WO2014126937A1 - Suspension arrays and multiplexed assays based thereon - Google Patents

Suspension arrays and multiplexed assays based thereon Download PDF

Info

Publication number
WO2014126937A1
WO2014126937A1 PCT/US2014/015880 US2014015880W WO2014126937A1 WO 2014126937 A1 WO2014126937 A1 WO 2014126937A1 US 2014015880 W US2014015880 W US 2014015880W WO 2014126937 A1 WO2014126937 A1 WO 2014126937A1
Authority
WO
Grant status
Application
Patent type
Prior art keywords
target
microparticles
segment
polynucleotide
segments
Prior art date
Application number
PCT/US2014/015880
Other languages
French (fr)
Inventor
Michael S. AKHRAS
Erik Pettersson
Ronald W. Davis
Nader Pourmand
Original Assignee
The Board Of Trustees Of The Leland Stanford Junior University
The Regents Of The University Of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6827Hybridisation assays for detection of mutation or polymorphism
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6834Enzymatic or biochemical coupling of nucleic acids to a solid phase

Abstract

A microparticle suspension assay useful in molecular diagnostics is disclosed. The assay comprises the use of a mixture of a first plurality (subpopulation) of beads and preferably a second plurality (subpopulation) of beads, wherein the beads have attached thereto multiple copies of a polynucleotide (oligo-tagged), said polynucleotide comprising the following segments: (i) a linker; (ii) an identifier segment; (iii) a target specific binding segment; and (iv) a segment for use in sequence determination; and a second, etc. number of bead subpopulations have attached thereto multiple copies of a polynucleotide as in (i) through (iv) above, except that different target identification sequences and different identifier sequences are used in each. The combined subpopulations of oligo-tagged beads are contacted with a sample containing target molecules (preferably nucleic acids), the beads binding to the target are separated from non-binding beads and the number of beads having the various identifier sequences is determined.

Description

SUSPENSION ARRAYS AND MULTIPLEXED ASSAYS BASED THEREON

CROSS-REFERENCE TO RELATED APPLICATIONS

The instant application claims the benefit of U.S. provisional application no.

61/764,820, filed February 14, 2013, the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with Government support under contract P01-35HG000205 awarded by the National Institutes of Health. The Government has certain rights in this invention.

REFERENCE TO SEQUENCE LISTING, COMPUTER PROGRAM,

OR COMPACT DISK

The instant application contains a Sequence Listing which has been submitted as an

ASCII text file and is hereby incorporated by reference in its entirety. This text file was created on January 29, 2014, named 3815_115_lPCT_sequence_listing_2_ST25.txt, and is

5,233 bytes in size.

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The present invention relates to the field of molecular diagnostics and to assays based on suspension arrays of microparticles (e.g. beads) bearing oligonucleotides.

RELATED ART

Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. That is, individual parts or methods used in the present invention may be described in greater detail in the materials discussed below, which materials may provide further guidance to those skilled in the art for making or using certain aspects of the present invention as claimed. The discussion below should not be construed as an admission as to the relevance of the information to any claims herein or the prior art effect of the material described. DNA microarray technology originally evolved from Southern blotting with the general principle of attaching DNA to a surface, and interrogating known DNA sequences. A major breakthrough was reported in 1995, with a miniaturized microarray designed for gene- expression profiling1, which has since been formatted to a multitude of applications to expand our molecular toolbox . Microarrays dominated the high-throughput genomic space until the advent of Next Generation Sequencing (NGS) in 20053'4 and, with increased speed and a continuous drop in cost-per-base-sequenced, NGS may eventually replace the need for probe and hybridization based technologies, such as Real-Time PCR and microarrays 5-"7. Until then, there is still need for arrayed signatures and targeted sequencing, and with novel approaches the benefits of both systems can be combined, as in, for example, Array-Seq 8 ' 9. Suspension array technology presented an embodiment of microarray technology in which the typical spotted planar array is replaced with microspheres with distinct optical properties that can move freely in a solution10'11. Benefits include i) ease of use, ii) low cost, iii) statistical superiority in data acquisition, iv) rapid hybridization kinetics, v) improved specificity and sensitivity, vi) multiple biomolecule testing (i.e. DNA and proteins), and vii) simplified array preparation. However, a major limitation lies in the relatively low array sizes due to limited optical combinations of the spheres. The nanoparticle-based bio-barcode is an ultra- sensitive suspension array variant, where coated nanoparticles are selected for via targets, with subsequent release of bio-barcode nucleic acids for downstream processing 12.

The NGS Roche 454 Sequencing technology4 combined Pyro sequencing 13 with emulsion-PCR14 sample preparation of randomly generated DNA libraries for massively parallel sequencing applications. In brief, emulsion PCR traps an individual bead coated with a primer together with one unique DNA target containing the corresponding priming segment. Via PCR, the sequence is cloned onto the bead in an emulsion with ideally only one template initially present. Each bead is immobilized onto a slide with picoliter sized wells and individually Pyrosequenced. Life Technologies Ion Torrent semiconductor

sequencing15'16 took a novel approach to this by replacing pyrosequencing chemistry with a direct electrical detection of polymerase mediated nucleotide incorporation events 15-"18. This approach is highly scalable and allows for large-scale production, thus offering a low-cost alternative, using CMOS chips that include both the micro wells and the biosensor.

SPECIFIC PATENTS AND PUBLICATIONS Miller et al. "Basic concepts of microarrays and potential applications in clinical microbiology," Clin. Microb. Reviews 22:611-633 (2009) describes basic microarray platforms including high-density bead arrays and suspension bead arrays. As described there, solution-based chemistries all take advantage of universal microspheres with nonspecific capture sequences. The first universal sequences used to tag microspheres were

ZipCode/cZipCode capture sequences originally used with SBCE in SNP (single nucleotide polymorphism) genotyping assays. The 25-bp ZipCode sequences are based on random genomic sequences from Mycobacterium tuberculosis. Additional sets of universal capture sequences have been developed, including those by Tm Biosciences (Toronto, Canada), Luminex Molecular Diagnostics, Inc. (Austin, Texas) (xTAG), and EraGen (Madison, WI) (EraCode).

Mirkin et al. US 6,974,66, entitled "Bio-barcodes based on oligonucleotide-modified nanoparticles," discloses oligonucleotides as biochemical barcodes for detecting multiple protein structures in one solution. The approach takes advantage of protein recognition elements functionalized with oligonucleotide strands.

BRIEF SUMMARY OF THE INVENTION

The following brief summary is not intended to include all features and aspects of the present invention, nor does it imply that the invention must include all features and aspects discussed in this summary. The present invention is described for convenience as a Sequencing Bead Array

(SB A), but generally comprises the use of microparticles, which may be beads, in a suspension array. The microparticles are initially prepared with multiple copies of single- stranded polynucleotides, and the method comprises analysis of certain sequences in those polynucleotides (e.g. "ID traces") in a specific series of steps that collects the microparticles that have bound to targets in the sample. Only beads that have bound to a target analyte are analyzed. The microparticles bearing polynucleotides are referred to as "oligo-tagged beads" for convenience, but the "bead" can be any microparticle that can be handled in the assay and linekd to a population of single-stranded polynucleotides. The microparticles can also be linked to antibodies as described in Figure 7 and Example 7. The invention comprises a protocol for library construction of a defined set of oligo-tagged beads, and SB A assay target detection for applied approaches to molecular diagnostics. In principle, it may be regarded as an improvement of microarray methodology implemented as a suspension array, where the detection principle is based on bead labels that are pre-formed, rather than labels that are present due to target binding. In addition, the microarray analog light intensity detection system has been replaced with a digital sequence trace readout approach using Next

Generation Sequencing (NGS) platforms, or other short read sequence methodologies, such as sequencing by hybridization. A model assay was designed that could distinguish among ten clinically relevant Human Papillomavirus (HPV) associated with cervical cancer progression. As compared with results from standardized genotyping methods, SBA offers a multi-parallel, rapid, robust, and affordable detection system that could be readily adapted for a multitude of microarray-like diagnostic assays including but not limited to i) genetic signature detections, ii) single nucleotide polymorphism (SNP) genotyping and other structural variations, and iii) immunogenic assays. An in-house software package, termed Sphix, is also described; it provides easy accessibility and interpretation of SBA data for iPhone, other iOS, Android and other handheld devices. The present invention comprises, in certain aspects, a composition for use in an assay comprising a microparticle comprised in a population of similar microparticles, wherein the microparticle has attached thereto multiple copies of a single-stranded polynucleotide, said polynucleotide comprising various segments arranged along and within the sequence of the polynucleotide: (a) a linker segment linking the polynucleotide to the microparticle; (b) one or more target specific binding segments for binding to a marked or tagged target as described below; (c) an identifier segment ("ID trace") for distinguishing one population of microparticles from another population of microparticles; and, optionally, (d) a segment for use in sequence determination, adjacent to the identifier segment. The segment (d) may be primer segment for use in a sequencing-by-synthesis reaction, and is adjacent to the ID trace, in that the ID trace contains the sequence of interest in the present methods. A composition for use in an assay of a target, comprising a microparticle comprised in a population of microparticles, wherein the microparticles have attached thereto multiple copies of single-stranded polynucleotides, said polynucleotides each comprising:

(a) a linker segment linking the single-stranded polynucleotide to the

microparticle;

(b) one or more target specific binding segments;

(c) an identifier segment identifying the population of microparticles bearing the single stranded polynucleotides and target specific binding segments; and, optionally,

(d) a segment for use in binding a sequencing primer, adjacent to the identifier segment, wherein the sequencing primer is used to sequence identifier segments in single- stranded polynucleotides in isolated microparticles that have bound to targets.

In certain aspects, the inventive composition may further comprise multiple populations, which can be defined as subpopulations within the overall population used in an assay procedure, wherein a microparticle has attached thereto multiple copies of a

polynucleotide, said polynucleotide comprising defined segments (a) through (d) as described above, inclusive, where segments (a) (linker) and (d) (primer) are identical to those in the first subpopulation of microparticles, and segments (b) (target) and (c) (ID trace) differ in sequence from sequences in other subpopulations of microparticles.

In certain aspects, the inventive composition may further comprise a composition further comprising a multiplicity of subpopulations of microparticles wherein the defined segments (b) and (c) differ among different subpopulations.

In certain aspects and embodiments, the polynucleotides of the present invention are arranged in the specific order 5' to 3' of (a)- (b)- (c)- (d). In certain aspects, the

polynucleotides of the present invention have segments with the order of 5' to 3' (a)- (c)- (d)- (b).

In certain aspects and embodiments, the polynucleotides of the present invention are covalently linked to the microparticle. In certain aspects and embodiments, the

polynucleotides are between about 50 and 200 nucleotides in size.

In certain aspects, the polynucleotides of the present invention may have multiple target specific binding segments. In certain aspects, the target specific segments have sequences that bind specifically to one genotype in a mixture of genotypes. In certain aspects, the genotype is selected from a genotype of a virus, a genotype of a tumor, an SNP genotype and a micro satellite genotype.

In certain aspects and embodiments, the composition comprises polynucleotides wherein the identifier segment is not more than 50 nucleotides in length. In certain aspects and embodiments the target binding segment is (also) not more than 50 nucleotides in length.

In certain aspects, the inventive composition may further comprise beads wherein a bead is a material that is one of glass, polymer, ceramic, metal, paramagnetic material, or carbon. The beads may all be the same material, since the distinguishing construct is based on the target-polynucleotide complex, not on any property of the bead itself. In certain aspects, the inventive composition may further comprise a bead that is a polymer that is one of acrylamide, polyethylene, polystyrene, and polymeric latex.

In certain aspects and embodiments the invention comprises a method for detecting a number of different target molecules in a mixture, comprising the steps of: providing a mixture of microparticles comprising a first subpopulation of microparticles and at least a second subpopulation of microparticles, wherein the microparticles have attached thereto multiple copies of a polynucleotide, said polynucleotide comprising: a linker for linking the polynucleotide to the microparticle; one or more target specific binding segments; an identifier segment for distinguishing one plurality of microparticles from other pluralities of microparticles; a segment for use in sequence determination; and the second subpopulation has attached thereto polynucleotides as above, where the linker and sequence determination segments are the same as those in the first subpopulation of microparticles, and the target specific binding segments and identifier segments are different in different subpopulations. Additional steps include attaching affinity tags to nucleic acids in the mixture in the sample; contacting nucleic acids with the complex formed with the affinity tag-bearing nucleic acids with microparticles from (a) and (b) under conditions allowing hybridization of nucleic acids having target nucleic acid sequences to hybridize to target identification sequences thereby forming microparticle-target complexes; separating microparticle-target complexes from microparticles that have not hybridized to targets by use of the affinity tag; and performing a sequence determination of identifier segments in microparticle target complexes that were hybridized to targets, whereby target nucleic acid sequences are detected. In certain aspects and embodiments of the present invention, the methods may comprise a method wherein the step of performing a sequence determination is carried out using microparticles separated on the basis of an affinity tag. In certain aspects and embodiments of the present invention, the separating step comprises contacting the tag with a magnetic microparticle comprising a material which binds the affinity tag. In certain aspects and embodiments of the present invention the affinity tag is biotin, which may be provided by biotinylating the nucleic acids in a sample, e.g. with biotinylated PCR primers.

In certain aspects and embodiments of the present invention the method comprises contacting the microparticle with a separate, streptavidin-coated microparticle, which pulls down the microparticle having a complex with biotin. In certain aspects and embodiments of the present invention the method comprises using a target identification sequence which is not more than 50 nucleic acids in length, and this lessens the sequence length needed for sequence detection. In certain aspects and embodiments of the present invention, the method comprises quantifying numbers of targets detected. This may be done with software such as Sphix, described below.

In certain aspects and embodiments of the present invention, there is provided a method for detecting a number different target molecules in a mixture, comprising the steps of: (a) providing a mixture of microparticles comprising a first subpopulation of

microparticles wherein the microparticles have attached thereto multiple copies of a polynucleotide, said polynucleotide comprising a 5' end bound to the microparticle and (i) a linker for linking the polynucleotide to the microparticle; (ii) an identifier segment for distinguishing one plurality of microparticles from other pluralities of microparticles; (iii) a target specific binding segment at the 3' end which binds to a target having a polynucleotide sequence extending beyond the 3' end, providing a target overhang; and (iv) a segment for use in sequence determination; and then adding to the mixture an affinity tag for separating microparticles in which the polynucleotide has bound to the target. In certain aspects and embodiments of the inventive method, the affinity tag is in the form of biotinylated nucleotides and the enzyme is a polymerase. In certain aspects and embodiments of the inventive method, the affinity tag is in the form of a probe, having an affinity tag on the probe, that binds to the target overhang, and the method comprises the step of adding the probe to the mixture. In certain aspects and embodiments of the present invention, the target specific binding segment is an aptamer. In certain aspects and embodiments of the present invention the aptamer binds to a target which is a protein.

In certain embodiments and aspects, the present invention comprises a population of microparticles in suspension, having covalently attached thereto a plurality of single- stranded polynucleotides, each polynucleotide having a linker segment, an identifier segment, and a target specific binding segment; and a polynucleotide target hybridized to at least one target specific binding segment on a microparticle, said polynucleotide target attached to an affinity tag. In certain aspects and embodiments of the present invention, the composition is one wherein the population of microparticles comprises polynucleotides having identifier segments that differ as between microparticles. In certain aspects and embodiments of the present invention, the composition is one wherein the population of microparticles comprises polynucleotides having target binding segments and targets that differ as between

microparticles. In certain aspects and embodiments of the present invention the composition is one further comprising a second microparticle bound to the affinity tag. In certain aspects and embodiments of the present invention, the composition is one wherein the affinity tag is biotin and the second microparticle comprises avidin.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram showing construction of an oligo-tagged bead, for use in the present sequencing bead array (SBA) assay.

Figure 2 is a schematic diagram showing an SBA assay workflow.

Figure 3A, 3B and 3C is a series of histograms where Figure 3A is a computer- generated histogram (termed a "Sphixogram") derived from six separately constructed 10- plex HPV libraries with error-bars depicting deviation range for each bead. Figure 3B is a Sphixogram from a pooled bead library representation of six bead libraries run in six replicates with error-bars depicting deviation range for each oligo-tagged bead. Figure 3C is a Sphixogram of a quintuple HPV plasmid co-infection run in six replicates with error-bars depicting deviation range for each oligo-tagged bead.

Figure 4A, 4B, 4C and 4D is a series of histograms where Figure 4A is a

Sphixogram of an SBA assay selection of two synthetic targets T-45 and T-59 present at equimolar amounts of 1-picomolar respectively, 1 : 1. Figure 4B is a Sphixogram of targets T- 45 and T-59 present at 1-picomolar and 100-femtomolar amounts respectively, 1: 10. Figure 4C is a Sphixogram of targets T-45 and T-59 present at 1-picomolar and 10-femtomolar amounts respectively, 1: 100. Figure 4D is a Sphixogram of targets T-45 and T-59 present at 1-picomolar and 1-femto molar amounts respectively, 1: 1000. Figure 5A, 5B, 5C, 5D, 5E, 5F and 5G is a series of representations of clinical sample results, where Figure 5A is a Sphixogram of SBA assay results from clinical sample OM-1530, showing a single infection of HPV-16. Figure 5B is a Sphixogram of SBA assay results from clinical sample OM-1668, showing a single infection of HPV-59. Figure 5C is a Sphixogram of SBA assay results from clinical sample OM-1741, showing a single infection of HPV-18. Figure 5D is a Sphixogram of SBA assay results from clinical sample OM-1848, showing a single infection of HPV-45. Figure 5E is a Sphixogram of SBA assay results from clinical sample OM-2215, showing a dual co-infection of HPV-16 and HPV-18. Figure 5F is a Sphixogram of SBA assay results from clinical sample OM-2258, showing a dual co- infection of HPV-16 and HPV-45. Figure 5G is a bubble chart that shows results from 20 patient samples listed vertically (genomic DNA extracts from cervical tumors) (including those Figure 5A-F). The horizontal axis identifies the HPV strains found, and the size of the bubble is semi-quantitative as to the amount of the strain found.

Figure 6A, 6B, 6C, 6D, 6E is a series of schematic drawings of alternative SBA assays. Figure 6A shows selective hybridization between a complementary unbiotinylated target 608 and a bead's oligo tag at the 3 '-end of the oligo tag, which allows for a

polymerization reaction 620 using biotinylated nucleotides, which is shown, where the biotin is at 612 at the end nucleotide. Figure 6B shows selective hybridization between a biotinylated complementary target (or tag) 613 and a bead's oligo tag at the 3 '-end could possibly ease steric hindrance, and allowing for a polymerization 622, which further strengthens the hybridization bond. Figure 6C shows selective hybridization of a

complementary target (or tag) 608 to an oligo tag at the 3 '-end, and selection via a biotinylated synthetic capture probe 624. Optionally the capture probe 624 could contain a 5 'phosphorylation and thus allow for a ligation to the 3 '-end of the oligo tags, allowing for stronger bond. Figure 6D shows that selection could be performed by an initial intermediary polymerization reaction 622 constructing a complementary sequence to the target template 608, which would allow for selection with downstream complementary capture probe 612. Figure 6E shows an alternative immunologic approach involving direct conjugation of aptamer 634 to the plurality oligo tags 601 at the 3' end.

Figure 7 shows an alternative immunologic approach involving direct conjugation of antibody 732 to the bead 710, which contains a plurality of oligonucleotides 701, i.e. the oligo-tags. An antigen 730 could then be assayed using selection/separation 714 based on a secondary antibody 740 recognizing a different epitope of antigen 730 and carrying a biotin molecule 745 for selection of the entire antibody-antigen-antibody-bead complex.

Figure 8 is a flow chart showing operation of software (Sphix) for analyzing data from the present assays.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Generally, nomenclatures utilized in connection with, and techniques of, cell and molecular biology and chemistry are those well known and commonly used in the art. Certain experimental techniques, not specifically defined, are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. For purposes of clarity, the following terms are defined below.

Ranges: For conciseness, any range set forth is intended to include any sub-range within the stated range, unless otherwise stated. As a non-limiting example, a range of 120 to 250 is intended to include a range of 120-121, 120-130, 200-225, 121-250 etc. The term "about" has its ordinary meaning of approximately and may be determined in context by experimental variability. In case of doubt, "about" means plus or minus 5% of a stated numerical value. The term "bead" is used herein in a very general sense to refer to a microparticle that is essentially inert in the assay, can be placed in a liquid suspension, and can support multiple macromolecules immobilized thereon. It will generally be on the order of 0.1-50 μιη in nominal diameter. For example, the nominal diameter of the bead could be 0.5 μιη, 0.75-1 μιη, 2 μιη, 1-3 μιη, or 20-50 μιη. It can be manufactured from various natural and synthetic materials, such as glass, polymer (e.g. polyethylene, polystyrene, and polymeric latex), ceramic, metal, paramagnetic material, or carbon. It could be either solid or hollow. The bead could also be referred to as microsphere, nanoparticle or a nanosphere. The term "bead" is used in the present description for convenience. Any one of a number of microparticle configurations could be used for the oligo-tagging or the pull-down steps in the system.

The term "magnetic bead" is used herein to refer to a small spherical particle comprising magnetic or paramagnetic material. Magnetic beads are commercially available, e.g. as Dynabeads® from Life Technologies.

The term "ap tamer" is used herein in its conventional sense to refer to an oligonucleic acid or peptide molecules that bind to a specific target molecule.

The term "oligo" or ' 'oli gonucleotide' ' is used in its customary sense to refer to a polynucleotide that is relatively short in length, e.g. from 3 to 1000 bases, or from 3 to 200 bases, or from 3 to 150 bases in length.

The term "nucleic acids" is used herein to refer to large biological molecules including DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). Nucleic acids are linear polymers (chains) of nucleotides. Each nucleotide consists of three components: a purine or pyrimidine nucleobase (sometimes termed nitrogenous base or simply base), a pentose sugar, and a phosphate group. The substructure consisting of a nucleobase plus sugar is termed a nucleoside. Nucleic acid types differ in the structure of the sugar in their nucleotides: DNA contains 2'-deoxyribose while RNA contains ribose (where the only difference is the presence of a hydroxyl group). Also, the nucleobases found in the two nucleic acid types are different: adenine, cytosine, and guanine are found in both RNA and DNA, while thymine occurs in DNA and uracil occurs in RNA.

The term "polynucleotide" is used in its customary sense to refer to a biopolymer composed of 3 or more nucleotide monomers covalently bonded in a chain. DNA

(deoxyribonucleic acid) and RNA (ribonucleic acid) are examples of polynucleotides with distinct biological function. As described below, the polynucleotides have pre-determined sequences and are single- stranded, preferably completely single- stranded. The term "hybridization" is used in reference to the pairing of complementary nucleic acids, A to T (A to U in RNAs), and G to C. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the Tm (melting temperature), of the formed hybrid, and the G:C ratio within the nucleic acids.

The phrase "target hybridization " or "target specific binding" is used herein to refer to the specific recognition of an analyte to be detected using the present SBA methods. Such binding will be based on specific recognition of a target, e.g. by sequence specific nucleic acid hybridization, or aptamer conformation, etc. as described below. In particular, the target may be bound while similar molecules are not bound, thereby allowing discrimination among similar targets.

The term "target" when used in reference to hybridization assays, such as target DNA of the present inventions, refers to the molecules (e.g., nucleic acid) to be detected. The target in some embodiments may be a protein, polysaccharide or other molecular species. If the target is not a nucleic acid, the target specific binding segment will be configured to bind as an aptamer. Thus, the "target" is sought to be sorted out from other molecules (e.g., isolated nucleic acid sequences) or is to be identified as being present in a sample through its specific interaction with the oligo-tagged beads. The term "ID trace" is used herein after to refer to a known sequence that is used in an oligonucleotide or polynucleotide to provide a unique identification of that polynucleotide and corresponding pre-formed microparticle. As an example, an ID trace in the form of a barcode has been described in connection with Khurana et al. "Method and apparatus using electric field for improved biological assays," US 8,277,628. Essentially, the barcode is an oligonucleotide of a predefined sequence. As described in detail below, the ID trace may be a polynucleotide sequence of sufficient length that it may be made unique among different subpopulations of beads.

The term "biotin" also known as "Coenzyme R" or "vitamin H" or "B7" refers to a small molecule with a chemical formula

Figure imgf000014_0001
which is also a water-soluble B vitamin. It is composed of a ureido (tetrahydroimidizalone) ring fused with a

tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring. Biotin is a coenzyme for carboxylase enzymes, involved in the synthesis of fatty acids, isoleucine, and valine, and in gluconeogenesis.

The term "avidin" refers to various forms of avidin, including a compound that is or derives from a 53000 dalton tetrameric protein originally purified from the bacterium

Streptomyces avidinii, or an egg-white protein, which binds tightly to a small molecule, biotin. Examples include, recombinant streptavidin and derivatives of streptavidin retaining biotin-binding regions. Further examples are given in "Modified avidin and streptavidin molecules and use thereof," EP 0871658 Bl.

The term "primer" is used herein to refer to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced (i.e., in the presence of oligonucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide.

The term "software" refers to a general term used to describe a collection of computer programs, procedures and documentation that perform a task on a computer system. The terms "NGS" and "next- eneration sequencing" are used in the conventional sense to refer to high throughput, massively parallel sequencing methodologies. Examples are given in Quail et al., "A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers," BMC Genomics 13:341, pp. 1-13 (24 July 2012).

OVERVIEW

Described below is a technology referred to for convenience as a Sequencing Bead Array (SB A) (Figure 1 and Figure 2). Assays are based on suspension arrays and can be used in particular methods as described below for target detection, interrogation and quantitative analysis of a number of different targets in a sample. Further, the assay can be used with specially designed software, exemplified here with the in-house software termed "Sphix."

Sphix is implemented in Ruby (http (colon slash slash)www(dot)ruby-lang.org) encompassing all tasks of the data-processing pipeline from initial upstream data retrieval to downstream final output of information and results. Sphix generates platform independent histograms, referred to as Sphixograms, in portable document format (PDF) for a user- friendly experience on laptops or handheld devices.

The present technology provides means to perform suspension array analysis for microarray-like screenings with the digital advantages of pre-formed amplified oligo-tagged beads, and NGS based readout, at an affordable end-user cost. SB A was used in the examples below for pathogen diagnostics of PCR products derived from genomic DNA extracts based on detection of unique biomarkers. SBA technology could be applied to a multitude of applications where conventional microarrays might otherwise be used, including but not limited to: i) pathogen diagnostics, ii) SNP detection, iii) gene expression profiling, iv) miRNA detection, v) comparative genomic hybridization (CGH), vi) chromatin

immunoprecipitation on chip (ChIP), vii) DNA adenine methyltransferase (DamID), viii) DNA barcode screenings, ix) alternative splicing (including fusion genes), and x) short tandem repeats (STR) typing and xi) tiling arrays 2 ' 19-"21.

SBA uses a suspension array of pre-formed and predetermined constructs, (described for convenience as "oligo-tagged beads") wherein targets that are recognized bind to those constructs, and such binding results in separation of the constructs through a property of the target. The property of the target may be a sequence and/or an attached tag. Thus there is provided a composition in the form of a plurality of beads (microparticles) that have a dense lawn, i.e. high copy number, of essentially identical oligonucleotides on each bead. These constructs may be formed by emulsion PCR process of synthetic oligonucleotide templates.

The present assay utilizes a sequence-based readout that replaces various forms of optical readout such as used in conventional microarrays. It can be implemented with label- free NGS digital readout of known sequence ID traces (i.e. barcodes). It can be implemented so that selected oligo-tagged beads can be placed directly into a sequencing machine. As an alternative to microarray applications, SBA offers the following advantages: i) highly scalable, ii) digitally quantitative, iii) specific and sensitive, and iv) rapid. In a proof-of-concept study, a model assay was developed for genotyping of HPV, formatted for the NGS Ion Personal Genome Machine (PGM). HPV is well known for the existence of multiple genotypes 22 and for the association of certain HPV infections with cervical cancer. Based on their oncogenic potential, HPV subtypes are classified as high-risk or low-risk HPVs. In one embodiment, the model SB A assay targets ten high-risk HPV genotypes commonly associated with cervical cancer progression 23 , and has been

successfully applied here to screen clinically relevant genomic DNA extracts from cervical tumor biopsies.

As shown in Figure 1, a bead 110 (which represents a sub-population of identical beads) has attached to it multiple, identical, single- stranded oligonucleotide tags, with one, 108, being shown. Each oligonucleotide tag 108 has four segments: a linker segment 100 proximal (adjacent) to the bead, for attachment of the oligo to the bead; the linker is shown in this example as a short nucleic acid sequence; other linkers may be used, depending on the nature of the bead, the assay application, and the arrangement of the segments; a target specific binding segment 102, dashed line, for binding to a specific target in a sample (e.g. a sequence complementary to a viral DNA target for specific binding by hybridization); an identification segment ("ID trace") 104, dotted line, having a sequence uniquely identifying the subpopulation of oligonucleotide tags associated with the target specific binding segment 102, and also having an optional "key" sequence if appropriate for the NGS sequencing method used; and a primer segment 106 for binding to a sequencing primer which is used at a later stage in the assay to sequence the oligo tag. The oligonucleotides 108 are preferably designed to be sequenced directly, on the bead, without further nucleic acid modification. That is, an NGS sequencing primer can attach directly to segment 106 and NGS may be used to sequence the oligonucleotides 108, at least as far as the ID trace 104. As illustrated in Figure 1, a number of oligo tags 108 are first prepared; then in step a, a bead 110 is introduced and the oligo-tags 108 are bound to the bead until, in step b, the bead has multiple single stranded oligo-tags 108 attached, where each oligo-tag comprises segments of linker- target specific binding segment- ID trace- primer segment. As discussed further below, this order may be varied; for example, the target- specific binding segment may be placed on the 3 '-end, or the oligo tags may be linked at the 5 '-end to the bead. In Figure 1, the oligo is attached at the 5' end to the bead, and therefore the primer-binding segment is at the distal end of the oligo and the target binding segment is adjacent to the linker segment. Typically about 800,000 oligo tags will be attached to a 2 μηι single bead ' . Actual oligo copy number depends on bead size and type and may vary between about 100 and 5,000,000. For example, for Roche 454 systems, the beads could be about 20-50 μιη and have about 1-5 million oligos on each bead. The beads in a population will be consistent in the approximate number of oligos attached.

Referring now to Figure 2, subpopulations of beads in a total population ("library") will differ among themselves as to target specific binding segments 102 and ID traces 104. The subpopulations of oligo-tagged beads prepared as described above in connection with Figure 1 are placed in a mixture with different subpopulations of beads in suspension, with the target DNA. Then beads that bear one or more target sequences that have hybridized to the target specific binding segment(s) are separated. In particular, as shown in Figure 2, oligo-tags are prepared and attached to beads as shown at 202 and discussed in connection with Figure 1. As shown at 204 and also in Figure 1, the oligo-tags are present as a population of identical oligos on a given bead; an initial number of oligo(s) may be attached, then amplified so as to create the population of identical oligos on a given bead. A number of identical oligo-tagged beads will form a subpopulation. If there are a number n of different possible targets possibly in the sample (e.g. 10 genotypes of HPV virus) there will be n (e.g. 10) different subpopulations of beads, with unique identifier sequences and unique target identification sequences in each subpopulation, but common primer segments and linker segments among the subpopulations. The subpopulations with different target segments are designated "x", "y", and "z" in Figure 2. After the desired population of oligo-tagged beads is formed, as shown at 208, a sample is mixed with the population of beads. The sample DNA in 209 has been previously amplified (however, sample DNA amplification is not required). As shown in the figure, the sample at 209 has only x and z species present. The target DNA in a sample is biotinylated to allow target DNA, hybridized to the target region of an oligo-tag on the beads, to also be bound to a streptavidin-coated magnetic bead 212 (or any streptavidin coated solid surface, e.g. a pipette tip or like). Thus, a three -part cluster is formed of the oligo-tagged bead plus the target plus a magnetic bead attached to the target through the biotin. As shown at 210, the target DNA complementary to the oligo-tags binds to the bead. The target DNA, being biotinylated, then can be complexed with streptavidin-coated beads 212 to form the above-mentioned clusters. The clusters are isolated in container 213 by separating the magnetic bead- linked complexes from the beads that are not linked to targets and, as a result, did not form a cluster with a magnetic bead. As shown, biotinylated target molecules containing x and z biomarkers hybridize to the oligo-tagged beads. The magnetic beads 212 are removed prior to sequencing and thus magnetic beads 212 are not shown in container 213. The bead 110 formed at the beginning at the assay is suitable for direct introduction into a bead-based sequencing machine, without any further preparation.

Biotin from the target complex may also be removed during the step of magnetic bead removal, as NaOH will remove both entities. The biotinylation of target molecules, done prior to contact with the oligo-tagged beads, can be conveniently carried out by PCR.

However, there are other ways to attach biotin such as employed with terminal transferase followed by polymerase extensions including biotinylated dNTPs. Biotinylation kits are also commercially available at Thermo Fisher Scientific, Inc. (Massachusetts, USA). In addition, alternative affinity molecules, i.e. digoxigenin (DIG) and anti-digoxigenin antibodies and other biotin-streptavidin similar labels also may be used to treat the sample nucleic acids. Once the clusters are isolated by magnetic separation, they are identified by the ID trace sequence. This can be done by a variety of NGS (next generation sequencing) methods. As shown at 214, sequencing primers that bind to the 3' priming segment are introduced to initiate a sequencing protocol as commercially available from Roche 454, Illumina, Ion Torrent/Life Technologies, etc. Only the "barcode" or ID trace needs to be sequenced. It is not necessary to sequence any part of the target DNA. The target DNA sequence is identified by virtue of its hybridization to the target specific binding segment on the oligonucleotides making up the corresponding oligo-tagged bead.

The sequencing step is carried out with sequencing primers 214. The oligo-tagged beads are themselves compatible with bead-based sequencing methods and may be introduced into a commercial sequencer such as an Ion Torrent PGM™ benchtop sequencer without further treatment. The sequencing need only cover a short sequence length

(equivalent to the length of the ID trace) and may be carried out in a variety of ways. As noted above, each ID trace is associated with a different subpopulation and target specific binding segment. The total number of beads used in a given assay depends on the detection resolution desired. As shown at 214, a sequencing primer complementary to the 3' primer segment 106 (Figure 1) is added at a high concentration to saturate all beads, and due to the high density of oligo tags on a given bead, each selected bead self-amplifies its own signal. Sequencing of the ID trace identifies oligo tags in parallel, in this case x and z in a 3:2 ratio.

The number of clusters (bead + target + bead) passing through the magnetic selection process can be counted; this allows for digital quantification of the targets. After the cluster has been formed and separated, magnetic beads are removed and the selected oligo-tagged beads are sequenced. Removal of the magnetic beads will generally also involve removal of the biotin-tagged target; only the oligo tags are sequenced. As shown at 216, data from the sequencing step is processed by software and presented in graphical form, with metadata (such as comparisons among different ID trace reads) in a conveniently viewable variety of computer formats, such as PDF. The illustrated device at 218 is a smart phone displaying quantitative results of x, y, and z (a histogram, or "Sphixogram"). It can be seen that there is a 3:2 ratio of x to z graphically displayed on the device, and only a background level of y shown in the display. The display is in the form of a histogram.

Genotyping The SBA assay experimentally demonstrated here contains target specific binding segments that distinguish between strains of HPV. There are over 100 strains of HPV, which can be identified by their divergent amino acid and nucleic acid sequences and which could be distinguished using the present methods. Similarly, the target specific binding segments could also be designed for genotype selection between other viruses, such as HIV, as described e.g. in Genotypic Testing for HIV-1 Drug Resistance, http(colon slash

slash)hivinsite.ucsf.edu /insite?page=kb-03-02-07.

HCV genotyping may also be carried out. This is recommended prior to the start of therapy as the HCV genotype predicts the likelihood of treatment response, influences antiviral dosing, and determines the optimal duration of treatment with pegylated-interferon alpha plus ribavirin (Hadziyannis et al., Ann Intern Med. 2004; 140(5):346-55). The genotype of a tumor is important due to the heterogeneity of many cancer cells. These genotypes may be in the form of single nucleotide polymorphisms (SNPs). See, for example, Kiyohara et al., "Genetic polymorphisms in the nucleotide excision repair pathway and lung cancer risk: a meta-analysis," Int J Med Sci. 2007 Feb 1;4(2):59-71. Further mutations that may be detected are described in the database of the Cancer Genome Project, http (colon slash slash) www (dot) sanger.ac.uk/ research/projects/cancergenome/. SNP detection may be carried out with target specific binding segments directed to the SNP of interest, as described, for example in Arnold et al., "SNP Detection," US 6,410,231.

The construction of a bead library The present bead libraries can be constructed using an emulsion PCR strategy to attach a dense lawn of oligo tags to the beads. This strategy is described in detail in the literature, for example, in Church et al. "Polony fluorescent in situ sequencing beads," US 7,425,431. The multi-parallel advantage allows for quick production of large sets of oligos and their simultaneous attachment to the beads. See, for details, e.g. Kim et al., "Polony Multiplex Analysis of Gene Expression (PMAGE) in Mouse Hypertrophic Cardiomyopathy," Science 316: 1481-1484 (2007).

The bead-polony method relies on a biological process of an enzyme producing the DNA, with increased risk of beads with suboptimal DNA copy number. In addition, the fundamental methodology behind emulsion PCR benefits a locked oligonucleotide design, with the priming-segment at the 3'-end of the clustered oligonucleotides on the beads.

Producing several libraries, and merging them (Figure 3B), can assist in lowering the variability parameters (Table 1) when considering larger scale production.

In the current design, the oligonucleotides used for the bead library were, on average, ~115-bp in length (Table 2). These oligomers add complexity in terms of costs and inherent need for extensive purification, and thus are produced in lower quantities. Variants with a shorter linker region (average size 85-bp) were also investigated and successfully

implemented, making the longer versions optional depending on library complexity and quality parameters desired.

In-house oligonucleotide synthesis was employed without extensive purification protocols; while sufficient for an initial proof-of -concept, the lower grade of quality accounts for variability in total concentration of full-length probes. This phenomenon is clearly reflected in the library distribution (Figure 3A), and thus the assay could be further optimized by more extensive purification of the oligonucleotide templates, and inclusion of internal normalization standards. The synthesized oligonucleotides, designed with the segments shown in Figure 1, i.e. linker, identifier segment-target-specific binding segment, and primer segment, were attached to beads in high copy number. The oligonucleotides may be attached directly to the beads by covalent linkage in a number of ways. For example, protocols for bead enrichment and bead capping are available from http(colon slash slash) www(dot)polonator.org/index.htm.

Different kinds of beads are applicable for making the oligo-tagged beads, including silica beads (e.g. those from Bangs Laboratories, Inc.), magnetic beads (e.g. those from

Invitrogen/Dynal), polymeric beads (e.g. those from Rapp Polymere GmbH). One may also use gold or gold coated spheres (10-100-nanometer, thiol group), avidin/streptavidin coated magnetic beads (<10 μιη, biotin group), TentaGel beads (Rapp Polymere GmbH, Germany, 1-100 μιη, 3, 10, 30 μιη, NH2 or OH conjugation chemistry), Sephadex beads (20-50, 40-120 μιη, carboxyl, NH2 conjugation chemistry. Detailed protocols may be found, e.g. in the Integrated Data Technologies pamphlet "Strategies for attaching oligonucleotides to solid supports," found online at http (colon slash slash)www (dot)

idtdna.com/pages/docs/technical-reports/strategies-for-attaching-oligonucleotides-to-solid- supports.pdf. The beads were obtained from Life Technologies, which uses a chemistry of molding acrylamide gel together with oligonucleotide primers (PB primer), which are then used as emulsion PCR templates. These gel-beads are available commercially from Life Technologies.

In an effort to achieve a more even probe distribution, double-stranded DNA templates were used. Notably, however, both single- stranded DNA template counterparts provided an adequate template source for cost-efficient high-throughput applications. Since the oligonucleotide tag templates have many common regions (Table 2), one could employ a ligation assembly strategy of smaller oligonucleotides to increase oligonucleotide quality and lower production cost 24 ' 25 , or produce longer oligonucleotides via a PCR derived strategy 26. The ID trace segment of the oligonucleotide is included for a rapid readout process and for future advantages, e.g. a multidimensional ID trace strategy 27 , i.e. a specific panel of biomarkers used with different patient specific sets of ID traces (e.g. patient A with biomarkers X, patient B with the same biomarkers X, etc.). This would lower the overall cost per sample by screening multiple patients on the same chip. Data sorting and interpretation would be nearly instantaneous when using Sphix algorithms with different ID trace information (dual barcode tags). Furthermore error-correcting barcodes 28 could partially compensate for sequencing induced errors, and thus lower the uncalled bead parameter (Table 1). Shorter ID traces would allow for shorter oligos attached to the beads, and more rapid sequencing runtimes with an overall performance enhancement of the assay.

The exemplified embodiment of the invention successfully operated at a 1-hour hybridization protocol. When performing reproducibility studies, clear differences were observed in the amount of beads loaded during reproduced identical target reporting sets (Table 1). However when focusing on the bead library fraction of total sequencing reads instead of actual read-count, the data was very robust (Figure 3A, Example 1).

In the sensitivity and dynamic range studies (Figure 4A -D), target concentrations could be distinguished in the 10-100-femtomolar ranges with upper oligo-tagged bead saturation at 100-femtomolar. With an estimated bead-loading efficiency of -10%, only a subset of the oligo-tagged beads can be detected. This fraction is dependent on total oligo- tagged bead population in the set, and could be further optimized with improved loading schemes and chips supporting more wells. Target-to-bead ratio is essential and will affect the dynamic range of the assay. Any given bead requires a minimum finite number of targets in order to be activated and selected. Targets in abundance will saturate the beads, which would allow for an increase in the upper saturation level by simply using more beads. Further enhanced assay sensitivity could be achieved by using bead libraries of a higher quality, optimum target-to-bead ratio, and by exploring alternative strategies for capturing the target- bead complexes. Future improvements in sequencing sensors could allow for nanoparticle- sized beads with lowered demand on numbers of clustered oligonucleotides. Such improvements would greatly increase target surface-area display and possibly target binding capacities, with sensitivity near the nanoparticle bio-barcode assay 12 , thus circumventing need for prior nucleic acid amplification. Sequencing of ID trace/readouts

The Ion 314 chip was used throughout the examples below, due to cost benefits. This is a commercially available product available from Ion Torrent, a Life Technologies Company. It is available as a kit that contains chips for 8 sequencing runs using the Ion Personal Genome Machine (PGM) System, a semiconductor-based sequencer. The Ion 314 chip detects polymerase-driven base incorporation and translates this information into digital form. It provides easy handling and loading of templated Ion Spheres (acrylamide beads), which are used in the sequencing machine. The oligo-tagged beads of the present invention can be directly loaded onto such a chip and be used for sequencing in a manner equivalent to the manufacturer's Ion Spheres. At present, the Ion PGM15'16 is compatible with the commercially available Ion 314 chips having -1.3 M wells, the Ion 316 chips having -6.3 M wells, and the Ion 318 chips having -11.3 M wells. The Ion Proton instrument works with a similar methodology to the Ion PGM instrument, and an SBA assay could be formatted for its use.

One key advantage with the SBA methodology is that it does not require novel instrumentation, and the general principal can instead be formatted to a range of existing instruments and technological readout methods. The current configuration is compatible with the 454 Life Sciences instruments GS FLX+, and GS Junior (http://www.454.com), the Life Technologies SOLID platform (htttp(colon slash slash) www.lifetechnologies.com), and upcoming technologies such as Fluorogenic Pyrosequencing , the DNAe Genalysis platform 30 (http(colon slash slash) dnae.co.uk), and the GenapSys GENIUS platform 31 (http(colon slash slash) www (dot) genapsys.com). The present system allows for a very high degree of multiplexing with digital quantification of each target while offering general nucleic acid readout. The oligo-tagged beads may complement PCR as a downstream step or possibly eliminate the need for upstream amplification of target sequences. The present system also shortens the time of analysis and the number of instruments and reagents required in an assay, due to multi- parallel processing capacity. The multiplexing can provide for at least 10 different subpopulations of microparticles, or at least 20 different subpopulations, at least 30 different subpopulations, 30-100 subpopulations, etc.

Possible applications range from simple verification of the presence of a set of nucleic acid fragments to more sophisticated in- vitro diagnostics (IVD). Such molecular diagnostics would aim for determining the nucleic acid profile and composition of a patient sample e.g. detection of infectious diseases or routine screening of genetic disease or cancer where a multiple set of biomarkers (10-10,000 or more). Infectious agents could be of bacterial or viral origin, where both detecting the presence as well as quantification of multiple viral species is of significant clinical value and of crucial importance in a marketable assay. Other molecular diagnostic applications include, tumor diagnostics, genetic disease traits, pharmacogenomics, forensic, and prenatal diagnostics. In alternative embodiments, such as described in Example 7, antigen/antibody (or similar) reporting systems may be used. EXAMPLES

EXAMPLE 1: Oligo-tagged bead library construction A library as constructed here contains an equimolar set of beads of different specificities with a lawn of oligonucleotides (also referred to as "oligo-tags") on it. Each bead contains a high copy number cluster of unique single-strand oligos, as shown e.g. in Figure 1 and Figure 2. For each targeted genotype, unique oligonucleotide with distinct target specific binding segments and a corresponding ID trace were used to generate libraries of beads. Also a longer /extended linker region 100 (Figure 1) is used to further enhance the library quality. Subpopulations were included to constitute the bead library. After evaluating different strategies for bead library construction, the fabrication was performed via an emulsion PCR strategy in order to clone the oligos onto the beads.

To investigate construction reproducibility, identical bead libraries were generated from six separate emulsion PCRs and sequenced individually to examine library variability (Figure 3A, and Table 1).

Table 1: Key quality parameters for a 10-plex HPV genotype specific SBA assay

Figure imgf000025_0001
b

avg. s.d.

1. Oligo-tagged bead 4.75 x 10s + 8.77 xl04

population

2. Polyclonality 53.49% + 2.00%

3. Low quality 19.86% + 4.78%

4. Uncalled oligo-tagged 2.52% + 0.32%

beads

5. Called oligo-tagged 24.14% + 3.00%

beads

6. Bead density N/A N/A

* Average (avg.) and standard deviations (s.d.) for assay parameters (1-6) (a) Library variability of six identical individually constructed bead libraries; (b) Six identical SBA assay quintuple HPV plasmid target selections using one bead library source.

The Ion Personal Genome Machine (PGM) software Torrent Suite automatically generates parameters 1-3 during a sequencing run, 4 and 5 are generated during Sphix analysis, and 6 was quantitatively estimated via fluorescent readout data. In other words, results from processing by Sphix renders parameters 4 and 5, i.e. Uncalled and Called oligo- tagged beads respectively (processed in block 804, Figure 8). Parameters 1-3 are given by the Ion Torrent Suite but could also be produced by Sphix (block 802, Figure 8) if given access to raw data by the sequencing instrument. Many factors contribute to library variability between sequencing runs, such as i) oligonucleotide template quality, ii) emulsion PCR construct yield, iii) sample source quality, iv) chip-loading, v) batches of kits and chips and vi) handling. Additional optimization of bead library quality, salt reaction conditions, and bead densities could further enhance overall assay performance and may allow for the development of a reproducible and rapid (<5 min) protocol. The bead density had a significant effect on the signal-to-noise ratio, which might be caused by activated (i.e. complexed with a target) beads clumping and falsely co-selecting unwanted neighboring beads, and or general steric hindrance due to high concentration of beads. However, the bead density calculations were based on fluorescent measurements and estimations, and should not be considered absolute values. Also, optical effects caused by the beads were not accounted for in the fluorescent measurements.

Oligo-tagged bead population (parameter 1) denotes the number of beads that yield a strong enough sequencing read signal. Polyclonality (parameter 2) denotes filtering of oligo- tagged beads with mixed sequence reads due to beads containing two or more different sequences. Low quality (parameter 3) denotes filtering of sequence reads with low signal quality and other abnormalities. Uncalled oligo-tagged beads (parameter 4) denote obtained sequence reads that do not match any of the ID traces (caused by sequencing errors and alike). Called oligo-tagged beads (parameter 5) denote obtained sequence reads that match ID traces. Bead density (parameter 6) denotes a quantitative estimate of the amount of beads/μΐ for a constructed bead library. Bead density measurements were not performed for column (b) of Table 1.

The emulsion PCR reaction generated approximately 100 μΐ of bead library with an estimated average bead density of -5.93 xlO5 beads/μΐ. A fixed bead library volume of 10 μΐ was used per SBA reaction (i.e. total of -5.93 xlO6 beads). However, the average bead population in a sequencing run was only 6.75 xlO5, resulting in a loading efficiency of only -10%, and, accordingly loss of resolution. In an ideal case for a 10-plex library, each bead type would constitute 10% of all the called beads. The observed average bead frequency range was 6.72% to 12.75%, attributable to differences in sequence composition, template concentrations, and handling.

In addition, three different template resources (Table 2) were investigated: i) a synthetic single-stranded DNA oligonucleotide with a linker sequence complementary to the attached bead primer (the positive strand), ii) its reverse-complement strand (the negative strand), and iii) complementary strands annealed to form a double- stranded DNA template. All template variants were able to generate bead libraries containing the correct sequence composition; the double- stranded template was used for this assay.

A variety of oligonucleotide template concentrations were explored; however, it proved difficult to lower the polyclonality factor, which is an inherent problem with the emulsion PCR method. By pooling the ready-made libraries, library composition could be evened out, and is a recommended strategy for standardized testing (Figure 3B). Once produced, the library was refrigerated and stored for up to one month. Bead libraries sequenced directly after production and those sequenced within a one-month time frame demonstrated little or no degradation in quality, which could be tracked via fluorescence measurements of the bead-clustered oligonucleotides and via sequencing runs.

The oligonucleotides [100 μΜ] were synthesized in-house (Stanford Genome

Technology Center, Palo Alto, CA), and subsequently pooled with corresponding reverse complement in IX STE-buffer [10 mM Tris (pH 7.6), 50 mM NaCl, and 0.1 mM EDTA] . Duplex templates were annealed in the GeneAmp PCR system 9700 (Life Technologies, Carlsbad, CA) at 95°C for 2 minutes followed by slow cooling until room temperature was reached. Following the hybridization step, all template duplexes were pooled at equimolar concentrations, and further diluted to [32 pM] to ultraPURE H20 (Life Technologies,

Carlsbad, CA). The diluted pool later served as the bead library template for emulsion PCR. The emulsion PCR was performed using the Ion One Touch DL System with the Ion

OneTouch 200 Template Kit v2 DL (Life Technologies, Carlsbad, CA), using ~2-micron acrylamide beads15'16, in order to be compatible with the Ion PGM instrument (Life

Technologies, Carlsbad, CA). The automated Ion OneTouch ES (Life Technologies,

Carlsbad, CA) emulsion PCR library enrichment and cleanup procedure yields approximately 100-μ1 of bead library stock in IX Wash/Storage Buffer [PBS, and 0.2% Tween-20 (pH 7.7)].

For each SBA assay reaction, a fixed amount of 10-μ1 of bead library was used. Bead density estimates of the constructed bead libraries, were based on fluorescently based quantitative measurements using a single- stranded DNA assay on the Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA). Each constructed bead library was measured in three replicates of 3-μ1, with three independent measurements. Furthermore the fluorescent measurements provided means for tracking degradation of the bead-clustered

oligonucleotides for storage purposes, which was notable post one-month time frame. Quality control was also performed by sequencing the native libraries with the Ion PGM instrument when using the Ion 314 chips with the Ion PGM 200 Sequencing Kit (Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. The six bead libraries (Figure 3A, and Table 1) were later merged at equimolar concentrations to lower variability and refrigerated for up to one month (Figure 3B).

EXAMPLE 2: Oligo-tagged bead library construction for HPV detection A model 10-plex SB A assay was designed to genotype the ten high-risk HPV genotypes (HPV- 16, HPV- 18, HPV-33, HPV-35, HPV-39, HPV-45, HPV-52, HPV-56,

HPV-58, and HPV-59) commonly associated with cervical cancer progression 23. A 10-plex HPV bead library was constructed for multiplex genotyping of PCR products derived from the nested PCR product of general PCR primer-sets PGMY09/11 and GP5+/6+32. With an alignment of GenBank sequences for respective HPV genotypes (HPV-16; HQ644299, HPV- 18; GQ180792, HPV-33; HQ537707, HPV-35; JX129488, HPV-39; M62849, HPV-45;

EF202156, HPV-52; HQ537739, HPV-56; EF177181, HPV-58; HQ537768, and HPV-59; EU918767) genotype-specific hybridization segments were designed. The hybridization segments were chosen from the candidate GP5+/6+ sequences based on i) 20-25-bp of length, ii) melting temperatures (Tm) of 52°C < Tm < 61°C, and iii) having a GC content of 45% < GC < 60%. Sequences with hairpins or more than 5 single repeats were eliminated. To ensure relative uniqueness, all sequences were compared to one another using megaBLAST (v 2.2.10) to eliminate any with an 80% match in 20 bases. Cloned HPV-plasmids for HPV-16, HPV-18, and HPV-45 were kindly provided by

Dr. E.M. de Villiers (DKFZ, Heidelberg, Germany); HPV-33, and HPV-39 from Dr. M. Favre (Institute Pasteur, Paris, France); HPV-35, and HPV-56 DNA from Dr. A. Lorincz (Digene Corporation, Gaithersburg, MD, USA), HPV-52 DNA from Dr. W. Lancaster (Wayne State University School of Medicine, Detroit, ML USA), and HPV-58, and HPV-59 DNA from Dr. T. Matsukura (National Institute of Health, Tokyo, Japan). The HPV plasmid concentrations were normalized at 100-ng/ml using an ND-100 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA). PCR amplification was performed using the nested PCR with general primers PGMY09/11 and GP5+/6+ 32 according to a previously described protocol 33 , with an initial plasmid DNA concentration of 100-ng/ml. The GP6+ primer carried a 5 '-end biotin molecule for cleaning and selection purposes. A magnetic workstation, the Magnatrix 1200 (NorDiag, Biotrin International Ltd., Dublin, Ireland) was employed for automating the lab work. Running custom-made scripts, the robot generated single-stranded templates of the PCR-amplicons. M-270 beads (Life Technologies, Carlsbad, CA) were used for immobilizing the DNA followed by magnetic collection and washing, initially in IX TE- buffer [10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA] followed by thorough washes with purified H20 [18.2 ΜΩ-cm at 25 °C] (EMD Millipore, Billerica, MA) and NaOH [0.1 M] with constant mixing. Finally, the single stranded products were released by heating to 80°C for 1 second in purified H2034.

Oligonucleotide templates for the emulsion PCR construction of a 10-plex HPV SB A assay targeting HPV genotypes HPV-16, HPV-18, HPV-33, HPV-35, HPV-39, HPV-45, HPV-52, HPV-56, HPV-58, and HPV-59 are listed in Table 2.

Table 2: HPV Oligonucleotide templates for emulsion PCR construction

Figure imgf000030_0001
HPV-59 TACTGAGCTA (SEQ ID CACACACAGAAAGATTGGTGCTGCG NO: 20) (SEQ ID NO: 21)

Linker ATCACCGACTGCCCATAGAGAGGCTGAGACTGCCAAGGCACAC segment AGGGGATAGG

(SEQ ID NO: 22) (common to all beads)

The oligonucleotides are represented in the 5 '-3' directionality, and are

compartmentalized into five different sections. Sequence follows assembly of compartments, i.e. 5'-Priming segment - Key - ID trace X - Target specific binding segment X - Linker segment -3'. Each oligonucleotide was synthesized as both the strand displayed in the table, and its reverse complementary strand to form duplex DNA templates. The priming segment decodes where the sequence primer attaches. The Key is for the Ion Personal Genome Machine (PGM) sequencer decoding and quality control purposes during the sequencing run. The ID trace is unique to each oligo-tagged bead subset directed to a particular biomarker target. A subset of 10 out of a pre-defined library of 153 Multiplex Identifiers (MIDs) from 454 sequencing GS FLX Titanium chemistry was used to provide ID traces for this proof-of- concept study. The GS FLX system is a commercial product supplied by 454 Life Sciences, a Roche Company. The Titanium chemistry describes kits supplied for emulsion PCR preparation of sequencing libraries for use with the 454 product. Further details may be obtained at http (colon slash slash) www (dot)454.com/products/gs-flx-system/index.asp. The target specific binding segment of the oligo tag hybridizes to a genotype- specific sequence present in the LI gene of the HPV-genome. The linker was used to clone the fragments onto the beads during the emulsion PCR step. EXAMPLE 3: HPV SBA assay

The SBA assay is executed by; i) target specific hybridization binding to activate the oligo-tagged bead, ii) oligo-tagged bead immobilization when linked to magnetic beads, iii) magnetic bead selection of activated oligo-tagged beads (i.e. oligo-beads complexed with target DNA containing an affinity molecule), and iv) release of selected beads. A 10-μ1 aliquot of merged bead library stock was further diluted to a total of 30-μ1 in IX Annealing Buffer [PBS, supplemented with 0.2% Tween-20 (pH 7.7)]. Target DNA was added in 10-μ1 aliquot in IX Annealing Buffer to make the total reaction volume to 40-μ1. The mixture was sonicated at maximum intensity for 10 seconds using the desktop water bath and sonicator B2500ADTH (VWR, Radnor, PA). Sonication ensures maximum mixture and target exposure. Target hybridization was accomplished using an initial heating at 95 °C for 2 minutes with a GeneAmp PCR system 9700, then immediately transferred to a Thermomixer R & MTP Microblock (Eppendorf, Hamburg, Germany) programmed at 70 °C, followed by slow cooling to 45 °C, with constant shaking at 1300 rpm to ensure the beads avoided sedimentation for maximum target exposure.

The activated beads were then incubated at room temperature for 5 minutes with continuous mixing with 13-μ1 streptavidin coated paramagnetic MyOne Cl-beads (Life Technologies, Carlsbad, CA) in IX Annealing Buffer. Magnetic selection was performed manually with IX Wash/Storage Buffer and IX Elution buffer [0.1 M NaOH and 0.1% Tween-20] while using a Dynal Invitrogen bead separation magnetic rack (Life Technologies, Carlsbad, CA). The final product was further washed and concentrated with IX Annealing Buffer to yield a total volume of 3 μΐ ready for sequencing primer annealing and sequencing readout with Ion PGM instrument when using the Ion 314 chips with the Ion PGM 200 Sequencing Kit according to manufacturer instructions. The Sequencing runs were programmed at a constant of 100 flows for all reactions to ensure full-length ID traces.

EXAMPLE 4: Genotyping of clinical samples

The main objective was to screen samples with unknown viral loads and profiles using PCR products derived from authentic clinical samples. Standardized nested PCR using general primer sets PGMY09/11 and GP5+/6+ 32 was performed on 20 samples (extracted genomic DNA from cervical tumor samples).

Genomic DNA extracts from 20 tumors from different females were obtained commercially (Oncomatrix, Inc., San Marcos, CA) with accession codes OM-1078, OM- 1272, OM-1299, OM-1301, OM-1452, OM-1464, OM-1530, OM-1569, OM-1668, OM- 1741, OM-1751, OM-1848, OM-1854, OM-1967, OM-1980, OM-2006, OM-2059, OM- 2215, OM-2257, and OM-2258. The samples were PCR amplified using the nested PCR with general primers PGMY09/11 and GP5+/6+ 32 ' 33 , with an initial sample concentration of 40 ng/ml genomic DNA. The Magnatrix 1200 was programmed to generate single-stranded templates of the PCR-amplicons. As a comparator, PCR-amplicons were directly sequenced by Pyro sequencing ' . The presence of HPV was confirmed in 19 of the 20 samples, and genotype results achieved by the two methods were in agreement, with the exception for samples OM-2215 and OM- 2258 (Figure 5E and 5F). A dual co-infection of HPV-16 and HPV-18 in OM-2215, and a dual co-infection of HPV-16 and HPV-45 in OM-2258 were detected with the SBA assay, whereas Pyrosequencing only detected the dominant genotype in each sample (HPV-18 and HPV-45 respectively). This was due to a higher sensitivity, and dynamic range for the SBA assay. To confirm these findings, separate PCR screens were performed employing genotype- specific primers for HPV-16, HPV-18 and HPV-45, which in all cases generated the correct products with accurate sequence information. Among the remaining 18 samples, ten were positive for HPV-16 (OM-1078, OM-1272, OM-1301, OM-1464, OM-1530 (Figure 5A), OM-1751, OM-1967, OM-2006, OM-2059, OM-2257), four for HPV-18 (OM-1452, OM- 1741 (Figure 5C), OM-1854, OM-1980), one for HPV-45 (OM-1848, Figure 5D), two for HPV-59 (OM-1569, OM-1668 (Figure 5B), and one sample returning as HPV- negative (OM-1299), using either method. Variability in SBA assay yield was exacerbated by differences in PCR product yield, reflecting the original quantity of viral genome present. However since the 20 clinical samples were analyzed using bead libraries derived from different emulsion PCR constructs (due to their sheer number and material needed), and run in separate sequencing rounds, library variability effects must be considered. Figure 5G shows the relative amounts of the various HPV strains in the particular patient samples.

In the cases of OM-2215 and OM-2258, PCR-amplicon Pyrosequencing24'35 detected only a single (dominant) genotype, while the SBA detected two genotypes in each sample (HPV-16 and HPV-18 in OM-2215 and HPV-16, and HPV-45 in OM-2258). To confirm these findings, secondary PCR amplifications were performed using the genotype specific Multiple Sequencing Primer (MSP) MSP- 16, MSP- 18 and MSP-45 in separate reactions together with GP6+35, followed by Pyrosequencing using MSP-16, MSP-18 and MSP-45 as sequencing primers.

EXAMPLE 5: Data management and interpretation Experimental data generated by the Ion Personal Genome Machine (PGM) software the Torrent Suite 2.2 (Life Technologies, Carlsbad, CA) was automatically retrieved from the Torrent Server using HTTParty (http (colon slash slash) httparty.rubyforge.org/) including FASTQ sequence data and sequencing run metrics of importance. Parsing the FASTQ-file was performed using modules from BioRuby36. Sphix performs barcode de-multiplexing by matching a given set of 10-bp ID traces with the sequence at the very beginning (5 '-end) of each read, followed by binning and digital quantification of beads. Only perfect matches are scored, counted and added to the results matrix. Based on the results, a Sphixogram was generated, revealing the genotype profile of the sample. The Sphixogram, together with accompanying metrics and metadata was communicated via Prawn (http(colon slash slash)prawn.majesticseacreature.com/) a PDF-writer tool available via RubyGems.

The software tool (an in-house software solution), Sphix was developed to complement the SBA assay with effective condensation of experimental data, and to easily communicate output information in an understandable format (Figure 8). Sphix handles all data processing, starting with data acquisition from the Torrent Server, provides algorithmic profiling of the sample, and produces a comprehensive, structured report of analyzed results. Sphix generates platform-independent bar-histograms, referred to as Sphixograms, in a portable document format (PDF) for a user-friendly experience on laptops or handheld devices. Sphix was implemented in Ruby (http (colon slash slash) www.ruby-lang.org) encompassing all tasks of the data-processing pipeline from initial upstream data retrieval to downstream final output of information and result. The tool was customized for current requirements and in-house set-up, and was written with the aim of facilitating future improvements, e.g. compensating for differences in bead distribution 21 (Figure 3A).

Using Sphix, SBA assay screens were carried out on a total of 20 samples (genomic DNA extracts from cervical tumors) (Figure 5). The bead bars in the Sphixograms are represented as frequencies of total called oligo-tagged beads. The Sphixogram figure dimensions are formatted for easy viewing on an iPhone display (or similar). Positive genotype calls are colored black, and the highest bar is set at a fixed bar height, and used as normalization for all bar heights within a sample. In positive samples, all bars with representing fractions above 15% are colored, rendering co-infections in samples OM-2215 and OM-2258 obvious, while background noise in all samples remains in white. OM-1299 was negative for all ten investigated HPV genotypes; i.e. no peak signal was distinguishable from background noise. For sensitive visualization, the bar heights of sample OM-1299, were set to approximately average background peaks from the other samples. Metadata values (defined in Table 1) are as follows for each Sphixogram: Sample reporter polyclonality low Uncalled reporters Called reporters population quality

OM- 62851 38806 10553 1863 (3.0%) 11629 (18.5%) 1530 (61.7%) (16.8%)

OM- 231781 143349 48263 4189 (1.8%) 35980 (15.5%) 1668 (61.8%) (20.8%)

OM- 53660 32735 12659 967 (1.8%) 7299 (13.6%) 1741 (61.0%) (23.6%)

OM- 121564 65536 24360 1996 (1.6%) 29672 (24.4%) 1848 (53.9%), (20.0%)

OM- 98503 59800 18194 1596 (1.6%) 18913 (19.2%) 2215 (60.7%) (18.5%)

OM- 308226 177725 41921 5650 (1.8%) 82930 (26.9%)

2258 (57.7%) (13.6%),

Results were called as: ten single infections for HPV-16 (OM-1078, OM-1272, OM- 1301, OM-1464, OM-1530, OM-1751, OM-1967, OM-2006, OM-2059, and OM-2257), four single infections for HPV-18 (OM-1452, OM-1741, OM-1854, and OM-1980), two single infections for HPV-59 (OM-1569, and OM-1688), one single infection for HPV-45 (OM- 1848), one dual co-infection for HPV-16, and HPV-18 (OM-2215), one dual co-infection for HPV-16, and HPV-45 (OM-2258), and one genotype-negative sample (OM-1299). EXAMPLE 6: SB A assay validation and performance

In the current bead library design, 10-bp ID traces were used in order to call the reads. The Ion Personal Genome Machine (PGM) Torrent Suite software workflow requires a 4-bp key sequence that is used for read indexing for filtering and quality assessments. For de novo sequencing applications the Torrent Suite dispenses a pre-defined custom flow order of nucleotides, wherein 4 flows make one cycle. Ruby scripting was used to establish a theoretical minimum dispensation order for a set of 153 ID traces. The minimum number of dispensations was determined to be 10 cycles (i.e. 40 flows), corresponding to an estimated runtime of 10 minutes. However, to cover reads that were shorter than expected, we chose to use 100 flows, corresponding to a runtime of -37 minutes.

The SBA assay methodology was divided into four events; i) target hybridization to activate the oligo-tagged beads, ii) oligo-tagged bead immobilization, iii) magnetic bead selection of activated oligo-tagged beads, and iv) release of selected oligo-tagged beads. The SBA assay experimental settings were optimized with synthetic targets and PCR products derived from plasmids with cloned HPV DNA fragments (henceforth referred to as HPV plasmids). Target-to-bead ratios were explored by combing a fixed amount of bead library beads (-5.93 xlO6) with 1-picomolar target molecules in variable reaction volumes of 5 - 40 μΐ, (equivalent of -1.48 xlO5 - 1.19 xlO6 beads/ μΐ). The higher bead densities rendered high background signals, while a less dense reaction volume produced a stronger signal-to-noise ratio.

Target hybridization temperature profiles were explored ranging in temperature from 37 - 50°C in 5-minute to 24-hour protocols. Annealing times less than one hour resulted in reproducibility difficulties, and thus a 1-hour protocol was chosen, as little or no

improvements were observed with longer protocols. A higher annealing temperature also resulted in lowered observed background noise. Oligo-tagged bead selection was explored using both a manual approach (with a handheld magnetic -rack), and an automated robotic system; the automated variant offered uniform reproducibility and ease-of-use, and was therefore chosen for all detailed experiments.

PCR products derived from HPV plasmids of the ten targeted HPV genotypes were individually detected with the SBA assay, and results were validated by PCR-amplicon Pyrosequencing24'35. Patients may carry multiple HPV infections and, though rare, have shown to carry as many as five subtypes34. Therefore, the SBA assay was further deployed to screen pools of PCR products derived from different HPV plasmids. Equimolar pooling of PCR product from the five high-risk HPV genotypes HPV-16, HPV-18, HPV- 33, HPV- 45, and HPV-59 (1/5 component each) was used as a target source to simulate a quintuple infection. The experiment was repeated six times to also investigate reproducibility of the assay, and in all cases all five HPV subtypes were successfully detected at bead- saturated fractions (Figure 3C, and Table 1). Oligo-tagged bead selection increased polyclonality and low quality filtering, while the percentage of uncalled and called oligo-tagged beads decreased. This reflects the background noise effect, due to unwanted selection of low quality and polyclonal beads containing a multitude of target specific binding segments. Due to variability in total bead population in separate sequencing runs, genotype profiling should instead be viewed by comparing the frequencies of called oligo-tagged beads rather than absolute calls, making it a very robust and reproducible assay.

In order to investigate the sensitivity of the SBA assay, target-to-bead ratio was explored using a fixed bead density, while varying the amount of targets from 1-picomolar to 1-femtomolar molecules. The goal was to confidently reach a minimum detect level of 1- picomolar target molecules, similar to the standard yield of a 35-cycle PCR product. In multiple HPV co-infections, the viral load of each contributing genotype can differ by orders of magnitude24'35. The dynamic range capability of the assay was investigated employing a two-target detection model in which two synthetic targets, T-45 and T-59 (corresponding to biomarker sequences HPV-45, and HPV-59, respectively) were present at various concentrations; T-45 was present at a fixed amount of 1-picomolar molecules, while T-59 ranged from 1-picomolar down to 1-femtomolar molecules through a ten-fold dilution series (Figure 4). An upper bead saturation level of 100-femtomolar, and a lower detection limit of 10-femtomolar were observed. As shown, SBA is essentially 2-logs more sensitive than a standard PCR product yield, and 1-log more sensitive than the Pyrosequencing HS 96 instrument 37 produced by Pyrosequencing AB, for sequencing in a research laboratory.

Graphical Sphixogram representations of a 10-plex HPV bead library, and SBA assay selections of a quintuple HPV plasmid co-infection is shown in Figure 3A, 3B, and 3C. The y-axis represents frequency of total called beads, and error-bars detail frequency range for each oligo-tagged bead. Figure 3A Library variability of sequencing data derived from six individually constructed libraries. Figure 3B is a graphical Sphixogram from a pooled library representation of the six bead libraries in Figure 3A, when run in six replicates. Pooling libraries lowers the variability parameters when considering large-scale productions. Figure 3C Results from six quintuple-target selections of equimolar amounts of PCR-products derived from HPV plasmids HPV- 16, HPV- 18, HPV-33, HPV-45, and HPV-59. The uneven distribution of saturated beads is carried over from the distribution of the original bead libraries in Figure 3A and Figure 3B. Figure 4 shows the sensitivity and dynamic range of the SBA assay in a simulated co- infection selection performed with different amounts of targets. The y-axis represents frequency of total called oligo-tagged beads. Figure 4A is a Sphixogram of an SBA assay selection of two synthetic targets T-45 and T-59 (corresponding to biomarker regions in HPV-45 and HPV-59 respectively) present in equimolar amounts of 1-picomolar respectively (1: 1). Figure 4B is a Sphixogram of targets T-45 and T-59 present at 1-picomolar and 100- femtomolar amounts respectively (1: 10), corresponding to the observed bead upper saturation limit for the described embodiment of the SBA assay. Figure 4C is a Sphixogram of targets T-45 and T-59 present at 1-picomolar and 10-femtomolar amounts respectively (1: 100), corresponding to the observed lower detection limit for the described embodiment of the SBA assay. Figure 4D is a Sphixogram of targets T-45 and T-59 present at 1-picomolar and 1-femtomolar amounts respectively (1: 1000). T-59 can no longer be distinguished from the background signal.

Figure 5 shows a sub-set of Sphixograms of SBA assay screens from 20 samples (genomic DNA extracts from cervical tumors). The bead bars in the Sphixograms are represented as frequencies of total called oligo-tagged beads. HPV distribution is shown below:

Figure imgf000038_0001
% % % % % % %

2258 33.88 1.13% 2.08 2.04 2.11 48.46 1.97 3.28 3.4% 1.64%

% % % % % % %

Figure 5A shows that in sample OM-1530, a single infection of HPV-16 was clearly observed. Figure 5B shows that in sample OM-1668, a single infection of HPV-59 was clearly observed. Figure 5C shows that in sample OM-1741, a single infection of HPV-18 was clearly observed. Figure 5D shows that in sample OM-1848, a single infection of HPV- 45 was clearly observed. Figure 5E shows that in sample OM-2215, a double co-infection of HPV-16 and HPV-18 was clearly observed. HPV-18 was detected at a saturated level, while HPV-16 is a sub-infection at a lower concentration, which was not distinguishable with PCR- amplicon Pyrosequencing readout24'35. Figure 5F shows that in sample OM-2258, a double co-infection of HPV-16 and HPV-45 was clearly observed. HPV-45 was detected at a saturated level, while HPV-16 is a sub-infection at a lower concentration, which was not distinguishable with PCR-amplicon Pyrosequencing readout24'35.

EXAMPLE 7: Alternative embodiments

Figure 6 and Figure 7 illustrate a number of alternative embodiments of the present assay.

Referring now to Figure 6, in contrast to the scheme outlined in Figure 2, the oligo 601 that forms the plurality of synthetic oligonucleotides (oligo-tags), viewed from the 5'- end, here consists of: i) a general linker segment 600 for bead coupling; ii) a target unique ID trace 602, iii) a general primer segment 604, and in Figure 6A through Figure 6D, iv) a target specific binding segment 606. For purposes of illustration, a primer is shown in position adjacent to the primer segment 604 that it would hybridize to during sequencing. The oligos 601 are attached to the bead 610, which may be a bead or a branched polynucleotide for linking the plurality of oligos 601. In each case, as before, an oligo-tag is present on the microparticle in multiple copies. In each case, the oligo-tag is oriented with the 5 '-end bound to the bead, and the primer segment 604 and ID trace 602 are proximal to the target recognition segment 606 at the distal end of the oligo-tag. As shown by the arrows at 614, in each case Figure 6A through Figure 6E, a biotin or another affinity molecule is used to bind a solid phase, e.g. a bead or coated surface used for separation based on the formation of a complex between a target molecule and a target segment on the bead.

An affinity molecule e.g. biotin 612 is incorporated to be bound to the 3'-end of the oligo by different mechanisms as shown in alternative embodiments of Figure 6A through Figure 6D. Figure 6E and Figure 7, by way of contrast, illustrate embodiments where separation takes place by aptamer binding (Figure 6E) or antibody binding (Figure 7).

In Figure 6A, an embodiment of oligo-tag and assay design is shown where hybridization of the target recognition segment 606 to a complementary target 608 allows for a polymerization reaction 620 (illustrated by arrows) using biotinylated nucleotides. This creates covalently bonded biotin molecules to be incorporated at the 3' end of the tag only if target hybridization has taken place. This possibly enables a stronger selection, and furthermore allowing for single-base interrogations, e.g. SNP detection by allele specific extension procedures. If the target template is longer, the polymerization could continue further generating a longer complementary strand as far as the oligo-tagged bead segment (dotted segment) with multiple biotin oligo-tagged bead activating molecules, given that a fraction of the dNTPs in the reaction are biotinylated.

Figure 6B shows an embodiment where selectively hybridizing a biotinylated complementary target 613 to the target recognition segment 606 in a bead- clustered oligonucleotide at the 3 '-end could possibly ease steric hindrance. Also, by including multiple target specific binding segments 606 (replicates or different targets), the process can be made more sensitive, with the possibility of binding multiple-targets. This also allows for a polymerization reaction 622 at the 3 '-end that serves to provide a longer target- tag duplex and thus stronger target binding.

In the embodiment shown in Figure 6C, the oligo tag target segment 606 at the 3' end of the oligo-tag hybridizes to its complementary target 608 to create a 5' overhang at the end of the target 608. Then a biotinylated synthetic capture probe 624 complementary to the target sequence downstream (5') of the target segment is added to the complex to provide to the complex the biotin affinity molecule 612 which is on the capture probe 624. This approach could be further combined with ligation of the fragments via a ligase-enzyme to form a covalent bond. Combining a single-base specific polymerase extension of the 3 '-end (or a complete gap-fill) with ligation to a 5 '-end phosphorylated biotinylated probe would allow for further interrogation of the bases in the juxtaposition of the ends, e.g. SNP detections or else24'27.

An embodiment described in Figure 6D shows that selection could be performed by an initial intermediary polymerization reaction 622 constructing a complementary sequence (arrows and black dashed line) to the target template. The biotin of the affinity molecule could then be provided by hybridization or any of the other strategies outlined above (Figure 6A through Figure 6C), targeting a known downstream region of the target template (black dashed line) recently complementary constructed by polymerization and hence covalently joined to the bead. In Figure 6D, a biotinylated probe is added and allowed to hybridize to the newly synthesized strand. Optionally in Figure 6B, 6C, and 6D the biotinylated capture probe could be directly conjugated to the magnetic bead, thus obsoleting the need for a biotin- labeling scheme.

In another embodiment, the target fragment 209 (Figure 2), 608 (Figure 6A, 6C, 6D) and 613 (Figure 6B) may carry a conjugated antibody from an upstream antibody based selection scheme. This method may take advantage of a method known as proximity ligation, as described e.g. at Fredriksson S, et al., "Protein detection using proximity-dependent DNA ligation assays," Nat Biotechnol. 2002 May;20(5):473-7, Hammond et al., "Profiling Cellular Protein Complexes by Proximity Ligation with Dual Tag Microarray Readout," PLoS One. 2012; 7(7): e40405, published online 2012 July 10, and Weiner, US 2011/0143995, "Protein Capture, Detection and Quantitation," published June 16, 2011. Briefly, as reported by

Hammond et al., the proximity ligation assay (PLA) is an immunoassay utilizing so-called PLA probes - affinity reagents such as antibodies modified with DNA oligonucleotides - for detecting and reporting the presence of proteins either in solution or in situ. When two PLA probes bind the same or two interacting target molecules, the attached oligonucleotides are brought in close proximity.

An alternative immunologic approach would be the use of an aptamer 634 at the 3' end of the oligo-tag, as illustrated in Figure 6E. Target recognition takes place via the aptamer 634 at the 3' (distal) end of the oligo-tag. The aptamer used may be a peptide or an oligonucleotide, as is known in the art. Aptamers may be designed as described, e.g. in Gold et al., "Nucleic acid ligands," US 5,696,249, issued December 9, 1997. Peptide aptamers may be prepared, e.g., as described in Colas et al., "Targeted modification of intracellular compounds," US 2003/0143626 published July 31, 2003. Referring now to Figure 7, the oligo 701 that forms the plurality of synthetic oligonucleotides (oligo-tags), viewed from the 5'-end, here consists of: i) a general linker segment 700 for bead coupling; ii) a target unique ID trace 702, and iii) a general primer segment 704. In the embodiment of Figure 7, an antibody 720 is conjugated directly to the bead 710 that also contains the plurality of oligonucleotides 701. An antigen 730 could be assayed using selection/separation 714 based on a secondary antibody 740 carrying a biotin or another affinity molecule 745, which is used to bind a solid phase, e.g. a coated bead or surface used for separation based on the formation of a complex between a target and a segment on the bead.

In addition, alternative oligo-tag constructs may be employed. For example, the linker may be modified in a number of ways, and need not be an oligonucleotide sequence. Or, it may be engineered to include a restriction site. The linker may be extended so that in addition to containing a bead linker it will include a restriction fragment for digesting. In brief, once the SBA assay target selection has occurred, the bead is essentially creating self-amplification as a result of the beads selected (multiple copy numbers etc.). By digesting the restriction fragment the fragments are all released from the beads, and washed away. What remains then is a ready selected and prepared library suitable for MiSeq (Illumina personal sequencer) and other applications.

An example of the linker can be the following (SEQ ID NO: 23):

5 ' -TTTTTTTTTTTTUUUAATGATACGGCGACC ACCGAGATCT

(5'-PolyT spacer - restriction site - ILMN adaptor sequence P5).

In the example above, UUU is used for single stranded DNA restriction cutting, but could be performed in a multitude of ways. EXAMPLE 8: Data analysis methods

As noted above, the present methods and materials produce a quantitative readout of microparticles (beads) that have bound to a target and, as a result were separated from the mixture. The separated beads are analyzed by determining the sequence of the ID trace of the oligos on the bead. This is done, for example, by massively parallel sequencing (NGS). The sequence reads are conventionally communicated to the user by software that is provided with a sequencing machine. The present invention includes specialized software that converts sequence reads to numbers and a certain microparticle once identified is counted and may contain multidimensional information (e.g. patient X and target/pathogen Y). As noted above, this has been termed Sphix software and the graphical output that it can produce is termed a Sphixogram. Referring now to Figure 8, the present software inputs assay sequence read data, e.g. from an NGS machine. This is subjected to a sequence quality check, as shown in block 802; followed by examination to see if the oligo-tagged bead matching is successful. That is, if an input sequence matches a ID trace (ID trace may include multidimensional data, i.e. a bead type from patient/sample X and a targeting pathogen Y), it is shown at block 804 a comparison against a user input data set of ID traces. It results in a data structure in block 806 containing matched and quantified target sequences, i.e. information regarding microparticles (beads) present in the sample, ready for downstream visualization and formatting for a user- friendly output, e.g. graph 808 (Figure 5). As indicated, the software may be implemented on a computer with Internet or wireless access for display on a hand held device.

REFERENCES:

1. Schena, M., Shalon, D., Davis, R. W. & Brown, P. O. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467-470 (1995).

2. Miller, M. B. & Tang, Y. W. Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology. Clinical Microbiology Reviews 22, 611-633 (2009).

3. Shendure, J. Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome. Science 309, 1728-1732 (2005).

4. Margulies, M. et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature (2005).doi: 10.1038/nature03959

5. Shendure, J. The beginning of the end for microarrays? Nat Meth 5, 585-587 (2008).

6. Pettersson, E., Lundeberg, J. & Ahmadian, A. Generations of sequencing technologies. Genomics 93, 105-111 (2009).

7. Stahl, P. L. & Lundeberg, J. Toward the Single-Hour High-Quality Genome. Annu. Rev. Biochem. 81, 359-378 (2012).

8. Pettersson, E. et al. Allelotyping by massively parallel pyrosequencing of SNP-carrying trinucleotide threads. Hum. Mutat. 29, 323-329 (2008).

9. Myllykangas, S., Buenrostro, J. D., Natsoulis, G., Bell, J. M. & Ji, H. P.

Efficient targeted resequencing of human germline and cancer genomes by oligonucleotide- selective sequencing. Nat Biotechnol 29, 1024-1027 (2011).

10. Nolan, J. P. & Sklar, L. A. Suspension array technology: evolution of the flat- array paradigm. Trends Biotechnol. 20, 9-12 (2002).

11. Dunbar, S. A. Applications of Luminex® xMAP™ technology for rapid, high- throughput multiplexed nucleic acid detection. Clinica Chimica Acta 363, 71-82

(2006) .

12. Giljohann, D. A. et al. Gold Nanoparticles for Biology and Medicine. Angew. Chem. Int. Ed. 49, 3280-3294 (2010).

13. Nyren, P. The history of pyrosequencing. Methods Mol. Biol. 373, 1-14

(2007) .

14. Dressman, D., Yan, H., Traverso, G., Kinzler, K. W. & Vogelstein, B. Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc. Natl. Acad. Sci. U.S.A. 100, 8817 (2003).

15. Rothberg, J. M. et al. An integrated semiconductor device enabling non- optical genome sequencing. Nature 475, 348-352 (2012).

16. Merriman, B., R D Team, I. T. & Rothberg, J. M. Progress in Ion Torrent semiconductor chip based sequencing. Electrophoresis 33, 3397-3417 (2012).

17. Pourmand, N. et al. Direct electrical detection of DNA synthesis. Proc. Natl. Acad. Sci. U.S.A. 103, 6466-6470 (2006).

18. Anderson, E. P. et al. A System for Multiplexed Direct Electrical Detection of

DNA Synthesis. Sens Actuators B Chem 129, 79-86 (2008).

19. Hoheisel, J. D. Microarray technology: beyond transcript profiling and genotype analysis. Nat Rev Genet 7, 200-210 (2006).

20. Pierce, S. E. et al. A unique and universal molecular barcode array. Nat Meth 3, 601-603 (2006).

21. Pourmand, N. et al. Branch migration displacement assay with automated heuristic analysis for discrete DNA length measurement using DNA microarrays. Proc. Natl. Acad. Sci. U.S.A. 104, 6146 (2007).

22. de Villiers, E.-M., Fauquet, C, Broker, T. R., Bernard, H.-U. & Hausen, zur, H. Classification of papillomaviruses. Virology 324, 17-27 (2004).

23. Doorbar, J. Molecular biology of human papillomavirus infection and cervical cancer. Clinical Science 110, 525 (2006).

24. Akhras, M. S. et al. PathogenMip Assay: A Multiplex Pathogen Detection Assay. PLoS ONE 2, e223 (2007).

25. Jensen, M. A., Jauregui, L. & Davis, R. W. A Rapid, Cost-Effective Method of Assembly and Purification of Synthetic DNA Probes >100 bp. PLoS ONE 7, e34373 (2012).

26. Krishnakumar, S. et al. A comprehensive assay for targeted multiplex amplification of human DNA sequences. Proc. Natl. Acad. Sci. U.S.A. 105, 9296- 9301 (2008).

27. Akhras, M. S. et al. Connector inversion probe technology: a powerful one- primer multiplex DNA amplification system for numerous scientific applications. PLoS ONE 2, e915 (2007). 28. Hamady, M., Walker, J. J., Harris, J. K., Gold, N. J. & Knight, R. Error- correcting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nat Meth 5, 235-237 (2008).

29. Sims, P. A., Greenleaf, W. J., Duan, H. & Xie, X. S. Fluorogenic DNA sequencing in PDMS microreactors. Nat Meth 8, 575-580 (2011).

30. Chan, W. P., Premanode, B. & Toumazou, C. An Integrated ISFETs

Instrumentation System in Standard CMOS Technology. Solid-State Circuits, IEEE Journal of 45, 1923-1934 (2010).

31. Esfandyarpour, H., Zheng, B., Pease, R. F. W. & Davis, R. W. Structural optimization for heat detection of DNA thermo sequencing platform using finite element analysis. Biomicrofluidics 2, 24102 (2008).

32. Gravitt, P. E. et al. Improved amplification of genital human papillomaviruses. J. Clin. Microbiol. 38, 357-361 (2000).

33. Fuessel Haws, A. L. et al. Nested PCR with the PGMY09/11 and GP5+/6+ primer sets improves detection of HPV DNA in cervical samples. Journal of

Virological Methods 122, 87-93 (2004).

34. Holmberg, A. et al. The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures. Electrophoresis 26, 501-510 (2005).

35. Gharizadeh, B. et al. Sentinel-base DNA genotyping using multiple sequencing primers for high-risk human papillomaviruses. Molecular and Cellular

Probes 20, 230-238 (2006).

36. Goto, N. et al. BioRuby: bioinformatics software for the Ruby programming language. Bioinformatics 26, 2617-2619 (2010).

37. Gharizadeh, B. et al. Methodological improvements of pyrosequencing technology. Journal of Biotechnology 124, 504-511 (2006).

CONCLUSION

The above specific description is meant to exemplify and illustrate the invention and should not be seen as limiting the scope of the invention, which is defined by the literal and equivalent scope of the appended claims. Any patents or publications mentioned in this specification are intended to convey details of methods and materials useful in carrying out certain aspects of the invention which may not be explicitly set out but which would be understood by workers in the field. Such patents or publications are hereby incorporated by reference to the same extent as if each was specifically and individually incorporated by reference and contained herein, as needed for the purpose of describing and enabling the method or material referred to.

Claims

CLAIMS What is claimed is:
1. A composition for use in an assay of a target in a sample, comprising a microparticle comprised in a population of microparticles, wherein the microparticles have attached thereto multiple copies of single-stranded polynucleotides, said polynucleotides each comprising:
(a) a linker segment linking the single-stranded polynucleotide to the
microparticle;
(b) one or more target specific binding segments;
(c) an identifier segment identifying the population of microparticles bearing the single stranded polynucleotides and target specific binding segments; and, optionally,
(d) a segment for use in binding a sequencing primer, adjacent to the identifier segment, wherein the sequencing primer is used to sequence identifier segments in single- stranded polynucleotides in isolated microparticles that have bound to targets.
2. The composition of claim 1 wherein the population is divided into
subpopulations of microparticles wherein a microparticle has attached thereto multiple copies of a polynucleotide, said polynucleotide comprising defined segments (a) through present segment (d) inclusive, where segments (a) and (d) are identical in different subpopulations and segments (b) and (c) differ in sequence among different subpopulations.
3. The composition of claim 2 further comprising at least 10 subpopulations of microparticles wherein the defined segments (b) and (c) differ among each different subpopulation.
4. The composition of claim 1, 2, or 3 wherein the polynucleotide has segments in the order 5' to 3' of (a) to (b) to (c) to (d), (d) being present.
5. The composition of claim 1, 2, or 3 wherein the polynucleotide has segments in the order of 5' to 3' (a) to (c) to (d) to (b), (d) being present.
6. The composition of claim 1, 2, or 3 wherein said polynucleotides are covalently linked to the microparticle.
7. The composition of claim 1, 2, or 3 wherein said polynucleotides are between about 50 and 200 nucleotides in size.
8. The composition of claim 1, 2, or 3 having multiple target specific binding segments in a polynucleotide.
9. The composition of claim 1, 2, or 3 wherein the target specific segments have sequences that bind specifically to one genotype in a mixture of genotypes within the sample.
10. The composition of claim 9 wherein the genotype is selected from a genotype of a virus, a genotype of a tumor, an SNP genotype and a micro satellite genotype.
11. The composition of claim 1, 2, or 3 wherein the identifier segment is not more than 50 nucleotides in length.
12. The composition of claim 1, 2, or 3 wherein the bead is a material that is one of glass, polymer, ceramic, metal, paramagnetic material, or carbon.
13. The composition of claim 12 wherein the bead is a polymer that is one of acrylamide, polyethylene, polystyrene, and polymeric latex.
14. The composition of claim 1, 2, or 3 wherein the target binding segment is not more than 50 nucleotides in length.
15. A method for detecting a number of different target molecules in a mixture, comprising the steps of:
(a) providing a population of microparticles divided into a first subpopulation of microparticles and at least a second subpopulation of microparticles, wherein the microparticles have attached thereto multiple copies of a polynucleotide, said polynucleotide comprising segments:
(i) a linker for linking the polynucleotide to the microparticle;
(ii) one or more target specific binding segments;
(iii) an identifier segment for distinguishing one plurality of microparticles from other pluralities of microparticles; and
(iv) an optional segment for use in sequence determination; and
(b) the second subpopulation has attached thereto polynucleotides as in (a) segments (i) through (iv) inclusive, where (i) and (iv) are identical to those in the first subpopulation of microparticles, and segments (ii) and (iii) differ in sequence from sequences in other populations of microparticles;
(c) attaching affinity tags to nucleic acids in the mixture in the sample;
(d) contacting nucleic acids from step (c) with microparticles from (a) and (b) under conditions allowing hybridization of nucleic acids having target nucleic acid sequences to hybridize to target identification sequences thereby forming microparticle-target complexes;
(e) separating microparticle-target complexes from microparticles that have not
hybridized to targets by use of the affinity tag;
(f) performing a sequence determination of identifier segments (ii) in microparticle target complexes that were hybridized to targets in step (d), whereby target nucleic acid sequences are detected.
16. The method of claim 15 wherein the step of performing a sequence determination is carried out using microparticles separated in step (e).
17. The method of claim 15 or 16 wherein the separating of step (e) comprises contacting the tag with a magnetic microparticle comprising a material which binds the affinity tag.
18. The method of claim 15 or 16 wherein the affinity tag is biotin.
19. The method of claim 15 or 16 wherein the separating of step (e) comprises contacting the microparticle with a streptavidin-coated microparticle.
20. The method of claim 15 or 16 wherein the target identification sequence is not more than 50 nucleic acids in length.
21. The method of claim 15 or 16 further comprising the step of quantifying numbers of targets detected.
22. A method for detecting a number of different target molecules in a mixture, comprising the steps of:
(a) providing a mixture of microparticles comprising a first subpopulation of microparticles wherein the microparticles have attached thereto multiple copies of a polynucleotide, said polynucleotide comprising a 5' end bound to the microparticle and:
(i) a linker for linking the polynucleotide to the microparticle;
(ii) an identifier segment for distinguishing one plurality of microparticles from other pluralities of microparticles;
(iii) a target specific binding segment at the 3' end which binds to a target having a polynucleotide sequence extending beyond the 3' end, providing a target overhang; and
(iv) a segment for use in sequence determination; and (b) adding to the mixture an affinity tag for separating microparticles in which the polynucleotide has bound to the target.
23. The method of claim 22 wherein the affinity tag is in the form of biotinylated nucleotides and the enzyme is a polymerase.
24. The method of claim 22 or 23 wherein the affinity tag is in the form of a probe, having an affinity tag on the probe, that binds to the target overhang, and the method comprises the step of adding the probe to the mixture.
25. The method of claim 22 or 23 wherein the target specific binding segment is an aptamer.
26. The method of claim 22 wherein the aptamer binds to a target which is a protein.
27. A composition comprising:
(a) a population of microparticles in suspension, having covalently attached thereto a plurality of polynucleotides, each polynucleotide having a linker segment, an identifier segment, and a target specific binding segment; and
(b) a polynucleotide target hybridized to at least one target specific binding segment on a microparticle, said polynucleotide target attached to an affinity tag.
28. The composition of claim 27 wherein the population of microparticles comprises polynucleotides having identifier segments that differ as between microparticles.
29. The composition of claim 27 or 28 wherein the population of microparticles comprises polynucleotides having target binding segments and targets that differ as between microparticles.
30. The composition of claim 29 further comprising a second microparticle bound to the affinity tag.
31. The composition of claim 30 wherein the affinity tag is biotin and the second microparticle comprises avidin.
PCT/US2014/015880 2013-02-14 2014-02-11 Suspension arrays and multiplexed assays based thereon WO2014126937A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US201361764820 true 2013-02-14 2013-02-14
US61/764,820 2013-02-14

Publications (1)

Publication Number Publication Date
WO2014126937A1 true true WO2014126937A1 (en) 2014-08-21

Family

ID=51354502

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/015880 WO2014126937A1 (en) 2013-02-14 2014-02-11 Suspension arrays and multiplexed assays based thereon

Country Status (1)

Country Link
WO (1) WO2014126937A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160265069A1 (en) * 2013-08-28 2016-09-15 Cellular Research Inc. Massively parallel single cell analysis
US9708659B2 (en) 2009-12-15 2017-07-18 Cellular Research, Inc. Digital counting of individual molecules by stochastic attachment of diverse labels
US9727810B2 (en) 2015-02-27 2017-08-08 Cellular Research, Inc. Spatially addressable molecular barcoding
US9905005B2 (en) 2013-10-07 2018-02-27 Cellular Research, Inc. Methods and systems for digitally counting features on arrays

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040101835A1 (en) * 2000-10-24 2004-05-27 Willis Thomas D. Direct multiplex characterization of genomic dna
US20060240416A1 (en) * 2000-06-21 2006-10-26 Sukanta Banerjee Multianalyte molecular analysis using application-specific random particle arrays
US20070026438A1 (en) * 2005-06-28 2007-02-01 Smith Douglas R Methods of producing and sequencing modified polynucleotides
US20070207456A1 (en) * 2006-02-14 2007-09-06 The Board Of Trustees Of The Leland Stanford Junior University Multiplexed assay and probes for identification of HPV types
US20080269068A1 (en) * 2007-02-06 2008-10-30 President And Fellows Of Harvard College Multiplex decoding of sequence tags in barcodes

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060240416A1 (en) * 2000-06-21 2006-10-26 Sukanta Banerjee Multianalyte molecular analysis using application-specific random particle arrays
US20040101835A1 (en) * 2000-10-24 2004-05-27 Willis Thomas D. Direct multiplex characterization of genomic dna
US20070026438A1 (en) * 2005-06-28 2007-02-01 Smith Douglas R Methods of producing and sequencing modified polynucleotides
US20070207456A1 (en) * 2006-02-14 2007-09-06 The Board Of Trustees Of The Leland Stanford Junior University Multiplexed assay and probes for identification of HPV types
US20080269068A1 (en) * 2007-02-06 2008-10-30 President And Fellows Of Harvard College Multiplex decoding of sequence tags in barcodes

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9708659B2 (en) 2009-12-15 2017-07-18 Cellular Research, Inc. Digital counting of individual molecules by stochastic attachment of diverse labels
US10047394B2 (en) 2009-12-15 2018-08-14 Cellular Research, Inc. Digital counting of individual molecules by stochastic attachment of diverse labels
US9845502B2 (en) 2009-12-15 2017-12-19 Cellular Research, Inc. Digital counting of individual molecules by stochastic attachment of diverse labels
US9816137B2 (en) 2009-12-15 2017-11-14 Cellular Research, Inc. Digital counting of individual molecules by stochastic attachment of diverse labels
US10059991B2 (en) 2009-12-15 2018-08-28 Cellular Research, Inc. Digital counting of individual molecules by stochastic attachment of diverse labels
US20160265069A1 (en) * 2013-08-28 2016-09-15 Cellular Research Inc. Massively parallel single cell analysis
US9637799B2 (en) 2013-08-28 2017-05-02 Cellular Research, Inc. Massively parallel single cell analysis
US9598736B2 (en) 2013-08-28 2017-03-21 Cellular Research, Inc. Massively parallel single cell analysis
US9567645B2 (en) 2013-08-28 2017-02-14 Cellular Research, Inc. Massively parallel single cell analysis
US9567646B2 (en) 2013-08-28 2017-02-14 Cellular Research, Inc. Massively parallel single cell analysis
US10131958B1 (en) 2013-08-28 2018-11-20 Cellular Research, Inc. Massively parallel single cell analysis
US9905005B2 (en) 2013-10-07 2018-02-27 Cellular Research, Inc. Methods and systems for digitally counting features on arrays
US10002316B2 (en) 2015-02-27 2018-06-19 Cellular Research, Inc. Spatially addressable molecular barcoding
US9727810B2 (en) 2015-02-27 2017-08-08 Cellular Research, Inc. Spatially addressable molecular barcoding

Similar Documents

Publication Publication Date Title
Nallur et al. Signal amplification by rolling circle amplification on DNA microarrays
Plongthongkum et al. Advances in the profiling of DNA modifications: cytosine methylation and beyond
Zhao et al. Isothermal amplification of nucleic acids
Van Dijk et al. Ten years of next-generation sequencing technology
Demidov Rolling-circle amplification in DNA diagnostics: the power of simplicity
Sanders et al. Evaluation of digital PCR for absolute DNA quantification
Fan et al. Highly parallel genomic assays
US20090202984A1 (en) Single molecule nucleic acid sequence analysis processes and compositions
Shen et al. Multiplexed quantification of nucleic acids with large dynamic range using multivolume digital RT-PCR on a rotational SlipChip tested with HIV and hepatitis C viral load
US20120220494A1 (en) Compositions and methods for molecular labeling
US20140121116A1 (en) System and Methods for Detecting Genetic Variation
Kennedy et al. Detecting ultralow-frequency mutations by Duplex Sequencing
US20130157870A1 (en) Methods for obtaining a sequence
Miotke et al. High sensitivity detection and quantitation of DNA copy number and single nucleotide variants with single color droplet digital PCR
US20150376609A1 (en) Methods of Analyzing Nucleic Acids from Individual Cells or Cell Populations
US20120157322A1 (en) Direct Capture, Amplification and Sequencing of Target DNA Using Immobilized Primers
WO2006110855A2 (en) Methods for determining sequence variants using ultra-deep sequencing
WO1993010267A1 (en) Rapid assays for amplification products
WO2013130674A1 (en) Compositions and kits for molecular counting
WO2011142836A2 (en) Assays for the detection of genotype, mutations, and/or aneuploidy
Konry et al. Microsphere-based rolling circle amplification microarray for the detection of DNA and proteins in a single assay
US20040091879A1 (en) Nucleic acid sequence detection using multiplexed oligonucleotide PCR
JP2005143492A (en) Method for detecting target nucleic acid sequence
US20030113781A1 (en) Capture moieties for nucleic acids and uses thereof
Tjong et al. Amplified on-chip fluorescence detection of DNA hybridization by surface-initiated enzymatic polymerization

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14751243

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct app. not ent. europ. phase

Ref document number: 14751243

Country of ref document: EP

Kind code of ref document: A1