Toward Clinical Proteomics on a Next-Generation Sequencing

LETTER pubs.acs.org/ac

Toward Clinical Proteomics on a Next-Generation Sequencing Platform Daniel J. Turner,†,^ Robin Tuytten,‡ Kris P.F. Janssen,§ Jeroen Lammertyn,§ Jan Wuyts,‡ Jeroen Pollet,§ Sven Eyckerman,‡,z Clive Brown,‡,^ and Koen Kas*,‡ †

Sequencing Technology Development, Wellcome Trust Sanger Institute, Cambridge CB10 1SA, United Kingdom Pronota nv, Technologiepark 4, B-9052 Zwijnaarde/Ghent, Belgium § BIOSYST-MeBioS, Katholieke Universiteit Leuven, B-3001 Leuven, Belgium ‡

bS Supporting Information ABSTRACT: We report the first next generation sequencing (NGS) application to identify and quantify proteins. Customization of protein specific aptamers enabled direct conversion of serum protein information into NGS read outs. The intrinsic ability of aptamer sequencing to highly multiplex protein detection and quantification, together with the prospect of DNA sequencing further evolving into a commodity technology, could constitute the core of a novel, universal diagnostics paradigm.

employ “nonequilibrium capillary electrophoresis of equilibrium mixtures” (NECEEM)15 and we show that the modified aptamers still preserve their aptamer functionality. Finally, we combine NECEEM and NGS to translate clinical serum immunoglobulin E (IgE) levels into aptamer counts, uniquely demonstrating the viability of an aptamer sequencingbased protein diagnostics platform.

N

ext-generation DNA sequencing technology (NGS) has established a novel paradigm in polynucleotide sequencing; the processing of millions of clonally amplified DNA molecules in parallel enables diverse sequencing applications to be performed on a genome-wide scale.1-5 The massive parallelization capabilities intrinsic to NGS also bring a quantitative dimension to sequencing.6 We hypothesized that NGS could also identify and quantify proteins, by the quantitative sequencing of suitable ligands. Aptamers are short single stranded oligonucleotides that act as affinity probes for a wide range of substrates, e.g., proteins, small molecules, or cells.7-9 Because an aptamer's nucleotide sequence can be considered a unique “barcode” for its respective target, quantitative sequencing of an aptamer after elution from its target substrate will unequivocally identify and quantify that target. Since the molecular recognition element and the reporting probe are the same entity, aptamer sequencing would also obviate the need for composite constructs typical for antibodybased assays. The immense parallelization capabilities of NGS make it conceivable to perform massive multiplexing or profiling, both in terms of number of analytes as well as in the number of samples processed simultaneously.10 As aptamer sequences are typically shorter than 60 nucleotides,11 this makes them perfectly compatible with short-read NGS platforms.12 Here, we demonstrate that NGS aptamer sequencing allows identification and quantification of proteins (Figure 1, left). First, we modified a number of protein-specific aptamers with an adapter sequence, making them compatible with the Illumina Genome Analyzer II (GA), and we used these to verify that multiplexed quantification and identification of aptamers was possible. Second, in order to use an aptamer as a quantitative protein reporter, it is necessary to separate aptamer-protein complexes from free aptamers after incubation and binding.13,14 For this, we r 2010 American Chemical Society

’ EXPERIMENTAL SECTION NGS Library Preparation. Aptamers were synthesized by Thermo Fischer Scientific (Ulm, Germany). The library preparation rendering the studied aptamers (Table 1) compatible with Illumina NGS technology constituted three distinct steps (a) aptamer phosphorylation, (b) 50 adapter ligation, and (c) PCR amplification. To enable aptamer quantifications, aptamer samples were always supplemented with an internal reference: an Illumina PhiX sequencing library was always added. Detailed protocols are available in the Supporting Information. Sequencing. For NGS analysis, seven aptamer mixtures were loaded on seven separate lanes of an eight lane single read Illumina flow cell, leaving one lane for a PhiX-only control. The flow cell was then further processed and sequenced on an Illumina GAII instrument, in accordance with the manufacturer's instructions. Data Processing. The fastq files generated by the sequencer were converted into fasta files. Then, a custom approximate pattern-matching16 script allowed up to three insertions, deletions, or substitutions in the aptamer sequence. In the same way, Received: October 8, 2010 Accepted: November 30, 2010 Published: December 10, 2010 666

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(a) confirmation of APT1 affinity by means of NECEEM, (b) Kd determination, (c) APT1 vs original “Wiegand” aptamer, and (d) CE assisted fraction collection of APT1-IgE complex. Comprehensive descriptions of these experiments are given in the Supporting Information.

Figure 1. (Left) Generic diagnostics application: An aptamer collection containing an excess of aptamers with established affinities against the analytes of interest (A1) is used to incubate the sample under investigation (B1). All different aptamer-analyte complexes are separated from the excess (unbound) aptamers (C1) after which the bound aptamers are analyzed by NGS (D1). The analytes of interest are then identified and quantified by decoding and counting the nucleotide sequences of their respective aptamers. (Right) Generic aptamer evolution: A random aptamer library (A2) is incubated with sufficient analyte of interest (B2). All aptamer-analyte complexes are separated from the unbound aptamers (C2). Nucleotide sequences of the binding aptamers are obtained by NGS (D2). In silico sequence analysis of initial binders allows reiteration of the process with an excess aptamer library enriched in binders. This time, the separation step (C2) will discriminate bad binders from good binders. NGS will now provide information about nucleotide sequences and relative aptamer occurences; information which again will improve the constitution of the starting aptamer library. Such an evolution process, driven by early available sequence information, is envisioned to rapidly identify novel analyte-aptamer pairs. As a result, rapid expansion of the analytical capabilities of the diagnostics applications is also to be expected.

the number of sequences originating from the PhiX genome was counted. The relative abundance of aptamer sequences was obtained by dividing the former by the latter. Aptamer Multiplexing. To demonstrate the quantification and multiplexing capabilities of NGS, mixtures of aptamers were studied twice. For experiment (a), aptamers were individually prepared and only then mixed according to the ratios of Table 2. In experiment (b), the different aptamers mixtures (Table 2) were prepared directly from the purchased material after which the mixtures were subjected to the library preparation. A detailed description of the setups of experiments (a) and (b) can be found in the Supporting Information. NECEEM. The capillary electrophoresis experiments were performed on a Beckman P/ACE 2200 CE-LIF system (BeckmanCoulter). Four distinct NECEEM-based experiments were done

’ RESULTS AND DISCUSSION Four DNA aptamers, each reported17-20 to be specific for a different protein, were concatenated with an Illumina adapter sequence at the 30 terminus (Table 1). A 50 adapter was added at a later stage, during the library preparation process (Supporting Information: NGS library preparation). The IgE specific aptamer (APT1) was further functionalized with a fluorescent label at the 30 end to allow for detection during the NECEEM experiments. First, we sequenced mixtures containing the four proteinspecific aptamers in different proportions (Table 2). To allow normalization and direct comparison of the sequencing results from the different mixtures, a fixed amount of an Illumina PhiX174 control library was added to all mixtures as an internal reference (Supporting Information: Aptamer multiplexing). The aptamers were mixed either after (a) or before (b) the preparation of the aptamer library. In this way, any biases in resulting sequences can be attributed to the sequencing reaction,21 or to the aptamer library preparation. The library preparation inevitably contains a PCR amplification step, which is a demonstrated source of bias.22-24 In experiment (a), the four individual aptamers were mixed immediately before sequencing (Table 2; Supporting Information: Table S-1). A direct proportional relationship between the theoretical APT/PhiX ratios and the experimental APT/PhiX ratios was obtained (Supporting Information: Sequencing; Data processing; Figure 2a). For these dilution series, slight deviations from perfect linear correlations are apparent. Their stochastic appearance indicates that these arise from imperfections in the library preparation, rather than from the sequencing process itself. The consistently lower sequence count for APT1 is likely to be caused by the fluorophore on APT1, leading to overestimation of APT1 during the library preparation (Supporting Information: Aptamer Multiplexing). Overall, experiment (a) demonstrates that multiplexed identification and quantification of aptamers by NGS is possible (Supporting Information: Table S-2), with good accuracy, good linearity, and minimal introduction of quantitative biases. In experiment (b), the four aptamers were mixed prior to the library preparation procedure required for Illumina sequencing. Again seven mixtures were prepared in such a way as to generate the same APT/PhiX ratios (Table 2; Supporting Information: Table S-3). The direct correlations shown (Figure 2b) confirm that the newly developed library preparation procedure enables quantitative analyses of aptamer mixtures on NGS. The PCR amplification of the Illumina library preparation procedure is likely to be responsible for the divergence of the different “dose-response” curves.23,24 With amplification-free library preparations, as already developed for the sequencing of double stranded DNA24 and single stranded RNA,25 improved library preparation robustness and aptamerindependent digitalization should be feasible. Omitting the PCR amplification will also further enhance the analytical capabilities of the Illumina Genome Analyzer, as each cycle of amplification introduces redundancy, reduces parallelism by a factor of ∼2, and thus compromises the dynamic range and/or resolution available. Having demonstrated that NGS was capable of quantitative, multiplexed aptamer sequencing, we sought to use the proteinspecific affinity of aptamers to transcribe both the identity and 667

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Table 1. Sequences of Aptamers Used17-20,a DNA-sequence

a

affinity against

APT1

50 -GGGGCACGTTTATCCGTCCCTCCTAGTGGCGTGCCCC-adapter-FAM

human IgE

APT2

50 -GGTTGGTGTGGTTGG-adapter

thrombin

APT3

50 -AGGCTACGGCACGTAGAGCATCACCATGATCCT-adapter

platelet derived growth factor-BB

APT4

50 -GCGAAGGCACACCGAGTTCATAGTATCCAAGATCGGA-adapter

human basic fibroblast growth factor

adapter

50 -AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG

Concatenation of the adapter sequence at the 30 -terminus renders them compatible with Illumina GA sequencing technology.

Table 2. Theoretical Aptamer Loadsa lane

APT1

APT2

APT3

APT4

PhiX

1

1

0.5

200

100

10

5

100

50

200

2

2

1

100

50

40

20

30

15

200

3

5

2.5

50

25

200

100

5

4

2.5

200

Phix Control Lane

5

10

5

40

20

20

10

50

25

200

6 7

20 50

10 25

30 20

15 10

50 100

25 50

20 10

10 5

200 200

8

100

50

10

5

30

15

40

20

200

a

Left values: theoretical aptamer loads per Illumina flow cell lane; values are expressed relative to the lowest aptamer load applied (APT1, Lane 1). Right values: theoretical aptamer loads per lane; values are expressed relative to the theoretical amount of PhiX control sequences available per lane (%).

the quantity of proteins into a sequencing readout. This required that the adapter-modified aptamers retained the protein-specific affinity of the nonmodified aptamers. We re-evaluated the affinity of APT1, the adapter-modified IgE aptamer, toward IgE by NECEEM15 (Supporting Information: Figure S-1). High affinity was confirmed, showing that concatenation of the sequencingcompatible adapter does not impair APT1's aptamer capabilities (Kd = ca. 15 nM for APT1, Kd = ca. 65 nM for the unmodified aptamer).17 Next, samples containing different protein concentrations were incubated with a fixed amount of APT1, after which the bound APT1-protein fraction was purified via the same capillary electrophoresis setup. The bound APT1 correlated well with the IgE levels in the original samples (Supporting Information: Figure S-1b), demonstrating that quantitative protein information can be measured on next-generation DNA sequencers using aptamers. Finally, having successfully used the combination of NECEEM with NGS to analyze protein concentrations, we simulated a clinical application by directly determining endogenous IgE in serum. IgE serum levels are typically determined to identify patients with allergic diseases, as serum IgE levels correlate with allergic disease severity.26 A serum sample was incubated with an excess amount of aptamer APT1, after which the protein-bound APT1 was purified and collected. This procedure was repeated for the same sample after supplementing it with varying amounts of exogenous human IgE (standard addition method). The different preparations were then processed for sequencing. Figure 3 proves that aptamer sequencing can be used to determine the identity and concentration (fraction bound) of an analyte initially present in blood, the most complex clinical background.

’ CONCLUSION Here, we show that the range of NGS applications reaches far beyond mere DNA and RNA sequencing. Customization of aptamers with affinities against specific proteins enables direct conversion of protein information into a second-generation NGS readout, making NGS applicable to quantitative protein analyses. “Third-generation” sequencing approaches might even alleviate this modification requirement,27 which would be expected to improve the accuracy of this approach even further. We also envision that the identification of novel aptamers could be substantially accelerated following the fusion of aptamer technology with NGS. Typically, functional aptamers are identified from a random oligonucleotide library via an iterative process of selection, ultimately resulting in a small number of aptamers with high affinity to the target(s).8,28 It is only then that the final aptamer sequences are elucidated. Integration of NGS early in the workflows will provide full sequence information of all early binders. This information can fuel in silico sequence-fitness analyses29 which will rationalize and accelerate aptamer evolution (Figure 1, right). Very recently, Cho et al. demonstrated that NGS can indeed speed up the selection process dramatically: even without full exploitation of the in silico sequence optimization possibilities, aptamers were identified with binding affinities higher than those obtained by traditional cloning approaches.30 Accelerated generation of libraries of high affinity aptamers against proteins, metabolites, or any other analyte will in turn strengthen the position of NGS as a truly generic analytical detector: the superior analytical power residing in the massive multiplexing capabilities will become more and more relevant when suitably large collections of functional aptamers become available. It is self-evident that a number of challenges and preconditions still need resolution in order to transform the here reported proof of concept into the just depicted novel analytical paradigm. One challenge coincides with one of the cornerstones of the concept. The fact that aptamers combine the affinity probe and the identification tag in the same entity is the feature fundamental for the simple and direct determination of analytes. This asset is only valid when the aptamers are truly specific to their targets. Classically, this kind of high specificity is achieved, at the expense of assay simplicity, by combining two independent affinity probes, as is the case in enzyme linked immunosorbent assays (ELISA). Integration of generic strategies to tackle off-target interactions would be required in the further development of sensitive aptamer sequencing-based diagnostics. While considered nontrivial, Gold et al. recently overcame this challenge of “identifying a second element of specificity beyond binding a second ligand” with the selection of aptamers with slow dissociation rates.31 In this context, the application of NECEEM for both aptamer affinity purification (diagnostics) and aptamer evolution 668

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Figure 2. Parallel NGS of four aptamer dilution series. (a) The different aptamers were mixed according to the ratios in Table 2 just prior to quantification and amplification on an Illumina flow cell. Read outs relate only to the performance of the actual sequencing process (experiment (a)). Linear relationships were obtained for all four aptamers (r2 > 0.99). (b) The different aptamers were mixed according to the ratios in Table 2 prior to the sample preparation procedure. Readouts relate to combined impact of sample preparation and the actual sequencing (experiment (b)) and show again good precisions (0.94 < r2 e 0.99).

compares favorable. Being a homogeneous in-solution separation technique, the majority of off-target interactions are excluded since these usually occur at solid-liquid interfaces. On top, NECEEM has the unique ability to select for aptamers with slow dissociation constants.32 Because the mobility of aptamer-target complexes can easily be tuned in capillary electrophoresis setups,20 we are confident that our workflow can be made compliant with highly multiplexed aptamer-protein purifications. Currently, the most important hurdle for this novel analytical concept is still the cost and speed of NGS. Yet, with present sequencing efficiency improvements of 4 logs in 4 years33 and, e.g., optics-free (i.e., cheaper hardware and reagents), semiconductor-based sequencing technologies on the horizon (Ion torrent, IBM), it is legitimate to expect that cost and sequencing speed issues will vaporize in the near future. By showing the intrinsic ability of aptamer sequencing to detect and quantify proteins in a highly multiplexed fashion, together with the prospect of nucleotide sequencing further evolving into a commodity technology,34 we firmly believe that we have established the foundation of a novel, fully comprehensive diagnostics platform able to measure DNA, RNA, proteins, and metabolites.

Figure 3. Serum levels of IgE: the endogenous level (“0” nM) and three levels of IgE supplemented serum, determined by NGS via quantitative sequencing of IgE bound APT1. Serum was incubated with excess of APT1 (500nM), the APT1-IgE complex purified by means of capillary electrophoresis, and the bound APT1 decoded by means of quantitative sequencing on the Illumina GAII analyzer. (a) Technical repeats were conducted both at the level of the capillary electrophoresis (CE1 and CE2) and at the level of aptamer sequencing (NGS1 and NGS2). Small changes in separation dynamics between experiments impacted the yield from the timed collections of the APT1-IgE fraction; a bias fully accounted for by the calibration curves as obtained with the standard addition procedure. The dedicated NGS sample prep procedure showed good interexperiment robustness as obvious from the error bars. (b) In order to enable direct comparison of data generated during the different technical repeats on CE, all data of (a) were normalized. This normalization was performed for each CE run of the standard addition series by dividing all response values by the average response for all concentration levels analyzed during the run. This enabled the construction of the normalized standard addition curve presented in (b). This resulted, after correction for a 10-fold dilution factor, in an endogenous IgE concentration of ∼24 nM, which falls well within the allergic response range.

’ ASSOCIATED CONTENT

bS

Supporting Information. NGS library preparation, Aptamer multiplexing, NECEEM. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Fax: þ32 9 241 11 69. 669

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Present Addresses

Diakoumakos, K. D.; Dominguez-Fernandez, B.; Earnshaw, D. J.; Egbujor, U. C.; Elmore, D. W.; Etchin, S. S.; Ewan, M. R.; Fedurco, M.; Fraser, L. J.; Fajardo, K. V. F.; Furey, W. S.; George, D.; Gietzen, K. J.; Goddard, C. P.; Golda, G. S.; Granieri, P. A.; Green, D. E.; Gustafson, D. L.; Hansen, N. F.; Harnish, K.; Haudenschild, C. D.; Heyer, N. I.; Hims, M. M.; Ho, J. T.; Horgan, A. M.; Hoschler, K.; Hurwitz, S.; Ivanov, D. V.; Johnson, M. Q.; James, T.; Jones, T. A. H.; Kang, G. D.; Kerelska, T. H.; Kersey, A. D.; Khrebtukova, I.; Kindwall, A. P.; Kingsbury, Z.; Kokko-Gonzales, P. I.; Kumar, A.; Laurent, M. A.; Lawley, C. T.; Lee, S. E.; Lee, X.; Liao, A. K.; Loch, J. A.; Lok, M.; Luo, S. J.; Mammen, R. M.; Martin, J. W.; McCauley, P. G.; McNitt, P.; Mehta, P.; Moon, K. W.; Mullens, J. W.; Newington, T.; Ning, Z. M.; Ng, B. L.; Novo, S. M.; O'Neill, M. J.; Osborne, M. A.; Osnowski, A.; Ostadan, O.; Paraschos, L. L.; Pickering, L.; Pike, A. C.; Pike, A. C.; Pinkard, D. C.; Pliskin, D. P.; Podhasky, J.; Quijano, V. J.; Raczy, C.; Rae, V. H.; Rawlings, S. R.; Rodriguez, A. C.; Roe, P. M.; Rogers, J.; Bacigalupo, M. C. R.; Romanov, N.; Romieu, A.; Roth, R. K.; Rourke, N. J.; Ruediger, S. T.; Rusman, E.; Sanches-Kuiper, R. M.; Schenker, M. R.; Seoane, J. M.; Shaw, R. J.; Shiver, M. K.; Short, S. W.; Sizto, N. L.; Sluis, J. P.; Smith, M. A.; Sohna, J. E. S.; Spence, E. J.; Stevens, K.; Sutton, N.; Szajkowski, L.; Tregidgo, C. L.; Turcatti, G.; vandeVondele, S.; Verhovsky, Y.; Virk, S. M.; Wakelin, S.; Walcott, G. C.; Wang, J. W.; Worsley, G. J.; Yan, J. Y.; Yau, L.; Zuerlein, M.; Rogers, J.; Mullikin, J. C.; Hurles, M. E.; McCooke, N. J.; West, J. S.; Oaks, F. L.; Lundberg, P. L.; Klenerman, D.; Durbin, R.; Smith, A. J. Nature 2008, 456, 53–59. (23) Polz, M. F.; Cavanaugh, C. M. Appl. Environ. Microbiol. 1998, 64, 3724–3730. (24) Kozarewa, I.; Ning, Z. M.; Quail, M. A.; Sanders, M. J.; Berriman, M.; Turner, D. J. Nat. Methods 2009, 6, 291–295. (25) Mamanova, L.; Andrews, R. M.; James, K. D.; Sheridan, E. M.; Ellis, P. D.; Langford, C. F.; Ost, T. W. B.; Collins, J. E.; Turner, D. J. Nat. Methods 2010, 7, 130–U63. (26) Corry, D. B.; Kheradmand, F. Nature 1999, 402, B18–B23. (27) Branton, D.; Deamer, D. W.; Marziali, A.; Bayley, H.; Benner, S. A.; Butler, T.; Di Ventra, M.; Garaj, S.; Hibbs, A.; Huang, X. H.; Jovanovich, S. B.; Krstic, P. S.; Lindsay, S.; Ling, X. S. S.; Mastrangelo, C. H.; Meller, A.; Oliver, J. S.; Pershin, Y. V.; Ramsey, J. M.; Riehn, R.; Soni, G. V.; Tabard-Cossa, V.; Wanunu, M.; Wiggin, M.; Schloss, J. A. Nat. Biotechnol. 2008, 26, 1146–1153. (28) Berezovski, M. V.; Musheev, M. U.; Drabovich, A. P.; Jitkova, J. V.; Krylov, S. N. Nat. Protoc. 2006, 1, 1359–1369. (29) Knight, C. G.; Platt, M.; Rowe, W.; Wedge, D. C.; Khan, F.; Day, P. J. R.; McShea, A.; Knowles, J.; Kell, D. B. Nucleic Acids Res. 2009, 37, e6. (30) Cho, M.; Xiao, Y.; Nie, J.; Stewart, R.; Csordas, A. T.; Oh, S. S.; Thomson, J. A.; Soh, H. T. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 15373–15378. (31) Gold, L.; Ayers, D.; Bertino, J.; Bock, C.; Bock, A.; Brody, E.; Carter, J.; Cunningham, V.; Dalby, A.; Eaton, B.; Fitzwater, T.; Flather, D.; Forbes, A.; Foreman, T.; Fowler, C.; Gawande, B.; Goss, M.; Gunn, M.; Gupta, S.; Halladay, D.; Heil, J.; Heilig, J.; Hicke, B.; Husar, G.; Janjic, N.; Jarvis, T.; Jennings, S.; Katilius, E.; Keeney, T.; Kim, N.; Kaske, T.; Koch, T.; Kraemer, S.; Kroiss, L.; Le, N.; Levine, D.; Lindsey, W.; Lollo, B.; Mayfield, W.; Mehan, M.; Mehler, R.; Nelson, M.; Nelson, S.; Nieuwlandt, D.; Nikrad, M.; Ochsner, U.; Ostroff, R.; Otis, M.; Parker, T.; Pietrasiewicz, S.; Resnicow, D.; Rohloff, J.; Sanders, G.; Sattin, S.; Schneider, D.; Singer, B.; Stanton, M.; Sterkel, A.; Stewart, A.; Stratford, S.; Vaught, J.; Vrkljan, M.; Walker, J.; Watrobka, M.; Waugh, S.; Weiss, A.; Wilcox, S.; Wolfson, A.; Wolk, S.; Zhang, C.; Zichi, D. Nature Precedings 2010, http://hdl.handle.net/10101/npre.2010.4538.1. (32) Drabovich, A. P.; Berezovski, M.; Okhonin, V.; Krylov, S. N. Anal. Chem. 2006, 78, 3171–3178. (33) Mukhopadhyay, R. Anal. Chem. 2009, 81, 1736–1740. (34) Blow, N. Nat. Methods 2008, 5, 267–273.

^

Oxford Nanopore Technologies, Oxford OX4 4GA, United Kingdom. z Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium.

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dx.doi.org/10.1021/ac102666n |Anal. Chem. 2011, 83, 666–670