METHOD FOR DETECTING ANALYTES OF VARYING ABUNDANCE

Abstract

The present invention provides a method of detecting multiple analytes in a sample, wherein said analytes have varying levels of abundance in the sample, said method comprising: (i) providing multiple aliquots from the sample; and (ii) in each aliquot, detecting a different subset of the analytes by performing a separate multiplex assay for each aliquot, wherein the analytes in each subset are selected based on their predicted abundance in the sample.

Claims

1. A method of detecting multiple analytes in a sample, wherein said analytes have varying levels of abundance in the sample, said method comprising: (i) providing multiple aliquots from the sample; and (ii) in each aliquot, detecting a different subset of the analytes by performing a separate multiplex assay for each aliquot, wherein the analytes in each subset are selected based on their predicted abundance in the sample.

2. The method of claim 1, wherein the analyte is a non-nucleic acid analyte.

3. The method of claim 1 or 2, wherein the analyte is or comprises a protein.

4. The method of any one of claims 1 to 3, wherein in each aliquot the analytes are detected by detecting a reporter nucleic acid molecule specific for each analyte.

5. The method of claim 4, wherein the reporter nucleic acid molecules are generated in the multiplex detection assay performed for each aliquot.

6. The method of claim 4 or 5, wherein the reporter nucleic acid molecules are amplified by PCR, and preferably are detected by nucleic acid sequencing.

7. The method of claim 6, wherein one or more adapters for sequencing are added to the reporter nucleic acid molecules in one or more amplification and/or ligation steps.

8. The method of claim 6 or 7, wherein the reporter nucleic acid molecules are subjected to at least a first PCR reaction to add at least a first adaptor for nucleic acid sequencing.

9. The method of claim 8, wherein the PCR products from the first PCR reaction are subjected to a second PCR reaction to add a second adaptor for nucleic acid sequencing.

10. The method of any one of claims 6 to 9, wherein at least one PCR reaction is run to saturation.

11. The method of any one of claims 1 to 10, wherein the reaction products of the separate multiplex assays or, where said reaction products are nucleic acid molecules, amplification products thereof, are pooled to create a first pool, and are amplified in the first pool.

12. The method of claim 11, wherein the reaction products of the multiplex assays are reporter nucleic acid molecules, and the method comprises: amplifying the reporter nucleic acid molecules in first PCR reactions performed separately on each individual aliquot to generate first PCR products, pooling the first PCR products from individual aliquots to create a first pool, and performing a second PCR reaction on the first pool.

13. The method of claim 11 or 12, wherein different amounts of the reaction products or amplification products thereof are added to the first pool.

14. The method of any one of claims 11 to 13, wherein the method is performed in parallel for multiple different samples separately to generate reaction products, or amplification products thereof, for each sample, and wherein for each sample a separate first pool is created and a sample index is added to the products in the first pool by an amplification and/or ligation reaction.

15. The method of claim 14, wherein the separate first pool created for each sample comprises first PCR products, and wherein a sample index is added to the first PCR products in the second PCR reaction which is performed on the first pool for each sample.

16. The method of claim 14 or 15, wherein the indexed first pools generated for each sample are pooled together to create a second pool for performing nucleic acid sequencing.

17. The method of any one of claims 6 to 16, wherein the PCR reaction comprises an internal control for each aliquot.

18. The method of any one of claims 4 to 17, wherein the reporter nucleic acid molecule is generated in a proximity probe detection assay, in particular a proximity extension assay (PEA).

19. The method of any one of claims 4 to 16, wherein the reporter nucleic acid molecule comprises at least one barcode sequence, and detection of the reporter nucleic acid molecule comprises detecting the at least one barcode sequence, optionally in conjunction with a sample index, preferably wherein the reporter nucleic acid molecule comprises a combination of barcode sequences from the nucleic acid domains of a pair of proximity probes, and detection of the reporter nucleic acid molecule comprises detection of the combination of barcode sequences.

20. The method of any one of claims 1 to 19, wherein the sample is a plasma or serum sample.

21. The method of any one of claims 18 to 20, wherein the analytes are detected 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 nucleic acid 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 for each panel separate aliquots of the sample are provided for the detection of a different subset of the analytes in the group; and 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.

22. The method of claim 21, for detecting analytes from different samples, wherein the PCR products generated by amplification of the reporter nucleic acid molecules generated for each sample are provided with a sample index; and wherein the PCR products generated from each different sample using the same panel of proximity probe pairs are pooled into a panel pool for nucleic acid sequencing, the PCR products generated using each panel being pooled into separate panel pools; and wherein each panel pool is sequenced separately.

23. The method of any one of claims 7 to 22, wherein said nucleic acid sequencing is massively parallel DNA sequencing.

Description

BRIEF DESCRIPTION OF FIGURES

[0189] 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.

[0190] 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.

[0191] FIG. 3 shows the resulting counts (correctly paired barcodes) on a Log.sub.10 scale for 367 assays in one plasma sample. A comparison is made between contacting the sample with a probe pool comprising all 367 assays and contacting the sample with the same set of probes divided into four abundance blocks. The counts for assays in Blocks A and B have increased significantly compared to the assays with lower counts when not using abundance blocks, allowing higher detection of the corresponding assays. Counts for Block D have correspondingly decreased, mitigating the loss of flow cell real estate, compared to the assays with higher counts when not using abundance blocks.

[0192] FIG. 4 shows the resulting counts (correctly paired barcodes) on a linear scale for 367 assays in one plasma sample. A comparison is made between contacting the sample with a probe pool comprising all 367 assays and contacting the sample with the same set of probes divided into four abundance blocks. The counts for assays in Blocks A and B have increased significantly compared to the assays with lower counts when not using abundance blocks, allowing higher detection of the corresponding assays. Counts for Block D have correspondingly decreased, mitigating the loss of flow cell real estate, compared to the assays with higher counts when not using abundance block.

[0193] FIG. 5 shows boxplots of the results in 54 plasma samples contacted with a probe pool of 372 assays divided into four abundance blocks and sorted by the median count within a block. The abundance blocks allow detection of wide ranges of protein abundance between the samples without sacrificing detection, or risk the lower ranges of a assay with high variation between samples falling below a robust detection of counts. The dashed line indicates 100 counts as a threshold for enough detection of counts.

EXAMPLES

Example 1—Exemplary Experimental Protocol

Step 1—Sample Preparation and Incubation

[0194] 16 Aliquots from each of 48 to 96 plasma samples are incubated with each of up to 16 proximity probe pools (four abundance blocks for each of four 384-probe pair panels) in 96-well or 384-well incubation plates. [0195] Samples may be pre-diluted 1:10, 1:100, 1:1000 and 1:2000 for those probe pools containing assays that require it. [0196] 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. [0197] 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. [0198] If using the above-mentioned pipetting robot, volumes may be decreased to 0.2 μl sample and 0.6 μl incubation mix (5× reduction).

[0199] 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 Volume Incubation 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 PCR1 Amplification

[0200] Extension and amplification are performed using Pwo DNA polymerase. PCR1 is performed using common primers for amplification of all extension products.

[0201] 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 PCR1 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 preamplification (50° C. 20 min, 95° C. 5 min, (95° C. 30s, 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. The forward common primer comprises the Illumina P5 sequencing adapter sequence (SEQ ID NO: 1).

TABLE-US-00006 TABLE 6 PCR1 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 PCR1 solution 10.0 2.0 DNA Polymerase (1-10 U/μl) 1.0 0.2 Incubation mix 4.0 0.8 Total 100.0 20.0

TABLE-US-00007 TABLE 7 PCR1 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 “P5” primer 10.00 μM Reverse primer 10.00 μM

Step 3—Pooling Abundance Blocks

[0202] PCR1 products from each of the four abundance blocks from a 384-probe pair panel are pooled together. This results in up to four PCR1 pools per sample, one for each 384-probe pair panel.

[0203] Different volumes can be taken from each block to even out the relative levels of assays between the blocks. Pooling of PCR1 products can be performed manually, or by pipetting robot.

Step 4—PCR2 Indexing

[0204] 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 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 sample index, for easy identification and data processing. A forward common primer comprising the “IIlumina P5” sequencing adapter sequence (the same forward primer as used in PCR1) is provided in the PCR2 solution.

[0205] Each PCR1 pool is contacted with PCR2 solution containing the forward common primer, a single reverse (sample 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).

[0206] 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-00008 TABLE 8 PCR2 Reaction Mix Reagent Volume (μl) MilliQ water 14.96 PCR2 solution 2.0 DNA Polymerase (1-10 U/μl) 0.04 Sample index primer solution 2.0 Pooled PCR1 reactions 1.0 Total 20.0

TABLE-US-00009 TABLE 9 PCR2 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 “P5” Primer 5.00 μM

TABLE-US-00010 TABLE 10 Sample Index Primer Solution Component Concentration Tris base 1.948 mM Tris-HCl 8.052 mM EDTA 1 mM Sample index “P7” primer 5.00 μM

Step 5—End Pool

[0207] 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)

[0208] 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.

[0209] Depletion of the PCR2 primers means that this purification step may not be necessary.

Step 7—Quality Control (Optional)

[0210] 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

[0211] 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

[0212] 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. [0213] “Matching barcodes” represent interactions between two paired PEA probes. The count is relative to the number of interactions in the PEA. [0214] Counts for each assay and sample must be normalised using the internal reference controls to be able to compare between samples. [0215] Each of the four abundance blocks has its own internal reference control.

[0216] 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 2—PEA with and without Abundance Blocks

[0217] A multiplex PEA was performed (using probes comprising antibodies conjugated to nucleic acid domains having the structure described in Version 6, above) to detect 367 proteins in plasma samples. Each probe contained a unique barcode sequence. A proximity probe pool comprising all 367 assays was incubated with the samples, and as a comparison, 4 aliquots from each of the plasma samples were incubated with each of 4 proximity probe pools (four abundance blocks comprising the 367 assays) in 96-well or 384-well incubation plates.

[0218] The PEA was performed as described above, except Step 3 was omitted for the proximity probe pool without abundance blocks. During amplification of the extension products, P5 and P7 sequencing adapters were added to each end of the products, along with a unique sample index for reporter nucleic acids from each different sample, and all extension products sequenced by massively parallel DNA sequencing, employing the reversible dye terminator sequencing technique using an Illumina NovaSeq platform. The extension product resulting from the probe pool with 367 assays and extension products resulting from the pooled abundance blocks totaling 367 assays were sequenced at separate times in separate flow cells.

[0219] Results for one of the plasma samples can be seen in FIG. 3 and FIG. 4. The table below shows the ratio between the highest assay (counts) and lowest assay (counts) with and without using abundance blocks in the same plasma sample. The ratios in the abundance blocks are significantly lower than the ratio of the full pool of 367 assays, meaning the readouts of these assays use the flow cell real estate in a more optimal way (more counts for low abundance assays, fewer counts for high abundance assays).

TABLE-US-00011 Number Highest Lowest of Assay Assay High/Low Assays Count Count ratio Pool 367 381 749 13 28 441   Abundance A 59 149 065 243 614 Blocks B 138  60 007 456 132 C 110  78 685 563 140 D 60 104 851 1394  75

Example 3—PEA with Abundance Blocks on Samples with Assays of Varying Abundance

[0220] A multiplex PEA was performed (using probes comprising antibodies conjugated to nucleic acid domains having the structure described in Version 6, above) to detect 372 proteins in 54 plasma samples. Each probe contained a unique barcode sequence. 4 aliquots from each of the plasma samples were incubated with each of 4 proximity probe pools (four abundance blocks comprising the 372 assays) in 96-well or 384-well incubation plates.

[0221] The PEA was performed as described above. During amplification of the extension products, P5 and P7 sequencing adapters were added to each end of the products, along with a unique sample index for reporter nucleic acids from each different sample, and all extension products sequenced by massively parallel DNA sequencing, employing reversible dye terminator sequencing technique using an Illumina NovaSeq platform.

[0222] The results in FIG. 5 show that protein targets with a wide abundance range can be detected in the samples, without sacrificing the lower ranges of proteins with high variation in samples, or assays with relatively low abundance over all 54 samples, due to signal decrease (counts below a robust amount. e.g. 100 counts).