Single-molecule DNA-mapping and whole- of individual cells

Rodolphe Mariea,1, Jonas N. Pedersena, Loic Bærlocherb, Kamila Koprowskac,d, Marie Pødenphanta,Celine´ Sabatele, Maksim Zalkovskijf, Andrej Mironovf, Brian Bilenbergf, Neil Ashleyc,d, Henrik Flyvbjerga, Walter F. Bodmerc,d, Anders Kristensena, and Kalim U. Mirg

aDepartment of Micro- and Nanotechnology, Technical University of Denmark, 2800 Kongens Lyngby, Denmark; bFasteris SA, CH-1228 Plan-les-Ouates, Switzerland; cCancer and Laboratory, Weatherall Institute of Molecular , University of Oxford, Oxford OX3 9DS, United Kingdom; dDepartment of , University of Oxford, Oxford OX3 7DQ, United Kingdom; eDiagenode SA, 4102 Seraing (Ougree),´ Belgium; fNIL Technology ApS, 2800 Kongens Lyngby, Denmark; and gXGenomes, Boston, MA 02134

Edited by Joseph R. Ecker, Howard Hughes Medical Institute and The Salk Institute for Biological Studies, La Jolla, CA, and approved September 17, 2018 (received for review March 16, 2018) To elucidate cellular diversity and clonal in tissues and single-molecule optical mapping of DNA from individual cells tumors, one must resolve genomic heterogeneity in single cells. has not previously been demonstrated. The critical challenge is To this end, we have developed low-cost, mass-producible micro-/ to couple single-cell genome extraction with a means of han- nanofluidic chips for DNA extraction from individual cells. These dling the DNA at the appropriate length scales and performing chips have modules that collect genomic DNA for sequencing the mapping reaction in an integrated device. Stained single or map genomic structure directly, on-chip, with denaturation– genomic DNA molecules can become patterned according to renaturation (D-R) optical mapping [Marie R, et al. (2013) Proc Natl their AT/GC content by partial denaturation and renaturation Acad Sci USA 110:4893–4898]. Processing of single cells from the (D-R) (11). As the DNA double helix opens up, the intercalat- LS174T colorectal cancer cell line showed that D-R mapping of sin- ing dye leaves the AT-rich regions, thus creating dark spots along gle molecules can reveal structural variation (SV) in the genome the DNA strand (see SI Appendix and Fig. 1D). A high degree of of single cells. In one experiment, we processed 17 fragments stretching, typically above 90%, provided by a flow-stretch device covering 19.8 Mb of the cell’s genome. One megabase-large frag- allows imaging of D-R patterns at diffraction-limited resolution. ment aligned well to 19 with half its length, while D-R mapping identifies structural variations (SVs) relative to a the other half showed variable alignment. Paired-end single- in single copies of the genome (11), cell sequencing supported this finding, revealing a region of and this makes it a unique candidate for optical mapping of single complexity and a 50-kb deletion. Sequencing struggled, how- cells. ever, to detect a 20-kb gap that D-R mapping showed clearly in a megabase fragment that otherwise mapped well to the Results reference at the pericentromeric region of chromosome 4. Peri- Using valve-less polymer devices that can be easily mass- centromeric regions are complex and show substantial sequence produced by injection moulding (e.g., Fig. 1A), we have homology between different , making mapping of sequence reads ambiguous. Thus, D-R mapping directly, from a single molecule, revealed characteristics of the single-cell genome Significance that were challenging for short-read sequencing. We report optical mapping of DNA from a single cell. Notably, nanofluidics | single cell | DNA | sequencing | optical mapping we demonstrate isolation of single cells, DNA extraction, and optical mapping, all within a single integrated micro- equencing of the of individual isolated cells or /nanofluidic device. Single-cell optical mapping is less complex Snuclei has become well established (1, 2) and is used to than sequencing, which we performed after whole-genome resolve intercellular variations in a heterogeneous population amplification of DNA extracted from a single cell isolated on- of cells from tissues to tumours (3–5). However, it is diffi- chip. In some cases, optical mapping was more efficient than cult to accurately delineate the long-range structure of the sequencing at detecting structural variation. As single-cell genome by sequencing alone because of short read lengths analysis can address genomic heterogeneity within a tumor, and variant detection based on alignment to a reference, it may prove useful for the selection of cancer . which discards a substantial proportion of reads (6). By con- Thus, optical mapping of the long-range features of single- trast, single-molecule optical mapping of DNA (7–9) provides cell genomes and sequencing of the short-range features may a sweeping view of megabase lengths of the genome (10–13). become complementary tools for the analysis of tumors. Several schemes exist to locate sequence information along the length of stretched DNA, and some have been combined with Author contributions: R.M., A.K., and K.U.M. designed research; R.M., K.K., M.P., M.Z., A.M., and N.A. performed research; R.M., J.N.P., L.B., H.F., W.F.B., and K.U.M. analyzed superresolution microscopy (14, 15). In early realizations of opti- data; and R.M., J.N.P., L.B., K.K., M.P., C.S., M.Z., A.M., B.B., N.A., H.F., W.F.B., A.K., and cal mapping, DNA molecules were stretched on a surface (7, K.U.M. wrote the paper.y 16). More recently, nanoconfinement has emerged as a pow- Conflict of interest statement: R.M., J.N.P., A.K., and K.U.M. filed patents. K.U.M. declares erful technique to stretch DNA molecules uniformly and in a that XGenomes is developing nucleic acid sequencing technologies.y controlled manner without attaching them to a surface (17). This article is a PNAS Direct Submission.y Das et al. (18) were able to image sequence-specific fluores- Published under the PNAS license.y cent optical maps in nanochannels with a cross-section below Data deposition: The single-cell sequencing data are available at the European the persistence length of DNA. The combination of nanoconfine- Archive (accession nos. ERS2168085–ERS2168123), https://www.ebi.ac.uk/ena/ nent with additional mechanisms such as entropic recoil (19–21), browse.y hydrodynamic drag (22, 23), mechanical confinement (24), or 1 To whom correspondence should be addressed. Email: [email protected] thermophoresis (25) can enhance the degree of stretching. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Although genomic DNA extracted from individual cells has 1073/pnas.1804194115/-/DCSupplemental.y been stretched in micro- (26) and nanofluidic (27) devices, Published online October 15, 2018.

11192–11197 | PNAS | October 30, 2018 | vol. 115 | no. 44 www.pnas.org/cgi/doi/10.1073/pnas.1804194115 Downloaded by guest on October 2, 2021 Downloaded by guest on October 2, 2021 ai tal. et Marie 1B (Fig. population S1 a from isolat- cells and single trapping ing for architecture microfluidic a implemented .Ads flgtwsue onc h chromo- the nick to used was light of dose Appendix , A (SI S6). buffer DNA and lysis fluores- The the S5 the to trap. Figs. adding YOYO-1 by a dye uniformly in intercalating stained polymer captured cent then the was was into nucleus cells cell the fed single in We a 2. Fig. wherein in device depicted is device this of 1B Fig. in shown design as across low cells as 15× was single at bulk the 30% in in found dropout variants allelic heterozygous high-quality 10×; all as low as depth from microflu- ing contamination 2% the than DNA; of less nonhuman of was use 97% there for samples; the successful single-cell was without preparation cells environment: gave idic single outlets the processing compared in results with sequencing collection single-cell single DNA whole-genome isolating and superior for extraction device DNA our using cells, (SI C1), cells Fluidigm of (e.g., population devices S3 a cells from Fig. single obtained and out- Appendix, that from (MDA) the to comparable amplification in sequence was collected displacement The and sequencing. multiple DNA lysis Illumina for The alkaline port trap. by feeding separate let extracted the a then stopped occupied We cell was a traps. individual microchannel with a funnel-shape each of device of when side polymer series the the a toward cells into having steered S2) that cancer Fig. design colorectal Appendix, flow LS174T the (SI single from line in cells from cell of shown DNA population design cell a of the single fed Sequencing We by a enabled from 1D). was DNA (Fig. cells architec- genomic device an the the to of alternatively, within mapping or, sequencing D-R for 1C) for for (Fig. architecture cell ture device single an the a to of from device outside DNA the genomic within the aligned harvesting be can design the of map silico in an with compared is map D-R The mapping. for used pattern D-R the stretched constitute DNA is captured (see the DNA is genome along genomic cell reference segments single and light A and cycle, dark (B) heating–cooling renaturation, (red). a device by flow-stretch composition a AT/GC and to (green), according channel patterned meandering and a (C extracted (blue), visualized. is trap and cell DNA a trapping, comprises hydrodynamic connectors by 12 with device a-chip 1. 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Fig. 2. Device operation to realize the workflow shown in Fig. 1D. (A) The device comprises a main channel with inlets for cells and buffer, a cell trap connecting the main channel to the outlets. On the outlet side, a flow stretch device is placed. (B) Cells are introduced, and a single cell is captured in the trap. The flow is stopped when a cell occupies the trap. (C) Lysis buffer is introduced to remove the cytoplasm. The lysis solution contains YOYO-1 to stain the DNA of the nucleus. (D) The stained nucleus is illuminated with blue light (480 nm) to induce photonicking. (E) Buffer with BME is introduced to prevent further photonicking. (F) Same buffer to which protease K is added is introduced to release the genomic DNA by proteolysis. (G) When the DNA reaches the meandering channel, temperature is raised to partially denaturate the DNA before the device is cooled again. (H) DNA molecules are introduced to the flow-stretch device by electrophoresis and (I) stretched by a flow of buffer at a pressure of 80 mbar. (J) Brightfield image of the flow-stretch device indicating the buffer flow (dotted line) and the position of the DNA (red line). The nanoslit is 450 µm across.

molecules extracted from the single cell and its matching to the we cannot pinpoint the nature of the SV (11). Insertions and in silico-generated melting map of a reference genome. Two of deletions down to 5 kb can be detected with high confidence (for the molecules that did not match anywhere along the reference the SV detection sensitivity of D-R mapping, see SI Appendix, genome lacked a pattern that was rich enough in features to map section 3). Significant SV was detected on molecule 17 by D-R back to the genome (see SI Appendix, Fig. S21). This complication mapping (SI Appendix, Table S1); half of the 1 Mb-long molecule might arise because all molecules were denatured at a single tem- mapped poorly to Chr19:8.5 to 9.1 Mb (SI Appendix, Fig. S22 perature. This temperature had been found to yield informative and Fig. 4A). This is consistent with the LS174T whole-genome D-R maps for most molecules. But a uniformly GC-rich region sequencing data. The read-depth analysis suggests a 50-kb dele- can appear featureless and bright at this temperature, while a uni- tion at 8.687 to 8.737 Mb, which is likely homozygous, as well as a formly AT-rich region can appear featureless and dark (see SI 50-kb complex region around 8.87 Mb characterized by a higher Appendix, section 2E). The two other molecules that did not find read depth but with a poor mapping of the reads (Fig. 4 B and a match in the reference genome did contain sufficient pattern- C). In this case, single-cell D-R mapping data and bulk sequenc- ing for mapping to the reference; our inability to find a match for ing concur and point to a complex region. However, the poor these molecules, or even parts of the molecules, suggests that they agreement of the D-R map with the reference genome leading came from the fraction of the genome that remains uncharted or is to a high χ2-value background does not allow the SV detection subject to extreme structural change (29), which renders it unmap- by D-R mapping alone (SI Appendix, section 2B). pable and is therefore not represented in the reference genome We found two segments clearly missing in molecule 12 (8 and (SI Appendix, section 2C). 12 kb) at 51.012 and 51.513 Mb. Their absence could not be A number of the molecules that mapped to the genome had detected in the bulk or the single-cell data. However, we found peaks in their χ2 values that coincide with line shifts larger than a 20-kb region limited by two regions of reads with an incorrect a few kilobases in their plots of the genome versus D-R map insert size centered at the position of interest, 51.012 Mb. At the position (SI Appendix, Figs. S12–S20). These peaks indicate poor other position of interest, in one of the single-cell sequencings, alignment to the reference; however, if no line shift is detected, we see two deletions smaller than a kilobase positioned at 23 kb

A C

D

B E

Fig. 3. Extraction and mapping of DNA from a trapped cell. (A) Proteolysis liberates the genomic DNA from the trapped nucleus under flow (arrow). (Scale bar, 10 µm.) (B) DNA is collected as a 10-nL plug of solution in a section of the device where D-R is performed. (Scale bar, 10 µm.) (C) Single field-of-view of a segment of a stretched DNA molecule. (Scale bar, 10 µm.) (D) Full D-R map of a molecule is stitched together from five fields-of-view. (E) Match between the D-R map (pixel 900 to 1100) and a position on Chr7 of computer-simulated whole-genome melting map.

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GENETICS ENGINEERING shows that our approach can provide answers in regions where section: It is 4.3 µm wide at the bottom, 6 µm at middle depth, and 8 µm at sequencing does not. the top with a depth of 33 µm. The cell trap connects the feeding channel with the outlet channels, which consist of two meandering channels: one Discussion for waste and the other (green box in Fig. 1B) leading to the outlet and the A substantial proportion of somatic SVs in cancer genomes are flow-stretch device (red box in Fig. 1B). The flow-stretch device consists of a 20 µm-wide, 450 µm-long, 100 nm-deep cross-shaped nanoslit that connects complex events and are rich in interchromosomal rearrange- the outlet channel to three other microchannels (11). ments (29, 31). Short-read sequencing does not easily reveal mid- to long-range complex structural changes without a substan- Optical Mapping Device Fabrication. The device is fabricated by replicating tial effort (32). By contrast, the megabase-range a nickel shim using injection molding of TOPAS 5013 (TOPAS) as previously views of the genome provided by single-cell D-R optical mapping reported (36, 37). Briefly, a silicon master is produced by UV lithography and detect such changes. reactive ion etching. A 100-nm NiV seeding layer is deposited and nickel We have shown that D-R mapping can determine that a region is electroplated to a final thickness of 330 µm. The Si master is chemi- of the genome (on Chr19) in a LS174T cell has a ∼0.6-Mb region cally etched away in KOH. Injection molding is performed using a melt ◦ ◦ that matches poorly to the reference and that our single-cell temperature of 250 C, a mould temperature of 120 C, a maximum hold- sequencing data are consistent with this. However, D-R map- ing pressure of 1,500 bar for 2 s, and an injection rate varying between 20 cm3/s and 45 cm3/s. Finally, a 150 µm TOPAS foil is used to seal the device ping of a megabase fragment from another single cell shows the by a combined UV and thermal treatment under a maximum pressure of absence of a 20-kb segment on Chr4, which is not picked up 0.51 MPa. The surface roughness of the foil is reduced by pressing the foil at by single-cell read-depth or read pair sequence analysis. How- 140◦C and 5.1 MPa for 20 min between two flat nickel plates electroplated ever, single-cell sequencing does suggest that this is a complex, from silicon wafers before sealing the device. This ensures that the lid of unstable region of the genome that sequencing has not been able the device is optically flat, allowing for high-NA optical microscopy. The to chart accurately. Computational reconstruction of the region device is mounted on an inverted fluorescence microscope (Nikon T2000) by paired reads is unproductive because the region is not well equipped with an air objective (50×/0.8), an oil objective (60×/1.40), and mapped in the reference and has a high degree of similarity with an EMCCD camera (Photometrics cascade II 512). Fluids are driven through other parts of the genome. By contrast, with D-R mapping, we the device using a pressure controller (MFCS, Fluigent) at pressures in the 0 to 10 mbar range. The device is primed with ethanol, and then degassed are able to directly visualize the structure of the fragment and FACSFlow Sheath Fluid (BD Biosciences) is loaded in all microchannels but unequivocally assign its genome location by comparing its D- the microchannel connecting the flow-stretch device. Instead, a buffer suit- R melting map with an in silico-generated melting map of the able for single-molecule imaging and electrophoresis (0.5× TBE + 0.5% v/v human reference genome (SI Appendix, section 2); most of the Triton-X100 + 1% v/v β-mercaptoethanol, BME) is loaded in the channels of megabase fragments match with high confidence to Chr4, but the flow-stretch device. This buffer prevents DNA sticking in the nanoslit if the reference genome is an accurate representation of this of the flow-stretch device and suppresses electroosmotic flow that can region, a 20-kb segment is clearly deleted in this fragment or counteract the introduction of DNA to the nanoslit later in the experiment. translocated to another location. Because of the visual nature of the D-R experimental data, there is little doubt that what we are Cells. LS174T colorectal cancer cells are cultured in Dulbecco’s modified looking at is real and not an artifact. The LS174T cell line has Eagle’s medium (DMEM; Gibco) with 10% fetal bovine serum (FBS; Autogen- Bioclear UK Ltd.) and 1% penicillin/streptomycin (Lonza) before freezing at a 45,X karyotype, and spectral karyotyping does not reveal any a concentration of 1.7 106 cells per milliliter in 10% DMSO in FBS. After large-scale rearrangements (33, 34). Moreover, bulk SNP anal- thawing, cell suspension is mixed 1:1 with FACSFlow buffer, centrifuged ysis of Chr19 does not find microdeletions (SI Appendix, Table at 28.8 × g (A-4-44, Eppendorf) for 5 min, and resuspended in FACSFlow. S4). The single-cell D-R mapping might therefore be providing Finally, the cells are stained with 1 µM Calcein AM (Invitrogen) and loaded information on an uncommon variant. Single-cell D-R mapping in the chip at 0.35 106 cells per milliliter. So out of the 7,000 cells loaded, the thus expands the tools available to detect genomic heterogene- first cell trapped is analyzed. ity, and such a capability might help provide deeper insight into the structure and dynamics of a genomic region and may aid the Optical Mapping Device Operation. The protocol for operating the device is mapping of disease . sketched in Figs. 1D and 2 A–I. First, cells and buffer are introduced simul- The modest throughput of our current device design is bal- taneously, aligning the cells along the side wall of the microchannel where the trap is located. A cell is captured and kept in the trap for a buffer flow anced by the fact that we obtain a map from just one molecule. through the trap up to 30 nL/min. Cell doublets are present in the cell cul- While other nanofluidic platforms may currently be able to pro- ture and may be captured in the cell trap. In this case, the experiment is cess more molecules, the optical maps generated rely intrinsically aborted. The lysis buffer composed of 0.5× TBE + 0.5% v/v Triton-X100 + on the data from several overlapping molecules and thus do 0.1 µM YOYO-1 (Invitrogen) is loaded in one of the inlets and injected at not have the true single-molecule sensitivity required to map 10 nL/min through the trap for 10 min (see SI Appendix). Then, the solution genomic DNA from single cells. Moreover, analysis based on is exchanged to a buffer without YOYO-1 in all wells to stop the stain- individual megabase molecules means that we can resolve the ing. Next, the cell nucleus is exposed to blue excitation light at a dose of 2 long-range structure of haplotypes in a diploid genome. With the 1 nW/(µm) for up to 300 s, causing a partial photonicking of the DNA application of our full automation method (35), it will be possi- (see SI Appendix). Then, the buffer is changed to a solution containing BME (0.5× TBE + 0.5% v/v triton-X100 + 1% v/v BME), and the excitation ble to apply D-R optical mapping to whole single-cell genomes. of the fluorescence lamp is lowered to the minimum intensity that still Single-cell D-R mapping will then have the potential to simply allows fluorescence imaging. Finally, the temperature is raised to 60◦C, and and inexpensively screen whole-genome SV between cells as a the proteolysis solution (0.5× TBE + 0.5% v/v Triton-X100 + 1% v/v BME + complement or alternative to sequencing. 200 g/mL) is introduced, pushing the lysate through the trap. In experiments where a denaturation pattern is created on DNA extracted from a single Materials and Methods cell, the DNA staining is modified. Thus, the lysis buffer contains 0.04 µM Optical Mapping Device Design. The device architecture for optical mapping YOYO-1 instead of 0.1 µM. In addition, the device is heated further after is summarized in Fig. 1B. Microchannels are designed to accommodate cells the proteolysis for 10 min at 80◦C before rapid cooling to room temper- from a human cancer cell line with a typical diameter of 15 µm (SI Appendix, ature by moving the oil immersion objective in place. The temperature at Fig. S2), so the microfluidic network has minimal depths and widths of which the D-R pattern is created is highly dependent on the dye loading 33 µm. The device comprises an inlet for cells and an inlet for buffer (Fig. and the ionic strength of the buffer and should be adjusted by steps of 2◦C 1B) that merge into a single channel to feed the single-cell trap (blue box if necessary. DNA travels through the meandering channel as the temper- in Fig. 1B). At the intersection between the cell and buffer inlets, cells get ature is lowered, and an oil immersion objective is moved into place for aligned along the side wall of the feeding channel where the trap is located. single-molecule imaging (60×/1.4, with a 1.5× lens giving a 180-nm pixel The trap is a simple constriction dimensioned to capture cells from a human size). DNA fragments are introduced from the microchannel to the nanoslit cancer cell line. The constriction for cell trapping has a trapezoidal cross- of the flow-stretch device using electrophoresis by applying a voltage of 5

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Gawad single-cell 2. with evolution cancer Delineating (2015) NE Navin 1. opeeadacrt env euneasml ftecmlxaglp tauschii aegilops complex the of assembly sequence genome. novo de accurate and complete device. nanofluidic a in 4898. molecule DNA single analysis. resolution. nanometre channels. mapping. optical by constructed chromosomes chemistry. terminator Nature heterogeneity. 342:632–637. . the Med ehlrnfrs-ietdcikchemistry. click methyltransferase-directed Lett Nano frame. interface. ihsqec pcfi ursetprobes. fluorescent specific sequence with channels. 100-nm analysis. interfaces. micro-nanofluidic at DNA of 7:296fs29. Biomicrofluidics 501:355–364. rcNt cdSiUSA Sci Acad Natl Proc rcNt cdSiUSA Sci Acad Natl Proc LSOne PLoS rcNt cdSiUSA Sci Acad Natl Proc Science 12:3861–3866. a e Genet Rev Nat a Methods Nat 265:2096–2098. rcNt cdSiUSA Sci Acad Natl Proc 8:e55864. ) n eltashv emtysmlrt h trap the to similar geometry a have traps cell and µm), Nature hmSci Chem 9:044114. mgn n apn r efre spreviously as performed are mapping and Imaging 17:175–188. 13:833–836. 456:53–59. 107:10848–10853. 104:2673–2678. 1:453–460. 107:13294–13299. IAppendix SI neprmns hr N sprepared is DNA where experiments, In aoLett Nano 101:10979–10983. uli cd Res Acids Nucleic uli cd Res Acids Nucleic h eiei etdwt degassed with wetted is device The Science rcNt cdSiUSA Sci Acad Natl Proc 12:1597–1602. nti eie eight device, this On S1. Fig. , 262:110–114. -ieconstrictions). µm-wide 38:e177. 42:e50. 110:4893– c Transl Sci Science 1 onvk-hfrK,e l 21)Peetto flreDAmlclsfranalysis for molecules DNA large of Presentation (2013) al. et KL, Kounovsky-Shafer 21. 9 oisnJ,e l 21)Itgaiegnmc viewer. genomics Integrative (2011) al. with et in algorithm JT, melting Robinson DNA DNA of Speed-up 39. human (2003) E single-molecule Hovig TK, of Jenssen F, mapping Liu E, Tøstesen Optical (2015) 38. al. et PF, Østergaard 37. cells. single of analysis number copy -wide in (2012) variations al. structural et somatic T, Baslan of mechanisms 32. Diverse (2013) al. et L, Yang single 31. a pericentromeric human in of structure mosaic acquired The (2000) rearrangement E Eichler genomic S, Schwartz J, Massive Horvath (2011) 30. cancer al. single from et extracted PJ, genomes Stephens human of 29. Sequencing (2018) al. et R, Marie 28. templating. nanoscale by lens-induced Convex DNA (2014) al. methylated et DJ, of Berard selection 24. and analysis flow. Real-time elongational (2012) an al. in et dynamics BR, polymer Single Cipriany (1997) 23. S Chu D, Smith T, Perkins 22. 6 toP eso ,Kitne ,Lre B(01 neto oddnnflii chips: nanofluidic molded Injection (2011) NB Larsen device. A, Kristensen stretching F, molecule Persson single-DNA P, Utko a of 36. Automation (2015) al. et KT, Sørensen 35. of subsets additional suggests karyotyping Spectral (2001) al. and et karyotype WM, Longitudinal Abdel-Rahman (1980) 34. B Kahan M, Chao M, Siciliano C, Kaye L, Rutzky 33. using genomics cell single for platform a human of Development of (2015) analysis al. and et stretching S, extraction, Mahshid Microfluidic single- 27. (2012) a with al. measured et DNA JJ, on Benitez forces 26. 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