressable heating element <NUM> (e.g. a resistor) underneath some or all of the wells <NUM>. The heating element <NUM> is able to raise the temperature of the well <NUM> underneath which it is situated without significantly affecting the temperature of any adjacent wells <NUM>. The entire plate <NUM> may be cooled by exposure to air (e.g., by use of a heat sink), cooled fluids, or by use of a heat pump. In some implementations, the plate <NUM> may be fabricated with a Peltier device underneath each well <NUM>. These Peltier devices function as the separately-addressable heating elements <NUM> and also cool the wells <NUM> in order to provide the temperature changes needed for PCR.

PCR is performed in the wells <NUM> and the quantity of DNA generated in each well <NUM> may be measured using any suitable technique such as by detecting fluorescence of an intercalating dye. The quantity of DNA in each well <NUM> may vary due to differences in the amplification efficiency of the primer pairs. The quantity of DNA may be detected in real time as PCR proceeds.

If the wells <NUM> are equipped with separately-addressable heating elements <NUM>, additional PCR cycles may be added selectively to those wells <NUM> with low quantities of DNA. For example, any wells <NUM> for which the quantity of DNA is determined to be less than a threshold value may receive additional cycles of PCR. PCR may be continued in those wells with lower quantities of DNA until all the wells <NUM> in the plate <NUM> have approximate the same quantity of DNA.

For example, after a standard cycle of PCR amplification the quantity of DNA in the well <NUM> with the highest quantity of DNA may be set as the threshold. No further PCR is performed for the wells <NUM> with this quantity of DNA. However, for all the wells <NUM> with lower quantities of DNA (e.g., as detected by lower fluorescence levels) PCR is continued either for a set number of cycles or until real-time detection indicates that the quantity of DNA is the same or approximately the same as the threshold. The ability to separate the control number PCR amplification cycles for individual wells <NUM> makes it possible to provide copy-normalize DNA quantities in all of the wells <NUM> in a given plate <NUM>.

The contents of the wells <NUM> can be combined after normalization and analyzed using multiplex sequencing. The beads <NUM> may be discarded or cleaned and reused. After PCR the amplification products in the wells <NUM> may be cleaned and/or sorted by size using any of the techniques discussed above in association with system <NUM> shown in <FIG>.

<FIG> shows process <NUM> for generating samples of DNA with copy-normalized DNA quantities for multiplex sequencing. This process <NUM> may be implemented, for example, using either of the systems shown in <FIG> and <FIG>.

At operation <NUM>, one or more random-access requests are received. The random-access requests may be received by one or more computer systems that manages a DNA data storage system. The random-access requests may be requests for specific sets of digital data such as specific computer files.

At operation <NUM>, a DNA pool and primer pair are identified for each request. If there are multiple requests one or more DNA pools and multiple primer pairs may be identified in response to those random-access queries. The DNA data storage system contains strands of DNA organized into one or more DNA pools. Each strand of DNA is synthetically created according to a schema that includes both a payload region and flanking primer binding sites. Amplification with PCR primers that hybridize to the primer binding sites creates many copies of the payload region which can then be sequenced and decoded to obtain the digital data specified in the random-access request. The digital data is correlated to a specific DNA pool and primer pair by the computer systems (e.g., by using a lookup table).

At operation <NUM>, a plurality of isolated reaction volumes are created. Each of the isolated reaction volumes comprises a portion of one of the DNA pools, a primer pair, and PCR master mix. The PCR master mix may also contain a dye such as an intercalating fluorescent dye. The number of isolated reaction volumes created may depend on the number of random-access requests received at <NUM>. In an implementation, there is one isolated reaction volume created for each random-access request. Thus, each isolated reaction volume will contain a unique combination of a portion of DNA from a DNA pool and a primer pair. In other implementations, multiple isolated reaction volumes that contain the same combination of DNA and primer pair may be created either intentionally or unintentionally.

The isolated reaction volumes may be formed as microdroplets such as a water-in-oil emulsion or as a calcium alginate emulsion. Water-in-oil emulsions may be formed using a T-junction as described above.

Isolated reaction volumes may alternatively be formed as wells in a plate. The primer pairs may be provided by functionalizing each of the plurality of beads with a single primer pair. The plurality of beads are placed into a plurality of wells in a plate. The size and shape of the beads in the wells may be such that each of the plurality of wells is sized to hold at most a single one of the plurality of beads. This provides a single, unique primer pair to each isolated reaction volume.

At operation <NUM>, the plurality of isolated reaction volumes are thermocycled under conditions suitable for PCR. Persons of ordinary skill in the art will readily understand how to perform PCR including selection of a specific series of temperature changes and number of cycles.

At operation <NUM>, the quantity of DNA is measured in some or all of the plurality of isolated reaction volumes. Any suitable technique for measuring DNA may be used. For example, the quantity of DNA may be measured by measuring the fluorescence of a dye such as an intercalating fluorescent dye.

At operation <NUM>, the quantity of DNA in a selection of the isolated reaction volumes is normalized prior to multiplex sequencing. The selection of isolated reaction volumes may, in some implementations, include all of the isolated reaction volumes such as all of the microdroplets or all of the wells. Copy-normalization may be performed by performing additional PCR cycles or by batching samples with similar quantities of DNA.

If copy-normalization is performed by additional PCR cycles, process <NUM> proceeds to operation <NUM> where it is determined if the quantity of DNA in individual ones of the isolated reaction volumes is less than a threshold value. The quantity of DNA may be determined and compared to a threshold value for each of the isolated reaction volumes. The threshold value may be predetermined based on previous experience. The threshold value may be derived from a measured value of the individual ones of the isolated reaction volumes. For example, the threshold value may be the quantity of DNA in the one of the plurality of isolated reaction volumes that has the highest quality of DNA. As a further example, the threshold value may be defined relative to the quantity of DNA in the one of the plurality of isolated reaction volumes that has the highest quantity of DNA (e.g. example <NUM>%, <NUM>%, <NUM>%, of the highest quantity).

For an individual one of the isolated reaction volumes, if the quantity of DNA is not less than the threshold value, process <NUM> proceeds from operation <NUM> to operation <NUM>. If, however, the quantity of DNA is less than the threshold value, process <NUM> proceeds from operation <NUM> back to operation <NUM> where thermocycling is continued. The thermocycling may be continued by returning individual microdroplets to a reaction chamber where they are subj ect to further rounds of heating and cooling to continue the PCR. The thermocycling may be continued for DNA in wells of a plate by providing additional heating and cooling cycles to those wells without performing the same heating and cooling on the entire plate.

If copy-normalization is provided by batching, process <NUM> proceeds from operation <NUM> to operation <NUM>. At operation <NUM>, individual ones of the plurality of isolated reaction volumes that have a quantity of DNA within a range of values are batched into the same multiplex sequencing run. The values used for the range of values may be defined in advance or based on measured DNA quantities. Two ranges of values (e.g., from <NUM> to a threshold and from the threshold to infinity) may be used to batch the isolated reaction volumes into two batches. Similarly, a larger number of ranges may be used to divide the isolated reaction volumes into three or more different batches. Batching may be performed, for example, by using microfluidics and/or cell-sorting techniques such as electrostatic sorting.

At operation <NUM>, the copy-normalized amplification products are provided to a multiplex DNA sequencer. All of the DNA from each of the isolated reaction volumes is mixed together when provided to the multiplex DNA sequencer. Because the quantity of DNA from each of the isolated reaction volumes, which correspond to separate random-access requests, is normalized (i.e., the same or approximately the same) the DNA strands corresponding to each random-access request are represented approximately equally in the flow cell of the multiplex DNA sequencer.

The multiplex DNA sequencer generates output strings which represent the order of nucleotide bases in the DNA strands present in the flow cell. The strength of the signals generated by reading the DNA corresponding to the random-access requests are approximately equal because of copy-normalization, so the multiplex DNA sequencer is able to generate sequence output in which there is approximately equal depth of coverage for each sample. This creates accurate sequence output without consuming necessary reagents or using bandwidth of the multiplex DNA sequencer to generate additional, unnecessary coverage depth.

The techniques discussed above are based on measured quantities of DNA in individual reaction volumes. As mentioned earlier, one source of variation for the quantities of DNA created by PCR amplification is variations in primer efficiency. Some features of primer efficiency may be identified, or at least estimated, based only on the sequence of nucleotides in a primer pair. Thus, it is possible to preemptively modify aspects of the random-access techniques and systems discussed above based on knowledge of the relative efficiency of the primer pairs being used to respond to a plurality of random-access requests.

Primer efficiency is related to amplification efficiency. If the quantity of DNA doubles during each cycle of a PCR reaction than amplification efficiency is <NUM>%. Primer efficiency values may also be represented as a percentage based on the effect a given primer pair has on amplification efficiency. Thus, if amplification efficiency is <NUM>% with a highly efficient primer pair but under the same conditions amplification efficiency is only <NUM>% with a different primer pair, then this different primer pair is said to have <NUM>% primer efficiency value. Persons of ordinary skill in the art are aware of techniques for calculating primer efficiency values such as by creation of a standard curve. Some commercially available thermocyclers are also able to automatically calculate primer efficiency values.

Primer efficiency values may be identified for each primer pair that can be used to query a DNA pool. Because of the design of the DNA molecules placed into each DNA pool, the primer pairs used in response to random-access requests are known. Once primer efficiency values are available for each of the primer pairs, they may be stored in electronic format such as in a table, database, etc..

One way that primer efficiency may be used is by identifying a primer efficiency value for a particular primer pair and then adjusting the number of isolated reaction volumes that contain the particular primer pair based on the primer efficiency value. The number of individual, reaction volumes created for a random-access request may be inversely proportional to the primer efficiency value of the primer pair. As the primer efficiency decreases a greater number of isolated reaction volumes are created with that primer pair.

The number of microdroplets containing a particular primer pair may be adjusted based on the primer efficiency value. More microdroplets containing a low-efficiency primer pair can be created. Thus, some combinations of DNA and primer pairs may be present in only a single microdroplet, but others may be present in two, three, or more microdroplets. When the primer pair is provided on a bead, the number of beads functionalized with a particular primer pairs may be adjusted based on the primer efficiency value. The number of beads that have primer pairs with low primer efficiency values may be increased so that there may be two, three, or more wells in a plate filled with beads coated with the same primer pair. Although the quantity of DNA in each isolated reaction volume is not changed, there are more isolated reaction volumes containing the same amplification products which are combined prior to sequencing thereby increasing the total quantity of DNA provided to a DNA sequencer.

Wells filled with beads that provide low-efficiency primer pairs may be subject to additional rounds of thermocycling (possibly absent any measurement of DNA quantity) so that the final quantity of amplification products is similar to that of other wells. There are multiple possible techniques to identify which wells contain a specific bead. Individual beads may be placed into specific wells using microfluidics and laboratory robotics and the location recorded. Beads that are functionalized with low-efficiency primer pairs may also be marked either by functionalization with other molecules that are identifiable (e.g. dyes, radioactive tags, etc.) or the bead itself may be different (e.g., in color, radioactivity, quantity of ferromagnetic material, etc.). Those wells containing the beads functionalized with low-efficiency primer pairs may receive additional cycles of PCR in inverse proportion to the primer efficiency (i.e., the lower the efficiency the greater the number of PCR cycles).

Another technique for adjusting processing based on primer efficiency values includes adjusting the relative concentrations or amounts of the components of the PCR reaction. The relative concentrations may be changed by increasing or decreasing the amount of any or all of the components of the PCR reaction. The quantity of DNA may be adjusted by changing the quantity of DNA drawn from the DNA pool used (e.g., a larger quantity of DNA may be used with low-efficiency primer pairs). The quantity of DNA drawn from the pool may be adjusted by taking a larger or smaller volume of sample from the DNA pool. If the volume of the sample is changed, the volume of another component (e.g., a buffer) may be increased or decreased by an equal amount to maintain a constant volume. Alternatively, if the sample drawn from the DNA pool is diluted prior to mixing with other reagents, the extent of the dilution may be changed in order to obtain a greater lesser quantity of DNA. The quantity of the primer pair itself may be adjusted by providing more or fewer molecules of the primers (e.g., the quantity of each primer in the primer pair may be increased in inverse proportion to the primer efficiency). Additionally, the concentration of the PCR master mix may be changed based on the primer efficiency values.

<FIG> is a computer architecture diagram showing an illustrative computer hardware and software architecture for a computing device. In particular, the computer <NUM> illustrated in FIG. <NUM> can be used to control either of the random-access systems <NUM>, <NUM> shown in <FIG> and <FIG> as well as to control a DNA sequencer <NUM>.

The computer <NUM> includes one or more processing units <NUM>, a memory <NUM>, that may include a random-access memory <NUM> ("RAM") and a read-only memory ("ROM") <NUM>, and a system bus <NUM> that couples the memory <NUM> to the processing unit(s) <NUM>. A basic input/output system ("BIOS" or "firmware") containing the basic routines that help to transfer information between elements within the computer <NUM>, such as during startup, can be stored in the ROM <NUM>. The computer <NUM> further includes a mass storage device <NUM> for storing an operating system <NUM> and other instructions <NUM> that represent amplification programs and/or other types of programs such as, for example, instructions to implement the random-access module <NUM>. The mass storage device <NUM> can also be configured to store files, documents, and data such as, for example, sequence data that is obtained from a DNA sequencer <NUM>.

The mass storage device <NUM> is connected to the processing unit(s) <NUM> through a mass storage controller (not shown) connected to the bus <NUM>. The mass storage device <NUM> and its associated computer-readable media provide non-volatile storage for the computer <NUM>. Although the description of computer-readable media contained herein refers to a mass storage device, such as a hard disk, solid-state drive, CD-ROM drive, DVD-ROM drive, or USB storage key, it should be appreciated by those skilled in the art that computer-readable media can be any available computer-readable storage media or communication media that can be accessed by the computer <NUM>.

Communication media includes computer-readable instructions, data structures, program modules, or other data in a modulated data signal such as a carrier wave or other transport mechanism and includes any delivery media. The term "modulated data signal" means a signal that has one or more of its characteristics changed or set in a manner so as to encode information in the signal. By way of example, and not limitation, communication media includes wired media such as a wired network or direct-wired connection, and wireless media such as acoustic, radio frequency, infrared and other wireless media. Combinations of any of the above should also be included within the scope of computer-readable media.

By way of example, and not limitation, computer-readable storage media can include volatile and non-volatile, removable and non-removable media implemented in any method or technology for storage of information such as computer-readable instructions, data structures, program modules or other data. For example, computer-readable storage media includes, but is not limited to, RAM <NUM>, ROM <NUM>, EPROM, EEPROM, flash memory or other solid-state memory technology, CD-ROM, digital versatile disks ("DVD"), HD-DVD, BLU-RAY, <NUM> Ultra BLU-RAY, or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store the desired information and which can be accessed by the computer <NUM>. For purposes of the claims, the phrase "computer-readable storage medium," and variations thereof, does not include waves or signals per se or communication media.

According to various configurations, the computer <NUM> can operate in a networked environment using logical connections to a remote computer(s) <NUM> through a network <NUM>. The computer <NUM> can connect to the network <NUM> through a network interface unit <NUM> connected to the bus <NUM>. It should be appreciated that the network interface unit <NUM> can also be utilized to connect to other types of networks and remote computer systems. The computer <NUM> can also include an input/output (I/O) controller <NUM> for receiving and processing input from a number of other devices, including a keyboard, mouse, touch input, an electronic stylus (not shown), or equipment such as a DNA sequencer <NUM> and/or a random-access system <NUM>, <NUM>. Similarly, the input/output controller <NUM> can provide output to a display screen or other type of output device (not shown).

It should be appreciated that the software components described herein, when loaded into the processing unit(s) <NUM> and executed, can transform the processing unit(s) <NUM> and the overall computer <NUM> from a general-purpose computing device into a special-purpose computing device customized to facilitate the functionality presented herein. The processing unit(s) <NUM> can be constructed from any number of transistors or other discrete circuit elements, which can individually or collectively assume any number of states. More specifically, the processing unit(s) <NUM> can operate as a finite-state machine, in response to executable instructions contained within the software modules disclosed herein. These computer-executable instructions can transform the processing unit(s) <NUM> by specifying how the processing unit(s) <NUM> transitions between states, thereby transforming the transistors or other discrete hardware elements constituting the processing unit(s) <NUM>.

Encoding the software modules presented herein can also transform the physical structure of the computer-readable media presented herein. The specific transformation of physical structure depends on various factors, in different implementations of this description. Examples of such factors include, but are not limited to, the technology used to implement the computer-readable media, whether the computer-readable media is characterized as primary or secondary storage, and the like. For example, if the computer-readable media is implemented as semiconductor-based memory, the software disclosed herein can be encoded on the computer-readable media by transforming the physical state of the semiconductor memory. For instance, the software can transform the state of transistors, capacitors, or other discrete circuit elements constituting the semiconductor memory. The software can also transform the physical state of such components to store data thereupon.

As another example, the computer-readable media disclosed herein can be implemented using magnetic or optical technology. In such implementations, the software presented herein can transform the physical state of magnetic or optical media, when the software is encoded therein. These transformations can include altering the magnetic characteristics of particular locations within given magnetic media. These transformations can also include altering the physical features or characteristics of particular locations within given optical media, to change the optical characteristics of those locations. Other transformations of physical media are possible, with the foregoing examples provided only to facilitate this discussion.

In light of the above, it should be appreciated that many types of physical transformations take place in the computer <NUM> to store and execute the software components presented herein. It also should be appreciated that the architecture shown in <FIG> for the computer <NUM>, or a similar architecture, can be utilized to implement many types of computing devices such as desktop computers, notebook computers, servers, supercomputers, gaming devices, tablet computers, and other types of computing devices known to those skilled in the art. For example, the computer <NUM> may be wholly or partially integrated into one or both of the DNA sequencer <NUM> and the random-access system <NUM>, <NUM>. It is also contemplated that the computer <NUM> might not include all of the components shown in <FIG>, can include other components that are not explicitly shown in <FIG>, or can utilize an architecture different than that shown in <FIG>.

The computer <NUM> may include a random-access module <NUM> that can control formulation of the inputs to a random-access system <NUM>, <NUM> and additionally control operation of the random-access system <NUM>, <NUM> itself. For example, random-access requests for digital data received by the computer <NUM> may be translated into a DNA pool and primer pair by the random-access module <NUM>. As mentioned above, this translation may be performed by using a look-up table or other record of correlation. The random-access module <NUM> may also generate instructions to control microfluidic devices (e.g., Puddle) and/or laboratory robotics. The random-access module <NUM> may further control modifications to random-access protocols based on primer efficiency values.

The DNA sequencer <NUM> may be any conventional or later-developed type of DNA sequencing technique. Common sequencing techniques include dideoxy sequencing reactions, NGS, and nanopore sequencing. Classic dideoxy sequencing reactions (Sanger method) use labeled terminators or primers and gel separation in slab or capillary electrophoresis.

NGS refers to any of a number of post-classic Sanger type sequencing methods which are capable of high throughput, multiplex sequencing of large numbers of samples simultaneously. Current NGS sequencing platforms are capable of generating reads from multiple distinct nucleic acids in the same sequencing run.

Nanopore sequencing uses a small hole, a "nanopore," on the order of <NUM> nanometer in diameter. Immersion of a nanopore in a conducting fluid and application of a potential across it results in a slight electrical current due to conduction of ions through the nanopore. The amount of current which flows is sensitive to the size of the nanopore. As a DNA molecule passes through a nanopore, each nucleotide on the DNA molecule obstructs the nanopore to a different degree. Thus, the change in the current passing through the nanopore as the DNA molecule passes through the nanopore represents a reading of the DNA sequence.

The terms "a," "an," "the" and similar referents used in the context of describing the invention are to be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. The terms "based on," "based upon," and similar referents are to be construed as meaning "based at least in part" which includes being "based in part" and "based in whole," unless otherwise indicated or clearly contradicted by context. The terms "portion," "part," or similar referents are to be construed as meaning at least a portion or part of the whole including up to the entire noun referenced. As used herein, "approximately" or "about" or similar referents denote a range of ± <NUM>% of the stated value.

For ease of understanding, the processes discussed in this disclosure are delineated as separate operations represented as independent blocks. However, these separately delineated operations should not be construed as necessarily order dependent in their performance. The order in which the processes are described is not intended to be construed as a limitation, and unless other otherwise contradicted by context any number of the described process blocks may be combined in any order to implement the process or an alternate process. Moreover, it is also possible that one or more of the provided operations is modified or omitted.

Claim 1:
A method of preparing copy-normalized oligonucleotide samples for multiplex sequencing comprising:
identifying (<NUM>) one or more oligonucleotide pools (<NUM>) and multiple primer pairs (<NUM>) responsive to a random-access request for digital data encoded onto DNA strands in the one or more oligonucleotide pools, wherein each random-access request corresponds to a different primer pair;
creating (<NUM>) a plurality of isolated reaction volumes (<NUM>, <NUM>) each comprising a portion of one of the oligonucleotide pools (<NUM>), one of the identified primer pairs (<NUM>), and polymerase chain reaction, PCR, master mix (<NUM>), wherein the isolated reaction volumes prevent interaction between components in separate isolated reaction volumes;
thermocycling (<NUM>) the plurality of isolated reaction volumes under conditions suitable for PCR, wherein different PCR reactions occur in isolated reaction volumes comprising different primer pairs during thermocycling;
measuring (<NUM>) a quantity of oligonucleotides in individual ones of the plurality of isolated reaction volumes; and
normalizing (<NUM>) the quantity of oligonucleotides in a selection of the plurality of isolated reaction volumes prior to multiplex sequencing;
decode a read from the multiplex sequencer to reproduce the digital data in the random access request.