(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`5 April 2012 (05.04.2012)
`
`PCT
`
`I IIIII IIIIIIII II IIIIII IIIII IIIII IIIII IIII I II Ill lllll lllll lllll lllll lllll llll 1111111111111111111
`
`(10) International Publication Number
`WO 2012/042374 A2
`
`(51) International Patent Classification:
`CI2Q 1/68 (2006.01)
`
`(21) International Application Number:
`
`(22) International Filing Date:
`29 September 201 l (29.09.2011)
`
`PCT/IB20l l/002438
`
`(72) Inventors; and
`(71) Applicants : TAIPALE, Anssi Jussi Nikolai [FUSE];
`Molinvagen 12, 168 50 Bromma (SE). LINNARSSON,
`Sten [SE/SE]; Vindragarvagen IO, S-11750 Stockholm
`(SE).
`(74) Agents: MEWBORN ELLIS LLP et al.; 33 Gutter Lane,
`London EC2V SAS (GB).
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`(Sl)
`
`English
`English
`
`(30) Priority Data:
`I016608.0
`I022 l l l.7
`
`I October 20IO (Ol.l0.20IO)
`30 December 20IO (30.12.2010)
`
`GB
`GB
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
`CA,CH,CL,CN,CO,CR,CU,CZ,DE,DK,DM,DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
`
`[Continued on next page]
`
`(54) Title: METHOD OF DETERMINING NUMBER OR CONCENTRATION OF MOLECULES
`
`(57) Abstract: A method of determining the number or concen(cid:173)
`tration of molecules in a sample, a method of tracking a substance
`in a reaction or system, and applications of such methods. Use of
`nucleic acid as a tracer.
`
`------
`. · (cid:173)- -- -- -- -- --==- -l labeling
`--~
`-· ..._
`- --
`~ -(cid:173)
`-..=--
`l amplification,
`-~= ~•r
`normalization
`-~ -:a=c=
`a ----e ~ -...,
`• • • • • • • • • . -• --· ------· -
`~ ----..__ --------=
`..= -
`
`i:,...- -
`
`' . ) -
`
`! sequencing
`
`Approx.19
`(Poisson)
`
`FIGURE 1
`
`2
`
`3
`
`4
`
`---
`
`- ----;
`
`;;;;;;;;;;;;; -;;;;;;;;;;;;;; -
`---;;;;;;;;;;;;;;
`;;;;;;;;;;;;;; --;;;;;;;;;;;;;;
`!!!!!!!! --
`
`i
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`FOUNDATION EXHIBIT 1062
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`WO 2012/04 23 7 4 A2 I IIIII IIIIIIII II IIIIII IIIII IIIII IIIII IIII I II Ill lllll lllll lllll lllll lllll llll 1111111111111111111
`
`SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM,
`NO, NZ, OM, PE, PG, PH, PL, PT, QA, RO, RS, RU,
`RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ,
`GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, Declarations under Rule 4.17:
`ZM,ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD,
`RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ,
`DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT,
`LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS,
`
`-
`
`ofinventorship (Rule 4.17(iv))
`
`Published:
`
`without international search report and to be republished
`upon receipt of that report (Rule 48.2(g))
`
`ii
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`METHOD OF DETERMINING NUMBER OR CONCENTRATION OF MOLECULES
`
`5
`
`The present invention relates to methods for determining the number or concentration of entities
`
`in a sample. In particular, the present invention relates to methods for determining the number
`
`or concentration of molecules, e.g. biomolecules such as nucleic acid, in a sample.
`
`Advances in molecular biology have made it relatively simple to identify different DNA or RNA
`
`1 O
`
`species and to copy them. Identification of nucleic acid species can be accomplished by
`
`sequencing, i.e. reading the DNA sequence (Maxam and Gilbert, 1977; Sanger and Coulson,
`
`1975); currently millions of molecules can be sequenced in a single day using massively parallel
`
`sequencing (Margulies et al,. 2005; Shendure et al., 2005). Efficient copying of DNA-molecules
`
`of arbitrary sequence was made possible by molecular cloning (Cohen et al., 1973), and the
`
`15
`
`polymerase chain reaction (Saiki et al,. 1985).
`
`Changes in the relative abundance of a large number of different sequences between two or
`
`more samples can in turn be measured using microarray hybridization (Schema et al., 1995)
`
`and/or tag sequencing e.g. EST sequencing (Okubo et al., 1992) or sequencing of
`
`20
`
`concatenated mRNA-derived tags (Velculescu et al 1995). However, determining the relative
`
`abundance of two different species and/or the absolute number of molecules present in a single
`
`sample has proven much more challenging.
`
`Measuring accurate concentrations for multiple different mRNAs simultaneously is difficult
`
`25
`
`because different mRNA species are present in wildly different concentrations in cells.
`
`Differences in concentration between high-abundance mRNA and low abundance mRNA can
`
`range over six to ten orders of magnitude (see for example Holland et al., 2002). Microarrays
`
`have relatively low specificity and linear range, and massively parallel sequencing/molecule
`
`counting suffers from the fact that hundreds of thousands of abundant molecules need to be
`
`30
`
`counted before even a single molecule from a rare species is found. Accuracy of measurement
`
`requires that a relatively large number of molecules of each species is counted. Measuring a
`low-abundance mRNA (1 ppm) to an accuracy of ±20% requires counting approximately 108
`molecules. The range of concentrations can be made more even by subtractive hybridization
`
`(Patanjali et al., 1991; Zhulidov et al., 2004), but this results in loss of information about the
`
`35
`
`original concentrations of the mRNA species.
`
`Similarly, methods to amplify the signal by making exact copies of molecules necessarily
`
`destroy information about the absolute number of molecules in the original sample, as digital
`
`copies derived from two identical molecules are identical. Deriving the absolute number of
`
`40 mRNA or DNA-molecules in the original sample requires precise information about how many
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`copies of each molecule exist after amplification and/or subtractive hybridization. As efficiency
`
`of all known DNA-copying and subtraction processes are dependent on the sequence and
`
`length of DNA, determining the number of copies that exist after these procedures is error-prone
`
`and not feasible for a large number of different DNA-species. In addition, the cumulative nature
`
`5
`
`of the amplification error makes it very difficult to accurately measure mRNA abundance in
`
`samples derived from small amount of starting material. No current method exists to determine
`
`absolute number of molecules from complex mixtures efficiently.
`
`It is theoretically possible to count the absolute number of RNA or DNA-molecules by single
`
`10 molecule sequencing (Ozsolak et al., 2010). Using single molecule sequencing to count
`
`absolute number of molecules requires a large number of tag counts, as all molecules need to
`
`be counted. In addition, as samples from different sources have identical sequences, it is not
`
`currently possible to effectively multiplex samples in direct single-molecule sequencing.
`
`15
`
`This invention relates to counting the absolute number of entities of a species of interest. The
`
`invention is based upon a realisation that, while directly counting an absolute number of entities
`
`is a challenging and in some cases virtually impossible prospect, it can be relatively
`
`straightforward to determine how many different types of entity are present in a sample. For
`
`example, as discussed above, molecules having different sequences in a sample can be
`
`20
`
`detected by sequencing, and techniques such as amplification, subtractive hybridisation and/or
`
`normalisation of a sample can be used to process the sample before sequencing so that the
`
`presence of each different molecule can be reliably and confidently determined.
`
`The present invention relates to counting, measuring or determining the absolute number of
`
`25
`
`entities of a species of interest in a sample by ensuring that the entities of that species of
`
`interest differ from each other, and determining the absolute number of the different entities. By
`
`ensuring that the entities of the species of interest are detectably different, this allows the
`
`absolute number of entities of the species of interest to be determined based on the number of
`
`different entities of that species.
`
`30
`
`Entities of a species of interest can be modified to render them different, e.g. by labelling or
`
`other modification as described in more detail below. This facilitates determination of the
`
`absolute number of those entities of the species of interest, since one can determine how many
`
`differently modified entities of the species are present, and from this information the number of
`
`35
`
`original entities of the species of interest in the sample can be derived.
`
`Alternatively, where a population of entities of a species of interest comprises some entities of
`
`the species of interest which are the same and some which differ from one another (for
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`example, where the species of interest is nucleic acid, and a population comprises examples of
`
`the same and different nucleotide sequences), it is possible to ensure that the species of
`
`interest differ from one another by taking a sample from that population, where the sample size
`
`is sufficiently small that the entities of the species of interest in the sample differ from one
`
`5
`
`another (e.g. where each nucleic acid molecule in the sample has a different nucleotide
`
`sequence). The absolute number of entities of the species of interest in the sample can then be
`
`determined by determining the number of different entities of the species of interest in the
`
`sample. The step of sampling to ensure that the entities of the species of interest differ from
`
`one another represents a "bottlenecking" approach, to restrict the number of entities of the
`
`10
`
`species of interest which are subsequently amplified and is subsequently performed.
`
`Use of bottlenecking is of particular value in the context of a method in which the entities of the
`
`species of interest in the sample are fragments of larger entities in a starting population, where it
`
`is desired to know the relative proportions of the larger entities. By amplifying a limited sample
`
`15
`
`of fragments in which each fragment is different, the absolute number of those different
`
`fragments can be determined. Following detection of each different fragment, the fragment can
`
`be assigned to a larger entity, i.e. the method can comprise a step of determining the larger
`
`entity from which each fragment is derived. This provides information on the relative number of
`
`the different larger entities in the starting population. It is useful in a method comprising
`
`20
`
`karyotyping, to determine relative number of different chromosomes.
`
`Alternatively, or additionally, one can ensure that the species of interest are different from one
`
`another by providing or generating a plurality of different entities, e.g. randomly different
`
`molecular structures. These can, for example, be employed in situations such as tracing or
`
`tracking of substances or fluid flow. Thus, where the entities of the species of interest are mixed
`
`~
`
`with a substance to be monitored, samples may be taken after downstream processing, and the
`
`amount (e.g. number of entities, or concentration) of the substance present in the sample may
`
`be accurately estimated based on the absolute number of entities of the species of interest in
`
`the sample.
`
`25
`
`30
`
`The invention provides a method of determining the absolute number of entities of a species of
`
`interest in a sample, comprising:
`
`ensuring that the entities of the species of interest in the sample differ from each other,
`
`and
`
`35
`
`determining the absolute number of the different entities of the species of interest.
`
`The method may comprise:
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`(i) ensuring that the entities of that species of interest in the sample differ from each
`
`other,
`
`(ii) amplifying the different entities of the species of interest in the sample, and
`
`(iii) determining the number of different entities of the species of interest after
`
`5
`
`amplification, or determining the number of different amplicons,
`
`thereby determining the number of entities of the species of interest in the sample.
`
`Number or concentration can be determined, these being interconvertible based on the volume.
`
`10
`
`Amplification is performed on the entities in the sample. The sample contains the entities of the
`
`species of interest to be counted, which in some cases may be present in modified form in the
`
`sample, e.g. as labelled entities or conjugates.
`
`The amplification of the different entities of the species of interest in the sample provides a
`
`15
`
`library of amplicons, each amplicon being a copy of an entity of the species of interest in the
`
`sample. The number of different amplicons is representative of the number of entities of the
`
`species of interest in the sample before amplification. Thus, the number or concentration of
`
`entities of the species of interest in the sample can be determined based on the number or
`
`concentration of different amplicons.
`
`20
`
`The invention provides a way to preserve or store information about the absolute number of
`
`entities of a species of interest in a sample (e.g. the number of instances of a particular nucleic
`
`acid sequence, or the total number of nucleic acid molecules), so that this information is not lost
`
`during processing of the sample by methods such as amplification, subtractive hybridisation and
`
`25
`
`normalisation. By ensuring that the entities of the species of interest in a sample are different
`
`from each other before such processing steps are carried out, the absolute number of entities of
`
`the species of interest in the sample remains equal to the number of different entities of the
`
`species of interest, even after amplification. Processing of a sample (e.g. by amplification,
`
`normalisation and/or subtractive hybridisation) may change the relative proportions of entities of
`
`30
`
`the species of interest and of other entities in the sample. But, provided that all entities of the
`
`species of interest in the sample were different before amplification, the number of different
`
`entities of the species of interest after amplification is representative of the absolute number of
`
`entities of the species of interest in the sample before amplification. Thus, a step of ensuring
`
`that the entities of the species of interest are different from one another in a sample bestows a
`
`35
`
`form of "molecular memory" on the sample, so that information about the absolute number of
`
`entities of a species of interest in that sample is stored, and is not destroyed by subsequent
`
`changes in the absolute and relative proportions of species in the sample.
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`In a first aspect, the invention provides a method for determining the number or concentration of
`
`entities of a species of interest in a sample, the method comprising modifying each entity of the
`
`species of interest to provide a plurality of differently-modified entities, and determining the
`
`number or concentration of entities of the species of interest in the sample, based on the
`
`5
`
`number of differently-modified entities.
`
`The number of differently modified entities is representative of the absolute number of entities of
`
`the species of interest which were present in the original sample. Changing the entities of the
`
`species of interest into differently-modified entities means that techniques for determining the
`
`10
`
`number or concentration of different species can be used to determine how many (or what
`
`concentration of) entities of the species of interest were originally present in the sample. As
`
`discussed already above, such techniques are generally easier and faster than attempting to
`
`directly determine the absolute number of molecules such as nucleic acids. The invention thus
`
`provides an elegant solution to the problem of determining absolute molecular number, and
`
`15
`
`facilitates counting the number of entities of a species of interest in a sample, by enabling the
`
`number of differences to be determined instead of directly counting the number of the species of
`
`interest. The invention therefore has particular use for counting entities in situations where it
`
`would be more difficult or time consuming to count the entities directly, compared with
`
`determining how many differently modified entities are present. Furthermore, the methods of
`
`20
`
`the invention may be applied in parallel, where they provide a powerful way to determine the
`
`absolute number of many millions of individual species of interest in parallel, using multiplex
`
`techniques, and find use in a variety of different applications, as will be explained in more detail
`
`below.
`
`25
`
`The entities may be any items to be counted, provided that they can be differently modified in
`
`order to apply the techniques of the invention. In embodiments of the invention which do not
`
`require entities to be modified, e.g. where bottlenecking is used to ensure that entities of a
`
`species of interest differ from each other in a sample, then it is not necessary that the entities
`
`are differently modified, although it may be preferable to do so, and to use modifying and
`
`30
`
`bottlenecking approaches in combination. Entities which are especially suitable are those to
`
`which a label can be attached with relatively stable stoichiometry and then the labelled entity
`
`purified from free label. Typically, the entities are molecules, e.g. biomolecules such as nucleic
`
`acids, proteins, polysaccharides or lipids. The entities may be molecular complexes comprising
`
`two or more molecules, which may be two or more biomolecules belonging to the same class or
`
`35
`
`to different classes of biomolecule (classes of biomolecules in this context include nucleic acids,
`
`proteins, polysaccharides and lipids). Alternatively, the entities of a species of interest may be
`
`comprised in a molecule, rather than being separate molecules. For example, the species of
`
`interest may be a specific nucleotide sequence comprised in a chromosome, and the number of
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`copies or repeats of that sequence in the chromosome may be determined by the method of the
`
`present invention.
`
`In the following discussion the term "molecule" is used, but as set out above, it is intended that
`
`5
`
`the invention is not necessarily limited to the determination of the number or concentration of
`
`molecules.
`
`The molecules of the species of interest may be modified by labelling, that is, by providing the
`
`molecules of the species of interest with variant labels to provide a plurality of variant molecules,
`
`1 O
`
`or conjugates.
`
`Accordingly, a first development of the first aspect of the invention provides a method for
`
`determining the number or concentration of molecules of a species of interest in a sample, the
`
`method comprising labelling each molecule of the species of interest with a label, wherein each
`
`15
`
`label is selected from a group of different labels, to provide a plurality of conjugates, each
`
`conjugate comprising a label attached to a molecule of the species of interest, and determining
`
`the number or concentration of molecules of the species of interest in the sample, based on the
`
`number of different conjugates.
`
`20
`
`Further preferred or optional features of the invention will now be set out. These may be
`
`applied singly or in any combination with any aspect or development of the invention described
`
`herein, unless the context demands otherwise.
`
`The label is a marker, tag, adapter, part, sequence or structure that serves to distinguish a
`
`25 molecule of the species of interest from another molecule of the species of interest.
`
`The label may be a chemical group or moiety, which may be a polymer, e.g. a polymer
`
`comprising a sequence of different units. The label is preferably a nucleic acid (i.e. a
`
`polynucleotide), which may be DNA or RNA, or may be a modified nucleic acid such as PNA,
`
`30
`
`LNA or L-DNA. In some embodiments of the present invention, modified nucleic acids may be
`
`preferable for use as labels due to their relatively high chemical stability. A nucleic acid label
`
`may be a double-stranded or single-stranded nucleic acid
`
`A nucleic acid label may be at least 2 nucleotides in length. Preferably, a nucleic acid label is at
`
`35
`
`least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19 or 20 nucleotides in length, and may
`
`optionally be up to 20, 25, 50 or up to 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000
`
`nucleotides in length. A nucleic acid may be 2-100, 2-50, 2-25, 2-20, 4-20, 4-1 O or 4-8
`
`nucleotides in length. Preferably a nucleic acid is 4-20 nucleotides in length.
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`Preferably, the label is a polymer having a random monomer sequence. A random polymer
`
`sequence is a sequence comprising more than one type of monomer, in which the sequence of
`
`types of monomer is not predetermined by controlled steps during synthesis of the polymer, but
`
`5
`
`depends only on random collisions between reactant monomers and/or between reactant
`
`monomers and the nascent polymer. Accordingly, the method of the present invention may
`
`comprise a step of generating a group of polymers (such as nucleic acids, or polynucleotides)
`
`having random sequences of nucleotides. The generation of a random polymer may comprise
`the use of N random monomers of B different types, which results in BN different possible
`
`1 O
`
`polymers. The generation of a random polynucleotide may comprise the use of N random
`
`15
`
`20
`
`nucleotides comprising 4 different bases, which results in 4N different polynucleotides. For
`
`example, for a label which is a random polynucleotide synthesised from nucleotide monomers
`
`comprising one of four different bases, which label is ten nucleotides in length, there are
`4 10 = 1,048,576 different possible sequences. If such a label is twenty nucleotides in length,
`there are 420 == 1012 (i.e. approximately a trillion) different possible sequences i.e. 1012 different
`possible labels.
`
`Preferably, the group of different labels comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
`
`14, 15, 16, 17, 18, 19,20,21,22,23,24,25,26,27,28,29,30,31,32, 33,34, 35,36,37,38,
`39,40, 50, 60, 70,80, 90,100,150,200,250,500, 1000,2000, 5000, 10,000,50,000, 100,000,
`, 1011, 1012
`500,000,106,107,108
`, 1010
`, 1015 or 1018 different labels. Identical labels may be
`, 109
`present in the group. There is a statistical probability that such identical labels will be
`
`generated, if the labels are generated randomly e.g. comprising random monomer units as
`
`discussed above. Accordingly, the group of different labels may comprise two or more labels
`
`25
`
`that are identical to each other.
`
`The methods of the present invention may comprise labelling each molecule of a species of
`
`interest with a label, wherein each label is selected from a group of different labels. It may be
`
`the case that each label that is selected from the group of different labels may be different from
`
`30
`
`every other label that is selected from the group of different labels. If the group of different
`
`labels comprises two or more identical labels, then it may be the case that a label selected from
`
`the group of different labels is the same as another label selected from the group of different
`
`labels.
`
`35
`
`The invention provides a method of determining the number or concentration of entities of a
`
`species of interest in a sample, the method comprising:
`
`providing the sample from a population of entities of the species of interest, wherein the
`
`entities of the species of interest in the sample differ from each other,
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`amplifying the different entities of the species of interest in the sample to provide a
`
`library of amplicons, then
`
`determining the number or concentration of different amplicons, and thereby determining
`
`the number or concentration of entities of the species of interest in the sample.
`
`5
`
`The population may comprise identical and different entities of the species of interest. The size
`
`of sample taken may be adjusted such that the entities of the species of interest in the sample
`
`differ from each other.
`
`10
`
`The sample may be provided by physically separating the sample from the population, e.g. as
`
`an aliquot. Alternatively, the sample may be provided in same reaction mixture, for example by
`
`ensuring that amplification can only be performed on some of the entities of the species of
`
`interest in the population, e.g. by using a limited number of adaptors or label molecules. The
`
`provision of a sample represents a bottlenecking step.
`
`15
`
`The method may comprise the bottlenecking step in combination with a modifying step. For
`
`example, the method may comprise:
`
`modifying entities of the species of interest in a population to provide a plurality of
`
`differently~modified entities,
`
`20
`
`providing the sample from the population, wherein the entities of the species of interest
`
`in the sample differ from each other,
`
`amplifying the different entities of the species of interest in the sample to provide a
`
`library of amplicons, then
`
`determining the number or concentration of different amplicons, and thereby determining
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`25
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`the number or concentration of entities of the species of interest in the sample.
`
`The modifying step provides a plurality of differently modified-entities, so that at least some
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`entities of differently modified, e.g. all or almost all entities may be differently modified.
`
`Optionally, there may be some entities in the population which are identically modified, or which
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`30
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`are not modified, so that the population comprises some identical and some different entities of
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`the species of interest. For example, this may be the case where the modifying comprises
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`labelling molecules in the population with fewer labels than the number of molecules. In such a
`
`situation, the step of providing a sample can reduce the number of identical entities of the
`
`species of interest, e.g. the number of identically modified entities of the species of interest, so
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`35
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`that the entities of the species of interest in the sample differ from each other. Alternatively, the
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`step of modifying entities of the species of interest can be performed such that all of the entities
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`of the species of interest differ from each other, in which the entities of the species of interest
`
`will also differ from each other in the sample that is provided from the population.
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`IPR2019-00634
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`PCT/IB2011/002438
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`9
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`As discussed elsewhere herein, it is not essential that absolutely all entities of the species of
`
`interest are rendered mutually distinguishable, since the method can still be applied when
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`almost all entities of the species of interest differ. For example, at least about 90%, 95%, 98%,
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`5
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`99 %, 99.9 % or 99.99 % of the entities of the species of interest may differ from each other.
`
`In accordance with the second aspect of the present invention, there is provided a method of
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`tracking a population of entities of a species of interest that comprises providing a plurality of
`
`entities that are different from each other, as set out in more detail below. The plurality of
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`1 O
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`different molecules may be polymers e.g. nucleic acids, which may be synthesised such that
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`they are each different from each other, for example such that they have different sequences
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`from each other (e.g. 20bp random sequence). In this context the different sequence structures
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`of the nucleic acids are effectively labels, even though no step of attaching a label is performed.
`
`15 Methods in accordance with the second aspect of the invention may comprise generating a first
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`plurality of first molecules and generating a second plurality of second molecules, as discussed
`
`below. The first and second molecules may be nucleic acids, which may be synthesised so that
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`they have molecule-identifying sequence (e.g. 20bp random sequence, or label) and a plurality(cid:173)
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`identifying sequence (e.g. 20bp predetermined sequence) which is different between different
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`20
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`pluralities of molecules, but may be the same within a particular plurality of molecules.
`
`Different labels may differ from each other in terms of length and/or sequence. The labels may
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`be intentionally made different from each other by more than one monomer (e.g. more than one
`
`bases) to improve unambiguous detection of the labels and to decrease the effect of
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`25
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`amplification and sequencing errors. The advantages of using labels of different lengths, e.g.
`
`to allow detection of the different labels by mass spectrometry, is further discussed below.
`
`The species of interest is the defined physical entity to be counted, e.g. a specific or defined
`
`molecular structure. The species of interest may be a class of biomolecule, such as nucleic
`
`30
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`acid, or protein. The species of interest may be a specific protein (i.e. polypeptide). Preferably
`
`the species of interest is a nucleic acid comprising or consisting of a specific nucleotide
`
`sequence. A nucleic acid species of interest may be DNA, for example cDNA, or may be RNA,
`
`for example mRNA (messenger RNA) or miRNA (microRNA). A nucleic acid species of interest
`
`may be single-stranded or double-stranded.
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`35
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`The species of interest may be a distinct type, a single type, or an individual kind, of molecule or
`
`other entity in the sample. The species of interest may be a nucleic acid comprising or
`
`consisting of a nucleotide sequence that is distinct in the sample (e.g. a specific nucleotide
`
`sequence that is present only in a sub-set of the nucleic acids in the sample}, and the method of
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`FOUNDATION EXHIBIT 1062
`IPR2019-00634
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`PCT/IB2011/002438
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`the invention is used to determine the number of nucleic acid molecules in the sample
`
`comprising or consisting of that distinct nucleotide sequence. A molecule of a species of
`
`interest which is a nucleic acid comprising a specific nucleotide sequence may be a
`
`chromosome, or chromosome fragment, which comprises a specific nucleotide sequence of a
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`5
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`gene of interest and/or which comprises a specific nucleotide sequence of a mutation of
`
`interest. The method of the invention allows the number or concentration of chromosomes
`
`(which may differ from each other) that comprise that specific nucleotide sequence to be
`
`determined. A species of interest which is a nucleic acid consisting of a specific nucleotide
`
`sequence may be a messenger RNA. The method of the invention allows the number or
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`1 O
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`concentration of mRNAs that consist of a specific nucleotide sequence to be determined.
`
`The species of interest may be a defined sequence or structure occurring within a larger
`
`sequence or structure, for example a defined sequence of monomers comprised within a
`
`polymer. The species of interest may be a specific nucleotide sequence, or region, comprised
`
`15
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`in a genomic sequence or chromosome. Different sequences in the same chromosome can be
`
`individually and separately counted, as explained in more detail below. This is relevant for
`
`example for copy number variation and deletion analysis where number of sequences within
`
`different parts of the same molecular structure (chromosome) are analyzed. Thus the present
`
`invention encompasses determining the number of copies of a sequence of interest in a sample.
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`20
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`A chromosome may have regions that are duplicated (2 or more copies of the same sequence),
`deleted (0 copies) or translocated (0 copies of original sequence, 1 or more copi