Laure Duboeuf

Louis De Barros

Maria Kakurina

Yves Guglielmi

Frederic Cappa

Benoit Valley

Aseismic deformations perturb the stress state and trigger induced seismicity during injection experiments

Keywords:

Fluid injections can trigger seismicity even on faults that are not optimally oriented for reactivation, suggesting either sufficiently large fluid pressure or local stress perturbations. Understanding how stress field may be perturbed during fluid injections is crucial in assessing the risk of induced seismicity and the efficiency of deep fluid stimulation projects. Here, we focus on a series of in-situ decametric experiments of fluid-induced seismicity, performed at 280 m depth in an underground gallery, while synchronously monitoring the fluid pressure and the activated fractures movements. During the injections, seismicity occurred on existing natural fractures and bedding planes that are misoriented to slip relative to the background stress state, which was determined from the joint inversion of downhole fluid pressure and mechanical displacements measured at the injection. We then compare this background stress with the one estimated from the inversion of earthquake focal mechanisms. We find significant differences in the orientation of the stress tensor components, thus highlighting local perturbations. After discussing the influence of the gallery, the pore pressure variation and the geology, we show that the significant stress perturbations induced by the aseismic deformation (which represents more than 96 % of the total deformation) trigger the seismic reactivation of fractures with different orientations.

I.

Introduction

The last decades have shown an large increase of the seismicity induced by fluid injections in the context of industrial activities in oil and gas extraction, geothermal energy production, or CO 2 sequestration [START_REF] Ellsworth | Injection-Induced Earthquakes[END_REF][START_REF] Elsworth | Understanding induced seismicity[END_REF][START_REF] Healy | The Denver EarthquakeS[END_REF][START_REF] Rutqvist | The geomechanics of CO 2 storage in deep sedimentary formations[END_REF]. In fact, between 2008 and 2014, the massive injection of waste-water in the US mid-continent has led to a ~10-fold increase in the seismicity rate for events with M w >3.5 [START_REF] Ellsworth | Increasing seismicity in the U. S. midcontinent: Implications for earthquake hazard[END_REF]. Similarly, seismicity has been observed during different geothermal activities like in Reykjanes, Iceland [START_REF] Gudhnason | Analysis of seismic activity on the western part of the Reykjanes Peninsula, SW Iceland[END_REF], Basel, Switzerland [START_REF] Deichmann | Earthquakes Induced by the Stimulation of an Enhanced Geothermal System below Basel (Switzerland)[END_REF], Soult-sous-Forêts, France [START_REF] Cuenot | Analysis of the Microseismicity Induced by Fluid Injections at the EGS Site of Soultz-sous-Forêts (Alsace, France): Implications for the Characterization of the Geothermal Reservoir Properties[END_REF] or Pohang, South Korea [START_REF] Grigoli | The November 2017 Mw 5.5 Pohang earthquake: A possible case of induced seismicity in South Korea[END_REF]. Moreover, a causal link has been established between fluid injections and the occurrence of aseismic motion [START_REF] Cornet | Seismic and Aseismic Slips Induced by Large-scale Fluid Injections[END_REF][START_REF] Evans | Permeability creation and damage due to massive fluid injections into granite at 3.5 km at Soultz: 1. Borehole observations[END_REF], Guglielmi et al. 2015[START_REF] De Barros | Fault structure, stress, or pressure control of the seismicity in shale? Insights from a controlled experiment of fluid-induced fault reactivation[END_REF][START_REF] Duboeuf | Aseismic motions drive a sparse seismicity during fluid injections into a fractured zone in a carbonate reservoir[END_REF]. Aseismic motions were observed at large scale in geothermal fields, like for example in Soultz-Sous-Forêts, France [START_REF] Calò | Large-scale aseismic motion identified through 4-D P-wave tomography[END_REF][START_REF] Cornet | Seismic and Aseismic Slips Induced by Large-scale Fluid Injections[END_REF], in Geysers, California [START_REF] Martínez-Garzón | Stress tensor changes related to fluid injection at The Geysers geothermal field, California[END_REF] or in Brawley, California (Wei et al., 2015). Several observations and in-situ experiments suggested that fluid injections primarily induced important aseismic motions, and seismicity by stress transfer (Guglielmi et al. 2015[START_REF] Duboeuf | Aseismic motions drive a sparse seismicity during fluid injections into a fractured zone in a carbonate reservoir[END_REF][START_REF] De Barros | Seismicity and fault aseismic deformation caused by fluid injection in decametric in-situ experiments[END_REF][START_REF] Eyre | The role of aseismic slip in hydraulic fracturing-induced seismicity[END_REF], De Barros et al. 2019).

The number and magnitude of the induced earthquakes, as well as the seismic/aseismic partitioning [START_REF] Avouac | From geodetic imaging of seismic and aseismic fault slip to dynamic modeling of the seismic cycle[END_REF][START_REF] De Barros | Energy of injection-induced seismicity predicted from in-situ experiments[END_REF][START_REF] Cornet | Seismic and aseismic motions generated by fluid injections[END_REF] depend on the stress state on the faults [START_REF] Bhattacharya | Fluid-induced aseismic fault slip outpaces pore-fluid migration[END_REF][START_REF] Galis | Induced seismicity provides insight into why earthquake ruptures stop[END_REF][START_REF] Snee | State of stress in Texas: Implications for induced seismicity[END_REF][START_REF] Wynants-Morel | Stress Perturbation From Aseismic Slip Drives The Seismic Front During Fluid Injection In A Permeable Fault[END_REF] among other parameters. At relatively shallow depth, the stress field may be assessed by borehole measurements [START_REF] Zang | Stress Field of the Earth's Crust[END_REF]. For example, [START_REF] Zoback | Well bore breakouts and in situ stress[END_REF] estimated the in-situ stress orientation using borehole breakout. Combined with observation of drilling-induced tensile fractures, the minimum horizontal stress magnitude can be determined [START_REF] Hickman | Stress orientations and magnitudes in the SAFOD pilot hole[END_REF], whereas density logs allow computing the vertical stress. [START_REF] Haimson | ISRM suggested methods for rock stress estimation-part 3: hydraulic fracturing (HF) and/or hydraulic testing of pre-existing fractures (HTPF)[END_REF] proposed a protocol (HTPF protocol) to determine the stress tensor from leak-off tests conducted on pre-existing natural fractures. Because they require measurements in boreholes, such stress determination methods are technically challenging at large depth and they may be influenced by the stress perturbations in the borehole nearfield. Alternatively, the inversion of earthquake focal mechanisms allows the computation of the principal stress components orientation (e.g. [START_REF] Martínez-Garzón | Sensitivity of stress inversion of focal mechanisms to pore pressure changes[END_REF], with a resolution depending on the number and spatial distribution of the seismic events.

In fluid injection areas, a discrepancy between the stress states obtained by hydraulic and seismic methods is often observed [START_REF] Dorbath | The stress field at Soultz-sous-Forêts from focal mechanisms of induced seismic events: Cases of the wells GPK2 and GPK3[END_REF][START_REF] Schoenball | Change of stress regime during geothermal reservoir stimulation[END_REF]. In Soultz-sous-Forêts geothermal field, this difference might be explained by the scale of the stimulated volume, as focal mechanism inversion covers a larger stimulated volume than the one considered using borehole measures [START_REF] Dorbath | The stress field at Soultz-sous-Forêts from focal mechanisms of induced seismic events: Cases of the wells GPK2 and GPK3[END_REF]. Nevertheless, in the same case study, [START_REF] Schoenball | Change of stress regime during geothermal reservoir stimulation[END_REF] suggest that the stress states computed from focal mechanisms are not representative of the initial stress state existing before the injection. They conclude that borehole determination methods as well as focal mechanisms-based methods may reveal local stress perturbations.

Stress perturbations observed during fluid injections can be induced by a range of physical features and processes including geological heterogeneities, fluid overpressure or aseismic motion. The geological heterogeneities influence -in particular fault zones -was underlined, among others [START_REF] Shamir | Stress orientation profile to 3.5 km depth near the San Andreas Fault at Cajon Pass, California[END_REF]Valley & Evans, 2010), by [START_REF] Faulkner | Slip on'weak'faults by the rotation of regional stress in the fracture damage zone[END_REF], who showed that the contrast of mechanical properties across a fault zone induces a rotation of the stress field. Studies also show that fluids can trigger the reactivation of structures mis-oriented to slip in the regional stress state, as for example the main fault on which Ubaye Vallée (France) seismicity occurred [START_REF] Leclère | Reactivation of a strike-slip fault by fluid overpressuring in the southwestern French-Italian Alps[END_REF]. Among others studies, [START_REF] Altmann | Pore pressure stress coupling in 3D and consequences for reservoir stress states and fault reactivation[END_REF] and [START_REF] Kim | Study on the ratio of pore-pressure/stress changes during fluid injection and its implications for {CO2} geologic storage[END_REF] underlined the role of high stimulation pore-pressure in perturbating the stress state in a large stimulated volume. Using a geomechanical modeling approach, [START_REF] Jeanne | Geomechanical simulation of the stress tensor rotation caused by injection of cold water in a deep geothermal reservoir[END_REF] related stress perturbations deduced from focal mechanisms inversions [START_REF] Martínez-Garzón | Stress tensor changes related to fluid injection at The Geysers geothermal field, California[END_REF] to changes in fluid injection rates in the Geysers geothermal field (USA). Finally, [START_REF] Schoenball | Change of stress regime during geothermal reservoir stimulation[END_REF] suggested that an important aseismic motion induced a stress-state rotation during the fluid injections in the GPK2 borehole in Soultz-Sous-Forêts.

Characterizing local stress perturbations is thus a key for proper design of a successful stimulation program and for mitigating the risk of induced seismicity in engineering fluid manipulations in deep reservoirs. In this paper, we compare stress state deduced from earthquakes focal mechanisms with the one deduced from borehole hydraulic tests. These methods, which have been demonstrated individually in several field studies, are combined here to infer the stress within the same stimulation volume and during the same fluid injection tests.

We use data from a controlled induced seismicity experiment at a decameter scale conducted at 280 m depth in a horizontal gallery intersecting a fractured limestone reservoir. The seismicity induced by 11 injection tests was previously analyzed by [START_REF] Duboeuf | Aseismic motions drive a sparse seismicity during fluid injections into a fractured zone in a carbonate reservoir[END_REF]. Here, we focus on the focal mechanisms in order to determine which geological structures were re-activated during the injections. The earthquake mechanisms are then inverted to reconstruct the stress state. In parallel, the injection borehole displacements continuously monitored during the tests give insights on the stimulated fractures movements which are coupled to stimulation pressures to estimate the static stress tensor using a hydromechanical approach. We compare the hydraulic-based and the focal-mechanism-based stress estimations and we discuss the influence of the tunnel, geology and fluid injection in the observed differences. We conclude that the large aseismic motion measured during these experiments could have triggered significant local stress rotations, resulting in reactivation of misoriented structures.

II.

Experimental settings a. Experimental settings and previous results

The experimental site is located in the Southeast of France sedimentary basin (Figure 1, [START_REF] Jeanne | Dissimilar properties within a carbonate-reservoir's small fault zone, and their impact on the pressurization and leakage associated with {CO2} injection[END_REF], within the Low Noise Underground Laboratory (LSBB, http://lsbb-new.prod.lamp.cnrs.fr) in which galleries allow a direct access to unaltered cretaceous limestone. We conducted the experiments in a horizontal gallery at 280 m depth where five 20-m long vertical boreholes were drilled in a fractured zone (Figure 1). Eleven fluid injection tests were performed from boreholes B2 and B3 (Figure 1) in different geological structures (fractures, faults, bedding planes) using a hydromechanical probe called SIMFIP [START_REF] Guglielmi | ISRM Suggested Method for Step-Rate Injection Method for Fracture In-Situ Properties (SIMFIP): Using a 3-Components Borehole Deformation Sensor[END_REF]. The probe was composed of two inflatable packers surrounding a 2.4 m-long injection chamber centered on the selected geological structures that we aimed to reactivate during the fluid injection. The probe continuously measured the water pressure, the water temperature and the 3D borehole wall deformation at the injection point, with an accuracy of 0.001 MPa, 0.1 °C, and ~5 μm respectively. A high-resolution network of 24 accelerometers and 9 geophones assured the seismic monitoring. Sensors, located at 4 to 35 m from the injections both on the gallery floor and in the boreholes, scanned a frequency range from 10 Hz to 5 kHz. Thus, induced seismicity, fluid pressure and flowrate, and mechanical deformations were continuously monitored during the injections [START_REF] Duboeuf | Aseismic motions drive a sparse seismicity during fluid injections into a fractured zone in a carbonate reservoir[END_REF]. Here, we present a brief summary of the data processing and interpretation, detailed in [START_REF] Duboeuf | Aseismic motions drive a sparse seismicity during fluid injections into a fractured zone in a carbonate reservoir[END_REF]. Out of the eleven tests, seven tests (tests 1, 4, 5, 6, 7, 8 and 10) did not induce significant seismicity. We focus on the four tests (tests 2, 3, 9, 11) in which 215 seismic events were identified. 137 events, with moment magnitude lying between -4 and -3.1, were absolutely and relatively located within 1.5 m accuracy. They were found at 1 to 12 m from the injections, suggesting a lack of events at the injection point. Moreover, irreversible deformation was measured for all tests at the injection point. A comparison between the seismic energy and the aseismic deformation shows that more than 96% of the induced deformation occurs aseismically. Both the number and the magnitude of seismic events strongly vary among the tests. Such discrepancy was related to the injected volume when corrected by the amount of aseismic deformations [START_REF] De Barros | Energy of injection-induced seismicity predicted from in-situ experiments[END_REF]. Moreover, the seismic event distance-to-the-injection variation with injection time does not follow a conventional fluid diffusion law, suggesting the presence of additional effects from other mechanisms related to the observed strain or stress perturbations. From these observations, [START_REF] Duboeuf | Aseismic motions drive a sparse seismicity during fluid injections into a fractured zone in a carbonate reservoir[END_REF] proposed that fluid injections drive an aseismic motion that triggers the seismicity. The experiments took place within the damaged zone of a kilometric normal fault, oriented N030°-85°, that cuts 1-to-5 m thick carbonate layers [START_REF] Jeanne | Dissimilar properties within a carbonate-reservoir's small fault zone, and their impact on the pressurization and leakage associated with {CO2} injection[END_REF]. This 20 m thick fractured zone contains secondary faults and 1-to-10 m long fractures (Jeanne et al., 2012). The core samples and the boreholes optical logs (Figure 1b) allowed identifying four main families of geological structures intersecting the injection intervals (Figure 2.a) : ( 1) bedding planes oriented N110° to 135° with a dip angle between 20° and 35°SW (BP), (2) fractures oriented N10° to 30° and dipping 70° E or W (F1), (3) fractures oriented N90° with a dip from 20° to 50°S (F2) and ( 4) fractures oriented N0° and dipping 45°E (F3).

b. Geology of the stimulated fracture zone

Without considering the structure extensions, these families represent (BP) 26.8 %, (F1) 41.4 %, (F2)

22 %, and (F3) 9.8 % of the identified geological structures (Figure 2.a). All the tests are crossed by F1 structures (Table 1). BP family is not observed in test 3. Finally, Tests 9 and 11 contained few F2 and F3 structures, respectively.

III. Stress state estimation from hydromechanical measurements

The stress state using aseismic fracture displacements and water pressure was computed following the protocols on dislocation analysis during fluid injection and its application to stress inversion developed by Kakurina et al. (2020aKakurina et al. ( , 2020b)). The protocol is based on picking the main slip vectors on the test interval fractures, from the continuous SIMFIP borehole displacements recorded during a stimulation test. The main slip vectors correspond to the highest measured slip rates. They may or may not correspond to a seismic event. The rake and plunge of these vectors on the reactivated fracture planes is the starting point for estimating the stress state from a single injection test. The continuous pressure and flowrate records of the SIMFIP probe allow to estimate the normal stress of the activated fracture. It is assumed that the slip vector and the normal opening corresponds to the same activated fracture. The vertical stress exerted on the activated fracture is determined independently by calculating the overburden stress at the depth of the fracture using known density of the carbonate formation [START_REF] Cochard | Analyse des propriétés réservoirs d'une série carbonatée microporeuse fracturée: approches multi-échelle sédimentologiques, diagénétiques et mécaniques intégrées[END_REF]. The protocol then searches for the reduced stress tensors, i.e. principal stress orientation and relative magnitude, that are compatible with the slip direction on the reactivated fracture. These reduced stress tensors define the shape of the stress ellipsoids. To determine the magnitude, i.e. sizes of the stress ellipsoids, the protocol searches for the stress ellipsoids that fit the ratio of the normal and vertical stresses on the fracture. The final stress tensors that match all the measurements are resampled by the Monte Carlo method to estimate uncertainties [START_REF] Walker | The 2012 Brawley swarm triggered by injection-induced aseismic slip[END_REF].

Slip vectors, for the Tests 2, 3, 9 and 11, are oriented 319°/49°, 299°/55°, 105°/69° and 299°/56°, respectively, and are aligned with the structures F1 (Figure 3). Except for Test 3, these slip events occurred before the seismicity. The normal stress from the flowrate-vs-pressure curve was estimated as 1.7, 4.5, 3.2 and 1.7 MPa for Tests 2, 3, 9 and 11, respectively. The vertical stress of 5.7-5.8 MPa was calculated considering the limestone average density of 2.4 g/cm 3 and 280 m depth for Tests 2, 3, 9 and 11 (Figure 3). The principal stress orientation and the magnitude between all the tests and with the background stress previously estimated by Guglielmi et al. (2015) give the following full stress tensor : the maximum principal stress 𝜎𝜎 1 = 6 ± 0.4 MPa is sub-vertical and dips 80°S ± 5; 𝜎𝜎 2 = 5 ± 0.5 MPa is sub-horizontal and oriented N20° ± 20°; and 𝜎𝜎 3 = 3 ± 1 MPa is sub-horizontal and oriented N110° ± 20° (Guglielmi et al. 2015).

IV. Stress state estimation from focal mechanisms a. Method

The focal mechanisms were determined by inverting the first motion polarities of the P-waves. The signal-to-noise ratio allows an unambiguous picking of this polarity on at least 8 stations for 58 events. The inversion was performed using HASH software [START_REF] Hardebeck | A New Method for Determining First-Motion Focal Mechanisms[END_REF], 2003) which integrates errors on the observed polarities, on the event locations (azimuth and take-off angles), and on the velocity model. A set of several possible focal mechanisms is generated using a grid-search on the focal sphere. The preferred focal mechanism is the average of all the possible azimuths, dips and rakes. Uncertainties on the nodal planes are estimated from the root mean square deviation between the preferred focal mechanism and the possible set of solutions.

Even though earthquakes induced by fluid injections may have a non-double-couple component in their focal mechanisms [START_REF] Julian | Non-double-couple earthquakes 1[END_REF][START_REF] Rutledge | Faulting Induced by Forced Fluid Injection and Fluid Flow Forced by Faulting: An Interpretation of Hydraulic-Fracture Microseismicity, Carthage Cotton Valley Gas Field, Texas[END_REF], we neglected it and assumed a double-couple (DC) mechanism as the non-DC component is usually observed to be only a very small percentage of the rupture [START_REF] Godano | Etude théorique sur le calcul des mécanismes au foyer dans un réservoir et application à la sismicité de la saline de Vauvert (Gard)[END_REF] and the DC component inversion has shown to be stable even if a small volumetric component is neglected [START_REF] Šílený | Approximate retrieval of the point source in anisotropic media: numerical modelling by indirect parametrization of the source[END_REF].

The earthquake focal mechanisms indicate the fault plane orientation and the slip direction. Assuming the stress field is homogeneous within the considered rock volume and the seismic events slip in the direction of the shear stress on the fault plane, focal mechanisms are used to determine the principal stress field directions by minimizing the difference between the shear stress direction and the seismic event slip directions [START_REF] Hardebeck | Damped regional-scale stress inversions: Methodology and examples for southern California and the Coalinga aftershock sequence[END_REF].

Here, we used a MATLAB package called MSATSI for MATLAB Spatial And Temporal Stress Inversion [START_REF] Hardebeck | Damped regional-scale stress inversions: Methodology and examples for southern California and the Coalinga aftershock sequence[END_REF][START_REF] Lund | Calculating horizontal stress orientations with full or partial knowledge of the tectonic stress tensor[END_REF][START_REF] Martínez-Garzón | Spatiotemporal changes, faulting regimes, and source parameters of induced seismicity: A case study from The Geysers geothermal field[END_REF]. It is based on the Formal Stress Inversion (FSI) method, which is solved by a linearized least-square inversion (Martínez-Garzón, [START_REF] Martínez-Garzón | MSATSI: A MATLAB Package for Stress Inversion Combining Solid Classic Methodology, a New Simplified User-Handling, and a Visualization Tool[END_REF]. A damped inversion is applied on the focal mechanism dataset, which can be spatially and temporally gathered into smaller subsets. The inversion provides the principal stress orientations and the uncertainties, estimated using a bootstrap resampling method.

b. Focal mechanisms and geological structures

The focal mechanism of 58 events (i.e. 42% of the located events), with magnitude ranging between -3. In order to identify the fault planes from the secondary nodal planes, we compare the focal mechanisms (Figure 2.b) with the geological structures identified in the area (Figure 2.a). Assuming a reduced uncertainty of 20° on the azimuths and 15° on the dip, 31 of the focal mechanisms have one nodal plane that can be linked to one of the main geological families (Figure 2.b). These nodal planes, interpreted as fault planes are related to geological structures as following:

• 7 events (i.e. 22.6 % of the events with identified fault planes) occurred on BP,

• 10 events (i.e. 32.2 %) on F1,

• 6 events (i.e. 19.4 %) on F2,

• 8 events (i.e. 25.8 %) on F3.

Therefore, focal mechanisms highlight that all known geological structures have been re-activated during the experiment, whatever their orientations toward the stress state. The comparison between the percentage of reactivated fractures with the percentage of identified fractures (Figure 5.a) shows that the reactivation percentage of structures F1, F2 and BP is roughly proportional to their percentage of existence in the medium. Nevertheless, F3, which is less present in the medium shows a larger percentage of reactivation, as discussed later when comparing fracture orientation and stress state. The large heterogeneity in the distribution of the second nodal plane for F1, F2 and F3 families suggests a heterogeneity in the slip motions which is confirmed by the rake distribution (Figure 2.d).

c. Stress state

From the focal mechanisms, we then aim at determining the stress state (Table 2). First, all the focal mechanisms are inverted together assuming a homogeneous stress field (Figure 6.a). The large uncertainties on the principal stress underlines a poorly constrained solution. In order to test the solution stability, random perturbations are applied on the focal mechanisms by adding uncertainties up to 35° for the strike and up to 15° for the dip. Then, the stress state inversion is performed 100 times on catalogs with those random perturbations. The mean solution is almost (± 5°) identical to the first solution, which validates it. Thus, the inversion leads to the following stress orientation:

• the maximum stress 𝜎𝜎 1 (blue dots, Figure 6.a) is subvertical and dips 80°E. However, it could be more horizontal because of the poor azimuth constraint in the East-West direction. • The intermediate stress, 𝜎𝜎 2 (green dots), is sub-horizontal and oriented N260° ± 45°.

• The minimum principal stress 𝜎𝜎 3 (red dots) is also sub-horizontal and oriented North-South (N175° ± 45°). This stress state therefore shows an inversion of the two horizontal components, compared to the stress state determined by Guglielmi et al. (2015) and by the hydro-mechanical inversion (Figure 3). Such a 90° rotation could indicate a change between 𝜎𝜎 2 and 𝜎𝜎 3 -i.e. (𝜎𝜎 2 ; 𝜎𝜎 3 ) become(𝜎𝜎 3 ; 𝜎𝜎 2 )-, which will be likely if their magnitudes were closed. Here, the large difference between 𝜎𝜎 2 and 𝜎𝜎 3 magnitudes (3 and 5 MPa, respectively, Guglielmi et al. (2015)) requires a strong perturbation of either their amplitude or their orientation. Moreover, this solution is not well-constrained, as highlighted by the very large uncertainties. A possible explanation can be found into a non-homogeneous stress field in the experimental area. We therefore look for spatial variations of the stress field.

The state-of-stress is thus computed at 5 locations along the North-South main trend of events location (Figure 6.b, 1 and 4). Events are gathered on points located at 2, 6, 10, and 14 m depth and on B2 or B3 boreholes, according to their closest location. A variation of the stress tensor orientation with depth is observed:

• at 2 and 6 m depth, 𝜎𝜎 1 is sub-vertical while 𝜎𝜎 3 and 𝜎𝜎 2 are sub-horizontal, approximately oriented N180° ± 45° and N90° ± 30°, respectively. These stress orientations are quite similar to the one obtained from the inversion of all focal mechanisms. This is consistent with the fact that 64 % of the seismicity occurred between the gallery floor and 8 m depth. • at 10 m depth, 𝜎𝜎 1 rotates from subvertical to N135° ± 20°, dipping ~55° ± 7° SE. 𝜎𝜎 2 and 𝜎𝜎 3 rotate to N290° ± 20°, dipping ~45° ± 7° NW and to N40° ± 30° sub-horizontal, respectively. • at 14 m depth, 𝜎𝜎 1 is oriented N140° ± 20° dipping 40° ± 10°SW. 𝜎𝜎 2 is sub-horizontal N240° ± 30° and 𝜎𝜎 3 dips 85° ± 5°NW. We then determine the stress state individually at Tests 2, 3, 9 and 11, using a minimum of 6 focal mechanisms to perform each inversion (Figure 6.c). Tests 2 (7.5 m depth), 9 (14 m depth) and 11 (3.5 m depth) display stress states identical to the stress states previously deduced et 6, 14 and 2 m depth (Figure 6.b), respectively. Stress determined for the Test 3 differs from the one determined at 10 m, but it is badly constrained. We conclude that stress-state computed by event locations or by tests sequence are similar, probably because most of the events gather around their injection area (Figures 1 and4).

The shallower stress states and Tests 2 and 11 show a 90° rotation of the horizontal stress compared to the stress field inferred from geomechanical data; and the deepest test (9) highlights a 𝜎𝜎 1 and 𝜎𝜎 3 inversion in addition of the 90° rotation of 𝜎𝜎 2 . The stress state appears to be heterogeneous within the experimental site and characterized changes with depth and/or tests.

V. Comparison between the focal mechanism and the hydromechanical approaches for the estimation of stress

Stress deduced from focal mechanisms inversion differs from stress deduced from displacementpressure inversion (hereafter referred as geomechanical stress state). We observe a heterogeneity of the seismic-deduced principal stress orientations with depths and/or with the injection tests. In comparison, the principal stress orientations deduced from the hydromechanical approach are very consistent between tests.

a. Reactivation fractures and geomechanical stress field

We first consider the "geomechanical" stress state to calculate the potential for slipping on preexisting geological structures using equation 1 [START_REF] Sibson | A note on fault reactivation[END_REF]:

𝑅𝑅 = 1+ 𝜇𝜇 𝑠𝑠 𝑐𝑐𝑐𝑐𝑐𝑐𝑚𝑚𝑐𝑐(𝜃𝜃 𝑅𝑅 ) 1-𝜇𝜇 𝑠𝑠 𝑐𝑐𝑚𝑚𝑐𝑐(𝜃𝜃 𝑅𝑅 ) = 𝜎𝜎 1 ′ 𝜎𝜎 3 ′ = 𝜎𝜎 1 -𝑃𝑃 𝑓𝑓 𝜎𝜎 3 -𝑃𝑃 𝑓𝑓 , (1)
where 𝑃𝑃 𝑓𝑓 is the pore pressure, 𝜇𝜇 𝑠𝑠 the friction coefficient (here, 0.4 as determined by Jeanne et al. (2012) and 𝜃𝜃 𝑅𝑅 the angle between the fault plane and 𝜎𝜎 1 axis. 𝜃𝜃 𝑅𝑅 is computed for all structure families, and equation ( 1) solutions are plotted in Figure 5.b, which indicates whether structures are well, unfavorably or extremely mis-oriented to slip in the regional state-of-stress.

Within this stress field, the intermediate stress (𝜎𝜎 2 ) direction belongs to F1 and F3 fault planes (with angles of 0° and 13°, respectively). Thus, with 𝜃𝜃 𝑅𝑅 angles of about 23° and 44° and a ratio R of ~2 (equation 1), F1 and F3 structures are well oriented to slip and a low pore pressure of ~0.7 MPa is required to induce slips. It may explain why they are the most reactivated structures (~32 % and ~26 %, respectively, Figure 5.a). However, although these structures should slip in a normal motion, only half of the observed mechanisms are normal (Figures 2.d and4).

Figure 5.b indicates that the other geological structures are either unfavorably oriented to slip, like F2, or extremely misoriented, like BP. The angles 𝜃𝜃 𝑅𝑅 of about 70° and 65° lead to R ratio of -10 and 8.3 for BP and F2 respectively. Moreover, 𝜎𝜎 3 is within BP and F2 fault planes, as the angle between the fault planes and 𝜎𝜎 3 direction is of 5.7° and 11°, respectively. Rupture should be therefore driven by 𝜎𝜎 1 and 𝜎𝜎 2 . In this case, applying equation 1 leads to a pore pressure of 5.1 MPa and 4.9 MPa, respectively for BP and F2, in order to reach failure, which is in the range of the maximum injected pressure during Tests 9 and 11 (5.9 MPa). Considering the fluid pressure decays with the distance to the injection point, it is not likely to be above 5.1 MPa where events occurred (1.5 to 12 m to the injection). Finally, BP and F2 structures are also reactivated during Test 2 during which the maximum injection pressure is 4.8 MPa. Consequently, fluid pressure during the injections was not high enough to induce failures on those structures under the assumption of a constant and homogeneous stress state, suggesting that perturbation of stress is required to allow misoriented fractures to slip.

The stress state that applies on all fractures are represented in a shear stress versus normal stress plot, together with the Mohr-Coulomb criterion (Figure 1c and Figure 5c). The normal and shear stress are computed using the Cauchy equation [START_REF] Jaeger | Fundamentals of rock mechanics[END_REF]) within the regional stress-state of Guglielmi et al. (2015). It confirms our analysis, showing that while F1 and F3 are relatively well-oriented to slip into the geomechanical stress state, BP and F2 present a strong misorientation for reactivation. Therefore, a fluid pressure of ~5 MPa is required to reach the rupture on these structures.

b. Tunnel and Spatial heterogeneity of the stress field

As the experiments were performed close to a gallery, the tunnel might have influenced local perturbations of the stress state in the so-called Excavation Damage Zone (EDZ, [START_REF] Shen | The disturbed zone around tunnels in jointed rock Masses[END_REF][START_REF] Eberhardt | Numerical modelling of three-dimension stress rotation ahead of an advancing tunnel face[END_REF][START_REF] Kaiser | Mining-induced stress change and consequences of stress path on excavation stability -a case study[END_REF]). In the EDZ, the vertical stress can be reduced, yielding to a subvertical 𝜎𝜎 3 and a sub-horizontal 𝜎𝜎 1 . However, the computed stress state highlights sub-horizontal 𝜎𝜎 3 and sub-vertical 𝜎𝜎 1 . This ~0.5m thin damage zone observed on cores might not be enough to perturbate stress in these hard carbonate rocks even relatively close to the gallery. Consequently, the cavity presence cannot explain the stress state changes with depth.

In addition, fluid injections are realized in the damaged zone of a normal fault, which presents a high geological heterogeneity. [START_REF] Faulkner | Slip on'weak'faults by the rotation of regional stress in the fracture damage zone[END_REF] showed that fault structures could drive to a rotation of the stress field, because of the heterogeneities of mechanical properties across the fault zone. However, in our experiments, the injections were all at a similar, relatively large distance (~20 m) from the fault core. So the same stress fault perturbation should influence all the tests. At a smaller scale, similar geological structures are identified at the different injection points. It not likely that they may induce different stress state at a few meters distance. Therefore, the stress heterogeneity may be associated to the different tests, and not to the seismicity locations. As most of the events gather around the injection points, the differences due to the different tests are also visible with depths.

c. Scales effect between the seismic and geomechanical approaches?

The seismic mechanisms correspond to small, decimeter-scale ruptures [START_REF] De Barros | Energy of injection-induced seismicity predicted from in-situ experiments[END_REF][START_REF] Duboeuf | Aseismic motions drive a sparse seismicity during fluid injections into a fractured zone in a carbonate reservoir[END_REF]. The seismic method relates the stress to dislocations on these very localized activated structures, most of them being meters away from the injection source. The geomechanical approach associates the local direct measurement of the slip on a fracture at the injection source with the injection pressure. It appears that whatever the orientation of the activated structure is, there is a consistent estimation of the stress tensor's orientation and magnitude compared to the seismic approach which highlights a high variability. Main difference is that the geomechanical approach uses slow rupture which correspond to relatively large displacement magnitudes representative of plurimeter fracture pressurized patches. The seismic approach relies on micrometric displacement on small decimeter scale rupture patches given the low magnitude of the events [START_REF] Huang | Illuminating the Rupturing of Microseismic Sources in an Injection-Induced Earthquake Experiment[END_REF]. This might explain why there is much more variability of the stresses deduced from seismicity since they may be more sensitive to local heterogeneities such as discontinuities roughness, friction and critical slip distances. Besides our previous work (Guglielmi et al. 2015[START_REF] Duboeuf | Aseismic motions drive a sparse seismicity during fluid injections into a fractured zone in a carbonate reservoir[END_REF][START_REF] De Barros | Seismicity and fault aseismic deformation caused by fluid injection in decametric in-situ experiments[END_REF] showed that aseismic displacements were preceding seismicity, and that events were triggered at and beyond the fluid pressure front. This was related to the shear stress perturbation at the front (Wynants-Morel et al., 2020), which might be the one deduced from the current focal mechanisms approach.

d. Aseismic motion

In addition -as neither the fluid pressure nor the presence of the geological heterogeneities directly explain the reactivation of misoriented structures -the large aseismic deformation which continuously varies during the test sequence is an obvious link between stress deduced from seismicity and stress deduced from pressure and displacement. Tests 2, 9 and 11, which contain the misoriented BP and F2 structures, are characterized by more than 96 % of aseismic motion. This aseismic deformation induces stress perturbations at least tens of meters around the pressurized zone and through mixed mode rupture mechanisms (Guglielmi et al. 2015, Wei et al. 2015[START_REF] De Barros | Seismicity and fault aseismic deformation caused by fluid injection in decametric in-situ experiments[END_REF], 2019, Cappa et al. 2019).

At the scale of our experiments, induced seismicity may thus be representative of the stress perturbation induced by aseismic movements on preexisting fractures. Oppositely, the stress state determined from pressure-displacement data might figure a stress state representative of the scale of the stimulated pressurized volume. For the different tests, the amplitude and the direction of the aseismic deformation differ, as the structures in which the fluid is injected differs. It is likely to explain why the reconstructed stress field varies among tests. As the stress field is perturbated in response to the aseismic motion, structures that were misoriented in the regional stress field may, then, be well oriented to slip, with a limited fluid pressure. This has been suggested in Soultz-Sous-Forêts geothermal field, when fluid injections were performed in GPK2 borehole. [START_REF] Schoenball | Change of stress regime during geothermal reservoir stimulation[END_REF] proposed that the stress state change deduced from induced earthquakes was due to the strong aseismic motion mentioned by [START_REF] Calò | Large-scale aseismic motion identified through 4-D P-wave tomography[END_REF]. Similarly, in the Brawley geothermal field (California, USA), a strong aseismic motion was recorded on geodetic instruments preceding the seismicity occurrence and specially two large earthquakes with magnitude greater than 5 (Wei et al. 2015).

We here propose that fluid injections trigger the aseismic motion which then induces stress perturbation. These stress perturbations allow reactivating fractures with different orientations. Consequently, a significant part of the measured induced seismicity may reveal local stress perturbations more or less distant from the fluid injections. Therefore, for small event magnitudes, the stress state inferred from the focal mechanisms may not be representative of the regional initial stress state, but its heterogeneity may highlight the presence of strong aseismic deformation. At a reservoir scale, heterogeneities in the stress field may also suggest that aseismic deformations trigger seismicity, as suggested by [START_REF] Schoenball | Change of stress regime during geothermal reservoir stimulation[END_REF] during an injection in Soultz-Sous-Forêts. However, while small events may show heterogeneous mechanisms due to stress perturbations, large earthquakes, with size comparable or larger than the size of the pressurized zone, are likely to be compatible with the regional stress state. Therefore, stress heterogeneities may be visible at very local scales for small magnitude events and they may be masked when averaged at the full reservoir scale. Such statement should be however validated and tested on different reservoir injections by comparing stress-state determined from hydro-mechanical measures in deep boreholes with stress state from seismic mechanisms at different scales.

VI. Conclusion

Here, we studied the seismicity and stress perturbations during a series of fluid-injections performed at 280 m depth in selected geological structures of a carbonate fractured reservoir zone. Comparing the seismic mechanisms with the main geological structures observed in the area, we observed that all fractures families were reactivated, including those that are badly oriented toward the regional stress state. The stress states reconstructed from the inversion of focal mechanisms always differ from the one determined through hydro-mechanical measurements at the locations of the injection. When using event subsets, we observed that the reconstructed stress states vary with depth and/or tests. The fluid pressure or the geological heterogeneities cannot explain the heterogeneity of the stress field and the reactivation of misoriented structures deduced from the induced earthquakes focal mechanisms. Therefore, we propose that the strong aseismic deformation, precisely measured at the injection, temporarily modifies the stress state, which allows seismic events to be triggered on a large variety of structures, initially misoriented, inside and around the stimulated volume. The perturbed stress fields deduced from seismic mechanisms might then be a qualitative probe of aseismic deformation.

Figure 1 (

 1 Figure 1 (a) Location of the injection tests and seismicity in a North-South cross-section, adapted from Duboeuf et al. 2017.The colored stars and dots indicate the fluid injection and seismic locations, respectively, colored by test number (Tests 1 in cyan, 2 in red, 3 in green, 5 in grey, 6 in white, 7 in dark grey, 8 in pink, 9 in dark blue, 10 in orange and 11 in black). The 5 vertical boreholes are represented by the blue lines. The inset on the left shows the location of the LSBB facility. (b) is a core sample, showing the BP (dark blue) and F1 (cyan) structures where Test 2 was performed between 6.2 m and 8.6 m depth. The red rectangle shows the position of the injection chamber. (c) Stress-state of the known geological structures (colored stars) in a 3-D Mohr-Coulomb diagram. The grey line represents the rupture for a friction coefficient of 0.4 within the geological stressstate(Guglielmi et al. 2015) and the 3 half-circles the stress tensor.

Figure 2

 2 Figure 2 Fault planes and rake distribution, colored by the main geological families: BP (blue), F1 (cyan), F2 (green), and F3 (orange). (a) shows the poles of the main geological structures crossing the injection intervals. (b) shows the poles of the nodal planes of the seismic mechanisms. The colored dots and squares show the interpreted fault and the secondary nodal planes, respectively. Mechanisms without clear geological interpretation are left in grey. (c) is identical to (b) but gathered by fracture families. (d) presents the rake histogram colored by geological structure families. Note that stereographic projections are presented from the lower-hemisphere view, with equal angles.

Figure 3

 3 Figure3Stress inversion from the hydromechanical data of tests 2, 3, 9 and 11. Upper line shows orientation of the mean principal stress directions (stars) and principal stress uncertainties (small points) determined using the Monte Carlo method. The displacement vector picked in the measurements and projected on the test activated plane. Lower line shows the magnitudes of the principal stresses with the 95 % confidence interval (line) and the mean principal stress magnitude (stars). Blue color corresponds to 𝜎𝜎 1 , green to 𝜎𝜎 2 , and red to 𝜎𝜎 3 .

   were computed (Figures 2.b.c.d and 4). The mean uncertainty on the nodal plane orientation reaches ~35°. Focal mechanisms show a wide variety of solutions, particularly between the injection tests. They are dominated by reverse motions (Figures2.d and 4) but they also show normal and strike-slip motions (Figure4).

Figure 4

 4 Figure 4 Focal mechanisms solution colored by test number (with Tests 2 in red, 3 in green, 9 in dark blue and 11 in black) in (a) a map view, and within the (b) depth-north direction. The figure (a) shows the focal mechanisms with a beach ball representation. Colored dots indicate the event locations. The white and grey areas show the gallery floor and walls. The grey circles show the borehole locations. The figure (b) presents the focal mechanisms into the Depth-North direction. The colored stars indicate the injection test locations. The grey plane shows the gallery floor. Finally, the main identified geological structures that intersect the injection intervals are shown as colored planes (blue for the BP, cyan for the F1, green for F2, and orange for F3).

Figure 5

 5 Figure 5 (a) Percentage of reactivated fractures in function of the percentage of fractures into the medium. (b) Stress ratio versus the reactivation angle 𝜃𝜃 𝑅𝑅 between 𝜎𝜎 𝑚𝑚𝑚𝑚𝑚𝑚 axis and the fault plane for the four main fracture families (stars) that are reactivated. This figure is adapted from[START_REF] Sibson | A note on fault reactivation[END_REF], with 𝜃𝜃 𝑅𝑅 * the optimal reactivation angle and the grey area showing the unfavorably fault orientation. In both panels, BP is dark blue, F1 is cyan, F2 is green and F3 is orange. (c) Stress state of the known geological structures (stars) and seismic events with a determined focal mechanism (dots) in a 3-D Mohr-Coulomb diagram. The grey line represents the rupture for a friction coefficient of 0.4 within the geomechanical stress state(Gugliemi et al. 2015) and the 3 half-circles the stress tensor.

Figure 6

 6 Figure 6 Stress states from the focal mechanism inversion. The stress fields are determined by inverting (a) all focal mechanisms; (b) focal mechanisms gathered by common locations, in a North-Depth cross-section; (c) focal mechanisms gathered by injection tests. The principal stress directions are indicated by the diamond symbol with the specific color: blue for 𝜎𝜎 1 , green for 𝜎𝜎 2 , and red for 𝜎𝜎 3 . The smallest points are the output of the bootstrap resampling used to determine the uncertainty range, while the diamonds show the best solution. To compare with from the stress states obtained from hydromechanical computation, the principal stress directions are represented by the star symbol.

Table 1 :

 1 Summary of injection depth, identified geological structures, maximum injection fluid pressure and percentage of aseismicity per seismic injection test[START_REF] Duboeuf | Aseismic motions drive a sparse seismicity during fluid injections into a fractured zone in a carbonate reservoir[END_REF]).

	Test	Test	Geological structures	Maximum	Aseismic
	injection	Location		injection	percentage (%)
		(depth -borehole)		pressure (MPa)
	Test 2	7.5 m depth -B2	BP, and F1 structures.	4.86	97-99.9
	Test 3	9.5 m depth -B2	F1 structures	5.3	80-99.9
	Test 9	14 m depth -B3	BP, F1 and F2	5.93	98-99.9
			structures
	Test 11	3.5 m -B3	BP, F1 and F3	5.89	94-99.9
			structures

Table 2

 2 Regional stress state and stress states determined using the full set of focal mechanisms, or subsets gathered by depth or test. Dipping angles are considered as subhorizontal when smaller than 10°.

	Stress state	𝜎𝜎 1	𝜎𝜎 2	𝜎𝜎 3
	Regional, computed by	Subvertical, dips 80° Subhorizontal,
	Guglielmi et al. 2015		N20° ± 20°

Acknowledgments

We would like to thank the Agence Nationale de la Recherche (ANR) and Total SA company who funded this research through projects HYDROSEIS (ANR-13-JS06-0004-01) and HPMS-Ca (Albion PI. G. Massonat), respectively. We also thank NORSAR (Kjeller Norway) for the use of Mstudio, a NORSAR in-house software package, to generate the Figure 4.b.

Data from the experiments are available upon requests at debarros@geoazur.unice.fr VII.


Julien Fassy

Caroline Lacoux

Sylvie Leroy

Latifa Noussair

Sylvain Hubac

Aurélien Degoutte

Georges Vassaux

Vianney Leclercq

David Rouquié

Charles-Hugo Marquette

Martin Rottman

Patrick Touron

Antoinette Lemoine Corbel

Jean-Louis Herrmann

Pascal Barbry

Jean-Louis Nahon

Laure-Emmanuelle Zaragosi

Ph.D Bernard Mari
email: mari@unice.fr..

Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples

Introduction

To control the pandemic and monitor virus propagation of SARS-CoV-2 in real time, extensive testing is necessary. Although alternatives are available [START_REF] Marca | Testing for SARS-CoV-2 (COVID-19): a systematic review and clinical guide to molecular and serological in-vitro diagnostic assays[END_REF][START_REF] Ghaffari | COVID-19 Serological Tests: How Well Do They Actually Perform[END_REF], viral load detection from nasopharyngeal or saliva samples is still the most appropriate method to identify SARS-CoV-2 carriers. The current diagnostic testing methods recommended by the Centers for Diseases Control (CDC) and the World Health Organization (WHO) are based on a traditional RT-qPCR assay, with validated primers [START_REF] Lieberman | Comparison of Commercially Available and Laboratory-Developed Assays for In Vitro Detection of SARS-CoV-2 in Clinical Laboratories[END_REF].

However, the availability of this assay has been a major hurdle in the orderly and efficient management of the pandemic because of reagent shortages, as well as material and staff limitations.

A rapid processing of the samples is also a crucial factor. To further stem the spread of coronavirus disease 2019 (COVID-19), a series of innovative approaches have been recently proposed [START_REF] Pettit | All In': a pragmatic framework for COVID-19 testing and action on a global scale[END_REF].

Notably, to face material shortage and reduce processing times, two kinds of process optimization should be undertaken: [START_REF] Marca | Testing for SARS-CoV-2 (COVID-19): a systematic review and clinical guide to molecular and serological in-vitro diagnostic assays[END_REF] multiplexing sample and PCR probes, as well as [START_REF] Ghaffari | COVID-19 Serological Tests: How Well Do They Actually Perform[END_REF] reducing the steps in sample preparation.

Most approved RT-qPCR assays are "one-step" kits that should be performed on standard real-time thermocyclers. When sample pooling [START_REF] Lohse | Pooling of samples for testing for SARS-CoV-2 in asymptomatic people[END_REF] is not used, throughput is quite limited and the use of alternative systems, such as the Biomark TM HD device from Fluidigm may overcome this limitation.

The Biomark TM HD device is a nanofluidic automated real-time PCR system that exploits the microfluidic technology through the use of dynamic arrays of integrated fluidic circuits (IFCs). Using, for example, the 192.24 Integrated Fluid Circuit (IFC), 192 samples can be processed in parallel with 24 independent sets of probes, allowing increased confidence in viral RNA detection as additional SARS-CoV-2 probes can be included. Probes to detect supplementary targets such as other RNA viruses, or host response genes can also be included. This flexibility presents a clear added value for both clinical monitoring of viral and bacterial pathogens as well as for research studies [START_REF] Olwagen | Performance of the Biomark HD real-time qPCR System (Fluidigm) for the detection of nasopharyngeal bacterial pathogens and Streptococcus pneumoniae typing[END_REF][START_REF] Goecke | Subtyping of Swine Influenza Viruses Using a High-Throughput Real-Time PCR Platform[END_REF]. Another advantage of this system resides in the low amount of reagents needed compared to classical realtime PCR machines. The reaction volume is down to the 10 nL-scale compared to the 10 µL scale in classical qPCR, thus reducing reagent requirement, an important point in times of shortages.

The second improvement resides in the reduction of the number of steps for sample preparation. RT-qPCR detection starts with viral RNA extraction. This is time consuming for laboratories that are not equipped with high-throughput automated systems. It is also reagent consuming, and as March 2020, laboratories have suffered from major RNA extraction kit shortages. Several teams have proposed RNA extraction-free protocols. The resulting improvement in processing times were balanced by a loss in sensitivity. The comparison of these different studies is made difficult by the great variabilities of the protocols. This includes parameters such as the type of collection media, the use of additives such as detergents, heat-inactivation, or selection of a particular RT-qPCR kit [START_REF] Bruce | Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step[END_REF][START_REF] Smyrlaki | Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR[END_REF][START_REF] Beltrán-Pavez | Accuracy of a RT-qPCR SARS-CoV-2 detection assay without prior RNA extraction[END_REF][START_REF] Fomsgaard | An alternative workflow for molecular detection of SARS-CoV-2 -escape from the NA extraction kit-shortage[END_REF][START_REF] Grant | Extraction-free COVID-19 ( SARS-CoV-2 ) diagnosis by RT-PCR to increase capacity for national testing programmes during a pandemic[END_REF][START_REF] Hasan | Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA[END_REF][START_REF] Fukumoto | Efficacy of a novel SARS-CoV-2 detection kit without RNA extraction and purification[END_REF][START_REF] Wei | Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction[END_REF][START_REF] Ranoa | Saliva-Based Molecular Testing for SARS-CoV-2 that Bypasses RNA Extraction[END_REF].

In 2 of these studies, measurements on a large set of clinical samples demonstrated the effectiveness of direct RT-PCR assays with or without heat inactivation on various transport medium (VTM) [START_REF] Bruce | Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step[END_REF][START_REF] Smyrlaki | Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR[END_REF]. Srivatsan et al. proposed to collect dry swabs and elute them in Tris-EDTA (TE) to eliminate collection medium dilution and variation, and to bypass the RNA extraction step [START_REF] Srivatsan | Preliminary support for a "dry swab, extraction free" protocol for SARS-CoV-2 testing via RT-qPCR[END_REF]. This simplified protocol was as sensitive as a conventional protocol (sample collection in VTM). They also evaluated the addition of detergents (IGEPAL, Triton X-100 and Tween-20) that facilitates virus inactivation in the elution medium. Addition of all three detergents resulted in a loss of sensitivity.

In the present study, we evaluated the use of a high-throughput real-time PCR device, the Biomark TM HD, to increase throughput, flexibility in probe inclusion, and decrease reagent consumption, together with an optimized protocol for SARS-CoV-2 RNA detection without RNA extraction.

Materials and methods

Primers and probes for the detection of viral and cellular genes:

All the DNA primers and probes used in this study are listed and described in Table 1 and2. Positive control plasmid:

Positive control plasmid containing the full length nucleocapsid coding sequence (N) was purchased at Integrated DNA Technologies (2019-nCoV_N_Positive Control; cat. no. 10006625). Plasmid was supplied at a final concentration of 200 000 copies/μL in IDTE pH=8 buffer, and has been used to evaluate both the detection efficiency and sensitivity of the Biomark HD System Analysis.

In vitro Transcription:

The synthetic transcript for the 2019-nCoV N coding sequence (N transcript) was generated using the 2019-nCoV_N_Positive Control plasmid. The template for the in vitro transcription was produced by PCR: briefly, 6 ng of plasmid was used to amplify the entire coding sequence of the N gene using: T7-For-sens (ATATAATACGACTCACTATAGGATGTCTGATAATGGACCC; T7 promoter sequence underlined) and Rev-sens (TTAGGCCTGAGTTGAGTC) as primers and the Q5-High Fidelity DNA polymerase (New England Biolabs, cat. no. M0491S). Thermal cycling conditions were: 98°C for 30 s, 40 cycles of 98°C for 10 s, 52°C for 30 s, 72°C for 1 min followed by 72°C for 2 min (SimpliAmp Thermal Cycler, ThermoFisher Scientific). The size of the PCR product was verified on a 1.5% agarose gel in 0.5X TAE and further purified using the QIAquick PCR purification kit (Qiagen, cat. 28106). Template was used in the in vitro transcription reaction using the T7 RiboMAXExpress Large Scale Production System (Promega, cat. no. P1320) according to manufacturer's instructions. The in vitro transcript was extracted using phenol chloroform isoamyl followed by a chloroform wash and further precipitated using 300 mM sodium acetate in absolute ethanol at -20°C, over-night. Sample was centrifugated at 4°C for 30 min and washed twice with 70% cold ethanol. The RNA pellet was dried at RT for 5 min and resuspended in 100 µL water. Garches Hospital (AP-HP): Purified RNA samples (n=92) from nasopharyngeal swabs with known COVID-19 status were obtained from the Garches Hospital following an ISO 15189 certified lab protocol previously described [START_REF] Touron | A mobile DNA laboratory for forensic science adapted to coronavirus SARS-CoV-2 diagnosis[END_REF] and used to validate the BIOMARK TM HD protocol. Additional frozen VTMs from 55 patients with known COVID-19 status collected in 1-2 mL of saline buffer were obtained to set up the protocol. All samples were recorded for traceability on the basis of a unique barcode identifier.

Bioesterel laboratories: Samples (18 in virucide VTM, 2 in non-virucide VTM, Lingen) were stored overnight at 4°C and processed the next day. A generic consent form was signed by the patients, allowing the utilization of the samples for scientific research. All samples were anonymized and their COVID-19 status was recorded.

RNA extraction:

Both miRNeasy Serum/Plasma Advanced Kit (Qiagen, cat. no. 217204) and QIAamp Viral RNA Mini Kit (Qiagen, cat. no. 52906) were used for Total RNA extraction from clinical samples according to manufacturer's instructions and using the Qiacube (Qiagen) apparatus. Final elution was performed in 20 µL water and 60 µL of AVE buffer for the miRNeasy Serum/Plasma Advanced Kit and QIAamp Viral RNA Mini Kit, respectively. Total RNA (containing miRNAs) were directly used for further analysis or stored at -20°C.

Detergent treatments of clinical samples:

All clinical samples were handled in a biosafety level 2 laboratory and under biological safety cabinet using the adapted biosafety personal protective and respiratory equipment, according to the recommendations of the French society of microbiology (https://www.sfm-microbiologie.org/). The following detergents were used: Triton X-100 (Sigma, cat. no. T9284-100ML), Brij™35 (Millipore, cat.

No. 1.01894.0100), Tween-20 (Sigma, cat. no. P7949-500ML), Brij™O10 (Sigma, cat. no. P6136-100G) and Poly Ethylene Glyco-600 (Aldrich, cat no. 202401-250G). They were included alone or in combination to prepare a 2X detergent master mix that was added to clinical transport media samples. A 10 mg/mL Proteinase K (Sigma P4850) solution was mixed extemporaneously with the 2X detergent master mix to reach a 2 mg/mL final concentration. Heating treatments were carried out at 95°C for 5 min in a thermo-cycler apparatus (SimpliAmp Thermal Cycler, ThermoFisher Scientific).

Treatment using the Quick Extract TM DNA Extraction Solution (Lucigen, QE09050) was performed using the same volume of reagent and sample, according to the manufacturer's instructions.

Mixtures were then incubated at 95°C for 5 min or at 60°C for 10 min in a thermo-cycler. TE buffer was prepared using 10 mM Tris-HCl pH7.0, 2 mM EDTA, 20 µg/mL yeast tRNA with or without 0.5% Triton X-100.

Detection of mRNA expression protocol using the Biomark HD System Analysis Reverse Transcription using the Fluidigm Reverse transcriptase:

Extracted RNA (2 µL) or processed clinical samples were reverse transcribed using the Reverse Transcription Master Mix kit according to the manufacturer's instructions (catalog number # PN 100-6297). Thermal cycling conditions were: 25°C for 5 min, 42°C for 30 min, 85°C for 5 min.

Reverse transcription using the Vilo SuperScript IV step: 2.5 µL of processed clinical samples were used in a reverse transcription reaction using the SuperScript IV Vilo Master Mix (ThermoFisher Scientific, cat. no. 11756500) following the manufacturer's instructions. Reactions were incubated at 25°C for 10 min, 55°C for 10 min, 85°C for 5 min cDNA pre-amplification step: 2.5 µL of cDNA were preamplified using the Preamp Master Mix kit (Fluidigm, cat. no. PN 100-5744) according to the manufacturer's instructions: 1 µL of Preamp Master Mix was combined with 1.25 µL of Pooled Taqman assay mix and 2.5 µL of cDNA in a 5 µL total volume reaction. The Pooled Taqman assay (180 nM) used in these reactions was prepared from an intermediate pooled Taqman assay solution (6.7 µM Forward and Reverse primers, 1.7 µM probes). Thermal cycling conditions were: 95°C for 2 min followed by 20 cycles of 95°C for 15 s, 60°C for 2 min. After each preamplification reaction, samples were diluted 1:5 by adding nuclease-free water up to 25 µL.

One step "RT-Preamplification":

One step "RT-Preamplification reactions" were performed using the Cells Direct One-Step qRT-PCR kit (Invitrogen, cat. no. 46-7200) according to a protocol adapted from the Reverse Transcription-Specific Target Amplification (Fluidigm). Briefly 5.5 µL of total RNA were mixed with 6.25 µL the Pooled Taqman assay, 0.5 µL Superscript III RT/ Platinum Taq, 12.5 µL 2x Reaction mix and nucleasefree water to 25 µL total volume. Thermal cycling conditions were: 42°C for 15 min, 95°C for 2 min followed by 15 to 20 cycles of 95°C for 15 s, 60°C for 2 min. Then, samples were diluted 1:5 using nuclease-free water up to 125 µl.

Real time qPCR using Biomark HD System: PCR was performed following Gene Expression Standard TaqMan Assays protocol (Fluidigm cat n° 100-6170 C1), using a 10X assays mix and a pre-sample mix prepared separately. The 10x assays mix was prepared by mixing 2 µL of combined Primer (Forward/Reverse 6.7uM, Probe 1.7 µM) and 2ul 2X Assay Loading Reagent (Fluidigm PN 100-7611) to a final volume of 4 µL (per reaction). The presample mix was prepared by mixing 2 µL TaqMan Universal PCR Master Mix (2X) (Life Technologies PN 4304437) and 0.2 µL 20X GE Sample Loading Reagent (Fluidigm PN 100-7610) and 1.8 µL preamplified cDNA to a final volume of 4 µL.

Then, 3 µL of 10x assays mix and of pre-sample mix are transferred into the 192.24 IFC, loaded into the Biomark IFC controller RX and transferred to the Biomark HD apparatus. Thermal cycling conditions were as follows: 50°C for 120 s, 95°C for 600 s followed by 20 cycles of 95°C for 15 s, 60°C for 1min.

Detection of microRNA-expression protocol using the Biomark HD System Analysis

RNAs (1 µL) were processed using the miScript 2 RT kit (QIAGEN cat n° 218161). Preamplification was performed using 5 µL of diluted cDNA using the mi Script microfluidics PreAMP kit (QIAGEN, cat n° 331455) and following the 384-plex preamplification cycling conditions (see manufacturer's protocol). QPCR was performed following miScript microfluidics PCR kit (QIAGEN cat n° 331431) with commercially-available primers (miScript Primer assays). Six µL of assays mix and sample mix were prepared as described in the manufacturer's protocol and 5 µL of each are loaded onto the 96.96 IFC plates (Fluidigm cat n° BMK-M-96.96). The list of microRNAs (miRNAs) tested in this study is presented in Table S1.

Biomark HD System Analysis:

Exhaustive protocols are available at: https://dx.doi.org/10.17504/protocols.io.bd3ii8ke (Two-Step qPCR Protocol) https://dx.doi.org/10.17504/protocols.io.bnx4mfqw (One-Step qPCR Protocol) Fluidigm HD System qPCR Analysis: qPCR results were analyzed using the Real Time PCR analysis software provided by Fluidigm.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software. Results are given as mean ± SEM. Two-tailed unpaired Student's t-test was used for single comparisons and one-way ANOVA followed by Bonferroni post hoc test was used for multiple comparison. P-value < 0.05 was considered significant.

Results

Performance and validation of a Biomark-HD SARS-CoV-2 qPCR assay

We first set up a classical Fluidigm qPCR assay protocol that combines all primers/probe sets in a single reaction. This system requires a pre-amplification step after cDNA synthesis. This is due to the low volume of the IFC reaction chamber (9nl). As a result and compared to the traditional RTqPCR protocol (cDNA synthesis and qPCR), three consecutive reaction steps (RT, pre-amplification and qPCR) are required in the classical Biomark-HD assay.

To evaluate the efficiency of the qPCR, we first assessed the test sensitivity using a range of synthetic viral N transcript dilutions with the US CDC primers/probe set N1. We found that the N transcript detection limit was seven transcript copies per reaction (Fig. 1A). Linear regression showed a good correlation across Cq value and dilution series of N transcript (R²=0.9846) (Fig. 1A). Similar sensitivity experiments were performed with additional primers/probes from the US CDC and by diluting the transcript in TE buffer without (Fig. S1A) or with (Fig. S1B) a RNA purification step. Dilution of a SARS-CoV-2 positive clinical sample also indicated a large dynamic range of detection of 6 orders of magnitude for N1 and the E (19) primers/probe (Fig. 1B). We then tested the combination of various primers/probe sets during the pre-amplification reaction, showing that 2 distinct mixes (mix #1: N1, E, human RNP; mix #2: ORF1ab, E, N and human RNP) gave similar data on clinical samples (Fig. 1C).

We finally evaluated the performance of the test on a set of 18 samples from positive patients showing a large range of Cq values. The initial reference analysis was performed using the Elitech Genefinder TM COVID-19 detection kit, comprising 3 sets of viral primers/probe targeting the same viral genes as the mix #2. We noticed excellent correlations between both methods for the detection of the 3 transcripts with R 2 above 0.95 (Fig. 1D), only affected by some high-range samples (i.e. Cq below 5), which displayed a saturation on Biomark. Similarly, we also evaluated the performance of the test using the probe of the viral N gene on a set of 92 purified RNA samples of known status, comprising 15 positive samples, previously analyzed using the GeneFirst COVID-19 detection kit (comprising two sets of primers/probe including the N target). We plotted the correlation for the Cq obtained for the detection of the N transcript using both methods, showing a R 2 of 0.97, with no false positive detected on the remaining 77 negative samples (Fig. S2).

Optimization of the test Additional tests were performed to reduce handling and reaction times by optimizing cycle numbers, elongation times and primer concentrations in the preamplification reaction (Fig. S3). In another optimization, we combined RT and pre-amplification into a one-step reaction, using the Cells Direct One-Step qRT-PCR kit (ThermoFisher Scientific). We compared this method to the classical Fluidigm RT-qPCR on a set of 18 clinical samples with a wide range of Cq values. Very similar Cq values were obtained with both methods for the cellular (RNP) and for 2 viral (E and N) primers/probe sets. The detection limit for ORF1ab gene was even improved using the Cells Direct One-Step qRT-PCR kitbased protocol (Fig. 2A). Overall, this protocol offers the possibility to run 192 samples with 24 couples of primers/probe sets (using the Fluidigm 192. as well as inflammatory / antiviral response genes (CXCL8, IL1A, IL1B, IL6, IFNB1, IFIT1). A set of miRNAs (see Supplemental Table 1 for detail) was also tested (Fig. 3A). We divided the patient samples (n=72) into groups, according to their viral load (strong, medium, weak or negative). ACE2 and TMPRSS2 were detected in all samples, regardless of the viral status, with Cq values close to those for RNP, suggesting they could represent valid human control genes to assess the quality and the presence of epithelial cells in the samples (Fig. 3B-C). The signal obtained for the inflammation and interferon responses was more heterogeneous (Fig. 3B). We particularly noticed a strong signal (in the range of 5 Cq) for IFIT1 in the 3 samples with the highest viral load (Cq values < 10 for the viral probes) (Fig. 3C). Additionally, we evaluated the possibility to quantify miRNA levels in the same samples. A large number of miRNAs were detected in most of the samples (examples in Fig. 3B-C).

Overall, our results demonstrate that cellular markers can be easily quantified in nasopharyngeal swabs and may provide useful information to refine the diagnosis or prognosis of COVID-19 patients.

Direct qPCR detection of SARS-CoV-2 using inactivating lysis buffers-based protocols Direct SARS-CoV-2 diagnostic methods have been described, mainly performing RT-qPCR directly on crude or heat-inactivated subject samples [START_REF] Bruce | Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step[END_REF][START_REF] Smyrlaki | Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR[END_REF][START_REF] Beltrán-Pavez | Accuracy of a RT-qPCR SARS-CoV-2 detection assay without prior RNA extraction[END_REF]. An additional refinement would be to improve the sensitivity and safety of these protocols by the use of RT-qPCR-compatible inactivating lysis buffers containing detergents such as Triton X-100, a standard non-ionic detergent widely used for inactivation of enveloped viruses such as SARS-CoV [START_REF] Darnell | Evaluation of inactivation methods for severe acute respiratory syndrome coronavirus in noncellular blood products[END_REF][START_REF] Colavita | Evaluation of the inactivation effect of Triton X-100 on Ebola virus infectivity[END_REF]. We first compared a regular extractionbased method with a direct RT-qPCR protocol on a synthetic N transcript spike in TE buffer in the presence or the absence of 0.5% Triton X-100 and confirmed that Triton X-100 did not interfere with the efficiency of the RT-qPCR steps (Fig. S4A). We then performed two consecutive nasopharyngeal swab samplings on a COVID-19 diagnosed patient, using either a regular VTM or a TE buffer containing 0.5% Triton X-100 followed or not by a heating step at 65°C for 10 min. As expected, the presence of Triton X-100 and the heating process did not affect the detection of the human RNP transcript for both RT-qPCR methods. Conversely, while the Triton X-100-lysis buffer / heating process slightly improved the signal for the virus N1 primers/probe using the regular extractionbased protocol, this treatment strongly inhibited the direct RT-qPCR method for the same cellular transcript (Fig. S4B), indicating that 0.5% Triton X-100-lysis buffers are not compatible for a sensitive direct SARS-CoV-2 RT-qPCR assay.

We then tested the use of additional detergents and emulsifiers (Tween-20, Brij™-35, Brij™ O10), alone or in combination with polyethylene glycol (PEG600), in the presence or the absence of a treatment with proteinase K (PK), followed by heat inactivation. The experiment was performed on 2 clinical samples of known COVID-19 status, sampled in a commercial VTM medium, aliquoted and then treated in parallel using 16 distinct detergent conditions in the presence or absence of PK, for a total of 64 experimental conditions (Fig. 4 andS5). Additional controls included the crude and heatinactivated samples, the use of Quick Extract TM DNA Extraction Solution (QE), which has been recently proposed as an alternative method to extraction [START_REF] Sentmanat | One-step RNA extraction for RT-qPCR detection of 2019-nCoV[END_REF][START_REF] Ladha | A 5-min RNA preparation method for COVID-19 detection with RT-qPCR[END_REF] as well as a regular extractionbased protocol (positive control). Three and five sets of SARS-CoV-2 (Fig. 4A) and human (Fig. 4B) primers/probe, respectively, were used in the same assay on a 192.24 dynamic array. In these conditions, a direct assay on a crude sample resulted in a 9 to 15-Cq increase, depending on the viral primers/probe set, when compared to the extraction method, with an even more pronounced effect when samples were heated 5 min at 95°C (Fig. 4A). While the addition of 0.5% Triton X-100 alone was similar to the crude direct assay condition, the combination of Triton X-100 with the emulsifier Brij™ O10 (Oleth-10) improved the detection for all viral primers/probes, with a drop of around 5 Cq compared to Triton X-100 alone. All other detergents used alone (Tween-20, Brij™ O10, Brij™-35) gave a similar signal as 0.5% Triton X-100 alone. Of note, the addition of PK resulted in an improvement of sensitivity in the presence of various detergents including Tween-20, Brij™ O10 and Brij™-35 while addition of PEG had no beneficial effect (Fig. 4 and Fig. S5).

Based on these results, we selected two of the best direct detergents based assays (Triton X-100 / Brij™ O10 and Tween-20 / PK) and compared the sensitivity of these methods with a crude direct assay and the extraction method on additional clinical samples. For all direct assays, all samples were heat-inactivated at 95°C (5 min). Moreover, we used a set of 17 clinical samples from SARS-CoV-2diagnosed patients collected in saline solution with a wide range of Cq values. To control pH conditions and limit RNA degradation, all samples were diluted in TE buffer. We plotted the relationships between Cq obtained for the detection of the N and ORF1ab primers/probe sets for the based assays. However, the detection sensitivity still appeared quite high with 11 to 14 positive samples detected. Overall, these data suggest that a direct qPCR method using saline as a VTM and a basic TE buffer followed by a 5 min inactivation step at 95°C can efficiently support detection of SARS-CoV2.

Discussion

The present paper describes a reliable and flexible multiplex nanofluidic qPCR system-based protocol to detect SARS-CoV-2. Its versatile format makes it easily adaptable to multiple other pathogens and / or host cellular markers. The same run allows to test in parallel viral and host RNAs, including miRNAs. We show a high concordance between this method and clinically approved traditional qPCR tests. This assay addressed some of the challenges of RT-qPCR assays, including analyzing a larger number of reactions per run, making the assay more cost-effective and less time-consuming. Further, IFCs dynamic arrays not only reduces the reaction volume from about 10 μL down to the 10 nL scale, but allows large multiplexing as well as increased parallelization throughput of qPCR reactions.

The flexibility of this platform may be used in biomarker studies aiming at predicting at diagnosis the severity (requirement or not for hospitalization/intensive care) or the length (development of socalled "long-COVID") of the disease. Several studies have depicted an elevated innate and adaptive immune activation in severe COVID-19 patients [START_REF] Blanco-Melo | Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19[END_REF][START_REF] Zhou | Heightened Innate Immune Responses in the Respiratory Tract of COVID-19 Patients[END_REF][START_REF] Lucas | Longitudinal analyses reveal immunological misfiring in severe COVID-19[END_REF], and a differential immune phenotype in moderate versus severe disease after the second week of infection [START_REF] Lucas | Longitudinal analyses reveal immunological misfiring in severe COVID-19[END_REF][START_REF] Hadjadj | Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients[END_REF]. The Biomark HD offers the possibility to extend the number of genes tested to biomarkers including pro-inflammatory cytokines, chemokines, interferons, tissue repair genes and miRNAs by nasopharyngeal swabs or in saliva.

An important limitation of current tests is the requirement for RNA extraction that constitutes an obstacle to scale-up the capacity of testing both in term of time and cost. Several groups have explored methods to circumvent RNA extraction by performing RT-qPCR directly on crude or heatinactivated clinical samples [START_REF] Bruce | Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step[END_REF][START_REF] Smyrlaki | Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR[END_REF][START_REF] Beltrán-Pavez | Accuracy of a RT-qPCR SARS-CoV-2 detection assay without prior RNA extraction[END_REF]. Overall these methods show that testing for SARS-CoV-2 infection can be performed without RNA extraction, with a limited loss in accuracy for determining negative and positive cases. While this procedure is simple and attractive, it might be improved by the addition of detergents to facilitate viral capsid lysis to release genomic RNA and also directly inactivate the virus to facilitate sample handling and safety. Several methods and commercial kits have developed approaches to lyse efficiently mammalian cells and directly perform RT-qPCR or RNA-seq libraries. However, these methods are not fully optimized for virus lysis, which requires increased concentrations of detergents. Some studies indicate that Triton X-100, widely used in virus inactivation procedures, or Tween-20 may slightly improve or at least not interfere with the RT-qPCR SARS-CoV-2 direct testing of nasopharyngeal swabs or saliva [START_REF] Smyrlaki | Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR[END_REF][START_REF] Ranoa | Saliva-Based Molecular Testing for SARS-CoV-2 that Bypasses RNA Extraction[END_REF]. However, these initial reports clearly mentioned that additional efforts were needed to optimize direct RT-qPCR assays on detergent-inactivated samples. We evaluated here the use of several detergents and emulsifiers (Triton X-100, Tween-20, Brij™-35, Brij™ O10), alone or in combination, in the presence or the absence of a treatment with PK or polyethylene glycol (PEG) followed by heat inactivation to assess their compatibility with a direct reverse transcription enzymatic step. This initial screening indicated that the addition of each of these detergents, alone, did not improve the assay compared to a direct assay performed on a crude commercial VTM sample. By contrast, the addition of PK improved the detection with all the detergents tested, in agreement with a recent study [START_REF] Lalli | Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric reverse-transcription loop-mediated isothermal amplification[END_REF]. Of note, a combination of Triton X-100 with Brij™ O10, in the absence of PK, had a similar effect, by decreasing the Cqs for the 3 SARS-CoV-2 primers/probes. (Fig. 4A) Brij™ O10 contains Oleth-10, a polyoxyethylene oleyl ether used in aqueous emulsions that may increase solubilization of the viral capsid or contribute to stabilization of the emulsion. When we tested the three best direct protocols on a cohort of samples collected in saline solution, we could not confirm the detection improvement observed in the screening assay using both lysis buffers (Tween-20 / PK and Triton X-100 / Brij™ O10, Fig. 5). This discrepancy may arise from the fact that the screened samples were collected in a commercial VTM while the cohort experiment was performed on biopsies collected in saline buffer.

This difference suggests a complex interaction between the various constituents of this VTM (pH, nature of the medium, presence of albumin, gelatin, anti-bacterial agents, for examples), and the different detergents and emulsifiers tested. Further developments are definitely required to fine tune specific combinations of detergents and emulsifiers with specific VTMs. In any case, our data clearly indicate that a direct qPCR method using saline as a VTM and a basic TE buffer followed by a 5 min inactivation step at 95°C shows almost identical performance to that of a classical extraction-based assay, allowing notably the identification of samples with high Cq values (Fig. 5). Considering that SARS-CoV-2 remains detectable in phosphate buffer for up to a month when stored at various temperatures [START_REF] Rodino | Evaluation of Saline, Phosphate-Buffered Saline, and Minimum Essential Medium as Potential Alternatives to Viral Transport Media for SARS-CoV-2 Testing[END_REF], our data supports the use of PBS, as an alternative to VTM for direct SARS-CoV-2 testing. We further confirm and refine previous studies showing that testing for SARS-CoV-2 infection can be performed with simplified protocols omitting RNA extraction steps without major loss in accuracy [START_REF] Bruce | Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step[END_REF][START_REF] Smyrlaki | Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR[END_REF]. Based on the data presented here, the simplest sample collection and preparation for direct RT-qPCR COVID-19 test would be to sample the swab into a small volume (around 0.5 mL) of TE buffer at a pH of 7.0 to limit dilution of the virus, supplemented with yeast tRNA or RNAse inhibitors to increase RNA stability. This type of buffer was recently shown to have an excellent capacity to preserve the SARS-CoV-2 signal [START_REF] Smyrlaki | Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR[END_REF][START_REF] Rodino | Evaluation of Saline, Phosphate-Buffered Saline, and Minimum Essential Medium as Potential Alternatives to Viral Transport Media for SARS-CoV-2 Testing[END_REF]. Lysis buffers containing detergents would provide the possibility to fully inactivate the virus, allowing a rapid and safe handling of the clinical samples, but our data indicate that their efficiency may vary depending on clinical sample processing and should be further optimized.

We propose that such a direct qPCR procedure may be especially useful for massively scaling up SARS-CoV-2 testing. We believe that this protocol can be very efficiently coupled with pooling approaches [START_REF] Perchetti | Pooling of SARS-CoV-2 samples to increase molecular testing throughput[END_REF], to screen asymptomatic individuals in communities at risk. It would provide an additional tool to enhance testing capacity and affordability across the world, as widely recommended by the Health Community worldwide, such as the "all-in" approach to testing recently proposed (4).

Table 1. List of primers/probe for virus detection. TaqMan probes are labeled at the 5'-end with the reporter molecule 6-carboxyfluorescein (FAM) and with the quencher, Blackhole Quencher 1 (BHQ1) at the 3'end.

Country

  Clinical samples:Pulmonology department, Nice University Hospital: 20 clinical samples from study participants were collected as part of the ELISpot study (ClinicalTrials.gov Identifier: NCT04418206). Participants were enrolled after signing written informed consent. Ethics committee approval was obtained from the "Comité de Protection des Personnes Sud Méditerranée V" (registration # 2020-AO1050-39) on April 22, 2020. Nasopharyngeal swabs were collected in ESwab™ (COPAN) transport medium (2 mL), stored at 4°C between sample collection and transport to the laboratory. One COVID-19 confirmed patient was collected consecutively using 2 different transport media: ESwab TM or a Tris-EDTA (TE: 10 mM Tris HCl pH 7.0, 2 mM EDTA, 20 µg/mL yeast tRNA) buffer.

  24 IFC) corresponding to 4608 end-points in less than 3 hours. Alternative RNA extraction method using the miRNeasy Serum/Plasma Advanced Kit (Qiagen) To limit the possible impact of a shortage of viral RNA extraction kits, we compared the performance of the QIAamp Viral mini kit to that of the miRNeasy Serum/Plasma Advanced Kit. A set of 18 SARS-CoV-2 positive samples with a wide range of Cq values was extracted using both protocols. The 36 resulting RNA samples were run in parallel on the BIOMARK-HD using 3 human and 3 viral primers/ probe sets. Identical Cq values were obtained for the human probe (RNP) with both extraction protocols. A slight gain of signal (around 1-2 Cq) was measured after QIAamp Viral purification for the 3 viral primer/probes (E, N and ORF1ab) (Fig. 2B). These data suggest that the QIAamp Viral RNA mini Kit slightly increases the efficiency of viral transcript extraction, but the miRNeasy Serum/Plasma Advanced Kit represents an acceptable alternative solution in case of shortage. Use of the BIOMARK TM HD-based protocol to analyze the host Response to SARS-CoV-2 infection at the mRNA and miRNA levels Exploiting the potential for multiplexing of the BIOMARK TM HD-based technology, we included sets of human primers/probe to evaluate the expression of genes required for viral entry (ACE2, TMPRSS2),

2 =0

 2 Biomark assay and the GeneFirst COVID-19 detection kit (Fig5). As expected, an excellent correlation was obtained when comparing the RNA extraction-based Fluidigm protocol with the reference assay , all samples detected). Conversely to the data obtained with the commercial VTM, the best direct protocol corresponded to the crude assay (TE), approaching a very similar performance to that obtained with the extraction-based assay (R samples detected). The R 2 values dropped in a dramatic way for the two direct detergents

  Figure 1. Performance and validation of the Biomark-HD SARS-CoV-2 qPCR assay.

Figure 2 .

 2 Figure 2. Optimization of the assay.

Figure 3 .

 3 Figure 3. Use of the Biomark-based protocol to analyze the host Response to SARS-CoV-2 infection

Figure 4 .

 4 Figure 4. Detection of viral and cellular genes using inactivating lysis buffers-based protocols.

Figure 5 .

 5 Figure 5. Correlation of the detection of two viral genes (N, ORF1ab) obtained using a commercial

  et al. Fig.5

Table 2 .

 2 List of primers/probe for cellular genes detection. TaqMan probes are labeled at the 5'-end with the reporter molecule 6-carboxyfluorescein (FAM) and with the quencher, Blackhole Quencher 1 (BHQ1) at the 3'end.

	Target	Name	Name used in this study	Sequence 5' to 3'
		CDC-RP-F		AGATTTGGACCTGCGAGCG
	RnaseP	CDC-RP-R	RNP	GAGCGGCTGTCTCCACAAGT
		CDC-RP-P		TTCTGACCTGAAGGCTCTGCGCG
		TMPRSS2 Forward		TATAGCCTGCGGGGTCAAC
	TMPRSS2	TMPRSS2 Reverse	TMPRSS2	CACTCGGGGGTGATGATGG
		TMPRSS2 Probe		TCAAGCCGCCAGAGCAGGATCGT
		ACE2 Forward		GGCTCCTTCTCAGCCTTGTT
	ACE2	ACE2 Reverse	ACE2	GGTCTTCGGCTTCGTGGTTA
		ACE2 Probe		TGCTGCTCAGTCCACCATTGAGG
		IL1-a Forward		CATTGGCGTTTGAGTCAGCA
	IL1a	IL1-a Revrse	IL1a	CATGGAGTGGGCCATAGCTT
		IL1-a Probe		GTCAAGATGGCCAAAGTTCCAGACA
		IL1b Forward		CAGAAGTACCTGAGCTCGCC
	IL1b	IL1b Reverse	IL1b	AGATTCGTAGCTGGATGCCG
		IL1b Probe		CCAGGACCTGGACCTCTGCCC
		CXCL8 Forward		TGGACCCCAAGGAAAACTGG
	CXCL8	CXCL8 Reverse	CXCL8	TTTGCTTGAAGTTTCACTGGCA
		CXCL8 Probe		GTGCAGAGGGTTGTGGAGAAGTTT
		IL6 Forward		TGCAATAACCACCCCTGACC
	IL6	IL6 Reverse	IL6	GTGCCCATGCTACATTTGCC
		IL6 Probe		TGCCAGCCTGCTGACGAAGC
		IFNb1 Forward		AGTAGGCGACACTGTTCGTG
	IFNb1	IFNb1 Reverse	IFNb1	GCCTCCCATTCAATTGCCAC
		IFNb1 Probe		TGCTCTCCTGTTGTGCTTCTCCA
		IFIT1 Forward		GATCTCAGAGGAGCCTGGCTAA
	IFIT1	IFIT1 Reverse	IFIT1	TGATCATCACCATTTGTACTCATGG
		IFIT1 Probe		CAAAACCCTGCAGAACGGCTGCC

Acknowledgments: Supported by funds from the "Centre National de la Recherche Scientifique" (CNRS), the "Université Côte d'Azur", the French "French Defence Innovation Agency -Agence de l'Innovation de Défense " (project "Safe and direct COV-2 qPCR Test") and the Département des Alpes Maritimes (COVID-19 Health program). JF is supported by the Cancéropole PACA and CL is supported by Plan Cancer 2018 « ARN non-codants en cancérologie: du fondamental au translationnel » (number 18CN045). The Biomark equipment was funded by Canceropole PACA and France Génomique (Commissariat aux Grands Investissements: ANR-10-INBS-6 09-03, ANR-10-INBS-09-02). We thank our colleagues from the IPMC and the Délégation CNRS Côte d'Azur for their help and support, especially Catherine Lecalvez, Michel Bordes, Nadine Pagliano and Simon Szmidt for administrative support and gratefully acknowledge the staff from the Département de Pneumologie of the Nice Hospital, especially Jennifer Griffonet and Charlotte Maniel. We also thank the technical support of the UCA GenomiX platform and MICA imaging facility of the University Côte d'Azur. We are grateful to Bayer SAS for loan of equipment and reagents as well as to Dr Pol-Henri Guivarch and Alexandre Romain (Agence Régionale De Santé PACA) for their support and interesting discussion.

We are also grateful to the "Propagate" consortium, notably Syril D. Pettit, Emily A. Bruce, Jason W Botten and Keith R. Jerome for their support and fruitful discussion.