Analyte Detection Method Employing Concatemers

Abstract

Methods of detecting DNA sequences from multiple pools comprising at least one species of DNA molecule comprise combining the pools to form a combination pool; in the combination pool, generating at least one linear DNA concatemer containing one DNA molecule from each pool, wherein a position of each DNA molecule within the concatemer correlates to the pool from which the DNA molecule originated; and sequencing the concatemers, thereby detecting the DNA sequence of each DNA molecule at each position in each concatemer, wherein each detected DNA sequence is assigned to the pool from which its DNA molecule originated based upon its position within the concatemer.

Claims

1. A method of detecting DNA sequences from multiple pools, wherein each pool comprises at least one species of DNA molecule, the method comprising: (i) combining the pools to form a combination pool; (ii) in the combination pool, generating at least one linear DNA concatemer containing one DNA molecule from each pool, wherein a position of each DNA molecule within the concatemer correlates to the pool from which the DNA molecule originated; and (iii) sequencing the concatemers, thereby detecting the DNA sequence of each DNA molecule at each position in each concatemer, wherein each detected DNA sequence is assigned to the pool from which its DNA molecule originated based upon its position within the concatemer.

2. The method of claim 1, wherein the method comprises, prior to step (i), in each pool, joining to each DNA molecule of the pool a first end sequence, and, when the number N of multiple pools is greater than two, for at least N-2 pools, joining to each DNA molecule of each N-2 pool, a second end sequence, wherein each end sequence is different from the other end sequences and each end sequence of each pool is configured to join to one end sequence in one other pool to form the linear DNA concatemers.

3. The method of claim 1, wherein each DNA molecule is an amplicon generated in a DNA amplification reaction.

4. The method of claim 1, wherein each DNA molecule is a reporter DNA molecule specific for an analyte, and sequencing of each reporter DNA molecule results in detection of the corresponding analyte.

5. The method of claim 4, wherein the reporter DNA molecules are generated by a multiplex detection assay performed on a sample; and the method comprises performing multiple multiplex detection assays on one or more samples, in order to detect multiple analytes in each sample, and each multiplex detection assay yields a pool of reporter DNA molecules.

6. The method of claim 5, wherein each multiplex detection assay comprises a first PCR which generates a respective first PCR product; and wherein the first PCR products are modified by a second PCR, in order to prepare the first PCR products for concatenation, wherein the second PCR generates the multiple pools of DNA molecules.

7. The method of claim 5, wherein the detection assay is a proximity extension assay, comprising an extension step that generates the reporter DNA molecules, and an amplification step in which the reporter DNA molecules are amplified, and the extension and amplification steps take place within a single PCR.

8. The method of claim 7, wherein the multiple multiplex proximity extension assays are performed on the same sample; and wherein each proximity extension assay comprises detecting analytes using pairs of proximity probes, each proximity probe comprising: (i) an analyte-binding domain specific for an analyte; and (ii) a nucleic acid domain, wherein both probes within each pair comprise analyte-binding domains specific for the same analyte, and each probe pair is specific for a different analyte, and wherein each probe pair is designed such that on proximal binding of the pair of proximity probes to their respective analyte the nucleic acid domains of the proximity probes interact to generate a reporter DNA molecule; wherein at least 2 panels of proximity probe pairs are used, each panel being for the detection of a different group of analytes, and each multiplex proximity extension assay uses one panel of proximity probe pairs; wherein (a) within each panel, every probe pair comprises a different pair of nucleic acid domains; and (b) in different panels the probe pairs comprise the same pairs of nucleic acid domains; and wherein the product of each panel of proximity probe pairs forms one of the multiple pools.

9. The method of claim 1, wherein concatenation is performed by USER assembly or Gibson assembly.

10. The method of claim 9, wherein the method comprises performing a PCR on each pool using assembly primers, wherein all the DNA molecules in one pool are amplified using the same primer pair, and a different primer pair is used for amplification in each pool, and wherein each primer of the primer pairs comprises a unique assembly site which is complementary to one unique assembly site in one other pool; and wherein in step (ii), the PCR products of each pool are joined to the PCR products of different pools via their complementary assembly sites, thereby generating the linear concatemers.

11. The method of claim 10, wherein concatenation is performed by USER assembly, and each assembly site comprises multiple uracil residues.

12. The method of claim 10, wherein: (a) each DNA molecule is a reporter DNA molecule specific for an analyte and obtained by performing multiple multiplex proximity extension assays, the multiple multiplex proximity extension assays generating the multiple pools of reporter DNA molecules, wherein the reporter DNA molecules in each pool comprise universal primer binding sites at their 3′ and 5′ termini; (b) the linear concatemers are formed by USER assembly comprising: (i) processing the PCR products in each pool to generate 3′ overhangs comprising the assembly sites; (ii) combining the pools; and (iii) generating the multiple linear DNA concatemers, the PCR products of each pool being joined to the PCR products of different pools having complementary 3′ overhangs; and (d) sequencing the concatemers, thereby identifying the analytes detected in each proximity extension assay; wherein the analytes detected in each proximity extension assay are identified based on the combination of the sequence of each reporter DNA molecule and its position within its concatemer.

13. The method of claim 1, wherein the linear DNA concatemers are subjected to a PCR to add at least a first sequencing adaptor to the concatemers.

14. The method of claim 13, wherein in the PCR a first sequencing adaptor is added to one end of the concatemers, and a second sequencing adaptor is added to the other end of the concatemers.

15. The method of claim 1, wherein the linear DNA concatemers are subjected to a PCR to add at least a first sequencing primer binding site to the concatemers.

16. The method of claim 15, wherein in the PCR a first sequencing primer binding site is added at one end of the concatemers, and a second sequencing primer binding site is added at the other end of the concatemers.

17. The method of claim 1, wherein: (I) multiple sets of pools are individually combined and a separate concatenation reaction performed for each set of pools, yielding multiple concatenation reaction products; (II) a unique index sequence is added to each concatenation reaction product by PCR; (III) the concatenation reaction products are combined; and (IV) the concatemers are sequenced, and the index sequence identifies the set of pools from which each concatemer originates.

18. The method of claim 17, wherein in the PCR a first index sequence is added at one end of the concatemers, and a second index sequence is added at the other end of the concatemers.

19. The method of claim 18, wherein the concatemers are subjected to a single PCR, in which a sequencing adaptor, a sequencing primer binding site, and an index sequence are added to both ends of each concatemer.

20. The method of claim 19, wherein the PCR to which the concatemers are subjected yields products comprising, at each end, from 5′ to 3′, a sequencing adaptor, a sequencing primer binding site, and an index sequence.

21. A method of detecting multiple analytes in one or more samples, comprising: (i) performing multiple multiplex detection assays on one or more samples, in order to detect multiple analytes in each sample, wherein each multiplex detection assay is a proximity extension assay comprising an extension step that generates reporter DNA molecules, and an amplification step in which the reporter DNA molecules are amplified, wherein the extension and amplification steps take place within a single PCR and yield a pool of amplified reporter DNA molecules, each reporter DNA molecule being specific for an analyte, (ii) performing a PCR on each pool using assembly primers, wherein all the reporter DNA molecules in one pool are amplified using the same primer pair, and a different primer pair is used for amplification in each pool, and wherein each primer of the primer pairs comprises a unique assembly site which is complementary to one unique assembly site in one other pool; (iii) combining the PCR products of each pool to form a combination pool; (iv) in the combination pool, forming by USER assembly linear DNA concatemers containing a PCR product of one reporter DNA molecule from each pool, wherein a position of each PCR product of a reporter DNA molecule within the concatemer correlates to the pool from which the reporter DNA molecule originated; (v) subjecting the concatemers to a single PCR in which a sequencing adaptor, a sequencing primer binding site, and an index sequence are added to both ends of each concatemer; and (vi) sequencing the concatemers, thereby identifying the analytes detected in each proximity extension assay based on the combination of the sequence of each reporter DNA molecule and its position within its concatemer.

22. A kit comprising: (i) multiple proximity probe pairs, wherein each proximity probe comprises: an analyte-binding domain specific for an analyte; and a nucleic acid domain, wherein in each pair, the nucleic acid domain of one proximity probe comprises a first universal primer binding site and a barcode sequence 3′ thereof, and the nucleic acid domain of the other proximity probe comprises a second universal primer binding site and a barcode sequence 3′ thereof, wherein both probes within each pair comprise analyte-binding domains specific for the same analyte, and each probe pair is specific for a different analyte, and wherein each probe pair is designed such that on proximal binding of the pair of proximity probes to their respective analyte the nucleic acid domains of the proximity probes interact to generate a reporter DNA molecule; (ii) a first primer pair, wherein the primers are designed to bind the first and second universal primer binding sites; (iii) a set of assembly primer pairs suitable for preparing DNA molecules for directed assembly by USER assembly or Gibson assembly into a linear concatemer, wherein each primer comprises, from 5′ to 3′, an assembly site and a hybridisation site, and in each primer pair the hybridisation sites are designed to bind the first and second universal primer binding sites; (iv) enzymes suitable for assembling DNA fragments by USER assembly or Gibson assembly, wherein the enzymes are suitable for use in the same means of DNA assembly as the assembly primer pairs; and (v) a second primer pair, wherein each primer comprises a sequencing adaptor, a sequencing primer binding site, an index sequence and a hybridisation site, wherein the hybridisation sites are designed to bind the assembly sites of the assembly primers designed to form the ends of the linear concatemer; and wherein the first primer in the pair comprises a first sequencing adaptor, a first sequencing primer binding site and a first index sequence, and the second primer in the pair comprises a second sequencing adaptor, a second sequencing primer binding site and a second index sequence.

Description

DESCRIPTION OF THE FIGURES

[0334] FIG. 1 shows a schematic representation of six different versions of proximity extension assays, described in detail above. The inverted ‘Y’ shapes represent antibodies, as an exemplary proximity probe analyte-binding domain.

[0335] FIG. 2 shows a schematic representation of examples of extension controls which may be used in proximity extension assays. Parts A-F show suitable extension controls for use in versions 1-6 of FIG. 1, respectively. In parts B-E, different possible extension controls for use in versions 2-5 of FIG. 1, respectively, are shown in options (i) and (ii). The legend for FIG. 1 also applies to FIG. 2.

[0336] FIG. 3 shows a comparison of normalised count number obtained by two PEA protocols, using 4 probe panels to assay a plasma sample. Normalised counts obtained using an “index inside” concatenation protocol are compared to normalised counts obtained using a method not including concatenation. A high correlation between the normalised counts obtained using the two protocols is seen (R=0.91).

[0337] FIG. 4 shows a comparison of normalised count number for IL-8 specifically from the assays compared in FIG. 3. A high correlation between the normalised counts obtained using the two protocols is seen for each panel (R=0.97-0.99).

[0338] FIG. 5 shows a comparison of normalised count number obtained by two PEA protocols, using 4 probe panels to assay a plasma sample. Normalised counts obtained using an “index inside” concatenation protocol are compared to normalised counts obtained using an “index outside” concatenation protocol. A high correlation between the normalised counts obtained using the two protocols is seen (R=0.98).

[0339] FIG. 6 shows a comparison of normalised count number for IL-8 specifically from the assays compared in FIG. 5. A high correlation between the normalised counts obtained using the two protocols is seen for each panel (R=0.99-1.00).

[0340] FIG. 7 shows a schematic representation of a method as disclosed herein, and depicts the generation of a concatemer comprising a PCR amplicon from each of 4 pools, A, B, C and D. Each pool comprises amplicons from a set of assays. PCR amplicons in each pool are generated by PCR1. A single amplicon from each pool is shown. In PCR2 the amplicons are provided with defined end sequences, which permit directed concatenation, using assembly primers. The assembly primers comprise a 5′ primer (“pool-specific” portion) which comprises the defined end sequence, and a 3′ primer hybridisation site (“universal” portion) which hybridises to the amplicon. A star (*) indicates a complementary sequence to the corresponding letter. For example, the sequence labelled “A*” is complementary to the sequence labelled “A.” The ends are digested. The digested products from pools A, B, C and D are pooled (combined), and ligated to generate a concatemeric product. PCR3 is performed to add sequencing adaptors to the ends.

EXAMPLES

Example 1—Exemplary Experimental Protocol

Step 1—Sample Preparation and Incubation

[0341] Sixteen aliquots from each of 48 to 96 plasma samples are incubated with one of each of 16 proximity probe sets (four abundance blocks from each of four 384-probe pair panels) in 96-well or 384-well incubation plates. [0342] Samples may be pre-diluted 1:10, 1:100, 1:1000 and 1:2000 for those probe panels/groups containing assays that require it. [0343] Dilution and dispensing of plasma samples into incubation solution can be performed manually, or by pipetting robot e.g. LabTech's Mosquito® HTS. Incubation solution is dispensed into the wells of the plate. [0344] 1 μl of sample is added to 3 μl of incubation mix at the bottom of each well, the plate is sealed with adhesive film, spun at 400×g for 1 minute at room temperature and incubated overnight at 4° C. [0345] If using the above-mentioned pipetting robot, volumes may be decreased to 0.2 μl sample and 0.6 μl incubation mix (5× reduction).
The tables below give exemplary reagent formulations. Other components may be included, for example other blocking agents in the probe solutions.

TABLE-US-00001 TABLE 1 Sample Diluent and Negative Control Solution Component Concentration NaCl 8.01 g/l KCl 0.2 g/l Na.sub.2HPO.sub.4 1.44 g/l KH.sub.2PO.sub.4 0.2 g/l BSA 1 g/l

TABLE-US-00002 TABLE 2 Incubation Mix 4 μl 0.8 μl Incubation Incubation Volume Volume Reagent Volume (μl) Volume (μl) Incubation Solution 2.40 0.48 Forward Probe Solution 0.30 0.06 Reverse Probe Solution 0.30 0.06 Sample 1.00 0.20 Total 4.0 0.8

TABLE-US-00003 TABLE 3 Incubation Solution Component Concentration Triton X-100 1.70 g/l NaCl 8.01 g/l KCl 0.2 g/l Na.sub.2HPO.sub.4 1.44 g/l KH.sub.2PO.sub.4 0.2 g/l EDTA Na-salt 1.24 g/l BSA 8.80 g/l Blocking-probes Mix 0.199 g/l GFP 1-5 pM

TABLE-US-00004 TABLE 4 Forward Probe Solution Component Concentration NaCl 8.01 g/l KCl 0.2 g/l Na.sub.2HPO.sub.4 1.44 g/l KH.sub.2PO.sub.4 0.2 g/l EDTA Na-salt 1.24 g/l Triton X-100 1 g/l BSA 1 g/l Probes 1-100 nM per probe

TABLE-US-00005 TABLE 5 Reverse Probe Solution Component Concentration NaCl 8.01 g/l KCl 0.2 g/l Na.sub.2HPO.sub.4 1.44 g/l KH.sub.2PO.sub.4 0.2 g/l EDTA Na-salt 1.24 g/l Triton X-100 1 g/l BSA 1 g/l Probes 1-100 nM per probe Detection Control 6.4-1188 fM Extension Control 75-10686 fM

Step 2—Proximity Extension and Reporter Molecule Amplification

[0346] Extension and amplification are performed using Pwo DNA polymerase. The PCR is performed using common primers for amplification of all extension products. (See, for example, PCR1 in FIG. 7)

[0347] The incubation plate (from step 1) is brought to room temperature and centrifuged at 400×g for 1 minute. The extension mix (comprising ultrapure water, DMSO, Pwo DNA polymerase and reaction solution) is added to the plate, and the plate is then sealed, briefly vortexed and centrifuged at 400×g for 1 minute, then placed in a thermal cycler for the PEA reaction and amplification (50° C. 20 min, 95° C. 5 min, (95° C. 30 s, 54° C. 1 min, 60° C. 1 min)×25 cycles, 10° C. hold). Preferably, a dispensing robot may be used to dispense the extension mix into the plate, e.g. the Thermo Scientific™ Multidrop™ Combi Reagent Dispenser.

TABLE-US-00006 TABLE 6 PEA PCR Reaction Mix 4 μl 0.8 μl Incubation Volume Incubation Volume Reagent Volume (μl) Volume (μl) MilliQ water 75.0 15.00 DMSO (100%) 10.0 2.00 Reaction Solution 10.0 2.0 DNA Polymerase 1.0 0.2 (1-10 U/μ1) Incubation mix 4.0 0.8 Total 100.0 20.0

TABLE-US-00007 TABLE 7 Reaction Solution Component Concentration Tris base 168.40 mM Tris-HCl 31.47 mM MgCl.sub.2 hexahydrate 10.00 mM dATP 2.00 mM dCTP 2.00 mM dGTP 2.00 mM dTTP 2.00 mM Forward primer 10.00 μM Reverse primer 10.00 μM

Step 3—Pooling Abundance Blocks

[0348] PCR products from each of the abundance blocks from each 384-probe pair panel from each sample are pooled together. This results in four mixtures (pools) of PCR products per sample, one for each 384-probe pair panel. Each pool in this case is thus a mixture, or collection, of PCR products which corresponds to a panel of proximity probes, or in other words, a panel of assays performed on a sample. The pool is made up of the PCR products derived from four abundance blocks (i.e. there are four abundance blocks for each panel. Each block corresponds to a set of assays, based on the relative abundances of the analytes under test in each assay).

[0349] Different volumes can be taken from each abundance block to even out the relative numbers of assays between the blocks. Pooling of PCR products can be performed manually, or by pipetting robot.

Step 4—Amplification with Assembly Primers

[0350] For each mixture of PCR products (i.e. the product of each 384-probe pair panel) from each sample, a separate second PCR is performed using assembly primers for USER assembly. This is depicted as PCR2 in FIG. 7. Each assembly primer comprises a “pool-specific” portion, which comprises or provides the defined end sequence to be added to the amplicon and a “universal” portion that hybridises to the amplicon; the universal portion, and its complementary binding site, are shared between the amplicons of different pools. A set of USER assembly primers is used for the various panel products of each sample. An exemplary set of assembly primers is shown in the table below (as shown, each primer has a unique assembly site, which with the exception of the terminal assembly sites have a neighbouring complementary site, and each of the forward and reverse hybridisation sites are, respectively, the same). One pair of assembly primers is used for amplification of the products of each panel (which corresponds to each pool) from a sample, e.g. using the exemplified primers, for each sample Pair A is used for panel 1, Pair B for panel 2, Pair C for panel 3 and Pair D for panel 4 (corresponding to pools 1-4 as depicted in FIG. 7). The products of the first PCR are added to a second PCR mix (comprising Taq polymerase, dNTPs, universal buffer and assembly primers in ultrapure water) and PCR is performed: 95° C. 3 min, (95° C. 30 sec, 45° C. 30 sec, 72° C. 1 min)×5 cycles, (95° C. 30 sec, 65° C. 30 sec, 72° C. 1 min)×10 cycles, 10° C. hold.

TABLE-US-00008 TABLE 8 Second PCR Mix Reagent Volume Polymerase Buffer (20X stock) 0.5 μl dNTPs (25 mM of each) 0.08 μl Taq polymerase (5 U/μl) 0.05 μl MilliQ Water 4.87 μl Assembly Primers (5 μM of each) 2.5 μl PEA-PCR Product (0.1 μM) 2 μl Total Volume: 10 μl

TABLE-US-00009 TABLE 9 Assembly Primers Pair A Forward 5′ CCUCUGCUGCUCUCAUUGUCGCTCTTCCGATCT 3′ SEQ ID NO: 5 Pair A Reverse 5′ ACACUGUACGUTAGAGACTCCAAGC 3′ SEQ ID NO: 6 Pair B Forward 5′ ACGUACAGUGUCGCTCTTCCGATCT 3′ SEQ ID NO: 7 Pair B Reverse 5′ AGCUCAAUCCUTAGAGACTCCAAGC 3′ SEQ ID NO: 8 Pair C Forward 5′ AGGAUUGAGCUCGCTCTTCCGATCT 3′ SEQ ID NO: 9 Pair C Reverse 5′ ACAGACUUACUTAGAGACTCCAAGC 3′ SEQ ID NO: 10 Pair D Forward 5′ AGUAAGUCUGUCGCTCTTCCGATCT 3′ SEQ ID NO: 11 Pair D Reverse 5′ GUGCGUGCAUGAUCCUACUTAGAGACTCCAAGC 3′ SEQ ID NO: 12 Assembly sites are underlined. Uracil residues for USER assembly are highlighted in bold.

Step 5—Digestion

[0351] The products of Step 4 are digested to degrade the uracil-containing assembly sites, leaving 3′ overhangs at the end of each PCR product. The product of each separate second PCR is digested separately. The second PCR products are added to USER enzymes and incubated at 37° C. for 60 to 120 minutes.

TABLE-US-00010 TABLE 9 Digestion Mix Reagent Volume Enzyme Buffer (20X) 1 μl Endo VIII (10 U/μl) 1 μl UDG (1 U/μl) 1 μl Second PCR Product (1.25 μM) 10 μl Total Volume: 13 μl

Step 6—Concatenation

[0352] The digested products of each PEA panel (each panel representing a pool of products from four abundance blocks) from each sample are combined and ligated to generate a concatemer comprising a product from each panel of the sample in question. The products are concatenated in the order defined by the complementary overhangs generated from the assembly sites. In the example above, where Panel 1 was amplified with assembly primer pair A, Panel 2 with assembly primer pair B, Panel 3 with assembly primer pair C and Panel 4 with assembly primer pair D, the products of the panels are concatenated in the order Panel 1-Panel 2-Panel 3-Panel 4.

TABLE-US-00011 TABLE 10 Ligation Mix Reagent Volume ATP (10 mM) 1 μl T4 Ligase (400 U/μl) 1 μl Pooled Digested Product (240 nM) 8 μl Total Volume: 10 μl

Step 7—Attachment of Sequencing Adaptors

[0353] For Illumina sequencing, sequencing adaptors are added to both ends of each concatemer. This is performed in a third PCR (depicted as PCR3 in FIG. 7), which is also used to add sequencing primer binding sites and index sequences to identify the sample from which each concatemer derives. The primers for the third PCR comprise, from 5′ to 3′, a sequencing adaptor (e.g. the P5 and P7 adaptors, mentioned above), a sequencing primer binding site (e.g. Rd1SP and Rd2SP binding sites, mentioned above), an index sequence and the hybridisation site.

[0354] Ligated concatemers are added to a third PCR mix comprising Taq polymerase, primers, buffer and dNTPs, and amplified: 95° C. 3 min, (95° C. 30 sec, 60° C. 30 sec, 72° C. 1 min)×5 cycles, (95° C. 30 sec, 65° C. 30 sec, 72° C. 1 min)×15 cycles, 10° C. hold.

TABLE-US-00012 TABLE 11 Third PCR Mix Reagent Volume MilliQ Water 5.5 μl Polymerase Buffer (20X) 1 μl dNTP Mix (2.5 mM of each) 0.8 μl Taq Polymerase (5 U/μl) 0.05 μl Forward Primer (100 μM) 0.1 μl Reverse Primer (100 μM) 0.1 μl Ligation Product (1.92 nM) 2 μl Total Volume: 10 μl

Step 8—Sequencing

[0355] Concatemers are pooled and then sequenced using an Illumina platform (e.g. the NoveSeq platform). By generating concatemers comprising reporter DNA molecules from four panels, the throughput of each sequencing run is increased four-fold.

Step 9—Data Output

[0356] Barcode (from each reporter DNA molecule) and index (from each concatemer) sequences are identified in the data, counted, summed and aligned/labeled according to a known barcode-assay-sample key. [0357] “Matching barcodes” represent interactions between two paired PEA probes. The count is relative to the number of interactions in the PEA. [0358] Counts for each assay and sample must be normalised using the internal reference controls to be able to compare between samples. [0359] Each abundance block has its own internal reference control.

Example 2—Reference Example of Method without Concatenation

[0360] This reference protocol is disclosed in co-pending application PCT/EP2021/058008. In this protocol, steps 1 to 3 were performed as in Example 1. Thereafter the protocol was as follows:

Step 4—PCR2 Indexing

[0361] A primer plate containing 48 to 96 reverse primers is provided (generally one primer in each well of a 96-well plate). Each reverse primer comprises the “IIlumina P7” sequencing adapter sequence (SEQ ID NO: 2) and a sample index barcode. A unique barcode sequence is used for PCR1 products (i.e. the products of the PCR performed in Step 2) from each different sample. Preferably each of the up to four PCR1 pools comprising the same plasma sample (one for each 384-probe pair panel) receive the same index sequence, for easy identification and data processing. A forward common primer comprising the “Illumina P5” sequencing adapter sequence (the same forward primer as used in PCR1) is provided in the PCR2 solution.

[0362] Each PCR1 pool is contacted with PCR2 solution containing the forward common primer, a single reverse (index) primer from the primer plate, and a DNA polymerase (Taq or Pwo DNA polymerase). Amplification is performed by PCR until primer depletion (95° C. 3 min, (95° C. 30 s, 68° C. 1 min)×10 cycles, 10° C. hold).

[0363] The theoretical end concentration of pooled PCR1 product is 1 μM (all primers used). PCR1 amplicons are diluted 1:20 dilution for PCR2, giving a starting concentration of 50 nM in each PCR2 reaction. The concentration of each PCR2 primer is 500 nM. PCR2 primer depletion should therefore occur after 3.3 cycles (10-fold amplification).

TABLE-US-00013 TABLE 8 PCR2 Reaction Mix Reagent Volume (μl) MilliQ water 14.96 PCR2 solution 2.0 DNA Polymerase (1-10 U/μ1) 0.04 Sample index primer solution 2.0 Pooled PCR1 reactions 1.0 Total 20.0

TABLE-US-00014 TABLE 9 PCR2 Solution Component Concentration Tris base 168.40 mM Tris-HCl 31.47 mM MgC1.sub.2 hexahydrate 10.00 mM dATP 2.00 mM dCTP 2.00 mM dGTP 2.00 mM dTTP 2.00 mM Forward “P5” Primer 5.00 μM

TABLE-US-00015 TABLE 10 Index Primer Solution Component Concentration Tris base 1.948 mM Tris-HCl 8.052 mM EDTA 1 mM Index “P7” primer 5.00 μM

Step 5—End Pool

[0364] All 48 to 96 indexed sample pools belonging to the same 384-probe pair panel are pooled together, adding the same volume from each sample. This yields up to four final pools (or libraries), one for each 384-probe pair panel.

Step 6—Purification and Quantification (Optional)

[0365] The libraries are purified separately using magnetic beads, and purified libraries' total DNA concentration is determined using qPCR with a DNA standard curve. AMPure XP beads (Beckman Coulter, USA), which preferentially bind longer DNA fragments, may be used in accordance with the manufacturer's protocol. The AMPure XP beads bind the long PCR products but do not bind short primers, thus enabling purification of the PCR product from any remaining primers.

[0366] Depletion of the PCR2 primers means that this purification step may not be necessary.

Step 7—Quality Control (Optional)

[0367] A small aliquot of each (purified) library is analysed on an Agilent Bioanalyser (Agilent, USA), in accordance with the manufacturer's instructions, to confirm successful DNA amplification.

Step 8—Sequencing

[0368] Libraries are sequenced using an Illumina platform (e.g. the NoveSeq platform). Each of the up to four libraries (from each 384-probe pair panel) is run in a separate “lane” of a flow cell. Depending on the size and model of flow cell and sequencer used, the up to four libraries may be sequenced in parallel or sequentially (one after the other) in different flow cells.

Step 9—Data Output

[0369] Barcode (from each reporter nucleic acid molecule) and sample index (from the sample index primers) sequences are identified in the data, counted, summed and aligned/labeled according to a known barcode-assay-sample key. [0370] “Matching barcodes” represent interactions between two paired PEA probes. The count is relative to the number of interactions in the PEA. [0371] Counts for each assay and sample must be normalised using the internal reference controls to be able to compare between samples. [0372] Each of the four abundance blocks has its own internal reference control.
Each 384-probe pair panel is separated based on the lane it is read out in. Each panel comprises the same 96 sample indexes and the same 384 barcode combinations and internal reference controls.

Example 3—Sequencing of Concatenated and Unconcatenated Reporters

[0373] Three reaction protocols were compared:

[0374] 1. A protocol as described above in Example 1 (referred to as “Index Inside”).

[0375] 2. A protocol as described above in Example 1, with the exception of a difference in the primers used for the third PCR. In protocol 2, the primers for the third PCR were arranged differently to in Example 1. Specifically, the primers for the third PCR comprised, from 5′ to 3′, a sequencing adaptor, an index sequence, a sequencing primer binding site and the hybridisation site (i.e. the order of the index sequence and the sequencing primer binding site is reversed, referred to as “Index Outside”).

[0376] 3. A protocol as described in Example 2.

[0377] For each of the three protocols, eight plasma samples were tested and compared. Each sample was assayed using four panels of PEA probes, each of which contained 372 probe pairs. Each of the panels included a probe pair for detection of IL-8. After sequencing, all matched barcode reads (counts) within each abundance block were normalized against an internal control. The normalised barcode counts generated by each protocol were compared.

[0378] A comparison of the normalised counts obtained from protocols 1 and 3 for one sample (sample 7) is shown in FIG. 3. The figure shows a high correlation (R.sup.2=0.91) between the normalised counts obtained with the two different protocols (and similar R.sup.2 values were obtained for the other seven samples as well), showing that the two different protocols generate approximately the same number of normalised barcode counts for each probe pair used to assay the sample. The normalised counts obtained from protocols 1 and 2 for the same sample were also compared, as shown in FIG. 5. The figure shows a very high correlation (R.sup.2=0.98) between the normalised counts obtained with the two different protocols (and similar R.sup.2 values were obtained for the other 7 samples as well), showing that there is essentially no difference between the performance of the “index inside” and “index outside” protocols.

[0379] The normalised counts from the different protocols for IL-8 were also specifically compared. The counts for IL-8 obtained from each assay panel using protocols 1 and 3 for each of the 8 samples were compared, as shown in FIG. 4. The figure shows a high level of correlation between the normalised counts obtained with the two methods (R.sup.2 values between 0.97 and 0.99 for the four different assay panels). The same comparison was made for normalised counts obtained using protocols 1 and 2, as shown in FIG. 6. The figure shows a very high level of correlation between the normalised counts obtained with the two methods (R.sup.2 values between 0.99 and 1 for the four different assay panels).

[0380] These results show that very similar results are obtained when assaying a sample using a PEA method comprising a concatenation step as provided herein, as when using the earlier method in which each reporter DNA molecule is individually sequenced. If a sample contains a high or low level of a particular target protein (e.g. IL-8), this is correctly identified in all of the three protocols tested. As detailed above, concatenation allows a significant improvement in throughput of the method, and these results show that the improvement in throughput is obtained without any loss of accuracy.